A Phase 2 Study of MLN0128 (TAK-228) in Relapsed and/or Refractory Acute Lymphoblastic Leukemia (ALL)
Phase: PHASE2
Conditions: B Acute Lymphoblastic Leukemia, B Acute Lymphoblastic Leukemia With t(9;22)(q34.1;q11.2); BCR-ABL1, B Acute Lymphoblastic Leukemia, Philadelphia Chromosome Negative, Recurrent Adult Acute Lymphoblastic Leukemia, Refractory Adult Acute Lymphoblastic Leukemia, T Acute Lymphoblastic Leukemia
Sponsor: National Cancer Institute (NCI)

NCI Protocol #: 9775 
Local Protocol #: MC1482 
NCI Version Date: October 2, 2017 
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A Phase 2 Study of MLN0128 (TAK-228) in Relapsed and/or Refractory Acute 
Lymphoblastic Leukemia (ALL) 
 
 
 
Summary of Changes 
 
MCCC Addendum 3 
 
Investigator Initiated Revisions: 
 
# 
Section 
Revision 
1. 
Global 
Updated protocol headers to the new NCI version date. 
2. 
Title 
EDDOP / Early Drug Development Opportunity Program 
 
 

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A Phase 2 Study of MLN0128 (TAK-228) in Relapsed and/or Refractory Acute 
Lymphoblastic Leukemia (ALL) 
 
 
Corresponding Organization: LAO-MN026 / Mayo Clinic Cancer Center LAO 
 
 
Principal Investigator: 
Aref Al-Kali, M.D. 
 
Mayo Clinic  
 
200 First Street SW 
 
Rochester, MN 55905 
 
Telephone: (507) 284-2511 
 
Telefax: (507) 284-5280 
 
Email:  alkali.aref@mayo.edu 
 
 
 
Participating “Rostered” Organizations  
LAO-11030 / University Health Network Princess Margaret Cancer Center LAO 
LAO-CA043 / City of Hope Comprehensive Cancer Center LAO 
LAO-CT018 / Yale University Cancer Center LAO 
LAO-MA036 / Dana-Farber - Harvard Cancer Center LAO 
LAO-MD017 / JHU Sidney Kimmel Comprehensive Cancer Center LAO 
LAO-NC010 / Duke University - Duke Cancer Institute LAO 
LAO-NJ066 / Rutgers University - Cancer Institute of New Jersey LAO 
LAO-OH007 / Ohio State University Comprehensive Cancer Center LAO 
LAO-PA015 / University of Pittsburgh Cancer Institute LAO 
LAO-TX035 / University of Texas MD Anderson Cancer Center LAO 
LAO-NCI / National Cancer Institute LAO 
EDDOP / Early Drug Development Opportunity Program 
 
 
 
 
 
 
 
 
 
 

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Co-Investigators: 
Cristina Correia, Ph.D.* 
 
Scott H. Kaufmann, M.D., Ph.D. 
 
Mark Litzow, M.D. 
 
Joel M. Reid, Ph.D. * 
 
Andrea Wahner Hendrickson, M.D.* 
 
*Responsible for correlative studies only 
 
 
 
Project Manager 
Statistician 
Sanna McKinzie, M.S. 
Pamela Atherton, M.S. 
Mayo Clinic 
Mayo Clinic 
Telephone: (507) 538-6646 
Telephone: (507) 284-0660 
Email: mckinzie.sanna@mayo.edu 
Email: atherton.pamela@mayo.edu 
 
 
Clinical Trials.gov identifier: NCT02484430 
 
 
NCI-Supplied Agent:  MLN0128 (TAK-228) (NSC# 768435) 
 
IND Sponsor: DCTD, NCI  
 
 
 
Protocol Type / Version # / Version Date: 
 
Original  / Version 1 / May 4, 2015 
Revision / Version 2 / July 1, 2015 
Revision / Version 3 / July 14, 2015 
Revision / Version 4 / August 5, 2015 
Revision / Version 5 / September 10, 2015 
Revision / Version 6 / September 29, 2015 
Revision / Version 7 / May 26, 2016 
Revision / Version 8 / June 21, 2016 
Revision / Version 9 / July 12, 2016  
 
Revision / Version 10 / September 9, 2016 
 
Activation on CTSU October 20, 2016 used Version 10 dated September 9, 2016 
 
Revision / MCCC Addendum 1 / May 9, 2017  
Revision / MCCC Addendum 2 / June 29, 2017 
Revision / MCCC Addendum 3 / October 2, 2017 

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SCHEMA 
 
 
 
 
 
 
 
 
Cycle length = 28 days 
  
Responders = CR/CRi/PR/MLFS 
 
 
 
 
  
 

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TABLE OF CONTENTS 
SCHEMA .........................................................................................................................................4 
TABLE OF CONTENTS .................................................................................................................5 
1. 
OBJECTIVES ......................................................................................................................7 
1.1 
Primary Objectives...................................................................................................7 
1.2 
Secondary Objectives...............................................................................................7 
1.3 
Tertiary Objectives...................................................................................................7 
2. 
BACKGROUND .................................................................................................................8 
2.1 
Acute Lymphoblastic Leukemia ..............................................................................8 
2.2 
MLN0128 (TAK-228) .............................................................................................8 
2.3 
Rationale ................................................................................................................14 
2.4 
Correlative Studies Background ............................................................................16 
3. 
PATIENT SELECTION ....................................................................................................19 
3.1 
Eligibility Criteria ..................................................................................................19 
3.2 
Exclusion Criteria ..................................................................................................21 
3.3 
Inclusion of Women and Minorities ......................................................................22 
4. 
REGISTRATION PROCEDURES ...................................................................................22 
4.1 
Investigator and Research Associate Registration with CTEP ..............................22 
4.2 
Site Registration .....................................................................................................23 
4.3 
Patient Registration ................................................................................................25 
4.4 
General Guidelines.................................................................................................26 
5. 
TREATMENT PLAN ........................................................................................................26 
5.1 
Agent Administration.............................................................................................27 
5.2 
General Concomitant Medication and Supportive Care Guidelines ......................28 
5.3 
Duration of Therapy ...............................................................................................29 
5.4 
Duration of Follow Up ...........................................................................................30 
5.5 
Criteria for Removal from Study ...........................................................................30 
6. 
DOSING DELAYS/DOSE MODIFICATIONS ...............................................................30 
7. 
ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS ............................33 
7.1 
Comprehensive Adverse Events and Potential Risks List (CAEPR).....................34 
7.2 
Adverse Event Characteristics ...............................................................................37 
7.3 
Expedited Adverse Event Reporting ......................................................................38 
7.4 
Routine Adverse Event Reporting .........................................................................40 
7.5 
Secondary Malignancy...........................................................................................40 
7.6 
Second Malignancy ................................................................................................41 
8. 
PHARMACEUTICAL INFORMATION ..........................................................................41 
8.1 
MLN0128 (TAK-228) (NSC # 768435) ................................................................41 

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8.2 
Agent Ordering and Agent Accountability ............................................................42 
9. 
BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES ........................................43 
9.1 
Biomarker Studies ..................................................................................................43 
9.2 
Laboratory Correlative Studies ..............................................................................54 
10. 
STUDY CALENDAR .......................................................................................................59 
11. 
MEASUREMENT OF EFFECT........................................................................................60 
11.1 
Antitumor Effect ....................................................................................................60 
12. 
DATA REPORTING / REGULATORY REQUIREMENTS ...........................................61 
12.1 
Study Oversight .....................................................................................................61 
12.2 
Data Reporting .......................................................................................................62 
12.3 
Collaborative Agreements Language .....................................................................64 
13. 
STATISTICAL CONSIDERATIONS...............................................................................66 
13.1 
Study Design/Endpoints.........................................................................................66 
13.2 
Sample Size/Accrual Rate ......................................................................................68 
13.3 
Stratification Factors ..............................................................................................69 
13.4 
Analysis of Secondary/Tertiary Endpoints ............................................................69 
13.5 
Safety Analysis ......................................................................................................70 
13.6 
Data Monitoring .....................................................................................................71 
13.7 
Reporting and Exclusions ......................................................................................71 
REFERENCES ..............................................................................................................................72 
APPENDIX A: PERFORMANCE STATUS CRITERIA .............................................................77 
APPENDIX B: STUDY MEDICATION CALENDAR................................................................78 
APPENDIX C: PATIENT DRUG INFORMATION HANDOUT AND WALLET CARD ........79 
APPENDIX D: PHARMACOKINETIC BLOOD SAMPLES LIST ............................................81 
APPENDIX E: ADDITIONAL INFORMATION REGARDING PROPOSED BIOMARKER 
STUDIES ...........................................................................................................................84 
 
 
 

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1. 
OBJECTIVES 
 
1.1 
Primary Objectives 
 
1.1.1 To determine the CR/CRi rate when MLN0128 (TAK-228) is administered to adult 
patients with relapsed/refractory ALL. 
 
1.2 
Secondary Objectives 
 
1.2.1 To determine the overall response rate (CR, CRi/PR/morphologic leukemia free state 
[MLFS]). 
 
1.2.2 To determine the CR/CRi duration when MLN0128 (TAK-228) is administered to adult 
patients with relapsed/refractory ALL 
 
1.2.3 To determine the frequency of proceeding to allogeneic SCT for patients with 
relapsed/refractory ALL who achieve a response on MLN0128 (TAK-228) 
 
1.2.4 To determine the overall survival for relapsed/refractory ALL patients on MLN0128 
(TAK-228) 
 
1.3 
Tertiary Objectives 
 
1.3.1 To examine the pharmacokinetics of MLN0128 (TAK-228) in ALL patients. 
 
1.3.2 To assess whether phosphorylation of the mTOR substrate 4EBP1 decreases in leukemic 
blasts harvested from the bone marrow on day 8 compared to baseline. 
 
1.3.3 To assess in an exploratory fashion whether MLN0128 (TAK-228) enhances expression 
of the pro-apoptotic proteins BCL2L11 (Bim)* and BBC3 (Puma) in marrow ALL cells. 
 
1.3.4 To assess in an exploratory fashion whether MCL1 levels decrease in blast cells during 
MLN0128 (TAK-228) treatment.   
 
1.3.5 To assess in an exploratory fashion whether a) the phospho-protein pattern at baseline or 
b) MLN0128 (TAK-228)-associated changes in the phospho-protein pattern differs 
between ALL samples that respond to therapy and those that do not. 
 
 *Please note that Human Gene Organization (HUGO) names have been provided throughout 
this protocol at the request of CTEP reviewers to allow them to track the protocol.  To aid 
scientific and clinical reviewers who might be unfamiliar with the HUGO names, we have 
provided in parentheses throughout the protocol the conventional names of the proteins that are 
being described or analyzed. 
 
 
 

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2. 
BACKGROUND 
 
2.1 
Acute Lymphoblastic Leukemia 
 
Acute lymphoblastic leukemia (ALL) is one of the most aggressive adult leukemias.  
Each year more than six thousand are diagnosed with ALL in the US (1).  One of the 
most commonly used regimens for ALL typically is hyper-CVAD, a regimen consisting 
of cycles of high dose cyclophosphamide, vincristine, doxorubicin and dexamethasone 
alternating with methotrexate and cytarabine, with or without the addition of a Bcr/abl 
kinase inhibitor depending upon the presence or absence of a t(9;22) chromosomal 
translocation (2, 3).  Despite this aggressive treatment, more than half of these patients 
will not survive past four years from diagnosis.  Accordingly, there has been substantial 
interest in testing new therapeutic agents for ALL, including various monoclonal 
antibodies, NOTCH1 inhibitors, hypomethylating agents such as decitabine, folic acid 
antagonists, flavopiridol, bortezomib, and mTOR inhibitors (4, 5).  
 
Despite advances in management, relapsed ALL is very resistant to available therapies.  
In a retrospective study, O’Brien et al reported an 18% complete remission (CR) rate and 
a 3-month median survival rate for relapsed ALL treated at MD Anderson with various 
regimens (6).  Moreover, because the median age at onset for adult ALL is roughly age 
65 (7), a substantial proportion of patients are not candidates for rigorous therapy such as 
hyper-CVAD.  Therefore, novel and targeted therapies are needed for this group of 
patients.  Finally, patients who relapse after allogeneic bone marrow transplant pose a 
difficult therapeutic dilemma, coupled with graft versus host disease and/or difficulty to 
find a second donor, making them suitable candidates for clinical trials of novel agents. 
 
2.2 
MLN0128 (TAK-228) 
 
2.2.1 Mechanism of Action 
 
MLN0128 (TAK-228) is an inhibitor of the mechanistic target of rapamycin (mTOR).  
This serine/threonine kinase is a major integrator of various types of signals within cells 
(8). Two distinct complexes exist, mTOR complex 1 and 2 (TORC1 and TORC2) (9). 
TORC1 phosphorylates p70 S6 kinase and eukaryotic translation initiation factor 4E 
(eIF4E) binding protein 1 (4EBP1) to regulate protein synthesis and, indirectly, 
progression from G1 into S phase, while TORC2 phosphorylates AKT on Ser473, 
contributing to activation of this kinase and affecting survival pathways. MLN0128 
(TAK-228) (previously called MLN0128; INK-128) has been shown to target both 
mTORC1 and mTORC2 (10-14), which is thought to provide improved inhibition of 
mTOR and enhanced cytotoxicity in lymphoid malignancies as described below. 
 
MLN0128 (TAK-228) inhibited mTOR kinase in vitro with an IC50 of 1.1 nM.  It was 
100- to 1000-fold less potent against class I, class II and class III phosphatidylinositol-3 
(PI3) kinase activities as well as PI4 kinase isoforms (15).  Among a panel of 222 protein 
kinases, TAK-228 inhibited only 4 kinases (DNA-PK, PDGFRα, Flt3 and CK1ε) in 
addition to mTOR by 80% at 1 µM. 

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2.2.2 Nonclinical Studies 
 
Preclinical efficacy studies (15): MLN0128 (TAK-228), like other dual mTOR inhibitors, 
inhibits proliferation in a wide range of human tumor cell lines in vitro.  In particular, 
when examined in 105 cell lines, MLN0128 (TAK-228) inhibited growth with IC50 
values ranging from 1 nM (SW620 colorectal cancer cells) to 2700 nM (HepG2 
hepatoma cells).  
 
Immunoblotting studies in PC-3 prostate cancer, T47D breast cancer, 786-O renal cell 
carcinoma lines and multiple myeloma cell lines demonstrated that MLN0128 (TAK-
228) inhibits phosphorylation of the translation inhibitor 4EBP1 (an mTORC1 substrate) 
and the serine/threonine kinase AKT (Ser473, an mTORC2 substrate) in intact cells, with 
almost complete inhibition at 100 nM (10, 16).  In contrast, the allosteric mTORC1 
inhibitor rapamycin inhibited phosphorylation of ribosomal S6 (downstream of another 
mTORC1 substrate p70 S6 kinase) but had little impact on either 4EBP1 or AKT Ser473 
phosphorylation.  Likewise, when administered to mice bearing U87G glioblastoma 
xenografts, MLN0128 (TAK-228) inhibited phosphorylation of 4EBP1, ribosomal 
protein S6 and AKT Ser473 at 1, 2, 4 and 8 hours, although some recovery of signal was 
observed at 24 h. 
 
In vivo efficacy studies in mice bearing of human tumor xenografts, including U87MG 
(PTEN-mutant glioblastoma), A549 (KRAS/LKB1-mutant NSCLC), ZR-75-1 (PTEN-
mutant breast cancer), MDA-MB361 (PIK3CA/HER2-mutant breast cancer), 786-O 
(PTEN/VHL-mutant renal cell carcinoma) and HEC-1 (PIK3CA/KRAS-mutant 
endometrial cancer) MLN0128 (TAK-228) demonstrated strong tumor growth inhibition 
at tolerable oral doses ranging from 0.15 mg/kg/d to 3 mg/kg every other day or 3 mg/kg 
every week.   
 
Preclinical toxicology studies: Preclinical toxicology studies were performed in rats and 
monkeys.  The dose-limiting toxicities in single-agent toxicology studies in both species 
were GI distress, decreased activity, hypothermia, decreased food intake, hyperglycemia, 
hyperinsulinemia, and lymphoid depletion.  
 
In 28-day GLP-compliant toxicology studies, rats treated with 3.0 mg/kg MLN0128 
(TAK-228) exhibited reduced body weights compared to controls; increases in aspartate 
aminotransferase (AST) that was attributed to nonspecific muscle injury (creatine kinase 
was also higher in affected animals); increases in glucose and insulin; decreases in 
leukocytes, lymphocytes, and eosinophils; and microscopic changes in the bone marrow   
(hypocellularity), thymus (lymphoid necrosis), testes (degeneration/atrophy), 
epididymides (oligospermia), nonglandular stomach (epithelial degeneration/ulceration/ 
hyperplasia; males only), and lungs (alveolar histiocytosis). 
 
In 28-day GLP-compliant toxicology studies in cynomolgus monkeys, MLN0128 (TAK-
228)-related effects observed at 0.30 mg/kg included decreased body weight, appetite, 
activity, and body temperature, abnormal posture, and skin cold to touch.  Two of 10 

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monkeys treated at this dose level had to be euthanized in day 18 and 19.  Clinical 
chemistry changes included increased serum AST, creatine kinase MM isoform, 
cholesterol, triglycerides, and insulin, and decreased serum sodium, potassium, and 
phosphorous.  Hematology parameter alterations consisted of decreased lymphocytes 
(females only) and increased leukocytes and neutrophils, with sporadic increases in 
monocytes and eosinophils.  Microscopic changes at 0.15 and 0.3 mg/kg were seen in the 
thymus, spleen, lymph nodes, gut-associated lymphoid tissue (0.30 mg/kg only), bone 
marrow, adrenal glands, salivary glands, pancreas, GI tract, and skin (0.3 mg/kg only).  
Specific MLN0128 (TAK-228)-related microscopic findings in the lymphoid organs, 
including gut-associated lymphoid tissue, were multicentric lymphoid depletion and 
splenic red pulp depletion.  At 0.30 mg/kg, the spleen had moderate to severe depletion of 
RBCs and hematopoietic cells.  These changes correlated with decreased spleen weights 
in males at 0.30 mg/kg and decreased thymus weights in females at 0.15 and 0.30 mg/kg.  
Other microscopic changes included mixed cell depletion in bone marrow; minimal to 
moderate bilateral cortical hyperplasia in adrenals (thought to be stress induced); acinar 
cell secretory depletion in the pancreas, parotid and mandibular salivary glands (thought 
possibly due to decreased food consumption); erosion/ulceration, edema, hemorrhage, 
and acute inflammation of the cardia and pylorus of the stomach; and mild and sporadic 
changes in the duodenum, jejunum, colon, and rectum.  At 0.3 mg/kg, some monkeys 
exhibited mild to moderate erosion/ulceration and minimal to moderate epidermal 
hyperplasia of the skin on the face, neck, and forelimbs.  Electrocardiography 
demonstrated no MLN0128 (TAK-228)-related effects, consistent with lack of significant 
findings in a GLP-compliant cardiovascular safety pharmacology study conducted in 
conscious monkeys. 
 
2.2.3 Clinical Studies  
 
MLN0128 (TAK-228) has been evaluated in two single-agent oral dose-finding studies, 
one in patients with advanced solid malignancies and one in patients with relapsed and/or 
refractory multiple myeloma, non-Hodgkin lymphoma, and Waldenstrom’s 
macroglobulinemia.  
 
In INK128-001, single-agent MLN0128 (TAK-228) has been administered to patients 
with advanced solid tumors in 28-day cycles according to four schedules:  q day, q week, 
3 consecutive days q week, and 5 consecutive days q week.  The maximum tolerated 
doses were 6 mg q day, 40 mg q week, 16 mg q day x 3 each week, and 10 mg q day x 5 
each week (15).  
 
Treatment related adverse events, serious adverse events, and events causing withdrawal 
from study INK128-001 (15): In this study, ≥ 1 treatment-emergent adverse event 
(TEAE) was reported for 139 (98%) of 142 treated patients.  Across the dosing groups, 
the most frequent TEAEs were hyperglycemia (65% of patients), nausea (63% of 
patients), and vomiting (54%).  In general, TEAEs were mostly Grade 1 or 2 and 
manageable with supportive care and/or interruption or dose reduction of study drug.  
Severity ≥ Grade 3 TEAEs (SAEs) that were reported in 6% or more of patients, 
regardless of causality, were hyperglycemia (13% of patients), asthenia (8%), anemia 

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(7%), and hypophosphatemia (6%) and lymphopenia (6%).  As of the clinical database 
cutoff date, SAEs had been reported for 58 patients (41%), with the most common terms 
being mucosal inflammation (4%), asthenia (3%), pneumonia (3%), abdominal pain 
(2%), stomatitis (2%), and renal failure (2%).  Of the 142 patients treated in Study 
INK128-001 as of the clinical data cutoff, 29 (20%) discontinued because of 1 or more 
AEs, which included rash (6 patients), mucosal inflammation (4 patients), asthenia (3 
patients), hyperglycemia (2 patients), nausea (2 patients), performance status decreased 
(2 patients), pruritus (2 patients), and acute renal failure (2 patients).  Of the total 40 AEs 
present at study withdrawal, 21 events (53%, including 11 nonserious AEs) were reported 
as severity Grade 3, and 4 SAEs were Grade 5 (fatal).  
 
A death on INK128-001 due to ventricular arrhythmia and cardiac arrest was assessed as 
possibly related to MLN0128 (TAK-228).  After discussion with the FDA, study C31002, 
a phase 1 single-arm study to evaluate the effect of a single dose of 40 mg MLN0128 
(TAK-228) on the QT/QTc interval was initiated in patients with advanced solid tumors.  
After completing the per-protocol PK/ECG/cardiac contractility monitoring, the patients 
continued MLN0128 (TAK-228) 30 mg QW with continued cardiac monitoring.  The 
study results showed that treatment with MLN0128 (TAK-228) was not associated with 
clinically meaningful effects on the overall electrocardiographic safety profile, and that 
ECHO/MUGA at screening is not required on MLN0128 (TAK-228) trials.  Takeda and 
CTEP have agreed that ECG monitoring should be performed on patients prior to 
entering MLN0128 (TAK-228) trials, at the time of completing treatment with MLN0128 
(TAK-228), and periodically during the trial when clinically appropriate.     
 
In INK128-002, which was reported by Ghobrial et al. (17) MLN0128 (TAK-228) was 
administered orally to 37 patients in two different schedules, either daily or three days 
on/four days off every week schedule with 28-day cycles.  The maximum tolerated dose 
(MTD) was 6 mg for the daily schedule and was not defined in the 3 days on/four days 
off schedule.  Common side effects included nausea (56%), fatigue (49%), 
hyperglycemia (38%), thrombocytopenia (36%), diarrhea (28%), anorexia (23%), emesis 
(23%), stomatitis (21%), anemia (21%), and constipation (18%). 27% discontinued drug 
due to an adverse event.  Among 27 patients evaluable for confirmed responses, 1 
multiple myeloma patient (7 mg QD) achieved a major response lasting 2 months, and 13 
myeloma patients (2 at 2 mg QD, 4 each at 4 mg QD and 7 mg QD, 2 at 9 mg QDx3d 
QW, and 1 at 12 mg QDx3d QW) and 2 Waldenstrom’s macroglobulinemia patients (at 6 
mg QD and 9 mg QDx3d QW) had stable disease. 
 
Subsequent experience with the daily schedule has indicated that MLN0128 (TAK-228) 
at 6 mg/day is intolerable for prolonged administration in some patients (L. A. Doyle, 
personal communication).  Accordingly, the recommended phase 2 dose is 5 mg/day for 
daily administration. 
 
2.2.4 Nonclinical Pharmacokinetics  
 
Preclinical pharmacokinetic studies were examined in mice, rats, dogs, and monkeys.  
After intravenous administration, MLN0128 (TAK-228) displayed a volume of 

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distribution of 1.4 (dogs) to 14.3 liters/kg (rats) and a terminal half-life of 1.1 (mice) to 
18.9 hours (dogs).  These studies also demonstrated that MLN0128 (TAK-228) is rapidly 
absorbed from the GI tract and displays high bioavailability (64-73%) in all species tested 
except rats (15).  MLN0128 (TAK-228) crossed the blood-brain barrier poorly, with 
plasma:brain concentrations of 5:1 in mice receiving a single oral dose (15). 
 
MLN0128 (TAK-228) binding to plasma proteins was examined across species.  Plasma 
protein binding ranged from 47% (dogs) to 70.4% (humans).  MLN0128 (TAK-228) 
inhibited both drug exporters and drug importers, but only at concentrations that far 
exceed the anticipated clinical concentrations.  In particular, MLN0128 (TAK-228) 
inhibited the exporter ABCG2 (BCRP)with an IC50 of 52 micromolar and the drug 
importers OCT1 and OCT2 with IC50s of 19-28 micromolar and 1.9 micromolar, 
respectively (15). 
 
2.2.5 Clinical Pharmacokinetics 
 
Pharmacokinetic analysis performed in conjunction with the INK128-001 study 
demonstrated rapid oral absorption, with a Cmax at approximately 2 h, and a mean 
terminal half-life in the plasma of 6-8 hours across dose levels (15).  After the initial 
dose, exposures (measured as the area under the plasma concentration vs. time curve) 
were dose-proportional over the range of 2 to 40 mg.  MLN0128 (TAK-228) did not 
accumulate to any appreciable extent with repeated oral dosing. 
 
Pharmacokinetic analysis performed in conjunction with the INK128-002 study in 
hematological malignancies yielded similar results (15).  After oral administration, Cmax 
was achieved within 1-2 hours.  Once again the mean plasma half-life was 8 hours and a 
dose-dependent increase in exposure was observed.  The mean plasma Cmax at the MTD 
of 6 mg/d was 46 ng/ml on cycle 1/day 1 and 31 ng/ml on cycle 2/day 1, corresponding to 
plasma levels of 150 and 100 nM, respectively.  These results again indicate that 
MLN0128 (TAK-228) did not accumulate to any appreciable extent during daily oral 
dosing.  
 
Metabolism in humans 
Incubation of MLN0128 with human liver microsomes results in a single metabolite (M1) 
that is equipotent with the parent agent in inhibiting mTOR kinase.  While M1 represents 
as much as 30% of circulating drug in rats, it represents only 4% of circulating drug in 
humans and is not felt to contribute significantly to MLN0128 (TAK-228) 
pharmacodynamic effects (15). 
 
The capacity for MLN0128 (TAK-228) to interact with the cytochrome P450 drug 
metabolizing machinery has been examined (15).  In a study using recombinant human 
isozymes, CYP2C9, 2C19, and 3A4 metabolized MLN0128 in vitro, whereas CYP1A2, 
2B6, and 2C8 did not. MLN0128 (TAK-228) (up to 10 µ M) did not induce CYP1A2, 
2B6, and 3A4 activity and expression in human hepatocytes.  MLN0128 (TAK-228) did 
not inhibit CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, or 3A4/5 in human liver microsomal 
incubations. 

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Potential for drug interactions 
Based on the high concentrations required to inhibitor drug transporters, as well as the 
lack of induction or inhibition of commonly studied cytochrome P450 isoenzymes, 
MLN0128 (TAK-228) is felt to have limited potential for drug-drug interactions at the 
anticipated therapeutic concentrations (15).  
 
2.2.6 Rationale for Proposed Starting Dose 
 
Preclinical studies have demonstrated that prolonged mTOR inhibition (at least 72 hours) 
is required to see signs of apoptosis induction in ALL cell lines by dual 
mTORC1/mTORC2 inhibitors in vitro (18).  Upon administration of these inhibitors to 
mice bearing human lymphoid xenografts, xenograft regression is not evident until days 
6-8 but continues throughout the course of treatment (18).  These observations, along 
with studies in murine Ph+ ALL showing efficacy of daily dosing (12), prompt this trial 
of MLN0128 (TAK-228) dosed on a daily schedule. 
 
Preclinical studies in the Kaufmann laboratory have demonstrated that uptake of the 
TORC1/TORC2 dual inhibitor OSI-027 is diminished in three of four lymphoid lines 
selected for resistance by continuous exposure.  These lines are cross-resistant to 
MLN0128 (TAK-228).  The uptake defect is reversed by drug removal for 3 days.  These 
observations lead to the proposal to administer MLN0128 (TAK-228) for 21 of every 28-
day cycle. 
 
2.2.7 Milled MLN0128 (TAK-228) Drug Substance (DS, process B)  
 
In order to allow more predictable absorption of MLN0128 (TAK-228) after oral 
administration and to allow scale-up manufacturing of MLN0128 (TAK-228) capsules, 
Millennium/Takeda developed a new milled formulation of the agent.  The physical 
milling step during the granulation process controls particle size distribution of 
MLN0128 (TAK-228).  In order to observe whether this milling step altered the safety 
and PK profile of MLN0128 (TAK-228), the company performed in vivo studies with PK 
analysis of milled MLN0128 (TAK-228).  These studies indicated that the milled 
formulation may result in faster absorption with possibly higher maximum concentration 
(Cmax), which could result in a different safety profile, compared to the previous 
unmilled API capsules. In rats, the mean AUC milled/unmilled was 2.97    while in 
monkeys, it was     he Cmax ratios were    
Takeda developed new MLN0128 (TAK-228) capsules containing milled active 
pharmaceutical ingredient (API) for clinical studies in 1 mg, 3 mg, and 5 mg strengths.  
Patients receiving the milled formulation were added onto ongoing studies C31001 and 
C31002, as well as a new study MLN0128-1004, to determine the PK parameters of 
milled and unmilled MLN0128 in patients administered single doses under both fasted 
and fed conditions, with various treatment cohorts including daily and weekly 
administration of milled MLN0128 (TAK-228) 
The recommended dose of milled MLN0128 (TAK-228) was evaluated in 17 patients of 

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MLN0128-1004, with PK, safety, and tolerability assessed.  Six patients were given a 4 
mg QD dose of milled MLN0128 (TAK-228) and 3 patients had observed DLT (rash, 
appetite loss and fatigue).  A dose of 3 mg QD was given to 11 patients with only 1 DLT 
(decreased platelets) observed.  The 3 mg QD dose of MLN0128 (TAK-228) was 
declared the RP2D, and was generally well tolerated and demonstrating objective 
responses in patients.   
 
