Conditions: Recurrent Lung Squamous Cell Carcinoma Stage IV Lung Squamous Cell Carcinoma AJCC v7
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 34
Healthy volunteers: no

Brief summary:
This phase II trial studies how well sapanisertib works in treating patients with lung cancer that is stage IV or has come back (recurrent) and has a mutation in the NFE2L2, KEAP-1, or KRAS gene. Damage to these genes may cause the cancer to grow. Sapanisertib may stop this from happening by blocking enzymes.

Detailed description:
PRIMARY OBJECTIVE:

I. Evaluate the overall response rate of the TORC1/TORC2 inhibitor sapanisertib (MLN0128 \[TAK-228\]) in stage IV squamous cell lung cancers or KRAS mutant lung cancers harboring NFE2L2 or KEAP1 mutations.

SECONDARY OBJECTIVES:

I. To evaluate the median progression free survival of patients in each cohort. II. To explore the feasibility of performing reverse phase protein array analysis (RPPA) in paired snap-frozen core biopsies from patients in this study prior to MLN0128 (TAK-228) dosing and during week 2 of treatment.

III. To describe the effectiveness of MLN0128 (TAK-228) in suppressing activation of mTOR and PI3K signaling through the exploratory RPPA analysis.

OUTLINE:

Patients receive sapanisertib orally (PO) once daily (QD) on days 1-28. Cycles repeat every 28 days in the absence of disease progression or unacceptable toxicity.

After completion of study treatment, patients are followed up for 4 weeks.

Arms:
arm 1: Patients receive sapanisertib PO QD on days 1-28. Cycles repeat every 28 days in the absence of disease progression or unacceptable toxicity.

Interventions: intervention 1: Laboratory Biomarker Analysis intervention 2: Pharmacological Study intervention 3: Sapanisertib
Intervention descriptions: intervention 1: Correlative studies intervention 2: Correlative studies intervention 3: Given PO

Protocol document:
Prot_SAP_000.pdf: 
i 
 
SUMMARY OF CHANGES - Protocol 
 
For Protocol Amendment to: A Phase 2 Study of MLN0128 (TAK-228) in Patients with 
Advanced Non-Small Lung Cancers Harboring NFE2L2 and KEAP-1 Mutations 
 
NCI Protocol #: 9780 
Local Protocol #: 15-249 
 
NCI Version Date: 05/14/2020 
Protocol Date: 05/14/2020 
 
 
# 
Page(s) 
Change 
1. Throughout 
Editorial, formatting and grammatical changes were made throughout the 
protocol. 
2. Throughout 
NCI Version Date has been updated to 05/14/2020 throughout the protocol. 
3. 4-8 
The following clinicians have been added as co-investigators: 
 
Andrew Chow, MD, PhD 
Adam Schoenfeld, MD 
Stuart Lichtman, MD 
Neil Shah, MBBS 
Andrew Laccetti, MD, MS 
Julia Brockway-Marchello, MD 
 
The following clinicians have been removed as co-investigators: 
 
Han Xiao, MD 
Sree Chalasani, MD 
Parisa Momtaz, MD 
 
 


 
 
1 
 
NCI Protocol #: 
9780 
 
Local Protocol #: 
15-249 
 
ClinicalTrials.gov Identifier: NCT02417701  
 
TITLE: A Phase 2 Study of MLN0128 (TAK-228) in Patients with Advanced Non-Small 
Cell Lung Cancers Harboring NFE2L2 and KEAP1 Mutations 
 
Coordinating Center: 
Memorial Sloan Kettering Cancer Center 
 
Principal Investigator: 
Paul K. Paik, MD 
Memorial Sloan Kettering Cancer Center 
1275 York Ave. 
New York, NY 10065 
Phone: 646-888-4202 
Email: paikp@mskcc.org 
 
Co-investigators: 
Mark G. Kris, MD 
Memorial Sloan Kettering Cancer Center 
1275 York Ave. 
New York, NY 10065 
Phone: 646-888-4205 
Email: krism@mskcc.org 
 
Gregory J. Riely, MD, Ph.D. 
Memorial Sloan Kettering Cancer Center 
1275 York Ave. 
New York, NY 10065 
Phone: 646-888-4199 
Email: rielyg@mskcc.org 
 
Matthew Hellmann, MD 
Memorial Sloan Kettering Cancer Center 
1275 York Ave. 
New York, NY 10065  
Phone: 646-888-4863 
Email: hellmanm@mskcc.org 
 
Helena Yu, MD 
Memorial Sloan Kettering Cancer Center 
1275 York Ave. 
New York, NY 10065 
Phone: 646-888-4274 
Email: yuh@mskcc.org 
 


 
 
2 
 
Alexander Drilon, MD 
Memorial Sloan Kettering Cancer Center 
1275 York Ave. 
New York, NY 10065 
Phone: 646-888-4206 
Email: drilona@mskcc.org 
 
Piro Lito, MD, PhD 
Memorial Sloan Kettering Cancer Center 
1275 York Ave. 
New York, NY 10065 
Phone: 646-888-4873 
Email: litop@mskcc.org 
 
Jamie Chaft, MD 
Memorial Sloan Kettering Cancer Center 
1275 York Ave. 
New York, NY 10065 
Phone: 646-888-4545 
Email: chaftj@mskcc.org 
 
Marjorie G. Zauderer, MD 
Memorial Sloan Kettering Cancer Center 
1275 York Ave. 
New York, NY 10065 
Phone: 646-888-4656 
Email: zauderem@mskcc.org 
 
Charles Rudin, MD, PhD 
Memorial Sloan Kettering Cancer Center 
1275 York Ave. 
New York, NY 10065 
Phone: 646-888-4527 
Email: rudinc@mskcc.org 
 
Stephen R.Veach,.MD 
Memorial Sloan Kettering Cancer Center 
1275 York Ave.  
New York, NY 10065 
Phone: 646-888-4633 
Email: veachs@mskcc.org 
 
Michelle S. Ginsberg, MD 
Memorial Sloan Kettering Cancer Center 
1275 York Avenue 
New York, NY 10065 


 
 
3 
 
Phone: 212-639-7292 
Email: ginberm@mskcc.org 
 
Bob Li, MD 
Memorial Sloan Kettering Cancer Center 
1275 York Ave. 
New York, NY 10065 
Phone: 646-888-4201 
Email:lib1@mskcc.org 
 
Robert Daly, MD 
Memorial Sloan Kettering Cancer Center 
1275 York Ave. 
New York, NY 10065 
Phone: 646-888-4203 
Email: dalyr1@mskcc.org 
 
Kathryn Arbour, MD 
Memorial Sloan Kettering Cancer Center 
1275 York Ave. 
New York, NY 10065 
Phone: 646-449-1775 
Email: arbourk@mskcc.org 
 
Wei-Chu (Victoria) Lai, MD 
Memorial Sloan Kettering Cancer Center 
1275 York Ave. 
New York, NY 10065 
Phone: 646-449-1983 
Email: laiw@mskcc.org 
 
James Fetten, MD 
Memorial Sloan Kettering Cancer Center 
1275 York Ave. 
New York, NY 10065 
Phone: 646-888-4236 
Email: fettenj@mskcc.org 
 
Michael Offin, MD 
Memorial Sloan Kettering Cancer Center 
1275 York Ave. 
New York, NY 10065 
Phone: 646-449-1778 
Email: offinm@mskcc.org 
 
 


 
 
4 
 
Andrew Chow, MD 
Memorial Sloan Kettering Cancer Center 
1275 York Ave. 
New York, NY 10065 
Phone: 646-761-1523 
Email: chowa1@mskcc.org 
 
Adam Schoenfeld, MD 
Memorial Sloan Kettering Cancer Center 
1275 York Ave. 
New York, NY 10065 
Phone: 646-608-2227 
Email: schoenfa@mskcc.org  
 
Loren Michel, MD 
Memorial Sloan Kettering Cancer Center 
480 Red Hill Road 
Middletown, NJ 07748 
Phone: 848-225-6113 
Email: michell@mskcc.org 
 
Afsheen Iqbal, MD 
Memorial Sloan Kettering Cancer Center 
136 Mountain View Blvd 
Basking Ridge, NJ 07920 
Phone: 908-542-3383 
Email: iqbala@mskcc.org 
 
Avni Desai, MD 
Memorial Sloan Kettering Cancer Center 
650 Commack Road 
Commack, NY 11725 
Phone: 631-623-4253 
Email: desaia@mskcc.org 
 
Juliana Eng, MD 
Memorial Sloan Kettering Cancer Center 
650 Commack Road 
Commack, NY 11725 
Phone: 631-623-4249 
Email: engj@mskcc.org 
 
John Fiore, MD 
Memorial Sloan Kettering Cancer Center 
650 Commack Road 
Commack, NY 11725 


 
 
5 
 
Phone: 631-623-4247 
Email: fiorej@mskcc.org 
 
Jia Li, MD 
Memorial Sloan Kettering Cancer Center 
650 Commack Road 
Commack, NY 11725 
Phone: 631-623-4252 
Email: lij1@mskcc.org 
 
Stuart Lichtman, MD 
Memorial Sloan Kettering Cancer Center 
650 Commack Road 
Commack, NY 11725 
Phone: 631-623-4250 
Email: lichtmas@mskcc.org 
 
Azadeh Namakydoust, MD 
Memorial Sloan Kettering Cancer Center 
480 Red Hill Road 
Middletown, NJ 07748 
Phone: 848-225-6114 
Email: namakydoust@mskcc.org 
 
Colette Owens, MD 
Memorial Sloan Kettering Cancer Center 
480 Red Hill Road 
Middletown, NJ 07748 
Phone: 848-225-6112 
Email: owensc@mskcc.org 
 
Marina Shcherba, DO 
Memorial Sloan Kettering Cancer Center 
480 Red Hill Road 
Middletown, NJ 07748 
Phone: 848-225-6119 
Email: shcherbm@mskcc.org 
 
Neil Shah, MBBS 
Memorial Sloan Kettering Cancer Center 
480 Red Hill Road 
Middletown, NJ 07748 
Phone: 646-422-4468 
Email: shahn6@mskcc.org 
 
 


 
 
6 
 
Arlyn Apollo, MD 
Memorial Sloan Kettering Cancer Center 
1101 Hempstead Turnpike 
Uniondale, NY 11553 
Phone: 516-559-1520 
Email: apolloa@mskcc.org 
 
Pamela Drullinsky, MD 
Memorial Sloan Kettering Cancer Center 
1101 Hempstead Turnpike 
Uniondale, NY 11553 
Phone: 516-559-1521 
Email: drullinp@mskcc.org 
 
Zoe Goldberg, MD 
Memorial Sloan Kettering Cancer Center 
1101 Hempstead Turnpike 
Uniondale, NY 11553 
Phone: 516-559-1522 
Email: goldberz@mskcc.org 
 
Kenneth Ng, MD 
Memorial Sloan Kettering Cancer Center 
1101 Hempstead Turnpike 
Uniondale, NY 11553 
Phone: 516-559-1524 
Email: ngk@mskcc.org 
 
Tiffany Troso-Sandoval, MD 
Memorial Sloan Kettering Cancer Center 
1101 Hempstead Turnpike 
Uniondale, NY 11553 
Phone: 516-559-1525 
Email: torso-st@mskcc.org 
 
Oscar Lahoud, MD 
Memorial Sloan Kettering Cancer Center 
1101 Hempstead Turnpike 
Uniondale, NY 11553 
Phone: 516-256-3995 
Email: lahoudo@mskcc.org 
 
Ryan Sugarman, MD 
Memorial Sloan Kettering Cancer Center 
1101 Hempstead Turnpike 
Uniondale, NY 11553 


 
 
7 
 
Phone: 516-559-1312 
Email: sugarmar@mskcc.org 
  
Julia Brockway-Marchello, MD 
Memorial Sloan Kettering Cancer Center 
1101 Hempstead Turnpike 
Uniondale, NY 11553 
Phone: 516-559-1330 
Email: brockwaj@mskcc.org 
 
Louise Ligresti, MD 
Memorial Sloan Kettering Cancer Center 
225 Summit Avenue 
Montvale, NJ 07645 
Phone: 908-542-3379 
Email: ligrestl@mskcc.org 
 
Ping Gu, MD 
Memorial Sloan Kettering Cancer Center 
225 Summit Avenue 
Montvale, NJ 07645 
Phone: 201-775-7183 
Email: gup@mskcc.org 
 
Daniel Danila, MD 
Memorial Sloan Kettering Cancer Center 
225 Summit Avenue 
Montvale, NJ 07645 
Phone: 646-422-4612 
Email: danilad@mkscc.org 
 
Rui Wang, MD 
Memorial Sloan Kettering Cancer Center 
225 Summit Avenue 
Montvale, NJ 07645 
Phone: 201-775-7445 
Email: wangr5@mskcc.org 
 
Isabel Preeshagul, MD 
Memorial Sloan Kettering Cancer Center 
225 Summit Avenue 
Montvale, NJ 07645 
Phone: 201-775-7444 
Email: preeshai@mskcc.org 
 
 


 
 
8 
 
Anuja Kriplani, MD 
Memorial Sloan Kettering Cancer Center 
225 Summit Avenue 
Montvale, NJ 07645 
Phone: 201-775-7443 
Email: kriplana@mskcc.org 
 
Andrew Laccetti, MD, MS 
Memorial Sloan Kettering Cancer Center 
225 Summit Avenue 
Montvale, NJ 07645 
Phone: 646-422-4683 
Email: laccetta@mskcc.org 
 
Andrew Plodkowski, MD 
Memorial Sloan Kettering Cancer Center 
300 East 66th Street 
New York, NY 10065 
Phone: 646-888-4502 
Email: plodkowa@mskcc.org 
 
Etay Ziv, MD, PhD 
Memorial Sloan Kettering Cancer Center 
410 East 68th Street 
New York, NY 10065 
Phone: 212-639-3312 
Email: zive@mskcc.org 
 
 
Statistician:  
 
 
 
Study Coordinator: 
Glenn Heller 
Thomas Borkowski 
485 Lexington Avenue 
885 2nd Avenue 10th Floor 
New York, NY 10017 
New York, NY 10017 
646-888-8235 
646-449-1764 
hellerg@mskcc.org 
borkowst@mskcc.org 
 
Responsible Research Nurse: 
 
Responsible Data Manager: 
Linda Ahn 
Thomas Borkowski 
300 East 66th Street 
885 2nd Avenue, 10th Floor 
New York, NY 10065 
New York, NY 10017 
646-888-4202 
646-449-1764 
ahnl@mskcc.org                                                    borkowst@mskcc.org 
 
 
 
 


 
 
9 
 
NCI-Supplied Agent(s): MLN0128 (TAK-228) (NSC # 768435) 
 
IND Sponsor: DCTD, NCI 
 
Protocol Type / Version # / Version Date: Original / Version #1 / Version Date: 02/05/2015 
Protocol Type / Version # / Version Date: Original / Version #2 / Version Date: 04/08/2015 
Protocol Type / Version # / Version Date: Original / Version #3 / Version Date: 11/17/2015 
Protocol Type / Version # / Version Date: Original / Version #4 / Version Date: 01/15/2016 
Protocol Type / Version # / Version Date: Original / Version #5 / Version Date: 04/06/2016 
Protocol Type / Version # / Version Date: Original / Version #6 / Version Date: 08/01/2016 
Protocol Type / Version # / Version Date: Amended/Version #7 / Version Date: 10/31/2016 
Protocol Type / Version # / Version Date: Amended/Version #8 / Version Date: 03/15/2017 
Protocol Type / Version # / Version Date: Amended/Version #9 / Version Date: 07/24/2017 
Protocol Type / Version # /Version Date: Amended/Version #10/ Version Date: 09/08/2017 
Protocol Type / Version # /Version Date: Amended/Version #11/ Version Date: 10/31/2017 
Protocol Type / Version # /Version Date: Amended/Version #12/ Version Date: 02/01/2018 
Protocol Type / Version # /Version Date: Amended/Version #13/ Version Date: 03/02/2018 
Protocol Type / Version # /Version Date: Amended/Version #14/ Version Date: 04/05/2018 
Protocol Type / Version # /Version Date: Amended/Version #15/ Version Date: 05/08/2018 
Protocol Type / Version # /Version Date: Amended/Version #16/ Version Date: 06/08/2018 
Protocol Type / Version # /Version Date: Amended/Version #17/ Version Date: 12/03/2018 
Protocol Type / Version # /Version Date: Amended/Version #18/ Version Date: 09/23/2019 
Protocol Type / Version # /Version Date: Amended/Version #19/ Version Date: 10/25/2019 
Protocol Type / Version # /Version Date: Amended/Version #20/ Version Date: 05/14/2020 
 
 
 
 
 
 
 
 


 
10 
 
 
SCHEMA 
 
Study Title: 
A Phase 2 Study of MLN0128 (TAK-228) in Patients with Advanced Non-
Small Cell Lung Cancers Harboring NFE2L2 and KEAP-1 Mutations 
Study Objectives: 
Primary Objective: 
• Evaluate the overall response rate of the TORC1/TORC2 
inhibitor MLN0128 (TAK-228) in NFE2L2 or KEAP1 
mutant Stage IV squamous cell lung cancers and KRAS 
mutant lung cancers harboring KEAP1 or NFE2L2 
alterations. 
Secondary Objectives: 
• To evaluate median progression free survival 
• Explore the feasibility of performing reverse phase protein array 
analysis (RPPA) in paired snap-frozen core biopsies from patients 
in this study prior to MLN0128 (TAK-228) dosing and 1 week 
afterwards. 
• Describe the effectiveness of MLN0128 (TAK-228) in 
suppressing activation of mTOR and PI3K signaling through the 
exploratory RPPA analysis. 
Patient population: 
Stage IV or recurrent squamous cell lung cancer or KRAS mutant lung 
cancer patients who have progressed after at least first-line 
chemotherapy 
Number of 
patients: 
40 patients 
Inclusion Criteria: 
1.  Refer to Section 3.1 of the protocol 
Exclusion Criteria: 
1.  Refer to Section 3.2 of the protocol 
Study Drug: 
MLN0128 (TAK-228) 


 
11 
 
 
Study Design: 
This protocol is a phase 2, single institution study of MLN0128 
(TAK-228) in patients with stage IV or recurrent squamous cell 
lung cancer or KRAS mutant lung cancer harboring either an 
NFE2L2 or KEAP1 mutation who have progressed after at least 
first-line chemotherapy. 
The study will enroll three cohorts: 1) squamous cell lung cancer with 
NFE2L2 mutations; 2) squamous cell lung cancer with KEAP1 
mutations; 3) KRAS mutant lung cancer with concomitant NFE2L2 or 
KEAP1 aberration. A Simon two- stage design will be used to assess the 
primary endpoint of radiographic objective response rate, with a 5% 
undesirable response rate (H0) and a 40% ideal response rate (HA) 
(α=0.1, power=90%), for each cohort.   Five patients in each cohort will 
be enrolled into the first stage of the design.  If ≥1 patient in any cohort 
develops a response, then an additional 5 patients will be added for the 
second stage.  ≥2 patients must respond out of the final cohort of 10 
patients to consider MLN0128 (TAK-228) worthy of further 
investigation. 
Patients will be treated as follows: MLN0128 (TAK-228) 3mg p.o. 
daily on a continuous schedule. 
Toxicity will be graded according to the National Cancer Institute 
Common Terminology Criteria for Adverse Events (NCI CTCAE), 
version 4.0. Response to therapy will be assessed by interval imaging 
with CT scan of the chest after every 2 cycles with response evaluated 
per RECIST 1.1. 
Correlative Studies 
Molecular testing through MSK-IMPACT will be performed to 
identify KRAS mutations and both NFE2L2 and KEAP1 mutations as 
well as somatic changes in hundreds of other oncogenes and tumor 
suppressors. 
Reverse phase protein array analysis will be performed on optional paired 
tumor biopsies obtained before treatment begins and during week 2 of 
treatment. 


