A Phase Ib/II Safety and Efficacy Study of ABC294640 in Patients With Refractory or Relapsed Multiple Myeloma Who Have Previously Been Treated With Proteasome Inhibitors and Immunomodulatory Drugs
Phase: PHASE1
Conditions: Multiple Myeloma
Sponsor: RedHill Biopharma Limited

CONFIDENTIAL 
The information contained in this document is regarded as confidential, and may not be disclosed to another party unless such disclosure is 
required to initiate the study, to conduct study-related activities, or to comply with national, state, or local laws and regulations.  Written 
authorization from the coordinating site and sponsor is required for disclosure otherwise. 
PT-PhI-II v11.08.18 
A Phase Ib/II Safety and Efficacy Study of ABC294640 in Patients with Refractory or 
Relapsed Multiple Myeloma Who Have Previously Been Treated with Proteasome 
Inhibitors and Immunomodulatory Drugs. 
 
 
 
 
 
 
Sponsor:  
RedHill Biopharma Ltd., 21 Ha’arba’a St., Tel Aviv 64739, 
Israel 
Funding Source: 
NIH, Apogee Biotechnology Corporation and RedHill 
Biopharma Ltd. 
Protocol Source: 
PI - Duke Cancer Institute 
Duke IRB#: 
Pro00062304 
RedHill Study ID 
ABC-103 
IND# 
124650 
 
 
Original version: 
September 15, 2014 
Amended version 1: 
March 19, 2015 
Amended version 2: 
May 1, 2015  
Amended version 3: 
May 27, 2015 
Amended version 4: 
June 15, 2015 
Amended version 5: 
June 28, 2016 
Amended version 6: 
August 19, 2016 
Amended version 7: 
October 12, 2016 
Amended version 8: 
 
May 17, 2017 
 
Amended version 9: 
 
Amended version 10: 
 
Amended version 11: 
 
Amended version 12: 
October 5, 2017 
 
April 11, 2018 
 
December 4, 2018 
 
March 22, 2019 
 
 
Principal Investigator 
Yubin Kang, M.D. 
Associate Professor 
Division of Hematologic Malignancies 
and Cellular Therapy 
Department of Medicine 
Duke University Medical Center 
DUMC 3961 
Durham, NC 27710 
Phone:  (919) 668-2331 (Office) 
(919) 668-4532/(919) 681-7451 (Lab) 
Fax:  (919) 668-1091 (Clinic) 
         (919) 613-1319 (Lab) 
Email: yubin.kang@duke.edu 
 
Medical Director  
Terry Plasse, MD 
RedHill Biopharma Ltd 
Ha’arba’a St. 
Tel Aviv 64739 
Israel 
Phone: +972-50-588-7376 
Mobile: +1-917-913-7315 
Fax: +972-3-541-3144 
Email: Terry@redhillbio.com 
 
Charles D. Smith, Ph.D. 
President and CEO 
Apogee Biotechnology Corporation 
1214 Research Blvd, Suite 2014 
Hershey Center for Applied Research 
Hummelstown, PA  17036 
Phone: (717) 531-4758 
Fax: (717) 531-4758 
Email: cdsmith@apogee-biotech.com 
Co- Investigator(s) 
  
Taewoong Choi, MD 
Taewoong.Choi@duke.edu  
Cristiana Costa, DO 

 
 
 
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Cristiana.Costa@duke.edu  
Cristina Gasparetto, M.D. 
Cristina.Gasparetto@duke.edu  
Gwynn Long, M.D. 
Gwynn.Long@duke.edu  
 
 
Statistician 
Zhiguo Li, Ph.D. (Biostatistics) 
Assistant Professor 
Zhiguo.Li@duke.edu 
 
Study Coordinator: 
Ivy Belskie, RN 
ivy.belskie@duke.edu 
  
 

 
 
 
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Contents 
PROTOCOL SYNOPSIS ................................................................................................................................................................5 
DEFINITION OF TERMS USED................................................................................................. Error! Bookmark not defined. 
1. 
BACKGROUND ........................................................................................................................... 15 
1.1 
MULTIPLE MYELOMA ...................................................................................................... 15 
1.2 
SPHINGOLIPIDS, SPHINGOSINE KINASES AND CANCER ......................................... 15 
1.3 
Summary of ABC294640 Phase I Clinical Data in Solid Tumors ......................................... 18 
1.4 
Rationale for a clinical trial evaluating ABC294640 in patients with MM ........................... 20 
1.5  
Pre-clinical efficacy of ABC294640 for Multiple Myeloma .................................................... 20 
2. 
STUDY DESIGN .......................................................................................................................... 30 
2.1  
Study description .................................................................................................................... 30 
2.2  
Phase 1B ................................................................................................................................. 30 
2.3 
Phase 1B design ..................................................................................................................... 31 
2.4 
Definition of dose-limiting toxicities ..................................................................................... 32 
2.5 
Phase II ................................................................................................................................... 32 
3. 
STUDY PATIENTS ...................................................................................................................... 32 
4. 
ENROLLMENT AND TREATMENT ......................................................................................... 34 
4.1 
Screening ................................................................................................................................ 34 
4.2 
Phase IB: Dose escalation phase ............................................................................................ 35 
4.3 
Phase II ................................................................................................................................... 36 
4.4 
Safety confirmation and follow-up ........................................................................................ 37 
4.5 
Disease assessment ................................................................................................................ 37 
5. 
PK and PD Studies ......................................................................................................................... 37 
5.1 
Special Instructions related to administration of ABC 294640 on days of PK Sampling. ..... 37 
5.2 
 Pharmacodynamic and correlative assays ............................................................................. 38 
5.3 
Time points of sample collection ........................................................................................... 39 
5.4 
Sample collection and shipment ............................................................................................. 40 
5.5 
Assays .................................................................................................................................... 40 
6. 
STUDY DRUG: ABC294640 ....................................................................................................... 41 
6.1  
Formulation ............................................................................................................................ 41 
6.2  
Dosage and administration ..................................................................................................... 41 
6.3  
Storage ................................................................................................................................... 41 
6.4  
Labeling ................................................................................................................................. 42 
6.5  
Test article and accountability ............................................................................................... 42 
7. 
METHOD OF DETERMINING TREATMENT COMPLIANCE ................................................ 42 
7.1 
SAFETY PLAN ..................................................................................................................... 42 
7.2 
POST-TREATMENT EVALUATIONS ............................................................................... 43 
7.3 
 PATIENT DISCONTINUATION ........................................................................................ 44 
8. 
STATISTICAL CONSIDERATIONS ........................................................................................... 44 
8.1  
PRIMARY OBJECTIVE ....................................................................................................... 44 
8.2 
PRIMARY ENDPOINTS ....................................................................................................... 45 
8.3  
SECONDARY ENDPOINTS ................................................................................................ 45 
8.4  
DETERMINATION OF SAMPLE SIZE .............................................................................. 46 
8.5  
ANALYSIS PLANS .............................................................................................................. 46 
8.6  
INTERIM ANALYSES ......................................................................................................... 47 
8.7  
ANALYSIS POPULATIONS ................................................................................................ 48 
9. 
SAFETY MONITORING AND REPORTING ............................................................................ 48 

 
 
 
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9.1  
ADVERSE EVENTS ............................................................................................................. 48 
9.2  
SERIOUS ADVERSE EVENTS ........................................................................................... 49 
9.3  
ADVERSE EVENT ASSESSMENT ..................................................................................... 49 
9.4  
ADVERSE EVENT CAUSALITY ........................................................................................ 50 
9.5 
ADVERSE REACTION EXPECTEDNESS ......................................................................... 51 
9.6 
ADDITIONAL ADVERSE EVENT CONSIDERATIONS .................................................. 51 
9.7 
PROCEDURES FOR ELICITING, RECORDING, AND REPORTING ADVERSE 
EVENTS ................................................................................................................................ 52 
9.8  
Quality Control and Quality Assurance ................................................................................. 53 
9.9 
Audits ..................................................................................................................................... 53 
9.10  
Data Management and Processing ......................................................................................... 54 
10. 
ADMINISTRATIVE and ETHICAL CONSIDERATIONS ......................................................... 55 
10.1  
Regulatory and Ethical Compliance ....................................................................................... 55 
10.2 
 DUHS Institutional Review Board and DCI Cancer Protocol Committee ........................... 55 
10.3  
Informed Consent ................................................................................................................... 55 
10.4  
Study Documentation ............................................................................................................. 55 
10.5  
Privacy, Confidentiality, and Data Storage ............................................................................ 56 
10.6  
Data and Safety Monitoring ................................................................................................... 56 
10.7  
Protocol Amendments ............................................................................................................ 57 
10.8  
Records Retention .................................................................................................................. 57 
11. 
SPONSOR AND MULTI-SITE INVESTIGATOR REQUIREMENTS ...................................... 57 
11.1 
STUDY INITIATION ............................................................................................................ 57 
11.2 
STUDY COMPLETION ........................................................................................................ 58 
11.3 
INSTITUTIONAL REVIEW BOARD APPROVAL ............................................................ 58 
11.4 
QUALITY ASSURANCE ..................................................................................................... 59 
11.5 
DATA COLLECTION ........................................................................................................... 59 
12. 
GENERAL CONSIDERATIONS ................................................................................................. 59 
12.1 
Discontinuation of the Study .................................................................................................. 59 
12.2 
Routine Study Termination .................................................................................................... 59 
12.3 
Premature Study Termination ................................................................................................ 59 
12.4 
Changes to the Protocol ......................................................................................................... 59 
12.5 
Protocol Deviations for Emergency or Adverse Event .......................................................... 59 
12.6 
Source Documents ................................................................................................................. 60 
13. 
APPENDICES ............................................................................................................................... 60 
REFERENCES .............................................................................................................................................................................. 70 
 

 
 
 
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 PROTOCOL SYNOPSIS 
TITLE 
A Phase Ib/II Safety and Efficacy Study of ABC294640 in Patients with Refractory 
or Relapsed Multiple Myeloma Who have Previously Been Treated with Proteasome 
Inhibitors and Immunomodulatory Agents 
PHASE 
Ib/II 
STUDY 
RATIONALE 
ABC294640 [3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl) 
amide, hydrochloride salt] is an orally available inhibitor of the enzyme sphingosine 
kinase-2. SK2 is an innovative molecular target for anti-cancer therapy because of its 
critical role in sphingolipid metabolism, which is known to regulate tumor cell death and 
proliferation. Our preclinical studies demonstrate that SK2 is overexpressed in multiple 
myeloma (MM) cell lines and in human MM specimens. Additionally, inhibition of SK2 
by RNA interference or treatment with ABC294640 effectively promotes apoptosis in 
MM cell lines and inhibits the proliferation of primary human myeloma cells.  
Furthermore, ABC294640 reduces the expression of Mcl-1 and c-Myc by inducing their 
proteasome degradation.  ABC294640 effectively inhibited myeloma tumor growth in 
vivo in mouse xenograft models. Therefore, we hypothesize that ABC294640 will have 
therapeutic activity in refractory/relapsed MM patients. ABC294640 has recently 
completed the Phase I safety study and demonstrated an excellent safety profile. In this 
phase Ib/II study, ABC294640 will be assessed for its safety and efficacy in refractory or 
relapsed MM patients who are previously treated with proteasome inhibitors and 
immunomodulatory agents. 
STUDY DESIGN 
This is a Phase Ib/II safety and efficacy trial of single agent ABC294640 in refractory or 
relapsed MM. Cohorts of patients with refractory or relapsed MM who have previously 
been treated with proteasome inhibitors and immunomodulatory agents will receive 
increasing doses of oral ABC294640. HIV and hepatitis B and C serology screening will 
be performed during patient screening.  Patients with HIV infection or active hepatitis B or 
C infection with abnormal liver functions (i.e., LFTs > 2 x upper normal limits) will be 
excluded. The starting dosage for ABC294640 will be 250 mg BID (known to be safely 
tolerated as a single-agent), and the ABC294640 dose will be escalated to two additional 
dose cohorts of 500 and 750 mg BID using Bayesian model average continual 
reassessment method (BMA-CRM) for dose finding. It is expected that 18 patients will be
used to determine the MTD for ABC294640 in refractory or relapsed MM. 
Pharmacokinetic (PK) assessments of ABC294640 will be conducted on Day 1of Cycle 1. 
Pharmacodynamics (PD) assessments will be conducted on Day 1 of each cycle. Bone 
marrow biopsy will be obtained prior to the initiation of ABC294640, at the end of cycle #3 
and at the end of cycle #6. In addition to SPEP, UPEP and serum free light chain 
measurement, correlative studies will be performed to measure SK2 mRNA or enzyme 
activities, sphingosine metabolites, c-Myc, Mcl-1 and pS6 in CD138+ myeloma cells. 
OBJECTIVES – 
PHASE IB 
Primary Objectives 
• 
To assess safety and determine the maximum tolerated dose (MTD) of single agent 
ABC294640 in patients with refractory or relapsed MM who have been previously 
treated with proteasome inhibitors and immunomodulatory agents. 
Secondary Objectives 
• 
To assess the antitumor activity of single agent ABC294640 in patients with 
refractory or relapsed MM after 3 cycles of treatment. 
• 
To determine the pharmacokinetics of ABC294640 following administration of the 
drug. 

 
 
 
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• 
To describe the effects of ABC294640 on plasma levels of sphingosine 1-phosphate, 
IL-6, and other cytokines in patients with refractory or relapsed MM. 
• 
To assess pharmacodynamic markers (SK2 mRNA level or activity, sphingolipid 
metabolites, c-Myc, Mcl-1 and pS6) in bone marrow CD138+ myeloma cells. For 
patients who receive more than 3 cycles of treatment and have a repeated bone 
marrow biopsy, RNA sequencing and Assay for transposases accessible chromatin 
sequencing (ATAC seq) will be performed to assess the effects of ABC294640 on 
global gene and epigenetic expression. 
• 
To perform correlative studies including serum cell-free PINK1/PARK2 (mitophagy 
markers), cell free DNA for next generation sequencing (for signal pathway 
analyses), serum bone destruction/formation markers (TRAP, Osteocalcin) and bone 
markers and Lyn/Src gene expression in both CD138+ and CD138- cells. 
• 
To evaluate immunologic function and immunologic panels (NK, NK T, T, B cells 
and myeloid derived suppressive cells (MDSCs) and the immune co-signalling 
molecules) in bone marrow environment 
NUMBER OF 
PATIENTS  
(PHASE 1B) 
Up to eighteen patients will be enrolled in the phase IB portion of the trial. 
OBJECTIVES –
PHASE II 
Primary Objectives 
• 
Assess overall treatment response rate and overall survival in patients with relapsed 
or refractory MM treated with single-agent ABC294640. 
Secondary Objectives 
• 
To assess the treatment response of ABC294640 in patients with refractory or 
relapsed MM after 3 cycles of treatment. 
• 
  To determine if pharmacodynamic markers (SK2 mRNA or activity, sphingolipid 
metabolites, c-Myc, Mcl-1 and pS6) in bone marrow CD138+ myeloma cells predict 
tumor response to the treatment with ABC294640. 
• 
  To perform correlative studies including serum cell-free PINK1/PARK2 (mitophagy 
markers), cell free DNA for next generation sequencing (for signal pathway 
analyses), serum bone destruction/formation markers (TRAP, Osteocalcin), and bone 
gene markers and Lyn/Src gene expression in both CD138+ and CD138- cells. 
• 
To evaluate immunologic function and immunologic panels (NK, NK T, T, B cells 
and myeloid derived suppressive cells (MDSCs) and the immune co-signalling 
molecules) in bone marrow environment 
NUMBER OF 
PATIENTS 
(PHASE II) 
59 (may include 3 patients treated at MTD in Phase Ib, thus 56 additional patients) 
PATIENT 
ELIGIBILITY 
CRITERIA 
Inclusion criteria 
1. Patient must have a diagnosis of symptomatic multiple myeloma, who relapses or is 
refractory after previous treatment with a proteasome inhibitor (bortezomib or 
carfilzomib) and an immunomodulatory agent (thalidomide, lenalidomide or 
pamolidomide). 
2. Have measurable disease as defined by at least one of the following: 
 -  Serum monoclonal (M) protein ≥0.5 g/dl by protein electrophoresis  
-  >200 mg of M protein in the urine on 24-hour electrophoresis   
-  Serum immunoglobulin free light chain ≥10 mg/dL AND abnormal serum 
immunoglobulin kappa to lambda free light chain ratio 
-  Monoclonal bone marrow plasmacytosis ≥30%  
3. Voluntary signed and dated institutional review board (IRB) approved informed 
consent form in accordance with regulatory and institutional guidelines. 
4. Time interval from last systemic chemotherapy (not including low dose 

 
 
 
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dexamethasone) more than 2 weeks prior to initiation of ABC294640. Patients 
receiving high dose dexamethasone defined as 40mg dexamethasone a day for 4 days 
will need 2 weeks washout prior to initiation of ABC294640 
5. 18 years of age or older. 
6. ECOG performance status of 0-2. 
7. Acceptable liver function: 
- Bilirubin ≤ 1.5 times upper limit of normal (CTCAE Grade 1 baseline) 
- AST (SGOT), ALT (SGPT)  5 x ULN (CTCAE Grade 2 baseline) 
- Serum creatinine ≤1.5 XULN (CTCAE Grade 1 baseline) 
8. Acceptable hematologic status (with or without transfusion support): 
- Absolute neutrophil count  ≥1000 cells/mm3, 
- Platelet count ≥50,000 (plt/mm3),  
- Hemoglobin ≥9 g/dL. 
NOTE: If the patient’s bone marrow biopsy shows greater than or equal to 50% 
plasma cells, the platelet count should be >30,000 plt/mm3, Hemoglobin >8 g/dL 
and ANC >500 cells/mm3 (transfusion support or growth factor support is 
acceptable). 
9. Urinalysis: No clinically significant abnormalities. 
10. PT and PTT ≤ 1.5 X ULN after correction of nutritional deficiencies that may 
contribute to prolonged PT/PTT. 
11. As determined by the treating investigator, the patient must have well-controlled 
blood pressure, defined as systolic blood pressure <150mmHg and/or diastolic blood 
pressure <100 mmHg for the majority of measurements. 
12. A negative pregnancy test (if female of child bearing potential). 
13. For men and women of child-producing potential, willingness to use effective 
contraceptive methods during the study. 
 
Exclusion criteria 
1. Pregnant or nursing women. NOTE: Women of childbearing potential and men must 
agree to use adequate contraception (hormonal or barrier method of birth control; or 
abstinence) prior to study entry and for the duration of study participation. Should a 
woman become pregnant or suspect she is pregnant while participating in this study, 
she should inform her treating physician immediately. 
2. Patients who are currently participating in any other clinical trial of an investigational 
product. 
3. Major surgery within 30 days prior to start of treatment 
4. Treatment with intravenous antibiotics, antivirals, or antifungals within 14 days prior 
to start of treatment. 
5. Known human immunodeficiency virus infection 
6. Active hepatitis B or C infection with abnormal liver functions (i.e., LFTs > 2 x 
upper normal limits) 
7. Unstable angina or myocardial infarction within 4 months prior to start of treatment, 
NYHA Class III or IV heart failure, uncontrolled angina, history of severe coronary 
artery disease, severe uncontrolled ventricular arrhythmias, sick sinus syndrome, or 
electrocardiographic evidence of acute ischemia or Grade 3 conduction system 
abnormalities unless subject has a pacemaker 
8. Uncontrolled hypertension or uncontrolled diabetes within 14 days prior to start of 
treatment 
9. Nonhematologic malignancy within the past 3 years with the exception of 
a) adequately treated basal cell carcinoma, squamous cell skin cancer, or thyroid 
cancer; b) carcinoma in situ of the cervix or breast; c) prostate cancer of Gleason 
Grade 6 or less with stable prostate-specific antigen levels; or d) cancer considered 
cured by surgical resection or unlikely to impact survival during the duration of the 

 
 
 
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study, such as localized transitional cell carcinoma of the bladder 
10. Subjects with pleural effusions requiring thoracentesis or ascites requiring 
paracentesis within 14 days prior to initiation of ABC294640 
11. Any other clinically significant medical or psychiatric disease or condition, or social 
situation that, in the Investigator’s opinion, may interfere with protocol adherence or a 
subject’s ability to give informed consent 
TEST ARTICLE 
ADMINISTRATION 
ABC294640 will be administered orally twice a day (approximately 12 hours apart) 
continuously for 28 days. A cycle will be defined as 28 days. 
DEFINITION OF 
DOSE LIMITING 
TOXICITY 
Toxicities will be graded according to the NCI CTCAE version 4.0.3 criteria.  DLTs 
will be defined as any of the following events that are at least (possibly, probably, or 
definitely) attributable to ABC294640 during dose escalation in the phase 1 portion of 
the study. 
 
Non hematologic DLT is defined as: 
Any Grade ≥ 3 AE, with the following exceptions 
Symptomatic adverse events such as nausea, vomiting and diarrhea will not be 
considered dose limiting if they can be reduced to less than grade 3 within 72 hours 
with standard supportive measures such as antiemetics and antidiarrheals. 
 
Hematologic DLT is defined as: 
≥Grade 4 neutropenia or thrombocytopenia that lasts more than 7 days after the last 
dose of study drug; 
≥Grade 3 thrombocytopenia in the presence of ≥grade 3 hemorrhage of any organ/site; 
Any grade 5 hematologic toxicity   
TREATMENT 
REGIMENS 
The projected ABC294640 doses for the escalation phase are:  250, 500, and 750 mg BID 
as determined in the single agent trial for ABC294640. The dose will be given under 
fasting conditions or after a light to moderate meal. Every 28 days as a cycle. 
 
For phase Ib study, one patient will be enrolled per week per dose. Subsequent patients 
on each cohort may not be enrolled less than one week after the previous patient. Patients 
in each dose cohort must complete cycle 1 and undergo toxicity evaluation before the next 
dose level cohort is opened. The target toxicity probability is set as 
. We treat 
a cohort of 3 patients at each dose level. The following dose-finding algorithm will be 
employed. 
• 
Treat the first cohort of patients at the 250 mg BID dose level. 
• 
Suppose that the current dose level is 
. Denote the estimated probabilities of 
toxicity based on the accumulated data to be 
 for the 3 dose levels 
, respectively. Find the dose level 
 such that  
 
Then 
– 
if 
, de-escalate to dose level 
; 
– 
if 
, escalate to dose level 
; 
– 
otherwise, stay at dose level 
 for the next cohort of patients. 
• 
Once the maximum sample size is reached, the dose with the toxicity probability 
closest to 
 is declared as the MTD. 
If the lowest dose is still too toxic, as indicated by the fact that the posterior probability 

 
 
 
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that the rate of toxicity for the lowest dose is greater than 0.33 is > 0.9, the trial will be 
terminated early for safety. 
 