A significant and meaningful difference in tolerability was observed in the comparison of 
the MTDs between unmilled and milled MLN0128 (TAK-228) when administered QD.  
This difference in tolerability may be possibly explained due to the effect of food on the 
safety/tolerability of unmilled MLN0128 (TAK-228) in study IND128-001.  The 
GastroPlusTM simulation performed on study suggests that under fasting conditions, 
unmilled and milled MLN0128 (TAK-228) administrations result in comparable 
exposures to MLN0128 (TAK-228); whereas in the fed state, milled MLN0128 (TAK-
228) resulted in higher Cmax (1.5- to 2-fold higher) and earlier Tmax than unmilled 
MLN0128 (TAK-228) with comparable AUCs.  Consequently, a dose of 3 mg QD was 
chosen as the RP2D of milled MLN0128 (TAK-228) dose in empty stomach conditions.   
 
The RP2D for milled MLN0128 (TAK-228) on a weekly schedule was determined to be 
30 mg, the same weekly RP2D as seen for the older unmilled formulation.  Six patients 
treated at 30 mg weekly with the milled formulation did not demonstrate any DLT, but 
the agent was not escalated further.  No DLT had been demonstrated for milled 
MLN0128 (TAK-228) at the prior 20 mg QW dose as well. 
 
 
2.3 
Rationale 
 
A number of studies (19-24) have demonstrated that the rapalogs temsirolimus and 
everolimus, which inhibit TORC1, have clinical activity in lymphoid malignancies.  
Unfortunately, fewer than 5 patients with ALL were treated with rapamycin analogs in 
phase I clinical trials (25), so the single-agent activity of rapalogs in ALL is unknown.  
 
The rationale for the development of TORC1/TORC2 dual inhibitors has been 
extensively reviewed (26-33).  In brief, after binding to FKBP12, rapalogs inhibit 
TORC1 but do not directly inhibit TORC2.  As a result, there has been concern that 
rapalogs can activate survival pathways, either through increased TORC2-mediated 
phosphorylation of AKT or through derepression of insulin receptor substrate- (IRS-) 
mediated signaling to the PI3 kinase/AKT pathway in some cell types (34-36).  To 
overcome this potential limitation of rapalogs, agents that simultaneously target TORC1 
and TORC2 have been developed. OSI-027 and MLN0128 (TAK-228) are two such 
inhibitors.  Both are ATP-competitive inhibitors of TORC1 and TORC2, but MLN0128 
(TAK-228) is approximately 20-fold more potent. 
 
Preclinical studies performed in vitro and in vivo have demonstrated that dual mTOR 
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certain lymphoid neoplasms (18, 37).  When Gupta et al. compared the effects of dual 
mTOR inhibitor OSI-027 in cell lines derived from various lymphoid malignancies, 
including T-cell ALL (Jurkat, CEM, Molt3), B-cell ALL (Nalm6), diffuse large B cell 
lymphoma (HT, RL) and mantle cell lymphoma (Jeko, Mino), the ALL lines were 
consistently more sensitive than the other lymphoid cell lines in 5-day MTS assays (18).  
Subsequent experiments demonstrated that OSI-027, but not rapamycin, induced 
apoptosis in the ALL lines that was mediated by the upregulation of the pro-apoptotic 
BCL2 family members BCL2L11 (Bim) and BBC3 (Puma) (18).  Further investigation 
has shown that BCL2L11 (Bim) upregulation reflects transactivation of the BCL2L11 
(Bim) promoter downstream of mTORC2 inhibition, whereas BBC3 (Puma) upregulation 
reflects inhibition of mTORC1-mediated 4EBP1 phosphorylation and consequent 
inhibition of translation of a BBC3 (Puma) suppressor (14).  
 
Two studies performed in nude mice bearing Jeko (human mantle cell lymphoma) 
xenografts demonstrated marked tumor regressions, associated with induction of 
BCL2L11 (Bim) and BBC3 (Puma), after treatment with OSI-027 but not rapamycin 
(18). In contrast, the RL diffuse large B-cell lymphoma cell line, which contains a 
t(14;18) translocation that results in marked upregulation of a BCL2 protein with a gain-
of-function mutation (38), did not respond to OSI-027 in vivo (18). 
 
In further preclinical studies, clinical samples from patients with various lymphoid 
neoplasms were exposed to varying OSI-027 concentrations ex vivo and examined for 
viability at the end of 5 days. OSI-027 diminished MTS reduction in a variety of samples, 
including ALL, diffuse large B cell lymphoma, mantle cell lymphoma, marginal zone 
lymphoma, and Sezary syndrome, with ALL samples being among the most sensitive 
(18). In contrast, CLL and follicular lymphoma were highly resistant. 
 
In a more detailed study in primary ALL samples using the same assay (14), IC50 values 
ranged from 80-2000 nM for OSI-027 and 2-100 nM for MLN0128 (TAK-228). Clinical 
ALL isolates were generally as sensitive as Jurkat cells (14), an intermediate sensitivity 
T-cell ALL line (18).  Direct comparison of the effects of 250 nM MLN0128 (TAK-228) 
and 10 nM rapamycin (the highest sustainable concentrations in humans) revealed that 
MLN0128 (TAK-228) exhibited greater cytotoxic effects in all six  clinical ALL isolates 
treated with both agents.  
 
Further analysis indicated that similar biochemical effects were observed in ALL cell 
lines and primary ALL samples after treatment with mTORC1/mTORC2 dual inhibitors 
in vitro (14).  In particular, MLN0128 (TAK-228) abolished 4EBP1 phosphorylation in 
Jurkat T-cell ALL cells at 200 nM and in primary ALL cells at 62.5 nM in vitro (N. 
Vincelette, K. Peterson and S. Kaufmann, unpublished observations).  These observations 
provide part of the rationale for the correlative studies described in Sections 2.4 and 9 of 
this protocol 
 
In a separate study, MLN0128 (TAK-228) suppressed proliferation of B-ALL cell lines 
and reduced colony formation by primary human leukemia cells from adult and pediatric 
B-ALL patients in vitro (39).  In a syngeneic mouse model of lymphoid BCR-ABL 

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positive disease, MLN0128 (TAK-228) cleared leukemic growth.  In this and additional 
in vivo models, MLN0128 (TAK-228) was well tolerated and did not suppress 
endogenous bone marrow proliferation.  
 
Given the sensitivity of ALL cells and clinical ALL samples to TORC1/TORC2 dual 
inhibitors described above, as well as the limited treatment options for patients with 
relapsed ALL, the present study is a phase II trial designed to assess the activity of 
single-agent MLN0128 (TAK-228) in relapsed and refractory ALL.  
 
2.4 
Correlative Studies Background 
 
2.4.1 Plasma and Marrow Pharmacokinetics 
 
Rationale:  These studies are necessary to characterize the pharmacokinetics of 
MLN0128 (TAK-228) in patients with acute lymphoblastic leukemia, to provide a basis 
for understanding differences in toxicity and responses to MLN0128 (TAK-228) and to 
assess pharmacodynamic relationships with phosphorylated mTOR substrates and 
expression levels of BCL2L11 (Bim), BBC3 (Puma), MCL1 and BCL2. 
 
Assay description:  Plasma and marrow concentrations of MLN0128 (TAK-228) will be 
measured by lc/ms/ms methods developed in the Mayo Clinic Cancer Center 
Pharmacology Shared Resource.  
 
2.4.2 Phosphorylation of the mTOR Substrate 4EBP1 (Integrated Biomarker) 
 
Rationale: By comparing the phosphorylation status of 4EBP1 in marrows harvested 
prior to treatment and again on day 8, we will assess whether levels of MLN0128 (TAK-
228) sufficient to inhibit mTOR have been achieved in leukemic blasts in situ.  
Conversely, we have observed that Nalm6 B-cell ALL cells selected for resistance to 
mTORC1/mTORC2 dual inhibitors fail to exhibit inhibition of 4EBP1 phosphorylation 
upon treatment with OSI-027 or MLN0128 (TAK-228), suggesting that failure to inhibit 
the mTORC1/4EBP1 axis might represent a mechanism of resistance to this class of 
agent.  Thus, this assay is being performed to confirm mechanism of action of MLN0128 
(TAK-228) as well as assess a potential readout for resistance to this class of agent. 
 
Assay description:  Immunoblotting will be performed for phospho-Thr37,46-4EBP1 using 
techniques previously described by the Kaufmann laboratory (40, 41).  Whole cell lysates 
(42, 43) will be prepared from Ficoll/Hypaque-purified marrow mononuclear cells 
(assayed for blast percentage by morphological criteria).  Aliquots containing equal 
amounts of protein from cells harvested prior to treatment and on day 8 will be subjected 
to SDS-PAGE in adjacent wells of gels and probed for phospho-Thr37,46-4EBP1 and total 
4EBP1. Further details related to the assay are found in Section 9.1.3 of this protocol.   
 
2.4.3 Phosphorylation of the mTOR Substrate AKT Ser473 (Exploratory Biomarker) 
 

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Rationale: the assay for phosphorylated 4EBP1 examines inhibition of mTORC1.  As 
indicated in Section 2.3, mTORC1/mTORC2 dual inhibitors have been developed based 
on the rationale that inhibiting mTORC2 will prevent feedback-mediated upregulation of 
pro-survival pathways.  In addition, recent results have demonstrated that mTORC2 
inhibition plays a major role in the MLN0128 (TAK-228)-induced upregulation of the 
proapoptotic BCL2 family member BCL2L11 (Bim) (14).  By comparing the 
phosphorylation of AKT (Ser473) in marrows harvested prior to treatment and again on 
day 8, we will assess whether TORC2 has also been inhibited in leukemic blasts in situ.  
Thus, this assay is being performed to provide further proof of the mechanism of action 
of this agent in clinical ALL in situ. 
 
Assay description: Whole cell lysates prepared from Ficoll/Hypaque-purified marrow 
mononuclear cells prior to treatment and on day 8 will subjected to immunoblotting for 
phospho-Ser473-AKT and total AKT. Further details regarding the proposed assay are 
contained in Section 9.1.4. 
 
2.4.4 Expression of BCL2L11 (Bim), BBC3 (Puma), MCL1 and BCL2 (Exploratory 
Biomarkers) 
 
Rationale: Previous studies have demonstrated that the mTORC1/mTORC2 dual 
inhibitors induce apoptosis in vitro and in xenograft models by upregulating the pro-
apoptotic BCL2 family members BCL2L11 (Bim) and BBC3 (Puma) as well as, in some 
lymphoid lines, down regulating MCL1 (14, 18).  Importantly, blunting BCL2L11 (Bim) 
or BBC3 (Puma) upregulation diminishes mTORC1/mTORC2 dual inhibitor-induced 
apoptosis (14, 18).  By comparing expression of BCL2L11 (Bim), BBC3 (Puma) and 
MCL1 at the protein level by immunoblotting in marrows harvested prior to treatment 
and again on day 8, we will begin to assess whether the mechanisms of induction of 
apoptosis observed in preclinical studies are also observed in ALL in situ.  
 
Conversely, prior results have shown that BCL2 overexpression is a mechanism of 
resistance to this class of agent (14, 18).  Thus, assessment of pretreatment BCL2 as well 
as the degree of upregulation of BCL2L11 (Bim) and BBC3 (Puma) will begin to 
examine potential mechanisms of resistance as well. 
 
Assay description:  Immunoblotting will be performed for BCL2L11 (Bim), BBC3 
(Puma), MCL1 and BCL2 (40, 41).  As described in Section 2.4.2, whole cell lysates (42, 
43) will be prepared from Ficoll/Hypaque-purified marrow mononuclear cells (assayed 
for blast percentage by morphological criteria).  Aliquots containing equal amounts of 
protein from cells harvested prior to treatment and on day 8 will be subjected to SDS-
PAGE in adjacent wells of gels and probed with commercial antibodies to BCL2L11 
(Bim), BBC3 (Puma), MCL1, and BCL2.  Blots will be reprobed for histone H1, a 
protein that should be present in similar amounts in different diploid cells, to provide a 
loading control.  Further details regarding this assay can be found in Section 9.1.5 of this 
protocol.  If sufficient material remains after this assay, additional BCL2 family members 
not currently implicated in mTOR inhibitor action in ALL samples, such as BCL2L1 

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(Bcl-xL) and BCL2L2 (Bcl-w) that could in principle affect the outcome of treatment, 
will be considered for assessment in a similar fashion. 
 
2.4.5 Phosphoproteomic Analysis (Exploratory Biomarker) 
 
Rationale:  Prior publications have identified differences in the pattern of protein 
phosphorylation between cell lines that are sensitive to kinase inhibitors and similar cell 
lines that are resistant (e.g., 44, 45).  Preliminary studies in the Kaufmann laboratory 
have likewise demonstrated differences in protein phosphorylation at key nodes between 
parental Nalm6 B-cell ALL cells and Nalm6 cells selected for the ability to grow 
continuously in 8 µM OSI-027 (C. Correia, K. L. B. Knorr, S. Yun, A. E. Wahner 
Hendrickson, B. Madden, D. McCormick, S. Dasari, and S. H. Kaufmann, unpublished 
observations).  Thus, we hypothesize that pretreatment patterns of phosphorylation might 
differ between ALLs that respond to MLN0128 and those that do not. 
 
Assay description and analysis:  Bone marrow aspirates harvested prior to therapy and 
again on day 8 of treatment will be enriched for leukemic blasts on Ficoll-Hypaque 
gradients.  Whole cell lysates prepared as described in Sections 2.2 and 2.3 will be 
analyzed for a set of more than 60 key phosphorylations and 200 key regulatory proteins 
by reverse phase protein array (RPPA) in the M.D. Anderson RPPA Core Facility. 
Further information regarding this assay is found in Section 9.1.6. 
 
2.4.6 Transcriptional Profiling (Exploratory Biomarker) 
 
Rationale:  Recent publications have identified additional changes that correlate with 
resistance to mTOR dual inhibitors, including overexpression of eIF4E (46).  To search 
for changes such as this and provide an opportunity for further unbiased analysis of 
potential markers of MLN0128 sensitivity vs. resistance, samples (when cell number is 
sufficient) will be saved for transcriptional profiling to assess whether there is a particular 
gene expression profile that correlates with sensitivity vs. resistance to MLN0128 (TAK-
228). 
 
Assay description and analysis:  Bone marrow aspirates harvested prior to therapy and 
again on day 8 of treatment will be enriched for leukemic blasts on Ficoll-Hypaque 
gradients. Samples will be lysed in RNAlater (Qiagen) and stored at -80 °C until 
analyzed.  Further analysis will be performed only if the studies above do not yield robust 
testable hypotheses regarding responders vs. nonresponders. A further description of this 
assay is provided in Section 9.1.7. 
 
2.4.7 Prioritization of Cell-Based Correlative Studies 
 
The cell-based correlative studies are designed to provide a test of mechanism.  
According to current understanding, the killing of ALL cells by mTORC1/mTORC2 dual 
inhibitors depends upon the activation of two pathways, one involving drug-induced 
inhibition of mTORC1-mediated 4EBP1 phosphorylation, leading sequentially to 
decreased 4EBP1 phosphorylation, decreased eIF4E-facilitated translation of cMYC 

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mRNA, decreased c-MYC protein, increased BBC3 (Puma) mRNA stability and 
increased BBC3 (Puma) protein, and another pathway involving decreased mTORC2-
mediated phosphorylation of a transcription factor cascade leading to increased activation 
of the BCL2L11 promoter,  increased BCL2L11 (Bim) mRNA, and increased BCL2L11 
(Bim) protein.  One or both pathways are engaged in all clinical ALL samples exposed to 
MLN0128 (TAK-228) ex vivo to date.  To assure that the mechanism of action is 
thoroughly investigated in paired clinical samples, the order of priority will be to assay 
first those antigens that have been directly implicated in mTORC1/mTORC2 dual 
inhibitor induced killing in human ALL cells, i.e., phospho-4EBP1 (integrated biomarker, 
a marker of mTORC1 inhibition), phospho-Ser473-AKT (exploratory biomarker, a marker 
of mTORC2 inhibition), BBC3 (Puma) protein levels (exploratory biomarker, responsible 
for killing downstream of mTORC1 inhibition) and BCL2L11 (Bim) protein levels 
(exploratory biomarker, responsible for killing downstream of mTORC2 inhibition).  
Because some of the antibodies used in the RPPAs (notably anti-BBC3) are particularly 
problematic (recognize a band at the expected molecular weight in cells with targeted 
interruption of the BBC3 gene), we have elected to perform the analysis by 
immunoblotting (the technique approved by the NCI Biomarker Review Committee for 
the integrated biomarker) and will utilize the RPPA for confirmatory analysis of those 
antigens that are in common.  Addition analytes recommended by the CTEP Consensus 
Review, including phospho-p70S6 kinase (another biomarker of mTORC1 inhibition, but 
one that does not correlate with mTOR inhibitor killing[18]) and the anti-apoptotic 
proteins BCL2L1 (Bcl-xL) and BCL2L2 (Bcl-w), which might correlate with response 
but are not directly implicated in the mechanism of action of this class of compounds, 
have been prioritized lower.  Likewise, because mTOR inhibitors affect translation of 
mRNA particularly dramatically, protein studies have been prioritized above mRNA 
studies.  The proposed cell-based studies are presented above and in Section 9.1 in the 
order in which they are prioritized. 
 
 
3. PATIENT SELECTION 
 
3.1 
Eligibility Criteria  
 
3.1.1 WHO-defined acute lymphoblastic leukemia and either: 
 
• 
Relapsed after achieving remission 
 
• 
Refractory to therapy; 
 
• 
Newly diagnosed and ineligible for intensive chemotherapy induction 
 
Note:  Patients with T lineage and B lineage ALL are eligible for this trial.  Likewise, 
patients with Ph+ (as long as they are not candidate for other therapies for Ph+) 
and Ph- ALL are eligible. 
 
 

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3.1.2 Bone marrow blasts of at least 10%. 
 
3.1.3 At least 4 weeks away from any previous antineoplastic or investigational agent.  Patients 
may receive hydroxyurea or glucocorticoids for suppression of leukocytosis, but these 
must be stopped at least 24 h prior to initiation of therapy. 
 
3.1.4 Age ≥18 years.    
  
3.1.5 ECOG performance status ≤ 2 
 
3.1.6 Life expectancy of >2  months    
 
3.1.7 Patients must have normal organ function and metabolism as defined below (abnormal 
labs may be retested to determine if they normalize and meet this critieria): 
 
− 
Total bilirubin 
≤ 1.5 × institutional upper limit of normal 
− 
AST(SGOT)/ALT(SGPT) 
 
≤ 2.5 × institutional upper limit of normal 
− 
Creatinine 
 
≤ 1.5 × institutional upper limit of normal 
− 
Fasting blood glucose (FBG)       < 130 mg/dL 
− 
HbA1C  
 
< 7.0% 
 
3.1.8 Relapse after stem cell transplantation (SCT) is allowed but no active GVHD as per 
treating physician.  Also must not exceed the number of prior induction regimens listed 
above. SCT does not count as line of therapy. 
 
3.1.9 Negative serum pregnancy test result 
 
Note: The effects of MLN0128 (TAK-228) on the developing human fetus are unknown.  
For this reason and because mTOR inhibitors are known to be teratogenic, women of 
child-bearing potential and men must agree to use 1 highly effective method of 
contraception and 1 additional effective (barrier) method, at the same time, from the time 
of signing the informed consent through 90 days (or longer, as mandated by local 
labeling [e.g. USPI, SmPC, etc]) after the last does of study drug.  Should a woman 
become pregnant or suspect she is pregnant while she or her partner is participating in 
this study, she should inform her treating physician immediately.  Men treated or enrolled 
on this protocol must also agree to use highly effective barrier contraception prior to the 
study, for the duration of study participation, and 4 months after completion of MLN0128 
(TAK-228) administration. 
 
3.1.10 Ability to understand and the willingness to sign a written informed consent document. 
 
3.1.11 No prior therapy with mTOR inhibitors except for rapalog treatment as part of GVH 
prophylaxis or treatment. 
 
3.1.12 HIV infected patients (if HIV positive) 
 

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HIV infected individuals are eligible provided they meet all the protocol eligibility criteria 
in addition to the following: 
• No history of AIDS defining illness other than an historic CD4+ T-cell nadir < 
200/mm3. 
 
• Prior to leukemia diagnosis, the HIV disease was uncomplicated as evidenced by: 
o the  CD4+ T-cell counts were generally in excess of 300/mm3 
o the HIV viral loads were less than 200 copies/ml if on anti-HIV therapy. 
o If the HIV is newly diagnosed or there is no history of using anti-HIV 
therapy, there are no AIDS defining conditions or other HIV-related 
symptoms. 
o Zidovudine is not allowed as part of the anti-HIV therapy. 
 
3.1.13 Patients with diabetes controlled by diet or medication are allowed on trial.  Controlled 
diabetes is defined as FBG < 130 mg/kL in the context of this study. 
 
 
3.2 
Exclusion Criteria 
 
3.2.1 Patients who have had chemotherapy or radiotherapy ≤ 4 weeks prior to entering the 
study or those who have not recovered from adverse events due to agents administered 
more than 4 weeks earlier.  Treatment with glucocorticoids, hydroxyurea, and tyrosine 
kinase inhibitors is allowed up to 24 hour prior to initiation of therapy. 
 
3.2.2 Patients with WBC > 30,000 are not eligible to start therapy.  However, it is permissible 
to use glucocorticoids and/or hydroxyurea to diminish peripheral WBC to less than 
30,000 provided these agents are stopped at least 24 hours prior to the first dose of 
MLN0128 (TAK-228). 
 
3.2.3 Patients who are receiving any other investigational agents. 
 
3.2.4 Patients with known other active cancers.  Skin cancers (basal or squamous) are 
exempted. 
 
3.2.5 History of allergic reactions attributed to compounds of similar chemical or biologic 
composition to MLN0128 (TAK-228). 
 
3.2.6 Because of the reportedly low potential for interaction with agents that induce or inhibit 
cytochrome P450 enzymes (summarized in Section 2.2.5), there are no prohibitions of 
specific medications on the basis of anticipated drug-drug interactions.  
 

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3.2.7 Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection, 
symptomatic congestive heart failure, hypertension, unstable angina pectoris, cardiac 
arrhythmia, or psychiatric illness/social situations that would limit compliance with study 
requirements.  No ischemic myocardial or cerebrovascular event, placement of 
pacemaker, or pulmonary embolism within six months of receiving first dose of 
MLN0128 (TAK-228).   
 
3.2.8 Any patient receiving chronic corticosteroid administration prior to study enrollment is 
ineligible. 
 
3.2.9    Baseline prolongation of the rate-corrected QT interval (QTc) > 480 milliseconds or 
history of congenital long QT syndrome or Torsades de pointes.  
 
3.2.10   Concomitant administration of any proton pump inhibitor (PPI) is not permitted during 
the study.  Patients receiving PPI therapy before enrollment must stop using the PPI for 7 
days before their first dose of study drugs. 
 
   
3.3 
Inclusion of Women and Minorities 
 
Both men and women of all races and ethnic groups are eligible for this trial. 
 
 
 
4. 
REGISTRATION PROCEDURES  
 
4.1 
Investigator and Research Associate Registration with CTEP 
 
4.1.1 CTEP Registration Procedures 
 
Food and Drug Administration (FDA) regulations and National Cancer Institute (NCI) 
policy require all investigators participating in any NCI-sponsored clinical trial to register 
and to renew their registration annually. 
 
Registration requires the submission of: 
 
• a completed Statement of Investigator Form (FDA Form 1572) with an original 
signature 
• a current Curriculum Vitae (CV) 
• a completed and signed Supplemental Investigator Data Form (IDF) 
• a completed Financial Disclosure Form (FDF) with an original signature 
 
Fillable PDF forms and additional information can be found on the CTEP website at 
http://ctep.cancer.gov/investigatorResources/investigator_registration.htm.  
 

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For questions about Investigator Registration, please contact the CTEP Investigator 
Registration Help Desk by email at pmbregpend@ctep.nci.nih.gov. 
 
4.1.2 CTEP Associate Registration Procedures / CTEP-IAM Account 
 
The Cancer Therapy Evaluation Program (CTEP) Identity and Access Management 
(IAM) application is a web-based application intended for use by both Investigators (i.e., 
all physicians involved in the conduct of NCI-sponsored clinical trials) and Associates 
(i.e., all staff involved in the conduct of NCI-sponsored clinical trials). 
 
Associates will use the CTEP-IAM application to register (both initial registration and 
annual re-registration) with CTEP and to obtain a user account. 
 
Investigators will use the CTEP-IAM application to obtain a user account only. (See 
CTEP Investigator Registration Procedures above for information on registering with 
CTEP as an Investigator, which must be completed before a CTEP-IAM account can be 
requested.) 
 
An active CTEP-IAM user account is needed to access all CTEP and CTSU (Cancer 
Trials Support Unit) websites and applications, and is critical to the conduct of this study, 
including document access, patient enrollment, and clinical data submission. 
 
Additional information can be found on the CTEP website at 
http://ctep.cancer.gov/branches/pmb/associate_registration.htm. 
 
For questions about Associate Registration or CTEP-IAM Account Creation, please 
contact the CTEP Registration Help Desk: ctepreghelp@ctep.nci.nih.gov 
 
 
4.2 
Site Registration 
 
This study is supported by the NCI Cancer Trials Support Unit (CTSU). 
 
Each investigator or group of investigators at a clinical site must obtain IRB approval for 
this protocol and submit IRB approval and supporting documentation to the CTSU 
Regulatory Office before they can be approved to enroll patients.   Assignment of site 
registration status in the CTSU Regulatory Support System (RSS) uses extensive data to 
make a determination of whether a site has fulfilled all regulatory criteria including but 
not limited to: an active Federal Wide Assurance (FWA) number, an active roster 
affiliation with the Lead Network or a participating organization, a valid IRB approval, 
and compliance with all protocol specific requirements. 
 
Sites participating on the NCI CIRB initiative that are approved by the CIRB for this 
studyare not required to submit  IRB approval documentation to the CTSU Regulatory 
Office.  For sites using the CIRB, IRB approval information is received from the CIRB 
and applied to the RSS in an automated process.  Signatory Institutions  must submit a 

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Study Specific Worksheet for Local Context (SSW) to the CIRB (via IRB Manager) to 
indicate their intention to open the study locally.  The CIRB’s approval of the SSW is 
then communicated to the CTSU Regulatory Office for compliance in the RSS.  In order 
for the SSW approval to be processed, the Signatory Institution must inform the CTSU 
which CIRB-approved institutions aligned with the Signatory Institution are participating 
in the study.  
 
4.2.1 Downloading Regulatory Documents 
 
Site registration forms may be downloaded from the 9775 protocol page located on the 
CTSU Web site.  Permission to view and download this protocol is restricted and is based 
on person and site roster data housed in the CTSU RSS.  To participate, Investigators and 
Associates must be associated with the lead or participating protocol organization in the 
RSS.  
 
• Go to https://www.ctsu.org and log in using your CTEP IAM username and 
password 
• Click on the Protocols tab in the upper left of your screen  
• Either enter the protocol number in the search field at the top of the protocol tree 
or  
• Click on the By Lead Organization folder to expand, then select LAO-MN026 
and protocol # 9775 
• Click on LPO Documents, select the Site Registration documents link, and 
download and complete the forms provided. (Note: For sites under the CIRB 
initiative, IRB data will automatically load to RSS.) 
 
4.2.2 Requirements for Protocol 9775 Site Registration 
 
• CTSU Transmittal Sheet (optional) 
• IRB approval (For sites not participating via the NCI CIRB; local IRB 
documentation, an IRB-signed CTSU IRB Certification Form, Protocol of Human 
Subjects Assurance Identification/IRB Certification/Declaration of Exemption 
Form, or combination is accepted ) 
 
4.2.3 Submitting Regulatory Documents 
 
Submit required forms and documentsto the CTSU Regulatory Office, where they will be 
entered and tracked in the CTSU RSS.  Regulatory Submission Portal: www.ctsu.org  
(members’ area) → Regulatory Tab →Regulatory Submission 
 
When applicable, original documents should be mailed to: 
CTSU Regulatory Office 
1818 Market Street, Suite 1100 
Philadelphia, PA 19103 
 

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Institutions with patients waiting that are unable to use the Portal should alert the CTSU 
Regulatory Office immediately at 1-866-651-2878 in order to receive further instruction 
and support. 
 
4.2.3 Checking Site Registration Status 
 
Sites can check the status of their registration packets by querying the Site Registration 
subtab of the members’ section of the CTSU Web site.  (Note: Sites will not receive 
formal notification of regulatory approval from the CTSU Regulatory Office.) 
• Go to https://www.ctsu.org and log in using your CTEP IAM username and 
password 
• Click on the Regulatory tab at the top of your screen 
• Click on the Site Registration sub tab 
• Enter your 5-character CTEP Institution Code and click on Go 
Note: If possible, please allow three working days for site registration approval before 
attempting to enroll your first patient. 
 
4.3 
Patient Registration 
 
4.3.1 OPEN / IWRS 
 
Patient enrollment will be facilitated using the Oncology Patient Enrollment Network 
(OPEN).  OPEN is a web-based registration system available to users on a 24/7 basis.  It 
is integrated with the CTSU Enterprise System for regulatory and roster data interchange 
and with the Theradex Interactive Web Response System (IWRS) for retrieval of patient 
registration/randomization assignment.  Patient enrollment data entered by Registrars in 
OPEN / IWRS will automatically transfer to the NCI’s clinical data management system, 
Medidata Rave. 
 
The OPEN system will provide the site with a printable confirmation of registration and 
treatment information.  Please print this confirmation for your records.  
 
4.3.2 OPEN/IWRS User Requirements 
 
OPEN/IWRS users must meet the following requirements: 
• Have a valid CTEP-IAM account (i.e., CTEP username and password). 
• To enroll patients or request slot reservations:  Be on an ETCTN Lead or 
Participating Organization roster with the role of Registrar. 
• To access cohort management:  Be identified to Theradex as the “Client Admin” 
for the study.  
• Have regulatory approval for the conduct of the study at their site. 
 
Prior to accessing OPEN/IWRS, site staff should verify the following: 

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• All eligibility criteria have been met within the protocol stated timeframes.  Site 
staff should use the registration forms provided on the CTSU web site as a tool to 
verify eligibility. 
• If applicable, all patients have signed an appropriate consent form and HIPAA 
authorization form. 
 
4.3.3 OPEN/IWRS Questions? 
 
Further instructional information on OPEN is provided on the OPEN tab of the CTSU 
website at https://www.ctsu.org or at https://open.ctsu.org.  For any additional questions 
contact the CTSU Help Desk at 1-888-823-5923 or ctsucontact@westat.com.  
 