 
12 
 
 
TABLE OF CONTENTS 
 
SCHEMA .......................................................................................................................................... 10 
1. OBJECTIVES ............................................................................................................................... 15 
 
1.1 Primary Objectives ........................................................................................................ 15 
 
1.2 Secondary Objectives .................................................................................................... 15 
2. BACKGROUND .......................................................................................................................... 15 
 
2.1 Squamous cell lung cancer and NFE2L2/KEAP1 mutations ........................................ 15 
 
2.2 KRAS mutant lung cancers harboring alterations in the NFE2L2/KEAP1 pathway ..... 17 
 
2.3 MLN0128 (TAK-228) ................................................................................................... 18 
 
2.4 Rationale ....................................................................................................................... 23 
 
2.5 Correlative Studies Background .................................................................................... 27 
3. PATIENT SELECTION ............................................................................................................... 27 
 
3.1 Eligibility Criteria ......................................................................................................... 27 
 
3.2 Exclusion Criteria .......................................................................................................... 29 
 
3.3 Inclusion of Women and Minorities ............................................................................. 30 
4. REGISTRATION PROCEDURES ............................................................................................... 31 
 
4.1 Investigator and Research Associate Registration with CTEP ...................................... 31 
 
4.2 Site Registration ............................................................................................................ 32 
 
4.3 MSK Patient Registration Process ................................................................................ 32 
 
4.4 Informed Consent Procedures ....................................................................................... 32 
 
4.5 General Guidelines ........................................................................................................ 33 
5. TREATMENT PLAN ................................................................................................................... 33 
 
5.1 Agent Administration .................................................................................................... 33 
 
5.2 General Concomitant Medication and Supportive Care Guidelines .............................. 34 


 
13 
 
 
5.3 Duration of Therapy ...................................................................................................... 35 
 
5.4 Duration of Follow Up .................................................................................................. 36 
 
5.5 Criteria for Removal from Study .................................................................................. 36 
6. DOSING DELAYS/DOSE MODIFICATIONS ........................................................................... 36 
7. ADVERSE EVENTS: LIST AND REPORTING REQUIREMENTS ........................................ 50 
 
7.1 Comprehensive Adverse Events and Potential Risks List (CAEPR) ............................ 50 
 
7.2 Adverse Event Characteristics ....................................................................................... 53 
 
7.3 Expedited Adverse Event Reporting ............................................................................. 54 
 
7.4 Routine Adverse Event Reporting ................................................................................. 56 
 
7.5 Secondary Malignancy .................................................................................................. 56 
 
7.6 Second Malignancy ....................................................................................................... 57 
8. PHARMACEUTICAL INFORMATION ..................................................................................... 57 
 
8.1 MLN0128 (TAK-228) ................................................................................................... 57 
9. BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES .................................................... 59 
 
9.1 Integral Laboratory or Imaging Studies ........................................................................ 59 
 
9.2 Exploratory/Ancillary Correlative Studies .................................................................... 60 
10. STUDY CALENDAR ................................................................................................................. 62 
11. MEASUREMENT OF EFFECT ................................................................................................. 63 
 
11.1 Antitumor Effect – Solid Tumors ................................................................................ 63 
12. DATA REPORTING / REGULATORY REQUIREMENTS .................................................... 69 
 
12.1 Data Reporting ............................................................................................................ 69 
 
12.2 CTEP Multicenter Guidelines ..................................................................................... 69 
 
12.3 Collaborative Agreements Language .......................................................................... 69 
13. STATISTICAL CONSIDERATIONS ........................................................................................ 71 
 
13.1 Study Design/Endpoints .............................................................................................. 71 


 
14 
 
 
13.2 Sample Size/Accrual Rate ........................................................................................... 72 
 
13.3 Reporting and Exclusions............................................................................................ 73 
14. PROTECTION OF HUMAN SUBJECTS .................................................................................. 73 
 
14.1 Privacy ........................................................................................................................ 74 
REFERENCES .................................................................................................................................. 75 
APPENDIX A PERFORMANCE STATUS CRITERIA ............................................................... 76 
APPENDIX B ELIGIBLE NFE2L2, KEAP1 AND KRAS MUTATIONS .................................... 77 
APPENDIX C BIOASSAY TEMPLATES .................................................................................... 85 
APPENDIX D PATIENT PILL DIARY ........................................................................................ 89 
APPENDIX E  PATIENT GLUCOSE MONITORING LOG ....................................................... 92 
APPENDIX F  LIST OF RELEVANT CYTOCHROME P450 INHIBITORS AND INDUCERS ... 94 
 
 


 
15 
 
 
1. OBJECTIVES 
 
1.1 
Primary Objectives 
 
1. Evaluate the overall response rate of the TORC1/TORC2 inhibitor MLN0128 
(TAK-228) in Stage IV squamous cell lung cancers or KRAS mutant lung 
cancers harboring NFE2L2 or KEAP1 mutations 
 
1.2 
Secondary Objectives 
 
1. To evaluate the median progression free survival of patients in each cohort 
2. To explore the feasibility of performing reverse phase protein array analysis 
(RPPA) in paired snap-frozen core biopsies from patients in this study prior to 
MLN0128 (TAK-228) dosing and during week 2 of treatment. 
3. To describe the effectiveness of MLN0128 (TAK-228) in suppressing activation 
of mTOR and PI3K signaling through the exploratory RPPA analysis. 
 
 
2. BACKGROUND 
 
2.1 
Squamous cell lung cancer and NFE2L2/KEAP1 mutations 
 
Non-small cell lung cancer remains the leading cause of cancer related mortality in the US and 
worldwide. Squamous cell lung cancer accounts for approximately 25% of all non-small cell 
lung cancer cases. In the United States, 55,000 patients are diagnosed with squamous cell lung 
cancer per year, with approximately half of these patients harboring metastatic disease at 
diagnosis. Patients with advanced squamous cell lung cancers generally have a poor prognosis 
with a median survival of 12 months. Furthermore, these patients have fewer therapeutic options 
than those with other histologies. Pemetrexed, which has greater efficacy in non-squamous 
NSCLC, is not FDA approved for patients with squamous cell lung cancers. Because of rare 
cases of life threatening hemoptysis in early phase clinical trials, bevacizumab is similarly not 
FDA approved for patients with squamous cell lung cancers. Most pressing of all is the current 
absence of druggable molecular targets in squamous cell lung cancers. For instance, activating 
mutations in EGFR that render lung tumors sensitive to erlotinib and afatinib and ALK 
rearrangements that sensitize to treatment with crizotinib do not occur in SQCLCs.1,2 Taken 
together, there remains a high unmet medical need for new treatments for squamous cell lung 
cancer. 
 
Nrf2, the gene product of NFE2L2, is a transcription factor that binds to antioxidant response 
elements (AREs) contained in the promoters of a number of antioxidant genes and drug efflux 
pumps.  Keap1, the product of KEAP1, sequesters Nrf2 to the cytoplasm and is an adapter for the 
Cul3-dependent E3 ubiquitin ligase, which targets Nrf2 for degradation.3   During times of 
oxidative stress, cysteine residues are modified on Keap1, leading to a structural change that 
releases Nrf2 for nuclear translocation.  Nrf2-/- mice are susceptible to carcinoma formation in 


 
16 
 
response to chemical mutagens, notably benzo[a]pyrene, which is a carcinogen found in tobacco 
smoke.  In patients with SQCLCs, NFE2L2 mutations occur exclusively in a single N-terminal 
region coined Neh2, which is the canonical Keap1 binding domain (aa.1-80).  Mutations in  
KEAP1 are sporadic but occur in functional domains; 50% occur in the C-terminal Nrf2 binding 
domains; the other 50% occur in the N-terminal BTB and IVR domains that regulate Nrf2 
activity.  Collectively, these genes are mutated in ~20-30% of SQCLC patients, circumscribing 
one of the largest SQCLC subtypes described to date. 
 
Shibata et al. previously characterized the oncogenic potential of activating mutations in 
NFE2L2.  HEK293 cells expressing Neh2-domain mutant NFE2L2 cDNAs exhibit increased cell 
proliferation and colony formation and develop sporadic tumors in orthotopic mouse models 
(Figure 1 and 2).4   These tumors were universally poorly differentiated carcinomas with high 
mitotic activity and a propensity for local invasion, although metastases were also observed. 
Signaling experiments demonstrated an upregulation in S6 kinase activity and not MAPK, which 
was supported by gene set enrichment analysis of NFE2L2 mutant cells that identified mTOR as 
a significantly upregulated pathway.  Further experiments isolated a small G protein, RagD, as 
the transcriptional target of Nrf2 which induces activation of mTOR signaling.  More recent data 
from Bendavit and colleagues has also shown that Nrf2 can bind directly to the MTOR promoter 
sequence.6 
 
 
 
 
Figure 1. Increased anchorage independent growth in 
HEK293 cells expressing mutant Nrf2. Ctrl=wild type 
Nrf2; TR1/TR2=Nrf2 T80R; LF1/LF2=Nrf2 L30F. 
Figure 2. Spontaneous tumor formation in Nrf2 
mutant xenograft models.  Ctrl=wild type Nrf2; 
T80R=Nrf2 T80R clone; L30F=Nrf2 L30F clone. 


 
17 
 
 
Importantly, both rapamycin and BEZ235 (a PI3K/TORC1 inhibitor) were cytotoxic to two squamous cell 
lung cancer cell lines (LK2 and EBC1) that endogenously harbor canonical (Neh2 domain) NFE2L2 
mutations but not WT cell lines (Figure 3).  BEZ235 also abolished tumor growth in an associated 
xenograft model of LK2 SQCLC cells (Figure 4).  While Nrf2 signaling has not been a formal focus of 
the MLN0128 (TAK-228) clinical development program, it is worth noting that of the 5 human tumor cell 
line xenografts in which MLN0128 (TAK-228) was found to be effective, one, the KRAS-mutant A549 
NSCLC cell line, also harbors a KEAP1 G333C mutation (which occurs in one of the 6 Nrf2-binding 
Kelch domains) and exhibits KEAP1 loss of heterozygosity (loss of 19p13.2).3   A549 cells are also 
associated with increased Nrf2 nuclear localization compared to KEAP1 WT cells. The specific KEAP1 
G333C mutation is also unable to abolish Nrf2-mediated ARE reporter activity, supporting its functional 
impact in A549 cells. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2.2 
KRAS mutant lung cancers harboring alterations in the NFE2L2/KEAP1 pathway  
 
In keeping with the observation that the KRAS mutant A549 NSCLC cell line harboring KEAP1 loss of 
heterozygosity is associated with increased Nrf2 nuclear localization, recent data from Romero and 
colleagues5 have elucidated a role for Nrf2 activation in KRAS mutant lung cancer.  In sum, they showed that 
KEAP1 loss in a KRAS-driven lung cancer model hyperactivates Nrf2 leading to resistance to multiple 
oxidative stress agents.  This phenotype could be rescued by the addition of antioxidant treatments or 
exogenous reconstitution of Nrf2.  Loss of KEAP1 also accelerated tumorigenesis and growth (Figure 5A-D).  
A CRISPR-Cas9-based genetic screen further identified dependence on the glutamine transporter SLC1A5.  
Taken together, these data suggest that KRAS mutant lung cancer harboring events that activate Nrf2 via 
NFE2L2 or KEAP1 alterations would also be rational targets for inhibition of this pathway.  Pre-clinical data 
generated by us in Section 2.3 demonstrates that TAK228 monotherapy does indeed have anti-tumor efficacy 
in this context.  Existing data published by TCGA and us (through MSK-IMPACT) shows that the proportion 
of KRAS mutant lung cancer patients who tumors harbor concomitant alterations in KEAP1 or NFE2L2 is 
relatively high, at 25.6%.  Of the 405 patients with KRAS mutant lung cancers sequenced at MSK since 2014, 
102 patients had KEAP1 alterations, 25% of which were nonsense or frameshift deletion mutations.  2.22% 
had canonical mutations in the NFE2L2 Neh2 domain. 
 
Figure 4. Inhibition of tumor growth in Nrf2 mutant LK2 
mouse xenografts treated with BEZ235. 
Figure 3. Decreased cell viability in Nrf2 mutant 
squamous cell lung cancer cell lines (LK2 and 
EBC1) treated with rapamycin and the 
PI3K/mTOR inhibitor BEZ235. 


 
18 
 
 
 
 
 
 
 
 
 
 
 
2.3 
MLN0128 (TAK-228) 
 
Mechanism of Action 
 
MLN0128 (TAK-228) is an orally available inhibitor that has demonstrated potent and selective inhibition 
of mTOR kinase (Investigator's Brochure, 2012). MLN0128 (TAK-228) exerts a dual mechanism of action 
targeting both TORC1 and TORC2 (TORC1/2) complexes. MLN0128 (TAK-228) is in the category of 
ATP-competitive inhibitors, as it competes with ATP for binding to TORC1/2 active sites. 
 
In vitro antitumor activity 
MLN0128 (TAK-228) selectively and potently inhibited mTOR kinase, demonstrating 50% inhibition at 
the concentration of 1.1 nM (IC50). Relative to mTOR, MLN0128 (TAK-228) was 
>100-fold less potent as an inhibitor of Class I (PI3 kinase isoforms α, β, γ, δ (Table 1), class II (PI3KC2α 
and PI3K2Cβ), and Class III (VPS34) PI3K family members, as well as PI3Kα and PI3Kβ. 
 
Table 1. Comparison of MLN0128 (TAK-228) inhibition power against mTOR and a family of PI3 
kinases 
 
Recombinant Enzyme 
IC50 (nM) 
PI3Kα 
254 
PI3Kβ 
4183 
PI3Kγ 
221 
PI3Kδ 
223 
mTOR 
1.1 
IC50:  Drug concentration at which 50% inhibition is achieved (values represent the 
averages of 16-25 repeat experiment 
 
 
MLN0128 (TAK-228) (1 mcM) also inhibited (>80%) biochemical activity of five kinases (mTOR, DNA-
Figure 5 (a) Micro-CT quantification of total tumor volume (mm3) for tumors from sgKeap1.4 or sgTom mice (n = 5 and 3, respectively) at 4 
and 5 months after infection. Whiskers represent minimum and maximum values, upper and lower perimeters represent the interquartile 
distance, and midlines represent mean values. (b) Combined quantification of tumor burden (total tumor area/total lung area) in KP mice after 
infection with pSECC lentiviruses. Left, tumor burden of mice infected with control sgTom (n = 3) or sgKeap1.2 (n = 7) at 21 weeks after 
infection. Right, tumor burden in mice infected with control sgTom (n = 6) or sgKeap1.4 (n = 5) at 21 weeks after infection. Asterisks indicate 
statistical significance obtained from comparing KP-sgKeap1 samples to KP-sgTom samples. (c) Distribution of histological tumor grades in 
KP mice 21 weeks after infection with pSECC lentiviruses expressing control (sgTom KP, n = 7 mice) or sgKeap1.2 (KP; n = 3 mice). (d) 
Quantification of phosphorylated histone H3 (pHH3)-positive nuclei per squared millimeter of tumor for assessment of the mitotic index of 
tumor cells from lung tumors in KP mice at 21 weeks after infection with pSECC lentiviruses expressing control (sgTom; n = 14 tumors) or 
sgKeap1.2 (n = 50 tumors). 


 
19 
 
PK, PDGFRα, Flt3, and CK1 epsilon kinases) out of a panel of 222 protein kinases.  MLN0128 (TAK-
228) inhibited ligand binding of 10 receptor and intracellular protein kinases including (ACVR1, 
BMPR1B, CSF1R, CSNK1D, CSNK1E, DDR1, MEK1, MEK2, PDGFRβ, and RIPK2) out of a panel of 
402 distinct kinases. MLN0128 (TAK-228) displayed cellular inhibition of TORC1 and TORC2 pathways 
with IC50 less than 10 nM. 
 
In Vivo Studies: 
 
Pharmacokinetic (PK) studies of MLN0128 (TAK-228) have been investigated in a number of animal 
models, including mice, rats, dogs, and monkeys.  A summary of the non-clinical PK are shown in Table 
4-3 from the Investigator’s Brochure v.7, below. 
 
 
Drug Metabolism and Pharmacokinetics: TAK-228 was rapidly absorbed after PO administration to mice, 
rats, dogs, and monkeys,with high oral bioavailability.  TAK-228 displayed dose-proportional plasma 
exposures, a moderate propensity to cross the blood-brain barrier, and was modestly bound (70.5%) to 
human plasma proteins.  TAK-228 did not inhibit P-glycoprotein, but did inhibit breast cancer-resistance 
protein (BCRP), organic cation transporter (OCT)1 and OCT2. 
 
Recently completed in vitro metabolism experiments in human hepatocytes using 14C-labeled TAK-228 
suggest that TAK-228 is metabolized primarily via CYP1A2 (approximately 31%-40%), with a minor 
contribution from CYP3A4 (approximately 11%-22%). These data suggest that TAK-228 is also 
metabolized by direct glucuronidation (approximately 22%) and an unidentified non-uridine diphosphate 
glucuronosyl transferase pathway (approximately 18%). The new data differ from the previous in vitro CYP 
phenotyping data obtained using recombinant CYP enzymes, which suggested the involvement of CYP2C9 
(approximately 35%), CYP2C19 (approximately 28%), and CYP3A4 (approximately 28%) in TAK-228 
metabolism. In addition, physiologically based PK modeling and simulation using the new metabolism data 
for TAK-228 suggest that the risk for a metabolism-based drug-drug interaction with TAK-228 appears to 
be low. Therefore, strong CYP1A2 inhibitors and CYP inducers should be administered with caution and at 
the discretion of the investigator during the study. 
 
Toxicology: Adverse events of TAK-228 in rats and monkeys included body weight loss, decreased activity, 
increased glucose and insulin levels, alterations in white blood cells, bone marrow and lymphoid depletion, 
thymic necrosis, oligospermia, testes degeneration/atrophy, nonglandular stomach epithelial 
degeneration/ulceration/hyperplasia, pancreatic islet degeneration and fibrosis, lens fiber degeneration with 
cataract correlate, adrenal cortex hypertrophy, pituitary atrophy secondary to body weight loss, liver 


 
20 
 
hepatocellular vacuolation, retinal dysplasia with or without optic nerve atrophy, and alveolar histiocytosis.  
TAK-228 was negative for genotoxicity in an in vitro bacterial mutagenesis (Ames) assay, an in vivo rat 
micronucleus assay, and an in vivo rat comet assay.  TAK-228 was negative for phototoxicity in the 3T3 
fibroblast assay. 
 
Safety Pharmacology: TAK-228 has a low potential to affect the human ether-a-go-go related gene (hERG) 
potassium ion channel and did not affect cardiovascular (CV) parameters in vivo in telemeterized monkeys. 
 
Clinical Development of MLN0128 (TAK-228) 
 
The safety and tolerability of single-agent MLN0128 (TAK-228) (TAK-228) has been evaluated in two 
phase 1 company-sponsored studies:  1) the first-in-human dose-finding study in patients with advanced 
solid malignancies (Study INK128-001; n=166) and 2) a dose-finding study in patients with relapsed or 
refractory MM and Waldenstrom macroglobulinemia (WM), and non- Hodgkin’s Lymphoma (NHL) 
(Study INK128-002; n=39). MLN0128 (TAK-228) (TAK-228) has been administered on a 28-day cycle 
via various dosing schedules, including the continuous daily (once a day; QD) regimen or the intermittent 
regimens, such as the once weekly (QW), the 3-day-on/4-day-off repeated weekly (QD×3d every week), 
and the 5-day-on/2-day-off repeated every week (QDx5d every week) schedules.  In addition, the 
company has initiated a phase 1b combination study of MLN0128 (TAK-228) with paclitaxel (± 
trastuzumab in patients with HER2+ and HER2- breast cancer) with advanced solid tumors (INK128-003; 
n=69). 
 
In the daily-dosing cohort of INK128-001, a total of 25 patients received dose-escalated MLN0128 (TAK-
228) daily using a standard 3+3 design. The four cohorts included 2 mg (n=3), 4 mg (n=7), 6 mg (n=7), 
and 7 mg (n=8). The MTD was 6 mg QD with a DLT of G3 rash in 1 out of 6 patients (Infante et al., 
AACR 2012). The MTD for the QDx3d QW dosing was 16mg; for the QDx5d QW dosing, 10mg; and 
for the QW dosing, 40mg. The recommended phase 2 dose of 3mg po qd is based on the reported MTD 
of 6mg po qd. 
 
Treatment-emergent serious adverse events (SAEs) were reported in 58 patients (41%) in INK128-001.  
The most common AEs were mucosal inflammation in 5, asthenia and pneumonia in 4, and abdominal 
pain, stomatitis, and renal failure in 3 each.  Most AEs were grade 1 and 2 and manageable with 
supportive care and/or dose interruption/reduction. Grade ≥ 3 events included hyperglycemia (13%), 
asthenia (8%), anemia (7%), and hypophosphatemia and lymphopenia (6%). Of 142 patients treated in 
INK128-001, 20% discontinued due to AEs. As of December 2013, 4 patients had died within 30 days of 
the last dose of drug, 3 from disease progression and 1 from ventricular fibrillation and cardiac arrest 
possibly related to study drug. 
 
 
Summary of Effects in Humans: 
 
TAK-228 has been studied in 2 different phase I clinical trials in patients with solid malignancies and an 
additional phase I trial in patients with hematologic malignancies utilizing different dosing schedules (QD, 
QW, QDx3d every week and QDx5d every week) in 28-day cycles (Infante et al., 2012; Ghobrial et al., 
2012; Tabernero et al., 2012). The original trials with TAK-228 used the original unmilled TAK-228 active 


 
21 
 
pharmaceutical ingredient (API); current manufacturing process produces milled TAK-228 API. The 
recommended phase 2 dose for unmilled TAK-228 for the daily dosing schedule of the unmilled formulation 
were determined at 5 mg/day and for a weekly dosing schedule at 30 mg/week. (Infante et al., 2012; 
Ghobrial et al., 2012). A dose limiting toxicity of grade 3 rash was reported in in these trials (Infante et al., 
2012).  
 
 
Safety: As of the clinical data cutoff (09 December 2014), a total of 335 patients had received ≥ 1 dose of 
study drug across studies. A total of 18 deaths that occurred within 30 days of the last study drug dose had 
been reported to the clinical database as of the data cutoff; of these events, 1 (cardiac arrest; Study 
INK128-001) was considered related to TAK-228. 
At least 1 treatment-emergent SAE, regardless of causality, had been reported in 125/335 patients (37%). 
Across the studies and regardless of causality or dosing regimen, the most common TEAEs included nausea, 
fatigue, hyperglycemia, vomiting, diarrhea, stomatitis, and decreased appetite. 
 