For phase II study, the patients will be treated with single agent ABC294640 at the MTD 
determined from the phase IB study until disease progression or intolerable toxicities.    

 
 
 
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PRESTUDY 
ASSESSMENTS 
Screening Period: 
• 
Informed consent 
• 
Eligibility determination 
 
Baseline Period (within 30 days of Treatment Day 1) 
• 
Complete medical history 
• 
Concomitant medication assessment 
• 
Baseline Review of Systems and AE documentation 
• 
Focused Physical examination (including vital signs, HEENT, 
heart/lung/chest/abdomen/skin and neurological assessment)  
• 
Vital signs (temperature, blood pressure, pulse rate, and respiratory rate) 
• 
ECOG Performance Status  
• 
Pregnancy test (for nonsterile women of childbearing potential within 2 days of 
Treatment Day 1) 
• 
12-lead electrocardiogram 
• 
Serum chemistry 
• 
Coagulation parameters 
• 
CBC with differential 
• 
Urinalysis 
• 
Bone marrow biopsy and aspirates (should be within 2 weeks of treatment day 1) 
• 
SPEP, IFE, UPEP, serum free light chain, LDH, beta-2 microglobulin 
• 
Bone skeletal survey 
TREATMENT 
ASSESSMENTS 
Patients will be monitored weekly in Cycle 1, biweekly for Cycles 2-4, and monthly for 
subsequent cycles. The patient assessments include: 
• 
Review of concomitant medications. 
• 
Adverse Events monitoring (patient diary, review of systems). 
• 
ECOG Performance Status. 
• 
Physical exam. 
• 
Weight. 
• 
Vital signs (temperature, blood pressure, pulse rate, and respiratory rate). 
• 
Serum chemistry. 
• 
CBC with differential. 
• 
Blood sample collection for pharmacodynamics (PD) analysis. 
• 
Urinalysis. 
• 
Coagulation parameters. 
 
Myeloma treatment response assessment 
• 
SPEP, UPEP, serum free light chain, and beta-2 microglobulin before each cycle. 
• 
Bone skeletal survey at screening, then every year or at the end of treatment. 
• 
Bone marrow biopsy and aspirates at the last day of cycle #3 and cycle #6 (± 7 
days). 
PHARMACO- 
KINETIC  
ASSESSMENTS 
On Day-1, Cycle-1: a blood sample will be drawn from each subject for PK analysis prior 
to drug administration. Subsequently, all subjects will receive the morning dose of 
ABC294640 after which PK sampling will be performed for 8 hours. Patients will take 
the second dose approximately 12 hours after the initial dose was taken.  

 
 
 
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CORRELATIVE 
STUDY AND 
PHARMACO- 
DYMANIC 
ASSESSMENTS 
Correlative studies 
- 
plasma PD analysis for sphingosine 1-phosphate will be performed at the beginning of 
each cycle prior to ABC294640 drug administration and on the PK samples drawn on 
Cycle 1 Day 1. Plasma IL-6 level will be measured at the beginning of each cycle 
prior to ABC294640 drug administration for the first 6 cycles. Up to 41 different 
cytokines will be measured using multiplex cytokine assay. 
- 
sphingolipid concentrations (ceramides, sphingosine and sphingosine 1-phosphate will 
be assessed on the bone marrow aspirate supernatants at the beginning of treatment, at 
the last day of cycle #3 and at the last day of cycle #6. 
- 
expression of sphingolipid signaling genes, SK2 mRNA level or activity, c-Myc, Mcl-
1 and pS6 will be measured in bone marrow CD138+ myeloma cells isolated from 
bone marrow aspirate at the beginning of treatment, at the last day of cycle #3 and at 
the last day of cycle #6 (if sufficient CD138+ myeloma cells can be obtained). For 
patients who receive more than 3 cycles of treatment and have a repeated bone 
marrow biopsy, RNA sequencing and Assay for transposases accessible chromatin 
sequencing (ATAC seq) will be performed to assess the effects of ABC294640 on 
global gene and epigenetic expression. 
 
- 
serum cell-free PINK1/PARK2 (mitophagy markers), cell free DNA for next 
generation sequencing (for signal pathway analyses), serum bone 
destruction/formation markers (TRAP, Osteocalcin), and bone gene markers and 
Lyn/Src gene expression in both CD138+ and CD138- cells. 
 
- 
Immunologic function and immunologic panels (NK, NK T, T, B cells and myeloid 
derived suppressive cells (MDSCs) and the immune co-signalling molecules) in bone 
marrow environment 
DATA 
COLLECTION 
Data will be captured in a secure, web-based, 21 CFR Part 11 compliant system.   
INVESTIGATIVE 
SITES 
This study will be conducted at: Duke University Medical Center, Duke Cancer Institute, 
Durham, NC 27710. The Phase Ib will be conducted at Duke University Division of 
Hematologic Malignancies and Cellular Therapy. The Phase II portion may be conducted 
at multiple sites managed by the Duke Cancer Network. 
STATISTICAL 
ANALYSIS 
The MTD for ABC294640 will be determined using a Bayesian model averaging 
continual reassessment method, and is the dose at which the estimated probability of 
toxicity is closest to the target probability 0.33 among all doses. The primary safety 
analysis will be conducted using descriptive statistics of the incidence of AEs (including 
DLTs and discontinuations due to AEs). Adverse events will be coded by body system 
and summary tables with incidence rates of AEs will be generated.  Descriptive statistics 
of AEs will be reported by doses and for subsets of patients with DLTs, patients who 
discontinue due to AEs, and patients with related AEs.  Severity, duration, investigator 
attributed relationship to treatment, and outcomes of AEs will be reported. 
 
For the Phase II portion of the study, 59 patients provides 80% power to detect a HR of 
0.67 for OS and an increase from 10% to 21% for ORR based on a one-sided α = 0.025, 
controlling for multiple tests. 
 
The primary analysis will be conducted after all patients are off treatment for at least 30 

 
 
 
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days.  A follow-up analysis, which will be an addendum to the primary study report, 
will be issued once all patients are off treatment for at least 2 years or have died, 
whichever comes first. 
DURATION OF 
STUDY 
Approximately 5 years (assuming Phase I accrues at Duke Division of Hematologic 
Malignancies and Cellular Therapy in 12 months, 56 additional Phase II patients accrue 
in 24 months, with 24 months follow-up after last patient is enrolled). 
End of study is defined as the time when all subjects have completed the follow-up period. 
 
DEFINITION OF TERMS USED 
 
oC 
degrees Centigrade 
oF 
degrees Fahrenheit 
ABC294640 
3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide,  
 
hydrochloride salt 
AE 
adverse event 
ALT 
alanine aminotransferase 
ANC 
absolute neutrophil count 
aPTT 
activated partial thromboplastin time (also PTT) 
API 
active pharmaceutical ingredient  
AST 
aspartate aminotransferase 
AUC 
area under the drug concentration over time curve 
BID 
twice per day 
BSA 
body surface area 
BUN 
blood urea nitrogen 
CBC 
complete blood count 
CFR 
Code of Federal Regulations 
CHF 
congestive heart failure 
CI 
combination index 
CPC 
Cancer Protocol Committee 
CR 
complete response 
CrCl 
Creatinine Clearance 
CRF 
Case Report Form 
CSR 
Clinical Study Report 
CT 
computer-assisted tomography 
CTCAE 
Common Toxicity Criteria adverse event  
CTRC 
Clinical and Translation Research Center 
CV 
cardiovascular 
CYP 
cytochrome P450 
DCI 
Duke Cancer Institute 
DCN 
Duke Cancer Network 
DLT 
dose limiting toxicity 
DMS 
N,N-dimethylsphingosine 
DVT 
deep venous thrombosis 
ECG 
electrocardiogram 
ECOG 
Eastern Cooperative Oncology Group 
eCRF 
electronic Case Report Form 
EGF 
epidermal growth factor 
FCBP 
Females of childbearing potential 
FDA 
Food and Drug Administration 
FISH 
fluorescent in situ hybridization 

 
 
 
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FLC 
free light chain 
G-CSF 
granulocyte colony stimulating factor 
GCP 
Good Clinical Practice 
GI 
gastrointestinal 
GLP 
Good Laboratory Practice 
GM-CSF 
granulocyte macrophage colony stimulating factor 
h 
hour(s) 
HED 
human equivalent dose 
hERG 
human ether-a-go-go related gene 
HIPAA 
Health Insurance Portability and Accountability Act 
HIV 
human immunodeficiency virus 
HNSTD 
highest nonseverely toxic dose 
IB 
Investigator Brochure 
ICH 
International Conference on Harmonization 
IC50 
concentration that inhibits by 50% 
IEC 
Independent Ethics Committee  
IL-1β 
interleukin 1-beta 
IL-6 
interleukin 6 
IND 
Investigational New Drug (Application) 
iNOS 
inducible nitric oxide synthase 
IRB 
Institutional Review Board 
IV 
intravenous 
Kg 
kilogram(s) 
LC/MS 
liquid chromatography with mass-spectrometry detection 
LDH 
lactate dehydrogenase 
mg 
milligram(s) 
min 
minute(s) 
mIU 
Milli International Units 
mL 
milliliter(s) 
MM 
multiple myeloma 
mm2 
millimeter(s) squared 
mm3 
millimeter cubed 
MR 
minimal response 
MTD 
maximum tolerated dose 
MRI 
magnetic resonance imaging 
NCI 
National Cancer Institute 
NFκB 
nuclear factor kappa-B 
NHL 
non-Hodgkin’s lymphoma 
NOAEL 
no adverse effect level 
ORR 
overall response rate 
p 
probability factor 
PBMC 
peripheral blood mononuclear cells 
PD 
progressive disease 
PDGF 
platelet-derived growth factor 
PFS 
progression-free survival 
PI 
Principal Investigator 
PK 
pharmacokinetic(s) 
PO 
per os (oral) 
PR 
partial response 
PSA 
prostate-specific antigen 
PT 
prothrombin 
PTT 
prothrombin time 
QIU 
Qualified Investigator Undertaking Form 

 
 
 
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RBC 
red blood cell 
S1P 
sphingosine 1-phosphate 
SAE 
serious adverse event 
SAP 
Statistical Analysis Plan 
sCR 
stringent complete response 
s.c. 
subcutaneous 
SD 
stable disease 
SEER 
Surveillance, Epidemiology, and End Results 
SEM 
standard error of the mean 
SK 
sphingosine kinase 
SK1 
sphingosine kinase-1 
SK2 
sphingosine kinase-2 
SPEP 
serum protein electrophoresis 
STD10 
severely toxic dose in 10% of animals 
TLS 
Tumor lysis syndrome 
TNFα 
tumor necrosis factor-alpha 
TTP 
time to tumor progression 
TUNEL 
terminal deoxynucleotidyl transferase dUTP nick end labeling  
ULN 
upper limit of normal 
UPEP 
urine protein electrophoresis 
VEGF 
vascular endothelial growth factor 
VGPR 
very good partial response 
w 
week 
WBC 
white blood cells 
 

 
 
 
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1. BACKGROUND 
 
1.1 MULTIPLE MYELOMA 
Multiple Myeloma (MM) is a malignant plasma cell disorder with no standard curative therapy1.  
Symptomatic MM is characterized by a clonal proliferation of plasma cells preceding clinical findings 
that include bone lesions, fractures, anemia, renal failure and hypercalcemia2. MM affects 4.3 per 
100,000 individuals yearly3 and accounts for about 1% of all cancers and 10% of all hematological 
malignancies in the United States2. 
 
For decades, low doses of melphalan and prednisone were the cornerstone of MM treatment. However, 
complete responses under this regimen are rare, and the median time for progression is not higher than 
15 months4,5. A first significant advance in the management of MM was the upfront use of high doses of 
melphalan with autologous hematopoietic stem cell transplantation (AHSCT). Such treatment has 
allowed for improved response rates, progression free survival and, in some trials, prolonged survival in 
MM6-9. High dose melphalan with AHSCT is considered a fundamental therapeutic modality to be 
explored by younger patients upfront (after response to conventional induction therapy) and/or at the 
time of disease progression. 
 
The advent of new “biological” agents in treatment regimens for MM has led to marked improvement in 
the depth and duration of the responses obtained. The immunomodulatory drugs (i.e., thalidomide and 
lenalidomide), along with the first proteasome inhibitor (i.e., bortezomib), have shown efficacy in the 
management of both newly diagnosed as well as relapsed and refractory MM patients1,10-12. Treatment of 
MM patients with combination regimens, containing one or more biological agents, followed by 
autologous HSC transplantation consolidation have resulted in the highest response rates ever reported in 
the management of newly diagnosed MM patients13-18.  
 
Despite the use of the “biological” agents and the incoporation of AHSCT, MM remains an incurable 
disease. Patients may relapse within months after autologous hematopoietic stem cell transplantation.  
Furthermore, nearly all MM patients will eventually develop resistance to currently available agents. 
There is an unmet medical need for the development of novel therapeutic agents for this disease. It is 
particularly important to develop new agents that do not share similar mechanism of action with 
proteasome inhibitors or immunomodulatory drugs because most of the refractory/relapsed MM patients 
would have exposed to those agents during their course of treatment. 
 
Role of bone marrow transplantation in multiple myeloma: Bone marrow transplant in myeloma is 
almost always autologous. Allogeneic hematopoietic stem cell transplant is currently only performed 
under study protocol and is associated with high rates of complications and mortality. Autologous bone 
marrow transplant is usually employed for patients whose myeloma is in remission after some form of 
induction chemotherapy, and is not curative. For patients in relapse or being refractory, the value of 
autologous bone marrow transplant is limited.  Our current study focuses on patients with relapse or 
refractory multiple myeloma.  Most of these patients would have already received bone marrow 
transplantation or they are not candidates for transplant ever due to age or comorbidities.  Please note 
that history of autologous transplant generally does not increase or decrease safety of later lines of 
therapy including clinical trials, since patients would have full count recovery in about one month after 
transplant. 
 
1.2 SPHINGOLIPIDS, SPHINGOSINE KINASES AND CANCER 
Sphingolipids are an extremely diverse group of water insoluble molecules that include ceramides, 
sphingoid bases, ceramide phosphates and sphingoid base phosphates.  In addition to supporting the 
structure and fluidity of the lipid bilayer, sphingolipid metabolites function as second messengers and 

 
 
 
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hormones, and regulate cytokine-mediated cell signaling19,20. Sphingolipids are involved in a wide range 
of biological and pathological events including inflammation, cell proliferation, apoptosis, angiogenesis, 
and transformation (reviewed in21-26).   More recently, sphingolipid metabolism is being increasingly 
recognized as a key pathway in tumor cell survival and in cancer biology27-34. 
 
Among sphingolipid metabolites, ceramide, sphingosine and sphingosine-1-phosphate (S1P) are the key 
players for their biophysiological functions.  Ceramide can be produced via hydrolyzation of 
sphingomyelin in response to stimuli such as cytokines and growth factors. Ceramide is further 
hydrolyzed to sphingosine.  Sphingosine is then rapidly phosphorylated by sphingosine kinases (SK) to 
S1P.   Ceramide and sphingosine are pro-apoptotic, inducing apoptosis in tumor cells without disrupting 
quiescent normal cells35-38. In contrast, S1P is mitogenic and anti-apoptotic.  A critical balance, i.e. a 
ceramide:S1P rheostat, is hypothesized to determine the fate of the cell28,39,40. 
 
Sphingosine Kinase (SK) is an innovative molecular target for anti-cancer therapy because of its critical 
role in sphingolipid metabolism, which is known to regulate cancer cell proliferation and activation.  SK 
is also a critical mediator of the actions of inflammatory cytokines and angiogenic growth factors.  
Therefore, inhibitors of SK are expected to have utility for the treatment of a variety of 
hyperproliferative, inflammatory and angiogenic diseases.  
 
 
1.2.1 
Sphingolipid metabolism in cancer 
 
The mechanisms and effects of the interconversion of sphingolipids have been the subjects of a growing 
body of scientific investigation.  Sphingomyelin is a building block for cellular membranes and also 
serves as the precursor for potent lipid messengers that have profound cellular effects.  As indicated in 
Figure 1, ceramide is produced by the hydrolysis of sphingomyelin in response to several growth 
stimulatory and/or inflammatory signals.  Ceramide induces apoptosis in tumor cells without disrupting 
quiescent normal cells.  Additionally, ceramide can be further hydrolyzed by the action of ceramidase to 
produce sphingosine, which is phosphorylated by sphingosine kinases (SK1 and SK2) to produce 
sphingosine 1-phosphate (S1P).  Studies in various cancer cell lines consistently demonstrate that S1P 
induces proliferation and protects cells from ceramide-induced apoptosis. Therefore, a critical balance, 
i.e. a ceramide / S1P rheostat, has been hypothesized to determine the fate of tumor cells.  In this model, 
the balance between ceramide and S1P determines whether a tumor cell proliferates or undergoes 
apoptosis.  Inhibition of SK activity causes the accumulation of ceramides along with the depletion of 
S1P, thereby driving cancer cells into apoptosis.  Many studies have shown that a variety of proliferative 
factors rapidly elevate SK activity.  Signaling via the Ras-MAP kinase pathway is dependent on SK 
activity, as are angiogenic processes such as cell motility, mitogenesis in smooth muscle cells and 
endothelial cell differentiation.  
 
In another aspect of tumor biology, sphingolipids regulate the sensitivities of cancer cells to anticancer 
drugs and ionizing radiation.  For example, ceramides enhance apoptosis in response to paclitaxel and 
etoposide.  Therefore, disruption of metabolism of ceramides to S1P is a new method to enhance drug 
and radiation sensitivity in cancer cells, including those that are resistant to traditional cytotoxic drugs 
such as multiple myeloma (MM). 

 
 
 
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1.2.2   Differential roles of SK1 and SK2  
 
Sphingosine kinase has 2 isoforms: SK1 and SK2. The relative roles of SK1 and SK2 in tumor biology 
have been of great interest to many investigators, and were a central issue in the selection of 
ABC294640 as a clinical agent. Space limitations preclude full discussion of this issue, but many studies 
have suggested different biological roles of the two SK isozymes33,41-45. Most studies have focused on 
SK1 because it is the predominant isozyme in most cells, and is upregulated in many cancers. The latter 
response is likely due to hypoxia because SK1 is HIF-regulated 46-49, making it difficult to discern 
whether overexpression is required to drive tumor growth or is a consequence of tumor growth. Target 
validation studies using cDNA transfection or RNA interference are inconsistent in ascribing dominance 
to either isozyme. For example, overexpression of SK1 has been shown to be oncogenic 50,51, while 
transfection with SK2 was originally reported to inhibit cell growth and to induce apoptosis 52. However, 
these effects of SK2 are not dependent on its catalytic activity, suggesting that the antiproliferative 
effects may be mediated by its BH3 domain 52. Consistent with this are the observations that 
physiological levels of SK2 do not inhibit DNA synthesis 53. Similarly, EGF is reported to induce the 
expression of SK1 but not SK2 in MCF-7 cells 54; however, it has also been reported that EGF activates 
SK2 in MDA-MB-453 cells 55. Thus additional work is needed to refine our knowledge about SK1 vs. 
SK2 biology. 
 
  
1.2.3    Development of SK inhibitors 
 
Because S1P is the direct mitogenic messenger, inhibition of its production 
should have antiproliferative effects on tumor cells, making SKs prime 
targets for inhibition. Research at Apogee has identified structurally novel 
“drug-like” inhibitors of SK1 and SK2 56-62, and ABC294640 was selected 
as the first compound in this program for clinical testing. Much of the 
pharmacology of ABC294640 61,62, as well as its antitumor 61,63-69 and anti-
inflammatory 58,59,70-72 activities have been published. Space limitations 
preclude full discussion of the properties and in vivo activities of ABC294640, but key points include: Ki 
for SK2 = 9 µM, competitive with respect to sphingosine; No inhibition of SK1 or panel of protein 
kinases; Depletes S1P and elevates ceramide in tumor cells; Plasma concentrations can reach >200 µM 
without toxicity; Good pharmacokinetics, oral bioavailability and biodistribution; No hematologic or 
major organ toxicity; Effective in multiple cancer models; Pharmacodynamic marker (plasma S1P) for in 
vivo and clinical testing; Therapeutically effective in multiple animal models of inflammation including: 
Figure 1. A simplified model of 
sphingolipid metabolism. Growth
stimulatory signals increase the 
activity of ceramidase and SK 
driving the production of S1P, 
which promotes cell proliferation,
angiogenesis and inflammation; 
while depleting the cells of 
ceramide, thereby inhibiting 
apoptosis.   
 
Cl
HN
O
N
Figure 2.  ABC294640

 
 
 
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Crohn’s Disease, ulcerative colitis, rheumatoid arthritis, osteoarthritis and ischemia / reperfusion injury; 
cGMP synthesis and formulation (gelatin caps) completed; cGLP toxicology studies in rats and dogs 
completed; IND application approved by FDA; Single-agent phase I clinical trial in patients with 
advanced solid tumors completed as discussed below; and Excellent correlation between mouse and dog 
PK parameters with human data from the clinical trial.  
 
1.3 Summary of ABC294640 Phase I Clinical Data in Solid Tumors 
 
1.3.1. Study design. The first-in-human phase I clinical trial of ABC294640 in patients with advanced 
solid tumors has completed enrollment at the Hollings Cancer Center at MUSC (ClinicalTrials.gov ID 
NCT01488513). ABC294640 was administered orally on a continuous schedule, with 28 days 
constituting a Cycle. Tumors were reimaged every two Cycles (8 weeks), and patients were allowed to 
continue receiving the drug if there is no disease progression by RECIST criteria. Primary endpoints for 
the trial were: identification of the Maximum Tolerated Dose (MTD); determination of the Dose 
Limiting Toxicities (DLTs); and evaluation of safety. Secondary endpoints were: determination of PK 
(Day 1 and Day 28 of Cycle 1); evaluation of PD effects; and assessment of antitumor activity. 
 