4.4 
General Guidelines 
 
Following registration, patients should begin protocol treatment within 7 days.  Issues 
that would cause treatment delays should be discussed with the Principal Investigator.  If 
a patient does not receive protocol therapy following registration, the patient’s 
registration on the study may be canceled.   
 
 
5. TREATMENT PLAN 
 
Patients will be screened for eligibility within 7 days of day 1.  Starting dose will be 3 mg daily 
for days 1-21 of every 28-day cycle.  Concomitant corticosteroid administration is not allowed 
for treatment of underlying malignancy (ALL) but is allowed for treatment emergent medical 
conditions that would ordinarily require corticosteroids, e.g., diffuse alveolar hemorrhage or 
severe treatment emergent dermatological conditions.  
 
Timing of treatment should be such that pretreatment and day 8 marrows can be shipped on 
Monday-Thursday as described in Section 9.2.   
 
All patients will receive one dose of clinically indicated intrathecal (IT) chemotherapy 
prophylactically within one week of starting study treatment.  For patients with malignant CSF 
and due to poor penetration of CSF by MLN0128 (TAK-228), patients can be treated with IT 
methotrexate or cytarabine per standard practice of participating institution.  At the time of ITl 
chemotherapy performed prior to study treatment and any clinically indicated subsequent IT 
chemotherapy performed during the study, up to a 2 mLsample of CSF will be collected  for 
MLN0128 (TAK-228) pharmacokinetics if possible.  
 
Responders (CR/CRi/PR/MLFS) to therapy at any time can proceed to consolidation therapy at 3 
mg orally daily for 21-days every 28-day cycle until disease progression and as long as they are 
tolerating MLN0128 (TAK-228).  Responders may go off study and  proceed to stem cell 
transplantation if candidate and donor are available at any time.  
 
Patients who achieve a PR with MLN0128 (TAK-228) will, by definition, have normalization of 

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platelet and neutrophil counts despite persistent identifiable blasts in the bone marrow.  It is 
unclear how long they might respond to MLN0128 (TAK-228) and whether they are at risk of 
rapid progression while on this agent.  Accordingly, all patients in PR after two cycles will be 
discussed with the study PI and CTEP monitor in a timely fashion to determine on a case by case 
basis whether continued treatment at same dose, escalation to a higher dose (per Section 6.0) or 
removal from study for an alternative regimen with a well described response rate in 
relapsed/refractory ALL is the best course of action 
 
Non-responders and PR after 2 cycles of therapy (with no evidence of significant drug-related 
toxicity) could proceed to dose level +1 (4 mg orally daily for 21 days every 28-day cycle).  
Non-responders and PR after 2 additional cycles (with no evidence of significant drug-related 
toxicity) of dose escalation (end of cycle 4) could proceed to dose level +2 (4 mg orally daily for 
28 days every 28-day cycle).  Non-responders after 2 cycles (end of cycle 6), will be taken off 
study.  
 
5.1 
Agent Administration 
 
5.1.1 MLN0128 (TAK-228) 
 
Patients will receive MLN0128 (TAK-228) orally daily on days 1-21 every 28-day cycle.  
Patients will complete a pill diary and return at each visit.  Leftover study medication will 
also be returned. 
  
Treatment will be administered on an outpatient basis, but inpatient status is allowed too.  
Reported adverse events and potential risks are described in Section 7.  Appropriate dose 
modifications are described in Section 6.  No investigational or commercial agents or 
therapies other than those described below may be administered with the intent to treat 
the patient's malignancy. 
 
Antiemetics and antidiarrheal agents are not required but may be administered as needed 
for symptom control. 
  
If severe emesis or mucositis prevents the patient from taking scheduled doses, that dose 
will be skipped.  If emesis occurs after study medication ingestion, the dose will not be 
readministered, and patients should resume dosing at the next scheduled time with the 
prescribed dosage.  Patients should record the occurrence of the emesis in their dosing 
diaries.  Under no circumstance should a patient repeat a dose or double-up doses. 
 
5.1.2 Intrathecal chemotherapy: 
 
5.1.2.1 For patients without known CNS involvement:   
 
Within one week (at most ten days) prior to start of therapy, patients will receive 
mandatory CSF prophylaxis consisting of an intrathecal regimen according to 
institutional or national guidelines, e.g., methotrexate 12 to 15 mg and/or cytosine 
arabinoside 40 mg or dexamethasone 4 mg or equivalent steroid dose.  In case of 

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anticipated safety risks caused by lumbar puncture, e.g., in case of thrombocytopenia, 
CSF prophylaxis may be omitted. 
 
5.1.2.2 For patients with CNS involvement: 
 
Intrathecal (IT) chemotherapy (methotrexate 12 to 15 mg and/or cytarabine 100 mg) 
can be given for patient with malignant CSF (involved with ALL by morphology or 
flow cytometry) due to poor penetration of MLN0128 (TAK-228) to CSF.  Treatment 
will be as per standard practice of each participating institution. 
 
5.2 
General Concomitant Medication and Supportive Care Guidelines 
 
As indicated in Section 2.2.5, MLN0128 (TAK-228) has low potential for interaction 
with other drugs based on its limited ability to induce or inhibit cytochrome P450 
isoenzymes.  Therefore, there are no proscriptions against particular concomitant 
medications.  
 
Supportive care: transfusions with packed RBC or platelets will be done as per each local 
participating institution guidelines. 
 
G-CSF: Treatment with filgrastim (5 mcg/kg SC daily until ANC > 5000) or 
Pegfilgrastim (6 mg SC x1) will be allowed if severe neutropenia exists or is suspected to 
occur.  The start date will be left to the judgment of the treating physician, but dates of 
administration are to be recorded in the case report forms. 
 
Management of Hyperglycemia 
 
On the basis of the clinical experience in TAK-228 trials, most episodes of 
hyperglycemia observed occurred within the first 60 days after initiation of treatment 
with TAK-228 and have been either Grade 1 or Grade 2, and have responded quickly to 
oral metformin.  Hyperglycemia has not been dose-limiting since the institution of a 
standard regimen for early treatment of hyperglycemia.  
 
All patients developing hyperglycemia during the study should have their glucose closely 
monitored by study staff.  The investigator may choose to continue close monitoring of 
patients who develop Grade 1 hyperglycemia (fasting glucose >ULN ≤160 mg/dL) or, 
alternatively, consider initiating treatment with an oral hypoglycemic agent, such as 
metformin.  All patients with ≥ Grade 2 hyperglycemia (fasting glucose >160 mg/dL) 
must be treated aggressively with oral hypoglycemic agents and/or insulin as clinically 
indicated.  The investigator should consult an endocrinologist, if needed, to aid in 
optimizing the patient’s hyperglycemia treatment plan. 
 
It is recommended that patients with elevated fasting blood glucose be initially treated 
with a fast acting insulin sensitizer such as metformin at 500 mg orally QD and titrate up 
to a maximum of 1000 mg orally BID as needed.  Concurrent addition to metformin of 
DPP-4 inhibitors (e.g., sitagliptin or vildagliptin) and/or insulin should also be 

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considered. Oral sulfonylureas (e.g., glipizide or glyburide) should be used with caution, 
due to the higher risk of inducing hypoglycemia in patients.  The dose of oral 
hypoglycemic agents should be adjusted in patients with renal insufficiency.  In addition, 
patients should be encouraged to follow a low carbohydrate diet once hyperglycemia is 
first observed.  
 
If any fasting serum glucose reading performed at the site indicates hyperglycemia (> 
ULN or ≥ 110 mg/dL), the study staff should first confirm that the patient was fasting at 
the time of blood specimen collection (ie, nothing by mouth for at least 8 hours before 
collection). 
 
In-Home Daily Fasting Glucose Monitoring 
 
In addition to obtaining fasting glucose levels at the clinic visits as outlined in the 
Schedule of Events, all patients  receiving TAK-228 will be given a glucometer to 
monitor their daily fasting blood glucose levels at home.  The level should be collected 
daily, predose on dosing days, and at approximately the same time each day. 
 
On Cycle 1 Day 1, the patient will be provided an in-home glucometer.  Patients should 
be trained on proper use of the glucometer and instructed to collect a daily fasting blood 
glucose level every morning (predose on dosing days), starting on Cycle 1 Day 2.  
Patients will be instructed to bring the glucometer with them to each study visit so that 
the data collected can be reviewed and recorded in the source documents.  Investigators 
will be responsible for reviewing the home glucose monitoring logs for hyperglycemia. 
 
The patient will be instructed to contact the site immediately if the value is abnormal (ie, 
≥150 mg/dL) for further instructions on the management of their hyperglycemia. 
Hyperglycemia observed during home glucose monitoring should be confirmed in the 
clinic. 
 
If no irregularities in the fasting blood glucose level are observed during a minimum of 2 
consecutive months, then the frequency of in-home fasting blood glucose testing can be 
reduced to a minimum frequency of once weekly, depending on the investigator’s 
judgment and approval.  Patients will continue to notify the investigator of fasting blood 
glucose levels that exceed 150 mg/dL and, if blood glucose levels are not well controlled 
or if the patient requires either oral hypoglycemic agents or insulin to control blood 
glucose levels, then the frequency of in-home testing of fasting blood glucose levels will 
be reinstated to daily. 
 
5.3 
Duration of Therapy 
 
In the absence of treatment delays due to adverse event(s), treatment may continue after 
response until one of the following criteria applies: 
 
• 
Disease progression at any time, 
 

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• 
Intercurrent illness that prevents further administration of treatment, 
 
• 
Unacceptable adverse event(s), 
 
• 
Patient decides to withdraw from the study,                                                                          
 
• 
General or specific changes in the patient's condition render the patient 
unacceptable for further treatment in the judgment of the investigator. 
 
• 
No response after 6 cycles of MLN0128 (TAK-228). Response includes 
CR/CRi/PR or MLFS on bone marrow  
 
• 
Dose delay of more than 1 cycle due to related toxicity or more than 2 cycles to 
unrelated toxicity 
 
• 
If responding, patient proceeds with allogeneic stem cell transplant. 
 
5.4 
Duration of Follow Up 
 
Patients will be followed for toxicity for 1 month after removal from study or until death, 
whichever occurs first.  Patients removed from study for unacceptable adverse event(s) 
will be followed until resolution or stabilization of the adverse event.  Patients will be 
followed for survival status by a telephone call every 3 months unless patient refuses.  
Patients will be followed periodically until death by using public health records, for 
example, a local newspaper. 
 
5.5 
Criteria for Removal from Study 
 
Patients will be removed from active treatment when any of the criteria listed in Section 
5.3 applies.  The reason for removal from active treatment and the date the patient was 
removed must be documented in the Case Report Form. 
 
 
6. 
DOSING DELAYS/DOSE MODIFICATIONS 
 
Dose de-escalation will be pursued if patients develop a significant related AE as below.  
 
Dose escalation could be chosen if patients fail to show a response (CR/CRi/ MLFS) and show 
no evidence of drug-related toxicity.  Dose escalation or re-escalation will be done after a dose 
has been tolerated for 2 cycles. 
 
Dose delays are allowed for a maximum of 1 cycle if patient develops a related toxicity and 2 
months if unrelated toxicity (disease related for example). 
 

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Dose Level 
MLN0128 (TAK-228) Dose 
-2 
2 mg orally Monday-Friday,  
weeks 1 - 3 of each 28-day cycle 
-1 
2 mg orally days 1 - 21 
0 
3 mg orally days 1 - 21 
+1 
4 mg orally days 1 - 21 
+2 
4 mg orally days 1 - 28 
 
Note:  All treatment modifications must be expressed as a specific dose or amount rather than as 
a percentage of the starting or previous dose. 
Nausea 
Management/Next Dose for MLN0128 (TAK-228) 
≤ Grade 1 
No change in dose 
Grade 2 
Hold until ≤ Grade 1. 
Resume at same dose level. 
Grade 3 
If experienced for ≤ 72 hours, hold MLN0128 (TAK-
228) until ≤ Grade 1, then resume MLN0128 (TAK-
228) without dose modification. 
 
If experienced for > 72 hours despite optimal therapy, 
hold MLN0128 (TAK-228) until ≤  Grade 1, then 
resume treatment with the dose of MLN0128 (TAK-
228) reduced by 1 level. 
Grade 4 
Off protocol therapy 
*Patients requiring a delay of > 4 weeks if related to MLN0128 (TAK-228) 
should go off protocol therapy. 
 
Vomiting 
Management/Next Dose for MLN0128 (TAK-228) 
≤ Grade 1 
No change in dose 
Grade 2 
Hold until ≤ Grade 1.   
Resume at same dose level. 
Grade 3 
If experienced for ≤ 72 hours, hold MLN0128 (TAK-
228) until ≤ Grade 1, then resume MLN0128 (TAK-
228) without dose modification.  
 
If experienced for > 72 hours despite optimal therapy, 
hold MLN0128 (TAK-228) until ≤ Grade 1, then 
resume treatment with the dose of MLN0128 (TAK-
228) reduced by 1 level. 
Grade 4 
Off protocol therapy 
*Patients requiring a delay of > 4 weeks if related to MLN0128 (TAK-228) 
should go off protocol therapy. 
 

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Diarrhea 
Management/Next Dose for MLN0128 (TAK-228) 
≤ Grade 1 
No change in dose 
Grade 2 
Hold until ≤ Grade 1.   
Resume at same dose level. 
Grade 3 
Hold* until < Grade 2.   
Resume at one dose level lower 
Grade 4 
Off protocol therapy 
*Patients requiring a delay of > 4 weeks if related to MLN0128 (TAK-228) 
should go off protocol therapy. 
 
**Patients requiring > two dose reductions should go off protocol therapy. 
Recommended management:  Loperamide antidiarrheal therapy 
Dosage schedule:  4 mg at first onset, followed by 2 mg with each loose 
motion until diarrhea-free for 12 hours (maximum dosage:  16 mg/24 hours) 
Adjunct anti-diarrheal therapy is permitted and should be recorded when 
used. 
 
Hyperglycemia 
Management/Next Dose for MLN0128 (TAK-228) 
≤ Grade 1 
No change in dose. Optimize blood sugar control. * 
Grade 2 
No change in dose.  Optimize blood sugar control. 
Grade 3 
Asymptomatic 
No change in dose. Optimize blood sugar control. 
Grade 3 
Symptomatic or >7 
days on therapy 
Hold** until < Grade 2.   
Resume at one dose level lower. 
Grade 4 
Off protocol therapy 
* If medical therapy is needed, metformin is preferred unless contraindicated.  
Sulfonylurea can be added if metformin does not control blood glucose level.  
If this fails to bring it to ≤ G1 within 1 month, then oral hypoglycemics can be 
stopped and insulin used. 
 
**Patients requiring > two dose reductions should go off protocol therapy. 
Hyperglycemia will be based on fasting glucose level.  
 
 
Thrombocytopenia 
and  neutropenia 
Management/Next Dose for MLN0128 (TAK-228) 
≤ Grade 1 
No change in dose 
Grade 2 
Hold until ≤ Grade 1.   
Resume at same dose level. 
Grade 3 
Hold* until < Grade 1.   
Resume at one dose level lower 
Grade 4 
Off protocol therapy 
This dose modification for thrombocytopenia and neutropenia only pertains to 

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Thrombocytopenia 
and  neutropenia 
Management/Next Dose for MLN0128 (TAK-228) 
patients in remission. Thrombocytopenia and neutropenia will not be 
considered dose limiting during remission induction. 
 
*Patients requiring a delay of >4 weeks if related to MLN0128 (TAK-228) 
should go off protocol therapy. This only will be counted if patients are in 
remission (CR). 
 
**Patients requiring > two dose reductions should go off protocol therapy. 
 
Non-hematological toxicity: 
 
Non-hematological toxicities of fatigue,  electrolyte abnormalities and abnormal liver function 
tests, including bilirubin, transaminases and alkaline phosphatase will only require dose 
modification if they are likely or definitely related to MLN0128 (TAK-228) and persist more 
than 7 days.  Neutropenic fever and infectious toxicities are only counted for if patients achieve 
remission and then develop these adverse events. 
 
Non-Hematological Toxicity 
Event 
Management/Next Dose for MLN0128 (TAK-228) 
≤ Grade 1 
No change in dose 
Grade 2 
Hold until ≤ Grade 1.   
Resume at same dose level. 
Grade 3 
Hold* until Grade 1.   
Resume at one dose level lower 
Grade 4 
Patients who develop Grade 4 nonhematologic 
toxicities (with the exception of isolated non-clinically 
significant laboratory values) should permanently 
discontinue study treatment, unless they derive clinical 
benefit, in which case they may be retreated at a 
reduced dose level after recovery to ≤ Grade 1 severity. 
**Patients requiring a delay of >4 weeks if related to MLN0128 (TAK-228) 
should go off protocol therapy. 
 
**Patients requiring > two dose reductions should go off protocol therapy. 
 
7. ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS  
 
Adverse event (AE) monitoring and reporting is a routine part of every clinical trial.  The 
following list of AEs (Section 7.1) and the characteristics of an observed AE (Section 7.2) will 
determine whether the event requires expedited reporting (via CTEP-AERS) in addition to 
routine reporting. 
 

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7.1 
Comprehensive Adverse Events and Potential Risks List (CAEPR) 
 
The Comprehensive Adverse Event and Potential Risks list (CAEPR) provides a single list of 
reported and/or potential adverse events (AE) associated with an agent using a uniform 
presentation of events by body system.  In addition to the comprehensive list, a subset of AEs, 
the Specific Protocol Exceptions to Expedited Reporting (SPEER), appears in a separate column 
and is identified with bold and italicized text.  The SPEER is a list of events that are protocol-
specific exceptions to expedited reporting to NCI via CTEP-AERS (except as noted below).  
Refer to the 'CTEP, NCI Guidelines: Adverse Event Reporting Requirements' 
http://ctep.cancer.gov/protocolDevelopment/adverse_effects.htm for further clarification.  
 
The CAEPR may not provide frequency data; if not, refer to the Investigator’s Brochure for this 
information. 
 
NOTE:  The highest grade currently reported is noted in parentheses next to the AE in the 
SPEER.  Report ONLY AEs higher than this grade expeditiously via CTEP-AERS.  If this 
CAEPR is part of a combination protocol using multiple investigational agents and has an AE 
listed on different SPEERs, use the lower of the grades to determine if expedited reporting is 
required. 
 
7.1.1 CAEPR for MLN0128 (TAK-228) 
 
Comprehensive Adverse Events and Potential Risks list (CAEPR) 
for 
MLN0128 (TAK-228) (INK128, NSC 768435) 
 
The Comprehensive Adverse Events and Potential Risks list (CAEPR) provides a single list of 
reported and/or potential adverse events (AE) associated with an agent using a uniform 
presentation of events by body system. In addition to the comprehensive list, a subset, the 
Specific Protocol Exceptions to Expedited Reporting (SPEER), appears in a separate column and 
is identified with bold and italicized text. This subset of AEs (SPEER) is a list of events that are 
protocol specific exceptions to expedited reporting to NCI (except as noted below).  Refer to the 
'CTEP, NCI Guidelines: Adverse Event Reporting Requirements' 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/aeguidelines.pdf for 
further clarification. Frequency is provided based on 252 patients. Below is the CAEPR for 
MLN0128 (TAK-228)(INK128). 
 
NOTE: Report AEs on the SPEER ONLY IF they exceed the grade noted in parentheses next to 
the AE in the SPEER.  If this CAEPR is part of a combination protocol using multiple 
investigational agents and has an AE listed on different SPEERs, use the lower of the grades to 
determine if expedited reporting is required. 
 
 
 
 
                                                                        

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Version 2.2, April 26, 2016 
 
 Adverse Events with Possible  
 Relationship to MLN0128 (TAK228) 
 (CTCAE 4.0 Term) 
[n= 311] 
 
  
 Specific Protocol 
Exceptions to Expedited 
Reporting (SPEER)  
 
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious (<3%)   
BLOOD AND LYMPHATIC SYSTEM DISORDERS 
  
 
Anemia 
 
 Anemia (Gr 2) 
CARDIAC DISORDERS 
  
 
 
Cardiac arrest 
  
 
 
Ventricular fibrillation 
  
GASTROINTESTINAL DISORDERS 
  
 
Abdominal pain 
 
 Abdominal pain (Gr 2) 
 
Constipation 
 
 Constipation (Gr 2) 
Diarrhea 
 
 
 Diarrhea (Gr 2) 
 
Dry mouth 
 
 Dry mouth (Gr 2) 
Mucositis oral 
 
 
 Mucositis oral (Gr 2) 
Nausea 
 
 
 Nausea (Gr 2) 
Vomiting 
 
 
 Vomiting (Gr 2) 
GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS 
  
 
Edema limbs 
 
  
Fatigue 
 
 
 Fatigue (Gr 2) 
 
Fever 
 
 Fever (Gr 2) 
 
General disorders and 
administration site 
conditions - Other 
(mucosal inflammation) 
 
 General disorders and 
administration site 
conditions - Other 
(mucosal inflammation) 
(Gr 2) 
INFECTIONS AND INFESTATIONS 
  
 
Urinary tract infection 
 
 Urinary tract infection (Gr 
2) 
INVESTIGATIONS 
  
 
Creatinine increased 
 
 Creatinine increased (Gr 2) 
 
 
Electrocardiogram QT 
corrected interval 
prolonged 
  
 
Platelet count decreased 
 
 Platelet count decreased 
(Gr 2) 
 
Weight loss 
 
 Weight loss (Gr 2) 
METABOLISM AND NUTRITION DISORDERS 
  

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Anorexia 
 
 
 Anorexia (Gr 2) 
 
Dehydration 
 
 Dehydration (Gr 2) 
Hyperglycemia 
 
 
 Hyperglycemia (Gr 2) 
 
Hypokalemia 
 
 Hypokalemia (Gr 2) 
 
Hypomagnesemia 
 
 Hypomagnesemia (Gr 2) 
 
Hypophosphatemia 
 
 Hypophosphatemia (Gr 2) 
MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS 
  
 
Arthralgia 
 
  
 
Back pain 
 
 Back pain (Gr 2) 
 
Pain in extremity 
 
  
NERVOUS SYSTEM DISORDERS 
  
 
Dizziness 
 
 Dizziness (Gr 2) 
 
Dysgeusia 
 
 Dysgeusia (Gr 2) 
 
Headache 
 
 Headache (Gr 2) 
PSYCHIATRIC DISORDERS 
  
 
Insomnia 
 
  
RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS 
  
 
Cough 
 
 Cough (Gr 2) 
 
Dyspnea 
 
 Dyspnea (Gr 2) 
 
 
Pneumonitis 
  
 
Respiratory, thoracic and 
mediastinal disorders - 
Other (oropharyngeal pain) 
 
  
SKIN AND SUBCUTANEOUS TISSUE DISORDERS 
  
Pruritus 
 
 
 Pruritus (Gr 2) 
Rash maculo-papular 
 
 
 Rash maculo-papular (Gr 
2) 
 
1This table will be updated as the toxicity profile of the agent is revised.  Updates will be 
distributed to all Principal Investigators at the time of revision.  The current version can be 
obtained by contacting PIO@CTEP.NCI.NIH.GOV.  Your name, the name of the investigator, 
the protocol and the agent should be included in the e-mail. 
 
Adverse events reported on MLN0128 (TAK228) trials, but for which there is insufficient 
evidence to suggest that there was a reasonable possibility that MLN0128 (TAK228) caused 
the adverse event: 
 
Cardiac disorders - Heart failure; Pericardial effusion; Sinus tachycardia 
Eye disorders - Blurred vision; Eye disorders - Other (visual acuity reduced); Eye pain; 
Photophobia 
Gastrointestinal disorders - Dyspepsia; Dysphagia; Esophagitis; Gastroesophageal reflux 
disease; Gastrointestinal disorders - Other (intestinal obstruction); Oral pain; Pancreatitis; Small 
intestinal obstruction; Small intestinal perforation; Toothache 

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General disorders and administration site conditions - Chills; Gait disturbance; Non-cardiac 
chest pain; Pain 
Hepatobiliary disorders - Gallbladder obstruction 
IMMUNE SYSTEM DISORDERS - Allergic reaction 
Infections and infestations - Kidney infection; Lung infection; Sepsis; Skin infection; Upper 
respiratory infection 
Injury, poisoning and procedural complications - Fall; Tracheal obstruction 
Investigations - Alanine aminotransferase increased; Aspartate aminotransferase increased; 
Blood bilirubin increased; Cholesterol high; Lymphocyte count decreased; Neutrophil count 
decreased; White blood cell decreased 
Metabolism and nutrition disorders - Hypercalcemia; Hypertriglyceridemia; 
Hypoalbuminemia; Hypocalcemia; Hyponatremia; Metabolism and nutrition disorders - Other 
(vitamin D deficiency) 
Musculoskeletal and connective tissue disorders - Flank pain; Generalized muscle weakness; 
Musculoskeletal and connective tissue disorder - Other (muscle spasms); Myalgia 
Neoplasms benign, malignant and unspecified (incl cysts and polyps) - Treatment related 
secondary malignancy 
Nervous system disorders - Intracranial hemorrhage; Nervous system disorders - Other (carotid 
artery occlusion); Tremor 
Psychiatric disorders - Anxiety; Confusion; Depression; Personality change 
Renal and urinary disorders - Acute kidney injury; Hematuria; Urinary tract pain 
Respiratory, thoracic and mediastinal disorders - Bronchopulmonary hemorrhage; Epistaxis; 
Hypoxia; Nasal congestion; Pleural effusion; Pneumothorax; Postnasal drip; Productive cough 
Skin and subcutaneous tissue disorders - Dry skin; Hyperhidrosis; Rash acneiform; Urticaria 
Vascular disorders - Flushing; Hypotension; Thromboembolic event 
 
 
Note: MLN0128 (TAK228) in combination with other agents could cause an exacerbation of any 
adverse event currently known to be caused by the other agent, or the combination may result in 
events never previously associated with either agent. 
 
 
7.2 
Adverse Event Characteristics  
 
• CTCAE term (AE description) and grade:  The descriptions and grading scales found 
in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 
4.0 will be utilized for AE reporting.  All appropriate treatment areas should have access 
to a copy of the CTCAE version 4.0.  A copy of the CTCAE version 4.0 can be 
downloaded from the CTEP web site 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm. 
 
• For expedited reporting purposes only:   
- 
AEs for the agent that are bold and italicized in the CAEPR (i.e., those listed in the 
SPEER column, Section 7.1.1) should be reported through CTEP-AERS only if the 
grade is above the grade provided in the SPEER. 

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- 
Other AEs for the protocol that do not require expedited reporting are outlined in 
section 7.3.4. 
 
• Attribution of the AE: 
- 
Definite – The AE is clearly related to the study treatment. 
- 
Probable – The AE is likely related to the study treatment. 
- 
Possible – The AE may be related to the study treatment. 
- 
Unlikely – The AE is doubtfully related to the study treatment. 
- 
Unrelated – The AE is clearly NOT related to the study treatment. 
 
7.3 
Expedited Adverse Event Reporting 
 
7.3.1 Expedited AE reporting for this study must use CTEP-AERS (CTEP Adverse Event 
Reporting System), accessed via the CTEP Web site (https://eapps-
ctep.nci.nih.gov/ctepaers).  The reporting procedures to be followed are presented in the 
“NCI Guidelines for Investigators: Adverse Event Reporting Requirements for DCTD 
(CTEP and CIP) and DCP INDs and IDEs” which can be downloaded from the CTEP 
Web site 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/adverse_events.htm
).  These requirements are briefly outlined in the tables below (Section 7.3.3). 
 
In the rare occurrence when Internet connectivity is lost, a 24-hour notification is to be 
made to CTEP by telephone at 301-897-7497.  Once Internet connectivity is restored, the 
24-hour notification phoned in must be entered electronically into CTEP-AERS by the 
original submitter at the site. 
 
7.3.2 CTEP-AERS is programmed for automatic electronic distribution of reports to the 
following individuals:  Study Coordinator of the Lead Organization, Principal 
Investigator, and the local treating physician.  CTEP-AERS provides a copy feature for 
other e-mail recipients.  
 
The Coordinating Center of the Lead Organization is responsible for submitting to the 
CTSU documentation of AEs that they deem reportable for posting on the CTSU protocol 
web page and inclusion on the CTSU bi-monthly broadcast. 
 
7.3.3 Expedited Reporting Guidelines 
 
Use the NCI protocol number and the protocol-specific patient ID assigned during trial 
registration on all reports. 
 
Note:  A death on study requires both routine and expedited reporting regardless of 
causality, unless as noted below.  Attribution to treatment or other cause must be 
provided. 
 
Death due to progressive disease should be reported as Grade 5 “Neoplasms benign, 
malignant and unspecified (incl cysts and polyps) - Other (Progressive Disease)” 

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under the system organ class (SOC) of the same name.  Evidence that the death was a 
manifestation of underlying disease (e.g., radiological changes suggesting tumor growth 
or progression: clinical deterioration associated with a disease process) should be 
submitted.  
 