Due to the cardiac death on study INK128-001, study C31002, a phase 1 single-arm study to evaluate the 
effect of a single dose of 40 mg 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 TAK-228 30 mg QW with continued cardiac monitoring.  The study results showed that treatment 
with TAK-228 was not associated with clinically meaningful effects on the overall electrocardiographic 
safety profile, and that ECHO/MUGA at screening was not required.   
 
 
Clinical Pharmacokinetics 
 
Pharmacokinetics: TAK-228 was well tolerated at the doses and schedules tested and showed a high oral 
bioavailability and dose-linear PK across all dosing regimens evaluated with exposures in the range of 
predicted biological activity (Infante et al., 2012 and Tabernero et al., 2012). TAK-228 exhibits fast oral 
absorption (time to reach Cmax [tmax], generally between 1-4 hours after dosing); has dose-linear PK, with 
a mean plasma half-life of approximately 8 hours; and does not accumulate meaningfully in plasma when 
dosed as frequently as once daily (QD)  
 
The PK of MLN0128 (TAK-228) has been evaluated in over 130 patients in ongoing studies. The mean 
steady-state plasma Cmax ranged from 52-232 nM.  No significant change in the PK parameters of 
MLN0128 (TAK-228) was observed on repeat dosing; the mean accumulation index of MLN0128 (TAK-
228) ranged from 0.7-1.7-fold following multiple doses. PK parameters in 25 patients with advanced solid 
tumors receiving MLN0128 (TAK-228) on the QD dosing are presented in Table 5.4 from the 
Investigator’s Brochure v.7. 
 


 
22 
 
 
Consistent with the QD dosing, MLN0128 (TAK-228) displayed dose-dependent PK on all three 
intermittent dosing regimens, with rapid absorption (Tmax ranging from 0.5-4 hours) and mean plasma t1/2 8 
hours (Tabernero et al., AACR 2012). 
 
Drug-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.  
 
Recently completed in vitro metabolism experiments in human hepatocytes using 14C-labeled TAK-228 
suggest that TAK-228 is metabolized primarily via CYP1A2 (approximately 31%-40%), with a minor 
contribution from CYP3A4 (approximately 11%-22%). These data suggest that TAK-228 is also 
metabolized by direct glucuronidation (approximately 22%) and an unidentified non-uridine diphosphate 
glucuronosyl transferase pathway (approximately 18%). The new data differ from the previous in vitro CYP 
phenotyping data obtained using recombinant CYP enzymes, which suggested the involvement of CYP2C9 
(approximately 35%), CYP2C19 (approximately 28%), and CYP3A4 (approximately 28%) in TAK-228 
metabolism. In addition, physiologically based PK modeling and simulation using the new metabolism data 
for TAK-228 suggest that the risk for a metabolism-based drug-drug interaction with TAK-228 appears to 
be low. Therefore, strong CYP1A2 inhibitors and CYP inducers should be administered with caution and at 
the discretion of the investigator during the study. 
 
 
Development of a Milled Formulation of TAK-228 
 
In order to allow more predictable absorption of TAK-228 after oral administration and to allow scale-up 
manufacturing of TAK-228 capsules, Millenium/Takeda developed a new milled formulation of the agent.  
The physical milling step during the granulation process controls particle size distribution of TAK-228.  In 


 
23 
 
order to observe whether this milling step altered the safety and PK profile of TAK-228, the company 
performed in vivo studies with PK analysis of milled 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. 
 
Takeda developed new 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, with various treatment 
cohorts including daily and weekly administration of milled TAK-228 
 
The recommended dose of milled TAK-228 was evaluated in 17 patients of MLN0128-1004, with PK, 
safety, and tolerability assessed.  Six patients were given a 4 mg QD dose of milled 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 TAK-228 was declared the RP2D, and was 
generally well tolerated and demonstrating objective responses in patients.   
 
The significant difference in tolerability observed in the comparison of the MTDs between unmilled and 
milled TAK-228 when administered QD may be possibly explained due to the effect of food on the 
safety/tolerability of unmilled TAK-228 in study IND128-001.  The GastroPlusTM simulation performed 
under fasting conditions on the trial demonstrated that unmilled and milled TAK-228 administration result 
in comparable exposures to TAK-228; whereas in the fed state, milled TAK-228 resulted in higher Cmax 
(1.5- to 2-fold higher) and earlier Tmax than unmilled TAK-228 with comparable AUCs.  Consequently, a 
dose of 3 mg QD was chosen as the RP2D of milled TAK-228 dose in empty stomach conditions.   
 
The RP2D for milled 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 TAK-228 at the prior 20 mg QW dose as well.  
 
TAK-228-1004, a phase I, open label study to evaluate the safety, tolerability, and pharmacokinetics of 
TAK-228 in combination with paclitaxel in adult patients with advanced non-hematological malignancies-), 
with the new milled API was used to determine the recommended phase 2 dose (RP2D) for TAK-228 
QD×3days per week in combination with paclitaxel.  The RP2D of milled TAK-228, given 3 consecutive 
days weekly, in combination with weekly paclitaxel at 80 mg/m2, was 6 mg.   
 
2.4 
Rationale 
 
While the existing data suggest that TORC1 inhibition may modestly inhibit Nrf2 signaling and cell 
viability in NFE2L2 mutant models, the activation of concurrent Akt signaling in NFE2L2 mutant cell lines, 
increased efficacy of BEZ235 in the above pre-clinical models, as well the prior experimental data 
demonstrating a bypass pathway through TORC2/Akt supports the use of dual TORC1/TORC2 inhibition. 
 
To strengthen the rationale for the use of MLN0128 (TAK-228) in squamous lung cancers harboring 
NFE2L2 and KEAP1 mutations, we performed cytotoxicity and signaling experiments in both NFE2L2 
mutant (LK-2) and KEAP1 mutant (H460, A549, H838) lung cancer cell lines treated with MLN0128 


 
24 
 
(TAK-228) and a number of rapalogs. We note that while LK-2 cells are squamous cell in histology, all 
KEAP1 mutant cell lines were either adenocarcinomas or large cell carcinomas (no KEAP1 mutant 
squamous cell lung cancer cell line exists to our knowledge). An Alamar blue assay was used to assess the 
effect of MLN0128 (TAK-228), rapamycin, everolimus, and deforolimus on cell viability in a dose 
response manner as shown below in   
Figure 5. 
 
 
 
 
Cell Line 
KEAP1 mutation 
NFE2L2 mutation KEAP1 CNV 
Co-mutations 
H460 
D236H 
WT 
diploid 
KRAS Q61H, PIK3CA E545K 
A549 
G333C 
WT 
loss 
KRAS G12S 
H838 
E444* 
WT 
diploid 
N/A 
SK-MES-1 
WT 
WT 
diploid 
N/A 
LK-2 
WT 
E79K 
N/A 
N/A 
 
Figure 5. Left- Heatmap of IC50 values for cytotoxicity using Alamar blue.  3 days after 
seeding, cells were exposed to Alamar blue for 24 hours prior to fixation and readout. 
IC50 for MLN0128 is shown.  IC50s for all rapalogs were ≥10μM for all cell lines tested. 
Right- Table lists genotype data for the cell lines used. 
 
 
 
No cytotoxic effect was observed for any of the rapalogs tested against any cell line at any drug 
concentration.  Conversely, MLN0128 (TAK-228) was associated with a low nanomolar IC50 
for cytotoxicity in NFE2L2 mutant LK2 cells and a relatively low IC50 for the KEAP1 null 
(KEAP1 G333C mutant + KEAP1 loss) A549 cells. Squamous cell lung cancer SK-MES-1 cells 
WT for NFE2L2 and KEAP1 showed no response to MLN0128 (TAK-228). With regard to the 
differential response in A549 cells compared to the two other KEAP1 mutant cell lines tested, 
loss of heterozygosity may potentiate response to drug.  It is worth reiterating that A549 cells 
were found to be sensitive to MLN0128 (TAK-228) by Millenium in their pre-clinical data as 
well.  Alternatively, not all mutations in KEAP1 may predict for sensitivity to drug inhibition. 


 
25 
 
 
 
Figure 6. Bar graph plot of 
total phospho-S6 (Ser240/244) 
staining intensity. Cell lines are 
represented by color scale: 
H460 in gray, A549 in blue, 
H838 in green, SK-MES-1 in 
red, and LK-2 in pink. 
Increasing drug concentration is 
symbolized by the triangles (0, 
0.001, 0.1 and 10 µM dose 
levels) 
 
 
 
 
 
 
 
 
 
 
 
 
 
To determine the downstream effect of each drug on mTOR signaling, cells were fixed and 
incubated with a phospho-S6 (Ser240/244) antibody after 2 hours of drug exposure. As seen in  
Figure 6, a dose-dependent decrease in S6 phosphorylation was generally seen across the cell 
lines in response to each drug.  Importantly, inhibition of S6 phosphorylation in LK-2 and A549 
cells was substantially greater in response to MLN0128 (TAK-228) compared to the rapalogs at 
100nM concentrations (Figure 7 and 8). 
 
 
 
Figures 7 and 8. Bar graph plot of relative phospho-S6 (Ser240/244) staining intensity at 100nM drug concentrations in NFE2L2 mutant LK-2 
cells (left) and KEAP1mutant A549 cells (right).  Data are shown relative to DMSO control. 
 
Taken together, these data suggest that MLN0128 (TAK-228) will be superior to the TORC1 
rapalogs at inhibiting downstream mTOR signaling and at inducing cell death in NFE2L2 mutant 
and a subset of KEAP1 mutant lung cancers, providing strong rationale for its use in patients 
with these oncogenic genotypes. 


 
26 
 
Cell line xenograft studies indeed bear this out in both our NFE2L2 mutant squamous cell lung cancer 
LK-2) and KRAS mutant/KEAP1 inactivated (A549) contexts (Figure 9 and Figure 10) where 
MLN0128 (TAK-228) treatment leads to a decrease in the size of generated tumors.   
 
 
Figure 9.  Differential anti-tumor effect of the TORC1/2 inhibitor MLN0128/TAK-228 against the rapalogs everolimus and 
deforolimus in an LK-2 NFE2L2 mutant squamous cell lung cancer xenograft.  Decrease in tumor volume was seen only in xenografts 
treated with MLN0128/TAK-228 (-55% tumor reduction). 
 
 
Figure 10.  Anti-tumor effect of the TORC1/2 inhibitor MLN0128/TAK-228 against an A549 KRAS mutant/KEAP1 null lung cancer 
xenograft.  MLN0128/TAK-228 led to a decrease in tumor volume not seen with the glutaminase inhibitor CB-839.
0
200
400
600
800
1000
1200
1400
1600
1800
6
9
13
16
21
23
Vehicle
MLN0128 1mg/kg po qd
MLN0128 3mg/kg po qd
Everolimus 3mg/kg po qd
AZD2014 2.5mg/kg po qd
Deforolimus 3mg/kg ip qd qow
0
20
40
60
80
100
120
140
160
180
200
13
16
20
23
27
Tumor volume (mm3)
Days
Vehicle
MLN0128 0.5mg/kg po qd
CB-839 100mg/kg po qd


 
27 
 
2.5 
Correlative Studies Background 
 
mTOR inhibition 
 
Inhibition of TORC1/2 signaling by MLN0128 (TAK-228) leads to changes in phosphorylation of the 
TORC1 downstream targets S6 and 4EBP1 (↓pS6 and p4EBP1) and a TORC2 target AKT (↓pAKT) in 
cell lines of various tumor types and genetic backgrounds, such as HER2-amplified trastuzumab-
sensitive and –resistant breast cancer cells (De et al., 2012), breast ductal T47D cell line with PI3KCA 
mutation (Investigator's Brochure), metastatic RCC (Wang et al., 2011), endometrial cell lines (Kessler 
et al., 2012), and prostate cancer line PC-3 with PTEN deletion (Investigator's Brochure).  Dual 
TORC1/2 inhibition mitigates the feedback activation of AKT, known to cause resistance to TORC1-
selective inhibitors such as rapamycin (De et al., 2012).  Its effect in squamous cell lung cancer models 
with NFE2L2 and KEAP1 mutations is shown in  Section 2.3, where a decrease in pS6 and p4EBP1 was 
also seen. 
 
Reverse phase protein array analysis (RPPA) 
 
There are a number of practical limits associated with pharmacodynamic testing in cancers where the 
majority of tissue material comes from small needle core biopsies. These include tumor 
heterogeneity necessitating microdissection, small total protein yields, the need to snap- freeze 
samples at -80C to preserve phosphoprotein stability, and the demand for a quantitative assay.  
Immunohistochemistry, which is the most readily available proteomic test, is hampered by its 
qualitative nature.  Western blotting, which can be quantitative depending on the specificity of the 
antibody used, has poor multiplex capabilities. 
 
This study incorporates reverse phase protein array (RPPA) analysis as an exploratory biomarker 
analysis to determine the pharmacodynamic effect of MLN0128 (TAK-228) in optional pre- and on-
treatment tumor biopsies.  RPPA is a highly multiplex quantitative proteomic platform that can test for 
both phosphorylated and non-phosphorylated proteins.  Dr. Lauren Byers at MD Anderson will 
perform RPPA on these optional paired tumor specimens. A panel of 156 validated antibodies (217 
total) is available for testing simultaneously, including key targets for this proposal (p4EBP1, pS6K, 
AKT pS463 and pT308). We will determine, in descriptive terms, changes in phosphorylation in these 
specific downstream targets in response to MLN0128 (TAK-228). 
 
Tumor samples will be snap-frozen in liquid nitrogen and stored at -80C.  This material will then be 
shipped frozen to Dr. Byers’ laboratory at MD Anderson for analysis. 
 
3. PATIENT SELECTION 
 
3.1 
Eligibility Criteria 
 
3.1.1 Patients must have histologically or cytologically confirmed stage IV or recurrent squamous 
cell lung cancer or KRAS mutant lung cancer that harbors any of the NFE2L2 mutations or 
KEAP1 mutations below.  Prioritized KEAP1 mutations are shown below, but any KEAP1 
mutation will be eligible. 


 
28 
 
 
Eligible and functionally validated Nrf2 amino acids/ mutations (Appendix B) 
NFE2L2 mutation sites 
Domain 
Function 
Validation 
aa.17-36 
DLG motif 
Keap1 binding site 
in silico and in vitro 
aa.76-82 
ETGE motif 
Keap1 binding site 
in silico and in vitro 
K44, K50, K52, K53, K56, K64, K68 
Lysine ubiquitination targets 
Nrf2 degradation 
in silico and in vitro 
Exon 2 deletion 
DLG/ETGE motifs 
Keap1 binding site 
in vitro 
 
Eligible and functionally validated Keap1 amino acids/mutations (Appendix B) 
KEAP1 mutation sites 
Domain 
Function 
Validation 
R380, R415, R483 
DGR-CTR 
Nrf2 binding residues 
in silico and in vitro 
Y525, Q530, N382, Y573, F577, Y334, S363, S602, S555 
DGR-CTR 
Nrf2 binding cavity 
in silico and in vitro 
G364C, G430C 
DGR-CTR 
Nrf2 binding cavity 
in silico and in vitro 
aa.315-624 
DGR-CTR 
Nrf2 binding domain 
in silico 
aa.96-100 
BTB HKVVL 
Keap1 dimerization 
in silico and in vitro 
C151, V155, V167 
BTB 
electrophile sensor 
in vitro 
D244, S243, C273, C288 
IVR 
electrophile sensor 
in vitro 
Any frameshift or nonsense mutation 
N/A 
N/A 
N/A 
 
 
3.1.2 Patients must have measurable disease, defined as at least one lesion that can be accurately 
measured in at least one dimension (longest diameter to be recorded for non- nodal lesions 
and short axis for nodal lesions) as ≥20 mm (≥2 cm) with conventional techniques or as ≥10 
mm (≥1 cm) with spiral CT scan, MRI, or calipers by clinical exam. See Section 11 for the 
evaluation of measurable disease. 
 
3.1.3 Patients must have completed at least 1 prior line of systemic therapy. Patients who have 
declined first line therapy or for whom first-line therapy would be clinically inappropriate, will 
be considered eligible for the trial. 
 
3.1.4 Age ≥18 years.  Because no dosing or adverse event data are currently available on the use 
of MLN0128 (TAK-228) in patients <18 years of age and because lung cancer is rare in this 
age group, children are excluded from this study. 
 
3.1.5 ECOG performance status ≤2 (Karnofsky ≥60%, see Appendix A). 
 
3.1.6 Life expectancy of greater than 3 months. 
 
3.1.7 Patients must have normal organ and marrow function as defined below: 
 
− 
leukocytes 
≥3,000/mcL 
− 
absolute neutrophil count 
≥1,500/mcL 
− 
platelets 
≥100,000/mcL 


 
29 
 
− 
total bilirubin 
within normal institutional limits 
− 
Fasting Serum Glucose 
≤ 130 mg/dL or HBA1C < 7.0% 
− 
AST(SGOT)/ALT(SGPT) 
≤2.5 × institutional upper limit of normal 
− 
creatinine 
within normal institutional limits OR 
− 
creatinine clearance 
≥50 mL/min/1.73 m2 for patients with elevated creatinine 
above institutional normal. 
 
3.1.8 Patients with controlled Diabetes are allowed on study. Controlled diabetes is defined as FBS 
< 130 mg/dL in the context of this study 
 
3.1.9 The effects of MLN0128 (TAK-228) on the developing human fetus are unknown. For this 
reason women of child-bearing potential and men must agree to practice 1 highly effective 
method of contraception and 1 additional effective (barrier) method, at the same time, prior to 
study through 90 days (or longer, as mandated by local labeling [eg, USPI, SmPC, etc;]) after 
the last dose 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.  Any woman who becomes pregnant while receiving MLN0128 (TAK-228) 
will be removed from the trial. 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 120 days after completion of MLN0128 (TAK-228) administration.  Men 
must agree not to donate sperm during the course of this study or within 120 days after 
receiving their last dose of study drug.  
 
3.1.10 Ability to understand and the willingness to sign a written informed consent document. 
 
3.1.11 Ability to swallow oral medications. 
 
3.1.12 Known HIV positive patients who meet the following criteria will be considered eligible: 
 
- 
CD4 count > 350 cells/mm3 
- 
Undetectable viral load 
- 
Maintained on modern therapeutic regimens utilizing non-CYP-interactive agents 
 
3.2 
Exclusion Criteria 
 
3.2.1 Patients who have had chemotherapy or radiotherapy within 2 weeks prior to the planned start 
of study treatment or those who have not recovered to baseline or less than grade 2 from 
adverse events from prior treatments. 
 
3.2.2 Patients who are receiving any other investigational agents. 
 
3.2.3 Patients with untreated CNS metastases.  Patients with treated CNS metastases who are off 
steroids are eligible. 
 
3.2.4 History of allergic reactions attributed to compounds of similar chemical or biologic 


 
30 
 
composition to MLN0128 (TAK-228). 
 
3.2.5 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, class III or IV heart failure, 
placement of pacemaker, or pulmonary embolism within six months of receiving first dose of 
MLN0128 (TAK-228).   
 
3.2.6 Baseline prolongation of the rate-corrected QT interval (QTc) > 480 milliseconds, or history of 
congenital long QT syndrome, or tosades de pointes. 
 
Pregnant women are excluded from this study because MLN0128 (TAK-228) is an mTOR 
agent with the potential for teratogenic or abortifacient effects.  Because there is an unknown 
but potential risk for adverse events in nursing infants secondary to treatment of the mother 
with MLN0128 (TAK-228), breastfeeding should be discontinued if the mother is treated with 
MLN0128 (TAK-228). 
 
3.2.7 Patients previously treated with an mTOR or PI3K inhibitor. 
 
3.2.8 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. 
 
3.2.9 Uncontrolled diabetes mellitus (Fasting plasma glucose > 130mg/dL despite optimal medical 
management of hyperglycemia) 
 
3.2.10 Known hepatitis B surface antigen-positive, or known or suspected active hepatitis C infection  
 
3.2.11 Patients receiving histamine H2 receptor antagonists before enrollment must stop using these 
medications for at least 24 hours before their first dose of study drug.  See section 5.2 for further 
details on the use of histamine H2 receptor antagonists. 
 
 
3.3 
Inclusion of Women and Minorities 
 
NIH policy requires that women and members of minority groups and their subpopulations be 
included in all NIH-supported biomedical and behavioral research projects involving NIH- defined 
clinical research unless a clear and compelling rationale and justification establishes to the satisfaction 
of the funding Institute & Center (IC) Director that inclusion is inappropriate with respect to the health 
of the subjects or the purpose of the research. Exclusion under other circumstances must be designated 
by the Director, NIH, upon the recommendation of an IC Director based on a compelling rationale and 
justification. Cost is not an acceptable reason for exclusion except when the study would duplicate 
data from other sources.  Women of childbearing potential should not be routinely excluded from 
participation in clinical research. Please see http://grants.nih.gov/grants/funding/phs398/phs398.pdf. 