1.3.2.  Safety data. Twenty-two patients were enrolled, as outlined in Table 1. Three patients (one at 250 
mg bid, and two at 500 mg bid) were unable to complete Cycle 1 due to complications from their 
disease, and were replaced. The first patient on study at 250 mg qd developed grade 4 hyperglycemia in 
the setting of rapidly progressing pancreas cancer; however, no other patients experienced possibly drug-
related hyperglycemia. Among the four patients enrolled at 750 mg bid, one ovarian cancer patient had 
dose-limiting grade 3 nausea and vomiting, and two patients were unable to complete Cycle 1 due to 
diverse possibly drug-related toxicities. The 750 mg bid dose level was declared not tolerable, and the 
500 mg dose level was expanded. One patient at the expanded 500 mg dose level was dose reduced at 
Cycle 3 due to grade 1 AEs. The other five patients at 500 mg bid tolerated the drug well, and the 500 
mg bid dose level was established as the recommended phase II dose. Across all dose levels, the most 
common drug-related toxicities were nausea, fatigue, vomiting, and diarrhea. In addition, 2 patients 
experienced psychiatric disorders including agitation/anxiety, mood changes, and/or hallucinations; and 
5 patients experienced grade 1-2 nervous system disorders, including dizziness, dysarthria, dysgeusia, 
dysesthesia, memory loss, muscle spasms, paraesthesias, somnolence, and/or spasticity that resolved 
upon discontinuation of ABC294640.   
 
 
 
 
 
Patient 
Study ID 
Dose 
Level 
Primary Histologic Diagnosis 
Number of 
Treatment Cycles 
DLT 
 
250 mg qd 
Pancreatic adenocarcinoma 
1 
Grade 4 
hyperglycemia 
2 
250 mg qd 
Cholangiocarcinoma 
19 
None 
3 
250 mg qd 
Rectal adenocarcinoma 
3 
None 
4 
250 mg qd 
Colon adenocarcinoma 
3 
None 
5 
250 mg qd 
Carcinoma of unknown primary 
5 
None 
6 
250 mg qd 
Urothelial carcinoma 
13 
None 
7 
250 mg bid 
Colon adenocarcinoma 
3 
None 
8 
250 mg bid 
Rectal adenocarcinoma 
1 
None 
9 
250 mg bid 
Colon adenocarcinoma 
4 
None 
10 
250 mg bid 
Hepatocellular carcinoma 
4 
None 
11 
500 mg bid 
Lung carcinoma 
3 
None 
12 
500 mg bid 
Bile duct cancer 
5 
None 
13 
500 mg bid 
Pancreatic adenocarcinoma 
1 
None 
14 
500 mg bid 
Pancreatic adenocarcinoma 
2 
None 
15 
750 mg bid 
Ovarian cancer 
1 
Grade 3 
nausea/vomiting 
16 
750 mg bid 
Pancreatic adenocarcinoma 
1 
None 
17 
500 mg bid 
Bladder cancer 
3 
None 
18 
750 mg bid 
Colon adenocarcinoma 
1 
None 
19 
500 mg bid 
Cholangiocarcinoma 
4 
None 
20 
None 
Lung carcinoma 
None 
None 
21 
750 mg bid 
Urothelial carcinoma 
2 
None 
22 
500 mg bid 
Adrenal cortical carcinoma 
1 
None 
 
 
 
1.3.3.   Efficacy data. Of the 21 ABC294640-treated patients, 11 (52%) completed at least 2 drug 
cycles and 7 (64%) of those had Stable Disease at that time. The number of treatment cycles is also 
indicated in Table 1. Subjects included a patient with advanced HCC who lived for 10 months after 

 
 
 
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completing 4 cycles, a patient with recurrent metastatic bladder cancer who received 12 cycles and is 
alive more than 2 years after starting the trial, and a patient with advanced cholangiocarcinoma who 
received 18 cycles (PFS for 72 weeks).  
 
1.3.4. Pharmacokinetic data. PK profiling was conducted on Days 1 and 28 of Cycle 1. The data from 
patients treated with 500 mg of ABC294640 are shown in Figure 3. After oral dosing, plasma 
concentrations of ABC294640 typically peak at 1-2 hours, and then decline with a half-time of clearance 
of ~4 hours. The peak plasma concentrations (Cmax) and AUCs within a cohort were generally similar. 
Notably, the Cmax and AUC values for most patients were not significantly different between Day 1 and 
Day 28, indicating a lack of metabolic adaptation to the drug. Importantly, Cmax levels of ABC294640 in 
patients receiving 500 mg of the drug were in the range expected to have therapeutic activity. 
Specifically, antitumor activity in mouse models occurs at ABC294640 doses of 25-50 mg/kg 73, which 
results in plasma Cmax levels of 9-20 µM. Patients receiving a 500 mg dose of ABC294640 had Cmax 
levels averaging 16.4 µM (Figure 3), with 9 of 12 profiles exceeding the threshold of 9 μM. Patients 
given 250 mg of ABC294640 exceeded the Cmax threshold approximately 50% of the time, while all 
patients receiving 750 mg of ABC294640 reached this level. Because the t1/2s are equal in mice and 
humans, the drug exposure in patients at 500 mg and higher dose levels is expected to be sufficient to 
inhibit SK2 in the tumors. 
 
 
Plasma Concentration After 500 mg Dose
0
10
20
30
0
5
10
15
20
25
Predicted
therapeutically
active Cmax
Time after Administration (Hr)
Plasma [ABC294640] (M)
Peak  Plasma Concentration
250 mg
500 mg
750 mg
0
10
20
30
40
50
Predicted
therapeutically
active Cmax
ABC294640  Dose
Plasma ABC294640 Cmax (M)
AUC
250 mg
500 mg
750 mg
0
50
100
150
200
ABC294640  Dose
Plasma ABC294640 AUC (M•hr)
 
 
1.3.5. Pharmacodynamic data. The most direct PD biomarker for inhibition of SK2 by ABC294640 is 
S1P in the target tissue. However, tumor samples were not obtained in the phase I clinical trial, so we 
measured S1P in plasma as a biomarker for inhibition of SK2. ABC294640 treatment caused rapid 
decreases in plasma S1P levels (Figure 4). In general, plasma S1P reached a minimum at 12 hr after 
ABC294640 treatment and recovered to baseline by 24 hr. This is consistent with the PK of the drug and 
supports the BID dosing schedule. The maximum S1P decreases for the 250, 500 and 750 mg cohorts 
were 51±6% (n=10), 46±9% (n=5) and 54±14% (n=2), respectively, indicating that ABC294640 
exposure attained with the 250 mg dose are sufficient to optimally inhibit S1P generation in the patients.  
Figure 3.  Pharmacokinetics of ABC294640 in humans.  Left Panel. Plasma was isolated from patients given 500 mg of ABC294640 
on either Day 1 or Day 28 of Cycle 1 and levels of ABC294640 were quantified using a GLP LC/MS method. Values represent the 
mean ± SEM for 7 data sets. Antitumor activity in mouse models occurs at ABC294640 doses ≥25 mg/kg, which provides plasma Cmax 
levels of 9 µM. The red line indicates the Cmax predicted to have antitumor activity based on the mouse tumor models. Middle Panel. 
Cmax values are shown for patient samples collected on either Day 1 or Day 28 (there is no change in Cmax or AUC in this period). The 
red line indicates the Cmax predicted to have antitumor activity based on the mouse tumor models. Right Panel. The AUCs for 
ABC294640 at each dose level are indicated. 

 
 
 
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0
10
20
30
0
20
40
60
80
100
120
140
160
250 mg
500 mg
750 mg
Time (hr)
Plasma [S1P] (% of time=0)
 
1.4 
Rationale for a clinical trial evaluating ABC294640 in patients with MM 
MM is characterized by the accumulation of malignant plasma cells that are defective in apoptotic cell 
death 74,75. Sphingolipid metabolism is being increasingly recognized as a key pathway in regulating 
myeloma cell survival, migration and adhesion, and responses to treatment. For instance, S1P up-
regulates Mcl-1 expression in myeloma cells and protects myeloma cells from apoptosis76. Additionally, 
S1P promotes myeloma cell adhesion and migration to bone marrow77. Consistent with the importance 
of S1P in myeloma pathogenesis, targeting S1P receptors using FTY720 induces apoptosis and 
overcomes drug resistance78. Furthermore, sphingolipid metabolites (i.e., ceramide) play a critical role in 
thalidomide-induced antiangiogenic effects 79. Importantly, SK mediates the survival and proliferation 
signal pathway induced by IL-6, the most critical cytokine in myeloma pathogenesis80. 
There is an unmet medical need for the development of novel therapeutic agents for MM. It is 
particularly important to develop new agents that do not share similar mechanism of action with 
proteasome inhibitors or immunomodulatory drugs because most of the refractory/relapsed MM patients 
would have exposed to those agents during their course of treatment. 
 
1.5  
Pre-clinical efficacy of ABC294640 for Multiple Myeloma 
We have published data indicating significant single-agent activity for ABC294640 (at clinically 
achievable plasma concentrations) in an established xenograft model for myeloma, including 
treatment for established tumors81.  The data are summarized below. 
 
1.5.1 Sphingosine Kinase 2 is over-expressed in myeloma cells.  
To determine the potential utility of targeting SKs for the treatment of MM, we measured the gene 
expression levels of SK1 and SK2 in myeloma cells.  We measured SK1 and SK2 mRNA expression in 
a publicly available myeloma microarray dataset, in myeloma cell lines, and in freshly isolated human 
bone marrow CD138+ myeloma cells (Fig. 5).  We downloaded the GSE6477 Affymetrix microarray 
dataset originated by Mayo Clinic.82,83 This dataset contained microarray mRNA gene profile on purified 
plasma cells isolated from normal control subjects (n=15) or newly diagnosed MM patients (n=73).  We 
generated the Robust Multi-array Average (RMA)- normalized gene expression data for SK1 and SK2 
and compared their expression levels between normal subjects and newly diagnosed MM patients.  As 
shown in Fig. 5A, SK2 expression was increased in MM patients compared to normal subjects 
(p=0.046), whereas there was no significant difference in SK1 expression level in plasma cells between 
MM patients and normal subjects.  
Figure 4. Effect of ABC294640 treatment on plasma 
S1P levels.  S1P concentrations were normalized to the 
Time 0 levels for individual patients at each dose of 
ABC294640.  S1P levels in patients receiving 250, 500 
mg and 750 mg of ABC294640 are indicated in blue, 
red and green, respectively. 

 
 
 
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SK1 and SK2 mRNA expression levels in two B cell lines (EBV-immortalized B cell line and ATCC B 
lymphocyte cell line) and seven myeloma cell lines (NCI-H929, OPM1, U266, RPMI-8226, RPMI-
8226-Dox40, MM.1R and MM.1S) were measured by quantitative RT-PCR.  As shown in Fig. 5B, the 
mRNA expression level of SK2 was higher than that of SK1 in all tested myeloma cell lines except 
RPMI-8226-Dox40 cells.  Furthermore, the mRNA expression level of SK2 was higher in all seven 
myeloma cell lines than that in the two B cell lines.    
 
We also determined the SK1 and SK2 mRNA gene expression in freshly isolated primary human bone 
marrow (BM) CD138+ MM specimens. CD138+ plasma cells were isolated from the BM aspirates of 
normal controls, MGUS (monoclonal gammopathy of undetermined significance) patients, or MM 
patients including amyloidosis patients.  No difference in SK1 mRNA expression was observed between 
these three populations of patients (data not shown).  Interestingly, SK2 gene expression was increased 
in the CD138+ cells in 10 out of 34 (29%) MM patients (Fig. 5C).  We performed additional subset 
analyses to determine whether SK2 mRNA expression correlated with myeloma disease stage, 
cytogenetic profile, M protein level or BM plasma cell number. No correlation was observed with these 
subset analyses (data not shown).   
Figure 5: SK2 was over-expressed in myeloma cells. A: SK1 and SK2 gene expression in GSE6477 Affymetrix microarray 
dataset. Publicly available Affymetrix microarray data set GSE6477 was downloaded and the RMA normalized gene expression data 
were generated.  The SK1 and SK2 expression level between plasma cells from normal subjects (blue bar; n=15) and purified CD138+ 
cells from newly diagnosed MM (red bar; n=73) were compared.  B: SK1 and SK2 expression in myeloma cell lines and B cell lines.  
RNA was extracted from two B cell lines (EBV-Immortalized B cells (IBC) and ATCC® CCL-156 B lymphocytes) and seven MM cell 
lines (NCI-H929, OPM1, U266, RPMI-8226, RPMI-8226-Dox40, MM.1R, and MM.1S) and RT-PCR was performed for SK1 and SK2. 
Relative SK1 and SK2 mRNA expression with respect to β-Actin was shown (Mean± SEM of 3 separate sets of experiments). C: SK2 
expression in primary human bone marrow CD138+ cells. Primary human CD138+ cells were isolated using CD138 enrichment kit 
from the BM aspirates of normal subjects (n=5), MGUS patients (n=6) and myeloma patients (n=34). SK2 gene expression was 
normalized against β-Actin control (Each dot represented one individual patient and the two solid circles represented amyloidosis 
patients). D: Sphingosine level in myeloma cell lines and B cell lines. Sphingosine was measured by HPLC in freshly prepared EBV-
Immortalized B cells, ATCC B lymphocytes, and 6 MM cell lines (NCI-H929, OPM1, U266, RPMI-8226, RPMI-8226-Dox40, and 
MM.1S). Data represented sphingosine concentration (pmole/1×106 cells) (Mean± SEM of one of 4 separate sets of experiments) (*: 
p<0.05; **: p<0.01). 

 
 
 
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SKs catalyze the phosphorylation of sphingosine to S1P and sphingosine derives from ceramide. To 
determine if the over-expression of SK2 in myeloma cells affects the levels of ceramides, sphingosine 
and S1P, mass spectrometry measurement of 14 different ceramides, sphingosine and S1P was 
performed in two B cell lines and six MM cell lines.  The levels of ceramides and S1P varied highly 
among MM cell lines and between B cell lines and MM cell lines (data not shown).  Interestingly, 
sphingosine level was lower in MM cells than that in B cells and the difference was statistically 
significant for 4 out of 6 MM cell lines we tested (Fig. 5D). The decrease in the level of sphingosine in 
MM is consistent with increased sphingosine kinase gene expression.  
 
1.5.2. SK2-specific shRNA inhibits myeloma proliferation and induces Caspase 3-mediated cell 
death.  
To determine the roles of SK2 in MM cell survival and proliferation, we used specific shRNA to 
knockdown SK2 expression in MM cells.  Lentiviral vector expressing SK2-specific shRNA or control 
shRNA was constructed and used to transduce MM cell lines.  Both SK2-specific shRNA and control 
shRNA effectively transduced MM cell lines as demonstrated by high level of DsRFP expression (Fig. 
6A).  SK2-specific shRNA decreased SK2 mRNA expression by ~80% (Fig. 6B).  SK2-specific shRNA 
effectively inhibited myeloma cell proliferation as measured by MTT assay (Fig. 6C).  To further 
determine the effect of SK2 on myeloma cell proliferation, we transduced OPM1 myeloma cells with 
SK2-specific shRNA or control shRNA.  We then labeled the cells with CellTrace Violet Cell 
Proliferation dye and measured dye fluorescence intensity 7 days later (Fig. 6D).  With cell division, the 
dye is diluted and the fluorescence intensity is reduced.  As shown in Fig. 6D, compared to control 
shRNA, SK2-specific RNA inhibited myeloma cell proliferation and division.  Additionally, we found 
that SK2-specific shRNA activated Caspase 3 (Fig. 6E). These data suggested that SK2 plays an 
important role in both cell proliferation and survival of myeloma cells, and thus provides a therapeutic 
target for the treatment of MM.     
 
 
 
 
 
 
Figure 6: SK2-specific shRNA inhibited cell 
proliferation and induced Caspase 3 activation 
in myeloma cells. OPM1 cells were transduced 
with lentiviruses expressing SK2-shRNA-DsRFP 
or control shRNA-DsRFP for 4h.  The cells were 
then washed and grew in regular culture medium 
for additional 48h. A: Fluorescent microscopy 
image showing DsRFP expression.  B: 
Expression of SK2 mRNA in SK2-shRNA- or 
control shRNA- transduced OPM1 cells. C: Cell 
proliferation by MTT assay.  Cell proliferation 
was measured using MTT assay at 0h and 48h 
following transduction. D: Cell proliferation by 
flow cytometry. OPM1 cells transduced with SK2-
shRNA or control shRNA were stained with 
CellTrace Violet Cell Proliferation dye and allowed 
to proliferate for 7 days. The dye fluorescence 
intensity was measured by flow cytometry. E: 
Activation of Caspase 3. OPM1 cells were 
transduced with SK2-shRNA viruses or control 
shRNA viruses. Forty-eight hrs later, the cells were 
stained with Fixable Live/Dead cell dye, then fixed 
and permeabilized, and stained with Caspase-3 
antibody. Caspase 3 intensity was gated on live cell 
population. Data were representative of 4 separate 
experiments. 

 
 
 
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1.5.3. SK2-specific inhibitor (ABC294640) inhibits myeloma growth in vitro.  
We next tested the effectiveness of the SK2-selective inhibitor (ABC294640) in killing myeloma cells in 
vitro. ABC294640 is the most advanced, non-lipid-based oral SK2 inhibitor and shows no inhibition for 
SK1 or panel of protein kinases.61 ABC294640 is currently undergoing single agent phase I/II clinical 
trial at our institute for solid tumors.  We treated 6 different MM cell lines with various concentrations of 
ABC294640 and found that ABC294640 inhibited myeloma cell growth, including steroid resistant 
MM.1R cells (Fig. 7A). Among the 6 MM cell lines we tested, OPM1 appears to be the most sensitive 
cell line to ABC294640 treatment whereas U266 is relatively resistant.  ABC294640 inhibited myeloma 
cell growth as early as 16 hours after exposure (Fig. 7B).  
 
To determine if ABC294640 induces cytotoxic or cytostatic effects on MM cells, we cultured MM cells 
with 30μM of ABC294640 and then quantified live cell numbers over time. As shown in Fig. 7C, 
ABC294640 exhibited cytotoxic effects on the majority (7 out of 8) of MM cell lines we tested, except 
for U266 cells. We further found that ABC294640 treatment induced apoptotic cell death in myeloma 
cells as demonstrated by Annexin V staining (Fig. 7D), Caspase 3 activation (Fig. 7E), Caspase 9 
activation, and PARP cleavage (Fig. 7F). ABC294640 induced growth arrest, but no apoptosis in U266 
cells (data not shown). 
 
 
 
 
 
 
 
 

 
 
 
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We also tested the anti-tumor effects of ABC294640 on primary human CD138+ myeloma cells. Human 
CD138+ myeloma cells were freshly isolated from myeloma patient’s BM aspirate and treated with 
various concentrations of ABC294640.  Cell proliferation was then measured by MTT assay. 
ABC294640 inhibited primary human myeloma cells with the same efficacy as with the most sensitive 
myeloma cell lines (OPM1) (Fig. 7G), demonstrating the potential clinical utility of ABC294640 in the 
treatment of MM.  
 
The effects of SK1 inhibitor, Myriocin (a natural product inhibitor of serine palmitoyltransferase), and 
FTY720 (a S1P receptor antagonist) on myeloma cell proliferation were investigated (Figure 8). 
Consistent with previous reports by others,78 FTY720 inhibited myeloma cell growth (Figure 8A).  No 
inhibitory effects of Myriocin or SK1 inhibitor on myeloma cells were observed (Figure 8B and 8C).    
These data again support the important role of SK2 in myeloma cell proliferation and survival. 
 
Figure 7: ABC294640 inhibited cell proliferation and induced apoptosis in MM cells. A: Dose-dependent inhibition of cell 
proliferation by ABC294640.  Six different MM cell lines were treated with various concentrations of ABC294640 for 48h and 
cell proliferation was measured by MTT assay (Mean± SEM of one of 3 separate sets of experiments). B: Time course of 
proliferation inhibition by ABC294640. Six different MM cells lines were treated with 30µM of ABC294640 or DMSO for 
various durations. Cell proliferation was analyzed by MTT assay at the time-points indicated (Mean± SEM of one of 3 separate sets 
of experiments). C: Cytotoxic effects of ABC294640 on MM cells. OPM1 cells were treated with 30µM of ABC294640 or 
DMSO and total live cells were quantified at the time-points indicated (Mean± SEM of 3 separate sets of experiments). D: 
Increased Annexin V+ cells by ABC294640.  OPM1 cells were treated with 30µM of ABC294640 or DMSO for 16h and cells 
were stained with Annexin V and 7-AAD and analyzed by flow cytometry. Data shown were representative of 3 separate sets of 
experiments. E: Caspase 3 activation. OPM1 cells were treated with 30µM of ABC294640 or DMSO for 16h and cells were then 
fixed, permeabilized, and stained Caspase3 antibody. F: Caspase 9 activation and PAPR cleavage. OPM1 cells were treated with 
30µM of ABC294640 (indicated as A) or DMSO control (indicated as D) for 3h, 6h, 9h and 12h and analyzed for PARP and 
cleaved PARP, full length Caspase-9 and cleaved Caspase -9 by western blot.  β-actin was used as loading control (data were 
representative of 3 separate sets of experiments). G: Inhibition of primary human CD138+ myeloma cells by ABC294640. 
Primary human CD138+ cells were freshly isolated using CD138 enrichment kit from the BM aspirates of myeloma patients and 
cultured in triplicate at 1×104 cells in 100 μl of RPMI1640 medium supplemented with 2 mM Glutamax and 10% fetal calf serum 
containing DMSO control or various concentrations of ABC294640 for 24h at 370C under 5% CO2. Cell proliferation was then 
measured by MTT assay. OPM1 cells were similarly treated for comparison (n=3; ID#: 7244, 7452 and 7476). 
Figure 8: Effects of 
FTY720, Myriocin and 
SK1-II on myeloma cell 
proliferation. MM cell 
lines were treated with 
various concentrations of 
FTY720 (A), Myriocin (B) 
for 48hr or SK1I (C) for 
24hr or 48hr, and cell 
proliferation was measured 
by MTT assay. 

 
 
 
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1.5.4. ABC294640 up-regulates Noxa expression and promotes proteasome degradation of Mcl-1. 
We performed extensive mechanistic studies to understand the pathways through which SK2 inhibition 
induces myeloma cell death. Extensive studies have demonstrated an essential role of Mcl-1in the 
survival of human myeloma cells.84,85 Mcl-1 is over-expressed in more than half of newly diagnosed 
MM patients and in ~81% of relapsed MM patients.86  Furthermore, recent studies suggested that SK1 
plays a key role in Mcl-1 expression induced by IL-6.76 Therefore, we examined if ABC294640 could 
affect the expression of Mcl-1 in MM. As shown in Fig. 9A, ABC294640 treatment led to down-
regulation of Mcl-1 protein expression in MM cells, with the largest reduction occuring in OPM1 cells, 
which is also the most sensitive cell line for inhibition of proliferation by ABC294640. ABC294640 
caused minimal changes in Mcl-1 expression in U266 cells. 
 