Late Phase 2 and Phase 3 Studies: Expedited Reporting Requirements for Adverse Events 
that Occur on Studies under an IND/IDE within 30 Days of the Last Administration of the 
Investigational Agent/Intervention1, 2 
FDA REPORTING REQUIREMENTS FOR SERIOUS ADVERSE EVENTS (21 CFR Part 312) 
NOTE:  Investigators MUST immediately report to the sponsor (NCI) ANY Serious Adverse Events, whether or not 
they are considered related to the investigational agent(s)/intervention (21 CFR 312.64) 
An adverse event is considered serious if it results in ANY of the following outcomes:   
1) 
Death 
2) 
A life-threatening adverse event  
3) 
An adverse event that results in inpatient hospitalization or prolongation of existing hospitalization for ≥ 24 
hours  
4) 
A persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions  
5) 
A congenital anomaly/birth defect.  
6) 
Important Medical Events (IME) that may not result in death, be life threatening, or require hospitalization 
may be considered serious when, based upon medical judgment, they may jeopardize the patient or 
subject and may require medical or surgical intervention to prevent one of the outcomes listed in this 
definition. (FDA, 21 CFR 312.32; ICH E2A and ICH E6). 
ALL SERIOUS adverse events that meet the above criteria MUST be immediately reported to the NCI via AdEERS 
within the timeframes detailed in the table below. 
Hospitalization 
Grade 1 
Timeframes 
Grade 2 
Timeframes 
Grade 3 Timeframes 
Grade 4 & 5 
Timeframes 
Resulting in 
Hospitalization ≥ 24 
hrs 
10 Calendar Days 
24-Hour 5 
Calendar Days 
Not resulting in 
Hospitalization ≥ 24 
hrs 
Not required 
10 Calendar Days 
NOTE:  Protocol specific exceptions to expedited reporting of serious adverse events are found in the Specific 
Protocol Exceptions to Expedited Reporting (SPEER) portion of the CAEPR 
Expedited AE reporting timelines are defined as: 
o 
“24-Hour; 5 Calendar Days” - The AE must initially be reported via AdEERS within 24 hours of learning of 
the AE, followed by a complete expedited report within 5 calendar days of the initial 24-hour report. 
o 
“10 Calendar Days” - A complete expedited report on the AE must be submitted within 10 calendar days of 
learning of the AE. 
1Serious adverse events that occur more than 30 days after the last administration of investigational 
agent/intervention and have an attribution of possible, probable, or definite require reporting as follows:  
Expedited 24-hour notification followed by complete report within 5 calendar days for: 
• 
All Grade 4, and Grade 5 AEs 
Expedited 10 calendar day reports for: 
• 
Grade 2 adverse events resulting in hospitalization or prolongation of hospitalization  
1. 
Grade 3 adverse events 
2For studies using PET or SPECT IND agents, the AE reporting period is limited to 10 radioactive half-lives, 
rounded UP to the nearest whole day, after the agent/intervention was last administered.  Footnote “1” above 
applies after this reporting period. 
Effective Date:  May 5, 2011 
 
 
 

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7.3.4 Additional Protocol-Specific Expedited Adverse Event Reporting Exclusions 
 
For this protocol only, the AEs/grades listed below do not require expedited reporting via 
CTEP-AERS.  However, they still must be reported through the routine reporting 
mechanism (Section 7.4): 
 
CTCAE SOC 
Adverse Event 
Grade 
Blood and 
lymphatic system 
disorders 
Anemia 
Grade 3 or 4 
 
 
 
 
Investigations 
White blood cell 
decreased 
Grade 3 or 4 
Lymphocyte count 
decreased  
Grade 3 or 4 
Neutrophil count 
decreased 
Grade 3 or 4 
Platelet count decreased 
Grade 3 or 4 
 
 
7.4 
Routine Adverse Event Reporting 
 
All Adverse Events must be reported in routine study data submissions.  AEs reported through 
CTEP-AERS must also be reported in routine study data submissions. 
 
Adverse event data collection and reporting, which are required as part of every clinical trial, are 
done to ensure the safety of patients enrolled in the studies as well as those who will enroll in 
future studies using similar agents.  AEs are reported in a routine manner at scheduled times 
during the trial using Medidata Rave.    For this trial the Adverse Event is used for routine AE 
reporting in Rave.   
 
7.5 
Secondary Malignancy 
 
A secondary malignancy is a cancer caused by treatment for a previous malignancy (e.g., 
treatment with investigational agent/intervention, radiation or chemotherapy).  A secondary 
malignancy is not considered a metastasis of the initial neoplasm.  
 
CTEP requires all secondary malignancies that occur following treatment with an agent under an 
NCI IND/IDE be reported via CTEP-AERS. Three options are available to describe the event:  
 
• Leukemia secondary to oncology chemotherapy (e.g., acute myelocytic leukemia 
[AML])  
• Myelodysplastic syndrome (MDS)  
• Treatment-related secondary malignancy  
 

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Any malignancy possibly related to cancer treatment (including AML/MDS) should also be 
reported via the routine reporting mechanisms outlined in each protocol.  
 
7.6 
Second Malignancy 
 
A second malignancy is one unrelated to the treatment of a prior malignancy (and is NOT a 
metastasis from the initial malignancy).  Second malignancies require ONLY routine adverse 
event reporting unless otherwise specified.  
 
8. 
PHARMACEUTICAL INFORMATION 
 
A list of the adverse events and potential risks associated with the investigational administered in 
this study can be found in Section 7.1. 
 
8.1 
MLN0128 (TAK-228) (NSC # 768435) 
 
Other Names: MLN0128, INK128, NSC# 768435 
 
Classification: mTOR inhibitor, TORC1/2  
 
CAS Registry Number: 1224844 
 
Molecular Formula: C15H15N7O  
M.W.: 309.3 
 
Approximate Solubility: MLN0128 (TAK-228) exhibits a pH-dependent aqueous solubility: at 
physiological pH the solubility is approximately 0.1 mg/mL and at or below pH 3 the solubility is 
greater than 15 mg/mL. 
 
Mode of Action: MLN0128 (TAK-228) is a non-rapamycin analog mTOR (mechanistic target of 
rapamycin) kinase inhibitor.  The mTOR kinase regulates cell growth, translational control, 
angiogenesis, and cell survival by integrating nutrient and hormonal signals.  The mTOR complex 
(TORC) is an intracellular point of convergence for a number of cellular signaling pathways.  
MLN0128 (TAK-228) is a potent and selective adenosine tri-phosphate (ATP)-competitive 
inhibitor of mTOR complex 1 and 2 (TORC1/2). 
 
Description: MLN0128 (TAK-228) drug substance is a white to off-white, crystalline powder. 
 
How Supplied: MLN0128 (TAK-228)  is supplied by Millennium Pharmaceuticals, Inc. and 
distributed by the Pharmaceutical Management Branch, CTEP/DCTD/NCI as size 2 hard gelatin 
capsules in the following strengths: 1 mg (white opaque color) and 3 mg (orange opaque color).  
The composition of the drug product consists of a blend of MLN0128 (TAK-228) drug substance, 
microcrystalline cellulose, and magnesium stearate.  Milled formulations will have a white label 
with a large watermark of the strength on the label.   
 
MLN0128 (TAK-228) capsules are packaged in 30-count, 60-cc high-density polyethylene 
(HDPE), white, opaque, round, tamper- and child-resistant bottles.   

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Storage: Capsules are to be stored in the original package between 15°C to 30°C, with allowed 
short-term excursions between 2°C and 40°C.   
 
Route of Administration: Orally, given 2 hours before or 1 hour after a meal.  Do not chew, open 
or manipulate the capsule in any way prior to swallowing.  Each dose should be taken with 8 ounces 
(240 mL) of water. 
 
Potential Drug Interactions:  Multiple human metabolizing enzymes are involved in the Phase I 
metabolism of MLN0128 (TAK-228).  When normalized for human liver content, the CYP 
isoforms CYP3A4, CYP2C9, and CYP2C19 appear to contribute to MLN0128 (TAK-228) 
metabolism.  MLN0128 (TAK-228) displayed low potential (IC50 > 25 μM) for inhibition of the 
major human CYP isoforms. 
 
Patient Care Implications: Women of childbearing potential should use effective methods of 
contraception during and through 90 days after the last dose of MLN0128 (TAK-228).  Men should 
use effective methods of contraception and not donate sperm during and through 120 days after the 
last dose of MLN0128 (TAK-228). 
 
Availability 
 
MLN0128 (TAK-228) is an investigational agent supplied to investigators by the Division of 
Cancer Treatment and Diagnosis (DCTD), NCI. 
 
MLN0128 (TAK-228) is provided to the NCI under a Collaborative Agreement between the 
Pharmaceutical Collaborator and the DCTD, NCI (see Section 12.3). 
 
 
8.2 
Agent Ordering and Agent Accountability 
 
8.2.1 NCI-supplied agents may be requested by the Principal Investigator (or their authorized 
designee) at each participating institution.  Pharmaceutical Management Branch (PMB) 
policy requires that agent be shipped directly to the institution where the patient is to be 
treated.  PMB does not permit the transfer of agents between institutions (unless prior 
approval from PMB is obtained).  The CTEP-assigned protocol number must be used for 
ordering all CTEP-supplied investigational agents.  The responsible investigator at each 
participating institution must be registered with CTEP, DCTD through an annual 
submission of FDA Form 1572 (Statement of Investigator), Curriculum Vitae, 
Supplemental Investigator Data Form (IDF), and Financial Disclosure Form (FDF).  If 
there are several participating investigators at one institution, CTEP-supplied 
investigational agents for the study should be ordered under the name of one lead 
investigator at that institution. 
 
In general, sites may order initial agent supplies when a subject is being screened for 
enrollment onto the study. 
 

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Active CTEP-registered investigators and investigator-designated shipping designees and 
ordering designees can submit agent requests through the PMB Online Agent Order 
Processing (OAOP) application (https://eapps-ctep.nci.nih.gov/OAOP/pages/login.jspx).  
Access to OAOP requires the establishment of a CTEP Identity and Access Management 
(IAM) account (https://eapps-ctep.nci.nih.gov/iam/) and the maintenance of an “active” 
account status and a “current” password.  For questions about drug orders, transfers, 
returns, or accountability, call or email PMB anytime.   
 
8.2.2 Agent Inventory Records – The investigator, or a responsible party designated by the 
investigator, must maintain a careful record of the receipt, dispensing and final 
disposition of all  agents received from PMB using the appropriate NCI Investigational 
Agent (Drug) Accountability Record Form (DARF) available on the CTEP forms page.  
Store and maintain separate NCI Investigational Agent Accountability Records for each 
agent, strength, formulation and ordering investigator on this protocol.  
 
8.2.3 Useful Links and Contacts 
• CTEP Forms, Templates, Documents:  http://ctep.cancer.gov/forms/ 
• NCI CTEP Investigator Registration:  PMBRegPend@ctep.nci.nih.gov 
• PMB policies and guidelines: 
http://ctep.cancer.gov/branches/pmb/agent_management.htm  
• PMB Online Agent Order Processing (OAOP) application: https://eapps-
ctep.nci.nih.gov/OAOP/pages/login.jspx  
• CTEP Identity and Access Management (IAM) account: https://eapps-
ctep.nci.nih.gov/iam/ 
• CTEP Associate Registration and IAM account help:  
ctepreghelp@ctep.nci.nih.gov  
• PMB email:  PMBAfterHours@mail.nih.gov 
• PMB phone and hours of service: (240) 276-6575 Monday through Friday 
between 8:30 am and 4:30 pm (ET) 
• IB Coordinator: IBCoordinator@mail.nih.gov 
 
 
9. BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES 
 
9.1 
Biomarker Studies 
 
9.1.1 Plasma Pharmacokinetics 
 
9.1.1.1 
Hypothesis and Rationale   
 
Hypothesis:  In those patients with inadequate drug exposure, inhibition of 4EBP1 
phosphorylation and other cellular effects of mTOR dual inhibitors assayed as 
described in Sections 9.1.2-9.1.6 will be limited. 
 

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Rationale:  These studies are necessary to characterize the pharmacokinetics of 
MLN0128 (TAK-228) in patients with acute lymphoblastic leukemia and to assess 
pharmacodynamic relationships with phosphorylated mTOR substrates and 
expression levels of BCL2L11 (Bim), BBC3 (Puma), MCL1 and BCL2. 
 
9.1.1.2 
Assay Validity and Appropriateness for Study 
 
Plasma and marrow concentrations of MLN0128 (TAK-228) will be measured by 
lc/ms/ms methods developed in the Mayo Clinic Cancer Center Pharmacology Shared 
Resource.  The Shared Resource, directed by Dr. Reid, is fully equipped to carry out 
pharmacologic investigations, including two exquisitely sensitive Xevo TQ-S mass 
spectrometers with Accuity UPLC systems to carry out the bioanalytical 
measurements, and licenses for Phoenix WINNONLIN, NONMEM and PsN Pirana 
to carry out standard and population PK/PD analyses.  The laboratory has developed 
or modified assays for more than 75 antitumor agents, served as a central analytical 
and pharmacokinetics resource laboratory for 9 published NCI-sponsored Phase I and 
Phase II trials with vorinostat (for example, 47, 48, 49), and serves as one of the 
pharmacology resource laboratories for the Children’s Oncology Group.  The 
approach to assay development and implementation of criteria for system suitability, 
quality assurance and ongoing evaluation of specific assay data are consistent with 
recommendations from two FDA-sponsored conferences on analytical methods 
validation (50, 51) that are described in a recent FDA guidance document "Guidance 
for Industry: Bioanalytical Method Validation" (52).  In particular, as we develop a 
specific HPLC or other method for measurement of drug and relevant metabolites, we 
incorporate standard assessments of drug extraction efficiency, sensitivity, linearity, 
precision, accuracy, storage stability and other parameters relevant to the particular 
method.  Assay performance is thoroughly validated before implementation in the 
clinical trial and monitored on a routine basis during the course of the clinical trial by 
inclusion of appropriate quality assurance samples.  The plasma concentration-time 
data will be will be analyzed by standard noncompartmental analysis using the 
program Phoenix WinNonlin (Pharsight Corp., St. Louis, MO) to determine Cmax, 
Tmax, half-life and accumulation.  If sufficient data are available, the data will also 
be analyzed by non-linear mixed effects modeling using the program NONMEM 
7.2.0 (ICON Development Solutions, Ellicot City, MD) to determine oral clearance, 
volume of distribution, sources of inter- and intra-subject variability, and 
pharmacodynamic relationships with phosphorylated mTOR substrates and 
expression levels of BCL2L11 (Bim), BBC3 (Puma), MCL1 and BCL2. 
 
9.1.1.3 
Performance Site   
 
The assay will be performed in the Pharmacology Shared Resource of the Mayo 
Clinic Cancer Center.  This lab has extensive experience with developing, validating 
and implementing assays for a variety of anticancer drugs, including mTOR dual 
inhibitors, as well as performing pharmacokinetic analysis after performance of the 
assays in preclinical and clinical studies. 
 

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9.1.1.4 
Justification of the Number of Patients and Specimens   
 
The purpose of the assay is to provide information about drug exposure that can be 
utilized for interpretation of pharmacodynamic and clinical response endpoints. 
Meaningful conclusions regarding the impact of drug exposure can only be drawn for 
those patients in whom the assays are performed.  Accordingly, the plan is to measure 
serum pharmacokinetics in all patients.  The number of samples (15 per patient) is 
required to assure sufficient data to allow calculation of standard pharmacokinetic 
parameters. 
 
9.1.1.5 
Description of Biological Samples Required for Assay 
 
This assay of serum MLN0128 (TAK-228) levels requires procurement of 6 cc of 
blood at 15 time points as described in detail in Section 9.2.1.2.  Risks of blood 
sample procurement include pain, infection and bleeding at the site of venous heparin 
lock placement as well as development of heparin-induced thrombocytopenia.  Sterile 
technique is utilized to minimize risk of infection.  With an adequate platelet count 
(10,000 or more) and local pressure, the risk of bleeding is low.  Heparin-induced 
thrombocytopenia is rare; and patients’ platelet counts will be monitored daily for 
several days after the venipuncture, with platelet transfusions mandated for 
thrombocytopenia as indicated in Section 5.2 (generally for bleeding or platelet count 
less than 10,000 unless a higher threshold is deemed appropriate by the treating 
physician because of fever, infection or invasive procedure).  It is conceivable that 
infections or bleeding could be life threatening, but the risk of death after 
venipuncture is considered extremely rare (estimated to be 1 in 50 million). 
 
9.1.2 Overview of Cell-Based Studies 
 
Cell-based studies in this section are presented in the order in which they are prioritized.  
The rationale for this prioritization, which is that proof-of-mechanism studies (including 
the integrated biomarker) take higher priority than studies outside the direct mechanism 
of action of this class of agents, is described in detail in Section 2.4.7.  Additional details 
regarding these assays, including sample processing at the assay site and 
performance characteristics of the assays, are found in Appendix D. 
 
9.1.3 Phospho-Thr37,46-4EBP1 analysis of pretreatment and day 8 bone marrow samples by 
immunoblotting (integrated biomarker) 
 
9.1.3.1 
Hypothesis and Rationale 
 
Hypothesis:  4EBP1 phosphorylation will be inhibited by MLN0128 (TAK-228) in 
situ. 
 
Rationale:  Previous studies have shown that mTOR dual inhibitors such as 
MLN0128 (TAK-228) and OSI-027 diminish the mTORC1-mediated 

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phosphorylation of the translation inhibitor 4EBP1, causing it to bind to and inhibit 
the translation factor eIF4E (14, 18).  In ALL cell lines the down regulation of 
4EBP1 produces resistance to mTOR dual inhibitors (14), demonstrating the critical 
role of this protein in killing by mTOR dual inhibitors.  We now propose to assess 
whether 4EBP1 has been dephosphorylated in leukemic blasts by therapeutically 
achievable MLN0128 (TAK-228) concentrations in situ as a proof of mechanism. 
 
9.1.3.2 
Assay Validity and Appropriateness for Study 
 
Immunoblotting will be performed to assess 4EBP1 phosphorylation at Thr37 and 
Thr46 in Ficoll-Hypaque enriched cells from pretreatment and day 8 bone marrow 
aspirates.  Alternative approaches to assessing 4EBP1 phosphorylation include 
ELISA, immunohistochemistry and 2-dimensional gel electrophoresis.  The major 
advantage of immunoblotting is the ability to assess the signal at the molecular 
weight of interest rather than assuming that a signal observed in ELISA is due to the 
antigen of interest.  While it is somewhat less quantitative than ELISA, the Kaufmann 
laboratory has demonstrated that blotting with a rabbit monoclonal antibody can 
detect differences in phospho-4EBP1 over a 64-fold range that encompasses the 
extent of phosphorylation observed in baseline ALL samples (Appendix C).  
Preliminary studies presented to the Biomarker Review Committee indicated a strong 
correlation (R2  > 0.94) between results observed when samples with a wide range of 
4EBP1 phosphorylation were run on separate SDS-polyacrylamide gels and probed. 
 
9.1.3.3 
Performance site   
 
The assay will be performed in the Kaufmann laboratory of the Mayo Clinic Cancer 
Center.  This lab has extensive experience with detecting changes in antigen content 
by immunoblotting (53, 54). 
 
9.1.3.4 
Data Supporting the Degree of Biomarker “Fit for Purpose”:   
 
Credentialing of the proposed immunoblotting assay is described in Appendix C.  A 
Standard Operating Procedure for the assay is also included in the Appendix per the 
instructions in the protocol template. 

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9.1.3.5 
Justification of the Number of Patients and Specimens   
 
The purpose of the assay is to determine whether MLN0128 (TAK-228) induces 
dephosphorylation of an mTORC1 target in leukemia cells in situ at clinically 
achievable concentrations.  After the signal for phospho-4EBP1 is quantitated, results 
will be examined graphically to assess the extent and frequency of down regulation as 
described in Section 13.4.6.  This will be the first opportunity to assess 4EBP1 
dephosphorylation in ALL after treatment with an mTOR dual inhibitor, so the extent 
and frequency of down regulation cannot be estimated from precedent data.  As a 
point of reference, however, complete dephosphorylation of 4EBP1 was observed in 
Jeko mantle cell lymphoma xenografts after treatment of mice with the mTOR dual 
inhibitor OSI-027, which induced regression of the xenografts (18). 
 
9.1.3.6 
Description of Biological Samples Required for Assay 
 
This assay of paired samples depends upon procurement of bone marrow aspirates 
prior to treatment and again on day 8 of therapy.  A pretreatment bone marrow 
aspirate (and biopsy) is considered standard of care for acute leukemia, so the 
pretreatment aspirate will simply involve the acquisition of an extra 3-5 cc of bone 
marrow at the time an aspirate is ordinarily obtained.  The day 8 marrow sample will 
require an extra bone marrow for research purposes only (to determine whether 
MLN0128 (TAK-228) is having its intended effect on mTOR-mediated signaling and 
downstream apoptotic pathways).  Risks of bone marrow aspiration include pain, 
infection and bleeding. Local anesthetic with or without conscious sedation is 
provided to minimize pain.  Sterile technique is utilized to minimize risk of infection.  
With an adequate platelet count (10,000 or more) and local pressure, the risk of 
bleeding is low.  It is conceivable that bleeding, infection or complications of the 
anesthesia could be life threatening, but the risk of death with the day 8 bone marrow 
aspirate is estimated to be on the order of one in a million. 
 
For Patients at Mayo Clinic Rochester only: 
To assess whether this alteration in signaling can also conceivably be detected at 
earlier timepoints and in more accessible cells, for patients treated at Mayo Clinic 
Rochester with a circulating blast count greater than 5000/um3, peripheral blood 
drawn prior to therapy and again at 24 h after the first dose of MLN0128 (TAK-228) 
will be assayed as described above.  To facilitate this analysis, two 7 ml EDTA tubes 
will be drawn prior to the first dose of MLN0128 (TAK-228) and at the time of the 
Cycle 1 Day 2 pharmacokinetics draw. 
 
9.1.3.7 
Comment on Feasibility of Detecting Multiple Antigens by Immunoblotting  
 
Techniques previously reported from the Kaufmann lab (55-57) allow the sequential 
detection of multiple antigens on the same filter without interference.  Once phospho-
4EBP1 has been successfully detected, we propose to use the same filters for 
detection of additional antigens, which are presented below in the order in which they 
will be examined.  Because we routinely detect 10-14 antigens on a duplicate pair of 

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filters (including 8 BCL2 family members, three phospho-proteins and the three 
corresponding total protein controls) if the molecular weights do not over lap and 
suitable secondary antibodies are available, we do not anticipate difficulty in 
completing the proposed immunoblotting with the available samples. 
 
9.1.4 Phospho-Ser473 Analysis of Pretreatment and Day 8 Bone Marrow Samples by 
Immunoblotting (Exploratory Biomarker) 
 
9.1.4.1 
Hypothesis and Rationale 
 
Hypothesis: AKT Ser473 phosphorylation will be inhibited by MLN0128 (TAK-228) 
in situ. 
 
Rationale:  Previous studies have shown that mTOR dual inhibitors such as 
MLN0128 (TAK-228) and OSI-027 diminish the mTORC2-mediated 
phosphorylation of the pro-survival kinase AKT on Ser473, leading to upregulation of 
the pro-apoptotic protein BCL2L11 (Bim) and induction of apoptosis (14, 18). In 
ALL cell lines the down regulation of the mTORC2 component Rictor reproduces 
these effects and down regulation of BCL2L11 (Bim) produces resistance to mTOR 
dual inhibitors (14), demonstrating the critical role of signaling cascade in killing by 
mTOR dual inhibitors. We now propose to assess whether AKT Ser473 has been 
dephosphorylated in leukemic blasts by therapeutically achievable MLN0128 (TAK-
228) concentration in situ as a proof of mechanism for this class of agent. 
 
9.1.4.2 
Assay Validity and Appropriateness for Study 
 
Immunoblotting will be performed to assess AKT phosphorylation on Ser473 in 
mononuclear cells isolated from pretreatment and day 8 bone marrow aspirates using 
Ficoll-Hypaque gradients. Alternative approaches to assessing AKT Ser473 
phosphorylation include ELISA, immunohistochemistry and 2-dimensional gel 
electrophoresis.  The major advantage of immunoblotting is the ability to assess the 
signal at the molecular weight of interest rather than assuming that a signal observed 
in ELISA is due to the antigen of interest.  While it is somewhat less quantitative than 
ELISA, the Kaufmann laboratory has demonstrated that blotting with a rabbit 
monoclonal antibody can detect differences in phospho-Akt over a 64-fold range that 
encompasses the extent of phosphorylation observed in baseline ALL samples 
(unpublished data similar to that in Appendix D for phospho-4EBP1). 
 
9.1.4.3 
Performance Site   
 
The assay will be performed in the Kaufmann laboratory of the Mayo Clinic Cancer 
Center.  This lab has extensive experience with detecting changes in antigen content 
by immunoblotting (53, 54). 
 

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9.1.4.4 
Data Supporting the Degree of Biomarker “Fit for Purpose”   
 
As indicated above, unpublished data using a commercially available anti-phospho-
Ser473-AKT antibody demonstrate a 32-fold dynamic range for detection of this 
antigen that encompasses the baseline antigen phosphorylation in clinical ALL 
specimens. 
 
9.1.4.5 
Justification of the Number of Patients and Specimens   
 
The purpose of the assay is to determine whether MLN0128 (TAK-228) induces 
dephosphorylation of an mTORC2 target in leukemia cells in situ at clinically 
achievable concentrations.  After the signal for phospho-Ser473-AKT is quantitated, 
results will be examined graphically to assess the extent and frequency of down 
regulation as described in Section 13.4.6.  This will be the first opportunity to assess 
AKT phosphorylation in ALL after treatment with an mTOR dual inhibitor, so the 
extent and frequency of down regulation cannot be estimated from precedent data.  
As a point of reference, however, complete dephosphorylation of AKT Ser473 was 
observed in Jeko mantle cell lymphoma xenografts after treatment of mice with the 
mTOR dual inhibitor OSI-027, which induced regression of the xenografts (18). 
 
9.1.5 Expression of BCL2L11 (Bim), BBC3 (Puma), MCL1 and BCL2 Proteins in 
Pretreatment and Day 8 Bone Marrow Samples by Immunoblotting (Exploratory 
Biomarkers) 
 
9.1.5.1 
Hypothesis and Rationale 
 
Hypothesis: Levels of BCL2L11 (Bim) and BBC3 (Puma) in ALL will increase 
during MLN0128 (TAK-228) treatment, whereas levels of MCL1 and possibly 
BCL2 will decrease. 
 
Rationale:  Previous studies have shown that mTOR dual inhibitors such as 
MLN0128 (TAK-228) and OSI-027 cause upregulation of the pro-apoptotic BCL2 
family members BBC3 (Puma) and BCL2L11 (Bim) in malignant lymphoid cells 
(14, 18).  In ALL cell lines the down regulation of BBC3 (Puma) or BCL2L11 (Bim) 
produces resistance to mTOR dual inhibitors (14, 18), demonstrating the critical role 
of these proteins in killing by mTOR dual inhibitors.  Conversely, the anti-apoptotic 
BCL2 family member MCL1 has been shown to decrease after treatment with 
mTOR dual inhibitors in certain malignant human lymphoid cell lines (18).  
Overexpression of eponymous BCL2 has also been shown to convey resistance to 
mTOR dual inhibitors (18).  Accordingly, we propose to assess whether BCL2L11 
(Bim) and BBC3 (Puma) have been upregulated or MCL1 down regulated in 
leukemic blasts by therapeutically achievable MLN0128 (TAK-228) concentration 
in situ as a further proof of mechanism for this class of agent. 
 

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9.1.5.2 
Assay Validity and Appropriateness for Study 
 
Immunoblotting will be performed to assess BCL2L11 (Bim), BBC3 (Puma), MCL1 
and BCL2 in mononuclear cells isolated from pretreatment and day 8 bone marrow 
aspirates using Ficoll-Hypaque gradients.  Alternative approaches to assessing 
expression of these proteins include ELISA, immunohistochemistry and qRT-PCR.  
The major advantage of immunoblotting is the ability to assess the signal at the 
molecular weight of interest rather than assuming that a signal observed in ELISA or 
immunohistochemistry is due to the antigen of interest.  The advantages of 
immunoblotting over qRT-PCR are i) the proteins that are regulating apoptosis are 
actually being measured rather than mRNA levels, which might not correlate with 
protein levels; and ii) all of these proteins persist in mitochondria as cells undergo 
apoptosis, whereas mRNA is rapidly degraded at the outset of apoptosis.  The 
Kaufmann laboratory has previously used this immunoblotting approach to examine 
differences in BCL2 family members among different cell lines (41) and pretreatment 
acute leukemia samples (58) as well as changes in BCL2, MCL1 and BCL2L11 
(Bim) during the course of acute leukemia (40) and lymphoma treatment (59). 
 
9.1.5.3 
Performance Site   
 
The assay will be performed in the Kaufmann laboratory of the Mayo Clinic Cancer 
Center.  This lab has extensive experience with detecting changes in antigen content 
by immunoblotting (53, 54). 
 
9.1.5.4 
Data Supporting the Degree of Biomarker “Fit for Purpose”  
 
Unpublished data using commercially available anti-MCL1 and anti-BCL2 antibodies 
demonstrate a 32-fold dynamic range for detection of these antigens. 
 
9.1.5.5 
Justification of the Number of Patients and Specimens   
 
The purpose of the assay is to determine whether MLN0128 (TAK-228) induces 
upregulation of BCL2L11 (Bim) and BBC3 (Puma) or down regulation of MCL1 and 
BCL2 in ALL cells in situ at clinically achievable concentrations.  After the signal for 
BCL2L11 (Bim), BBC3 (Puma), MCL1 and BCL2 is quantitated, results will be 
examined graphically to assess the extent and frequency of down regulation as 
described in Section 13.4.6.  This will be the first opportunity to assess these BCL2 
family members in clinical ALL after treatment with an mTOR dual inhibitor in vivo, 
so the extent and frequency of altered expression cannot be estimated from precedent 
data.  As a point of reference, however, induction of BBC3 (Puma) was observed in 
Jeko mantle cell lymphoma xenografts after treatment of mice with the mTOR dual 
inhibitor OSI-027, which induced regression of the xenografts (18). 
 
9.1.5.6 
Detection of Additional Antigens 
 

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In the consensus review of this Protocol, CTEP reviewers suggested that additional 
BCL2 family members also be examined, specifically noting that BCL2L1 (Bcl-xL) 
and BCL2L2 (Bcl-w) would be of interest.  These analytes are prioritized lower than 
the four BCL2 family members listed above because i) changes in BCL2L1 and 
BCL2L2 have not been directly implicated in the mechanism of cytotoxicity of 
MLN0128 (TAK-228) in ALL cells and ii) we accordingly have not characterized 
assays for these proteins to the same extent as we have the proof-of-mechanism 
studies and cannot comment on the dynamic range, fitness of purpose, or appropriate 
standards that will encompass the expression levels likely found in clinical ALL 
samples.   
 
 
9.1.6 Reverse Phase Protein Array for Protein Phosphorylations and Expression (Exploratory 
Biomarkers) 
 
9.1.6.1 
Hypothesis and Rationale 
 
Hypothesis: Changes in phosphorylation of mTOR substrates and complementary 
pathway components will change during the course of mTOR dual inhibitor 
treatment. 
 
Rationale:  Prior publications have identified differences in the pattern of protein 
phosphorylation between cell lines that are sensitive to kinase inhibitors and similar 
cell lines that are resistant (e.g., 44, 45). Preliminary studies in the Kaufmann 
laboratory have likewise demonstrated differences in protein phosphorylation at key 
nodes between parental Nalm6 ALL cells and Nalm6 cells selected for the ability to 
grow continuously in mTOR dual inhibitor (C. Correia, K. L. B. Knorr, S. Yun, A. E. 
Wahner Hendrickson, B. Madden, D. McCormick, S. Dasari, and S. H. Kaufmann, 
unpublished observations). Thus, we hypothesize that pretreatment patterns of 
phosphorylation might differ between ALLs that respond to MLN0128 (TAK-228) 
and those that do not. 
 