 
31 
 
 
 
 
4. REGISTRATION PROCEDURES 
 
4.1 
Investigator and Research Associate Registration with CTEP 
 
Food and Drug Administration (FDA) regulations require IND sponsors to select qualified 
investigators.  NCI policy requires all persons participating in any NCI-sponsored clinical trial 
to register and renew their registration annually.  To register, all individuals must obtain a 
CTEP Identity and Access Management (IAM) account (https://ctepcore.nci.nih.gov/iam).  In 
addition, persons with a registration type of Investigator (IVR), Non-Physician Investigator 
(NPIVR), or Associate Plus (AP) (i.e., clinical site staff requiring write access to OPEN or 
RAVE or acting as a primary site contact) must complete their annual registration using 
CTEP’s web-based Registration and Credential Repository (RCR) 
(https://ctepcore.nci.nih.gov/rcr).  Documentation requirements per registration type are 
outlined in the table below. 
 
 
An active CTEP-IAM user account and appropriate RCR registration is required to access all 
CTEP websites and applications.  Additional information can be found on the CTEP website at 
https://ctep.cancer.gov/investigatorResources/default.htm.  For questions, please contact the 
RCR Help Desk by email at RCRHelpDesk@nih.gov. 
 
 
 
 
 
Documentation Required 
IVR 
NPIVR 
AP 
A 
FDA Form 1572 
 
 
Financial Disclosure Form 
 
 
 
NCI Biosketch (education, training, 
employment, license, and certification) 
 
 
 
HSP/GCP training 
 
 
 
Agent Shipment Form (if applicable) 
 
CV (optional) 
 
 
 
 


 
32 
 
4.2 
Site Registration 
 
N/A 
 
 
4.3 
MSK Patient Registration Process 
 
Confirm in the electronic medical record that the patient has received the Notice of Privacy Practice.  
This must be obtained before the eligibility confirmation and obtaining of the research informed 
consent. 
 
Confirm eligibility as defined in the section entitled Patient Selection. 
 
Obtain informed consent, by following procedures defined in section entitled Informed Consent  
Procedures. 
 
During the registration process registering individuals will be required to complete a protocol 
specific Eligibility Checklist.  The individual signing the Eligibility Checklist is confirming whether 
or not the participant is eligible to enroll in the study.  Study staff are responsible for ensuring all 
institutional requirements necessary to enroll a participant to the study have been completed.  See 
related Clinical Research Policy and Procedure #401 (Protocol Patient Registration). 
 
 
4.4 
Informed Consent Procedures 
 
Before protocol-specified procedures are carried out, consenting professionals will explain full details 
of the protocol and study procedures as well as the risks involved to participants prior to their inclusion 
in the study. Participants will also be informed that they are free to withdraw from the study at any time. 
All participants must sign an IRB/PB-approved consent form indicating their consent to participate. 
Only those individuals designated on the protocol title page as consenting professionals may obtain 
informed consent. This consent form meets the requirements of the Code of Federal Regulations and 
the Institutional Review Board/Privacy Board of this Center. The consent form will include the 
following: 
 
1. The nature and objectives, potential risks and benefits of the intended study. 
2. The length of study and the likely follow-up required. 
3. Alternatives to the proposed study. (This will include available standard and 
investigational therapies. In addition, patients will be offered an option of supportive 
care for therapeutic studies.) 
4. The name of the investigator(s) responsible for the protocol. 
5. The right of the participant to accept or refuse study interventions/interactions and to 
withdraw from participation at any time. 
Before any protocol-specific procedures can be carried out, the consenting professional will fully explain 


 
33 
 
the aspects of patient privacy concerning research specific information. In addition to signing the IRB 
Informed Consent, all patients must agree to the Research Authorization component of the informed 
consent form 
 
Each participant and consenting professional will sign the consent form. The participant must 
receive a copy of the signed informed consent form. 
 
 
4.5 
General Guidelines 
 
Eligible patients will be entered on study centrally at Memorial Sloan Kettering Cancer Center by the 
Study Coordinator. 
 
Following registration, patients should begin protocol treatment within 5-7 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.  
The Study Coordinator should be notified of cancellations as soon as possible. 
 
 
5. TREATMENT PLAN 
 
5.1 
Agent Administration 
 
Treatment will be administered on an outpatient basis.  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. 
 
Regimen Description 
Agent 
Premedications; 
Precautions 
Dose 
Route 
Schedule 
Cycle 
Length 
MLN0128 
(TAK-
228) 
none 
3mg 
oral 
daily 
continuous 
28 days 
(4 weeks) 
 
Patients will be treated MLN0128 (TAK-228) 3mg oral daily continuously on an every 4 week 
schedule.  MLN0128 (TAK-228) will be taken at the same time every day.  The capsules should not 
be opened, chewed, or crushed.  The patient will be given a medication diary at the beginning of each 
cycle of therapy.  The medication diary, along with the pill bottles and any unused drug, will be 
returned to the clinic staff at the end of each cycle. 
 
In cases where a subject misses dosing at his/her dosing time, the subject may still take the dose within 
12 hours of the regular dosing time (subjects should not take 2 consecutive daily doses within 12 
hours of each other).  Subjects who vomit shortly after receiving MLN0128 (TAK-228) will not 


 
34 
 
receive a replacement dose.  If confirmed that the study drug has been vomited, the dose should be 
noted as having been missed. 
 
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. 
 
 
5.2 
General Concomitant Medication and Supportive Care Guidelines 
 
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. 
 
Strong CYP1A2 inhibitors and CYP inducers should be administered with caution, at the discretion of 
the investigator (see Appendix F). Alternative treatments, if available, should be considered. 
 
Histamine H2 receptor antagonists may be allowed, if needed provided that the histamine H2 receptor 
antagonist is not taken within 12 hours before and within 6 hours after study drug administration. 
Patients receiving histamine H2 receptor antagonists before enrollment must stop using these 
medications for at least 24 hours before their first dose of study drug. Examples of histamine H2 
receptor antagonists include ranitidine, famotidine, nizatidine, and cimetidine and nizatidine. Cimetidine, 
a moderate cytochrome P450 (CYP)1A2 inhibitor, is not recommended as a first choice H2 receptor 
antagonist. 
 
Because there is a potential for interaction of MLN0128 (TAK-228) with other concomitantly 
administered drugs through the cytochrome P450 system, the case report form must capture the 
concurrent use of all other drugs, over-the-counter medications, or alternative therapies. The Principal 
Investigator should be alerted if the patient is taking any agent known to affect or with the potential to 
affect selected CYP450 isoenzymes. 
 
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 


 
35 
 
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 (eg, sitagliptin or vildagliptin) 
and/or insulin should also be considered. Oral sulfonylureas (eg, 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 will be given a prescription for a glucometer to monitor their daily FBG 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 FBG 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 FBG 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 until one of the 
following criteria applies: 
 
• 
Disease progression (at the discretion of the PI, patients who continue to derive clinical benefit 


 
36 
 
may be treated beyond progression; see Section 5.3.1 for details), 
 
• 
Intercurrent illness that prevents further administration of treatment, 
 
• 
Unacceptable adverse event(s), 
 
• 
Patient decides to withdraw from the study, or 
 
• 
General or specific changes in the patient's condition render the patient unacceptable for 
further treatment in the judgment of the investigator. 
 
   5.3.1 Treatment Beyond Progression 
             
Patients can be treated beyond progression at the discretion of the PI for clinical benefit and 
will undergo scans per the usual schedule.  In these instances, the scan demonstrating RECIST 
PD will be used as a new baseline to determine further progression.  Patients will be removed 
from the study if RECIST PD is achieved relative to the new baseline scan unless otherwise 
discussed with the Principal Investigator, if there is intolerable toxicity or if the patient 
withdraws consent. 
 
 
5.4 
Duration of Follow Up 
 
Patients will be followed for 4 weeks 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. 
 
5.5 
Criteria for Removal from Study 
 
Patients will be removed from study when any of the criteria listed in Section 5.4 applies.  If a 
patient is removed for any reason other than disease progression, he or she will be replaced. The 
reason for study removal and the date the patient was removed must be documented in the Case 
Report Form.  Patients can, at the discretion of the PI, receive treatment beyond progression for 
clinical benefit (see section 5.3.1 for details). 
 
 
 
6. DOSING DELAYS/DOSE MODIFICATIONS 
 
Toxicities will be graded using the NCI CTCAE Version 4.0 and dose modifications of the study 
drugs will be made for hematologic and other toxicities. Patients experiencing study drug related 
toxicities that require a delay in dosing for >21 days will be discontinued from further 
participation in this study. In the event that patients must have treatment delayed with a treatment 
cycle due to toxicities, those doses held during a cycle will not be made up. 
 


 
37 
 
 
 
Dose Level 
MLN0128 (TAK-228) Dose 
0 
3 mg flat dose daily continuous 
-1 
2 mg flat dose daily continuous 
-2 
2 mg daily 5 days/ week 
-3 
1 mg 5 days/week 
-4 
Withdraw from study 
 
 
 
 
Treatment Modification for ML N0128 (TAK-228)-Related Adverse Events 
Event 
CTCAE. V4 
Grade 
Action to be Taken 
QTc prolongation 
≥ Grade 3 or > 
60ms change from 
baseline on at least 
two separate ECGs 
First Occurrence: 
• Omit MLN0128 (TAK-228) dose 
• Perform an analysis of potassium and 
magnesium, and if below lower limit of normal, 
correct with supplements to within normal 
limits. Concomitant medication usage must be 
reviewed. 
• Perform a repeat ECG within one hour of the 
first QTcF of 500 ms 
• If QTcF remains > 500 ms, repeat ECG as 
clinically indicated, but at least once a day until 
the QTcF returns to < 480 ms. Seek cardiologist 
input 
• Once QTcF prolongation has resolved, 
MLN0128 (TAK-228) may be restarted at a 
one lower dose level 
Second Occurrence: 
• Permanently discontinue patient from the study 
treatment 
Other Cardiac Events 
Grade 1-2 
Maintain dose level of MLN0128 (TAK-228) 
Grade 3 
Omit dose of MLN0128 (TAK-228) until resolved to 
≤ Grade 1 
• If resolved within 7 days, then resume at same 
dose level of MLN0128 (TAK-228). 
• If resolved in >7 days, resume at ↓1 dose level 
of MLN0128 (TAK-228). 
• If not resolved in 21 days, then discontinue 
patient from study treatment. 


 
38 
 
Grade 4 
Permanently discontinue patient from the study 
treatment 
Treatment Modification for ML N0128 (TAK-228)-Related Adverse Events 
Event 
CTCAE. V4 
Grade 
Action to be Taken 
ENDOCRINE 
Fasting Plasma 
Glucose (FPG) 
(2 hrs fasting) 
Grade 1 
Maintain dose level of MLN0128 (TAK-228), 
check FPG every week 
• Initiate or intensify with appropriate anti- 
diabetic treatment as per investigator’s 
discretion 
• Instruct patient to follow dietary guidelines 
according to local and/or institutional standards 
for management of diabetes mellitus (such as 
those provided by the American Diabetes 
Association) during the study 
• Consider use of oral anti-hyperglycemic therapy 
such as metformin 
Check FGP daily for 8 weeks, then continue 
checking at least every 1 week 


 
39 
 
Grade 2 
First Occurrence: 
• If signs or symptoms of hyperglycemia (for 
example, mental status changes, excessive thirst, 
polyuria), omit dose of MLN0128 (TAK-228) 
immediately and manage as for Grade 3 
hyperglycemia (below) 
• If asymptomatic, maintain MLN0128 (TAK-
228) dose and re-check FPG within 24 hours. If 
grade worsens or improves then follow specific 
grade recommendations: 
o If FPG remains at Grade 2: 
▪ Maintain dose level of MLN0128 
(TAK-228) 
▪ Initiate or intensify with appropriate 
anti-diabetic treatment 
▪ Instruct patient to follow dietary 
guidelines according to local and/or 
institutional standards for management 
of diabetes mellitus (such as those 
provided by the American Diabetes 
Association) during the study 
▪ Consider use of oral anti-hyperglycemic 
therapy such as metformin 
o If FPG does not resolve to ≤ Grade 1 within 7 
days after initiation/intensifying anti-diabetic 
treatment: 
▪ Omit dose of MLN0128 (TAK-228) if 


 
40 
 
 
Treatment Modification for ML N0128 (TAK-228)-Related Adverse Events 
Event 
CTCAE. V4 
Grade 
Action to be Taken 
not already done 
▪ Monitor FPG at least weekly until FPG 
resolves to ≤ Grade 1 
▪ Then re-start MLN0128 (TAK-228) and 
↓1 dose level 
▪ Continue with anti-diabetic treatment 
▪ Check FPG weekly for 8 weeks, then 
continue checking every 2 weeks 
Second and Subsequent Occurrence: 
• Maintain dose level of MLN0128 (TAK-228), 
re- check FPG within 24 hours. If grade worsens 
or improves then follow specific grade 
recommendations. If FPG remains at Grade 2: 
o Omit dose of MLN0128 (TAK-228) 
o Initiate or intensify with appropriate anti- 
diabetic treatment 
o Monitor FPG at least twice weekly until FPG 
resolves to ≤ Grade 1 
o Then re-start MLN0128 (TAK-228) and ↓1 
dose level 
o Continue with anti-diabetic treatment 
o Check FPG at least weekly for 8 weeks, then 
continue checking at least every 2 weeks 


 
41 
 
Grade 3 
Immediately omit MLN0128 (TAK-228), initiate 
or intensify medication with appropriate anti-
diabetic treatment, re-check FPG within 24 hours. 
If grade worsens or improves then follow specific 
grade recommendations. 
• If FPG remains at Grade 3: 
o Administer intravenous hydration and 
intervention for 
electrolyte/ketoacidosis/hyperosmolar 
disturbances as clinically appropriate 
o Continue to omit dose of MLN0128 (TAK-228) 
o Monitor FPG at least twice weekly until FPG 
resolves to ≤ Grade 1 
o If FPG resolves to ≤ Grade 1 in 7 days or less, 
then re-start MLN0128 (TAK-228) at the same 
dose level 
• If FPG remains greater than Grade 1 severity for 


 
42 
 
 
Treatment Modification for ML N0128 (TAK-228)-Related Adverse Events 
Event 
CTCAE. V4 
Grade 
Action to be Taken 
more than 7 days, then discontinue patient from 
study treatment: 
o Initiate or continue anti-diabetic treatment as 
appropriate 
o Instruct patient to follow dietary guidelines 
according to local and/or institutional standards 
for management of diabetes mellitus (such as 
those provided by the American Diabetes 
Association) during the study 
o Consider use of oral anti-hyperglycemic therapy 
such as metformin 
o Check FPG at least weekly for 8 weeks, then 
continue checking at least every 2 weeks 
For non-fasting plasma glucose >250-500mg/dL (> 
13.9 – 27.8 mmol/L) accompanied by signs/symptoms 
of hyperglycemia (for example, mental status changes, 
excessive thirst, polyuria), or presence of blood or urine 
ketones, omit MLN0128 (TAK-228) and follow 
guidance for management of Grade 3 fasting plasma 
glucose (FPG). 


 
43 
 
Grade 4 
Immediately omit dose of MLN0128 (TAK-228), 
initiate or intensify medication with appropriate anti- 
diabetic treatment, re-check within 24 hours, if 
confirmed Grade 4: 
• Administer intravenous hydration and 
intervention for 
electrolyte/ketoacidosis/hyperosmolar 
disturbances as clinically appropriate 
• Discontinue patient from the study. Instruct 
patient to follow dietary guidelines according to 
local and/or institutional standards for 
management of diabetes mellitus (such as those 
provided by the American Diabetes 
Association) during the study 
• Consider use of oral anti-hyperglycemic therapy 
such as metformin 
• Check FPG weekly for 8 weeks, then continue 
checking at least every 2 weeks if clinically 
indicated 


 
44 
 
 
Treatment Modification for ML N0128 (TAK-228)-Related Adverse Events 
Event 
CTCAE. V4 
Grade 
Action to be Taken 
For non-fasting plasma glucose >500 mg/dL (> 27.8 
mmol/L) accompanied by signs/symptoms of 
hyperglycemia (for example, mental status changes, 
excessive thirst, polyuria), or presence of blood or urine 
ketones, discontinue MLN0128 (TAK-228) and 
following guidance for management of Grade 4 fasting 
plasma glucose (FPG). 
GASTROINTESTINAL 
Mucositis oral 
Grade 1 
Maintain MLN0128 (TAK-228) dose level 
Grade 2 
Maintain MLN0128 (TAK-228) dose level if tolerable, 
if toxicity becomes intolerable omit dose of MLN0128 
(TAK-228) until resolved to Grade < 1, then restart at 
the same dose level 
Grade 3 
Omit dose of MLN0128 (TAK-228) until resolved to 
Grade < 1, then restart at ↓1 dose level of MLN0128 
(TAK-228), If not resolved in 21 days, then 
discontinue patient from study treatment. 
Grade 4 
Permanently discontinue patient from the study 
treatment 
GENERAL DISORDERS 
Fatigue (asthenia) 
Grade 1-2 
Maintain MLN0128 (TAK-228) dose level 
Grade 3 
Omit dose of MLN0128 (TAK-228) until resolved to 
≤ Grade 1, then: 
• If resolved in ≤ 7 days, maintain dose level of 
MLN0128 (TAK-228) 
• If resolved in > 7 days, ↓ 1 dose level of 
MLN0128 (TAK-228) 
• If not resolved in 21 days, then discontinue 
patient from study treatment. 
Grade 4 
Patients who develop Grade 4 nonhematological 
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. 
 
HEMATOLOGIC 
Neutropenia (ANC) 
Grade 1-2 
Maintain dose level of MLN0128 (TAK-228) 


 
45 
 
Grade 3 
Omit dose of MLN0128 (TAK-228) until resolved to 
< Grade 1 or baseline, then: 
• If resolved in < 7 days, maintain dose level of 
MLN0128 (TAK-228) 
• If resolved in > 7 days, resume treatment at ↓1 
dose level of MLN0128 (TAK-228) 
• 
If  not  resolved  in  21  days,  then  discontinue 


 
46 
 
 
Treatment Modification for ML N0128 (TAK-228)-Related Adverse Events 
Event 
CTCAE. V4 
Grade 
Action to be Taken 
patient from study treatment. 
Grade 4 
Permanently discontinue patient from the study 
treatment 
Febrile Neutropenia 
Grade 3 
Omit dose of MLN0128 (TAK-228) until resolved 
to Grade 1 or baseline, then: 
• If resolved in < 7 days, resume treatment at ↓1 
dose level of MLN0128 (TAK-228) 
• 
If resolved in > 7 days, discontinue MLN0128 
(TAK-228) 
Thrombocytopenia 
Grade 1-2 
Maintain dose level of MLN0128 (TAK-228) 
Grade 3 
Omit dose of MLN0128 (TAK-228) until resolved to 
≤ Grade 1 or baseline then: 
• If resolved in ≤ 7 days, resume treatment at ↓ 1 
dose level of MLN0128 (TAK-228) 
• If resolved in > 7 days, discontinue MLN0128 
(TAK-228) 
Grade 4 
Permanently discontinue patient from the study 
treatment 
HEPATIC 
Bilirubin 
Grade 1 
Maintain dose level of MLN0128 (TAK-228) 
Grade 2 (with ALT 
or AST grade 1 or 
2) 
Omit dose of MLN0128 (TAK-228) until resolved to < 
Grade 1 or baseline, then resume at the same dose level 
of MLN0128 (TAK-228) 
Grade 3 (with ALT 
or AST grade 1 or 
2) 
Omit dose of MLN0128 (TAK-228) until resolved to 
≤ Grade 1 or baseline, then: 
• If resolved in ≤ 7 days, maintain same dose 
level of MLN0128 (TAK-228) 
• If resolved in > 7 days but less than 21 days, 
resume treatment at ↓ 1 dose level of MLN0128 
(TAK-228). 
• 
If not resolved in 21 days, then discontinue 
patient from study treatment. 
Grade 4 
Permanently discontinue patient from the study 
treatment 


 
47 
 
 
Treatment Modification for MLN0128 (TAK-228)-Related Adverse Events 
Event 
CTCAE. V4 
Grade 
Action to be Taken 
AST or ALT 
Grade 1 or Grade 2 
(without total 
bilirubin elevation 
> grade 1) 
Maintain dose level of MLN0128 (TAK-228) 
Grade 3 (without 
total bilirubin 
elevation > grade 
1) 
Omit dose of MLN0128 (TAK-228) until resolved to 
≤ Grade 1 or baseline, then: 
• If resolved in ≤ 7 days, maintain dose level of 
MLN0128 (TAK-228) 
• If resolved in > 7 days, resume treatment at ↓1 
dose level of MLN0128 (TAK-228) 
• 
If not resolved in 21 days, then discontinue 
patient from study treatment. 
• 
Permanently discontinue study treatment if 
in combination with Grade 2 total bilirubin 
elevation when alternative causes cannot 
be identified (ie, Hy’s Law); 
Grade 4 
Permanently discontinue study treatment if in 
combination with Grade 2 total bilirubin elevation when 
alternative causes cannot be identified (ie, Hy’s Law). 
RENAL 
Creatinine 
Grade 1 
Maintain dose level of MLN0128 (TAK-228) 
Grade 2 
Omit dose of MLN0128 (TAK-228) until resolved to ≤ 
Grade 1 or baseline, then resume treatment at the same 
dose level. Patients will be instructed to increase their 
fluid intake until resolution to ≤ Grade 1 or baseline. 
Grade 3 
Omit dose of MLN0128 (TAK-228) until resolved to 
≤ Grade 1 or baseline then: 
• If resolved in ≤ 7 days, maintain dose level of 
MLN0128 (TAK-228) 
• If resolved in > 7 days, resume treatment at ↓ 1 
dose level of MLN0128 (TAK-228) 
• If not resolved in 21 days, then discontinue 
patient from study treatment. 
RESPIRATORY 
Grade 1 
Maintain dose level of MLN0128 (TAK-228) 