Protein expression is controlled by the rate of biosynthesis and degradation. We thus determined if 
ABC294640 affected Mcl-1 gene transcription.  MM cell lines were treated with ABC294640 for 16 hr 
and Mcl-1 mRNA was quantified by RT-PCR. Additionally, OPM1 cells were stably transduced with 
SK2-specific shRNA or control shRNA and Mcl-1 mRNA was measured. Inhibition of SK2 by either 
ABC294640 or shRNA did not affect Mcl-1 gene transcription (data not shown), suggesting that 
ABC294640 down-regulated Mcl-1 expression at the post-transcription level. We next tested if 
ABC294640 would increase the rate of Mcl-1 degradation. To this end, we treated OPM1 cells with 
DMSO or ABC294640 for 3 hrs, and then added cycloheximide to inhibit new protein synthesis.  Mcl-1 
protein levels were measured by immunoblot every hour for 4 hrs.  As shown in Fig. 9B, ABC294640 
treatment significantly increased Mcl-1 degradation.  
 
Proteasome degradation plays an important role in regulating protein stability. We thus tested if 
ABC294640 treatment promoted Mcl-1 degradation in a proteasome-dependent manner. OPM1 cells 
were treated with ABC294640 alone, proteasome inhibitor (either MG132 or bortezomib) alone or in 
combination of ABC294640 with proteasome inhibitor.  MG132 and bortezomib partially but 
reproducibly protected Mcl-1 from degradation induced by ABC294640 treatment (Fig. 9C).  These data 
suggested that ABC294640 at least in part increased Mcl-1 proteasome degradation.  
 
Noxa is a pro-apoptotic, Bcl-2 homolog (BH) 3-only member of the Bcl-2 family. Treatment with 
Bortezomib or arsenic trioxide induced up-regulation of Noxa while down-regulating Mcl-1.87,88 These 
studies and others89 suggested that Noxa and Mcl-1 form a complex and the Noxa/Mcl-1 ratio plays an 
important role in regulating apoptosis. We thus examined the effects of ABC294640 on Noxa 
expression.  ABC294640 treatment increased Noxa protein expression levels by 50% to 2 fold (Fig. 9D).  
Additionally, ABC294640 up-regulated Noxa mRNA expression in all eight MM cell lines we tested by 
at least 5 fold (Fig. 9E).  These data suggested that ABC294640 treatment shifted the Noxa/Mcl-1 
apoptosis rheostat towards favoring cell death.  
 

 
 
 
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1.5.5. ABC294640 down-regulates pS6 and promotes proteasome degradation of c-Myc. 
MM originates from post-germinal-center B cells and c-Myc is a class oncogene dysregulated in post-
germinal center malignancies including MM. Using specific FISH probes, translocation involving Myc 
was found in 19 of 20 MM cell lines and approximately 50% of advanced primary MM tumors90. c-Myc 
plays a critical role in the progression from MGUS to MM 91,92. The progression of MGUS to myeloma 
is associated with several-fold increase in MYC RNA expression 93. Overexpression of MYC leads to 
the development of myeloma phenotypes in the Vk*MYC mouse model 94 and synergizes with IL-6 in 
plasma cell tumor formation in mice95. Furthermore, targeting MYC using short hairpin RNA or a 
selective small molecule inhibitor of MYC-Max heterodimerization (10058-F4) induces myeloma cell 
death 96. These data suggest that myeloma cells are addicted to c-MYC activity, and that c-MYC is 
indispensable in myeloma development 93. Furthermore, constitutive activation of PI3K/AKT/mTOR 
pathway is a common event in MM pathogenesis and contributes to MM proliferation and survival.97  
Our unpublished data indicated that the downstream of mTOR pathway, i.e., pS6, was highly up-
Figure 9: ABC294640 enhanced Mcl-1 proteasome degradation and increased Noxa expression. A: ABC294640 down-regulated 
Mcl-1 protein expression. MM cells (OPM1, RPMI-8226, MM.1S, JK6L, and U266) were treated with 30µM of ABC294640 (A) or 
DMSO (D) for 16 hr and whole cell lysates were prepared and analyzed for Mcl-1 expression by Western blot. β-actin was used as the 
loading control. Data were representative of 3 separate sets of experiments.  B: A294640 increased Mcl-1 protein degradation. OPM1 
cells were treated with 30µM of ABC294640 or DMSO for 3 hr and then cycloheximide (100mg/ml) was added.  Cells were collected at 
each hour after cycloheximide treatment and whole cell lysate was prepared and analyzed for Mcl-1 expression by Western blot. β-actin 
was used as the loading control. Graph below represented the quantification of western blots. Western blots were quantified using ImageJ. 
Data were representative of 2 separate sets of experiments.  C: Proteasome inhibitor (MG132 and bortezomib) prevented the 
degradation of Mcl-1 by ABC294640. OPM1 and JK6L cells were treated with DMSO control buffer, proteasome inhibitor MG132 
(1µM) or Bortezomib (50nM) for 1 h, followed by treatment with DMSO or 30 µM of ABC294640 for additional 6h.  Whole cell lysate 
was prepared and analyzed for Mcl-1 expression by Western blot. Data were representative of 2 separate sets of experiments.  D: 
ABC294640 increased Noxa protein expression. OPM1, RPMI8226, MM.1S, and JK6L were treated with 30µM of ABC294640 (A) or 
DMSO (D) for 16 hr and whole cell lysates were prepared and analyzed for Noxa expression by Western blot.  E: ABC294640 induced 
Noxa gene expression. Eight MM cell lines were treated with 30µM of ABC294640 (A) or DMSO (D) for 16 hr and RNA was isolated 
and analyzed for Noxa gene expression by RT-PCR. Gene expression was normalized against β-actin internal control. Graphs represented 
the fold change of Noxa mRNA in ABC294640- treated MM cells lines compared to DMSO- treated cells. Data shown in the figure were 
representative of at least 2 separate sets of experiments. 

 
 
 
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regulated in nearly all myeloma patients’ bone marrow samples. Given the importance of c-Myc and pS6 
in MM cell survival, we examined the effects of ABC294640 on the expression of c-Myc and pS6 in 
MM cells. ABC294640 treatment significantly down-regulated the expression of c-Myc and pS6 (Fig. 
10A).  U266 has no detectable c-Myc expression by Western blot analysis. We performed additional 
studies similar to those described with Mcl-1 to understand the mechanisms through which ABC294640 
down-regulated c-Myc expression.  We found that ABC294640 or SK2-specific shRNA did not affect 
the rate of c-Myc gene transcription (data not shown). Cycloheximide study suggested that ABC294640 
increased c-Myc protein degradation (Fig. 10B).   MG132 protected c-Myc from ABC294640-induced 
c-Myc degradation, suggesting that ABC294640 enhances proteasome degradation of c-Myc (Fig. 10C).   
 
 
1.5.6. ABC294640 synergizes with Bcl-2 inhibitor in the killing of myeloma cells.  
We reasoned that combined chemotherapy incorporating drugs with different mechanisms of action is 
likely to be more effective in killing myeloma cells and could overcome drug resistance. To this end, we 
examined the anti-myeloma activity of ABC294640 in combination with other agents. We were 
particularly interested in combining ABC294640 with Bcl-2 inhibitor for the treatment of MM, because 
ABC294640 per se did not affect Bcl-2 expression (Fig. 11A). We treated MM cell lines with various 
concentrations of ABT-737 (a Bcl-2 inhibitor) in combination with 15µM of ABC294640. Cell 
proliferation was then measured by MTT assay.  Combining ABC294640 with ABT-737 led to greater 
inhibition of MM cell proliferation (Fig. 11B). Additional ABC294640 concentrations were tested with 
ABT-737 and the combination index (CI) value was calculated.   Fa-CI plot analysis demonstrated 
synergism between ABC294640 and ABT-737 in inhibiting myeloma cell proliferation (Fig. 11C).    
 
1.5.7. ABC294640 induces myeloma cell apoptosis in the presence of bone marrow stromal cells.  
BM stromal cells support the growth and survival of myeloma cells and confer them with drug 
resistance. We sought to test if ABC294640 could still effectively kill myeloma cells in the presence of 
BM stromal cells. We co-cultured e-GFP expressing myeloma cells with HS5 BM stromal cells. We then 
added ABC294640 to the co-culture system and measured Annexin V+ cells gated on the eGFP+ 
Figure 10: ABC294640 enhanced c-Myc proteasome degradation. A: ABC294640 down-regulated c-Myc and pS6 protein 
expression. MM cells (OPM1, RPMI-8226, MM.1S, JK6L, and U266) were treated with 30µM of ABC294640 (A) or DMSO (D) 
for 16 hr and whole cell lysates were prepared and analyzed for c-Myc and pS6 expression by Western blot. β-actin was used as 
the loading control. B: A294640 increased c-Myc protein degradation. OPM1 cells were treated with 30µM of ABC294640 or 
DMSO for 3 hr and then cyclohexamide (100mg/ml) was added.  Cells were collected at each hour after cyclohexamide treatment 
and whole cell lysate was prepared and analyzed for c-Myc expression by Western blot. β-actin was used as the loading control. 
Graph below represented the quantification of western blots using ImageJ.  C: Proteasome inhibitor (MG132) prevented the 
degradation of c-Myc by ABC294640. OPM1 and JK6L cells were treated with DMSO control buffer or MG132 (1µM) for 1 h, 
followed by treatment with DMSO or 30 µM of ABC294640 for additional 6h.  Whole cell lysate was prepared and analyzed for 
c-Myc expression by Western blot. Data shown in the figure were representative of at least 2 separate sets of experiments. 

 
 
 
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myeloma cells. ABC294640 did not induce apoptosis of HS5 BM stromal cells (Fig. 11D).  
Interestingly, as shown in Fig. 11D, the percentage of Annexin V+ myeloma cells following ABC294640 
treatment was quite similar in OPM1 cells alone and in OPM1 cells co-cultured with HS5 BM stromal 
cells.  These data suggested that ABC294640 could induce myeloma cell apoptosis even in the presence 
of BM stromal cells.  
 
Figure 11: ABC294640 acted synergistically with Bcl-2 inhibitor in inhibiting myeloma cell growth and induced myeloma 
cell apoptosis in the presence of bone marrow stromal cells. A. ABC294640 did not affect Bcl-2 expression. MM cells 
(OPM1, RPMI-8226, MM.1S, and U266) were treated with 30µM of ABC294640 (A) or DMSO (D) for 16 hr and whole cell 
lysates were prepared and analyzed for Bcl-2 expression by Western blot. B: Combination of ABC294640 and ABT-737 led to 
enhanced inhibition of cell proliferation. OPM1 cells were treated with various concentrations of ABT-737 in the absence or 
presence of ABC294640 (15 µM) for 48 hr and cell proliferation was measured by MTT assay. C:  FA-CI plots showing the 
synergistic effect of ABC294640 and ABT-737. Fa-CI plots for OPM1 cells revealed a synergistic inhibitory effect for 
ABC294640 15μM and ABT-737 at 0.1µM (indicated as 1), 0.3µM (indicated as 2) and 1µM (indicated as 3). In the Fa-CI plot, 
the dashed line [combination index (CI) =1] indicates an additive reaction between the two substances. Values below this dashed 
line imply synergism. D: ABC294640 induced myeloma apoptosis in the presence of bone marrow stromal cells. GFP 
expressing OPM1 cells were cultured on the monolayer of HS5 BM stromal cells and were treated for 8h with 30µM of 
ABC294640 or DMSO. The cells were stained with Annexin V and 7-AAD and Annexin V+ apoptotic cells were gated on GFP 
positive OPM1 cells.   

 
 
 
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1.5.8. ABC294640 suppresses MM tumor growth in mouse xenograft models. 
The in vivo anti-myeloma activity of ABC294640 was assessed using mouse xenograft models. We 
transduced MM.1S myeloma cells with lentiviral vector expressing luciferase and generated MM.1S cell 
line stably expressing luciferase.  We performed two series of in vivo experiments.  In the first series of 
experiments, the luciferase expressing MM.1S cells were injected via tail vein (Fig. 12A) or 
subcutaneously (Fig. 12B) into sublethally irradiated NOD/SCID IL-2γ (NSG) mice.  Two days after the 
tumor injection the mice were treated with ABC294640 (50mg/kg daily i.p.) or vehicle control buffer for 
30 days. In our second series of experiments, MM.1S cells were injected subcutaneously into irradiated 
(2.5 Gy) NSG mice.  ABC294640 treatment was started 14 days later when bioluminescence imaging 
showed tumor engraftment, and continued daily for ~1 month (Fig. 12C).  Tumor growth was monitored 
by bioluminescence imaging at the time points indicated. As shown in Fig. 12A-12C, ABC294640 
effectively inhibited myeloma growth in vivo. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 12: ABC294640 suppressed myeloma growth in vivo in mouse xenograft models. A: ABC294640 inhibited 
myeloma growth in vivo in intravenously administrated mouse xenograft model. NSG mice were sublethally irradiated (2.5 
Gy) and injected via tail vein 0.5x106 MM.1S myeloma cells stably expressing leuciferase. Two days later, the mice were divided 
randomly into 2 groups and treated either with ABC 294640 50mg/kg i.p. (A) or control vehicle (C; PBS+ 0.3% Tween-80) for 
30 days. Every 10 days the mice were imaged using Perkin Elmer Ivis 200 imager and Live Image software. B: ABC294640 
inhibited myeloma growth in vivo in subcutaneously inoculated mouse xenograft model. NSG mice were sublethally 
irradiated (2.5 Gy) and injected subcutaneously with 0.5x106 MM.1S myeloma cells stably expressing leuciferase. Two days 
later, the mice were treated either with ABC294640 50mg/kg i.p. (Mice indicated as “A”) or control vehicle (Mouse “C”) for 30 
days. Tumor growth was monitored by bioluminescence imaging. C: ABC294640 inhibited myeloma growth in vivo. NSG 
mice were sublethally irradiated (2.5 Gy) and injected subcutaneously with 0.5x106 MM.1S myeloma cells stably expressing 
leuciferase. Two weeks later, the mice were imaged using bioluminescence imaging and showed tumor engraftment (D0).  The 
mice were then treated with ABC294640 50mg/kg i.p. or control daily for 27 days (from D0 to D27). Tumor growth was 
monitored by bioluminescence imaging at time-points indicated (up to one week after the discontinuation of injection, i.e., D35). 
D: Schematic diagram of the mechanisms of action of ABC294640 in MM cells.   

 
 
 
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Our pre-clinical data justify continued development of ABC294640 as a therapy for patients with 
myeloma.  
 
1.5.9. The roles of SK2 in the regulation of mitophagy and in bone marrow microenvironment – our 
most recent studies. 
 
Mitophagy is the autophagy process of mitochondria. We recently found that suppressed mitophagy (i.e., reduced 
level of PINK1 and PARK2 expression) correlated with myeloma pathogenesis and was associated with worse 
survival in patients with multiple myeloma.  Furthermore, we found that SK2 plays a critical role in the regulation 
of mitophagy in multiple myeloma. More interestingly, when we injected VK*MYC myeloma cells into SK2 
knockout mice or wildtype mice.  SK2 knockout recipient mice did not develop myeloma whereas wildtype 
recipient mice did, suggesting a critical role of SK2 in the regulation of bone marrow microenvironment (such as 
osteoclasts, osteoblasts, macrophages or bone matrix). More recently, we found that SK2 knockout mice that did 
not develop myeloma had increased number of NK cells, NK T cells and B cells and reduced number of myeloid 
derived suppressive cells, compared to wildtype littermates that developed myeloma. These data implicated a role 
of SK2 in the regulation of anti-myeloma immunity. 
 
Thus, it would be important to perform correlative studies to understand the effects of ABC294640 on mitophagy, 
bone formation/destruction markers and on Lyn/Src gene expression. 
 
2. STUDY DESIGN 
This is a two-part study comprised of a Phase I Dose Escalation and Expansion portion and a Phase II 
Efficacy at MTD portion.  
 
Dexamethasone was commonly used in myeloma treatment.  After debating the effects of combining 
ABC294640 with dexamethasone in the study, it was decided to exclude the combination of 
dexamethasone for several considerations: 1) All the patients would have been treated with 
dexamethasone before and are likely resistant to dexamethasone when they relapse or become refractory. 
Thus, including dexamethasone offers little benefits while adding toxicities and side effects; 2) The 
effects of combining ABC294640 with dexamethasone are unknown and they could antagonize each 
other. In addition, dexamethasone could affect the mechanism of ABC294640; 3) Many agents such as 
Carfilzomib, Elotuzumab and pomalidomide were tested as a single agent without the combination of 
dexamethasone.  
2.1  
Study description 
The study will be conducted in two parts, an initial Phase Ib dose-finding study with dose escalation of 
the study drug ABC294640. The projected ABC294640 doses for the escalation phase are:  250, 500, 
and 750 mg BID as determined in the single agent trial for ABC294640. The dose will be given under 
fasting conditions or after a light to moderate meal.   
2.2  
Phase 1B 
 
Primary Objectives 
• 
To assess safety and determine the maximum tolerated dose (MTD) of single agent ABC294640 in 
patients with refractory or relapsed MM who have been previously treated with proteasome inhibitors 
and immunomodulatory agents. 
 
Secondary Objectives 
• 
To assess the antitumor activity of single agent ABC294640 in patients with refractory or relapsed 
MM after 3 cycles of treatment. 
• 
To determine the pharmacokinetics of ABC294640 following administration of the single drug. 
• 
To describe the effects of ABC294640 on plasma levels of sphingosine 1-phosphate, IL-6 and other 

 
 
 
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cytokines in patients with refractory or relapsed MM. 
• 
To assess pharmacodynamic markers (SK2 mRNA level or activity, sphingolipid metabolites, c-
Myc, Mcl-1 and pS6) in bone marrow CD138+ myeloma cells that may predict tumor response to 
ABC294640. For patients who receive more than 3 cycles of treatment and have a repeated bone 
marrow biopsy, RNA sequencing and Assay for transposases accessible chromatin sequencing 
(ATAC seq) will be performed to assess the effects of ABC294640 on global gene and epigenetic 
expression. 
• 
To perform correlative studies including serum cell-free PINK1/PARK2 (mitophagy markers), cell 
free DNA for next generation sequencing (for signal pathway analyses), serum bone 
destruction/formation markers (TRAP, Osteocalcin), and bone gene markers and Lyn/Src gene 
expression in both CD138+ and CD138- cells. 
• 
To evaluate immunologic function and immunologic panels (NK, NK T, T, B cells and myeloid 
derived suppressive cells (MDSCs) and the immune co-signalling molecules) in bone marrow 
environment. 
 
2.3 
Phase 1B design 
 
After signing informed consent, patients will complete the evaluation process to determine eligibility. 
Once a patient is determined to be eligible for the study, he/she will be enrolled in the dose escalation 
Phase Ib study testing 3 ABC294640 doses (250 mg, 500 mg, and 750 mg BID). A cycle of treatment is 
defined as 28 days.  One patient will be enrolled per week per dose. Subsequent patients on each cohort 
may not be enrolled less than one week after the previous patient. Patients in each dose cohort must 
complete Cycle 1 and undergo toxicity evaluation before the next dose level cohort is opened. 
 
We use the Bayesian model averaging continual reassessment method (BMA-CRM)98-100 for dose 
finding. In BMA-CRM, multiple CRM models are used, each equipped with a different skeleton and 
different skeletons represent different prior guesses of the toxicity profile of the drug. A discrete prior 
probability is assigned to each CRM model. The estimate of the toxicity probability at each dose level is 
the weighted average of the estimated probabilities at that dose level from the multiple CRM models, 
where the weight is the estimated probability of each model. BMA-CRM adaptively assigns a larger 
weight to a model of a better fit, and thus ensures the estimated probabilities to be always close to the 
best estimates among all the candidate models. 
 
Let 
 be ABC294640 dose levels 250 mg BID, 500 mg BID and 750 mg BID, respectively. Let 
 be the CRM probability model associated with the th skeleton 
, for 
. 
Under model 
, the toxicity probability at dose level  is assumed to be 
  where the 
’s are unknown parameters. Prior data suggest that the 
toxicity probability should fall in the intervals (0, 0.1), (0.25, 0.35), and (0.4, 0.6), for the 3 doses 
, respectively. Therefore, we use 3 skeletons, with specified probabilities 
 as 
(0.03, 0.25, 0.4), (0.06, 0.3, 0.5), and (0.09, 0.35, 0.6.), for 
 respectively. The prior 
distribution of 
 is taken as 
, for 
. Assume equal prior probabilities for the 3 models. 
Suppose that up to the current stage of the trial, 
 patients have experienced toxicity among 
 patients 
treated at dose level , for 
. Given observed data, use the method described previously98-100 to 
obtain the BMA-CRM estimate of the toxicity probability at dose level , which is denoted by 
, for 
 
 
The target toxicity probability is set as 
. We treat a cohort of 3 patients at each dose level.  
 
Individual patients may continue to receive therapy at the same dose of ABC294640 provided they do 
not experience unacceptable toxicity or clinical disease progression. No intrapatient dose escalation is 

 
 
 
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allowed. 
 
Patients will continue on therapy until the development of intolerable toxicity, withdrawal of patient 
consent or other event as outlined in patient discontinuation section 8.2.  Patients may remain on study 
until there is unequivocal evidence of clinical progression or the development of intolerable toxicities. 
Patients will have an End of Treatment visit 4-6 weeks after last dose of study  drug. 
 
2.4 
Definition of dose-limiting toxicities 
 
Toxicities will be graded according to the NCI CTCAE version 4.0.3 criteria.  Dose-limiting toxicities 
(DLTs) will be defined as any of the following events that are at least (possibly, probably, or definitely) 
attributable to ABC294640 during dose escalation. 
 
Non hematologic DLT is defined as: 
Any Grade ≥ 3 AE, with the following exceptions 
Symptomatic adverse events such as nausea, vomiting and diarrhea will not be considered dose limiting 
if they can be reduced to less than grade 3 within 72 hours with standard supportive measures such as 
antiemetics and antidiarrheals. 
 
Hematologic DLT is defined as: 
≥Grade 4 neutropenia or thrombocytopenia that lasts more than 7 days after the last dose of study drug; 
≥Grade 3 thrombocytopenia in the presence of ≥grade 3 hemorrhage of any organ/site; 
Any grade 5 hematologic toxicity   
 
 
2.5 
Phase II 
 
Primary Objectives 
• 
Assess overall treatment response rate and OS in patients with relapsed or refractory MM treated 
with single-agent ABC294640. 
 