9.1.6.2 
Assay Validity and Appropriateness for Study 
 
Reverse phase protein arrays (RPPAs) will be utilized to assess levels of 
phosphorylation of 60 key proteins, including 4EBP1, ribosomal protein S6, and 
AKT, as well as expression levels of 200 proteins, including BCL2, BCL2L1 (Bcl-
xL), MCL1, and BCL2L11 (Bim).  Alternative approaches to assessing expression of 
these proteins include ELISA, immunohistochemistry, immunoblotting and qRT-
PCR.  The major advantage of reverse phase protein array is the ability to assess the 
signals related to multiple antigens in a quantitative fashion. Like ELISA or 
immunohistochemistry, this method assumes that the signal is due to the antigen of 
interest; and there is no opportunity to rule out cross reactivity with an antigen of a 
different molecular mass.  On the RPPAs, for example, the BBC3 (Puma) signal will 
be unreliable because the commercial antibody used recognizes a band that is present 

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even in BBC3-/- human cells (K.L. Peterson and S.H. Kaufmann, unpublished 
observations).  The advantages of RPPA over qRT-PCR are that in RPPA the proteins 
and posttranslational modifications that are regulating apoptosis are actually being 
measured rather than mRNA levels, which might not correlate with protein levels.  
This analysis of key signaling pathways will be utilized to supplement the results of 
the more targeted immunoblotting assays. 
 
9.1.6.3 
Performance site   
 
After mononuclear cells are isolated on Ficoll-Hypaque gradients, whole cell lysates 
will be prepared in the Kaufmann laboratory of the Mayo Clinic Cancer Center.  
Based on preliminary results, the same lysates prepared for immunoblotting (see 
Section 9.1.2) are suitable for RPPAs.  The RPPAs will be probed and initial 
bioinformatic analysis performed in the RPPA Core Facility of the M.D. Anderson 
Cancer Center, which is a fee-for-service facility. 
 
9.1.6.4 
Data Supporting the Degree of Biomarker “Fit for Purpose” 
 
The reproducibility and reliability of this method, as well as comparison to the gold 
standard of Western blotting, has been published by the founders of the RPPA Core 
Facility at M.D. Anderson Cancer Center (60). 
 
9.1.6.5 
Justification of the Number of Patients and Specimens   
 
The purposes of this assay are: i) to confirm the results obtained in the assays 
described in Sections 9.1.2-9.1.4 using different antibodies and a different analytical 
platform and ii) to assess the status of other signaling pathways before and after 
exposure to MLN0128 (TAK-228) in situ. This will be the first opportunity to assess 
the performance of this assay in ALL after treatment with an mTOR dual inhibitor, so 
the extent and frequency of altered phosphorylation or expression of various proteins 
cannot be estimated from precedent data.  
 
9.1.6.6 
Plans for comparison of immunoblotting and RPPA results 
 
Immunoblotting and blot quantification will be performed as described in Appendix 
C. Comparison of the relative signal in day 1 and day 8 samples from individual 
patients (run on adjacent lanes of the same gel and compared to a standard curve) will 
allow a calculation of % change in signal between the two samples.  Likewise, 
comparison of the two samples (day 1 and day 8) subjected to RPPA will allow a 
calculation of a % change between day 1 and day 8.  To compare the assays for any 
particular analyte (e.g., the integrated biomarker phospho-Thr37,46-4EBP1), we will 
construct a scatter plot with the % change in the immunoblotting assay on the x axis 
and the % change in the RPPA assay on the y axis, inspect the distribution of the 
values, and calculate a Spearman correlation coefficient.  Because these assays have 
not been previously performed in this manner, we cannot estimate the value of that 

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Spearman correlation coefficient for any of the assays that will be in common, 
although we suspect it will be close 0 for BBC3, where the signal for bona fide BBC3 
protein (identified by comparing wild type and BBC3-/- cells) is detected by 
immunoblotting and a spurious cross-reactive antigen is detected in the RPPA assays.  
If correlation coefficients closer to 1.0 are observed in assays of other analytes in 
common between the two platforms, we will take this as a sign that the RPPA is a 
reasonable approximation of the immunoblotting assay and examine RPPA results for 
other antigens (not included in the immunoblotting) for potentially interesting 
patterns, e.g., differences in BCL2L2 (Bcl-w) expression that might not be feasible 
with the limited material available for immunoblotting. 
 
9.1.7 Transcriptional Profiling (Exploratory Biomarker) 
 
9.1.7.1 
Hypothesis and Rationale 
 
Hypothesis: Treatment with MLN0128 (TAK-228) will activate two transcriptional 
pathways related to apoptosis. 
 
Rationale:  Previous studies have shown that the mTOR dual inhibitor OSI-027 
activates two distinct transcriptional pathways in ALL cell lines during the course of 
upregulating BCL2L11 (Bim) and BBC3 (Puma) to induce apoptosis (14).  If 
sufficient material is available to allow isolation of RNA (after samples for 
immunoblotting and RPPAs have been harvested), samples will be subjected to RNA 
sequencing (RNAseq) to determine whether MLN0128 (TAK-228) concentrations 
achievable in vivo activate these same transcriptional pathways as a proof of 
mechanism study.  
 
9.1.7.2 
Assay Validity and Appropriateness for Study 
 
RNAseq will be performed using RNA isolated from Ficoll-Hypaque purified 
pretreatment and day 8 bone marrow cells to assess whether transcriptional changes 
observed in clinical ALL leading to BCL2L11 (Bim) and BBC3 (Puma) upregulation 
are also observed in clinical ALL treated in vivo.  Alternative approaches to assessing 
transcript levels include quantitative RT-PCR.  The major advantages of RNAseq are: 
i) message can be quantitated relative to total number of mRNA molecules rather than 
relative to an arbitrary standard, which might or might not change during the course 
of treatment; ii) the entire transcriptome is interrogated in a quantitative fashion one 
assay; and iii) the assay also provides sequence information that can be examined to 
search for alterations in splicing and mutations in various messages.  This is the same 
assay that was utilized to identify the transcriptional pathways that are altered in 
mTOR dual inhibitor-treated ALL cell lines, with subsequent confirmation of results 
by qRT-PCR (14). 
 
9.1.7.3 
Performance Site   
 

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RNA will be isolated in the Kaufmann laboratory of the Mayo Clinic Cancer Center. 
Library construction will take place in the Gene Expression Core of the Mayo Clinic 
Genome Research Facility; and sequencing of the library will take place in the 
Sequencing Core of the Mayo Clinic Genome Research Facility. 
 
9.1.7.4 
Data Supporting the Degree of Biomarker “Fit for Purpose”   
 
Results currently submitted for publication (14) document the reproducibility of the 
assay as well as its ability to identify changes in the transcriptome that result from 
alterations in transcription factors as a consequence of treatment of human ALL cell 
lines with mTOR dual inhibitors. 
 
9.1.7.5 
Justification of the Number of Patients and Specimens   
 
The purpose of the assay is to determine whether MLN0128 (TAK-228) induces 
transcriptional changes in ALL cells in situ that reflect its mechanism of cytotoxicity 
at clinically achievable concentrations.  This will be the first opportunity to assess 
changes in the transcriptome in clinical ALL after treatment with an mTOR dual 
inhibitor, so the extent and frequency of transcriptional changes cannot be estimated 
from precedent data. 
 
9.2 
Laboratory Correlative Studies 
 
9.2.1 MLN0128 (TAK-228) Pharmacokinetics – CSF 
 
9.2.1.1 
Patient Considerations 
 
Patients participating in this study will have intrathecal (IT) chemotherapy as 
clinically indicated care within 7 to 10 days of starting study treatment.  If possible, 
collect up to a 2 mL sample of CSF prior to the infusion of the IT chemotherapy.  If a 
patient has another clinically indicated IT chemotherapy performed at any subsequent 
time in the study, collect an additional 2 mL sample of CSF prior to the infusion of 
the IT chemotherapy, if possible. 
 
9.2.1.2 
CSF Collection Schedule 
 
Collect up to 2 mL of CSF prior to the infusion of the IT chemotherapy withun 7 to 
10 days of starting study treatment and prior to any subsequent clinically indicated IT 
chemotherapy performed at any time during the study if possible. 
 
9.2.1.3 
Handling of Specimens 
 
At Mayo Clinic Rochester only: Transport CSF sample on ice to Dr Reid’s lab. 
 
 
For all other sites: Transfer CSF sample to cryovial and freeze at -80º C.  Ship with 
PK serum samples. 
 
9.2.1.4 
Shipment of Samples 

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Ship CSF and PK samples on dry ice Monday – Thursday by FedEx First Overnight 
to the following address.  Please be certain to use the First Overnight service; should 
there be a concern about this, please contact the study principal investigator or the 
project manager.  
 
 
Dr Joel Reid, PhD 
 
Mayo Clinic 
 
Guggenheim 17-37 
 
200 First Street SW 
 
Rochester, MN 55905 
 
 
9.2.2  MLN0128 (TAK-228) Pharmacokinetics – Plasma 
 
9.2.2.1 
Patient Considerations 
Patients participating in this study will be asked to provide blood samples during 
Cycle 1 of treatment.  The morning dose of MLN0128 (TAK-228) must not be given 
until after pretreatment blood draw. 
 
9.2.2.2 
Blood Collection Schedule 
Blood samples (6 ml) will be drawn in green top sodium heparin tubes 30 minutes 
before drug administration on Days 1, 2 and 8, and at the following times after 
administering the oral dose on Day 1 and 8:  30 min, 1 hr, 2 hrs, 3 hrs, 4 hrs, and 8 
hrs.  Thus, 15 samples will be collected for a total of 90 ml whole blood.  Note that 
the windows for the pk samples are ± 10 min for the 30 minutes pre-drug sample; ± 5 
minutes for the 30 minute and 1 hour post-drug samples; ± 10 min for 2 hours post-
drug sample and  ± 15 min for  the 3, 4 and 8 hour post-drug samples.  It is more 
important to record time accurately than to perform the blood drawn exactly at the 
scheduled time. 
 
Note: The physicial exam and labs performed on Cycle 1 Day 1 may be performed 
the day prior in order to begin the pharmacokinetic sample collection early enough on 
Cycle 1 day 1 that an 8 hour pk sample is possible.  If an 8 hour pk draw is not 
possible, please collect a 7 hour or a 7 ½ hour sample and note the timepoint on the 
label of the tube and a collection draw worksheet.    
 
Please complete the pk sample blood draw list in Appendix D and include a copy 
with the samples at shipment.   
 
9.2.2.3 
Handling of Specimens 
Collect blood samples in sodium heparin (green-top) tubes and immediately cool in 
an ice water bath.  Isolate plasma within 20 minutes of collection. 
 
Blood will be subjected to centrifugation (2,000 rpm for 10 minutes) in a refrigerated 
centrifuge kept at 4°C.  Following centrifugation, plasma aliquots of at least 2 ml 
each should be transferred to 4 mL plastic cryovials, capped and immediately frozen 

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at -70°C.  
 
Tube labels must contain the following data:  patient identification code, date,  draw 
timepoint and actual time of draw.  Please complete the pk sample blood draw list in 
Appendix D and include a copy with the samples at shipment.   with the samples. 
 
9.2.2.4 
Shipment of specimens 
Ship CSF and serum pk samples on dry ice only Monday – Thursday by FedEx First 
Overnight to the following address.  Please be certain to use the First Overnight 
service; should there be a concern about this, please contact the study principal 
investigator or the project manager.    
 
Dr. Joel Reid, PhD 
Mayo Clinic 
Guggenheim 17-37 
200 First Street SW 
Rochester, MN 55905 
 
9.2.2.5 
Site(s) Performing the Pharmacokinetic Study 
 
Samples will be analyzed in the Pharmacology Core of the Mayo Clinic Cancer 
Center, which has developed validated assays for over 85 separate anticancer drugs 
during the course of its existence. 
 
9.2.3 Immunoblotting for Phosphorylation of 4EBP1 and Other Markers 
 
9.2.3.1 
Collection of Specimen(s):  Bone marrow aspirate collected before treatment and on 
day 8 ± 2 of treatment with MLN0128 (TAK-228) 
 
9.2.3.2 
Handling of Specimens(s): Place 2-5 cc of marrow aspirate in a 5 or 7 cc heparin 
(green top) tube.  Place the tube in a Styrofoam container with adequate padding.  
Between May and October add a 4 ºC cold pack to the container to prevent 
overheating during shipping.  
 
9.2.3.3 
Shipping of Specimen(s):  Ship specimen only Monday-Thursday by FedEx First 
Overnight to: 
 
Ms. Karen Flatten 
c/o Scott Kaufmann, M.D., Ph.D. 
Gonda 19-205B 
Mayo Clinic 
200 First Street S.W. 
Rochester, MN 55905 
 
Please call (507) 284-4304 or fax (507) 293-0107 Ms. Flatten on the day of shipment so 
that she will know a sample is coming for the next day. 

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9.2.3.4 
Site(s) Performing Correlative Study:  Kaufmann laboratory, Mayo Clinic, Rochester, 
Minnesota 
 
9.2.3.5 
Other marrow correlative studies:  Please note that phosphorylation of AKT (Section 
9.1.4), expression of BCL2 family members (Section 9.1.5), reverse phase protein 
array (Section 9.1.6) and transcriptional profiling (Section 9.1.7) will be performed 
on the same marrow aspirate specimens harvested for 4EBP1 phosphorylation. 
Separate specimens for these assays are not required. 
 
9.2.4 Immunoblotting for Phosphorylation of 4EBP1 and Other Markers 
 
 
9.2.4.1 
Collection of Specimen(s):  Bone marrow aspirate collected before treatment and on 
day 8 ± 2 of treatment with MLN0128 (TAK-228) 
 
9.2.4.2 
Handling of Specimens(s): Place 2-5 cc of marrow aspirate in a 5 or 7 cc heparin 
(green top) tube.  Place the tube in a Styrofoam container with adequate padding.  
Between May and October add a 4 ºC cold pack to the container to prevent 
overheating during shipping.  
 
9.2.4.3 
Shipping of Specimen(s):  Ship specimen only Monday-Thursday by FedEx First 
Overnight to the following address.  Please be certain to use the First Overnight 
service; should there be a concern about this, please contact the study principal 
investigator or the project manager. 
 
Ms. Karen Flatten 
c/o Scott Kaufmann, M.D., Ph.D. 
Gonda 19-205B 
Mayo Clinic 
200 First Street S.W. 
Rochester, MN 55905 
 
Please call (507) 284-4304 or fax (507) 293-0107 Ms. Flatten on the day of shipment 
so that she will know a sample is coming for the next day. 
 
 
9.2.4.4 
Site(s) Performing Correlative Study:  Kaufmann laboratory, Mayo Clinic, Rochester, 
Minnesota 
 
9.2.4.5 
Other marrow correlative studies:  Please note that phosphorylation of AKT (Section 
9.1.4), expression of BCL2 family members (Section 9.1.5), reverse phase protein 
array (Section 9.1.6) and transcriptional profiling (Section 9.1.7) will be performed 
on the same marrow aspirate specimens harvested for 4EBP1 phosphorylation. 
Separate specimens for these assays are not required. 
 

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9.2.5 
Assessment of 4EBP1 phosphorylation inhibition at earlier time points (Mayo Clinic 
Rochester only) 
 
9.2.5.1 
Collection of Specimen(s): If circulating blast count is greater than 5000,  draw two 7 
mL samples in purple topped EDTA tubes prior to first dose of MLN0182 (TAK-228) 
and at time of Cycle 1 Day 2 MLN0128 (TAK-228) PK draw.  Please call Karen 
Flatten to let her know that a sample will be arriving. 
 
9.2.5.2 
Handling of Specimen(s):  Hand carry specimen to Kaufmann lab at Mayo Clinic 
Rochester at Gonda 19-205B. 
 
9.2.5.3 
Site(s) Performing Correlative Study:  Kaufmann laboratory, Mayo Clinic, Rochester, 
Minnesota  
 

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10. 
STUDY CALENDAR 
Baseline evaluations are to be conducted within 1 week prior to start of protocol therapy.  Scans 
and x-rays must be done < 4 weeks prior to the start of therapy.  In the event that the patient’s 
condition is deteriorating, laboratory evaluations should be repeated within 48 hours prior to 
initiation of the next cycle of therapy.  After C1D15, a window of + 7 days for study procedures 
is permitted. 
 
 
Pre- 
Study 
C 1 
D 1 
C 1 
D2 
C 1 
D8 
C1 
D15 
C1 
D 22 
C2 
D1 
C3 
D1 
C4 
D1 
C5 
D1 
C6 
D1 
C6 
D28 
C7 D1  
and 
above 
Off 
Studyd 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Concurrent meds 
X 
X 
 
 
X 
 
X 
X 
X 
X 
X 
 
X 
X 
Physical exam 
X 
X 
 
 
X 
 
X 
X 
X 
X 
X 
 
X 
X 
Vital signs 
X 
X 
 
 
X 
 
X 
X 
X 
X 
X 
 
X 
X 
Height 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Weight 
X 
X 
 
 
 
 
X 
X 
X 
X 
X 
 
X 
X 
Performance status 
X 
X 
 
 
X 
 
X 
X 
X 
X 
X 
 
X 
X 
CBC w/diff, plts 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
HbA1c 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Serum chemistry 
X 
X 
 
X 
 
 
X 
X 
X 
X 
X 
 
X 
X 
Serum pregnancy 
test 
Xb 
 
 
 
 
 
 
 
 
 
 
 
 
 
EKG (as indicated) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Adverse event 
evaluation 
X 
X 
 
 
X 
 
X 
X 
X 
X 
X 
 
X 
X 
Bone marrow 
aspirate 
X 
 
 
Xc 
 
 
 
X 
 
X 
 
X 
 
X c 
CSFR 
Xe 
 
 
 
 
 
 
 
 
 
 
 
 
 
Research blood for 
PKR 
 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
Bone Marrow 
aspirate 
pharmacodynamic 
assaysR 
X 
 
 
X 
 
 
 
 
 
 
 
 
 
X 

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Mayo Clinic 
Rochester only: 
Research blood for 
pharmacodynamic 
assaysh, R 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
 
a: Albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, chloride, 
creatinine, fasting glucose, LDH, phosphorus, potassium, total protein, SGOT [AST], 
SGPT [ALT], sodium, uric acid. 
b: Serum pregnancy test (women of childbearing potential). 
c:   Aspirate only for research assay is collected. 
d:   1 month after end of treatment 
e:   CSF sample collected during clinically indicated intrathecal chemotherapy for pk analysis if 
possible.  If subsequent clinically indicated intrathecal chemotherapy is performed, a CSF 
sample is requested if possible. Collect up to 2 mL if possible, transfer to a cryovial and 
freeze at -80º C. Ship to Dr Reid’s lab with the blood pk samples. 
f:   Blood draws for pks are collected 30 minutes (± 10 minutes) before study drug 
administration on Days 1, 2 and 8, and at the following times after study drug on Day 1 and 
8:  30 minutes (± 5 minutes), 1 hour (± 5 minutes), 2 hours (± 10 minutes), 3 hrs, 4 hrs, and 
8 hours (all ± 15 minutes).  It is more important to record time accurately than to perform 
the blood draw exactly on time. 
g:   Cycle 1 Day 1 physical exam and labs may be performed the day prior to Cycle 1 Day 1 in 
order to start the pharmacokinetic samples collection early on Cycle 1 Day 1. 
h:   Blood draws for pharmacodynamics are collected anytime prior to the first dose of 
MLN0128 (TAK-228) and at Cycle 1 Day 2 with the PK draw. R:   Research funded 
 
11. MEASUREMENT OF EFFECT 
 
11.1 
Antitumor Effect  
 
11.1.1 Definitions 
 
Evaluable for toxicity.  All patients will be evaluable for toxicity from the time of their 
first treatment with MLN0128 (TAK-228). 
 
Evaluable for objective response.  All patients who have measurable disease present at 
baseline, have begun treatment, and have had their disease re-evaluated will be 
considered evaluable for response.  These patients will have their response classified 
according to the definitions stated below.  (Note:  Patients who exhibit objective disease 
progression prior to the end of cycle 1 will also be considered evaluable.) 
 
11.1.2 Response Criteria 
 
11.1.2.1 
Evaluation  
 
Complete hematologic response (CR)  

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Less than 5% blasts in a non-hypocellular marrow with a granulocyte count of 1 
x109/L (or above), and a platelet count of 100 x109/L (or higher) and absence of 
peripheral blood blasts with complete resolution of any extra medullary disease.  
The patient is in sustained CR if they have previously achieved a CR and continue to 
meet the CR criteria (at least 28 days). 
 
CR incomplete (CRi) 
is called if patient meets all CR criteria except for residual neutropenia (ANC<1 
x109/L) or thrombocytopenia (platelets<100 x109/L) 
 
Partial Response (PR):   
The presence of trilineage hematopoiesis in the bone marrow with recovery of ANC 
and platelet count to above levels, but with 5-25% bone marrow blasts and ≥50% 
decrease in bone marrow blast percentage from baseline. 
 
Morphologic leukemia-free state (MLFS) 
If bone marrow blasts <5%, absence of Auer rods blasts, absence of extra medullary 
disease without hematological recovery. 
 
No response (NR):  
Failure to achieve CR/CRi/PR/MLFS after 2 cycles of MLN0128 (TAK-228). 
 
Disease Progression: 
At least a 25% increase in the absolute number of circulating blasts in peripheral 
blood, or at least 25% increase in bone marrow blasts, or development of 
extramedullary disease. 
 
Relapse 
Disease recurrence after achieving CR. Disease recurrence is defined by blast 
≥ 5% in the bone marrow, or recurrence of peripheral blood  > 5% or extramedullary 
involvement. 
 
11.1.3 Duration of Response 
 
Duration of response is the duration of CR or CRi is measured from the time 
measurement criteria are first met for CR until the first date that recurrence is objectively 
documented.  
 
 
12. 
DATA REPORTING / REGULATORY REQUIREMENTS 
 
Adverse event lists, guidelines, and instructions for AE reporting can be found in Section 7.0 
(Adverse Events: List and Reporting Requirements). 
 
12.1 
Study Oversight 
 

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This protocol is monitored at several levels, as described in this section.  The Protocol Principal 
Investigator is responsible for monitoring the conduct and progress of the clinical trial, including 
the ongoing review of accrual, patient-specific clinical and laboratory data, and routine and 
serious adverse events; reporting of expedited adverse events; and accumulation of reported 
adverse events from other trials testing the same drug(s).  The Protocol Principal Investigator and 
statistician have access to the data at all times through the CTMS web-based reporting portal.   
 
The Protocol Principal Investigator will have conference calls with the Study Investigators to 
review accrual, progress, and pharmacovigilance every two months for the first year that the 
study is accruing patients and then every three months after the first year.  Decisions to proceed 
to the second stage of a Phase 2 trial will require sign-off by the Protocol Principal Investigator 
and the Protocol Statistician through IWRS and Medidata Rave. 
 
All Study Investigators at participating sites who register/enroll patients on a given protocol are 
responsible for timely submission of data via Medidata Rave and timely reporting of adverse 
events for that particular study.  This includes timely review of data collected on the electronic 
CRFs submitted via Medidata Rave. 
 
All studies are also reviewed in accordance with the enrolling institution’s data safety monitoring 
plan. 
 
 
12.2 
Data Reporting 
 
Data collection for this study will be done exclusively through Medidata Rave.  Access to the 
trial in Rave is granted through the iMedidata application to all persons with the appropriate 
roles assigned in the Regulatory Support System (RSS).  To access Rave via iMedidata, the site 
user must have an active CTEP IAM account (https://eapps-ctep.nci.nih.gov/iam) and the 
appropriate Rave role (Rave CRA, Read-Only, or Site Investigator) on either the lead protocol 
organization or participating organization roster at the enrolling site.   
 
Upon initial site registration approval for the study in RSS, all persons with Rave roles assigned 
on the appropriate roster will be sent a study invitation e-mail from iMedidata.  To accept the 
invitation, site users must log into the Select Login (https://login.imedidata.com/selectlogin) 
using their CTEP-IAM user name and password, and click on the “accept” link in the upper 
right-corner of the iMedidata page.  Please note, site users will not be able to access the study in 
Rave until all required Medidata and study specific trainings are completed.  Trainings will be in 
the form of electronic learnings (eLearnings), and can be accessed by clicking on the link in the 
upper right pane of the iMedidata screen.   
 
Users that have not previously activated their iMedidata/Rave account at the time of initial site 
registration approval for the study in RSS will also receive a separate invitation from iMedidata 
to activate their account.  Account activation instructions are located on the CTSU website, Rave 
tab under the Rave resource materials (Medidata Account Activation and Study Invitation 
Acceptance).  Additional information on iMedidata/Rave is available on the CTSU website 
under the Rave tab at www.ctsu.org/RAVE/ or by contacting the CTSU Help Desk at 1-888-823-

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5923 or by e-mail at ctsucontact@westat.com. 
 
12.2.1 Method 
 
This study will be monitored by the Clinical Trials Monitoring Service (CTMS).  Data 
will be submitted to CTMS at least once every two weeks via Medidata Rave (or other 
modality if approved by CTEP).  Information on CTMS reporting is available at:  
http://www.theradex.com/CTMS.  On-site audits will be conducted on an 18-36 month 
basis as part of routine cancer center site visits.  More frequent audits may be conducted 
if warranted by accrual or due to concerns regarding data quality or timely submission.  
For CTMS monitored studies, after users have activated their accounts, please contact the 
Theradex Help Desk at (609) 799-7580 or by email at ctms@theradex.com for additional 
support with Rave and completion of CRFs. 
 
12.2.2 Responsibility for Data Submission 
 
For ETCTN trials, it is the responsibility of the PI(s) at the site to ensure that all 
investigators at the ETCTN Sites understand the procedures for data submission for each 
ETCTN protocol and that protocol specified data are submitted accurately and in a timely 
manner to the CTMS via the electronic data capture system, Medidata Rave.   
 
Data are to be submitted via Medidata Rave to CTMS on a real-time basis, but no less 
than once every 2 weeks.  The timeliness of data submissions and timeliness in resolving 
data queries will be tracked by CTMS.  Metrics for timeliness will be followed and 
assessed on a quarterly basis.  For the purpose of Institutional Performance Monitoring, 
data will be considered delinquent if it is greater than 4 weeks past due. 
 
Data from Medidata Rave and CTEP-AERS is reviewed by the CTMS on an ongoing 
basis as data is received.  Queries will be issued by CTMS directly within Rave.  The 
queries will appear on the Task Summary Tab within Rave for the CRA at the ETCTN to 
resolve.  Monthly web-based reports are posted for review by the Drug Monitors in the 
IDB, CTEP.  Onsite audits will be conducted by the CTMS to ensure compliance with 
regulatory requirements, GCP, and NCI policies and procedures with the overarching 
goal of ensuring the integrity of data generated from NCI-sponsored clinical trials, as 
described in the ETCTN Program Guidelines, which may be found on the CTEP 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/adverse_events.htm
)  and CTSU websites. 
 
An End of Study CRF is to be completed by the PI, and is to include the recommended 
phase 2 dose (RP2D), and a description of any dose-limiting toxicities (DLTs).  CTMS 
will utilize a core set of eCRFs that are Cancer Data Standards Registry and Repository 
(caDSR) compliant (http://cbiit.nci.nih.gov/ncip/biomedical-informatics-
resources/interoperability-and-semantics/metadata-and-models).  Customized eCRFs will 
be included when appropriate to meet unique study requirements.  The PI is encouraged 
to review the eCRFs, working closely with CTMS to ensure prospectively that all 
required items are appropriately captured in the eCRFs prior to study activation.  CTMS 

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will prepare the eCRFs with built-in edit checks to the extent possible to promote data 
integrity. 
 
CDUS data submissions for ETCTN trials activated after March 1, 2014, will be carried 
out by the CTMS contractor, Theradex.  CDUS submissions are performed by Theradex 
on a monthly basis.  The trial’s lead institution is responsible for timely submission to 
CTMS via Rave, as above. 
 
Further information on data submission procedures can be found in the ETCTN Program 
Guidelines 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/adverse_events.htm
). 
 
12.3 
Collaborative Agreements Language 
 
The agent(s) supplied by CTEP, DCTD, NCI used in this protocol is/are provided to the NCI 
under a Collaborative Agreement (CRADA, CTA, CSA) between the Pharmaceutical 
Company(ies) (hereinafter referred to as “Collaborator(s)”) and the NCI Division of Cancer 
Treatment and Diagnosis.  Therefore, the following obligations/guidelines, in addition to the 
provisions in the “Intellectual Property Option to Collaborator”  
(http://ctep.cancer.gov/industryCollaborations2/intellectual_property.htm) contained within the 
terms of award, apply to the use of the Agent(s) in this study: 
 
1. Agent(s) may not be used for any purpose outside the scope of this protocol, nor can 
Agent(s) be transferred or licensed to any party not participating in the clinical study.  
Collaborator(s) data for Agent(s) are confidential and proprietary to Collaborator(s) and shall 
be maintained as such by the investigators.  The protocol documents for studies utilizing 
Agents contain confidential information and should not be shared or distributed without the 
permission of the NCI.  If a copy of this protocol is requested by a patient or patient’s family 
member participating on the study, the individual should sign a confidentiality agreement.  A 
suitable model agreement can be downloaded from: http://ctep.cancer.gov. 
 
2. For a clinical protocol where there is an investigational Agent used in combination with 
(an)other Agent(s), each the subject of different Collaborative Agreements, the access to and 
use of data by each Collaborator shall be as follows (data pertaining to such combination use 
shall hereinafter be referred to as "Multi-Party Data”): 
 
a. NCI will provide all Collaborators with prior written notice regarding the existence and 
nature of any agreements governing their collaboration with NCI, the design of the 
proposed combination protocol, and the existence of any obligations that would tend to 
restrict NCI's participation in the proposed combination protocol. 
 
b. Each Collaborator shall agree to permit use of the Multi-Party Data from the clinical trial 
by any other Collaborator solely to the extent necessary to allow said other Collaborator 
to develop, obtain regulatory approval or commercialize its own Agent. 

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c. Any Collaborator having the right to use the Multi-Party Data from these trials must 
agree in writing prior to the commencement of the trials that it will use the Multi-Party 
Data solely for development, regulatory approval, and commercialization of its own 
Agent. 
 