 
48 
 
Non-infectious 
Pneumonitis 
Grade 2 
Omit dose of MLN0128 (TAK-228) until resolved 
to Grade < 1, then: 
• Restart at ↓1 dose level of MLN0128 (TAK-228) 
• Discontinue MLN0128 (TAK-228) if failure 
to recover within 4 weeks 
Grade 3 
Omit dose of MLN0128 (TAK-228) until resolved 
to Grade < 1, then: 
• Consider re-initiating MLN0128 (TAK-228) at 
↓1 dose level of MLN0128 (TAK-228) 
• If toxicity recurs at Grade 3, discontinue 
MLN0128 (TAK-228) 


 
49 
 
 
Treatment Modification for MLN0128 (TAK-228)-Related Adverse Events 
Event 
CTCAE. V4 
Grade 
Action to be Taken 
Grade 4 
Permanently discontinue patient from the study 
treatment 
SKIN AND SUBCUTANEOUS TISSUE DISORDERS 
Rash (maculo-papular) Grade 1 
Maintain dose level of MLN0128 (TAK-228). 
Consider initiation appropriate skin toxicity therapy 
(such as antihistamines, topical corticosteroids) 
Grade 2 
Maintain dose level of MLN0128 (TAK-228). 
Initiate/intensify appropriate skin toxicity therapy 
(such as antihistamines, topical corticosteroids) 
Grade 3 
Omit dose of MLN0128 (TAK-228) until resolved 
to Grade ≤ 1, then: 
• If resolved in ≤ 7 days, maintain at same dose 
of MLN 0218 
• If resolved in > 7 days, resume at ↓1 dose level 
of MLN0128 (TAK-228) 
• If not resolved in 21 days, then discontinue 
patient from study treatment. 
Grade 4 
Patients who develop Grade 4 rash should permanently 
discontinue study treatment, unless they derive clinical 
benefit, in which case they may be retreated at a 
reduced dose level after recover to  Grade 1 severity. 
Grade 4 rash is defined as rash acneifom/ 
papulopustular with papules and/or pustules covering 
any % body surface area, which may or may not be 
associated with symptoms of pruritus or tenderness, 
and are associated with extensive superinfection with 
intravenous (IV) antibiotics indicated; life threatening 
consequences (NCI CTCAE Version 4.03, effective 
date 14 June 2010). 
INVESTIGATIONS 
Cholesterol high 
and/or 
hypertriglyceridemia 
Grade 1 
Maintain MLN0128 (TAK-228) dose level 
Grade 2 
Maintain dose if tolerable, if toxicity becomes 
intolerable, omit dose MLN0128 (TAK-228) until 
resolved to < 1, then restart MLN0128 (TAK-228) 
as the same dose level 
Grade 3 
Omit dose of MLN0128 (TAK-228) until resolved to 
Grade  < 1, then restart at ↓1 dose level of MLN0128 
(TAK-228) 
Grade 4 
Permanently discontinue patient from the study 
treatment 


 
50 
 
OTHER ADVERSE EVENTS 
Other AEs 
Grade 1-2 
Maintain MLN0128 (TAK-228) 
Grade 3 
Omit dose of MLN0128 (TAK-228) until resolved to 
≤Grade 1, then: 
• If resolved in ≤ 7 days, maintain at same dose 
of MLN 0218 
• If resolved in > 7 days, resume at ↓1 dose level 
of MLN0128 (TAK-228) 
• If not resolved in 21 days, then discontinue 
patient from study treatment. 
Grade 4 
Permanently discontinue patient from the study 
treatment 
 
Note: Omit dose for ≥ Grade 3 vomiting or 
Grade 3 nausea only if the vomiting or nausea 
 
 
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 the CTEP Adverse Event Reporting System (CTEP-AERS) in 
addition to routine reporting. 
 
7.1 
  Comprehensive Adverse Events and Potential Risks List (CAEPR) 
For 
MLN0128 (TAK-228, 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/aeguidel
ines.pdf for further clarification. Frequency is provided based on 390 patients. 
Below is the CAEPR for MLN0128 (TAK-228). 
 
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. 


 
51 
 
 
 
7.1.1 CAEPR for MLN0128 (TAK-228) 
Version 2.3, July 28, 20191 
 
 Adverse Events with Possible  
 Relationship to MLN0128 (TAK-228) 
 (CTCAE 5.0 Term) 
[n= 390] 
 
 
 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) 
 
Dyspepsia 
 
  
Mucositis oral 
 
 
 Mucositis oral (Gr 2) 
Nausea 
 
 
 Nausea (Gr 3) 
Vomiting 
 
 
 Vomiting (Gr 3) 
GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS 
  
 
Edema limbs 
 
   
Fatigue 
 
 
 Fatigue (Gr 3) 
 
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 
  
Anorexia 
 
 
 Anorexia (Gr 2) 
 
Dehydration 
 
 Dehydration (Gr 2) 
Hyperglycemia 
 
 
 Hyperglycemia (Gr 3) 
 
Hypokalemia 
 
 Hypokalemia (Gr 2) 
 
Hypomagnesemia 
 
 Hypomagnesemia (Gr 2) 
 
Hypophosphatemia 
 
 Hypophosphatemia (Gr 2) 
MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS 
  
 
Arthralgia 
 
  
 
Back pain 
 
 Back pain (Gr 2) 


 
52 
 
 
 Adverse Events with Possible  
 Relationship to MLN0128 (TAK-228) 
 (CTCAE 5.0 Term) 
[n= 390] 
 
 
 Specific Protocol 
Exceptions to Expedited 
Reporting (SPEER)  
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious (<3%) 
 
 
 
Pain in extremity 
 
  
NERVOUS SYSTEM DISORDERS 
  
 
Dizziness 
 
 Dizziness (Gr 2) 
 
Dysgeusia 
 
 Dysgeusia (Gr 2) 
 
Headache 
 
 Headache (Gr 2) 
PSYCHIATRIC DISORDERS 
  
 
Insomnia 
 
  
RENAL AND URINARY DISORDERS 
  
 
Acute kidney injury 
 
  
RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS 
  
 
Cough 
 
 Cough (Gr 2) 
 
Dyspnea 
 
 Dyspnea (Gr 2) 
 
Oropharyngeal pain 
 
  
 
 
Pneumonitis 
  
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 (TAK-228) trials, but for which there is insufficient evidence to 
suggest that there was a reasonable possibility that MLN0128 (TAK-228) caused the adverse event: 
 
BLOOD AND LYMPHATIC SYSTEM DISORDERS - Blood and lymphatic system disorders - Other 
(hyperviscosity syndrome); Blood and lymphatic system disorders - Other (Raynaud's phenomenon); Febrile 
neutropenia 
CARDIAC DISORDERS - Heart failure; Pericardial effusion; Sinus tachycardia; Ventricular arrhythmia 
EYE DISORDERS - Blurred vision; Eye pain; Photophobia; Vision decreased 
GASTROINTESTINAL DISORDERS - Abdominal distension; Colitis; Dysphagia; Esophagitis; Gastritis; 
Gastroesophageal reflux disease; Gastrointestinal disorders - Other (intestinal obstruction); Gastrointestinal 
disorders - Other (intestinal perforation);  Gastrointestinal disorders - Other (salivary hypersecretion); 
Hemorrhoids; Ileus; Oral pain; Pancreatitis; Small intestinal obstruction; Small intestinal perforation; 
Toothache 
GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS - Chills; Flu like symptoms; Gait 
disturbance; General disorders and administration site conditions - Other (groin pain); Malaise; Non-cardiac 
chest pain; Pain 
HEPATOBILIARY DISORDERS - Gallbladder obstruction 
IMMUNE SYSTEM DISORDERS - Allergic reaction 
INFECTIONS AND INFESTATIONS - Abdominal infection; Infections and infestations - Other (cystitis); 
Infections and infestations - Other (lower respiratory tract infection); Infections and infestations - Other 
(mucosal infection); Infections and infestations - Other (parotid gland); Kidney infection; Lung infection; 
Papulopustular rash; Sepsis; Skin infection; Upper respiratory infection 
INJURY, POISONING AND PROCEDURAL COMPLICATIONS - Fall; Fracture; Injury, poisoning and 


 
53 
 
procedural complications - Other (accidental overdose); Injury, poisoning and procedural complications - 
Other (postoperative fever); Injury, poisoning and procedural complications - Other (subdural hemorrhage); 
Tracheal obstruction 
INVESTIGATIONS - Alanine aminotransferase increased; Alkaline phosphatase increased; Aspartate 
aminotransferase increased; Blood bilirubin increased; Blood lactate dehydrogenase increased; Cholesterol 
high; GGT increased; Lipase increased; Lymphocyte count decreased; Neutrophil count decreased; White 
blood cell decreased 
METABOLISM AND NUTRITION DISORDERS - Acidosis; Hypercalcemia; Hyperkalemia; Hypernatremia; 
Hypertriglyceridemia; Hyperuricemia; Hypoalbuminemia; Hypocalcemia; Hyponatremia; Metabolism and 
nutrition disorders - Other (severe chronic malnutrition); Metabolism and nutrition disorders - Other (vitamin 
D deficiency)  
MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS - Bone pain; Chest wall pain; Flank pain; 
Generalized muscle weakness; Muscle cramp; Myalgia 
NEOPLASMS BENIGN, MALIGNANT AND UNSPECIFIED (INCL CYSTS AND POLYPS) - Neoplasms 
benign, malignant and unspecified (incl cysts and polyps) - Other (non-hodgkin lymphoma); Treatment 
related secondary malignancy 
NERVOUS SYSTEM DISORDERS - Ataxia; Intracranial hemorrhage; Lethargy; Nervous system disorders - 
Other (carotid artery occlusion); Nervous system disorders - Other (neuropathy peripheral); Paresthesia; 
Radiculitis; Stroke; Tremor 
PSYCHIATRIC DISORDERS - Anxiety; Confusion; Depression; Psychiatric disorders - Other (mental status 
changes) 
RENAL AND URINARY DISORDERS - Dysuria; Hematuria; Proteinuria; Renal and urinary disorders - 
Other (strangury) 
REPRODUCTIVE SYSTEM AND BREAST DISORDERS - Vaginal hemorrhage 
RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS - Bronchopulmonary hemorrhage; 
Epistaxis; Hiccups; Hypoxia; Nasal congestion; Pleural effusion; Pleuritic pain; Pneumothorax; Postnasal 
drip; Productive cough 
SKIN AND SUBCUTANEOUS TISSUE DISORDERS - Alopecia; Dry skin; Hyperhidrosis; Rash acneiform; 
Urticaria 
VASCULAR DISORDERS - Flushing; Hypertension; Hypotension; Thromboembolic event 
 
 
Note: MLN0128 (TAK-228) 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 5.0 will be utilized for AE 
reporting beginning April 1, 2018 (investigators may continue to collect 
and locally store AE data in CTCAE v4.0).  All appropriate treatment 
areas should have access to a copy of the CTCAE version 5.0. A copy 
of the CTCAE version 5.0 can be downloaded from the CTEP web site  
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.ht
m. 
 
• For expedited reporting purposes only: 
 
- AEs for the agent that are bold and italicized in the CAEPR (i.e., those listed in the 


 
54 
 
SPEER column, Section 7.1.1) should be reported through CTEP-AERS only if the 
grade is above the grade provided in the SPEER. 
 
• 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 Distribution of Adverse Event Reports 
 
CTEP-AERS is programmed for automatic electronic distribution of reports to the 
following individuals:  Principal Investigator and Adverse Event Coordinator(s) (if 
applicable) of the Corresponding Organization or Lead Organization, the local treating 
physician, and the Reporter and Submitter. CTEP-AERS provides a copy feature for 
other e-mail recipients. 
 
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.  Attribution to treatment or other cause must be provided. 
 
Death due to progressive disease should be reported as Grade 5 “Disease 
Progression” in the system organ class (SOC) “General disorders and administration 
site conditions.” Evidence that the death was a manifestation of underlying disease 


 
55 
 
(e.g., radiological changes suggesting tumor growth or progression: clinical 
deterioration associated with a disease process) should be submitted. 
 
Phase 1 and Early Phase 2 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/Intervention 1, 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 electronic 
submission within the timeframes detailed in the table 
below. 
Hospitalization 
Grade 1 and Grade 2 Timeframes 
Grade 3-5 
Timeframes 
Resulting in 
Hospitalization 
≥ 24 hrs 
10 Calendar Days 
24-Hour 5 Calendar 
Days 
Not resulting in 
Hospitalization 
≥ 24 hrs 
Not required 
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 submitted electronically 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 electronically 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 3, 4, and Grade 5 AEs 
Expedited 10 calendar day reports for: 
• 
Grade 2 AEs resulting in hospitalization or prolongation of hospitalization 
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 


 
56 
 
 
7.4 
Routine Adverse Event Reporting 
 
All Adverse Events must be reported in routine study data submissions. AEs reported 
expeditiously through CTEP-AERS must also be reported in routine study data 
submissions. 
 
SAEs requiring submission to the HRPP Office must be reported within 5 calendar days of 
learning of the event. SAEs not requiring submission to the HRPP Office must be saved in the 
regulatory binder within 30 calendar days of learning of the event. The report should contain the 
following information: 
 
The following fields in this section will pre-populate based on the protocol information entered in 
PIMS.   
• IRB Number  
• Protocol Title  
• Protocol Status  
• Sponsoring Department  
• Principal Investigator  
• Protocol Type  
• Protocol Category  
• Sponsor 
 
Data needing to be entered: 
• The date the adverse event occurred 
• The adverse event 
• Relationship of the adverse event to the treatment (drug, device, or intervention) 
• If the AE was expected 
• The severity of the AE 
• The intervention 
• Detailed text that includes the following 
o 
An explanation of how the AE was handled 
o 
A description of the subject’s condition 
o 
Indication if the subject remains on the study 
o 
If an amendment will need to be made to the protocol and/or consent form. 
 
The PI’s signature and the date it was signed are required on the completed report. 
 
 
 
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 


 
57 
 
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 expeditiously 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 
 
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 AE 
reporting unless otherwise specified. 
 
 
 
8. PHARMACEUTICAL INFORMATION 
 
A list of the adverse events and potential risks associated with the investigational agent 
administered in this study can be found in Section 7.1. 
 
 
8.1 
MLN0128 (TAK-228) 
 
8.1.1 MLN0128 (TAK-228) Drug Information and Availability 
 
 
Other Names: TAK-228, INK128 
 
Classification: mTOR inhibitor, TORC1/2  
 
CAS Registry Number: 
1224844 
 
Molecular Formula: C15H15N7O 
 
M.W.:  
309.3 
 
Approximate Solubility: 
MLN0128 exhibits a pH-dependant 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 is a non-rapamycin analog mTOR (mechanistic target of 


 
58 
 
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.  
ML0128 is a potent and selective adenosine tri-phosphate (ATP)-competitive inhibitor of mTOR 
complex 1 and 2 (TORC1/2). 
 
Description:  
MLN0128 drug substance is a white to off-white, crystalline powder. 
 
How Supplied: 
MLN0128 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 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 capsules are packaged in 30-count, 60-cc high-density polyethylene (HDPE), white, 
opaque, round, tamper- and child-resistant bottles.   
 
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: MLN0128 (TAK-228) should be taken on an empty stomach at least 2 
hours after food and do not eat or drink (except water) for at least one hour after taking MLN0128 
(TAK-228).  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. When normalized for human liver content, the CYP isoforms CYP3A4, 
CYP2C9, and CYP2C19 appear to contribute to MLN0128 metabolism.  MLN0128 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. 
 
Men should use effective methods of contraception and not donate sperm during and through 120 days 
after the last dose of MLN0128. 
 
 
8.1.2 MLN0128 (TAK-228) Ordering and Accountability 
 
8.1.2.1 
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 


 
59 
 
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. 
 
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 (240) 276-6575 Monday through Friday between 8:30 am and 
4:30 pm (ET) or email PMBAfterHours@mail.nih.gov anytime. 
 
8.1.2.2 
Agent Inventory Records – The investigator, or a responsible party designated by the 
investigator, must maintain a careful record of the inventory and disposition of all 
agents received from DCTD using the NCI Oral Drug Accountability Record Form 
(DARF).  (See the NCI Investigator’s Handbook for Procedures for Drug 
Accountability and Storage.) 
 
 
 
 
9. BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES 
 
9.1 
Integral Laboratory or Imaging Studies 
 
9.1.1 NFE2L2, KEAP1 and KRAS mutation assessment 
 
A detailed explanation of the rationale for targeting NFE2L2 and KEAP1 mutations in squamous 
cell lung cancers and KRAS mutant lung cancers harboring NFE2L2 or KEAP1 alterations with 
MLN0128 (TAK-228) can be found in Section 2.0. Briefly, determination of mutation status 
will be performed through prospective New York State Department of Health approved targeted 
exon sequencing of patient tumor samples in a CLIA laboratory (IMPACT testing).  Results are 
posted in the patient EMR and discussed in detail by the ordering oncologist.  About 70 patients 
will need to be tested to identify 10 NFE2L2 mutations. About 140 will need to be tested to 
identify 10 KEAP1 mutations, given the restriction in the types of KEAP1 mutations that are 
eligible.  As of February 2014, 64 patients, predominantly with stage IV disease, have been 
tested in the past year. We have identified 15 cases with NFE2L2 mutations and 10 with 
KEAP1 mutations.  These numbers are in keeping with our anticipated accrual rates for the 


 
60 
 
protocol. 
 
IMPACT testing will be performed under MSKCC’s clinical consent process and NFE2L2, 
KEAP1, and KRAS mutation results obtained from the medical records. 
 
9.1.1.1 
Collection of Specimen(s) 
NA 
9.1.1.2 
Handling of Specimens(s) 
NA 
9.1.1.3 
Shipping of Specimen(s) 
N/A 
9.1.1.4 
Site(s) Performing Correlative Study 
Memorial Sloan Kettering Cancer Center 
9.2 
Exploratory/Ancillary Correlative Studies 
 
9.2.1 Pharmacodynamic effect of MLN0128 (TAK-228) through reverse phase 
proteomic analysis 
 
There are a number of practical limits associated with pharmacodynamic assessment of targeted 
therapies in cancers where the majority of tissue material comes from small needle core biopsies. 
These include tumor heterogeneity, small total protein yields, a common requirement to snap- 
freeze samples at -80C to preserve phosphoprotein stability, and the paucity of quantitative 
assays to assess signaling changes.  Immunohistochemistry, which is the most readily available 
proteomic test, is hampered by its qualitative nature. Western blotting, which can be quantitative 
depending on the specificity of the antibody used, has poor multiplex capabilities. 
 
The pre-clinical rationale for assessing dynamic changes in mTOR signaling is detailed in  Section 
2.3.  This correlative study incorporates reverse phase protein array (RPPA) analysis as an 
exploratory biomarker test to determine the pharmacodynamic effect of MLN0128 (TAK-228) in 
optional pre- and on-treatment tumor biopsies.  RPPA is a highly multiplex quantitative 
proteomic platform that can test for both phosphorylated and non-phosphorylated proteins.  Dr. 
Lauren Byers at MD Anderson will perform RPPA on these optional paired tumor specimens. A 
panel of 156 validated antibodies (217 total) is available for testing simultaneously, including key 
targets for this proposal (p4EBP1, pS6K, AKT pS463 and pT308).  The endpoint of this study is 
feasibility, as it is uncertain as to whether the paired small needle biopsies will yield sufficient 
material, after microdissection, for analysis. We will determine, in an exploratory fashion, what 
changes in phosphorylation in these specific downstream targets occurs in response to MLN0128 


 
61 
 
(TAK-228), including percent changes in specific signaling molecules (pS6K, p4EBP1, pAKT as 
examples) and a qualitative assessment of pathway changes through 
the generation of heatmaps. 
 
9.2.1.1 
Collection of Specimen(s) 
 
Patients will be asked, at the time of initial informed consent to the study, to participate in this 
optional correlative study which will obtain a biopsy of their tumor before drug dosing and 
during week 2 (C1D8 +/- 7 days). Patients will have their biopsies snap-frozen in liquid nitrogen 
and stored in a -80C freezer. A total of up to 4 18-20 gauge needle cores will be obtained, 
depending on the feasibility as assessed by the physician performing the biopsy.  For resection 
specimens obtained through surgery, a minimum of 0.5 mm3 of tissue will be obtained. 
 
9.2.1.2 
Handling of Specimens(s) 
 
Specimens will be obtained through interventional radiology or surgery. The biopsy will be 
picked up by the research study assistant and placed immediately in a cryotube and stored in 
liquid nitrogen. 
 