Secondary Objectives 
• 
To assess the antitumor activity of ABC294640 in patients with refractory or relapsed MM after 3 
cycles of treatment. 
• 
To determine if pharmacodynamic markers (SK2 mRNA or activity, sphingolipid metabolites, c-
Myc, Mcl-1 and pS6) in bone marrow CD138+ myeloma cells predict tumor response to ABC294640. 
• 
To perform correlative studies including serum cell-free PINK1/PARK2 (mitophagy markers), 
cell free DNA for next generation sequencing (for signal pathway analyses), serum bone 
destruction/formation markers (TRAP, Osteocalcin), and bone gene markers and Lyn/Src gene 
expression in both CD138+ and CD138- cells. 
• 
To evaluate immunologic function and immunologic panels (NK, NK T, T, B cells and myeloid 
derived suppressive cells (MDSCs) and the immune co-signalling molecules) in bone marrow 
environment 
 
The Phase II portion of the study will commence once the MTD of ABC294640 has been identified, 
which will be the Phase II dose. 
 
3. STUDY PATIENTS 
 
Patients with relapsed or refractory multiple myeloma who have previously been treated proteasome 

 
 
 
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inhibitors or immunomodulatory agents are eligible for this trial, regardless of the patients’ stem cell 
transplant status.  Both patients who have had a transplant and patients who did not receive a transplant 
are eligible for the study. Potential study patients will be screened according to the Eligibility Criteria. 
Study-specific evaluations may be performed only after written informed consent has been obtained. 
 
3.1 
Eligibility criteria 
 
Inclusion criteria 
 
1. Patient must have a diagnosis of symptomatic multiple myeloma, who relapses or is refractory after 
previous treatment with a proteasome inhibitor (bortezomib or carfilzomib) and an immunomodulatory 
agent (thalidomide, lenalidomide or pomalidomide). 
 
2. Have measurable disease as defined by at least one of the following: 
  
-  Serum monoclonal (M) protein ≥0.5g/dl by protein electrophoresis  
 
-  >200 mg of M protein in the urine on 24-hour electrophoresis   
 -  Serum immunoglobulin free light chain ≥10 mg/dL AND abnormal serum immunoglobulin kappa 
to lambda free light chain ratio 
 
-  Monoclonal bone marrow plasmacytosis ≥30%  
 
3. Voluntary signed and dated institutional review board (IRB) approved informed consent form in 
accordance with regulatory and institutional guidelines. 
 
4. Time interval from last systemic chemotherapy (not including low dose dexamethasone) more than 2 
weeks prior to initiation of ABC294640. Patients receiving high dose dexamethasone defined as 40mg 
dexamethasone a day for 4 days will need 2 weeks washout prior to initiation of ABC294640. 
 
5. 18 years of age or older. 
 
6. ECOG performance status of 0-2. 
 
7. Acceptable liver function: 
 
- Bilirubin ≤ 1.5 times upper limit of normal (CTCAE Grade 1 baseline) 
 
- AST (SGOT), ALT (SGPT)  5 x ULN (CTCAE Grade 2 baseline) 
 
- Serum creatinine ≤1.5 XULN (CTCAE Grade 1 baseline) 
 
8. Acceptable hematologic status (with or without transfusion support): 
 
- Absolute neutrophil count ≥1000 cells/mm3, 
 
- Platelet count ≥50,000 (plt/mm3), 
 
- Hemoglobin ≥9 g/dL. 
NOTE: If the patient’s bone marrow biopsy shows greater than or equal to 50% plasma cells, the 
platelet count should be >30,000 plt/mm3, Hemoglobin >8 g/dL and ANC >500 cells/mm3 
(transfusion support or growth factor support is acceptable). 
 
9. 
Urinalysis: No clinically significant abnormalities. 
 
10. PT and PTT ≤ 1.5 X ULN after correction of nutritional deficiencies that may contribute to 
prolonged PT/PTT. 
 
11. As determined by the treating investigator, the patient must have well-controlled blood pressure, 

 
 
 
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defined as systolic blood pressure <150mmHg and/or diastolic blood pressure <100 mmHg for the 
majority of measurements. 
 
12. A negative pregnancy test (if female of child bearing potential). For men and women of child-
producing potential, willingness to use effective contraceptive methods during the study. 
 
Exclusion criteria 
 
1. Pregnant or nursing women. NOTE: Women of childbearing potential and men must agree to use 
adequate contraception (hormonal or barrier method of birth control; or abstinence) prior to study entry 
and for the duration of study participation. Should a woman become pregnant or suspect she is pregnant 
while participating in this study, she should inform her treating physician immediately. 
 
2. Patients who are currently participating in any other clinical trial of an investigational product. 
 
3. Major surgery within 30 days prior to start of treatment 
 
4. Treatment with intravenous antibiotics, antivirals, or antifungals within 14 days prior to start of 
treatment. 
 
5. Known human immunodeficiency virus infection 
 
6. Active hepatitis B or C infection with abnormal liver functions (i.e., LFTs > 2X upper normal limits) 
 
7. Unstable angina or myocardial infarction within 4 months prior to start of treatment, NYHA Class 
III or IV heart failure, uncontrolled angina, history of severe coronary artery disease, severe uncontrolled 
ventricular arrhythmias, sick sinus syndrome, or electrocardiographic evidence of acute ischemia or 
Grade 3 conduction system abnormalities unless subject has a pacemaker 
 
8. Uncontrolled hypertension or uncontrolled diabetes within 14 days prior to start of treatment 
 
9. Nonhematologic malignancy within the past 3 years with the exception of a) adequately treated basal 
cell carcinoma, squamous cell skin cancer, or thyroid cancer; b) carcinoma in situ of the cervix or breast; 
c) prostate cancer of Gleason Grade 6 or less with stable prostate-specific antigen levels; or d) cancer 
considered cured by surgical resection or unlikely to impact survival during the duration of the study, 
such as localized transitional cell carcinoma of the bladder 
 
10. Subjects with pleural effusions requiring thoracentesis or ascites requiring paracentesis within 14 
days prior to initiation of ABC294640 
 
11. Any other clinically significant medical or psychiatric disease or condition or social situation that, in 
the Investigator’s opinion, may interfere with protocol adherence or a subject’s ability to give informed 
consent 
 
4. ENROLLMENT AND TREATMENT 
 
4.1 
Screening  
The screening examination will take place between Day -30 and 0.  An informed consent must be signed 
by the patient before any study-specific screening procedure takes place.  Subject data to be collected at 
the Screening Examination includes:  
• 
Complete medical history 

 
 
 
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• 
Concomitant medication assessment 
• 
Baseline Review of Systems and AE documentation 
• 
Physical examination 
• 
Vital signs (temperature, blood pressure, pulse rate, and respiratory rate) 
• 
ECOG Performance Status  
• 
Pregnancy test (for nonsterile women of childbearing potential within 2 days of Treatment Day 1) 
• 
12-lead electrocardiogram 
• 
Serum chemistry 
• 
Coagulation parameters 
• 
CBC with differential 
• 
Urinalysis 
• 
Bone marrow biopsy and aspirates (should be performed within 2 weeks of treatment day 1) 
• 
SPEP, IFE, UPEP, serum free light chain, LDH, beta-2 microglobulin 
• 
Bone skeletal survey. 
 
Subjects likely to meet eligibility criteria will be offered participation in the study after the investigator 
verifies that there is a current available slot (phase 1).  Subjects will sign informed consent prior to any 
protocol associated procedure. Screening procedures will 1) ensure that subject meets all the eligibility 
criteria, 2) obtain disease assessment to allow efficacy measurements, 3) assess baseline toxicity, and 4) 
provide initial biological samples for pharmacodynamic and correlative studies. 
 
 
4.2 
Phase IB: Dose escalation phase 
 
The projected ABC294640 doses for the escalation phase are:  250, 500, and 750 mg BID as 
determined in the single agent trial for ABC294640. Drug doses are defined in Table 4-1.  The dose 
will be given under fasting conditions or after a light to moderate meal. One patient will be enrolled 
per week per dose. Subsequent patients on each cohort may not be enrolled less than one week after the 
previous patient. Patients in each dose cohort must complete Cycle 1 and undergo toxicity evaluation 
before the next dose level cohort is opened.  
 
Table 4-1   Dose Escalation Scheme 
 
Dose level 
ABC294640 Dose 
(mg AM / PM) 
Capsules (AM / PM) 
0 
250 / 250 
1 / 1 
1 
500 / 500 
2 / 2 
2 
750 / 750 
3 / 3 
 
Let the target probability of toxicity be 
 The following dose-finding algorithm will be 
employed. 
• Treat the first cohort of patients at the 250 mg BID dose level. 
 
• 
Suppose that the current dose level is 
. Denote the estimated probabilities of toxicity based on 
the accumulated data to be 
 for the 3 dose levels 
, respectively. Find the dose level 
 such that  
 
 Then 

 
 
 
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o 
if 
, de-escalate to dose level 
; 
o 
if 
, escalate to dose level 
; 
o 
otherwise, stay at dose level 
 for the next cohort of patients. 
• Once the maximum sample size is reached, the dose with the toxicity probability closest to 
 is 
declared as the MTD. 
If the lowest dose is still too toxic, as indicated by the fact that the posterior probability that the toxicity 
rate for the lowest dose is greater than 0.33 is >0.9, the trial will be terminated early for safety. 
 
Individual patients may continue to receive therapy at the same dose of ABC294640 provided they do 
not experience unacceptable toxicity or disease progression. No intrapatient dose escalation is allowed. 
 
Patients experiencing Grade 3 or 4 hematologic toxicity will have the study drug withheld according to 
the Dose Modification Plan outlined in Table 4-2.   If Grade 3 or 4 toxicity continues after 4 weeks, the 
patient will be removed from the study. The patient will have an End of Treatment visit 4-6 weeks after 
last dose of study drug. 
 
Table 4-2   Dose Modification Plan 
NCI CTC 4.0.3 
Criteria 
ABC294640 Dose 
Modification Instructions 
Need for discontinuation from study 
Hematologic toxicity (ANC 
and/or platelet) ≥Grade 3 
Hold study drug. Assess patient weekly. If 
the toxicity resolves to ≤ baseline by the 
beginning of subsequent week, restart 
study drug and reduce dose by 1 dose level 
(Table 4-1). Omitted doses are not made 
up. 
If the toxicity does not resolve to ≤ 
baseline after 4 weeks, the patient will 
be removed from the study. 
Other non- hematologic 
toxicity ≥3 
Hold study drug. Assess patient weekly. If 
the toxicity resolves to ≤ baseline by the 
beginning of subsequent week, restart 
study drug and reduce dose by 1 dose level 
(Table 4-1). Omitted doses are not made 
up. 
Subsequent cycle can be delayed for up 
to 2 weeks until toxicity is resolved to 
≤ baseline. If toxicity does not resolve 
to ≤ baseline after the extended 2 
weeks, discontinue patient from study. 
4.3 
Phase II 
 
Once the MTD for ABC294640 is found, there will be expansion of the MTD cohort so that a total of 59 
individuals will be treated at the MTD of ABC294640. The 3 phase Ib subjects taking the MTD will 
continue into Phase II portion of the study and will be included in the Phase II analysis.  Thus, 56 
additional patients will be enrolled in the Phase II portion of the study.  
 
For the phase II portion of the study, we have incorporated safety stopping rules into our design to ensure 
patient safety (Table below). The first column of the table gives the number of patients treated in each 
stage. The second column gives the minimum number of patients with unacceptable serious adverse 
event (SAE) required to stop the trial early for safety. The third column lists the proportion of subjects 
with related SAEs. Specifically, we will stop the trial early if there is significant toxicity defined as an 
unacceptable serious adverse event rate of grade 3 or 4 toxicities deemed possibly, probably or definitely 
related to study drug. We consider an acceptable level to be 15% (null hypothesis) and an unacceptable 

 
 
 
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rate to be 30% (alternative hypothesis). We will craft a stopping rule for early stopping for strong 
evidence that the SAE rate is 30% vs. 15%.  Given our sample size of 59 and based on 1000 simulations, 
this stopping rule yields a 83% chance of early termination if the true SAE rate is 0.30 and only a 4% 
chance of early stopping if the true SAE rate is 0.15. 
 
 
Total number of 
patients treated 
Number of patients 
with grade 3 or 4 
related SAEs 
 
Point estimate of 
SAE rate 
12 
8 
0.67 
24 
10 
0.42 
36 
11 
0.31 
48 
12 
0.25 
59 
13 
0.22 
 
 
4.4 
Safety confirmation and follow-up 
Individual patients may continue to receive therapy at the same dose provided they do not experience 
unacceptable toxicity or disease progression. No intrapatient dose escalation is allowed. 
 
Treatment will continue until documentation of clinically progressive disease, unacceptable side effects, 
or patient withdraws consent. The patient will have an End of Treatment visit 4-6 weeks after last dose of 
study drug. Patients will then be followed for survival every 3 months for 2 years following treatment 
discontinuation and then follow-up per standard practice. 
 
4.5 
Disease assessment  
 
Disease assessment will occur at the beginning of each cycle.  Full myeloma restaging and treatment 
response assessment will be performed at the end of Cycles 3 and 6 (+/- 7 days) and will consist of 
serum protein electrophoresis, serum and urine immunofixation, 24h urine protein electrophoresis, 
serum free light chains, beta-2 microglobulin, bone marrow aspiration and biopsy, complete blood 
counts and metabolic panel. Disease response will be categorized according to appendix E. 
 
5. PK and PD Studies 
 
5.1 
Special Instructions related to administration of ABC 294640 on days of PK Sampling.  
 
PK sampling will occur during Cycle 1 on Day 1. During these study time-points, administration of 
ABC294640 should only occur at the investigative site. 
 
Blood samples for PK will be drawn on Day 1 of Cycle 1 immediately before dosing and at 1, 2, 4, and 8 
hours after dosing. 
 
On Day-1 of Cycle-1, a blood sample will be drawn from each subject for PD and PK analysis prior to 
drug administration. Then, all subjects will receive their morning dose.  PK sampling will continue over 
an 8 hour period. Patients should take the afternoon dose approximately 12 hours after the morning dose.  
 

 
 
 
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In the event a patient vomits within 60 minutes after ingesting the investigational agent on Day 1 of Cycle 
1, the PK blood draw schedule will be suspended. The investigational agent will not be administered 
again. Prior to receiving the investigational agent the following morning, the patient is to be treated with 
adequate anti-nausea therapy (reference Appendix B for contraindicated medications) prior to study drug 
administration.   
 
5.2 
 Pharmacodynamic and correlative assays 
For our secondary objectives, we will perform pharmacodynamic assays to measure the effects of 
ABC294640 on S1P in the plasma and in the bone marrow as well as on the c-Myc and Mcl-1 
expression on myeloma cells.  The key end-points for our pharmacodynamics and mechanistic studies 
are: 
 
- 
Plasma PD analysis for sphingosine 1-phosphate (performed using samples collected during the 
PK sampling and at the beginning of each cycle prior to ABC294640 drug administration). Plasma IL-6 
level performed at the beginning of each cycle for the first 6 cycles. Up to 41 different cytokines will be 
measured using multiplex cytokine assay. 
  
- 
Sphingolipid concentrations (ceramides, sphingosine and sphingosine 1-phosphate (assessed on 
the bone marrow aspirate supernatants at the beginning of treatment, at the last day of cycle #3 and at the 
last day of cycle #6). For patients who receive more than 3 cycles of treatment and have a repeated bone 
marrow biopsy, RNA sequencing and Assay for transposases accessible chromatin sequencing (ATAC 
seq) will be performed to assess the effects of ABC294640 on global gene and epigenetic expression. 
 
-          Expression of sphingolipid signaling genes, SK2 mRNA or activity, c-Myc, Mcl-1 and pS6 
(measured in bone marrow CD138+ myeloma cells isolated at the beginning of treatment, at the last day 
of cycle #3 and at the last day of cycle #6) (if sufficient number of bone marrow CD138+ cells can be 
obtained) 
 
- To perform correlative studies including serum cell-free PINK1/PARK2 (mitophagy markers), cell 
free DNA for next generation sequencing (for signal pathway analyses), serum bone 
destruction/formation markers (TRAP, Osteocalcin), and bone gene markers and Lyn/Src gene 
expression in both CD138+ and CD138- cells. 
 
- To evaluate immunologic function and immunologic panels (NK, NK T, T, B cells and myeloid 
derived suppressive cells (MDSCs) and the immune co-signalling molecules) in bone marrow 
environment 
 
Pharmacodynamic assays intend to verify if the hypothesized effects of the study drugs are indeed 
occurring in vivo.  
 
Table 5-1 - Pharmacodynamic (PD) and correlative assays: 
Assay 
Material 
Time points 
Lab performing the assay 
PK study 
Peripheral 
blood plasma 
Day 1 of cycle 1 
immediately before dosing 
and at 1, 2, 4 and 8 hours 
after dosing 
Sent-out test (Alliance 
Pharma) 
S1P levels 
Peripheral 
blood plasma 
In the PK draws and at the 
beginning of each cycle 
prior to ABC294640 drug 
administration (up to 
cycle#6) 
Sent-out test (Alliance 
Pharma) 

 
 
 
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IL-6 level 
Peripheral 
blood plasma 
At the beginning of each 
cycle prior to ABC294640 
drug administration (up to 
cycle#6) 
Dr. Yubin Kang’s lab at 
Duke 
Cytokines (up to 41 
different cytokines) 
Peripheral 
blood plasma 
(using existing 
samples) 
In the PK draws (0, 4, 8 
hours on cycle 1 day 1 and 
beginning of each cycle) 
Dr. Yubin Kang’s Lab and 
Duke core facility 
Cell free PINK1/PARk2 
expression, Serum bone 
formation/destruction 
markers 
Peripheral 
blood plasma 
At the beginning of each 
cycle prior to ABC294640 
drug administration (up to 
cycle#6) 
Dr. Yubin Kang’s lab at 
Duke 
Cell free DNA for next 
generation sequencing 
Peripheral 
blood plasma 
At the beginning of each 
cycle prior to ABC294640 
drug administration (up to 
cycle#6) 
Performed at the discretion 
of PI and only if the funds 
are available. 
Ceramide, sphingosine, 
and S1P levels 
Bone marrow 
supernatant  
At the beginning of 
treatment, at the last day of 
cycle #3 and at the last day 
of cycle #6 
Sent-out test (Medical 
University of South 
Carolina) 
SK mRNA or activity (if 
sufficient number of 
bone marrow CD138+ 
cells can be obtained) 
Bone marrow 
CD138+ cells 
At the beginning of 
treatment, at the last day of 
cycle #3 and at the last day 
of cycle #6 
 
For mRNA, Dr. Kang’s lab. 
For SK activity, Dr. Charles 
Smith’s lab at Apogee 
Biotechnology Corp. 
RNA seq and ATAC seq 
for determination of the 
ABC294640 on global 
gene and epigenetic 
expression 
Bone marrow 
CD138+ cells 
We had one patient in phase 
I who had received >3 
cycles of treatment and had 
a repeated bone marrow 
biopsy. RNAseq and 
ATACseq will be only for 
this patient 
Dr. Yubin Kang’s lab and 
Duke Genomic Core facility 
Protein level of c-Myc, 
Mcl-1 and pS6 (Western 
blot) (if sufficient 
number of bone marrow 
CD138+ cells can be 
obtained) 
Bone marrow 
CD138+ cells 
At the beginning of 
treatment, at the last day of 
cycle #3 and at the last day 
of cycle #6 
 
Dr. Yubin Kang’s lab at 
Duke 
Gene expression 
(Lyn/Src, 
Osteoclast/osteoblast 
genes, mitophagy 
markers) 
Bone marrow 
CD138+ cells 
and CD138- 
cells 
At the beginning of 
treatment, at the last day of 
cycle #3 and at the last day 
of cycle #6 
 
Dr. Yubin Kang’s lab at 
Duke 
Immunologic function 
and immunologic panel 
(immune cells and co-
signaling molecules) 
Bone marrow 
aspirate 
At the beginning of 
treatment, at the last day of 
cycle #3 and at the last day 
of cycle #6 
 
Dr. Yubin Kang’s lab at 
Duke 
5.3 
Time points of sample collection 
Please see the Table 5-1 (pharmacodynamics and correlative assays) for the time points at which 

 
 
 
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samples will be collected. To compare with healthy control plasma samples, we will acquire up to 20 de-
identified plasma samples from healthy donors, collected prior to 12/04/2018 under the biorepository 
protocol Pro00006268, A Research Specimen Repository and Database to Evaluate Hematologic 
Malignancies. 
 
5.4 
Sample collection and shipment 
For peripheral blood samples, at each time point, a total of 8ml of blood will be collected (2x 4ml 
collected in K3EDTA tubes).  Blood samples will be centrifuged as soon as possible at 500 x g for 15 
min and the plasma will be transferred to cryovials (in 0.5 ml aliquots) and frozen at -800 C.  These 
samples will be periodically transferred (shipped on dry ice by FedEx) to Alliance Pharma for analysis.  
For bone marrow samples, a total of 10 ml of marrow aspirate will be collected in K3 EDTA tube(s). For 
samples collected at Duke during the daytime, the samples will be transported immediately to Dr. 
Kang’s lab.  For samples collected at nighttime, the samples can be stored at 40C refrigerator and 
transportation to Dr. Kang’s lab the following day.  The address for Dr. Kang’s laboratory: Room 4040, 
GSRB1, Duke University, phone: 919 668-4532. 
 
5.5 
Assays 
 
-- Sphingolipid metabolite measurement (ceramides, sphingosine and sphingosine-1-phosphate): 
 
Bone marrow aspirates will be centrifuged at 500 × g for 15 min and the supernatants will be collected.  
The samples will then be measured for sphingolipid metabolites related to the ceramide/S1P axis, 
including ceramides, sphingosine, and S1P. The sphingolipid metabolites will be measured using HPLC-
MS/MS as described by Dr. Bielawski et al101. The concentration of each sphingolipid metabolite will be 
normalized against total cellular phosphate and represented as pM/nM of cellular phosphate. 
 
-- SK activity assay 
 
The SK activity in cell lysates prepared as described below, will be measured using HPLC-based SK 
activity assay as described61. In brief, the cell lysates will be incubated with NBD-Sph in the isozyme-
selective assay buffers detailed below with 1 mg/ml fatty acid-free bovine serum albumin, 100 μM ATP, 
and 400 μM MgCl2. The product, i.e., NBD-S1P, is separated from NBD-Sph by HPLC with 
fluorescence detection. The ratio of NBD-S1P/(NBD-Sph + NBD-S1P) is used as a measure of SK 
activity. SK-isozyme selective assay buffers each contain 20 mM Tris, pH7.4, 5 mM EDTA, 5 mM 
EGTA, 3 mM β-mercaptoethanol, 5% glycerol, 1× protease inhibitors (Sigma-Aldrich) and 1× 
phosphatase inhibitors (Roche Diagnostics, Indianapolis, IN). For the SK1 assay buffer, 0.25% (final) 
Triton X-100 is added; and for the SK2 buffer, 1 M (final) KCl is added. Assays are run at room 
temperature, and then a 1.5 volume of methanol is added to terminate the kinase reaction. After 10 min, 
the samples are centrifuged at 20,000g to pellet the precipitated protein, and the supernatants are 
analyzed by HPLC. The assay will be performed at Apogee Biotechnology Corporation. 
 