3. Clinical Trial Data and Results and Raw Data developed under a Collaborative Agreement 
will be made available to Collaborator(s), the NCI, and the FDA, as appropriate and unless 
additional disclosure is required by law or court order as described in the IP Option to 
Collaborator (http://ctep.cancer.gov/industryCollaborations2/intellectual_property.htm).  
Additionally, all Clinical Data and Results and Raw Data will be collected, used and 
disclosed consistent with all applicable federal statutes and regulations for the protection of 
human subjects, including, if applicable, the Standards for Privacy of Individually 
Identifiable Health Information set forth in 45 C.F.R. Part 164. 
 
4. When a Collaborator wishes to initiate a data request, the request should first be sent to the 
NCI, who will then notify the appropriate investigators (Group Chair for Cooperative Group 
studies, or PI for other studies) of Collaborator's wish to contact them. 
 
5. Any data provided to Collaborator(s) for Phase 3 studies must be in accordance with the 
guidelines and policies of the responsible Data Monitoring Committee (DMC), if there is a 
DMC for this clinical trial. 
 
6. Any manuscripts reporting the results of this clinical trial must be provided to CTEP by the 
Group office for Cooperative Group studies or by the principal investigator for non-
Cooperative Group studies for immediate delivery to Collaborator(s) for advisory review and 
comment prior to submission for publication.  Collaborator(s) will have 30 days from the 
date of receipt for review.  Collaborator shall have the right to request that publication be 
delayed for up to an additional 30 days in order to ensure that Collaborator’s confidential and 
proprietary data, in addition to Collaborator(s)’s intellectual property rights, are protected.  
Copies of abstracts must be provided to CTEP for forwarding to Collaborator(s) for courtesy 
review as soon as possible and preferably at least three (3) days prior to submission, but in 
any case, prior to presentation at the meeting or publication in the proceedings.  Press 
releases and other media presentations must also be forwarded to CTEP prior to release.  
Copies of any manuscript, abstract and/or press release/ media presentation should be sent to 
email: ncicteppubs@mail.nih.gov 
 
The Regulatory Affairs Branch will then distribute them to Collaborator(s).  No publication, 
manuscript or other form of public disclosure shall contain any of Collaborator’s confidential/ 
proprietary information. 
 
 

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13. 
STATISTICAL CONSIDERATIONS 
 
13.1 
Study Design/Endpoints 
 
This is a phase II study in relapsed or refractory ALL patients treated with MLN0128 
(TAK-228). This study will use a one-stage design with an interim analysis to evaluate 
the efficacy of MLN0128 (TAK-228) in this patient population.  
 
13.1.1 Primary Endpoint  
The primary endpoint of this trial is complete response (CR, CRi) rate.  A complete 
response will be considered synonymous with “success”.  All patients meeting the 
eligibility criteria who have signed a consent form and have begun treatment will be 
evaluable for response. 
 
13.1.2 Primary Analyses Plan 
The interim and final analyses for this trial will commence at the time the patients have 
become evaluable for the primary endpoint.  Analyses at other time points will be a 
decision made by the Statistician and Study Chair, in accord with CCS Standard 
Operating Procedures, availability of data for secondary endpoints, and the level of data 
maturity.  It is anticipated that the earliest date in which the results will be made available 
via manuscript, abstract, or presentation format is when the last patient registered has 
been followed for at least 6 months. 
 
13.1.2.1 Definition of Primary Endpoint  
The primary endpoint of this trial is complete response rate.  A complete response is 
defined to be a CR or CRi noted as the objective status at any time during treatment.  
Response will be evaluated using all cycles of treatment.  All patients meeting the 
eligibility criteria who have signed a consent form and have begun treatment will be 
evaluable for response.   
 
13.1.2.2 Estimation 
The proportion of successes will be estimated by the number of successes divided by 
the total number of evaluable patients.  Ninety-five percent Duffy-Santner confidence 
intervals for the true success proportion will be calculated.   
 
13.1.2.3 Primary Endpoint Evaluable Patient Exception 
Patients not being treated at the safe dose level (see section 13.5) will be summarized 
separately for the primary endpoint and will not be included when evaluating the final 
decision rule.   
 
13.1.2.4 Decision Rule  
Previous studies in relapsed or refractory ALL have shown a CR rate ranging from 20 
to 47% (61, 62).  Responses can vary by factors such as first or second relapse and age.  
Due to the heterogeneity of this disease, a response rate of 10% with MLN0128 (TAK-
228) would not be of interest.  The largest success proportion where the proposed 
treatment regimen would be considered ineffective in this population is 10%, and the 

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smallest success proportion that would warrant subsequent studies with the proposed 
regimen in this patient population is 30%.  The following one-stage design with an 
interim analysis uses 26 evaluable patients to test the null hypothesis that the true 
success proportion in a given patient population is at most 10%.   
 
13.1.2.5 Interim Analysis  
Enter 11 evaluable patients into the study. If 1 or fewer successes are observed in the 
first 11 evaluable patients, we will consider this regimen ineffective in this patient 
population and terminate this study. Otherwise, if the number of successes is at least 2, 
we will continue accrual.  
 
13.1.2.6 Final Decision Rule 
Enter 26 evaluable patients into the study.  If 4 or fewer successes are observed in the 
first 26 evaluable patients, we will consider this regimen ineffective in this patient 
population.  If 5 or more successes are observed in the first 26 evaluable patients, we 
may recommend further testing of this regimen in subsequent studies in this population. 
 
13.1.2.7 Over Accrual  
If more than the target number of patients are accrued at the safe dose, the additional 
patients will not be used to evaluate the stopping rule or used in any decision making 
process; however, they will be included in final endpoint estimates and confidence 
intervals. 
 
NOTE: We will not suspend accrual at the interim analysis to allow the first 11 patients 
to become evaluable, unless undue toxicity is observed.  Given the limited overall 
sample size and the inclusion of an adverse events stopping rule, we feel it is ethical to 
not halt accrual for the interim analysis.  However, if accrual is extremely rapid, we 
may temporarily suspend accrual in order to obtain safety data on these patients before 
re-opening accrual to further patients. 
 
13.1.3  Power and Significance Level   
Assuming that the number of successes is binomially distributed, the significance level is 
.09, i.e. there is a 9% chance of finding the drug to be effective when it truly is not.  The 
probability of declaring that this regimen warrants further study (i.e. statistical power) 
and the probability of stopping at the interim analysis under various success proportions 
can be tabulated as a function of the true success proportion as shown in the following 
table. 
 
 

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If the true success proportion is... 
0.10 
0.15 
0.20 
0.25 0.30 
 
Then the probability of declaring that the regimen 
warrants further study is… 
0.09 
0.28 
0.52 
0.72 0.85 
and the probability of stopping at the interim 
analysis is ... 
 
0.70 
0.49 
0.32 
0.20 0.11 
               
13.1.4 Other Considerations  
Adverse events, quality/duration of response, and patterns of treatment failure observed 
in this study, as well as scientific discoveries or changes in standard care will be taken 
into account in any decision to terminate the study. 
 
13.2 
Sample Size/Accrual Rate 
 
13.2.1 Sample Size   
The one-stage design with an interim analysis to be utilized is fully described above. A 
maximum of 26 evaluable patients will be accrued onto this phase II study unless undue 
toxicity is encountered.  If there is undue toxicity in the initial safety cohort (see section 
13.5), an additional 6 patients will be accrued.  Further, we anticipate accruing an 
additional 3 patients to account for ineligibility, cancellation, major treatment violation, 
or other reasons.  Therefore, the maximum projected accrual is 35 patients. 
 
13.2.2 Accrual Rate and Study Duration  
The anticipated accrual rate is 1 evaluable patient per month.  Therefore, the accrual 
period for this study is expected to be approximately 3 years.  The final analysis can 
begin approximately 3.5 years after the trial begins, i.e. as soon as the last patient has 
been on treatment for 6 months.  
 
13.2.3 Inclusion of Women and Minorities 
 
13.2.3.1 This study will be available to all eligible patients, regardless of race, gender, or ethnic 
origin. 
 
13.2.3.2 There is no information currently available regarding differential effects of this regimen 
in subsets defined by race, gender, or ethnicity, and there is no reason to expect such 
differences to exist.  Therefore, although the planned analysis will, as always, look for 
differences in treatment effect based on racial and gender groupings, the sample size is 
not increased in order to provide additional power for subset analyses. 
 
13.2.1.3 The geographical region served by Mayo Clinic has a population that includes approxi-
mately 7% minorities.  Based on prior Mayo phase 2 studies involving similar disease 
sites, we expect about 7% of patients will be classified as minorities by race and about 

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55% of patients will be women.  Expected sizes of racial by gender subsets are shown 
in the following table: 
 
DOMESTIC PLANNED ENROLLMENT REPORT 
Racial 
Categories 
Ethnic Categories 
Total 
Not Hispanic or Latino 
Hispanic or Latino 
Female 
Male 
Female 
Male 
American 
Indian/ Alaska 
Native 
0 
0 
0 
0 
0 
Asian 
0 
0 
0 
0 
0 
Native 
Hawaiian or 
Other Pacific 
Islander 
0 
0 
0 
0 
0 
Black or 
African 
American 
1 
1 
0 
0 
2 
White 
18 
13 
1 
1 
33 
More Than 
One Race 
0 
0 
0 
0 
0 
Total 
19 
14 
1 
1 
35 
 
 
 
13.3 
Stratification Factors 
 
None.  
 
13.4 
Analysis of Secondary/Tertiary Endpoints 
 
13.4.1 The overall response rate will be estimated by the total number of complete or partial 
responses (CR, CRi, PR, or MLFS) divided by the total number of evaluable patients.  
All evaluable patients will be used for this analysis. Exact binomial 95% confidence 
intervals for the true overall response rate will be calculated.   
 
13.4.2 Duration of complete response (CR/CRi) is defined for all evaluable patients who have 
achieved a complete response as the time from the date at which the patient’s objective 
status is first noted to be a CR to the earliest date progression is documented.  The 
distribution of duration of complete response will be estimated using the method of 
Kaplan-Meier. 
 
13.4.3 The frequency of proceeding to allogeneic SCT after achieving response (CR, CRi, PR, 
or MLFS) to MLN0128 (TAK-228) will be estimated by the number of patients who 
proceed to allogeneic SCT after achieving response divided by the total number of 

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evaluable patients who achieved a response.  All evaluable patients who achieved a 
response will be used for this analysis.  Exact binomial 95% confidence intervals for the 
true overall response rate will be calculated.   
 
13.4.4 Overall survival time is defined as the time from registration to death due to any cause.  
The distribution of survival time will be estimated using the method of Kaplan-Meier.   
 
13.4.5 Adverse Events   
All eligible patients that have initiated treatment will be considered evaluable for 
assessing adverse event rate(s).  The maximum grade for each type of adverse event will 
be recorded for each patient, and frequency tables will be reviewed to determine patterns.  
Additionally, the relationship of the adverse event(s) to the study treatment will be taken 
into consideration. 
 
13.4.6 Correlative Analyses 
All analyses with respect to the correlative component of this study (Goal 1.2.5-1.2.9) are 
intended to be hypothesis-generating and descriptive in nature.  Due to the exploratory 
nature of the correlative endpoints, we will not adjust for multiple comparisons for these 
analyses. 
 
Descriptive statistics and simple scatterplots will form the basis of presentation of these 
data and any calculated changes from baseline.  Single sample t-tests, Wilcoxon signed 
rank tests or McNamar’s tests may be employed as appropriate.  Data may be compared 
between success and failure groups using two sample t-tests or Wilcoxon rank sum tests.  
Correlations between these laboratory values and other outcome measures will be carried 
out by standard parametric and nonparametric correlation procedures (Pearson’s and 
Spearman’s coefficients).   
 
13.5 
Safety Analysis  
 
13.5.1 A safety analysis will be performed after the first 6 patients have been accrued to the 
study on dose 0 (3 mg, orally days 1-21).  If 2 or more of the first 6 patients experience a 
dose-limiting toxicity (DLT) as defined below, then 6 patients will be enrolled on dose -1 
(2 mg, orally days 1-21).  If 2 or more of the second 6 patients experience DLT, then the 
study will be terminated. 
 
13.5.2 Toxicity will be measured per NCI-CTCAE version 4. DLT is defined as an adverse 
event possibly, probably, or definitely related to study treatment that meets one of the 
following: 
 
•    Any grade 4 drug-related non-hematological toxicity 
•    Any grade 3 drug-related non-hematological toxicity that does not resolve to < 
grade 3 within 3 days, with the following exceptions: 
o Grade 3 Bilirubin, AST, ALT, Alkaline phosphatase will be 
considered DLT only if it does not recover to <grade 3 within 7 days  
•    Myelosuppression: hypoplasia (bone marrow cellularity <10%) >50 days 
 

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13.6 
Data Monitoring 
 
The principal investigator(s) and the study statistician will review the study at least twice 
a year to identify accrual, adverse event, and any endpoint problems that might be 
developing.  The accrual and safety data provided by Theradex for this trial will be 
reviewed and monitored by the Mayo Clinic Cancer Center (MCCC) Data Safety 
Monitoring Board (DSMB) at least twice a year, or more frequently as warranted. 
 
13.6.1 Adverse Event Stopping Rule 
 
The stopping rules specified below are based on the knowledge available at study 
development.  We note that the Adverse Event Stopping Rule may be adjusted in the 
event of either (1) the study re-opening to accrual or (2) at any time during the conduct of 
the trial and in consideration of newly acquired information regarding the adverse event 
profile of the treatment(s) under investigation.  The study team may choose to suspend 
accrual because of unexpected adverse event profiles that have not crossed the specified 
rule below. 
 
Accrual will be temporarily suspended to this study if at any time we observe events 
considered at least possibly related to study treatment (i.e. an adverse event with attribute 
specified as  “possible”, “probable”, or “definite”) that satisfy any of the following: 
 
• If 3 or more out of the first 12 treated patients experience grade 4 non-
hematological toxicities at least possibly related to treatment excluding fatigue, 
fever, infection, and lab abnormalities (hyper- or hypokalemia, hypocalcemia, 
hyperuricemia, hypophosphatemia) that resolve within 7 days without 
jeopardizing patient safety. 
 
• If after the first 12 patients have been treated, 25% of patients experience grade 4 
non-hematologic toxicities at least possibly related to treatment excluding fatigue, 
fever, infection, and lab abnormalities (hyper- or hypokalemia, hypocalcemia, 
hyperuricemia, hypophosphatemia) that resolve within 7 days without 
jeopardizing patient safety. 
 
 
We note that we will review grade 4 and 5 adverse events deemed “unrelated” or 
“unlikely to be related”, to verify their attribution and to monitor the emergence of a 
previously unrecognized treatment-related adverse event. 
 
13.7 
Reporting and Exclusions 
 
13.7.1 Evaluation of Toxicity 
 
All patients will be evaluable for toxicity from the time of their first treatment with 
MLN0128 (TAK-228). 
 

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13.7.2 Evaluation of Response 
 
All patients included in the study must be assessed for response to treatment, even if there 
are major protocol treatment deviations or if they are ineligible.  Each patient will be 
assigned one of the following categories:  1) complete response, 2) partial response, 3) 
stable disease, 4) progressive disease, 5) early death from malignant disease, 6) early 
death from toxicity, 7) early death because of other cause, or 9) unknown (not assessable, 
insufficient data).  [Note:  By arbitrary convention, category 9 usually designates the 
“unknown” status of any type of data in a clinical database.] 
 
All of the patients who met the eligibility criteria (with the possible exception of those 
who received no study medication) should be included in the main analysis of the 
response rate.  Patients in response categories 4-9 should be considered to have a 
treatment failure (disease progression).  Thus, an incorrect treatment schedule or drug 
administration does not result in exclusion from the analysis of the response rate.  Precise 
definitions for categories 4-9 will be protocol specific.  
 
All conclusions should be based on all eligible patients.  Sub analyses may then be 
performed on the basis of a subset of patients, excluding those for whom major protocol 
deviations have been identified (e.g., early death due to other reasons, early 
discontinuation of treatment, major protocol violations, etc.).  However, these sub 
analyses may not serve as the basis for drawing conclusions concerning treatment 
efficacy, and the reasons for excluding patients from the analysis should be clearly 
reported.  The 95% confidence intervals should also be provided.  
 
 
 
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mTOR Inhibitiors Induce Bim-Dependent Apoptosis in Acute Lymphoblastic Leukmeia via 
Inhibition of eIF4E/eIF4G and Activation of NF-kB/Egr-1. Submitted. 2015. 
15. Investigator's Brochure MLN0128. 7th edition ed, 2014. 
16. ABT-767 Investigator's Brochure. 3rd ed. North Chicago, IL: AbbVie, Inc.; 2013. 
17. Ghobrial IM, Siegel D, Vij R, Wolf JL, Berdeja JG, Matous JV, et al. MLN0128 (INK128), 
an Investigational Oral Dual TORC1/2 Inhibitor, in Patients (pts) with Relapsed or 
Refractory Multiple Myeloma (MM), Non-Hodgkin's Lymphoma (NHL), or Waldenstrom 
Macroglobulinemia (WM): Preliminary Results From a Phase 1 Dose-Escalation Study. 
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18. Gupta M, Wahner Hendrickson A, Seongseouk Y, Han JJ, Schneider PA, Stenson M, et al. 
Dual TORC1/TORC2 Inhibition Diminishes Akt Activation and Induces Puma-Dependent 
Apoptosis in Lymphoid Malignancies. Blood. 2012;119:476-87. 
19. Witzig TE, Geyer SM, Ghobrial I, Inwards DJ, Fonseca R, Kurtin P, et al. Phase II trial of 
single-agent temsirolimus (CCI-779) for relapsed mantle cell lymphoma. Journal of 
Clinical Oncology. 2005;23:5347-56. 
20. Ansell SM, Inwards DJ, Rowland KM, Flynn PJ, Morton RF, Moore DF, Jr., et al. Low-
dose single-agent temsirolimus for relapsed mantle cell lymphoma:  A phase 2 trial in the 
North Central Cancer Treatment Group. Cancer. 2008;113:508-14. 
21. Zent CS, LaPlant BR, Johnston PB, Call TG, Habermann TM, Micallef IN, et al. The 
treatment of recurrent/refractory chronic lymphocytic leukemia/small lymphocytic 
lymphoma (CLL) with everolimus results in clinical responses and mobilization of CLL 
cells into the circulation. Cancer. 2010;116:2201-7. 
22. Johnston PB, Inwards DJ, Colgan JP, Laplant BR, Kabat BF, Habermann TM, et al. A 
Phase II trial of the oral mTOR inhibitor everolimus in relapsed Hodgkin lymphoma. Am J 
Hematol. 2010;85:320-4. 
23. Witzig TE, Reeder CB, Laplant BR, Gupta M, Johnston PB, Micallef IN, et al. A phase II 
trial of the oral mTOR inhibitor everolimus in relapsed aggressive lymphoma. Leukemia. 
2011;25:341-7. 

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24. Ansell SM, Tang H, Kurtin PJ, Koenig PA, Inwards DJ, Shah K, et al. Temsirolimus and 
rituximab in patients with relapsed or refractory mantle cell lymphoma: a phase 2 study. 
Lancet Oncol. 2011;12:361-8. 
25. Yee KW, Zeng Z, Konopleva M, Verstovsek S, Ravandi F, Ferrajoli A, et al. Phase I/II 
study of the mammalian target of rapamycin inhibitor everolimus (RAD001) in patients 
with relapsed or refractory hematologic malignancies. Clinical cancer research : an official 
journal of the American Association for Cancer Research. 2006;12:5165-73. 
26. Bjornsti MA, Houghton PJ. The TOR pathway: a target for cancer therapy. Nat Rev 
Cancer. 2004;4:335-48. 
27. Dancey J. mTOR signaling and drug development in cancer. Nat Rev Clin Oncol. 
2010;7:209-19. 
28. Benjamin D, Colombi M, Moroni C, Hall MN. Rapamycin passes the torch: a new 
generation of mTOR inhibitors. Nat Rev Drug Discov. 2011;10:868-80. 
29. Zoncu R, Efeyan A, Sabatini DM. mTOR: from growth signal integration to cancer, 
diabetes and ageing. Nat Rev Mol Cell Biol. 2011;12:21-35. 
30. Blachly JS, Baiocchi RA. Targeting PI3-kinase (PI3K), AKT and mTOR axis in 
lymphoma. Br J Haematol. 2014;167:19-32. 
31. Roti G, Stegmaier K. New Approaches to Target T-ALL. Front Oncol. 2014;4:170. 
32. Eyre TA, Collins GP, Goldstone AH, Cwynarski K. Time now to TORC the TORC? New 
developments in mTOR pathway inhibition in lymphoid malignancies. Br J Haematol. 
2014;166:336-51. 
33. Mohindra NA, Giles FJ, Platanias LC. Use of mTOR inhibitors in the treatment of 
malignancies. Expert Opin Pharmacother. 2014;15:979-90. 
34. Sengupta S, Peterson TR, Sabatini DM. Regulation of the mTOR complex 1 pathway by 
nutrients, growth factors, and stress. Molecular Cell. 2010;40:310-22. 
35. Sabatini DM. mTOR and cancer: insights into a complex relationship. Nat Rev Cancer. 
2006;6:729-34. 
36. Hsu PP, Kang SA, Rameseder J, Zhang Y, Ottina KA, Lim D, et al. The mTOR-regulated 
phosphoproteome reveals a mechanism of mTORC1-mediated inhibition of growth factor 
signaling. Science. 2011;332:1317-22. 
37. Neri LM, Cani A, Martelli AM, Simioni C, Junghanss C, Tabellini G, et al. Targeting the 
PI3K/Akt/mTOR signaling pathway in B-precursor acute lymphoblastic leukemia and its 
therapeutic potential. Leukemia. 2013. 
38. Smith AJ, Dai H, Correia C, Lee S-H, Takahashi R, Kaufmann SH. Noxa/Bcl-2 
Interactions Contribute to Bortezomib Resistance in Human Lymphoid Cells. J. Biol. 
Chemistry. 2011;286:17682-92. 
39. Janes MR, Vu C, Mallya S, Shieh MP, Limon JJ, Li LS, et al. Efficacy of the 
investigational mTOR kinase inhibitor MLN0128/INK128 in models of B-cell acute 
lymphoblastic leukemia. Leukemia. 2012. 
40. Kaufmann SH, Karp JE, Svingen PA, Krajewski S, Burke PJ, Gore SD. Elevated 
Expression of the Apoptotic Regulator Mcl-1 at the Time of Leukemic Relapse. Blood. 
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41. Dai H, Meng XW, Lee S-H, Schneider PA, Kaufmann SH. Context-dependent Bcl-2/Bak 
Interactions Regulate Lymphoid Cell Apoptosis. J. Biol. Chemistry. 2009;284:18311-22. 
42. Kaufmann SH, Okret SH, Wikström A-C, Gustafsson J-Å, Shaper JH. Binding of the 
Glucocorticoid Receptor to the Rat Liver Nuclear Matrix:  The Role of Disulfide Bond 

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Formation. J. Biol. Chemistry. 1986;261:11962-7. 
43. Kaufmann SH, Svingen PA, Gore SD, Armstrong DK, Cheng Y-C, Rowinsky EK. Altered 
Formation of Topotecan-Stabilized Topoisomerase I-DNA Adducts in Human Leukemia 
Cells. Blood. 1997;89:2098-104. 
44. Casado P, Alcolea MP, Iorio F, Rodriguez-Prados JC, Vanhaesebroeck B, Saez-Rodriguez 
J, et al. Phosphoproteomics data classify hematological cancer cell lines according to tumor 
type and sensitivity to kinase inhibitors. Genome Biol. 2013;14:R37. 
45. Casado P, Rodriguez-Prados JC, Cosulich SC, Guichard S, Vanhaesebroeck B, Joel S, et al. 
Kinase-substrate enrichment analysis provides insights into the heterogeneity of signaling 
pathway activation in leukemia cells. Sci Signal. 2013;6:rs6. 
46. Cope CL, Gilley R, Balmanno K, Sale MJ, Howarth KD, Hampson M, et al. Adaptation to 
mTOR kinase inhibitors by amplification of eIF4E to maintain cap-dependent translation. J 
Cell Sci. 2013. 
47. Galanis E, Jaeckle KA, Maurer MJ, Reid JM, Ames MM, Hardwick JS, et al. Phase II trial 
of vorinostat in recurrent glioblastoma multiforme: a north central cancer treatment group 
study. J.Clinical Oncol. 2009;27:2052-8. 
48. Fouladi M, Park JR, Stewart CF, Gilbertson RJ, Schaiquevich P, Sun J, et al. Pediatric 
phase I trial and pharmacokinetic study of vorinostat: a Children's Oncology Group phase I 
consortium report. J..Clinical Oncol. 2010;28:3623-9. 
49. Lee EQ, Puduvalli VK, Reid JM, Kuhn JG, Lamborn KR, Cloughesy TF, et al. Phase I 
study of vorinostat in combination with temozolomide in patients with high-grade gliomas: 
North American Brain Tumor Consortium Study 04-03. Clinical Cancer Research. 
2012;18:6032-9. 
50. Shah VP, Midha KK, Dighe S, McGilveray IJ, Skelly JP, Yacobi A, et al. Analytical 
methods validation: bioavailability, bioequivalence and pharmacokinetic studies. 
Conference report. European journal of drug metabolism and pharmacokinetics. 
1991;16:249-55. 
51. Shah VP, Midha KK, Findlay JW, Hill HM, Hulse JD, McGilveray IJ, et al. Bioanalytical 
method validation--a revisit with a decade of progress. Pharm Res. 2000;17:1551-7. 
52. Guidance for Industry: Bioanalytical Method Validation. U.S. Department of Health and 
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(CDER), Center for Veterinary Medicine (CVM). Draft Guidance, September 2013. In: 
Administration FaD, editor. 
53. Kaufmann SH, Svingen PA. Immunoblot Analysis and Band Depletion Assays. In: Bjornsti 
M-A, Osheroff N, editors. Protocols for DNA Topoisomerases: Humana Press; 1998. p. 
253-68. 
54. Svingen PA, Loegering DA, Rodriquez J, Meng XW, Mesner PW, Jr., Monks A, et al. 
Components of the Cell Death Machine and Drug Sensitivity of the NCI Cell Line Panel. 
Clinical Cancer Research. 2004;10:6807-20. 
55. Kaufmann SH, Ewing CM, Shaper JH. The Erasable Western Blot. Analytical 
Biochemistry. 1987;161:89-95. 
56. Kaufmann SH. Reutilization of Immunoblots After Chemiluminescent Detection. 
Analytical Biochemistry. 2001;296:283-6. 
57. Kaufmann SH. Reutilizaton of Western Blots After Chemiluminescent or Autoradiographic 
Detection. In: Walker JM, editor. Protein Protocols Handbook, 3rd edition. Totowa, N.J.: 
Humana Press; 2009. p. 785-801. 

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58. Kaufmann SH, Karp JE, Letendre L, Kottke TJ, Safgren S, Greer J, et al. Phase I and 
Pharmacological Study of Infusional Topotecan and Carboplatin in Relapsed and 
Refractory Leukemia. Clinical Cancer Research. 2005;11:6641-9. 
59. Witzig TE, Tang H, Micallef IM, Ansell SM, Ding H, Link BK, et al. A Multi-Institution 
Phase 2 Study of the Farnesyltransferase Inhibitor Tipifarnib (R115777) in Patients with 
Relapsed and Refractory Lymphomas. Blood. 2011;118:4882-9. 
60. Tibes R, Qiu Y, Lu Y, Hennessy B, Andreeff M, Mills GB, et al. Reverse phase protein 
array: validation of a novel proteomic technology and utility for analysis of primary 
leukemia specimens and hematopoietic stem cells. Molecular Cancer Therapeutics. 
2006;5:2512-21. 
61. Faderl S, Thomas DA, O'Brien S, Ravandi F, Garcia-Manero G, Borthakur G, et al. 
Augmented hyper-CVAD based on dose-intensified vincristine, dexamethasone, and 
asparaginase in adult acute lymphoblastic leukemia salvage therapy. Clin Lymphoma 
Myeloma Leuk. 2011;11:54-9. 
62. O'Brien S, Schiller G, Lister J, Damon L, Goldberg S, Aulitzky W, et al. High-dose 
vincristine sulfate liposome injection for advanced, relapsed, and refractory adult 
Philadelphia chromosome-negative acute lymphoblastic leukemia. J.Clinical Oncol. 
2013;31:676-83. 
 
 
 
 

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APPENDIX A: PERFORMANCE STATUS CRITERIA 
 
ECOG Performance Status Scale 
Grade 
Descriptions 
0 
Normal activity.  Fully active, able to carry on all pre-
disease performance without restriction. 
1 
Symptoms, but ambulatory.  Restricted in physically 
strenuous activity, but ambulatory and able to carry out 
work of a light or sedentary nature (e.g., light housework, 
office work). 
2 
In bed < 50% of the time.  Ambulatory and capable of all 
self-care, but unable to carry out any work activities.  Up 
and about more than 50% of waking hours. 
3 
In bed > 50% of the time.  Capable of only limited self-care, 
confined to bed or chair more than 50% of waking hours. 
4 
100% bedridden.  Completely disabled.  Cannot carry on 
any self-care.  Totally confined to bed or chair. 
5 
Dead. 

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APPENDIX B: STUDY MEDICATION CALENDAR             
 
STUDY # _______________ 
 
Patient’s Name __________________ Cycle # _____  Day 1 of Cycle ______ / ______ / ______ PATIENT ID # ______________ 
INSTRUCTIONS: 
• 
The capsules are to be swallowed intact, not less than 2 hours before or 1 hour after a meal.  Do not chew, open or manipulate the capsule in any way prior to 
swallowing. Each dose should be taken with 8 ounces (240 mL) of water. Doses are to be taken at about the same time each day.  If a dose is missed, it can be taken up to 
6 hours past the scheduled time.  If more than 6 hours past the scheduled time, the dose should not be taken and will not be made up.  Vomited doses will not be made 
up. 
• 
Please record the times that you take your study medications. Return this diary and the medication bottles (even if unopened or empty) along with any unused pills at 
your next visit. 
DAY OF WEEK: 
_____________ 
DAY OF WEEK: 
_____________ 
DAY OF WEEK: 
_____________ 
DAY OF WEEK: 
_____________ 
DAY OF WEEK: 
_____________ 
DAY OF WEEK: 
_____________ 
DAY OF WEEK: 
_____________ 
Day 1 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 2 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 3 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 4 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 5 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 6 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 7 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 8 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 9 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 10 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 11 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 12 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 13 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 14 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 15 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 16 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 17 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 18 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 19 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 20 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 21 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 22 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 23 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 24 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 25 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 26 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 27 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
Day 28 of Cycle    
 
# Pills: ______ 
 
__ :___ AM or PM 
 
Patient’s Signature ____________________________________________ 
Date __________________________

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APPENDIX C: PATIENT DRUG INFORMATION HANDOUT AND WALLET CARD 
 
Information for Patients, Their Caregivers, and Non-Study Healthcare Team on Possible 
Interactions with Other Drugs and Herbal Supplements  
 
The patient ____________________________ is enrolled on a clinical trial using the 
experimental study drug, MLN0128 (TAK-228).  This clinical trial is sponsored by the National 
Cancer Institute.  This form is addressed to the patient, but includes important information for 
others who care for this patient.  
 