9.2.1.3 
Shipping of Specimen(s) 
 
Specimens will be shipped in dry ice shippers to Dr. Lauren Byers at MD Anderson Cancer 
Center at 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-6363, email:  
byersl@mdanderson.org. 
 
Samples will be scheduled to arrive Monday-Friday between 9AM-4PM.  Dr. Byer’s laboratory 
will be notified in advance to ensure staff is available for receipt. A sample list in Excel format 
will be included in the dry ice shipper which lists each sample contained in the box. No PHI will 
be included in the Excel file; only deidentified codes will be provided. Samples will be shipped 
in batches at the end of study. 
 
9.2.1.4 
Site(s) Performing Correlative Study 
MD Anderson Cancer Center. 


 
62 
 
10. STUDY CALENDAR 
 
Schedules shown in the Study Calendar below are provided as an example and should be 
modified as appropriate. 
 
Baseline evaluations are to be conducted within 2 weeks prior to start of protocol therapy. 
Informed consent is to be obtained within 45 days prior to start of protocol therapy. Scans and x-
rays must be done <4 weeks prior to the start of therapy.  Protocol visits, assessments, including 
radiology assessments must be completed within +/- 7 days. 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. 
 
Pre- 
Study 
Wk 
1 
Wk 
2 
Wk 
3 
Wk 
4 
Wk 
5 
Wk 
6 
Wk 
7 
Wk 
8 
Wk 
9 
Wk 
10 
Wk 
11 
Wk 
12+ 
Off Studyc 
MLN0128 (TAK-228) 
A 
A 
A 
Informed consent 
X 
Demographics 
X 
Medical history 
X 
Concurrent meds 
X 
X --------------------------------------------------------------------------------------------- X 
Physical exam 
X 
X 
X 
X 
X 
X 
Vital signs 
X 
X 
X 
X 
X 
X 
Height 
X 
Weight 
X 
X 
X 
X 
X 
Performance status 
X 
X 
X 
X 
X 
CBC w/diff, plts 
X 
X 
X 
X 
X 
X 
Chemistrya 
X 
X 
X 
X 
X 
X 
EKG 
X 
X 
 
        
Adverse event evaluation 
X --------------------------------------------------------------------------------------------- X 
X 
Tumor measurements 
X 
Tumor measurements are repeated every 8 weeks.  Documentation (radiologic) must 
be provided for patients removed from study for progressive disease. 
X 
Radiologic evaluation 
X 
Radiologic measurements should be performed every 8 weeks. 
X 
B-HCG 
Xb 
Optional tumor biopsy 
X 
X 
A: 
MLN0128 (TAK-228): Dose as assigned 
a: 
Albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, chloride, creatinine, fasting glucose, LDH, phosphorus, 
potassium, total protein, SGOT [AST], SGPT [ALT], sodium, fasting lipid panel, amylase, lipase 
b: 
Serum pregnancy test (women of childbearing potential). 
c: 
Off-study evaluation. 
d: 
EKGs will be done at screening, pre-dose at week 5 and as needed  after week 5 


 
63 
 
11. MEASUREMENT OF EFFECT 
 
11.1 
Antitumor Effect – Solid Tumors 
 
For the purposes of this study, patients should be re-evaluated for response every 8 weeks.  In 
addition to a baseline scan, confirmatory scans should also be obtained 4 (not less than 4) weeks 
following initial documentation of objective response.   
 
Response and progression will be evaluated in this study using the new international criteria 
proposed by the revised Response Evaluation Criteria in Solid Tumors (RECIST) guideline 
(version 1.1) [Eur J Ca 45:228-247, 2009].  Changes in the largest diameter (unidimensional 
measurement) of the tumor lesions and the shortest diameter in the case of malignant lymph 
nodes are used in the RECIST criteria. 
 
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.  Only those patients who have measurable disease 
present at baseline, have received at least one cycle of therapy, 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.) 
 
Evaluable Non-Target Disease Response.  Patients who have lesions present at baseline 
that are evaluable but do not meet the definitions of measurable disease, have received at 
least one cycle of therapy, and have had their disease re-evaluated will be considered 
evaluable for non-target disease.  The response assessment is based on the presence, 
absence, or unequivocal progression of the lesions. 
 
11.1.2 Disease Parameters 
 
Measurable disease.  Measurable lesions are defined as those that can be accurately 
measured in at least one dimension (longest diameter to be recorded) as ≥20 mm (≥2 cm) 
by chest x-ray or as ≥10 mm (≥1 cm) with CT scan, MRI, or calipers by clinical exam. 
All tumor measurements must be recorded in millimeters (or decimal fractions of 
centimeters). 
 
Note:  Tumor lesions that are situated in a previously irradiated area might or might not 
be considered measurable. 
 
Malignant lymph nodes.  To be considered pathologically enlarged and measurable, a 
lymph node must be ≥15 mm (≥1.5 cm) in short axis when assessed by CT scan (CT scan 


 
64 
 
slice thickness recommended to be no greater than 5 mm [0.5 cm]). At baseline and in 
follow-up, only the short axis will be measured and followed. 
 
Non-measurable disease.  All other lesions (or sites of disease), including small lesions 
(longest diameter <10 mm [<1 cm] or pathological lymph nodes with ≥10 to <15 mm [≥1 
to <1.5 cm] short axis), are considered non-measurable disease. Bone lesions, 
leptomeningeal disease, ascites, pleural/pericardial effusions, lymphangitis 
cutis/pulmonitis, inflammatory breast disease, and abdominal masses (not followed by 
CT or MRI), are considered as non-measurable. 
 
Note:  Cystic lesions that meet the criteria for radiographically defined simple cysts 
should not be considered as malignant lesions (neither measurable nor non-measurable) 
since they are, by definition, simple cysts. 
 
‘Cystic lesions’ thought to represent cystic metastases can be considered as measurable 
lesions, if they meet the definition of measurability described above. However, if non- 
cystic lesions are present in the same patient, these are preferred for selection as target 
lesions. 
 
Target lesions. All measurable lesions up to a maximum of 2 lesions per organ and 5 
lesions in total, representative of all involved organs, should be identified as target 
lesions and recorded and measured at baseline. Target lesions should be selected on the 
basis of their size (lesions with the longest diameter), be representative of all involved 
organs, but in addition should be those that lend themselves to reproducible repeated 
measurements.  It may be the case that, on occasion, the largest lesion does not lend itself 
to reproducible measurement in which circumstance the next largest lesion which can be 
measured reproducibly should be selected. A sum of the diameters (longest for non- 
nodal lesions, short axis for nodal lesions) for all target lesions will be calculated and 
reported as the baseline sum diameters.  If lymph nodes are to be included in the sum, 
then only the short axis is added into the sum. The baseline sum diameters will be used 
as reference to further characterize any objective tumor regression in the measurable 
dimension of the disease. 
 
Non-target lesions.  All other lesions (or sites of disease) including any measurable 
lesions over and above the 5 target lesions should be identified as non-target lesions and 
should also be recorded at baseline.  Measurements of these lesions are not required, but 
the presence, absence, or in rare cases unequivocal progression of each should be noted 
throughout follow-up. 
 
11.1.3 Methods for Evaluation of Measurable Disease 
 
All measurements should be taken and recorded in metric notation using a ruler or 
calipers.  All baseline evaluations should be performed as closely as possible to the 
beginning of treatment and never more than 4 weeks before the beginning of the 
treatment. 


 
65 
 
 
The same method of assessment and the same technique should be used to characterize 
each identified and reported lesion at baseline and during follow-up. Imaging-based 
evaluation is preferred to evaluation by clinical examination unless the lesion(s) being 
followed cannot be imaged but are assessable by clinical exam. 
 
Clinical lesions  Clinical lesions will only be considered measurable when they are 
superficial (e.g., skin nodules and palpable lymph nodes) and ≥10 mm (≥1 cm) diameter 
as assessed using calipers (e.g., skin nodules).  In the case of skin lesions, documentation 
by color photography, including a ruler to estimate the size of the lesion, is 
recommended. 
 
Chest x-ray Lesions on chest x-ray are acceptable as measurable lesions when they are 
clearly defined and surrounded by aerated lung.  However, CT is preferable. 
 
Conventional CT and MRI This guideline has defined measurability of lesions on CT 
scan based on the assumption that CT slice thickness is 5 mm (0.5 cm) or less.  If CT 
scans have slice thickness greater than 5 mm (0.5 cm), the minimum size for a 
measurable lesion should be twice the slice thickness. MRI is also acceptable in certain 
situations (e.g. for body scans). 
 
Use of MRI remains a complex issue. MRI has excellent contrast, spatial, and temporal 
resolution; however, there are many image acquisition variables involved in MRI, which 
greatly impact image quality, lesion conspicuity, and measurement.  Furthermore, the 
availability of MRI is variable globally. As with CT, if an MRI is performed, the 
technical specifications of the scanning sequences used should be optimized for the 
evaluation of the type and site of disease.  Furthermore, as with CT, the modality used at 
follow-up should be the same as was used at baseline and the lesions should be 
measured/assessed on the same pulse sequence.  It is beyond the scope of the RECIST 
guidelines to prescribe specific MRI pulse sequence parameters for all scanners, body 
parts, and diseases.  Ideally, the same type of scanner should be used and the image 
acquisition protocol should be followed as closely as possible to prior scans. Body scans 
should be performed with breath-hold scanning techniques, if possible. 
 
PET-CT At present, the low dose or attenuation correction CT portion of a combined 
PET-CT is not always of optimal diagnostic CT quality for use with RECIST 
measurements.  However, if the site can document that the CT performed as part of a 
PET-CT is of identical diagnostic quality to a diagnostic CT (with IV and oral contrast), 
then the CT portion of the PET-CT can be used for RECIST measurements and can be 
used interchangeably with conventional CT in accurately measuring cancer lesions over 
time.  Note, however, that the PET portion of the CT introduces additional data which 
may bias an investigator if it is not routinely or serially performed. 
 
Ultrasound  Ultrasound is not useful in assessment of lesion size and should not be used 
as a method of measurement.  Ultrasound examinations cannot be reproduced in their 


 
66 
 
entirety for independent review at a later date and, because they are operator dependent, 
it cannot be guaranteed that the same technique and measurements will be taken from 
one assessment to the next.  If new lesions are identified by ultrasound in the course of 
the study, confirmation by CT or MRI is advised.  If there is concern about radiation 
exposure at CT, MRI may be used instead of CT in selected instances. 
 
Endoscopy, Laparoscopy The utilization of these techniques for objective tumor 
evaluation is not advised.  However, such techniques may be useful to confirm complete 
pathological response when biopsies are obtained or to determine relapse in trials where 
recurrence following complete response (CR) or surgical resection is an endpoint. 
 
Tumor markers  Tumor markers alone cannot be used to assess response.  If markers are 
initially above the upper normal limit, they must normalize for a patient to be considered 
in complete clinical response.  Specific guidelines for both CA-125 response (in recurrent 
ovarian cancer) and PSA response (in recurrent prostate cancer) have been published 
[JNCI 96:487-488, 2004; J Clin Oncol 17, 3461-3467, 1999; J Clin Oncol 26:1148-1159, 
2008].  In addition, the Gynecologic Cancer Intergroup has developed CA-125 
progression criteria which are to be integrated with objective tumor assessment for use in 
first-line trials in ovarian cancer [JNCI 92:1534-1535, 2000]. 
 
Cytology, Histology These techniques can be used to differentiate between partial 
responses (PR) and complete responses (CR) in rare cases (e.g., residual lesions in tumor 
types, such as germ cell tumors, where known residual benign tumors can remain). 
 
The cytological confirmation of the neoplastic origin of any effusion that appears or 
worsens during treatment when the measurable tumor has met criteria for response or 
stable disease is mandatory to differentiate between response or stable disease (an 
effusion may be a side effect of the treatment) and progressive disease. 
 
 
11.1.4 Response Criteria 
 
11.1.4.1 
Evaluation of Target Lesions 
 
Complete Response (CR):  Disappearance of all target lesions. Any pathological 
lymph nodes (whether target or non-target) must have reduction in short axis to <10 
mm (<1 cm). 
 
Partial Response (PR):  At least a 30% decrease in the sum of the diameters of target 
lesions, taking as reference the baseline sum diameters. 
 
Progressive Disease (PD):  At least a 20% increase in the sum of the diameters of 
target lesions, taking as reference the smallest sum on study (this includes the 
baseline sum if that is the smallest on study).  In addition to the relative increase of 
20%, the sum must also demonstrate an absolute increase of at least 5 mm (0.5 cm). 


 
67 
 
(Note: the appearance of one or more new lesions is also considered progressions). 
 
Stable Disease (SD):  Neither sufficient shrinkage to qualify for PR nor sufficient 
increase to qualify for PD, taking as reference the smallest sum diameters while on 
study. 
 
11.1.4.2 
Evaluation of Non-Target Lesions 
 
Complete Response (CR):  Disappearance of all non-target lesions and normalization 
of tumor marker level.  All lymph nodes must be non-pathological in size (<10 mm 
[<1 cm] short axis). 
 
Note:  If tumor markers are initially above the upper normal limit, they must 
normalize for a patient to be considered in complete clinical response. 
 
Non-CR/Non-PD:  Persistence of one or more non-target lesion(s) and/or 
maintenance of tumor marker level above the normal limits. 
 
Progressive Disease (PD): Appearance of one or more new lesions and/or 
unequivocal progression of existing non-target lesions. Unequivocal progression 
should not normally trump target lesion status.  It must be representative of overall 
disease status change, not a single lesion increase. 
 
Although a clear progression of “non-target” lesions only is exceptional, the opinion 
of the treating physician should prevail in such circumstances, and the progression 
status should be confirmed at a later time by the review panel (or Principal 
Investigator). 
 
11.1.4.3 
Evaluation of Best Overall Response 
 
The best overall response is the best response recorded from the start of the treatment 
until disease progression/recurrence (taking as reference for progressive disease the 
smallest measurements recorded since the treatment started). The patient's best 
response assignment will depend on the achievement of both measurement and 
confirmation criteria. 
 
For Patients with Measurable Disease (i.e., Target Disease) 
 
Target 
Lesions 
Non-Target 
Lesions 
New 
Lesions 
Overall 
Response 
Best Overall Response when 
Confirmation is Required* 
CR 
CR 
No 
CR 
>4 wks. Confirmation** 
CR 
Non-CR/Non- 
PD 
No 
PR 
CR 
Not evaluated 
No 
PR 


 
68 
 
PR 
Non-CR/Non- 
PD/not 
evaluated 
No 
PR 
>4 wks. Confirmation** 
SD 
Non-CR/Non- 
PD/not 
evaluated 
No 
SD 
Documented at least once >4 
wks. from baseline** 
PD 
Any 
Yes or No 
PD 
no prior SD, PR or CR 
Any 
PD*** 
Yes or No 
PD 
Any 
Any 
Yes 
PD 
 
See RECIST 1.1 manuscript for further details on what is evidence of a new lesion. 
** Only for non-randomized trials with response as primary endpoint. 
***  In exceptional circumstances, unequivocal progression in non-target lesions may be 
accepted as disease progression. 
Note:  Patients with a global deterioration of health status requiring discontinuation of 
treatment without objective evidence of disease progression at that time should be 
reported as “symptomatic deterioration.”  Every effort should be made to document the 
objective progression even after discontinuation of treatment. 
 
For Patients with Non-Measurable Disease (i.e., Non-Target Disease) 
 
Non-Target Lesions 
New Lesions 
Overall Response 
CR 
No 
CR 
Non-CR/non-PD 
No 
Non-CR/non-PD* 
Not all evaluated 
No 
not evaluated 
Unequivocal PD 
Yes or No 
PD 
Any 
Yes 
PD 
 
‘Non-CR/non-PD’ is preferred over ‘stable disease’ for non-target disease since SD is 
increasingly used as an endpoint for assessment of efficacy in some trials so to assign this 
category when no lesions can be measured is not advised 
 
11.1.5 Duration of Response 
 
Duration of overall response:  The duration of overall response is measured from the time 
measurement criteria are met for CR or PR (whichever is first recorded) until the first 
date that recurrent or progressive disease is objectively documented (taking as reference 
for progressive disease the smallest measurements recorded since the treatment started). 
 
The duration of overall CR is measured from the time measurement criteria are first met 
for CR until the first date that progressive disease is objectively documented. 
 
Duration of stable disease:  Stable disease is measured from the start of the treatment 
until the criteria for progression are met, taking as reference the smallest measurements 
recorded since the treatment started, including the baseline measurements. 
 


 
69 
 
11.1.6 Progression-Free Survival 
 
PFS is defined as the duration of time from start of treatment to time of progression or 
death, whichever occurs first. 
 
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 
Data Reporting 
 
12.1.1 Method 
 
This study will be monitored by the Clinical Data Update System (CDUS) Version 3.0. 
Cumulative protocol- and patient-specific CDUS data will be submitted electronically to 
CTEP on a quarterly basis, either by FTP burst of data or via the CDS web application. 
Reports are due January 31, April 30, July 31, and October 31.  Instructions for 
submitting data using the CDUS can be found on the CTEP Web site 
(http://ctep.cancer.gov/reporting/cdus.html). 
 
Note:  If your study has been assigned to CDUS-Complete reporting, all adverse events 
(both routine and expedited) that have occurred on the study and meet the mandatory 
CDUS reporting guidelines must be reported via the monitoring method identified above. 
If your study has been assigned to CDUS-Abbreviated reporting, no adverse event 
reporting (routine or expedited) is required to be reported via CDUS. 
 
12.1.2 Responsibility for Data Submission 
 
N/A 
 
12.2 
CTEP Multicenter Guidelines 
 
N/A 
 
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 


 
70 
 
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. 
 
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  


 
71 
 
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. 
 
13. STATISTICAL CONSIDERATIONS 
 
13.1 
Study Design/Endpoints 
 
Patient accrual is nearly complete for the three study cohorts: 1) squamous cell lung cancer with 
NFE2L2 mutations; 2) squamous cell lung cancer with KEAP1 mutations; 3) KRAS mutant lung 
cancer with concomitant NFE2L2 or KEAP1 aberration.  The primary endpoint is objective 
response and the observed response rates in the three cohorts are: (1) 2/9, (2) 0/5, and (3) 0/5.  
We propose to amend the protocol to accrue an additional 10 patients for cohorts 1 and 2 to 
obtain preliminary information on progression free survival and greater precision for the 
observed response rate. Although no responses were observed in the first 5 patients in cohort 2, 
the genomic heterogeneity of KEAP1 mutations in squamous cell lung cancer would indicate 
that further exploration is needed. Cohort 3 will be closed to accrual. The original study design  
is provided below. 
 
This study utilizes a Simon two-stage design applied to each of the mutation cohorts.  The 
primary endpoint of the study is objective response rate. The null and desired response rates are 
5% and 40%.  A desired response rate of 40% was selected based on past successful targeted 
therapy trials in lung cancer. Response rates in these biomarker-led studies are universally equal 
to or greater than first-line chemotherapy response rates of between 30-40%. In stage 1 of each 
mutation cohort, 5 patients will enter the study; if there are no responses, the study will be 
terminated early.  If 1 or more patients respond, enrollment will be extended to 10 patients in 
total.  At the end of stage 2, if 2 or more patients responded in either cohort, the trial will be 
declared effective and worthy of further testing for the respective cohort. This two-stage design 
yields 90% power to detect the difference at a 10% type I error rate. 
 
Overall response rate (CR+PR) will be calculated separately for each cohort, including exact 


 
72 
 
95% confidence intervals. Duration of overall response and duration of stable disease as 
described in Section 11.1.5 will be calculated and summarized. 
 
Secondary endpoints include PFS and the RPPA correlative biomarker analysis. PFS is defined 
from the start of treatment until progression or death, whichever occurs first. Patients will be 
censored at the time of the last on-study evaluation if they do not experience the event of interest. 
Median progression-free survival will be estimated in each cohort from start of treatment using 
the Kaplan-Meier method with a two-sided 95% confidence interval. 
The endpoint of the 
RPPA analysis is feasibility, defined as the ability to procure sufficient quantity and quality of 
tumor protein for testing.  The proportion of patients with sufficient quantity and quality will be 
calculated, including an exact 95% confidence interval. Single target signaling changes (decrease 
in pS6K, p4EBP-1 as examples) will be reported as percentages relative to the baseline pre- 
treatment tumor sample.  Larger scale pathway changes will qualitatively represented through 
heatmaps. 
 
13.2 
Sample Size/Accrual Rate 
 
Between 5-10 patients were initially  accrued in each mutation cohort arm for a total of 20 
patients. An additional 10 patients in cohorts 1 and 2 will be added for the extension phase of 
this study. Accrual rate is estimated at 1 patient in each arm every 2 months. 
 
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 
3 
3 
0 
0 
6 
Native Hawaiian or 
Other Pacific Islander 
0 
0 
0 
0 
0 
Black or 
African 
American
3 
3 
0 
0 
6 
White 
12 
12 
2 
2 
28 
More Than One Race 
0 
0 
0 
0 
0 
Total 
18 
18 
2 
2 
40 
PHS 398 / PHS 2590 (Rev. 08/12 Approved Through 8/31/2015) 
OMB No. 0925-0001/0002 


 
73 
 
13.3 
Reporting and Exclusions 
 
13.3.1 Evaluation of Toxicity 
 
All patients will be evaluable for toxicity from the time of their first treatment with 
MLN0128 (TAK-228). 
 