-- Western blot for measurement of c-Myc, Mcl-1 and pS6 
 
Bone marrow CD138+ cells will be isolated from the bone marrow aspirates of these patients using 
Histopaque-1077 (Sigma) gradient separation followed by human CD138 enrichment kit (EasySep™, 
StemCell Technologies). The purity of the human CD138+ cells is normally > 95%.  The protein 
expression of c-Myc, Mcl-1 and pS6 will be measured by Western blot as we described81. MM cells are 
harvested, washed with PBS, and re-suspended in lysis buffer containing 50 mM Tris-HCl pH 7.4, 150 
mM NaCl, 1mM EDTA, 1% Triton x100, 1% Sodium deoxycholate, and 0.1% SDS. The cells are 
further lysed by brief sonication. The lysates are centrifuged at high speed for 10min to remove the cell 
debris. Total protein is quantified using Dc protein estimation kit (Bio Rad) with BSA for standard 

 
 
 
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curve. Approximately 20μg protein is loaded and run on SDS PAGE. The proteins are transferred onto 
nitrocellulose membrane. The membrane is blocked with 5% milk in Tris-Buffered Saline containing 
0.05% of Tween 20 (TBST) and primary antibodies are applied with 1% BSA in TBST for overnight at 
40C with gentle rocking. The membrane is then probed with HRP-conjugated secondary antibody and 
developed using Pierce ECL substrate. 
 
--   Correlative biomarker studies 
 
For the measurement of cell free serum/plasma PINK1/PARK2 (mitophagy markers), cell free DNAs 
will be isolated from the plasma/serum using cell free DNA isolation columns. PINK1/PARK2 
expression will be quantitated by PCR. For the measurement of serum/plasma bone 
formation/destruction markers, TRAP, Osteocalcin, osterix and others will be measured by ELISA or 
western blot analysis. For signaling pathway analyses, cell free DNA will be isolated from plasma 
samples and subject to the next generation sequencing and pathway enrichment analyses. For the 
measurement of Lyn/Src, osteoclast function and macrophage, CD138+ and CD138- cells will be 
isolated from the bone marrow aspirate and gene expression will be measured by quantitative RT-PCR. 
For the measurement of immunologic function and immunologic panel, bone marrow aspirates (CD138+ 
and CD138- cells) will be cultured with antibodies and cell proliferation will be measured. Additionally, 
the bone marrow aspirate will be measured for NK cells, NK T cells, T (CD3, CD4, CD8), B cells, 
MDSCs and co-signaling molecules (PD-1, PD-L1, CTLA-4, TIGIT, TIM3 etc). For simultaneous 
measurement of up to 41 different cytokines, multiplex cytokine assay will be performed using existing 
samples at the Duke Immunology core facility. We had one patient in our phase I study who had 
received >3 cycles of ABC294640 treatment and had a repeated bone marrow biopsy. To determine the 
effects of ABC294640 treatment on gene and epigenetic expression, we will perform RNA seq and 
Assay for transposases accessible chromatin with high-throughput sequencing (ATAC seq) using the 
existing bone marrow samples on this patient. 
 
-- Healthy control plasma samples. To compare with healthy control plasma samples, we will use the 
plasma samples collected under the 00006268 biorepository protocol. 
 
6. STUDY DRUG: ABC294640 
 
ABC294640 is an orally available inhibitor of the enzyme sphingosine kinase-2. 
 
6.1  
Formulation 
 
ABC294640 Capsules contain 250 mg of the milled active pharmaceutical ingredient (API) ABC294640 
along with the excipients microcrystalline cellulose, USP/NF (Avicel® PH102, FMC) (FMC 
biopolymer) and colloidal silicon dioxide, NF (Cab-O-Sil® M5P). ABC294640 is a white to off-white 
powder and for clinical use it is encapsulated in white opaque hard gelatin capsules. 
 
6.2  
Dosage and administration 
 
The study drug will be administered approximately every 12 hours, under fasting conditions or after a 
light to moderate meal for 28 days according to the dose escalation and de-escalation guidelines outlined 
in Section 4.  A dose modification plan is also provided in Section 4. 
 
6.3  
Storage 
 

 
 
 
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Storage conditions: Store at room temperature, 15-30C (59-86F) in a secure, locked area, properly labeled 
and segregated from other materials. This storage area should only be accessible to authorized 
individuals. 
 
6.4  
Labeling  
 
The test article will be labeled with the sponsor name and address, description of contents and storage 
conditions, and will contain the statement “Caution: New Drug—Limited by Federal (or United States) 
law to investigational use.” 
 
6.5  
Test article and accountability 
 
The Investigator must maintain adequate records showing the receipt, dispensing, return, or other 
disposition of the test article including the date, quantity, batch or code number and identification of 
subjects (patient number and initials) who receive the test article. The Investigator will not supply the test 
article to any person except those named as sub-investigators and submitted to the local regulatory 
authority, designated staff and patients in this study. The Investigator will not dispense the test article 
from any sites other than those submitted to the local regulatory authority. The test article will not be 
relabeled or reassigned for use by other patients 
 
Drug accountability procedures to be followed by study staff and patients are outlined in Section 7. 
 
Upon completion of the study, unused supplies of the ABC294640 test article will be returned to the 
sponsor or destroyed as directed. 
 
7. 
 METHOD OF DETERMINING TREATMENT COMPLIANCE 
Patients will be provided a drug diary at the beginning of each cycle, i.e. when additional drug is 
dispensed to the patient. Patients will be instructed to complete the drug diary and record their daily 
administration of ABC294640 during the morning and evening timeframes.  Any missed or modified 
doses should be documented along with the reason for the modification.  Patients will be instructed to 
return their container(s) of ABC294640 at each clinic visit so that the delegated research team can 
conduct a pill count to assess patient compliance. The research team has the ability to withdraw a patient 
from the study if that patient demonstrates repeated protocol noncompliance. Noncompliance is defined 
as intentionally not taking ABC294640 for 1 week in the absence of side effects attributed to 
ABC294640.  
 
 
7.1 
SAFETY PLAN 
 
7.1.1. GENERAL PLAN TO MANAGE SAFETY 
A number of measures will be taken to ensure the safety of patients participating in this trial. These 
measures will be addressed through exclusion criteria and routine monitoring as follows. In addition, an 
early stopping monitoring plan is outlined in Section 8.6.2. 
 
Patients enrolled in this study will be evaluated clinically and with standard laboratory tests before and at 
regular intervals during their participation in this study. Safety evaluations will consist of medical 
interviews, recording of adverse events, physical examinations, blood pressure, and laboratory 
measurements (performed by local laboratories). Patients will be evaluated for adverse events (all 
grades), serious adverse events, and adverse events requiring study drug interruption or discontinuation 

 
 
 
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at each study visit for the duration of their participation in the study. Patients discontinued from the 
treatment phase of the study for any reason will be evaluated ~30 days (within 30–42 days) after last 
dose of study drug.  Patients who have an ongoing treatment-related Grade > 3 or serious adverse event 
at the time of discontinuation from study treatment will continue to be followed as per Section 7.2. 
 
Women of childbearing potential must have a negative pregnancy test prior to starting therapy and must 
use adequate contraceptive methods during and for 12 weeks after discontinuing study therapy. If a 
patient becomes pregnant despite precautions, she should be apprised of the potential risk of fetal 
morbidity or loss. 
 
7.1.2. CONCOMITANT AND EXCLUDED THERAPIES 
All supportive care measures consistent with optimal patient care will be given throughout the study. 
 
Use of anti-neoplastic or anti-tumor agents not part of the study therapy, including chemotherapy, 
radiation therapy*, immunotherapy, and hormonal anticancer therapy, is not permitted while 
participating in this study. Use of concurrent investigational agents is not permitted. Use of full dose 
warfarin is not permitted. 
*Localized radiation is allowed. 
 
Patients should receive full supportive care, including anti-emetic drugs, hematopoietic growth factors, 
transfusions of blood and blood products, fluid and electrolyte replacement, and antibiotics when 
appropriate.  Patients are not permitted to have received cancer chemotherapy, including any other 
investigational therapy, within two weeks prior to study entry. 
 
Allopurinol (in subjects at risk for TLS due to high tumor burden) is optional and will be prescribed at 
the Investigator’s discretion.  These subjects may receive allopurinol 300 mg PO daily. Vitamins and 
supplements should be recorded on the concomitant medication page.  All transfusions and/or blood 
product related procedures must be recorded on the appropriate form. 
 
Patients cannot receive concomitant therapy with drugs that are sensitive substrates of CYP450 1A2, 
3A4, 2C9, 2C19 or 2D6, or strong inhibitors or inducers of all major CYP450 isozymes (including 
grapefruit juice). 
 
A list of commonly used drugs that are that are sensitive substrates of CYP450 1A2, 3A4, 2C9, 2C19 or 
2D6, or strong inhibitors or inducers of all major CYP450 isozymes (with the half-life of each drug 
identified) is included as Appendix B. Patients who receive any of these medications on a regular basis 
should be instructed not to take them during their participation in this study.  Where possible, alternative 
medications should be prescribed or recommended.  If a patient is receiving a drug that is a sensitive 
substrate of CYP450 1A2, 3A4, 2C9, 2C19 or 2D6, or strong inhibitor or inducer of a major CYP450 
isozyme that cannot be stopped or replaced by another medication for the duration of the clinical study 
that patient should not be enrolled. 
 
Additionally, due to the protein binding affinity of ABC 294640, patients should not receive concomitant 
therapy with Coumadin or Coumadin derivatives. 
 
 
 
7.2 
POST-TREATMENT EVALUATIONS 
 
The visit at which a response assessment shows disease progression or the decision was made to 
discontinue treatment for other reasons, such as toxicity, will be considered the End of Treatment visit. 
 

 
 
 
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Patients completing their treatment should return for Post-30 day visit per Study Calendar Section 4.4. 
Patients who have an ongoing Grade ≥ 3 or serious adverse event that is at least possibly related to 
treatment will be contacted by the investigator or designee approximately every week until the event is 
resolved or determined to be irreversible. 
 
Patients will be followed for survival by clinic visit, review of medical record, and/or telephone call 
every 3 months from treatment discontinuation date, until 2 years post treatment discontinuation. 
 
7.3 
 PATIENT DISCONTINUATION 
 
Patients may discontinue study treatment at any time. Any patient who discontinues treatment will be 
asked to return to the study center to undergo treatment discontinuation assessments as outlined within 
Study Calendar Section 4.4 End of Treatment visit. The primary reason for discontinuation should be 
recorded.  
 
Subjects who meet the following criteria should be discontinued from study treatment: 
 
• 
Grade 4 hypertension or Grade 3 hypertension not controlled with medication 
• 
Nephrotic syndrome 
• 
Grade ≥ 2 pulmonary or CNS hemorrhage; any Grade 4 hemorrhage 
• 
Any grade arterial thromboembolic event that that cannot be treated with non-coumadin therapy, 
such as IVC filter, or LMW heparin 
• 
Grade 4 congestive heart failure 
• 
Gastrointestinal perforation 
• 
Tracheoesophageal fistula (any grade) or Grade 4 fistula 
• 
Grade ≥ 3 bowel obstruction that has not fully recovered despite medical or surgical intervention 
• 
Wound dehiscence requiring major medical or surgical intervention 
• 
Documented disease progression per Appendix E 
• 
Clinically significant deterioration of the patient’s condition 
• 
Patient noncompliance defined as intentionally not taking ABC294640 for 1 week in the absence of 
side effects attributed to ABC294640. 
• 
Adverse events requiring treatment discontinuation 
• 
Investigator determination that it is not in the patient’s best interest to continue participation 
• 
Pregnancy 
• 
Withdrawal of consent by patient 
8. 
STATISTICAL CONSIDERATIONS 
 
8.1  
PRIMARY OBJECTIVE 
 
Phase 1b: The primary objective of the phase 1b component of the clinical trial is to determine the 
maximum tolerated dose (MTD) and evaluate the safety of single agent ABC294640 in patients with 
relapsed or refractory multiple myeloma who have previously been treated with a proteasome inhibitor 
and an immunomodulatory agent. 
 
Phase 2: The primary objective of the phase 2 portion of the clinical trial is to evaluate the effects of 
single agent ABC294640 on treatment response and overall survival (OS) in patients with relapsed or 
refractory multiple myeloma who have previously been treated with a proteasome inhibitor and an 
immunomodulatory agent. 
 

 
 
 
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8.2 
PRIMARY ENDPOINTS 
 
8.2.1. 
Maximum tolerated dose 
 
The primary endpoint of the phase 1b portion of the trial is the maximum tolerated dose (MTD), 
determined using Bayesian model average continual reassessment method (BMA-CRM) for dose finding. 
 
8.2.2. 
Treatment response 
 
One of the primary endpoints for the phase 2 portion of the trial is treatment response after Cycle 3 of 
treatment as defined by the International Myeloma Working Group criteria. 
 
8.2.3. 
Overall survival 
The other primary endpoint for the phase 2 portion of the trial is OS defined as the time interval from 
trial enrollment to death due to any cause. Survival times will be censored for patients lost to follow-up 
or still alive at the trial’s termination. 
 
8.3  
SECONDARY ENDPOINTS 
 
8.3.1. 
Safety Endpoints 
Safety assessments will consist of monitoring and reporting adverse events (AEs) and serious adverse 
events (SAEs) that are considered related to study drug, all events of death, and any study specific issue 
of concern. 
 
Dose-limiting toxicity: For the phase 1b portion of the trial, toxicities will be graded according to 
the NCI CTCAE version 4.0.3 criteria.  DLTs will be defined as any events that are at least (possibly, 
probably, or definitely) attributable to ABC294640 during dose escalation. 
 
Non hematologic DLT is defined as: 
Any Grade ≥ 3 AE, with the following exceptions 
Symptomatic adverse events such as nausea, vomiting and diarrhea will not be considered dose limiting 
if they can be reduced to less than grade 3 within 72 hours with standard supportive measures such as 
antiemetics and antidiarrheals. 
 
Hematologic DLT is defined as: 
≥Grade 4 neutropenia or thrombocytopenia that lasts more than 7 days after the last dose of study drug; 
≥Grade 3 thrombocytopenia in the presence of ≥grade 3 hemorrhage of any organ/site; 
Any grade 5 hematologic toxicity   
 
 
8.3.2   PK, PD and correlative endpoints 
- 
 Plasma PD analysis for sphingosine 1-phosphate (performed using the PK samples and at the 
beginning of each cycle prior to ABC294640 drug administration). Plasma IL-6 performed at the 
beginning of each cycles for the first 6 cycles. Up to 41 different cytokines will be performed using 
multiplex cytokine assay. 
  
- 
Sphingolipid concentrations (ceramides, sphingosine and sphingosine 1-phosphate (assessed on the 
bone marrow aspirate supernatants at the beginning of treatment, at the last day of cycle #3 and at the last 
day of cycle #6) 
 
- 
      Expression of sphingolipid signaling genes, SK2 mRNA or activity, c-Myc, Mcl-1 and pS6 

 
 
 
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(measured in bone marrow CD138+ myeloma cells isolated at the beginning of treatment, at the last day 
of cycle #3 and at the last day of cycle #6) (if sufficient number of bone marrow CD138+ cells can be 
obtained). For patients who receive more than 3 cycles of treatment and have a repeated bone marrow 
biopsy, RNA sequencing and Assay for transposases accessible chromatin sequencing (ATAC seq) will 
be performed to assess the effects of ABC294640 on global gene and epigenetic expression. 
 
- Correlative studies including: serum cell-free PINK1/PARK2 (mitophagy markers), cell free DNA 
for next generation sequencing (for signal pathway analyses), serum bone destruction/formation markers 
(TRAP, Osteocalcin), and bone gene markers and Lyn/Src gene expression in both CD138+ and CD138- 
cells. 
 
- Immunologic function and immunologic panels (NK, NK T, T, B cells and myeloid derived 
suppressive cells (MDSCs) and the immune co-signalling molecules) in bone marrow environment. 
8.4  
DETERMINATION OF SAMPLE SIZE 
 
8.4.1. Phase 1b 
The phase 1b trial will explore three dose cohorts of ABC294640. Bayesian model average continual 
reassessment method (BMA-CRM) will be used for dose finding to determine the MTD.  One patient 
will be enrolled per week per dose. Subsequent patients on each cohort may not be enrolled less 
than one week after the previous patient.  Patients in each dose cohort must complete cycle 1 and 
undergo toxicity evaluation before the next dose level cohort is opened. Up to 18 patients will be used 
for phase 1b study.  The patients at the MTD will contribute to the total sample size for the phase 2 
portion of the trial. 
 
8.4.2. Phase 2 
Multiple myeloma patients who relapse or become refractory after receiving previous treatment with a 
proteasome inhibitor and an immunomodulatory agent usually achieve an response rate of less than 10% 
and survive less than a year with weekly dexamethasone salvage therapy.  Currently several 
investigational drugs have improved the overall response rate (ORR) to about 20-35% and prolonged the 
OS to 1.5 years to 2 years. We consider an ORR of 20% and a 6 month improvement in OS for patients 
treated with single ABC294640 to be sufficiently promising to warrant further drug development. 
 
We therefore design the phase 2 trial to test a null ORR of 10% versus an alternative of 20% and a null 
median OS of 12 months versus an alternative of 18 months – equivalent to a hazard ratio (HR) of 0.67 – 
assuming exponential survival, with approximately 36 months total accrual time (including 12 months of 
accrual for the phase 1b patients treated at the MTD), and 24 months of follow-up following accrual of 
the final patient. 
 
The exponential survival assumption is strong.  Assuming exponential survival, a test of the null 
hypothesis that median OS = 12 months versus the alternative that median OS = 18 months is equivalent 
to a test of a null hazard rate of 0.058 versus an alternative hazard rate of 0.039 (HR = 0.67). We will test 
this hypothesis based on a likelihood ratio test of the exponential hazard rate parameter. Fifty nine 
subjects yields 80% power to detect a HR of 0.67 based on the stated assumptions and one-sided α = 
0.025. Power was estimated using the Southwest Oncology Group’s one sample survival power and 
sample size calculator (http://www.swogstat.org/stat/public/one_survival.htm). This number of patients 
will yield 73% (or 80%) power to detect an increase of ORR from 10% to 20% (or 21%) with a one-sided 
chi-square test (α = 0.025). 
 
 
8.5  
ANALYSIS PLANS 
 

 
 
 
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8.5.1. Phase 1b 
The phase 1b trial will explore three dose cohorts of ABC294640 following the Bayesian model 
averaging continual reassessment design: 250 mg BID (dose 0), 500 mg BID (dose 1), and 750 mg BID 
(dose 2). The starting dose of 250 mg BID is known to be safely tolerated as a single agent in patients 
with solid tumors. A dose-limiting toxicity (DLT) is defined per section 2.4. The MTD is the dose at 
which the estimated toxicity probability based on all 18 patients is closest to the targeted probability 0.33 
among all doses. If the lowest dose (250 mg BID) is found to be too toxic, as indicated by the fact that 
the posterior probability that the toxicity rate for the lowest dose is greater than 0.33 is >0.9, then the 
trial will close and the drug declared unsafe. 
 
8.5.2. 
Safety analysis 
 
The primary safety analysis will be conducted using descriptive statistics of the incidence of adverse 
events (AEs), including DLTs and discontinuations due to AEs. Adverse events will be coded by body 
system, and summary tables with incidence rates of AEs will be generated. Descriptive statistics of AEs 
will be reported by dose and for subsets of patients with DLTs, patients who discontinue due to AEs, and 
patients with related AEs. Severity, duration, investigator attributed relationship to treatment, and 
outcomes of AEs will be reported. The DLT rate will be estimated with its 95% CI. 
 
8.5.3. 
Phase 2 
 
Survival times will be displayed graphically using a Kaplan-Meier curve. Median survival will be 
estimated, and the corresponding 95% CI constructed using Greenwood’s variance estimate (Hosmer and 
Lemeshow, 1999). Assuming exponential survival, a test of the null hypothesis that median OS = 12 
months versus the alternative that median OS = 18 months is equivalent to a test of a null hazard rate of 
0.058 versus an alternative hazard rate of 0.039 (HR = 0.67). We will test this hypothesis based on a 
likelihood ratio test of the exponential hazard rate parameter. The overall response rate will tested with a 
chi-square test, under a null hypothesis that the ORR is 10% and an alternative hypothesis that the ORR is 
greater than 10%. 
 
8.5.4. 
Analysis of secondary endpoints 
 
Treatment response will be determined using international multiple myeloma working group response 
criteria after 3 cycles and 6 cycles of treatment (Appendix E). Frequencies and rates of complete 
response (CR), partial response (PR), stable disease, and progressive disease will be reported. 
 
 
8.6  
INTERIM ANALYSES 
 
8.6.1. 
Efficacy and futility interim analyses 
 
An interim analysis is planned after the enrollment of 33 efficacy evaluable patients in the Phase II 
portion of the study. Response data collected after the completion of 3 cycles of treatment will be 
reviewed. If there are 4 or more responses (PR, VGPR, CR, sCR) to this therapy at this time point, an 
additional 26 efficacy evaluable patients will be enrolled. Otherwise, the trial is stopped early for futility. 
If the ORR is 10%, the probability of early stop is 0.58, but if the ORR is 20%, the probability that the 
trial is stopped early is 0.08.   
 
 
 
8.6.2  Safety stopping rules  

 
 
 
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We have incorporated safety stopping rules into our design to ensure patient safety (Table below) during 
our phase II portion of the study. The first column of the table gives the number of patients treated in 
each stage. The second column gives the minimum number of patients with unacceptable serious adverse 
event (SAE) required to stop the trial early for safety. The third column lists the proportion of subjects 
with related SAEs. Specifically, we will stop the trial early if there is significant toxicity defined as an 
unacceptable serious adverse event rate of grade 3 or 4 toxicities deemed possibly, probably or definitely 
related to study drug. We consider an acceptable level to be 15% (null hypothesis) and an unacceptable 
rate to be 30% (alternative hypothesis). We will craft a stopping rule for early stopping for strong 
evidence that the SAE rate is 30% vs. 15%.  Given our sample size of 59 and based on 1000 simulations, 
this stopping rule yields a 83% chance of early termination if the true SAE rate is 0.30 and only a 4% 
chance of early stopping if the true SAE rate is 0.15. 
 