These are the things that you as a healthcare provider need to know: Because of the low 
potential for interaction with agents that induce or inhibit cytochrome P450 enzymes, there are 
no prohibitions of specific medications on the basis of anticipated drug-drug interactions.  
  
To the patient: Take this paper with you to your medical appointments and keep the 
attached information card in your wallet.   
 
MLN0128 (TAK-228) may interact with other drugs which can cause side effects.  For this 
reason, it is very important to tell your study doctors of any medicines you are taking before you 
enroll onto this clinical trial.  It is also very important to tell your doctors if you stop taking any 
regular medicines, or if you start taking a new medicine while you take part in this study.  When 
you talk about your current medications with your doctors, include medicine you buy without a 
prescription (over-the-counter remedy), or any herbal supplements such as St. John’s Wort.  It is 
helpful to bring your medication bottles or an updated medication list with you. 
 
Many health care providers can write prescriptions.  You must tell all of your health care 
providers (doctors, physician assistants, nurse practitioners, pharmacists) you are taking part in a 
clinical trial.  
 
These are the things that you and they need to know:  
 
• Please be very careful!  Over-the-counter drugs (including herbal supplements) may 
contain ingredients that could interact with your study drug.  Speak to your doctors or 
pharmacist to determine if there could be any side effects.  
 
• Your regular health care provider should check a frequently updated medical reference or 
call your study doctor before prescribing any new medicine or discontinuing any 
medicine.  Your study doctor’s name is _____________________________________ 
and he or she can be contacted at _____________________________________. 

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STUDY DRUG INFORMATION WALLET CARD   
You are enrolled on a clinical trial using the experimental study  
drug MLN0128.  This clinical trial is sponsored by the NCI.   
 
It is very important to: 
➢ Tell your doctors if you stop taking any medicines or if you start 
taking any new medicines. 
➢ Tell all of your health care providers (doctors, physician assistants, 
nurse practitioners, or pharmacists) that you are taking part in a 
clinical trial. 
➢ Check with your doctor or pharmacist whenever you need to use an 
over-the-counter medicine or herbal supplement. 
➢ Before prescribing new medicines, your regular health care 
providers should go to a frequently-updated medical reference for 
a list of drugs to avoid, or contact your study doctor.   
 
➢ Your study doctor’s name is _____________________________ and 
can be contacted at _________________________________.  

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APPENDIX D: PHARMACOKINETIC BLOOD SAMPLES LIST  
 
9775 / MC1482: A Phase 2 Study of MLN0128 (TAK-228) in Relapsed and/or Refractory 
Acute Lymphoblastic Leukemia (ALL) 
 
Pharmacokinetic Blood Samples List 
Cycle 1 Day 1 
 
Date: 
 
Patient ID: 
 
Site Name and CTEP ID: 
 
Instructions: Please complete the scheduled times and the actual times the pk blood samples 
were collected below.  Please include a copy of this document with the samples at time of 
shipment.  
Sample Collection Times (All times hh:mm in 24 hour clock) 
Time Point 
Scheduled Time 
Actual Time 
30 minutes pre-dose 
(± 10 minutes) 
 
 
Administration of  
MLN0128 (TAK-228) 
___________ 
 
30 minutes post-dose 
(± 5 minutes) 
 
 
1 hour post-dose 
(± 5 minutes) 
 
 
2 hours post-dose 
(± 10 minutes) 
 
 
3 hours post-dose 
(± 15 minutes) 
 
 
4 hours post-dose 
(± 15 minutes) 
 
 
8 hours post-dose* 
(± 15 minutes) 
 
 
 
*Note: If unable to draw an 8 hour post dose sample, please specify what timepoint blood 
sample was collected. 

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9775 / MC1482: A Phase 2 Study of MLN0128 (TAK-228) in Relapsed and/or Refractory 
Acute Lymphoblastic Leukemia (ALL) 
 
Pharmacokinetic Blood Samples List 
Cycle 1 Day 2 
 
Date: 
 
Patient ID: 
 
Site Name and CTEP ID: 
 
Instructions: Please complete the scheduled times and the actual times the pk blood samples 
were collected below.  Please include a copy of this document with the samples at time of 
shipment.  
 
Sample Collection Times (All times hh:mm in 24 hour clock) 
Time Point 
Scheduled Time 
Actual Time 
30 minutes pre-dose 
(± 10 minutes) 
 
 
Administration of  
MLN0128 (TAK-228) 
___________ 
 
 
 
 
 

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9775 / MC1482: A Phase 2 Study of MLN0128 (TAK-228) in Relapsed and/or Refractory 
Acute Lymphoblastic Leukemia (ALL) 
 
Pharmacokinetic Blood Samples List 
Cycle 1 Day 8 
 
Date: 
 
Patient ID: 
 
Site Name and CTEP ID: 
 
Instructions: Please complete the scheduled times and the actual times the pk blood samples 
were collected below.  Please include a copy of this document with the samples at time of 
shipment.  
 
Sample Collection Times (All times hh:mm in 24 hour clock) 
Time Point 
Scheduled Time 
Actual Time 
30 minutes pre-dose 
(± 10 minutes) 
 
 
Administration of  
MLN0128 (TAK-228) 
___________ 
 
30 minutes post-dose 
(± 5 minutes) 
 
 
1 hour post-dose 
(± 5 minutes) 
 
 
2 hours post-dose 
(± 10 minutes) 
 
 
3 hours post-dose 
(± 15 minutes) 
 
 
4 hours post-dose 
(± 15 minutes) 
 
 
8 hours post-dose* 
(± 15 minutes) 
 
 
 
*Note: If unable to draw an 8 hour post dose sample, please specify what timepoint blood 
sample was collected. 
 
 

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APPENDIX E: ADDITIONAL INFORMATION REGARDING PROPOSED 
BIOMARKER STUDIES 
 
 
Per the instructions in the CTEP Protocol Template, this appendix is constructed to provide 
information on endpoint validation including additional background (as needed), description of 
the assay(s) used, materials and methods, and assay validation.  The integrated biomarker 
(phospho-Thr37,46-4EBP1 phosphorylation by immunoblotting in blasts from pretreatment and 
day 8 marrows) has previously been reviewed by the Biomarker Review Committee; and that 
material is provided here in case that review is not provided to reviewers of this protocol.  
Similar information about the exploratory biomarkers is provided in more abbreviated form per 
our interpretation of the template instructions.  
 
Biomarkers are provided in the order of their priority, highest to lowest.  The rationale for this 
priority is provided in Section 2.4.7 of the main body of the protocol. 
 
1. Phospho-Thr37,46-4EBP1 Analysis of Pretreatment and Day 8 Bone Marrow Samples by 
Immunoblotting 
 
1.1 Role in Trial: Integrated Biomarker.  This is the top priority assay and will be 
performed first on every available sample from the trial. 
 
1.2 Hypothesis and Rationale 
 
Hypothesis:  4EBP1 phosphorylation will be inhibited by MLN0128 (TAK-228) in 
situ. 
 
Rationale:  Previous studies have shown that mTOR dual inhibitors such as MLN0128 
(TAK-228) and OSI-027 diminish the mTORC1-mediated phosphorylation of the 
translation inhibitor 4EBP1, causing it to bind to and inhibit the translation initiation 
factor eIF4E (1, 2). In acute lymphocytic leukemia (ALL) cell lines, the down 
regulation of 4EBP1 produces resistance to mTOR dual inhibitors (2), demonstrating 
the critical role of this protein in killing by mTOR dual inhibitors.  We now propose to 
assess whether 4EBP1 has been dephosphorylated in leukemic blasts by 
therapeutically achievable MLN0128 concentrations in situ as a proof of mechanism. 
 
Results of these assays will not help individual patients on this trial but might be 
particularly useful to clinical investigators thinking about future development of this 
class of agents:  If ALL fails to respond and 4EBP1 remains phosphorylated, this 
might imply that the 4EBP1 pathway has been inadequately targeted at therapeutically 
achievable concentrations, e.g., because toxicities in normal tissues preclude adequate 
drug levels, transport-mediated resistance diminishes ALL drug uptake, or signaling 
alterations restore 4EBP1 phosphorylation in the absence of mTOR. On the other 

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hand, if 4EBP1 is extensively dephosphorylated but ALL fails to respond, this will 
suggest that clinical ALL is not dependent on this target, but would not preclude 
combining mTOR inhibition with other therapies. 
 
1.3 Assay Procedure 
 
The assay procedure is described in detail in the attached Standard Operating Procedure 
previously provided to the Biomarker Review Committee (pp. 16- 26 of this appendix).  
In brief, 4EBP1 phosphorylation will be assessed by immunoblotting as previously 
described (1).  Detailed procedures for immunoblotting have been previously published 
by the Kaufmann laboratory (3, 4).  Sources of critical reagents are identified in the 
following description.  
 
After sequential samples of bone marrow blasts (harvested prior to treatment and on 
day 8 of therapy) are subjected to SDS-polyacrylamide gel electrophoresis in adjacent 
wells using standard techniques and electrophoresis grade reagents from BioRad as 
previously described (3, 4), proteins will be transferred to Biotrace NT nitrocellulose 
(Pall #66485).  After blocking according to standard techniques (4), blots will be 
incubated overnight with rabbit anti-phospho-Thr36,47-4EBP1 monoclonal antibody 
236B4 (Cell Signaling Technology product #2855) diluted 1:1000 in buffer consisting 
of 5% bovine serum albumin (Sigma-Aldrich #A7906) in 150 mM NaCl-10 mM Tris-
HCl (pH 7.4 at 20 °C) containing 0.05% Tween 20, 1 mM sodium azide, 100 U/ml 
penicillin G, and 100 µg/ml streptomycin.  Blots will be washed, incubated with 0.125 
µg/ml horseradish peroxidase-coupled goat anti-rabbit IgG (KPL #074-1506) in 5% 
(w/v) powdered nonfat dry milk in 150 mM NaCl-10 mM Tris-HCl, washed again, 
reacted with Supersignal West Dura enhanced chemiluminescence reagents (Thermo 
#34075), and exposed to high sensitivity x-ray film (Phenix Research Products #F-
BX810).  
 
Because it is difficult to purify and reproducibly phosphorylate 4EBP1, aliquots of 
lysate from the Nalm6 human ALL cell line (authenticated by short tandem repeat 
profiling in the Mayo Advanced Genomics Technology Center) will be lyophilized in 
single-use aliquots and stored at -80 °C.  Serial dilutions of these lysates on each blot 
will serve as a positive control and provide a standard curve for quantitation.  This cell 
line was chosen because our studies have shown that it contains high levels of phospho-
Thr37,46-4EBP1 (see Fig. 2 below).  Blots exposed in the linear range will be scanned on 
an Epson 4870 scanner and quantitated using NIH Image version 1.63.  A complete 
description of the immunoblotting procedure, including sources of all reagents not 
specified above, is provided in the Standard Operating Procedure attached at the end of 
this appendix.    
 
1.4 Source of Samples   
 
Marrow mononuclear cells (typically at least 80% malignant lymphoid cells in ALL 
specimens—ref. 5, 6) will be harvested by Ficoll-Hypaque gradient centrifugation (7) 

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from bone marrow aspirates harvested prior to therapy and on day 8 ± 1 of treatment 
with single-agent MLN0128 (TAK-228).  Day 8 was chosen for the post-treatment 
sample based several considerations: 
• In published murine xenograft experiments (1), 4EBP1 dephosphorylation is 
detectable in xenografts at early time points after dual mTOR inhibitor treatment 
and maintained for the duration of drug exposure.  This observation is presented in 
Fig. 4 below.  On the other hand, induction of critical signaling steps downstream 
of 4EBP1 such as PARP cleavage, which could be assessed in the same specimens 
in an exploratory manner, is not consistently evident at day 3 in the xenograft 
experiments but is evident at day 6 (1). 
• Xenograft regression was not extensive until beyond day 8 (1). 
• Day 8 is a standard time for follow up appointments and toxicity assessment in 
patients who are being treated in the outpatient setting. 
• Picking an arbitrary time such as day 5 or day 6 will harm patient accrual to the 
trial and sample acquisition: If samples were required on day 5, then patients could 
only start treatment on Monday, Thursday or Friday so that day 5 marrow 
procurement and processing would fall on a weekday.  
 
1.5 Assay Performance Characteristics 
 
The following performance characteristics of the phospho-4EBP1 assay have been 
evaluated: 
 
1.51 Characterization of antibody used in these studies: A critical antibody for this 
procedure, a rabbit monoclonal from Cell Signaling Technology, Inc., 
recognizing phospho-Thr37,46-4EBP1, is supplied as a pre-titered reagent. In 
proprietary information provided to the Biomarker Review Committee, this 
antibody has been shown by ELISA to be specific for the phosphorylated peptide 
compared to the non-phosphorylated peptide.  Moreover, additional experiments 
have demonstrated that the antibody reacts with cells expressing 4EBP1 but not 
with 4EBP1 knockout cells (personal communication from Kimberly McCabe, 
Cell Signaling Technology, 11/19/14), further establishing specificity of the 
antibody.  The supplier’s practice of titering each lot of antibody before release 
helps assure that Western blots performed with the same dilution will yield 
similar results with different lots of antibody whenever the assay is run.  
 
1.52 Dynamic range of the assay:  To assess the dynamic range of the proposed assay, 
serial dilutions of the Nalm6 ALL cell line were subjected to immunoblotting.  
As indicated in Fig. 1, signal was detectable in undiluted sample and six 2-fold 
dilutions (64-fold range).  Moreover, this range encompassed the baseline 
phosphorylation of 4EBP1 observed in four ALL samples harvested from newly 
diagnosed ALL patients prior to therapy and processed as described above.   
 

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Figure 1.  Assessment of dynamic and linear ranges of phospho-4EBP1 blotting.  A, a sample 
containing 50 µg of total cellular protein from the human B-cell ALL Nalm6 line (lane 1) and 
serial 2-fold dilutions (lanes 2-8) were subjected to SDS-PAGE and probed with antibody that 
recognizes phospho-Thr37,46-4EBP1. Aliquots containing 50 µg of total cellular protein from four 
pretreatment human ALL samples were also contained on the blot (lanes 9-12).  Two different 
exposures of the same blot are shown. B, quantitation of integrated signals [area x (intensity – 
background)] of blots shown in panel A using NIH Image v1.63. Note that signals can be detected 
in a lysate over 64-fold range (lower panel of A, lanes 1-7), although the linear range is more 
limited (panel B). 
 
 
1.53 
Reproducibility of the blotting for phospho-4EBP1 and quantification:  Lysates 
prepared from 15 different malignant human lymphoid cell lines were subjected to 
SDS-PAGE, transferred to nitrocellulose on three separate days, and probed with 
replicate aliquots of anti-phospho-4EBP1 antibody as described above.  As 
indicated in Fig. 2, the blots were qualitatively similar. Quantification using NIH 
 
 
Figure 2.  Assessment of reproducibility of blotting for phospho-Thr37,46-4EBP1. Aliquots 
containing 50 µg of total cellular protein from the indicated human neoplastic lymphoid cell lines 
were subjected to SDS-PAGE followed by transfer to nitrocellulose on three consecutive days. 
Blots were probed with replicate aliquots of the same anti-phospho-4EBP1 antibody, visualized 
by enhanced chemiluminescence reagents, and exposed to x-ray film exactly as proposed for the 
clinical samples. After blots were scanned, the integrated signal in individual lanes [area x 
(intensity – background)] was quantitated using NIH Image. Numbers at left indicate Spearman 

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correlation coefficients for signals in corresponding lanes of the indicated blots. 
 
Figure 3. Inhibition of 4EBP1 Thr37,46 
phosphorylation in Jurkat cells. A, Jurkat cells 
were treated for 8 h with diluent or 1.25, 2.5, 5, 
10 or 20 µM OSI-027 or 10 nM rapamycin, then 
harvested for blotting.  From ref. (1). B, after 
Jurkat cells were treated with diluent (lanes 1 
and 5); OSI-027 at 5, 10 or 20 µM (lanes 2-4); 
MLN0128 (TAK-228) at 0.25, 0.5 or 1 µM 
(lanes 6-8); or 10 nM rapamycin (lane 9), whole 
cell lysates were subjected immunoblotting.  
 
 
Image revealed a correlation between integrated signals for each cell line on pairs of 
blots as indicated on the figure.  
 
1.54 Assessment of the reproducibility of 4EBP1 dephosphorylation: To assess whether 
4EBP1 phosphorylation is reproducibly inhibited upon exposure to dual 
mTORC1/mTORC2 inhibitors, multiple investigators in the Kaufmann laboratory 
assessed the impact of the dual mTORC1/mTORC2 inhibitor OSI-027 on 4EBP1 
phosphorylation in the ALL cell line Jurkat independently over a 5-year period using 
different batches of phospho-Thr37,46-4EBP1 antibody from the indicated supplier. Two 
of these assays are shown in Figs. 3A (lanes 2-6) and 3B (lanes 2-4) below. These 
results show that 4EBP1 dephosphorylation is detectable by different lab personnel 
using different lots of antibody, nitrocellulose, x-ray film, detection reagents and 
buffers as long as the Standard Operating Procedure is followed. 
 
1.55 Occurrence of the proposed 4EBP1 dephosphorylation in human neoplastic lymphoid 
cells after dual mTORC1/mTORC2 inhibitor treatment of xenografts in vivo:  To assess 
whether 4EBP1 phosphorylation would be inhibited by therapeutically achievable 
concentrations in vivo, mice bearing xenografts of the human mantle cell lymphoma 
cell line Jeko were treated with the maximum tolerated dose of OSI-027 for 3, 5 or 6 
days. Xenografts were snap frozen, then subjected to sample preparation and 
immunoblotting as described in Section 1.3 of this Appendix.  Results of this analysis 
demonstrated inhibition of 4EBP1 Thr37,46 phosphorylation in vivo in all samples from 
treated mice compared to pretreatment samples (Fig. 4). 
 
 
 
Figure 4.  Dephosphorylation of 4EBP1 Thr37,46 and AKT 
Ser473 in Jeko xenografts after OSI-027 treatment in vivo. 
Whole cell lysates prepared (8) from separate xenografts of 
untreated mice (lanes 1-3) or xenografts harvested 4 h after 
treatment with OSI-027 on day 3 (lanes 4, 5), day 5 (lanes 6-8) 
or day 6 (lanes 9, 10) were subjected to SDS-PAGE and probed 
with antibodies to the indicated antigen.   

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Figure 5.  Effect of dual mTOR inhibitors OSI-027 (OSI) and MLN0128 
(TAK-228) on 4EBP1 phosphorylation in clinical ALL samples exposed ex 
vivo. Clinical ALL specimens were subjected to Ficoll-Hypaque gradient 
centrifugation as described above.  Aliquots containing 5 x 106 cells were 
incubated for 48 h with 0.1% DMSO (lane 1) or dual mTORC1/mTORC2 
inhibitor at the indicated final concentration in the presence of the broad-spectrum 
caspase inhibitor Q-VD-OPh to prevent caspase-medicated changes in protein 
expression. At the end of the incubation, cells were sedimented, washed in serum-
free medium, lysed, and subjected to immunoblotting for phospho-Thr37,46-
4EBP1 or total 4EBP1 as described above. Because degradation of 4EBP1 is 
phosphorylation dependent (explaining the increase in drug-treated cells), c-Raf 
and lamin B1 were included as loading controls.  
 
1.56 Occurrence of 4EBP1 dephosphorylation in clinical ALL samples upon treatment 
with dual mTORC1/mTORC2 inhibitors ex vivo:  To assess whether the 
biochemical change previously shown to be critical for killing of ALL cells (2) 
could occur in clinical ALL, ALL specimens harvested from three newly diagnosed 
ALL patients were exposed to the dual mTORC1/mTORC2 inhibitors OSI-027 and 
MLN0128 (TAK-228) ex vivo. Results of this analysis indicated that 4EBP1 
phosphorylation was reduced to undetectable levels in all samples upon drug 
exposure ex vivo (Fig. 5A-C; see also Fig. 6B).  
 
1.57 Occurrence of 4EBP1 dephosphorylation in the range of MLN0128 (TAK-228) 
concentrations that are anticipated to be achievable in vivo: To assess the ability of 
MLN0128 (TAK-228) to inhibit 4EBP1 phosphorylation at submicromolar 
concentrations, Jurkat T cell ALL cells were treated for 24 h with various 
concentrations of MLN0128 (TAK-228) and blotted for 4EBP1. As indicated in 
Fig. 6A, 4EBP1 dephosphorylation was readily detectable at MLN0128 (TAK-228) 
concentrations of 100 nM and above. To assure that 4EBP1 dephosphorylation 
would occur over the same MLN0128 (TAK-228) concentration range in clinical 
ALL specimens, a clinical specimen from a patient with newly diagnosed ALL 
(distinct from those in Fig. 5) was treated with varying concentrations of MLN0128 
(TAK-228) for 48 h, harvested, and subjected to immunoblotting for 4EBP1. As 
indicated in Fig. 6B, diminished 4EBP1 phosphorylation was detectable at the 
lowest concentration tested, 62.5 nM. These results compare favorably with the 

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peak MLN0128 (TAK-228) serum concentration of ~130 nM achieved in phase I 
solid tumor trials at the 6 mg dose level (9). 
 
 
Figure 6.  Effect of various MLN0128 (TAK-228) concentrations on 4EBP1 
phosphorylation.  A, Log phase Jurkat cells were treated for 24 h with diluent (0.1% DMSO, 
lane 1) or MLN0128 (TAK-228) at the indicated concentration, lysed, and subjected to 
immunoblotting for phospho-Thr37,46-4EBP1 or total 4EBP1 as described above. B, replicate 
aliquots isolated from a pretreatment ALL specimen were treated for 48 h with 0.1% DMSO or 
the indicated concentration of MLN0128 (TAK-228) in the presence of 5 µM Q-VD-OPh, 
washed, and subjected to SDS-PAGE followed by blotting for the indicated antigen. 
 
 
2.  Phospho-Ser473 analysis of pretreatment and day 8 bone marrow samples by immunoblotting  
 
2.1 Note on probing blots for multiple antigens 
 
Techniques previously reported from the Kaufmann lab (4, 10, 11) allow the sequential 
detection of multiple antigens on the same filter without interference. Once phospho-
4EBP1 has been successfully detected, we propose to use the same filters for detection of 
additional antigens, which are presented below in the order in which they will be 
examined. Because we routinely detect up to 14 antigens on a duplicate pair of filters 
from cell lines (including eight BCL2 family members, three phospho-proteins and the 
three corresponding total protein controls), we do not anticipate difficulty in completing 
the proposed immunoblotting with the available samples. 
 
2.2 
Role in trial:  Exploratory biomarker 
 
2.3 
Hypothesis and rationale: 
 
Hypothesis: AKT Ser473 phosphorylation will be inhibited by MLN0128 (TAK-228) 
in situ. 
 
Rationale:  Previous studies have shown that mTOR dual inhibitors such as MLN0128 
(TAK-228) and OSI-027 diminish the mTORC2-mediated phosphorylation of the pro-
survival kinase AKT on Ser473 (e.g., Fig. 10 below), leading to upregulation of the 
pro-apoptotic protein BCL2L11 (Bim) and induction of apoptosis (1, 2). In ALL cell 
lines the downregulation of the mTORC2 component Rictor reproduces these effects 
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(2), demonstrating the critical role of signaling cascade in killing by mTOR dual 
inhibitors. We now propose to assess whether AKT Ser473 has been dephosphorylated 
in leukemic blasts by therapeutically achievable MLN0128 (TAK-228) concentration 
in situ as a proof of mechanism for this class of agent. 
 
2.4 Assay procedure: 
 
The assay procedure will be performed as described in Section 1.3 and the Standard 
Operating Procedure (pp. 16-26) except that immunoblotting will use Cell Signaling 
Technology rabbit monoclonal antibody D9E, which recognizes AKT when 
phosphorylated in Ser473 but not when unphosphorylated at this site (See p. 27 of this 
appendix). 
 
2.41 Characterization of antibody used in these studies: A critical antibody for this 
procedure, a rabbit monoclonal from Cell Signaling Technology, Inc., recognizing 
phospho-Ser473-AKT, is supplied as a pre-titered reagent. The supplier’s practice of 
titering each lot of antibody before release helps assure that Western blots 
performed with the same dilution will yield similar results with different lots of 
antibody whenever the assay is run. The inability of the antibody to recognize 
unphosphorylated AKT is documented by the supplier in their datasheet appended 
to the end of this Appendix. 
 
2.42  Reproducibility of the blotting for AKT Ser473 and quantification:  Lysates 
prepared from 15 different malignant human lymphoid cell lines were subjected 
to SDS-PAGE, transferred to nitrocellulose on two separate days, and probed 
with replicate aliquots of anti-phospho-Ser473 antibody as described above.  As 
indicated in Fig. 7, the blots were qualitatively quite similar.  Quantification of 
integrated signal density [area x (intensity – background)] using NIH Image J 
revealed a correlation between integrated signal on blot 1 and blot 2 for the 
various cell lines on the two blots, with  = 0.95 (p = 0.0004).  
 
2.43 Occurrence of the proposed AKT Ser473 dephosphorylation in human neoplastic 
 
 
 
Figure 7.  Reproducibility of blotting for phospho-Ser473-AKT. Aliquots containing 50 µg of 
total cellular protein from the indicated human neoplastic lymphoid cell line were subjected to 

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SDS-PAGE followed by transfer to nitrocellulose on two consecutive days. Blots were probed 
with replicate aliquots of the same anti-phospho-Ser473-AKT antibody, visualized by enhanced 
chemiluminescence reagents, and exposed to x-ray film exactly as proposed for the clinical 
samples.  
 
lymphoid cells after dual mTORC1/mTORC2 inhibitor treatment in xenografts 
in vivo:  To assess whether 4EBP1 phosphorylation would be inhibited by 
therapeutically achievable concentrations in vivo, mice bearing xenografts of the 
human mantle cell lymphoma cell line Jeko were treated with the maximum 
tolerated dose of OSI-027 for 3, 5 or 6 days. Xenografts were snap frozen, then 
subjected to sample preparation and immunoblotting as described in Section 1.3 
of this Appendix.  Results of this analysis demonstrated inhibition of AKT Ser473 
phosphorylation in vivo in all drug-exposed xenografts compared to pretreatment 
samples (Fig. 4). 
 
3. Expression of BCL2L11 (Bim), BBC3 (Puma), MCL1 and BCL2 proteins in pretreatment and 
day 8 bone marrow samples by immunoblotting 
 
3.1 Roles in study: exploratory biomarkers 
 
3.2 Hypothesis and rationale: 
 
Hypothesis: Levels of BCL2L11 (Bim) and BBC3 (Puma) in ALL will increase during 
MLN0128 (TAK-228) treatment, whereas levels of MCL1 and possibly BCL2 will 
decrease. 
 
Rationale:  Previous studies have shown that mTOR dual inhibitors such as MLN0128 
(TAK-228) and OSI-027 cause upregulation of the pro-apoptotic BCL2 family members 
BBC3 (Puma) and BCL2L11 (Bim) in malignant lymphoid cells (1, 2). In ALL cell lines, 
down regulation of BBC3 (Puma) or BCL2L11 (Bim) produces resistance to mTOR dual 
inhibitors (1, 2), demonstrating the critical role of these proteins in killing by mTOR dual 
inhibitors. Conversely, the anti-apoptotic BCL2 family member MCL1 has been shown 
to decrease after treatment with mTOR dual inhibitors in certain malignant human 
lymphoid cell lines (1). Overexpression of eponymous BCL2 has also been shown to 
convey resistance to mTOR dual inhibitors (1). Accordingly, we propose to assess 
whether BCL2L11 (Bim) and BBC3 (Puma) have been upregulated or MCL1 down 
regulated in leukemic blasts by therapeutically achievable MLN0128 (TAK-228) 
concentration in situ as a further proof of mechanism for this class of agent. 
 
3.3 Assay procedure and sources of critical reagents:  
 
The assay procedure will be performed as described in Section 1.3 and the Standard 
Operating Procedure included in this Appendix with three exceptions. First, the 
following antibodies will be used (data sheets included at the end of this Appendix):  
 
BCL2L11 (Bim):  C34C5 rabbit monoclonal antibody, Cell Signaling Technology 

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BBC3 (Puma): G-3 murine monoclonal antibody (sc-374223), Santa Cruz 
Biotechnology 
BCL2:  Clone 124 mouse anti-human BCL2 antibody (Dako) 
MCL1:  D35A5 rabbit monoclonal antibody, Cell Signaling Technology 
 
Second, appropriate secondary antibody will be utilized. Third, as standards for these 
studies, we currently plan to purify recombinant human BCL2 (12-14), MCL1 (12, 13), 
BCL2L11 (Bim) (15) and BBC3 (Puma) (16) and apply known amounts in a serial 
dilution to serve as a standard curve. 
 
3.4 
Assay performance characteristics 
 
The following performance characteristics of the assay have been evaluated: 
 
3.41 Characterization of antibodies used in these studies: Critical antibodies for these 
procedures are the antibodies listed above.  For the antibodies from Cell 
Signaling Technology, Inc., the supplier’s practice of titering each lot of 
antibody before release helps assure that Western blots performed with the same 
dilution will yield similar results with different lots of antibody whenever the 
assay is run. The absence of signal in lysates from gene-deleted cells (BBC3) or 
diminished signal in cells with siRNA/shRNA-mediated knockdown 
(BCL2L11, BCL2 and MCL1) has been documented in the Kaufmann laboratory 
for each of these antibodies (data not shown). 
 