13.3.2 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. Subanalyses 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 
subanalyses 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. 
 
14. 
PROTECTION OF HUMAN SUBJECTS 
 
Prior to enrollment of each patient, the risks, benefits, and objectives of the study will be 
reviewed with the participant, including a discussion of the possible toxicities, side effects, and 
alternative, non-protocol treatment options. The patients will be reminded that participation in 
this clinical trial is voluntary and the patient may withdraw consent at any time. 
Risks: The standard second-line chemotherapy for patients with metastatic/recurrent squamous 
cell lung cancers is docetaxel. There is a chance that this regimen is less effective than docetaxel. 
Treatment with MLN0128 (TAK-228) will not absolutely preclude later treatment with 
docetaxel however. 
Benefits: It is hoped that this regimen will prove to be more effective than docetaxel historically. 
If so, this would add an additional treatment option for patients with stage IV/recurrent squamous 


 
74 
 
cell lung cancers. 
Consent process: Participation in this trial is voluntary. All patients will be required to sign a 
statement of informed consent which must conform to MSK IRB guidelines. 
Costs: Patients will be charged for physician visits, routine laboratory and radiologic studies 
required for monitoring their condition. Patients will not be billed for the study drug or for the 
optional biopsies, or for testing performed on the optional biopsy specimens. 
Alternatives: Alternative treatment options include standard chemotherapy, best supportive care, 
or participation in other investigational studies. 
 
Confidentiality: Every effort will be made to maintain patient confidentiality. Research and 
hospital records are confidential. Patient's names or any other personally identifying information 
will not be used in reports or publications resulting from this study. Qualified monitors from 
MSK, the NCI, and the FDA may review patient records as required. 
Patient safety: Patients are monitored by physicians and oncology nurses who are very familiar 
with clinical trials. In the case of an adverse reaction, immediate medical attention is available. 
In the evenings and weekends, we have a 24 hour urgent care facility for outpatients. The PI or 
co-PIs will also be available at all times to organize any necessary intervention. 
Monitoring of data to ensure safety: this study is to be monitored by institutional IRB. This 
incorporates an independent data and safety monitoring committee established by arrangement 
with the National Cancer Institute. The analysis of safety will include all patients. Adverse 
events, including all toxic effects of treatment, will be tabulated individually and summarized by 
severity and causality. 
 
 
14.1 
Privacy 
 
It is the responsibility of the Research Staff to ensure that protocol subjects received the Center’s 
Notice of Privacy Practices. If the subject has not received one, MSK personnel must provide a 
Notice of Privacy Practices and obtain acknowledgment before the subject participates in the 
study. 
MSK’s Privacy Office may allow the use and disclosure of protected health information pursuant 
to a completed and signed Research Authorization form. The use and disclosure of protected 
health information will be limited to the individuals described in the Research Authorization 
form. A Research Authorization form must be completed by the Principal Investigator and 
approved by the IRB and Privacy Board (IRB/PB). 
 
The consent indicates that individualized de identified information collected for the purposes of 
this study may be shared with other qualified researchers. Only researchers who have received 
approval from MSK will be allowed to access this information which will not include protected 
health information, such as the participant’s name, except for dates. It is also stated in the 
Research Authorization that their research data may be shared with other qualified researchers. 
 


 
75 
 
REFERENCES 
 
1. TCGA Network: Comprehensive genomic characterization of squamous cell lung cancers. 
Nature 489:519-25, 2012 
2. Paik P, Berger M, Hasanovic A, et al: Multiplex testing for driver mutations in squamous cell 
lung cancers. J Clin Oncol:abstr 7505, 2012 
3. Singh A, Misra V, Thimmulappa RK, et al: Dysfunctional KEAP1–NRF2 Interaction in Non- 
Small-Cell Lung Cancer. PLoS Med 3:e420, 2006 
4. Shibata T, Saito S, Kokubu A, et al: Global Downstream Pathway Analysis Reveals a 
Dependence of Oncogenic NF-E2–Related Factor 2 Mutation on the mTOR Growth Signaling 
Pathway. Cancer Research 70:9095-9105, 2010 
5. Romero R, Sayin V et al.: Keap1 loss promoted Kras-drive lung cancer and results in 
dependence on glutaminolysis. Nature Med 23:1362-1368, 2017. 
6. Bendavit G, Aboulkassim T, Hilmi K, Shah S, Batist G: Nrf2 transcription factor can directly 
regulate mTOR: linking cytoprotective gene expression to a major metabolic regulator that 
generates redox activity. J Biol Chm 291(49):25476-25488, 2016. 


 
76 
 
APPENDIX A 
PERFORMANCE STATUS CRITERIA 
 
ECOG Performance Status Scale 
Karnofsky Performance Scale 
Grade 
Descriptions 
Percent 
Description 
0 
Normal activity.  Fully active, able 
to carry on all pre-disease 
performance without restriction. 
100 
Normal, no complaints, no evidence 
of disease. 
90 
Able to carry on normal activity; 
minor signs or symptoms of disease. 
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). 
80 
Normal activity with effort; some 
signs or symptoms of disease. 
70 
Cares for self, unable to carry on 
normal activity or to do active 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. 
60 
Requires occasional assistance, but 
is able to care for most of his/her 
needs. 
50 
Requires considerable assistance and 
frequent medical care. 
3 
In bed >50% of the time.  Capable 
of only limited self-care, confined 
to bed or chair more than 50% of 
waking hours. 
40 
Disabled, requires special care and 
assistance. 
30 
Severely disabled, hospitalization 
indicated.  Death not imminent. 
4 
100% bedridden. Completely 
disabled.  Cannot carry on any 
self-care.  Totally confined to bed 
or chair. 
20 
Very sick, hospitalization indicated. 
Death not imminent. 
10 
Moribund, fatal processes 
progressing rapidly. 
5 
Dead. 
0 
Dead. 


 
77 
 
APPENDIX B 
ELIGIBLE NFE2L2, KEAP1 AND KRAS MUTATIONS 
 
The below Appendix B provides support for the inclusion of the NFE2L2, KEAP1 and KRAS 
mutations in this study. 
 
 
Functional NFE2L2 and KEAP1 domains 
 
Nrf2, the transcription factor encoded by NFE2L2, is regulated primarily by 
degradation through a Keap1-dependent ubiquitin-proteasome pathway.  Analysis of the Nrf2 
protein more than a decade ago identified 6 highly conserved Neh (Nrf2-ECH homology) 
domains thought to be functionally important (Figure 1). Of these 6 domains, the N-terminal 
Neh2 domain (aa.1-98) was identified as the canonical Keap1 binding site.(1) Subsequent 
work showed that within the Neh2 domain, two evolutionarily conserved motifs were 
present- the DLG motif (aa.17-36) residing in the N-terminal region and the ETGE motif 
(aa.76-82) residing in the C-terminal portion of the domain. Seven lysine residues N- 
terminal of the ETGE motif at aa.44, 50, 52, 53, 56, 64, and 68 have also been shown to be 
necessary for Keap1-dependent ubiquitination and degradation of Nrf2.(2)  The importance 
of the DLG and ETGE motifs is presented in the next section. 
 
Keap1 contains a number of conserved domains the most important of which are 
Bric-a-Brac (BTB, aa.61-179), the intervening region (IVR, aa.179-315), a double glycine 
repeat or Kelch repeat (DGR, aa.315-598), and the C-terminal region (CTR, aa.598-624) 
(Figure 1).  The BTB domain is thought to assist in dimerization of Keap1.  The C-terminal 
DGR-CTR domain (aa.315-624) forms a six-bladed β-propeller structure and is the key Nrf2 
binding domain.  What follows is a summary of the functional data available for each 
domain. 
 
Keap1 DGR-CTR domain and the Nrf2 Neh2 domain are the critical Keap1/Nrf2 binding sites 
 
The physical interaction between the DGR-CTR domain in Keap1 and the Neh2 
domain in Nrf2 is essential in suppressing Nrf2 activity. Padmanabhan et al. performed a 
structural analysis of the DGR-CTR domains of mouse Keap1 and the DGR-CTR complex 
with Nrf2.(3)  Functionally, mutations in the CTR domain were found to impair Keap1- 
mediated repression of Nrf2 transcriptional activity.  Mutations in this region also altered 
localization of Nrf2, leading to increased nuclear accumulation consistent with loss of 
function of Keap1. 
 
A separate structural analysis of the DGR-CTR domain from Keap1 complexed with the 
Neh2 domain from Nrf2 confirmed that the binding domains within Neh2 resided in the 
DLG and ETGE motifs.(4) First, a probe of the binding stoichiometry, binding constant, 
enthalpy change, and entropy change between Neh2 and DGR-CTR showed a biphasic 
isotherm curve consistent with a two-site binding model.  Padmanabhan et al. demonstrated 
that a β hairpin comprised of aa.77-82 (corresponding to the C-terminal ETGE motif) is the 
high-affinity binding site for Keap1, and that it binds to the bottom of the β propeller formed 


 
78 
 
by DGR-CTR (3).   Within Keap1, Arg-380, Arg-415, and Arg-483 appear to bind 
specifically to the two acidic glutamates in the ETGE motif.(4) Consistent with this, 
mutations in R415 substituting lysine for arginine and R483 substituting glycine for arginine 
decreased Neh2 binding affinity by an order of magnitude(3). Other amino acids in the 
DGR-CTR domain cavity exposed to the ETGE β hairpin include Tyr-525 and Gln-530, Asn- 
382, Tyr-572, Phe-577, Tyr-334, Ser-363, Ser-602, and Ser-555. 
 
Tong et al. also found that a weaker binding site is present between aa.1-30 which 
contains the DLG motif.  A peptide containing the DLG motif spanning aa.17-36 elicited a 
spectral change in the 3 arginine residues in the DGR-CTR domain of Keap1, similar to what 
was observed with the ETGE peptide motif. This region also appears to assist in positioning 
the intervening lysine residues for efficient ubiquitination(5). 
 
In terms of KEAP1 mutations found in lung cancer, Padmanabhan identified a G364C 
and G430C mutation in a lung cancer patient sample and cell line, respectively.  Both of 
these mutant proteins impaired the ability of Keap1 to repress Nrf2 activity, co- 
immunoprecipitate with Nrf2, and sequester Nr2 in the cytoplasm relative to WT Keap1. 
Modeling these mutant forms of Keap1 within the murine DRG-CTR/Nrf2 structural model 
showed that G364C abolishes Keap1-Nrf2 interaction by altering the conformation of Ser- 
363.  The hydrophobic nature of cysteine was theorized to be unable to fit within the charged 
vicinity formed by the key arginine and serine residues in the DGR-CTR cavity. Binding 
was also abolished with the G430C mutation, presumably by a conformational change that 
prevents interaction with Glu-79 on Nrf2. 
 
 
 
Figure 1.  Key functional domains for Keap1 and Nrf2. The N-terminal Neh2 domain is the 
binding site for the negative regulator Keap1. This interaction occurs through the Keap1 C- 
terminal DGR-CTR domain and both N-terminal (DLG-motif), C-terminal (ETGE motif), 
and intervening domain (lysine residues, in bold).(4) 
 
 


 
79 
 
Keap1 BTB and IVR domains 
 
The Keap1 BTB domain spanning aa.61-179 is required for homodimerization of Keap1 
and for mediating the interaction between Keap1 and Cul3 (forming the E3-ubiquitin ligase 
complex that degrades Nrf2).(6)  Homodimerization appears to be necessary for Keap1 to 
repress Nrf2 function and to promote Nrf2 ubiquitination.(7) Substitution of the HKVVL 
sequence (aa.96-100) in the BTB domain with 5 alanine residues abrogated dimerization and 
did not rescue mice from juvenile mortality when crossed with a Keap1-/- background, 
suggesting that BTB mutations have a dominant negative effect. A separate group found 
similar results, with a dominant negative BTB construct inhibiting the ability of Keap1 to 
retain Nrf2 in the cytoplasm.(8) 
 
Lastly, the IVR domain contains 2 cysteine residues and the BTB domain 1 cysteine 
residue that are critical for Keap1 function, presumably as electrophile sensors (C151, C273, 
C288).  Mutant forms of these residues diminish Keap1 repression of Nrf2 reporter activity 
in a dominant negative fashion but do not interfere with Keap1 dimerization. 
 
Functionally validated NFE2L2 mutations 
 
The following table lists functionally validated amino acids/domains in Nrf2 based on 
review of the above literature.  Mutations in these amino acids should lead to an increase in 
Nrf2 activity (Table 1). 
 
Table 1- Functionally important Nrf2 amino acids and mutations 
NFE2L2 mutations 
Domain 
Function 
Validation 
aa.17-36 
DLG motif 
Keap1 binding site 
in silico and in vitro 
aa.76-82 
ETGE motif 
Keap1 binding site 
in silico and in vitro 
K44, K50, K52, K53, K56, K64, 
K68 
Lysine ubiquitination targets 
Nrf2 degradation 
in silico and in vitro 
Exon 2 deletion 
DLG/ETGE motifs 
Keap1 binding site 
in vitro 
 
Interestingly, a review of the TCGA squamous cell lung cancer (SQCLC) dataset and 
our own sequencing data show that virtually all mutations in NFE2L2 occur in these 
validated regions as shown in Table 2. 
 
Table 2- NFE2L2 mutations in SQCLCs (TCGA and MSK internal data) 
ID 
NFE2L2 mutation 
Functional domain 
Validated? 
SQ-MAP 
Q51H 
alpha helix 
No 
TCGA-34-2608 
D27H 
DLG motif 
Yes 
TCGA-34-2596 
D29G 
DLG motif 
Yes 
TCGA-46-3768 
D29H 
DLG motif 
Yes 
TCGA-34-5240 
D29H 
DLG motif 
Yes 
TCGA-66-2783 
D29N 
DLG motif 
Yes 


 
80 
 
TCGA-33-4586 
D29Y 
DLG motif 
Yes 
TCGA-22-5482 
F37del 
DLG motif 
Yes 
TCGA-18-3415 
G31A 
DLG motif 
Yes 
TCGA-66-2766 
G31A 
DLG motif 
Yes 
TCGA-51-4081 
L30F 
DLG motif 
Yes 
TCGA-46-3765 
L30F 
DLG motif 
Yes 
TCGA-37-4135 
Q26L 
DLG motif 
Yes 
TCGA-22-5473 
Q26P 
DLG motif 
Yes 
TCGA-18-3411 
R34G 
DLG motif 
Yes 
TCGA-34-5236 
R34P 
DLG motif 
Yes 
TCGA-18-4721 
R34Q 
DLG motif 
Yes 
TCGA-22-5477 
R34Q 
DLG motif 
Yes 
TCGA-66-2795 
R34Q 
DLG motif 
Yes 
TCGA-21-1071 
T80K 
DLG motif 
Yes 
TCGA-34-2608 
W24C 
DLG motif 
Yes 
SQ-MAP 
R34P 
DLG motif 
Yes 
SQ-MAP 
G31A 
DLG motif 
Yes 
SQ-MAP 
G10R 
DLG motif 
No 
SQ-MAP 
G31R 
DLG motif 
Yes 
SQ-MAP 
W24S 
DLG motif 
Yes 
SQ-MAP 
P30_31 insIDL 
DLG motif 
Yes 
SQ-MAP 
L23del 
DLG motif 
Yes 
SQ-MAP 
R34G 
DLG motif 
Yes 
SQ-MAP 
D29H 
DLG motif 
Yes 
SQ-MAP 
D27G 
DLG motif 
Yes 
SQ-MAP 
R34L 
DLG motif 
Yes 
TCGA-18-5592 
D77G 
ETGE motif 
Yes 
TCGA-56-5897 
E79Q 
ETGE motif 
Yes 
TCGA-66-2770 
E79Q 
ETGE motif 
Yes 
TCGA-22-5474 
E79Q 
ETGE motif 
Yes 
TCGA-39-5022 
E79Q 
ETGE motif 
Yes 
TCGA-60-2709 
G81S 
ETGE motif 
Yes 
TCGA-66-2787 
G81S 
ETGE motif 
Yes 
TCGA-51-4079 
G81V 
ETGE motif 
Yes 
SQ-MAP 
E79Q 
ETGE motif 
Yes 
SQ-MAP 
E79K 
ETGE motif 
Yes 
SQ-MAP 
E79Q 
ETGE motif 
Yes 
SQ-MAP 
E79Q 
ETGE motif 
Yes 
SQ-MAP 
K487E 
Neh1 
No 
SQ-MAP 
V105del 
Neh2 
No 


 
81 
 
Functionally validated KEAP1 mutations 
 
The following table lists the functionally validated amino acids/domains in Keap1 
based on literature review. Mutations in these amino acids should lead to a loss of Keap1 
function and increased Nrf2 activity (Table 3). 
 
Table 3 Eligible and functionally validated Keap1 amino acids/mutations  
KEAP1 mutation sites 
Domain 
Function 
Validation 
R380, R415, R483 
DGR-CTR 
Nrf2 binding residues 
in silico and in vitro 
Y525, Q530, N382, Y573, F577, Y334, S363, S602, S555 
DGR-CTR 
Nrf2 binding cavity 
in silico and in vitro 
G364C, G430C 
DGR-CTR 
Nrf2 binding cavity 
in silico and in vitro 
aa.315-624 
DGR-CTR 
Nrf2 binding domain 
in silico 
aa.96-100 
BTB HKVVL 
Keap1 dimerization 
in silico and in vitro 
C151, V155, V167 
BTB 
electrophile sensor 
in vitro 
D244, S243, C273, C288 
IVR 
electrophile sensor 
in vitro 
Any frameshift or nonsense mutation 
N/A 
N/A 
N/A 
 
Review of the TCGA SQCLC data and our own internal sequencing data show that 
mutations in functionally validated amino acid positions comprise 21% of all KEAP1 
mutations, including nonsense and frameshifts (Table 4). 
 
Table 4- KEAP1 mutations in SQCLCs (TCGA and MSK internal data) 
ID 
KEAP1 mutation 
Functional domain 
Validated? 
SQ-MAP 
Q563* 
DGR-CTR 
Yes/truncation 
SQ-MAP 
E213* 
IVR 
Yes/truncation 
SQ-MAP 
R260* 
IVR 
Yes/truncation 
TCGA-46-6025 
Q75* 
BTB 
Yes/truncation 
SQ-MAP 
I519fs 
DGR-CTR 
Yes/frame shift 
TCGA-33-4532 
N469fs 
DGR-CTR 
Yes/frame shift 
SQ-MAP 
E205fs 
IVR 
Yes/frame shift 
SQ-MAP 
G430C 
DGR-CTR 
Yes 
SQ-MAP 
Y572C 
DGR-CTR 
Yes 
SQ-MAP 
L153F 
BTB 
No 
SQ-MAP 
S102L 
BTB 
No 
SQ-MAP 
S144Y 
BTB 
No 
TCGA-18-3407 
V155F 
BTB 
No 
TCGA-22-4599 
V155F 
BTB 
No 
TCGA-43-2578 
V167F 
BTB 
No 
TCGA-21-1077 
D422N 
DGR-CTR 
No 
SQ-MAP 
E488D 
DGR-CTR 
No 
TCGA-18-5595 
E493D 
DGR-CTR 
No 


 
82 
 
TCGA-39-5036 
G423V 
DGR-CTR 
No 
TCGA-37-5819 
G480W 
DGR-CTR 
No 
TCGA-60-2722 
G480W 
DGR-CTR 
No 
TCGA-43-6143 
I506V 
DGR-CTR 
No 
SQ-MAP 
M456V 
DGR-CTR 
No 
SQ-MAP 
N414I 
DGR-CTR 
No 
 
TCGA-18-3409 
P318L 
DGR-CTR 
No 
SQ-MAP 
R320L 
DGR-CTR 
No 
SQ-MAP 
R320L 
DGR-CTR 
No 
TCGA-66-2773 
R320Q 
DGR-CTR 
No 
SQ-MAP 
R326C 
DGR-CTR 
No 
TCGA-66-2773 
R470C 
DGR-CTR 
No 
TCGA-60-2710 
R470C 
DGR-CTR 
No 
SQ-MAP 
T609S 
DGR-CTR 
No 
TCGA-66-2756 
V369L 
DGR-CTR 
No 
TCGA-51-4081 
V418L 
DGR-CTR 
No 
TCGA-66-2754 
W544C 
DGR-CTR 
No 
SQ-MAP 
H311R 
IVR 
No 
TCGA-60-2723 
L231V 
IVR 
No 
TCGA-33-4538 
L310P 
IVR 
No 
SQ-MAP 
R204L 
IVR 
No 
TCGA-18-5595 
R260Q 
IVR 
No 
TCGA-66-2777 
S224Y 
IVR 
No 
TCGA-37-4133 
S243C 
IVR 
No 
TCGA-39-5031 
R15L 
NTR 
No 
 
Functional KRAS mutations 
 
KRAS mutations occur in approximately 25% of all non-squamous NSCLCs.  Nearly 
all KRAS mutations occur in codons 12 and 13.  Mutations in these codons lead to constitutive 
activation of KRAS by inhibiting its GTPase activity.  This leads to activation of the MAP and 
PI3K/mTOR pathways.9  Induction of mutant KRAS isoforms is oncogenic, and has formed 
the basis of many of the transgenic lung cancer models in use today.10  These include models 
that have demonstrated, as noted above, dependence on the mTOR pathway and glutamine 
metabolism in the context of KEAP1 loss of function.11 The below table lists the frequency 
breakdown of common KRAS mutations in NSCLC based on MSK-IMPACT sequencing data 
from 1,668 patients: 
 
Mutation 
Frequency (N=458 KRAS mutations) 
G12C 
42% 
G12V 
15% 


 
83 
 
G12D 
13% 
G12A 
9% 
G13D 
4% 
Q61H 
4% 
G13C 
3.5% 
G12S 
2.4% 
 
KEAP1 co-alterations occur in 23% of all KRAS mutant lung cancer cases in the MSK-
IMPACT dataset, circumscribing a sizable population of patients with both alterations.  
Specific KRAS mutations associated with KEAP1 alterations include codon 12 alterations 
(G12A,C,S,V,D), codon 13 alterations (G13C,D,R) and codon 61 (Q61H).   
 