 
Total number of 
patients treated 
Number of patients 
with grade 3 or 4 
related SAEs 
Point estimate of 
SAE rate 
12 
8 
0.67 
24 
10 
0.42 
36 
11 
0.31 
48 
12 
0.25 
59 
13 
0.22 
 
 
 
8.7  
ANALYSIS POPULATIONS  
8.7.1 
Intent to treat population 
 
The efficacy analysis will be performed using the intent to treat (ITT) population that is composed of all 
patients enrolled and who has received at least one dose of study medication. For the analysis of tumor 
response, patients lost to follow-up prior to the 12-week evaluation will be considered failures. 
 
8.7.2 
Safety population 
 
The safety population is defined as all patients who receive at least one dose of study drug. 
 
9. 
SAFETY MONITORING AND REPORTING 
 
The PI is responsible for the identification and documentation of adverse events and serious adverse 
events, as defined below.  At each study visit, the PI or designee must assess, through non-suggestive 
inquiries of the subject or evaluation of study assessments, whether an AE or SAE has occurred. 
 
The investigator is responsible for ensuring that all AEs and SAEs that are observed or reported during 
the study is collected and reported to the FDA, appropriate IRB(s), DCN, and the sponsor in accordance 
with CFR 312.32 (IND Safety Reports). 
 
9.1  
ADVERSE EVENTS 
An adverse event (AE) is any untoward medical occurrence in a subject receiving study drug and which 
does not necessarily have a causal relationship with this treatment.  For this protocol, the definition of 

 
 
 
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AE also includes worsening of any pre-existing medical condition, except MM.  An AE can therefore be 
any unfavorable and unintended or worsening sign (including an abnormal laboratory finding), 
symptom, or disease temporally associated with the use of study drug, whether or not related to use of 
the study drug.  Abnormal laboratory findings without clinical significance (based on the PI’s judgment) 
should not be recorded as AEs.  But laboratory value changes that require therapy or adjustment in prior 
therapy are considered adverse events. 
 
From the time the subject initiates study treatment through the End of Treatment visit, all AEs must be 
recorded in the subject medical record and adverse events case report form. AEs will be followed until 
resolution, the condition stabilizes, the event is otherwise explained, or the subject is lost to follow-up. 
 
AEs will be assessed according to the CTCAE version 4.0.3 (Appendix A).  If CTCAE grading does not 
exist for an AE, the severity of the AE will be graded as mild (1), moderate (2), severe (3), life-
threatening (4), or fatal (5). 
 
Attribution of AEs will be indicated as follows: 
- 
Definite:  The AE is clearly related to the study drug 
- 
Probably:  The AE is likely related to the study drug 
- 
Possible:  The AE may be related to the study drug 
- 
Unrelated:  The AE is clearly NOT related to the study drug 
 
9.2  
SERIOUS ADVERSE EVENTS 
An AE is considered “serious” if in the opinion of the investigator it meets one or more of the following  
criteria: 
 
- 
Results in death  
- 
Is Life-threatening 
- 
Constitutes a congenital anomaly or birth defect 
- 
Is a  medically significant condition (defined as an event that compromises subject safety or may 
require medical or surgical intervention to prevent one of the three outcomes above). 
- 
Requires inpatient hospitalization or prolongation of existing hospitalization 
- 
Results in persistent or significant incapacity or substantial disruption to conduct normal life 
functions. 
9.3  
ADVERSE EVENT ASSESSMENT 
 
The study period during which all AEs and SAEs must be reported begins after initiation of study 
treatment and ends 30 days following the last administration of study treatment. After this period, 
investigators should only report SAEs that are attributed to prior study treatment. 
 
All AEs and SAEs whether volunteered by the subject, discovered by study personnel during 
questioning, or detected through physical examination, laboratory test, or other means will be reported 
appropriately. Each reported AE or SAE will be described by its duration (i.e., start and end dates), 
regulatory seriousness criteria if applicable, severity, suspected relationship to the study, outcome, and 
actions taken. 
 
A consistent methodology for eliciting AEs at all subject evaluation time points should be adopted. 
Examples of non-directive questions include: 
“How have you felt since your last clinical visit?” 
“Have you had any new or changed health problems since you were last here?” 
 
Pre-existing diseases or conditions will not be considered AEs unless there is an increase in the 

 
 
 
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frequency or severity, or a change in the quality, of the disease or condition. Progression of cancer also 
will not be considered an AE. 
 
 
9.4  
ADVERSE EVENT CAUSALITY 
 
To ensure consistency of AE and SAE causality assessments, investigators should apply the following 
general guideline: 
 
Definitely 
related 
An adverse event occurring in a plausible time relationship to drug 
administration, and which cannot be explained by concurrent disease 
or other drugs or chemicals. The response to withdrawal of the drug 
should be clinically plausible. The event must be definite 
pharmacologically or phenomenologically, using a satisfactory 
rechallenge procedure if necessary and feasible. 
Probably 
related 
An adverse event with a reasonable time sequence to administration, 
and which cannot be explained by concurrent disease or other drugs or 
chemicals, and which follows a clinically reasonable response on 
withdrawal.  Rechallenge information is not required to fulfill this 
definition. (Note: there are important exceptions when an adverse 
event does not disappear upon discontinuation of the drug, yet drug- 
relatedness clearly exists, (e.g. bone marrow suppression, fixed-drug 
eruptions, and tardive dyskinesias) 
Possibly related 
An adverse event with a reasonable time sequence to administration of 
the drug, but which could also be explained by concurrent disease or 
other drugs or chemicals. 
Information on drug withdrawal may be lacking or unclear. 
Not related 
An adverse event with a temporal relationship to drug administration 
which makes a causal relationship improbable, and in which other 
drugs, chemicals or underlying diseases provide plausible explanations. 
 

 
 
 
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9.5 
ADVERSE REACTION EXPECTEDNESS 
 
Adverse reactions are those adverse events considered possibly, probably or definitely related to study 
medication. 
 
Expected adverse reactions are those adverse events that are listed or characterized in the Package 
Insert or current Investigator Brochure. 
 
Unexpected adverse reactions are those not listed in the Package Insert (P.I.) or current Investigator 
Brochure (I.B.) or not identified. This includes adverse events for which the specificity or severity is not 
consistent with the description in the P.I. or I.B. For example, under this definition, hepatic necrosis 
would be unexpected if the P.I. or I.B. only referred to elevated hepatic enzymes or hepatitis. ) 
 
9.6 
ADDITIONAL ADVERSE EVENT CONSIDERATIONS 
 
a. 
Diagnosis vs. Signs and Symptoms 
If known at the time of reporting, a diagnosis should be reported rather than individual signs and 
symptoms (e.g., record only liver failure or hepatitis rather than jaundice, asterixis, and elevated 
transaminases). However, if a constellation of signs and/or symptoms cannot be medically characterized 
as a single diagnosis or syndrome at the time of reporting, it is acceptable to report the information that is 
currently available. If a diagnosis is subsequently established, it should be reported as follow-up 
information. 
 
b. Deaths 
All deaths that occur during the protocol-specified AE reporting period, regardless of attribution, will be 
reported to the appropriate parties. When recording a death, the event or condition that caused or 
contributed to the fatal outcome should be reported as the single medical concept. If the cause of death is 
unknown and cannot be ascertained at the time of reporting, report “Unexplained Death”. 
 
c. Preexisting Medical Conditions 
A preexisting medical condition is one that is present at the start of the study. Such conditions should be 
reported as medical and surgical history. A preexisting medical condition should be re-assessed 
throughout the trial and reported as an AE or SAE only if the frequency, severity, or character of the 
condition worsens during the study. When reporting such events, it is important to convey the concept 
that the preexisting condition has changed by including applicable descriptors (e.g., “more frequent 
headaches”). 
 
d. Hospitalizations for Medical or Surgical Procedures 
Any AE that results in hospitalization or prolonged hospitalization should be documented and reported as 
an SAE. If a subject is hospitalized to undergo a medical or surgical procedure as a result of an AE, the 
event responsible for the procedure, not the procedure itself, should be reported as the SAE. For example, 
if a subject is hospitalized to undergo coronary bypass surgery, record the heart condition that 
necessitated the bypass as the SAE. 
 
e. Pregnancy 
If a female subject becomes pregnant while receiving investigational therapy or within 12 weeks after the 
last dose of study drug, a report should be completed and expeditiously submitted to the sponsor, and 
CATO Pharmacovigilance. Follow-up to obtain the outcome of the pregnancy should also occur. 
Similarly, any congenital anomaly/birth defect in a child born to a female subject exposed should be 
reported as an SAE. 
 

 
 
 
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f. 
Post-Study Adverse Events 
The investigator should expeditiously report any SAE occurring after a subject has completed or discontinued 
study participation if attributed to prior investigational agent exposure. If the investigator should become aware 
of the development of cancer or a congenital anomaly in a subsequently conceived offspring of a female 
subject who participated in the study, this should be reported as an SAE. 
 
9.7 
PROCEDURES FOR ELICITING, RECORDING, AND REPORTING ADVERSE 
EVENTS  
 
Investigators must report all SAEs within 24 hours of first becoming aware of the event. 
 
The completed SAE report form should be faxed or emailed to the following recipients: 
 
The Duke Study Team: 
 
ATTN: Ivy Belskie, RN 
cc: Dr. Yubin Kang  
FAX: (919) 668-1091 
Email: Ivy.Belskie@duke.edu; Yubin.Kang@duke.edu  
 
AND 
All SAEs must be reported promptly to CATO Pharmacovigilance within 24 hours of first becoming 
aware of the event by sending to the pharmacovigilance email box: 
RedHill-PhV@cato.com   
 
Terry F Plasse, MD, Medical Director, RedHill Biopharma Ltd. is available for discussion should an 
investigator or other study staff members require consultation. 
 
Phone: +1-917-913-7315 OR +972-50-588-7376 
Email: Terry@Redhillbio.com  
 
 
At the time of first notification of an SAE, the following information, if available, should be provided by 
the site to Duke and the pharmacovigilance email: 
• 
Study protocol number 
• 
Site Number 
• 
Subject’s study number  
• 
Patient’s date of birth 
• 
Patient’s gender 
• 
Date of first dose of study treatment 
• 
Date of last dose of test article, if applicable 
• 
Unit dose and schedule of test article 
• 
Adverse event term 
• 
Time and date of occurrence of the event 
• 
A brief description of the event, outcome to date and any actions taken 
• 
The seriousness criteria (on) that were met 
• 
Concomitant medication at onset of the event 
• 
Relevant past history information 

 
 
 
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• 
Relevant laboratory test findings 
• 
Investigator’s opinion of the relationship to test article (refer to Section 11.6.4.7)  
• 
Investigator’s signature 
Any missing or additional relevant information concerning the SAE should be provided in a follow-up SAE 
report. 
 
The Investigator is required to comply with applicable regulations regarding the notification to the IRB. 
Acknowledgement of IRB review should be forwarded to the Duke study team. 
 
Follow-up Information 
After the initial SAE report, the investigator is required to follow and provide further information in 
regards to the subject’s condition. All SAEs should be followed until resolution, the condition stabilizes, 
the event is otherwise explained, or the subject is lost to follow-up. A follow-up SAE report form should 
be submitted within 24 hours of first awareness.  
 
9.8  
Quality Control and Quality Assurance 
 
This clinical research study will be monitored internally by the PI and externally by the sponsor or 
designee.  The PI and study staff will continuously monitor and tabulate adverse events at each study 
visit.  Appropriate reporting to the Duke University Medical Center IRB will be made.  If an unexpected 
frequency of Grade III or IV events occur, depending on their nature, action appropriate to the nature 
and frequency of these adverse events will be taken.  This may require a protocol amendment, dose de-
escalation, or potentially closure of the study.  The PI of this study will also continuously monitor the 
conduct, data, and safety of this study to ensure that: 
 
• 
Interim analyses occur as scheduled; 
• 
Stopping rules for toxicity and/or response are met; 
• 
Risk/benefit ratio is not altered to the detriment of the subjects; 
• 
Appropriate internal monitoring of AEs and outcomes is done; 
• 
Over-accrual does not occur; 
• 
Under-accrual is addressed with appropriate amendments or actions; 
• 
Data are being appropriately collected in a reasonably timely manner. 
 
9.8.1 Sponsor or designee Oversight 
RedHill Biopharma Ltd. (or its designee) will be responsible for activities such as monitoring, Study 
Trial Master File (TMF) maintenance, data querying and data transfers, pharmacovigilance, statistical 
analysis and clinical study report writing. The sponsor (or its designee) will also be responsible 
for FDA Reporting that includes:  Expedited Serious Adverse Events Review and Annual IND reports.  
 
9.9 
Audits 
 
The Duke School of Medicine Office of Audit, Risk and Compliance (OARC) may conduct confidential 
audits to evaluate compliance with the protocol and the principles of GCP.  The PI agrees to allow the 
OARC auditor(s) direct access to all relevant documents and to allocate his/her time and the time of the 
study team to the OARC auditor(s) in order to discuss findings and any relevant issues. 
 
OARC audits are designed to protect the rights and well-being of human research subjects. OARC audits 
may be routine or directed (for cause). Routine audits are selected based upon risk metrics generally 
geared towards high subject enrollment, studies with limited oversight or monitoring, Investigator 

 
 
 
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initiated Investigational Drugs or Devices, federally-funded studies, high degree of risk (based upon 
adverse events, type of study, or vulnerable populations), Phase I studies, or studies that involve 
Medicare populations. Directed audits occur at the directive of the IRB or an authorized Institutional 
Official. 
 
OARC audits examine research studies/clinical trials methodology, processes and systems to assess 
whether the research is conducted according to the protocol approved by the DUHS IRB. The primary 
purpose of the audit/review is to verify that the standards for safety of human subjects in clinical trials 
and the quality of data produced by the clinical trial research are met. The audit/review will serve as a 
quality assurance measure, internal to the institution. Additional goals of such audits are to detect both 
random and systemic errors occurring during the conduct of clinical research and to emphasize “best 
practices” in the research/clinical trials environment. 
 
9.10  
Data Management and Processing 
 
9.10.1   
Case Report Forms (CRFs) 
 
The electronic CRF will be the primary data collection document for the study.  The CRFs will be 
updated in a timely manner following acquisition of new source data.  Only approved study staff (i.e. 
Data managers, Data Coordinators), are permitted to make entries, changes, or corrections in the CRF. 
 
Any paper CRFs created for the study will have errors crossed out with a single line, and this line will 
not obscure the original entry.  Changes or corrections will be dated, initialed, and explained (if 
necessary).  The PI or authorized key personnel will maintain a record of the changes and corrections. 
 
In the electronic CRF, an audit trail will be maintained automatically by the electronic CRF management 
system.  Designated personnel will complete user training, as required or appropriate per regulations. 
 
9.10.2   
Data Management Procedures and Data Verification 
The sponsor or its designee will be responsible for clinical data management.  
 
The database will be reviewed and discussed prior to database closure, and will be closed only after 
resolution of all remaining queries.  An audit trail will be kept of all subsequent changes to the data. 
 
9.10.3   
Study Closure 
 
Following completion of the study, the PI will be responsible for ensuring the following activities: 
 
- 
Data clarification and/or resolution 
- 
Accounting, reconciliation, and destruction/return of used and unused study drugs 
- 
Review of site study records for completeness 
- 
Shipment of all remaining laboratory samples to the designated laboratories 

 
 
 
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10. 
 ADMINISTRATIVE and ETHICAL CONSIDERATIONS 
 
10.1  
Regulatory and Ethical Compliance 
 
This protocol was designed and will be conducted and reported in accordance with the International 
Conference on Harmonization (ICH) Harmonized Tripartite Guidelines for Good Clinical Practice, the 
Declaration of Helsinki, and applicable federal, state, and local regulations. 
10.2 
 DUHS Institutional Review Board and DCI Cancer Protocol Committee 
 
The protocol, informed consent form, advertising material, and additional protocol-related documents 
must be submitted to the DUHS Institutional Review Board (IRB) and DCI Cancer Protocol Committee 
(CPC) for review.  The study may be initiated only after the Principal Investigator has received written 
and dated approval from the CPC and IRB. 
 
The Principal Investigator must submit and obtain approval from the IRB for all subsequent protocol 
amendments and changes to the informed consent form.  The CPC should be informed about any 
protocol amendments that potentially affect research design or data analysis (i.e. amendments affecting 
subject population, inclusion/exclusion criteria, agent administration, statistical analysis, etc.). 
 
The Principal Investigator must obtain protocol re-approval from the IRB within 1 year of the most 
recent IRB approval.  The Principal Investigator must also obtain protocol re-approval from the CPC 
within 1 year of the most recent IRB approval, for as long as the protocol remains open to subject 
enrollment. 
10.3  
Informed Consent 
 
The informed consent form must be written in a manner that is understandable to the subject population.  
Prior to its use, the informed consent form must be approved by the IRB. 
 
The Principal Investigator or authorized key personnel will discuss with the potential subject the purpose 
of the research, methods, potential risks and benefits, subject concerns, and other study-related matters.  
This discussion will occur in a location that ensures subject privacy and in a manner that minimizes the 
possibility of coercion.  Appropriate accommodations will be made available for potential subjects who 
cannot read or understand English or are visually impaired.  Potential subjects will have the opportunity 
to contact the Principal investigator or authorized key personnel with questions, and will be given as 
much time as needed to make an informed decision about participation in the study. 
 
Before conducting any study-specific procedures, the Principal Investigator must obtain written 
informed consent from the subject or a legally acceptable representative.  The original informed consent 
form will be stored with the subject’s study records, and a copy of the informed consent form will be 
provided to the subject.  The Principal Investigator is responsible for asking the subject whether the 
subject wishes to notify his/her primary care physician about participation in the study.  If the subject 
agrees to such notification, the Principal Investigator will inform the subject’s primary care physician 
about the subject’s participation in the clinical study. 
10.4  
Study Documentation 
 
Study documentation includes but is not limited to source documents, case report forms (CRFs), 
monitoring logs, appointment schedules, study team correspondence with sponsors or regulatory 
bodies/committees, and regulatory documents that can be found in the DCI-mandated “Regulatory 

 
 
 
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Binder”, which includes but is not limited to signed protocol and amendments, approved and signed 
informed consent forms, FDA Form 1572, CAP and CLIA laboratory certifications, and clinical supplies 
receipts and distribution records. 
 
Source documents are original records that contain source data, which is all information in original 
records of clinical findings, observations, or other activities in a clinical trial necessary for the 
reconstruction and evaluation of the trial.  Source documents include but are not limited to hospital 
records, clinical and office charts, laboratory notes, memoranda, subjects’ diaries or evaluation 
checklists, pharmacy dispensing records, recorded data from automated instruments, copies or 
transcriptions certified after verification as being accurate copies, microfiches, photographic negatives, 
microfilm or magnetic media, x-rays, subject files, and records kept at the pharmacy, at the laboratories 
and at medico-technical departments involved in the clinical trial.  When possible, the original record 
should be retained as the source document.  However, a photocopy is acceptable provided that it is a 
clear, legible, and an exact duplication of the original document. 
 
A case report form (CRF) (including both electronic and paper CRFs) will be the primary data collection 
document for the study.  The CRFs will be updated within two weeks of acquisition of new source data.  
Only approved study staff (i.e. Data Managers, Data Coordinators), are permitted to make entries, 
changes, or corrections in the CRF.  For paper CRFs, errors will be crossed out with a single line, and 
this line will not obscure the original entry.  Changes or corrections will be dated, initialed, and 
explained (if necessary).  The Principal Investigator or authorized key personnel will maintain a record 
of the changes and corrections.  For electronic CRFs, an audit trail will be maintained by the electronic 
CRF management system. 
 
10.5  
Privacy, Confidentiality, and Data Storage 
 
The Principal Investigator will ensure that subject privacy and confidentiality of the subject’s data will 
be maintained.  Research Data Security Plans (RDSPs) will be approved by the appropriate institutional 
Site Based Research group. 
 
To protect privacy, every reasonable effort will be made to prevent undue access to subjects during the 
course of the study.  Prospective participants will be consented in an exam room where it is just the 
research staff, the patient and his family, if desired. For all future visits, interactions with research staff 
(study doctor and study coordinators) regarding research activities will take place in a private exam 
room. All research related interactions with the participant will be conducted by qualified research staff 
who are directly involved in the conduct of the research study. 
 
To protect confidentiality, subject files in paper format will be stored in secure cabinets under lock and 
key accessible only by the research staff. Subjects will be identified only by a unique study number and 
subject initials.  Electronic records of subject data will be maintained using dedicated databases which 
are located on secure file servers.  Access to electronic databases will be limited to Dr. Kang’s staff, 
DCN staff, RedHill staff and designated affiliates.     
 
Upon completion of the study, research records will be archived and handled per DUHS HRPP policy.     
 
Subject names or identifiers will not be used in reports, presentations at scientific meetings, or 
publications in scientific journals. 
10.6  
Data and Safety Monitoring 
 
Data and Safety Monitoring will be performed in accordance with the Duke Quality Assurance Plan. 

 
 
 
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10.7  
Protocol Amendments 
 
All protocol amendments must be initiated by the Principal Investigator and approved by the IRB prior 
to implementation.  IRB approval is not required for protocol changes that occur to protect the safety of 
a subject from an immediate hazard.  However, the Principal Investigator must inform the IRB and all 
other applicable regulatory agencies of such action immediately. 
 
Though not yet required, the CPC should be informed about any protocol amendments that potentially 
affect research design or data analysis (i.e. amendments affecting subject population, inclusion/exclusion 
criteria, agent administration, etc.). 
 
10.8  
Records Retention 
 
The Principal Investigator will maintain study-related records for the longer of a period of: 
 
- at least two years after the date on which a New Drug Application is approved by the FDA  
- at least two years after formal withdrawal of the IND associated with this protocol  
- at least six years after study completion 
 
11. 
SPONSOR AND MULTI-SITE INVESTIGATOR REQUIREMENTS 
 
Dr. Yubin Kang will be responsible for selecting qualified investigators, providing them with the 
information they need to conduct an investigation properly, ensuring proper monitoring of the 
investigation, ensuring that the investigation is conducted in accordance with the general investigational 
plan and protocol, and ensuring that FDA and all participating investigators are promptly informed of 
significant new adverse effects or risks with respect to the drug. 
 
11.1 
STUDY INITIATION 
 
Centers participating in the Phase II portion of the study cannot begin enrollment until an initiation letter 
has been issued from the DCN. Each center is required to participate in an initiation conference call. 
 