3.42 Dynamic range of the assay:  To assess the dynamic range of the proposed assay, 
serial dilutions of the Nalm6 ALL cell line were subjected to immunoblotting 
with antibodies to BCL2 and BCL2L11 (Bim). As indicated in Fig. 8, signals 
were detectable in undiluted sample and six 2-fold dilutions 64-fold range) for 
BCL2 and in the undiluted sample and four 2-fold dilutions (16-fold range) for 
BCL2L11 (Bim). MCL1 likewise had a 16- to 32-fold dynamic range. BBC3 
(Puma) could not be effectively assayed in these cells prior to treatment because 
its baseline levels are too low. Note, however, that we plan to utilize 
 
 
 
Figure 8.  Assessment of dynamic ranges 
of BCL2 and BCL2L11 (Bim) blotting.  A 
sample containing 50 µg of total cellular 
protein from the human B-cell ALL Nalm6 
line (lane 1) and serial 2-fold dilutions 
(lanes 2-7) were subjected to SDS-PAGE 
and probed with antibodies that recognize 
BCL2 (top) or Bim (bottom). The locations 
of the extra-long, long and short splice 
variants of Bim (17) are indicated. 
 

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Figure 9.  Assessment of reproducibility of blotting for BCL2 and BCL2L11 (Bim). Aliquots containing 
50 µg of total cellular protein from the indicated human neoplastic lymphoid cell line were subjected to SDS-
PAGE followed by transfer to nitrocellulose on three (A) or two (B) consecutive days. Blots were probed with 
replicate aliquots of the same antibodies, visualized by enhanced chemiluminescence reagents, and exposed to 
x-ray film exactly as proposed for the clinical samples.  
 
serial dilutions of purified BBC3 (Puma) protein rather than a healthy cell line 
as a loading control for this assay (see Section 3.3 above). 
 
3.43 Reproducibility of the blotting for BCL2 and BCL2L11 (Bim):  Lysates from 15 
malignant human lymphoid cell lines were subjected to SDS-PAGE, transferred 
to nitrocellulose on multiple separate days, and probed with replicate aliquots of 
anti-BCL2 and anti-BCL2L11 (Bim) antibody as described above.  As indicated 
in Fig. 9, the blots were qualitatively similar.  Quantification of integrated 
signals [area x (intensity – background)] using NIH Image revealed a strong 
correlation between the BCL2 signals obtained for each cell line on blot 1 vs. 
blot 2, blot 2 vs. blot 3 and blot 1 vs. blot 3 in A [ =  0.90 - 0.95 (p = 0.0004 – 
0.008)] or BCL2L11EL (BimEL) on blot 1 vs. blot 2 in B [ = 0.95 (p = 0.0004) 
in B].  
 
3.44 Assessment of BCL2L11 (Bim) and BBC3 (Puma) induction: To assess whether 
BCL2L11 (Bim) and BBC3 (Puma) are reproducibly upregulated upon exposure 
to dual mTORC1/mTORC2 inhibitors, multiple investigators in the Kaufmann 
laboratory assessed the impact of the dual mTORC1/mTORC2 inhibitor OSI-
027 on levels of these proteins in the ALL cell line Jurkat independently over a 
5-year period using different batches of BCL2L11 (Bim) and BBC3 (Puma) 
antibodies from the indicated supplier. Two of these assays are shown in Figs. 
10A (lanes 2-6) and 10B (lanes 2-4) on the next page. These results show that 
upregulation of BCL2L11 (Bim) and BBC3 (Puma) is detectable by different lab 
personnel using different lots of antibody, nitrocellulose, x-ray film, detection 
reagents and buffers as long as the procedure described in Section 1.3 of this 
Appendix is followed. 
 
 

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Figure 10.  Induction of BCL2L11 (Bim) and BBC3 (Puma) by OSI-027.  A, after Jurkat 
cells were treated for 48 h with diluent (lanes 1); OSI-127 at 1.25, 2.5, 5, 10 or 20 µM (lanes 
2-6); or 10 nM rapamycin (lane 7) in the presence of the caspase inhibitor Q-VD-OPh (5 
µM—ref. 18) to inhibit apoptosis, whole cell lysates were subjected to SDS-PAGE followed 
by immunoblotting with antibodies that recognize the indicated antigen. PARP1 served as a 
loading control.  B, C, in two separate experiments designed to assess various aspects of OSI-
027-induced signaling (2), control Jurkat cells were treated with diluent or 10 µM OSI-027 
for 48 h and harvested for immunoblotting for the indicated antigens. 
 
 
4. Reverse phase protein array for protein phosphorylations and expression (exploratory 
biomarkers) 
 
4.1 Hypothesis and rationale: 
 
Hypothesis: Phosphorylation of mTOR substrates and complementary pathway 
components will change during the course of mTOR dual inhibitor treatment. 
 
Rationale:  Prior publications have identified differences in the pattern of protein 
phosphorylation between cell lines that are sensitive to kinase inhibitors and similar cell 
lines that are resistant (e.g., 20, 21). Preliminary studies in the Kaufmann laboratory have 
likewise demonstrated differences in protein phosphorylation at key nodes between 
parental Nalm6 ALL cells and Nalm6 cells selected for the ability to grow continuously 
in mTOR dual inhibitor (C. Correia, K. L. B. Knorr, S. Yun, A. E. Wahner Hendrickson, 
B. Madden, D. McCormick, S. Dasari, and S. H. Kaufmann, unpublished observations). 
Thus, we hypothesize that pretreatment patterns of phosphorylation might differ between 
ALLs that respond to MLN0128 (TAK-228) and those that do not. 
 
4.2 Assay validity and appropriateness for study: 
 
Reverse phase protein arrays (RPPAs) will be utilized to assess levels of phosphorylation 
of 60 key proteins, including 4EBP1, ribosomal protein S6, and AKT, as well as 
expression levels of roughly 200 proteins, including BCL2, BCL2L1 (Bcl-xL),  

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Figure 11.  Biological replicates in RPPA samples 
run at M. D. Anderson Cancer Center Reverse 
Phase Protein Array facility.  Samples of control 
Jurkat cells harvested on separate days were prepared 
for reverse phase protein arrays.  Shown are 
normalized linear expression values across 260 
antigens.  The QC score for each antibody was >0.8.  
Between the two samples, variation averaged 10%.   
 
 
 
MCL1, and BCL2L11 (Bim), using lysates from pretreatment and day 8 bone marrow 
blasts. This analysis of key signaling pathways will be utilized to supplement the results 
of the more targeted immunoblotting assays described above. 
 
4.3 Performance site:   
 
Whole cell lysates will be prepared in the Kaufmann laboratory of the Mayo Clinic Cancer 
Center. Based on preliminary results, the same lysates prepared for immunoblotting are 
suitable for RPPAs.  The RPPAs will be probed and initial bioinformatic analysis 
performed in the RPPA Core Facility of the M.D. Anderson Cancer Center, which is a fee-
for-service facility. Secondary bioinformatic analysis will be performed at Mayo. 
 
4.4 Performance characteristics of the assay:  
 
The reproducibility and reliability of this method, as well as comparison to the gold 
standard of Western blotting, has been published by the founders of the RPPA Core Facility 
at M.D. Anderson Cancer Center (22). 
 
To further assess the reproducibility of the assay in our hands, samples of control Jurkat 
cells harvested on sequential days were submitted to the M.D. Anderson Cancer Center 
RPPA Core Facility. Quantitative results across 260 antigens were scored in each sample.  
Information returned from the Core Facility (Fig. 11) indicated a quality score of >0.8 for 
each antibody.  Variation between the replicates averaged 9.9%; and a correlation 
coefficient of 0.67 was observed between samples on day 1 and day 2.  
 
 
5. Transcriptional profiling (exploratory biomarker) 
 
5.1 Role in trial:  Exploratory biomarker 
 
5.2 Hypothesis and rationale: 
 
Hypothesis: Treatment with MLN0128 (TAK-228) will activate two transcriptional 

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pathways related to apoptosis. 
Rationale:  Previous studies have shown that the mTOR dual inhibitor OSI-027 activates 
two distinct transcriptional pathways in ALL cell lines during the course of upregulating 
BCL2L11 (Bim) and BBC3 (Puma) to induce apoptosis (2). If sufficient material is 
available to allow isolation of RNA (after samples for immunoblotting and RPPAs have 
been harvested), samples will be subjected to RNA sequencing (RNAseq) to determine 
whether MLN0128 (TAK-228) concentrations achievable in vivo activate these same 
transcriptional pathways as a proof of mechanism study.  
 
5.3 Assay methodology: 
 
RNAseq will be performed on pretreatment and day 8 bone marrow aspirates to assess 
whether transcriptional changes observed in clinical ALL leading to BCL2L11 (Bim) and 
BBC3 (Puma) upregulation are also observed in clinical ALL treated in vivo. In brief, after 
Ficoll purification, total RNA will be extracted from marrow mononuclear cells using 
Qiagen RNA extraction kit. After RNA sample quality is assessed in the Gene Expression 
Core of the Mayo Clinic Cancer Center by measuring RNA Integration Number (RIN), 
an Illumina TruSeq mRNA kit will be used to generate cDNA for next generation 
sequencing. RNAs will be poly-A selected and fragmented, then subjected to reverse 
transcription with random primers and second-strand synthesis to generate double-
stranded cDNA. Ends will be repaired and poly(adenyl)ated, followed by adaptor and 
index ligation. The cDNAs will then denatured and PCR-enriched to generate the final 
genomic library, which will be analyzed on an Illumina HiSeq 2000. Data will analyzed 
according to a Mayo Clinic developed protocol for analyzing RNA-Sequencing data, 
(http://www.biomedcentral.com/content/pdf/1471-2105-15-224.pdf). Each mRNA count 
number will be normalized to counts per million.  
 
5.4 Assessment of assay performance: 
 
To assess assay performance, on two successive days Jurkat T cell ALL cells were treated 
with diluent vs. 10 µM OSI-027 for 48 h in the presence of the broad spectrum caspase 
inhibitor Q-VD-OPh (to prevent apoptosis-associated RNA degradation), RNA was 
extracted and subjected to the RNA sequencing procedure described in the preceding 
paragraph. As indicated in Fig. 12, results obtained with the duplicate samples under each 
condition were highly reproducible, with R values of 0.99 in diluent-treated cells and 
0.97 in drug-treated cells, respectively. 
 

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Figure 12.  Reproducibility of RNAseq data.  Jurkat cells treated with diluent + 5 µM 
Q-VD-OPh (left) or 10 µM OSI-027 + 5 µM Q-VD-OPh for 48 h (right) were harvested 
on two successive days and subjected to RNAseq analysis as described in Section 5.3.  
Plotted are relative reads for each mRNA in the first vs. second sample.   
 
 
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Yun S, Vincelette ND, Knorr KLB, Schneider PA, Peterson KL, Flatten KS, et al. Dual 
mTOR Inhibitiors Induce Bim-Dependent Apoptosis in Acute Lymphoblastic Leukmeia 
via Inhibition of eIF4E/eIF4G and Activation of NF-kB/Egr-1. Under revision. 2015. 
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Kaufmann SH, Svingen PA. Immunoblot Analysis and Band Depletion Assays. In: 
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Kaufmann SH. Reutilizaton of Western Blots After Chemiluminescent or 
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edition. Totowa, N.J.: Humana Press; 2009. p. 785-801. 
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Kaufmann SH, Karp JE, Burke PJ, Gore SD. Addition of Etoposide to Initial Therapy of 
Adult ALL:  A Combined Clinical and Laboratory Study. Leukemia and Lymphoma. 
1996;23:71-83. 
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Gore SD, Rowinsky EK, Miller CB, Griffin C, Chen T-L, Borowitz M, et al. A Phase II 
"Window" Study of Topotecan in Untreated Patients with High Risk Adult Acute 
Lymphocytic Leukemia. Clinical Cancer Research. 1998;4:2677-89. 
7. 
English D, Andersen BR. Single-Step Separation of Red Blood Cells.  Granulocytes and 
Mononuclear Leukocytes on Discontinuous Density Gradients of Ficoll-Hypaque. Journal 
of Immunological Methods. 1974;5:249-52. 
8. 
Friedman HS, Dolan ME, Kaufmann SH, Colvin OM, Griffith OW, Moschel RC, et al. 
Elevated DNA Polymerasea, DNA Polymeraseb, and DNA Topoisomerase II in a 
Melphalan-Resistant Rhabdomyosarcoma Xenograft that is Cross-Resistant to 
Nitrosoureas and Topotecan. Cancer Research. 1994;54:3487-93. 
9. 
Investigator's Brochure MLN0128. 7th edition ed2014. 
10. 
Kaufmann SH, Ewing CM, Shaper JH. The Erasable Western Blot. Analytical 

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Biochemistry. 1987;161:89-95. 
11. 
Kaufmann SH. Reutilization of Immunoblots After Chemiluminescent Detection. 
Analytical Biochemistry. 2001;296:283-6. 
12. 
Dai H, Meng XW, Lee S-H, Schneider PA, Kaufmann SH. Context-dependent Bcl-2/Bak 
Interactions Regulate Lymphoid Cell Apoptosis. Journal of Biological Chemistry. 
2009;284:18311-22. 
13. 
Smith AJ, Dai H, Correia C, Lee S-H, Takahashi R, Kaufmann SH. Noxa/Bcl-2 
Interactions Contribute to Bortezomib Resistance in Human Lymphoid Cells. Journal of 
Biological Chemistry. 2011;286:17682-92. 
14. 
Dai H, Ding H, Meng XW, Lee S-H, Kaufmann SH. Contribution of Bcl-2 
Phosphorylation to Bak Binding and Drug Resistance. Cancer Research. 2013;73:6998-
7008. 
15. 
Dai H, Smith A, Meng XW, Schneider PA, Pang Y-P, Kaufmann SH. Transient Binding 
of an Activator BH3 Domain to the Bak BH3-Binding Groove Initiates Bak 
Oligomerization. Journal of Cell Biology. 2011;194:39-48. 
16. 
Dai H, Pang Y-P, Kaufmann SH. Evaluation of the BH3-Only Protein Puma as a Direct 
Bak Activator. Journal of Biological Chemistry. 2014;289:89-99. 
17. 
Puthalakath H, Huang DC, O'Reilly LA, King SM, Strasser A. The proapoptotic activity 
of the Bcl-2 family member Bim is regulated by interaction with the dynein motor 
complex. Mol Cell. 1999;3:287-96. 
18. 
Caserta TM, Smith AN, Gultice AD, Reedy MA, Brown TL. Q-VD-OPh, a broad 
spectrum caspase inhibitor with potent antiapoptotic properties. Apoptosis. 2003;8:345-52. 
19. 
Kaufmann SH, Svingen PA, Gore SD, Armstrong DK, Cheng Y-C, Rowinsky EK. Altered 
Formation of Topotecan-Stabilized Topoisomerase I-DNA Adducts in Human Leukemia 
Cells. Blood. 1997;89:2098-104. 
20. 
Casado P, Alcolea MP, Iorio F, Rodriguez-Prados JC, Vanhaesebroeck B, Saez-Rodriguez 
J, et al. Phosphoproteomics data classify hematological cancer cell lines according to 
tumor type and sensitivity to kinase inhibitors. Genome Biol. 2013;14:R37. 
21. 
Casado P, Rodriguez-Prados JC, Cosulich SC, Guichard S, Vanhaesebroeck B, Joel S, et 
al. Kinase-substrate enrichment analysis provides insights into the heterogeneity of 
signaling pathway activation in leukemia cells. Sci Signal. 2013;6:rs6. 
22. 
Tibes R, Qiu Y, Lu Y, Hennessy B, Andreeff M, Mills GB, et al. Reverse phase protein 
array: validation of a novel proteomic technology and utility for analysis of primary 
leukemia specimens and hematopoietic stem cells. Mol Cancer Ther. 2006;5:2512-21. 
 
 
 
 

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Division of Oncology Research  
  
Mayo Clinic – Rochester 
 
 
 
Technical Reviewer:    
Scott H. Kaufmann  
 
Date:   
 
 
Secondary Reviewer:   
Charles Erlichman  
 
Date:   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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TABLE OF 
CONTENTS 
 
1.0  PURPOSE……………………………………………………………………………3 
 
2.0  SCOPE……………………………………………………………………………….3 
 
3.0  ABBREVIATIONS………………………………………………………………….3 
 
4.0  INTRODUCTION……………………………………………………………………3 
 
5.0  ROLES AND RESPONSIBILITIES………….……………………………………..3 
 
6.0  EQUIPMENT REQUIRED………………………………………………………… 4 
 
7.0  OPERATING PROCEDURES………………………………………………………8 
 
BLOT INFORMATION SHEET (APPENDIX 1)………………………………………11 
 
 

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1.  PURPOSE 
  
To standardize methods of probing bone marrow mononuclear cells (BMMCs) from 
bone marrow samples with anti-phospho-Thr37,46-4EBP1 antibody to determine 
whether anticancer agents are inhibiting phosphorylation of this protein. 
  
2.  SCOPE 
  
This procedure is used by all lab personnel responsible for probing immunoblots for 
phospho-Thr37,46-4EBP1 antibody to assess this phosphorylated protein in BMMC 
from patients participating in clinical trials of chemotherapeutic agents to ensure 
consistency in the handling of these samples. 
  
3.  ABBREVIATIONS 
  
4-EBP1 – Eukaryotic initiation factor 4E binding protein 1 
BMMC – Bone marrow mononuclear cells 
ECL – Enhanced chemiluminescence 
PBS – Dulbecco’s calcium- and magnesium-free phosphate buffered saline 
RT – Room temperature (20-22 °C) 
SOP – Standard Operating Procedure 
TS – Tris-Saline 
  
4.  INTRODUCTION 
 
Consistent processing of immunoblots is very important for reliably assessing 
posttranslational modifications in clinical leukemia samples. This SOP describes in 
detail the procedures to probing immunoblots to assess the phosphorylation status of the 
translation inhibitor 4E-BP1. 
  
5.  ROLE AND RESPONSIBILITIES 
  
Principal Investigator/Supervisor 
The Principal Investigator/Supervisor oversees laboratory operations and 
supervises research personnel. S/he is responsible for probing of immunoblots. S/he is 
also responsible for ensuring proper training and certification of research personnel who 
probe blots according to this SOP. 
  
Certified blot processor 
A certified blot processor may be a research technologist, post-doctoral fellow, 

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graduate student or research scientist. 
 
5.1 The Principal Investigator/Supervisor is responsible for ensuring proper training of 
certified blot processors in processing of immunoblots according to this SOP. 
  
5.2 The certified blot processor is responsible for proper handling and 
documentation of blot processing according to this SOP. 
 
6.  MATERIALS AND EQUIPMENT REQUIRED 
 
6.1 Pipettors (20 µl, 200 µl, 1000 µl) and tips 
 
6.2 Orbital shakers (2, e.g., Lab-Line Model #3520) 
 
6.3 5 ml (Falcon #357543) and 10 ml (Falcon #357551) sterile pipettes, individually 
wrapped. 
 
6.4 50 ml (Falcon #352098) sterile polypropylene conical tubes 
 
6.5 Microfuge tubes, 500 µl (e.g., Sarstedt #72.699) 
 
6.6 Magnetic stir platform (e.g., Cole Parmer 9” x 9” stirrer ) 
 
6.7 Magnetic stir bars 
 
6.8 Beakers (100 ml, 250 ml and 1000 ml, plastic or glass) 
 
6.9 Graduated cylinders (25 ml, 100 ml and 250 ml, plastic or glass) 
 
6.10 Automatic pipettor (e.g., Drummond Scientific 4-000-040) 
 
6.11 Ziploc bags (quart size, Ziploc #868624) 
 
6.12 10X PBS (Invitrogen 14200-075) 
 
6.13 Deionized water (e.g., produced by properly maintained Barnsted Nanopure II) 
 
6.14 Powdered nonfat dry milk (HyVee #14616) 
 
6.15 Tween 20 (Sigma-Aldrich P1379-1L) 
 
6.16 NaCl (ACS grade, Fisher 5271-3) 
 
6.17 Sodium azide (Sigma-Aldrich S2002-100G) 
 
6.18 100X Penicillin/streptomycin (American Type Culture Collection 30-2300) 
 
6.19 Bovine serum albumin (Sigma-Aldrich A7906-100G) 
 
6.20 Primary antibody: Rabbit anti-phosphospho-Thr37,46-4EBP1 (monoclonal 236B4, 
Cell Signaling Technology product #2855S) 
 

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6.21 
Horseradish peroxidase-coupled anti-rabbit IgG (KPL #074-1506, 1 mg) 
Preparation of stock solution (per supplier instructions): 
6.21.1 
Add 0.5 ml sterile water to 1 mg powdered antibody 
6.21.2 
Gently invert vial until antibody is dissolved 
6.21.3 
Slowly add 0.5 ml glycerol, mixing antibody up and down in pipettor 
without introducing bubbles 
6.21.4 
Aliquot antibody into 25 µl aliquots in 500 µl microfuge tubes 
6.21.5 
Store tubes at -20 °C in freezer. (Do not use a frost-free freezer because 
the temperature is not stable.) 
6.21.6 
Transfer working tube to 4 °C refrigerator, where it is stable for 4 
weeks. 
 
6.22 1X PBS 
  
Preparation: 100 ml 10X PBS + 900 ml deionized water 
Storage: Stable for 30 days at room temp. 
  
6.23 TS buffer 
Composition: 150 mM NaCl, 10 mM Tris-HCl (pH 7.4) 
Preparation: Weigh 8.72 gm NaCl into 1 L beaker 
Add 800 ml RT deionized water 
Add 1.21 gm Tris base 
Adjust pH to 7.4 with concentrated HCl 
Bring final volume to 1000 ml 
Sterile filter into sterile bottle using bottle top filter. 
Storage: 22 °C (stable for 1 year) 
6.24 Blot wash buffer 
  
Composition: PBS-0.05% Tween 20 
  
Preparation: Weigh out 0.5 gm Tween 20 in 1 L beaker 
Add 100 ml 10X PBS 
Add 900 ml deionized water 
Stir on magnetic stirrer until Tween 20 is dissolved 
 

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Storage: Stable for 7 days at RT. 
 
 
6.25 Primary antibody dilution buffer 
  
Composition: 5% bovine serum albumin in TS buffer containing 0.05% Tween 
20, 100 U/ml penicillin G, 100 µg/ml streptomycin and 1 mM sodium azide. 
  
Preparation: 
Weigh out 0.10 gm Tween 20 
Place in 250 ml beaker 
Add 150 ml TS buffer 
Stir until Tween 20 is dissolved 
Weigh out 10 gm bovine serum albumin 
Add to TS-Tween 20 and stir until dissolved 
Add 2 ml Penicillin/streptomycin solution 
Weigh out 13 mg of sodium azide 
Add to TS-Tween 20-Penicillin/streptomycin solution 
Stir until dissolved 
Bring to 200 ml in a graduated 
cylinder. Storage: Stable for 30 days at 4 °C. 
6.26 Secondary antibody dilution buffer 
  
Composition: 3% (w/v) milk in 1X PBS 
  
Preparation: Weigh 0.75 gm powdered nonfat dry milk into 50 ml conical tube 
Add 2.5 ml 10X PBS 
Add 15 ml deionized water 
Dissolve milk by inverting tube 6-8 times gently 
Bring to final volume of 25 ml 
  
Storage: Prepare fresh for each blot. 
  
6.27 SuperSignal West Dura extended duration ECL substrate (Thermo #34075) 
  
6.28 ECL working solution 
  
Preparation:  Combine 2 ml of solution 1 + 2 ml of solution 2 from West Dura 
 

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ECL kit for each blot 
  
Storage: Use within 10 min of combining these solutions 
 
6.29 X-ray film processor (e.g., Kodak X-omat 2000) 
  
6.30 X-ray cassettes (e.g., Fisher Biotech FBXC 810) 
  
6.31 X-ray film (e.g., Denville Scientific E3018 8 x 10 in HyBlot CL or Phenix 
Research Products #F-BX810) 

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7.  OPERATING PROCEDURES 
 
 
7.1 Record blot ID number and data on Blot Information Sheet (Appendix 1). 
 
7.2 Each blot will be recorded separately. 
 
7.21 This will ensure that deviations from procedure will be documented. 
 
7.3 Blot rehydration 
 
7.3.1 Blots are stored dry. 
 
7.3.1 Place each blot in a separate 1 quart Ziploc bag. 
 
7.3.2 Using a sterile 10 ml pipet, add 15 ml TS buffer to bag. Zip the top. 
 
7.3.3 Make certain that the blot is completely submerged in the buffer. 
 
7.3.4 Incubate 5 min at room temperature. 
 
7.3.5 Pour TS buffer down sink. 
 
7.3.6 Using a 10 ml pipet and an automatic pipetter, remove any remaining 
buffer from the bag. 
 
7.4 Add primary antibody to blot 
 
7.4.1   Prepare an aliquot of antibody by adding 10 µl of undiluted anti-phospho- 
Thr37,46-4EBP1 antibody to 10 ml of primary antibody dilution solution in 
a 15 ml conical tube. Invert tube slowly 5 times to mix antibody. 
 
7.4.2 Pour contents of antibody tube into bag containing blot. 
 
7.4.3 Placing the bag with blot on a flat surface, gently work as much air as 
possible from the bag, then seal it. 
 
7.4.4 Fold top of bag over and affix with tape to prevent leakage while rocking. 
 
7.4.5 Incubate on orbital shaker at 200 rpm for 12-16 hours at 4 °C. 
 
7.5 Washing after primary antibody 
 
7.5.1 Remove antibody solution from bag with a 10 ml pipet. 
 
7.5.2 Because of the presence of sodium azide, which can react with copper in 
pipes to cause explosions if present in concentrated solutions, the removed 
antibody solution is to be discarded by flushing down a sink containing 
non-copper pipe along with at least 25 volumes of cold water (250 ml per  

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10 ml aliquot of antibody solution) per Mayo Clinic Department of 
Laboratory Medicine Procedure [PROC 032915.013] dated 04/07/2014. 
 
7.5.3 Add 100 ml of RT blot wash buffer to bag. 
 
7.5.4 Incubate on orbital shaker at RT for 15 min. 
 
7.5.5 Pour off blot wash buffer. 
 
7.5.6 Repeat step 7.5.3-7.5.5 for a total of 3 washes 
 
7.5.7 Add 100 ml 1X PBS. 
 
7.5.8 Incubate on orbital shaker at RT for 5 min. 
 
7.5.9 Pour off 1X PBS. 
 
7.5.10 Repeat steps 7.5.7-7.5.9 for a total of two washes. 
 
7.6 Preparation of secondary antibody solution (during last three washes in 
Section 7.5) 
 
7.6.1 Prepare fresh aliquot of RT secondary antibody dilution buffer. 
 
7.6.2 Transfer to 50 ml conical tube. 
 
7.6.3 Add 3 µl of stock horseradish peroxidase-coupled anti-rabbit IgG to 
secondary blotting buffer. 
 
7.6.4 Invert tube gently 8 times to mix solution 
 
7.7 Incubation of blot with secondary antibody 
 
7.7.1 Pour secondary antibody solution into Ziploc bag containing blot. 
 
7.7.2 Incubate on orbital shaker for 60 min at RT. 
 
7.8 Washing after secondary antibody 
 
7.8.1 Remove antibody solution from bag with a 10 ml pipet. 
 
7.8.2 Add 100 ml of RT blot wash buffer to bag. 
 
7.8.3 Incubate on orbital shaker at RT for 5 min. 
 
7.8.4 Pour off blot wash buffer. 
 
7.8.5 Repeat step 7.8.2-7.8.4 for a total of 2 washes 
 
7.8.6 Add 100 ml of RT blot wash buffer to bag. 
 
7.8.7 Incubate on orbital shaker at RT for 15 min. 
 

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7.8.8 Pour off blot wash buffer. 
 
7.8.9 Repeat step 7.8.6-7.8.8 for a total of 2 washes 
 
7.8.10  Add 100 ml RT wash buffer. 
 
7.8.11  Incubate on orbital shaker at RT for 5 min. 
 
7.8.12  Pour off blot wash buffer. 
 
7.8.13  Repeat steps 7.8.10-7.8.12 for a total of 2 washes. 
 
7.9.   Signal detection (per instructions that accompany the SuperSignal ECL 
kit) 
 
7.91 
Remove blot wash buffer from blot. 
 
7.92 
Add ECL working solution. 
 
7.93 
Incubate for 60 sec. at room temp. with manual gentle agitation. 
 
7.94 
Pour off solution. Do not remove the small amount of liquid adhering to 
the blot. 
 
7.95 
Seal bag, taking care to remove as much air as possible. 
 
7.96 
Immediately place in x-ray cassette and start timer counting for 3 minutes. 
 
7.97 
When timer gets to 3 minutes, remove film from x-ray cassette and insert into 
X-ray film 
developer. 
 
7.98 
Examining the 3 minute exposure, determine whether increased or 
decreased exposure of x-ray film is required and insert a new piece of film 
in the cassette. 
 
7.99 
Reset timer for desired length of exposure. 
 
7.910   Continue until you acquire a series of exposures, some of which show 
various gradations of signal in the first (most concentrated) few samples on 
the standard curve and some of which show gradations of signal in the last 
(least concentrated) samples on the standard curve. 
 
 

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APPENDIX 1:  BLOT INFORMATION SHEET 
PAGE 1 
 
Each blot must be accompanied by a completed Blot Information Sheet with each page 
dated and initialed. A separate Blot Information Sheet should be started for each blot. 
 
NOTE: 
Record times using military time (24-h designation), for example specify 
16:15 to indicate 4:15 PM. 
 
Lab Technician (Certification number):     
 
  
Date: 
  
 
Reviewer/Supervisor:    
 
 
  
Date: 
  
 
Clinical Protocol Number:    
 
 
Blot ID (date and clinical samples contained):    
 
 
Processing Records: 
 
1. 
Consumables and Equipment 
Primary antibody (Lot # and receipt date)    
  
Secondary antibody (Lot # and preparation date)    
 
 
Serial numbers: 
 
P-20 Pipetman:    
 P-200:    
 P-1000:    
 
°C shaker (Manufacturer and Serial #):    
  
RT shaker (Manufacturer and Serial #):    
 
  
 
2. Blot processing 
 
Time primary antibody added to blot (date and time):                                                                        
Time primary antibody removed from blot (date and time):                                                               
Time primary secondary antibody added to blot (date and time):                                                      
Time first x-ray film applied to blot (date and time):                                                                          
 
 
 
 

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DATE   
 

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