Integral Biomarker Inclusion Criteria 
 
Based on the preceding analysis, virtually all NFE2L2 mutations detected to date are 
predicted to be functionally relevant. This clustering of mutations in specific subdomains of 
a larger regulatory domain is reminiscent of hotspot mutations in other oncogenes.  Only 
mutations that occur in amino acids listed in Table 1 should be included in the proposed 
phase 2 study of MLN0128 (TAK-228). 
 
Eligible KEAP1 mutations will be prioritized, but not limited, to those alterations 
that occur in the amino acids listed in Table 3.  
 
Eligible KRAS mutations will include any missense mutation that occurs in codons 
12, 13, and 61.


 
84 
 
References 
1) Itoh K, Wakabayashi N, Katoh Y, Ishii T, Igarashi K, Engel JD, et al. Keap1 represses 
nuclear activation of antioxidant responsive elements by Nrf2 through binding to the 
amino-terminal Neh2 domain. Genes & Development. 1999;13:76-86. 
2) Zhang DD, Lo S-C, Cross JV, Templeton DJ, Hannink M. Keap1 Is a Redox-Regulated 
Substrate Adaptor Protein for a Cul3-Dependent Ubiquitin Ligase Complex. Molecular 
and Cellular Biology. 2004;24:10941-53. 
3) Padmanabhan B, Tong KI, Ohta T, Nakamura Y, Scharlock M, Ohtsuji M, et al. Structural 
Basis for Defects of Keap1 Activity Provoked by Its Point Mutations in Lung Cancer. 
Molecular Cell. 2006;21:689-700. 
4) Tong KI, Katoh Y, Kusunoki H, Itoh K, Tanaka T, Yamamoto M. Keap1 Recruits Neh2 
through Binding to ETGE and DLG Motifs: Characterization of the Two-Site Molecular 
Recognition Model. Molecular and Cellular Biology. 2006;26:2887-900. 
5) Tong KI, Padmanabhan B, Kobayashi A, Shang C, Hirotsu Y, Yokoyama S, et al. 
Different Electrostatic Potentials Define ETGE and DLG Motifs as Hinge and Latch in 
Oxidative Stress Response. Molecular and Cellular Biology. 2007;27:7511-21. 
6) Yamamoto T, Suzuki T, Kobayashi A, Wakabayashi J, Maher J, Motohashi H, et al. 
Physiological   Significance of Reactive Cysteine Residues of Keap1 in Determining Nrf2 
Activity. Molecular and Cellular Biology. 2008;28:2758-70. 
7) Suzuki T, Maher J, Yamamoto M. Select Heterozygous Keap1 Mutations Have a 
Dominant- Negative Effect on Wild-Type Keap1 In Vivo. Cancer Research. 
2011;71:1700-9. 
8) Zipper LM, Mulcahy RT. The Keap1 BTB/POZ Dimerization Function Is Required to 
Sequester Nrf2  in Cytoplasm. Journal of Biological Chemistry. 2002;277:36544-52. 
9) Bhattacharya S, Socinski MA, Burns TF. KRAS mutant lung cancer: progress thus far 
on an elusive therapeutic target. Clin Transl Med. 2015;4:35 
10) Engelman JA, Chen L, Tan X, et al. Effective use of PI3K and MEK Inhibitors to 
Treat Mutant K-Ras G12D and PIK3CA H1047T Murine Lung Cancer. Nat Med. 
2008 14(12):1351-1356. 
11) Romero R, Sayin VI, Davidson SM, et al. Keap1 loss promotes Kras-driven lung 
cancer and results in dependence on glutaminolysis. Nat Med 2017 23(11):1362-1368.


 
85 
 
APPENDIX C 
BIOASSAY TEMPLATES 
 
The below Appendix C describes the validation studies for the integral NFE2L2 and KEAP1 
mutation biomarker testing.  The validation study for KRAS mutations was previously published as 
part of the MSK-IMPACT validation study leading to its FDA approval in 2017 as an in vitro 
diagnostic test to identify genetic mutations across cancers.  Please refer to Cheng DT et al. J Mol 
Diagn 2015 17(3):251-64 (PMID25801521) for the validation study details.   
 
Validation Study of KEAP1 and NFE2L2 Mutations 
 
Accuracy Study 
 
To carry out this validation, we obtained a total of 23 positive tumor DNA samples (18 
KEAP1 and 5 NFE2L2) that had been previously genotyped by our “MSK-IMPACT (Integrated 
Mutation Profiling of Actionable Cancer Targets): A Hybridization-Capture Based Next 
Generation Sequencing Assay”. The samples were also studied by Sanger DNA sequencing. All 
DNA sequencing reactions were performed in both forward and reverse directions. 
Concordant results were obtained for all 23 samples by DNA sequencing and MSK- 
IMPACT assay. The results of the validation study are summarized in Tables 1 & 2. 
 
1) Accuracy study – KEAP1 mutations 
 
Table 1.A: Sample coverage (mean coverage across all exons) and total number of mutations 
called across all 340 genes, for samples used in validation of this clinical exon. 
 
Sample 
ID# 
Sample 
Coverage 
Total Number of Mutations 
called across all 340 genes 
Normal sample used 
1 
2242-14 
866 
15 
Matched 
2 
4488-14 
526 
5 
Matched 
3 
4673-14 
1149 
8 
Matched 
4 
4756-14 
749 
11 
Matched 
5 
4869-14 
467 
8 
Matched 
6 
4930-14 
172 
7 
Matched 
7 
5566-14 
654 
9 
Matched 
8 
6134-14 
897 
21 
Matched 
9 
6451-14 
729 
39 
Matched 
10 
6714-14 
591 
9 
Matched 
11 
6989-14 
540 
11 
Matched 
12 
7327-14 
422 
19 
Matched 
13 
7447-14 
143 
15 
Matched 
14 
7909-14 
667 
8 
Matched 
15 
8091-14 
365 
8 
Matched 
16 
8707-14 
508 
4 
Matched 
17 
8731-14 
714 
6 
Matched 
18 
8831-14 
1020 
9 
Matched 
 


 
86 
 
Table 1.B: List of mutations detected that correspond to the known KEAP1 variants for the 
tested samples. 
 
Sample 
ID# 
Gene 
Exon 
cDNA change 
AA 
change 
DP 
AD 
VF 
Normalized 
Coverage 
1 
2242-14 
KEAP1 
exon4 
c.1511A>G 
p.N504S 
813 
125 
0.15 
0.9 
2 
4488-14 
KEAP1 
exon6 
c.1801C>T 
p.R601W 
517 
167 
0.32 
1 
3 
4673-14 
KEAP1 
exon2 
c.295G>A 
p.V99M 
946 
395 
0.42 
0.8 
4 
4756-14 
KEAP1 
exon5 
c.1626_1627delGA 
p.E542fs 
801 
247 
0.31 
1.1 
5 
4869-14 
KEAP1 
exon4 
c.1486G>T 
p.E496X 
886 
223 
0.25 
1.9 
6 
4930-14 
KEAP1 
exon3 
c.932A>G 
p.H311R 
92 
39 
0.42 
0.5 
7* 
5566-14a 
KEAP1 
exon2 
c.16A>T 
p.R6W 
153 
55 
0.36 
0.2 
5566-14b 
KEAP1 
exon3 
c.994G>T 
p.G332C 
402 
127 
0.32 
0.6 
8 
6134-14 
KEAP1 
exon2 
c.592G>T 
p.E198X 
1020 
250 
0.25 
1.1 
9 
6451-14 
KEAP1 
exon3 
c.997G>T 
p.G333C 
661 
462 
0.7 
0.9 
10 
6714-14 
KEAP1 
exon3 
c.655G>A 
p.E219K 
486 
160 
0.33 
0.8 
11* 
6989-14a 
KEAP1 
exon2 
c.411T>G 
p.I137M 
581 
69 
0.12 
1.1 
6989-14b 
KEAP1 
exon2 
c.414_424delATTCGCCTACA 
p.E138fs 
634 
69 
0.11 
1.2 
6989-14c 
KEAP1 
exon3 
c.1103G>T 
p.C368F 
647 
152 
0.23 
1.2 
12 
7327-14 
KEAP1 
exon5 
c.1638delC 
p.F546fs 
507 
218 
0.43 
1.2 
13 
7447-14 
KEAP1 
exon2 
c.244C>T 
p.Q82X 
99 
23 
0.23 
0.7 
14 
7909-14 
KEAP1 
exon6 
c.1808G>T 
p.G603V 
637 
274 
0.43 
1 
15 
8091-14 
KEAP1 
exon3 
c.1006C>T 
p.R336X 
245 
51 
0.21 
0.7 
16 
8707-14 
KEAP1 
exon3 
c.1085G>C 
p.R362P 
418 
140 
0.33 
0.8 
17 
8731-14 
KEAP1 
exon3 
c.772G>T 
p.E258X 
491 
100 
0.2 
0.7 
18* 
8831-14a 
KEAP1 
exon2 
c.493G>A 
p.D165N 
1041 
362 
0.35 
1 
8831-14b 
KEAP1 
exon2 
c.574G>A 
p.E192K 
1006 
348 
0.35 
1 
*These samples had more than 1 mutation 
 
2) Accuracy study – NFE2L2 mutations 
 
Table 2.A: Sample coverage (mean coverage across all exons) and total number of 
mutations called across all 340 genes, for samples used in validation of this clinical exon. 
 
Sample 
ID# 
Sample 
Coverage 
Total 
Mutations 
Normal sample 
used 
1 
5155-14 
826 
10 
Matched 
2 
7894-14 
650 
10 
Matched 
3 
8045-14 
1254 
3 
Matched 
4 
8556-14 
1112 
11 
Matched 
5 
8586-14 
920 
56 
Matched 
 
 
 
Table 2.B: List of mutations detected that correspond to the known NFE2L2 variants for 
the tested samples. 


 
87 
 
 
Sample 
ID# 
Gene 
Exon 
cDNA change 
AA 
change 
DP 
AD 
VF 
Normalized 
Coverage 
1 
5155-14 
NFE2L2 
exon2 
c.75_76delinsAA 
p.Q26K 
1261 
716 
0.57 
1.5 
2 
7894-14 
NFE2L2 
exon2 
c.100C>G 
p.R34G 
1279 
120 
0.09 
2 
3 
8045-14 
NFE2L2 
exon2 
c.76C>A 
p.Q26K 
1213 
471 
0.39 
1 
4 
8556-14 
NFE2L2 
exon2 
c.100C>G 
p.R34G 
1639 
303 
0.18 
1.5 
5 
8586-14 
NFE2L2 
exon2 
c.88C>T 
p.L30F 
1378 
254 
0.18 
1.5 
 
Precision and Reproducibility Study 
The precision and reproducibility studies were extensively performed in the initial MSK- 
IMPACT validation according to NGS NYSDOH guidelines (attached). In addition, two DNA 
samples, one KEAP1 positive and one NFE2L2 positive, were studied in triplicate in the same 
run for the intra-assay precision study.  The same DNA samples were also studied over three 
different runs for the inter-assay reproducibility study.  Concordant results were obtained for 
both intra- and inter-assay reproducibility studies. Replicates tested within the same run were 
assigned different barcodes to ensure reproducibility of results regardless of sample/barcode 
combination. Each replicate was assigned a different barcode (bc). Differences in barcodes, 
coverage depth, allele depth (AD) and variant frequency (VF) for the known variant across intra- 
and inter- run replicates are listed in Tables 3.1 & 3.2. 
 
3.1) Precision and reproducibility studies of KEAP1 
 
Table 3.1A: Total number of mutations called across all 340 genes, for KEAP1 intra- (precision) 
and inter-run (reproducibility) replicates. 
 
Sample 
Total Number of Mutations called 
across all 340 genes 
Normal sample used 
7555-13-1A 
13 
Matched 
7555-13-1B 
13 
Matched 
7555-13-1C 
13 
Matched 
7555-13-2 
13 
Matched 
7555-13-3 
13 
Matched 
 
Table 3.1B-i: Differences in Illumina TruSeq barcode, coverage, allele depth and variant 
frequency for the known variant detected in each inter-run (reproducibility) replicate. 
 
Sample 
cDNA 
change 
AAchange 
Validation 
Run 
Barcode 
Coverage 
AD 
VF 
Normalized 
DP 
7555-13-1A 
c.811G>A 
p.V271M 
KEAP1 Intra 
bc34 
910 
483 
0.42 
1.26 
7555-13-1B 
c.811G>A 
p.V271M 
KEAP1 Intra 
bc36 
1011 
506 
0.42 
1.19 
7555-13-1C 
c.811G>A 
p.V271M 
KEAP1 Intra 
bc38 
1185 
688 
0.44 
1.33 
 
Table 3.1B-ii: Differences in Illumina TruSeq barcode, coverage, allele depth and variant 
frequency for the known variant detected in each intra-run (precision) replicate. 
 


 
88 
 
Sample 
cDNA 
change 
AAchange 
Validation 
Run 
Barcode 
Coverage 
AD 
VF 
Normalized 
DP 
7555-13-1A 
c.811G>A 
p.V271M 
KEAP1 Inter 
bc34 
910 
483 
0.42 
1.26 
7555-13-2 
c.811G>A 
p.V271M 
KEAP1 Inter 
bc24 
1078 
517 
0.40 
1.19 
7555-13-3 
c.811G>A 
p.V271M 
KEAP1 Inter 
bc43 
1044 
560 
0.43 
1.25 
 
3.2) Precision and reproducibility studies of NFE2L2 
 
Table 3.2A: Total number of mutations called across all 340 genes, for NFE2L2 intra- (precision) 
and inter-run (reproducibility) replicates. 
 
Sample 
Total Number of Mutations called 
across all 340 genes 
Normal sample used 
5155-14-1A 
10 
Matched 
5155-14-1B 
10 
Matched 
5155-14-1C 
10 
Matched 
5155-14-2 
10 
Matched 
5155-14-3 
10 
Matched 
 
Table 3.2B-i: Differences in Illumina TruSeq barcode, coverage, allele depth and variant 
frequency for the known variant detected in each inter-run (reproducibility) replicate. 
 
cDNA 
change 
AA 
change 
Validation 
Run 
Barcode 
Coverage 
AD 
VF 
Normalized DP 
5155-14-1A 
c.76C>A 
p.Q26K 
NEF2L2 Intra 
bc39 
702 
555 
0.53 
1.51 
5155-14-1B 
c.76C>A 
p.Q26K 
NEF2L2 Intra 
bc40 
693 
537 
0.51 
1.51 
5155-14-1C 
c.76C>A 
p.Q26K 
NEF2L2 Intra 
bc42 
621 
491 
0.52 
1.53 
 
Table 2.2B-ii: Differences in Illumina TruSeq barcode, coverage, allele depth and variant 
frequency for the known variant detected in each intra-run (precision) replicate. 
 
cDNA 
change 
AA 
change 
Validation 
Run 
Barcode 
Coverage 
AD 
VF 
Normalized DP 
5155-14-1A 
c.76C>A 
p.Q26K 
NEF2L2 Inter 
bc39 
702 
555 
0.53 
1.51 
5155-14-2 
c.76C>A 
p.Q26K 
NEF2L2 Inter 
bc38 
742 
545 
0.51 
1.45 
5155-14-3 
c.76C>A 
p.Q26K 
NEF2L2 Inter 
bc43 
596 
462 
0.55 
1.42 


 
89 
 
APPENDIX D 
PATIENT PILL DIARY 
 
A Study to Test if  MLN0128 (TAK-228) can Treat  Patients with Non-Small 
Cell Lung Cancers with NFE2L2 and KEAP1 Mutations 
 
Name:  
 
 
MRN:  
 
 
To be completed by NP: 
Number of pills dispensed: 
_ 1mg 
_ 3mg 
  Date:   
 
  
Daily dose of MLN0128 (TAK-228):   
mg Cycle : 
 
   
Pill bottle returned: circle Yes or N 
 
Instructions for patient: 
 
1. You will be given a pill diary to record the time and doses of MLN0128 (TAK-228) 
you take each day. This diary along with any leftover pills and the pill bottles should 
be brought to each visit with your study doctor. 
 
2. MLN0128 (TAK-228) should be taken once daily, at approximately the same time 
every day. 
 
3. Take MLN0128 (TAK-228) on an empty stomach at least 2 hours after a meal.  Do not 
eat or drink (except water) for at least 1 hour after taking MLN0128 (TAK-228). 
 
4. Each dose should be taken with a glass of water and consumed whole (no chewing).  
The capsules should not be opened, chewed, or crushed. If multiple capsules are 
required for each dose, they should be taken at the same time. 
 
5. If you miss a dose, you can still take that dose if it  has been less than 12 hours since 
you missed the dose. 
 
6. If you vomit after taking the pill, do not replace that dose.  If you can see that the pill 
has come up, mark the dose as missed in the comments. 
 
 


 
90 
 
 
Cycle  
 
Date 
Time (circle one) Amount (mg) 
Comments 
Day 1 
AM/PM 
Day 2 
AM/PM 
 
 
Day 3 
AM/PM 
Day 4 
AM/PM 
Day 5 
AM/PM 
Day 6 
AM/PM 
Day 7 
AM/PM 
Day 8 
AM/PM 
Day 9 
AM/PM 
Day 10 
AM/PM 
Day 11 
AM/PM 
Day 12 
AM/PM 
Day 13 
AM/PM 
Day 14 
AM/PM 
Day 15 
AM/PM 
Day 16 
AM/PM 
Day 17 
AM/PM 
Day 18 
AM/PM 
Day 19 
AM/PM 


 
91 
 
Day 20 
AM/PM 
Day 21 
AM/PM 
Day 22 
AM/PM 
Day 23 
AM/PM 
Day 24 
AM/PM 
Day 25 
AM/PM 
Day 26 
AM/PM 
Day 27 
AM/PM 
Day 28 
AM/PM 
 
Patient signature: 
 Date: 
/ 
/  
 
 
Consenting professional / NP signature: 
Date: / 
/   
 
 


 
92 
 
APPENDIX E  PATIENT GLUCOSE MONITORING LOG 
 
A Study to Test if MLN0128 (TAK-228) can Treat Patients with Non-Small 
Cell Lung Cancers with NFE2L2 and KEAP1 Mutations 
 
Patient Name:_________________________ 
 
Patient MRN:_________________________ 
 
Directions: 
• You will need to perform the glucose monitoring test on a daily basis, predose on 
dosing days, and at approximately the same time each day- prior to breakfast. 
You are required to fast for at least 8 hours prior to testing. Fasting means not 
eating or drinking any liquids (with the exception of water). 
 
• In the log, please write in the date, note the time you completed the glucose test, 
whether you fasted or not (fed state) before the test, and your glucose levels as 
provided by your glucometer. 
 
• Please 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. 
 
Site Contact Name:____________________ 
 
Site Contact: Number:__________________ 
 
 
Day 
Date 
Approximate time 
of Test (Circle 
AM or PM) 
Fasting or Fed 
(Circle One) 
Glucose Test 
Results 
(mg/dL) 
Day 1 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 2 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 3 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 4 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 5 
 
____:____ am / pm 
Fasting   or   Fed 
 


 
93 
 
Day 6 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 7 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 8 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 9 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 10 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 11 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 12 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 13 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 14 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 15 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 16 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 17 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 18 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 19 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 20 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 21 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 22 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 23 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 24 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 25 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 26 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 27 
 
____:____ am / pm 
Fasting   or   Fed 
 
Day 28 
 
____:____ am / pm 
Fasting   or   Fed 
 
 
Patient Signature:______________________  Date:_______________ 
 
Investigator Signature:__________________  Date:_______________ 


 
94 
 
APPENDIX F  
LIST OF RELEVANT CYTOCHROME P450 INHIBITORS  
AND INDUCERS 
 
 
 
Moderate CYP1A2 Inhibitors 
Cimetidine 
methoxsalen  
 
Strong CYP1A2 Inhibitors 
Fluvoxamine 
Ciprofloxacin 
 
Clinically Significant Enzyme Inducers 
Carbamazepine 
rifabutin 
St. John’s Wort 
Phenobarbital 
rifampin 
Phenytoin 
rifapentine  
 
 
Source: 
fda.gov/Drugs/DevelopmentApprovalProcess/DevelopmentResources/DrugInteractionsLabe
ling/ ucm093664.htm. 
Note that these lists are not exhaustive.