Before the start of this study and the shipment of ABC294640 to a participating center, the following 
documents must be on file at Duke Cancer Center. Participating centers will be responsible for 
forwarding the initiation documents to DCN. 
Initiation documents can be submitted via electronic mail to the regulatory coordinator at Duke. Please 
ensure that the site and study identifier are clearly identified. 
These documents are required to be submitted by each participating center: 
1. U.S. Food and Drug Administration (FDA) Form 1572, signed by the Principal Investigator at the 
participating center. 
2. The names of any sub-investigators at the participating center must appear on this form. Investigators 
must also complete all regulatory documentation as required by local regulations. This includes any 
required human subjects training required by the site’s local IRB. 
3. Current curricula vitae and documentation of professional licensure of the Principal Investigator and 
co-Investigators listed on the 1572. 
4. Resumes and human subject protections documentation (e.g. NIH, CITI) for all research personnel 

 
 
 
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(e.g. study coordinators, data managers and other research personnel). 
5. A signed and dated protocol signature page. 
6. Written documentation of IRB approval of protocol (identified by title, protocol version and date of 
approval) for each site. 
7. IRB approved study informed consent and HIPAA consent form, if applicable. HIPAA consent 
language can be included within the study informed consent. Please note that all informed consent 
forms should be reviewed and approved by the Duke Cancer Network office prior to submission to 
the site’s designated IRB. 
8. A signed Confidentiality Agreement. 
9. A signed Clinical Trial Agreement for each site. 
10. Laboratory certifications (CAP, CLIAs) and laboratory reference value ranges for each laboratory 
listed on the site’s 1572. 
11. Any Duke Cancer Network site specific forms as required. 
 
11.2 
STUDY COMPLETION 
 
The following data and materials are required by DCN before a study can be considered complete or 
terminated: 
 
1. 
Copies of protocol amendments and IRB approval/notification, if appropriate. 
2. 
Copies of the IRB final report, documentation of submission to the IRB. 
3. 
A summary of the study prepared by the Principal Investigator (Study report, manuscript and/or 
abstract). 
4. 
All regulatory documents (e.g., updated curriculum vitae for each Principal Investigator, updated 
U.S. FDA Form 1572 for each site). 
 
11.3 
INSTITUTIONAL REVIEW BOARD APPROVAL 
 
This protocol, the informed consent document, and relevant supporting information must be submitted to 
the IRB for review and must be approved before the study is initiated. In addition, any advertising 
materials, if applicable, must be approved by the IRB. The study will be conducted in accordance with 
U.S. FDA, applicable national and local health authority, and IRB requirements. 
 
The Principal Investigator at each site is responsible for keeping his/her IRB apprised of the progress of 
the study and of any changes made to the protocol as deemed appropriate, but in any case the IRB must 
be updated at least once a year. The Principal Investigator must also keep the IRB informed of any 
significant adverse events. 
 
Investigators are required to promptly notify their respective IRB of all adverse drug reactions that are 
both serious and unexpected. This generally refers to serious adverse events that are not already 
identified in the Investigator Brochure and that are considered possibly or probably related to the study 
drug by the investigator. Some IRBs may have other specific adverse event requirements to which 
investigators are expected to adhere.  If a center’s IRB does not require the review of all external adverse 
events, a written policy or statement outlining the IRB’s reporting requirements must be provided to 

 
 
 
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DCN. 
 
11.4 
QUALITY ASSURANCE 
 
The PI, sponsor or its designee will be responsible for quality assurance. Steps to assure the accuracy 
and reliability of data include the review of protocol procedures with the Investigator and 
associated personnel prior to the study, and periodic monitoring visits by the sponsor and/or its 
representative, and ongoing data quality reviews of received case report forms. Data will be 
reviewed for accuracy and completeness and any discrepancies will be resolved with the 
Investigator or designees as appropriate. 
 
11.5 
DATA COLLECTION 
 
Data collected will be entered into electronic CRFs managed by the sponsor or designee. The eCRF 
system is a 21CFR part 11 compliant system. The sponsor and its representatives will be provided access 
to the data collected, and for monitoring purpose, to source information, in accordance with institutional 
policy. 
 
12. 
 GENERAL CONSIDERATIONS 
 
12.1 
Discontinuation of the Study 
The Sponsor reserves the right to discontinue this study for safety or administrative reasons at any time. 
12.2 
Routine Study Termination 
The end of this study is defined as the date of the last visit of the last patient (last patient out or last 
patient last visit) participating in the study. Within 90 days of the end of the clinical study, the sponsor or 
designee will notify the IRB and regulatory authorities regarding routine termination of the study as 
required. 
 
12.3 
Premature Study Termination 
The study may be terminated prematurely for any reason and at any time by the sponsor, an IRB/IEC, or 
a Regulatory Authority. A decision to prematurely terminate the study is binding to all investigators of 
all study sites. If the study is terminated prematurely, all investigators have to inform their patients and 
take care of appropriate follow-up and further treatment of the patients to ensure protection of the 
patients’ interests. 
 
12.4 
Changes to the Protocol 
This protocol cannot be altered or changed except through a formal protocol amendment, which requires 
the written approval of the Sponsor. The protocol amendment must be signed by the Investigator and 
approved by the IRB or IEC before implementation. Protocol amendments will be filed with the 
appropriate regulatory agency(s) having jurisdiction over the conduct of the study. 
 
12.5 
Protocol Deviations for Emergency or Adverse Event 
An Investigator shall notify the Principal Investigator and the reviewing IRB of any deviation from the 
investigational plan to protect the life or physical well-being of a patient in an emergency. Such notice 
shall be given as soon as possible, but in no event later than five working days after the emergency 
occurred. Except in such an emergency, prior approval by the Principal Investigator is required for 

 
 
 
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changes or deviations from a plan, and if these changes or deviations affect the scientific soundness of 
the plan or the rights, safety, or welfare of patients, notification of the FDA and IRB in accordance with 
government regulations is also required. 
 
12.6 
Source Documents 
All information recorded in the CRF must be supported by corresponding source documentation. 
Examples of acceptable source documentation include, but are not limited to, hospital records, clinic and 
office charts laboratory notes, and recorded data from automated instruments, memoranda, and 
pharmacy dispensing records. For the proposed study, electronic records will be the primary source 
documentation, along with written reports for laboratory assays. 
 
During the study, select CRF data may be used as original data collection tools as long as a description 
of this documentation process is maintained in the Investigator’s study files. Before the study starts, a 
list identifying any data to be recorded directly on the CRFs (i.e., no prior written or electronic record of 
data) and considered to be source data will be provided. 
 
Clinical laboratory data required by the protocol will be electronically transferred from the central 
laboratory to the sponsor or its designee. A paper copy of the laboratory results will be provided to the 
study site and should be retained with each patient’s source data. 
 
13. 
 APPENDICES 
 

 
 
 
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APPENDIX A:  NATIONAL CANCER INSTITUTE COMMON TOXICITY CRITERIA, VERSION 4.03 
 
HTTP://WWW.EORTC.BE/SERVICES/DOC/CTC/CTCAE_4.03_2010-06-14_QUICKREFERENCE_5X7.PDF

 
 
 
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APPENDIX B:  LIST OF CYP3A4 INHIBITORS 
 
From http://www.georgetown.edu/departments/pharmacology/davetab.html 
 
The following are known inhibitors of CYP3A4: 
Delaviridine 
Indinavir 
Nelfinavir 
Ritonavir 
Saquinavir 
Amiodarone 
Cimetidine 
Ciprofloxacin 
Clarithromycin 
Diethyl-dithiocarbamate 
Diltiazem 
Erythromycin 
Fluconazole 
Fluvoxamine 
Gestodene ++ 
Grapefruit juice 
Itraconazole 
Ketoconazole 
Mifepristone 
Nefazodone 
Norfloxacin 
Norfluoxetine 
Mibefradil 
Troleandomycin 
Atazanavir 
Indinavir 
Telithromycin 
Voriconazole 
 
The following are known inducers of CYP3A4: 
Rifampicine 
Phenytoin 
Rifabutin 
Rifapentine 
Carbamazepine 
Phenobarbital 
St. John’s Wort 

 
 
 
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APPENDIX C: ECOG PERFORMANCE STATUS SCALE 
 
 
Grade 
Description 
0 
Fully active, able to carry on all pre-disease performance without 
restriction 
1 
Restricted in physically strenuous activity but ambulatory and able to 
carry out work of a light or sedentary nature, e.g., light housework or 
office work 
2 
Ambulatory and capable of all self-care but unable to carry out any 
work activities. Up and about >50% of waking hours 
3 
Capable of only limited self-care, confined to a bed or chair 
>50% of waking hours 
4 
Completely disabled.  Cannot carry on any self-care. Totally 
confined to bed or chair 
5 
Dead 

 
 
 
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APPENDIX D:  NEW YORK HEART ASSOCIATION GUIDELINES 
 
 
 
 
Class I 
People whose physical activity is not limited. Ordinary physical activity 
does not cause undue fatigue, heart palpitations, trouble breathing, or 
chest pain. 
Class II 
People who have some limitation on physical activity. They are 
comfortable at rest, but ordinary physical activity causes fatigue, heart 
palpitations, trouble breathing, or chest pain. 
Class III 
People who have a marked limitation on physical activity. They are 
comfortable at rest, but less-than-ordinary physical activity causes 
fatigue, heart palpitations, trouble breathing, or chest pain. 
Class IV 
People who are unable to carry on any physical activity without 
discomfort.  Symptoms may be present even at rest. If any physical 
activity is undertaken, discomfort is increased 

 
 
 
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APPENDIX E:  RESPONSE AND RELAPSE CRITERIA FOR MULTIPLE MYELOMA 
 
Response category 
Criteria 
Stringent complete response (sCR)   
 
CR as defined below plus  
• Normal FLC ratio   
• Absence of clonal cells in bone marrow by 
immunohistochemistry or immunofluorescence 
Complete response (CR) 
• Negative immunofixation of the serum and urine  and 
• • < 5% plasma cells in bone marrow  and 
• Disappearance of any soft tissue plasmacytomas. 
 
Very good partial response (VGPR) • Serum and urine M-protein detectable by immunofixation 
but not on electrophoresis or  
• 90% or greater reduction in serum M-protein  plus urine M-
protein level <100 mg per 24 hour. 
Partial response (PR) 
≥ 50% reduction of serum M-protein and reduction in 24-
hour urinary M-protein by ≥ 90% or to < 200 mg per 24 hour. 
• If the serum and urine M-protein are unmeasurable, a ≥ 50% 
decrease in the difference between involved and uninvolved 
FLC levels is required In place of the M-protein criteria  
• If serum and urine M-protein are unmeasurable, and serum 
free light assay is also unmeasurable, ≥ 50% reduction in 
bone marrow plasma cells is required in place of M-protein, 
provided baseline bone marrow plasma cell percentage was ≥ 
30%  
• In addition to the above listed criteria, if a plasmacytoma is 
present at baseline, ≥ 50% reduction in the size of soft tissue 
plasmacytomas is also required  
 

 
 
 
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Progressive Disease 
Progressive Disease requires any one or more of the following: 
Increase of ≥25% from baseline in 
• 
Serum M-component and/or (the absolute increase must be ≥0.5 
g/dl) 
• 
Urine M-component and/or (the absolute increase must be ≥200 
mg/24hour 
• 
Only in patients without measurable serum and urine M-protein 
levels: the difference between involved and uninvolved FLC 
levels. The absolute increase must be >10 mg/dl 
• 
Bone marrow plasma cell percentage: the absolute % must be 
≥1-% 
• 
Definite development of new bone lesions or soft tissue 
plasmacytomas or definite increase in the size of existing bone 
lesions or soft tissue plasmacytomas 
• 
Development of hypercalcemia (corrected serum calcium > 
11.5 mg/dl or 2.65 mmol/l) that can be attributed solely to the 
plasma cell proliferative disorder 
 
Clinical relapse 
Clinical relapse requires one or more of: 
Direct indicators of increasing disease and/or end organ dysfunction 
(CRAB features). It is not used in calculation of time to progression or 
progression-free survival but is listed here as something that can be 
reported optionally or for use in clinical practice 
1. Development of new soft tissue plasmacytomas or bone lesions 
2. Definite increase in the size of existing plasmacytmas or bone 
lesions. A definte increase is defined as a 50% (and at least 
1cm) increase as measured serially by the sum of the products 
of the corr-diameters of the measurable lesion. 
3. Hypercalcemia (> 11.5 mg/dl) [2.65 mmol/l] 
4. Decrease in hemoglobin of ≥ 2g/dl [1.25 mmol/l] 
5. Rise in serum creatinine by 2 mg/dl or more [177 micro mol/l 
or more] 

 
 
 
Pro00062304:  Ph Ib/II single agent ABC294640 in R/R MM 
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APPENDIX F:  STUDY CALENDAR 
PHASE Ib AND II PORTION - CLINICAL AND LABORATORY EVALUATIONS 
Activity 
 
Screening 
Phase b 
Cycle 1 a 
Cycle 2 
Cycle 3 
Cycle 4 
Subsequent 
cycles 
End of 
Treatmentu 
Follow-
upp 
W 1 W 
2 
W 3 W 
4 
W 
1 
W 
2 
W 3 W 4 W 
1 
W 
2 
W 
3 
W 
4 
W 
1 
W 
2 
W 
3 
W 
4 
Informed Consent c 
X 
 
Serum Pregnancy Test d 
X 
 
Study Registration e 
X 
 
History and Physical Examt 
Xs 
Xq X 
X 
X X 
X 
X 
X 
X 
X 
X 
X 
X 
ECOG Performance Status 
Xs 
Xq X 
X 
X X 
X 
X 
X 
X 
X 
X 
X 
 
Vital Signs 
X 
X 
X 
X 
X X 
X 
X 
X 
X 
X 
X 
X 
Assessment of 
Concomitant Medications 
Xs 
X 
X 
X 
X X 
X 
X 
X 
X 
X 
X 
X 
 
Serum chemistry f 
Xr,s 
Xq X 
X 
X X 
X 
X 
X 
X 
X 
X 
X 
X 
PT/INR and aPTT g 
Xs 
Xq X 
X 
X X 
 
X 
 
X 
 
X 
 
 
Complete blood count 
with differential h 
Xs 
Xq X 
X 
X X 
X 
X 
X 
X 
X 
X 
X 
X 
Screening HIV, HBV and HCV i 
X 
 
12 Lead ECG j 
X 
 
 
 
 
 
 
 
Echocardiogram k 
X 
 
 
Urinalysis 
Xs 
Xq  
 
 
X 
 
X 
 
X 
 
X 
X 
 
SPEP (serum protein 
Xs 
Xq  
 
 
X 
 
X 
 
X 
 
X 
X 
 
Serum free light chain 
Xs 
Xq  
 
 
X 
 
X 
 
X 
 
X 
X 
 
Urine monoclonal protein 
Xs 
Xq  
 
 
X 
 
X 
 
X 
 
X 
X 
 
IFE (immunofixation 
Xs 
Xq  
 
 
X 
 
X 
 
X 
 
X 
X 
 
Immunoglobulin profile 
Xs 
Xq  
 
 
X 
 
X 
 
X 
 
X 
X 
 
Beta-2 microglobulin 
Xs 
Xq  
 
 
X 
 
X 
 
X 
 
X 
X 
Metastatic bone survey l 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
Administration of ABC296460 
X 
X 
X 
X X X X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Adverse Events Assessment m 
X 
X 
X 
X X 
X 
X 
X 
X 
X 
X 
X 
Review Patient Diary and Pill Count 
X 
X 
X 
X X 
X 
X 
X 
X 
X 
X 
X 
Plasma PK n 
X 
  
Plasma PD v 
 
X 
 
 
 
X 
 
X 
 
X 
 
Xv 
 
Bone marrow biopsy o 
X 
 
X 
 
Xo 
 
Survival Follow-up 
X 

 
 
 
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Footnotes: 
a) Cycle = 28 days. For cycle 1 to 4, ± 3 days for all visits except cycle 1 day 1.  For cycle 5 and 
beyond, ± 7 days for all visits. 
b) Screening Evaluations must be completed within 30 calendar days prior to Treatment Day 1, except 
for bone marrow biopsy, which should be performed within 2 weeks prior to Treatment Day 1. 
c) Informed Consent must be signed within 30 days before Treatment Day 1, and will be updated and 
re-signed by Investigator and Subjects, as needed. 
d) Pregnancy test (for WOCBP) must be performed within 48 hours of first administration of study 
drug on Treatment Day 1. 
e) Investigator must confirm that patient will not have taken chemotherapy for at least 14 days prior to 
Treatment Day 1. 
f) Serum Chemistries will include: sodium, chloride, carbon dioxide, potassium, calcium, phosphorous, 
magnesium, BUN, creatinine, blood sugar (random), albumin, alkaline phosphatase, alanine 
aminotransferase, aspartate aminotransferase, and bilirubin (total, direct).  
g) INR and aPTT will be performed at screening, every week during cycle 1 and then the first week of 
each following cycle. 
h) Complete blood count will include hemoglobin, hematocrit, total leukocyte count including 
differential, platelets. 
i) Patients undergo HIV and hepatitis screening (HBVsAg, anti-HBs, anti-HBc; anti-HCV 
qualitative). 
j) ECGs will be obtained during screening. 
k) Performed at baseline and end of treatment, and if patient demonstrates signs or symptoms of 
myocardial degeneration. 
l) Metastatic bone survey: at the screening, then every year or at the end of treatment.  
m) Performed according to CTCAE version 4. 
n) Plasma for PK analysis ABC294640 alone will be collected Cycle 1, Day 1 immediately before 
dosing and at approximately 1, 2, 4, and 8, hours after dosing and will be sent to Dr. Kang’s lab 
and/or Apogee Biotechnology Corporation for analysis. 
o) Bone marrow biopsy must be obtained prior to initiation of treatment with ABC294640 (within 2 
weeks prior to the treatment), at the end of cycle 3 (± 7 days) and at the end of cycle 6 (± 7 days). 
For patients who discontinue therapy for any reason before completing 3 cycles, bone marrow 
biopsy will be obtained at the time of discontinuation or as soon as feasible thereafter. After cycle 6, 
the bone marrow biopsy will be performed at the discretion of the treating physician. 
p) If the patient was removed from treatment for toxicity, the patient should be followed with weekly 
clinic visit and laboratory studies as appropriate, at Duke Cancer Center or the patient’s local MD, 
until the AE has resolved to baseline. 
Phase II patients should be followed every 3 months for survival, for 2 years from the time of 
their End of Treatment visit. 
q) If Screening was performed within 1 week prior to C1D1, these procedures/labs do not need 
to be repeated. If urine monoclonal protein was performed within 14 days of C1D1, it will not 
need to be repeated.  
r)  Includes LDH at screening.s) Screening procedures/labs which can be assessed prior to drug 
administration may be completed on C1D1, if necessary. 
t) Physical exam at screening, and day 1 of each cycle will be a focused exam including vital signs, 
HEENT, heart/lung/chest/abdomen/skin and neurological assessment. Physical exam at all other 
time points will be symptom-directed.  
u) EOT should occur within 4-6 weeks after the last dose of study drug. 
v) PD samples will be collected on C1D1 prior to study drug administration and at 
approximately 1, 2, 4, and 8, hours after dosing along with PK samples and prior to 
ABC294640 drug administration on D1 of each subsequent cycle through C6. 

 
 
 
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APPENDIX G: DRUG DIARY 
ABC294640 Drug Diary (Page 67 of 3)          Cycle:____________Subject ID:______    Version 3, 03/22/2017 
Take _____ pills in the morning and _____ pills in the evening approximately 12 hours apart. (Example: 9:00am 
and 9:00 pm) 
Take the pills at least 1 hour before or 2 hours after eating.  Bring the pill bottle with any leftover pills (or if 
empty) and diary with you to each clinic visit. 
 
If you have questions, call _______________________ at the phone number: ___________________. My doctor’s name is: 
____________________. 
*To be completed by Research RN:     wk1  date reviewed: _______ time:_____ Initials:___        
wk2 date reviewed: _______ time:_____ Initials:_______ 
 
Week Day 
 
Date 
(DD-MMM-
YYYY) 
ABC294640 
Comment 
(include details and reason 
for missed doses, etc) 
# of 
Tablets 
Taken 
Exact Time 
Tablets 
Taken 
# of 
Tablets 
Taken 
Exact Time 
Tablets 
Taken 
___ mg 
HH:MM 
(AM) 
___ mg 
HH:MM 
(PM)           
1 
1  
 
 
 
 
 
2  
 
 
 
 
 
3  
 
 
 
 
 
4  
 
 
 
 
 
5  
 
 
 
 
 
6  
 
 
 
 
 
7  
 
 
 
 
 
2 
8  
 
 
 
 
 
9  
 
 
 
 
 
10  
 
 
 
 
 
11  
 
 
 
 
 
12  
 
 
 
 
 
13  
 
 
 
 
 
14  
 
 
 
 
 

 
 
 
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Page 68 of 76 
CONFIDENTIAL 
ABC294640 Drug Diary (Page 68 of 3)                Cycle:___________Subject ID:______     Version 3, 03/22/2017 
Take _____ pills in the morning and _____ pills in the evening approximately 12 hours apart. (Example: 9:00am 
and 9:00 pm) 
Take the pills at least 1 hour before or 2 hours after eating.  Bring the pill bottle with any leftover pills (or if 
empty) and diary with you to each clinic visit. 
 
If you have questions, call _______________________ at the phone number: ___________________. My doctor’s name is: 
____________________. 
*To be completed by Research RN:     wk3 date reviewed: _______ time:_____ Initials:___        
wk4 date reviewed: _______ time:_____ Initials:_______ 
Week Day 
 
Date 
(DD-MMM-
YYYY) 
ABC294640 
Comment 
(include details and 
reason for missed doses, 
etc) 
# of 
Tablets 
Taken 
Exact Time 
Tablets 
Taken 
# of  
Tablets 
Taken 
Exact Time 
Tablets 
Taken 
___ mg 
HH:MM 
(AM) 
____ mg 
HH:MM 
(PM) 
3 
15  
 
 
 
 
 
16 
 
 
 
 
 
 
17 
 
 
 
 
 
 
18 
 
 
 
 
 
 
19 
 
 
 
 
 
 
20 
 
 
 
 
 
 
21 
 
 
 
 
 
 
4 
22 
 
 
 
 
 
 
23 
 
 
 
 
 
 
24 
 
 
 
 
 
 
25 
 
 
 
 
 
 
26 
 
 
 
 
 
 
27 
 
 
 
 
 
 
28 
 
 
 
 
 
 

 
 
 
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If you need additional room to write your information or comments, please use the table below. 
Write any additional notes regarding your answers above.  
 
Date 
Additional Notes 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
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