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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                                                                                                                                                                                                                                                                                                           |                     |                  |
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| 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 8:  | May 17, 2017     |
| Amended version 1:                                                                                                                                                                                                                                                                                                                                     | March 19, 2015                                                                                                                                                                                                                                                                                                                                                              | Amended version 9:  | October 5, 2017  |
| Amended version 2:                                                                                                                                                                                                                                                                                                                                     | May 1, 2015                                                                                                                                                                                                                                                                                                                                                                 | Amended version 10: | April 11, 2018   |
| Amended version 3:                                                                                                                                                                                                                                                                                                                                     | May 27, 2015                                                                                                                                                                                                                                                                                                                                                                |                     |                  |
| Amended version 4:                                                                                                                                                                                                                                                                                                                                     | June 15, 2015                                                                                                                                                                                                                                                                                                                                                               | Amended version 11: | December 4, 2018 |
| Amended version 5:                                                                                                                                                                                                                                                                                                                                     | June 28, 2016                                                                                                                                                                                                                                                                                                                                                               | Amended version 12: | March 22, 2019   |
| Amended version 6:                                                                                                                                                                                                                                                                                                                                     | August 19, 2016                                                                                                                                                                                                                                                                                                                                                             |                     |                  |
| Amended version 7:                                                                                                                                                                                                                                                                                                                                     | October 12, 2016                                                                                                                                                                                                                                                                                                                                                            |                     |                  |
| 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 | 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) |                     |                  |
| Medical Director  Terry Plasse, MD RedHill Biopharma Ltd Ha'arba'a St. Tel Aviv 64739                                                                                                                                                                                                                                                                  | Taewoong Choi, MD Taewoong.Choi@duke.edu Cristiana Costa, DO                                                                                                                                                                                                                                                                                                                |                     |                  |
| CONFIDENTIAL                                                                                                                                                                                                                                                                                                                                           |                                                                                                                                                                                                                                                                                                                                                                             |                     |                  |
| The information contained in this document is regarded as confidential, and may not be disclosed to another party unless such disclosure is                                                                                                                                                                                                            |                                                                                                                                                                                                                                                                                                                                                                             |                     |                  |  
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 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  
| Contents                                                                                                                                                 |                                                                                                                                                                  |              |
|----------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------|
| DEFINITION OF TERMS USED .                                                                                                                               |                                                                                                                                                                  |              |
| BACKGROUND .                                                                                                                                             |                                                                                                                                                                  |              |
| 1.                                                                                                                                                       | M MULTIPLE MYELOMA                                                                                                                                               |              |
| 1.1                                                                                                                                                      |                                                                                                                                                                  |              |
| 1.2                                                                                                                                                      | SPHINGOLIPIDS, SPHINGOSINE KINASES AND CANCER                                                                                                                    |              |
| 1.3                                                                                                                                                      | Summary of ABC294640 Phase I Clinical Data in Solid Tumors                                                                                                       |              |
| Rationale for a clinical trial evaluating ABC294640 in patients with MM ............................20                                                   |                                                                                                                                                                  |              |
| 1.4                                                                                                                                                      |                                                                                                                                                                  |              |
| 1.5                                                                                                                                                      | Pre-clinical efficacy of ABC294640 for Multiple Myeloma ..................................................................20                                     |              |
| 2.                                                                                                                                                       | STUDY DESIGN .                                                                                                                                                   |              |
| 2.1                                                                                                                                                      | Study description                                                                                                                                                |              |
| 2.2                                                                                                                                                      | Phase 1B                                                                                                                                                         |              |
| 2.3                                                                                                                                                      | Phase 1B design                                                                                                                                                  |              |
| 2.4                                                                                                                                                      | Definition of dose-limiting toxicities .                                                                                                                         |              |
| 2.5                                                                                                                                                      | Phase II                                                                                                                                                         |              |
| 3.                                                                                                                                                       | STUDY PATIENTS .                                                                                                                                                 |              |
| 4.                                                                                                                                                       | ENROLLMENT AND TREATMENT .............................................................................................................34                         |              |
| 4.1                                                                                                                                                      | SCREENING                                                                                                                                                        |              |
| 4.2                                                                                                                                                      | Phase IB: Dose escalation phase ...............................................................................................................35                |              |
| 4.3                                                                                                                                                      | Phase II .                                                                                                                                                       |              |
| 4.4                                                                                                                                                      | Safety confirmation and follow-up .........................................................................................................37                    |              |
| 4.5                                                                                                                                                      | Disease assessment .                                                                                                                                             |              |
| 5.                                                                                                                                                       | PK and PD Studies                                                                                                                                                |              |
| Special Instructions related to administration of ABC 294640 on days of PK Sampling.....37                                                               |                                                                                                                                                                  |              |
| 5.1                                                                                                                                                      |                                                                                                                                                                  |              |
| 5.2                                                                                                                                                      | Pharmacodynamic and correlative assays ..........................................................................................38                              |              |
| Time points of sample collection ................................................................................................................39      |                                                                                                                                                                  |              |
| 5.3                                                                                                                                                      |                                                                                                                                                                  |              |
| 5.4                                                                                                                                                      | Sample collection and shipment                                                                                                                                   |              |
| ASSAYS ...............................................................................................................................................40 |                                                                                                                                                                  |              |
| 5.5                                                                                                                                                      | STUDY DRUG: ABC294640 ....................................................................................................................41                     |              |
| 6.                                                                                                                                                       | 6.1                                                                                                                                                              | Formulation  |
| 6.2                                                                                                                                                      | Dosage and administration                                                                                                                                        |              |
| 6.3                                                                                                                                                      | S Storage                                                                                                                                                        |              |
| 6.4                                                                                                                                                      | L Labeling ...................................................................................................................................................42 |              |
| Test article and accountability ...............................................................................................................42        |                                                                                                                                                                  |              |
| 6.5                                                                                                                                                      |                                                                                                                                                                  |              |
| 7.                                                                                                                                                       | METHOD OF DETERMINING TREATMENT COMPLIANCE                                                                                                                       |              |
| 7.1                                                                                                                                                      | SAFETY PLAN                                                                                                                                                      |              |
| 7.2                                                                                                                                                      | POST-TREATMENT EVALUATIONS ............................................................................................43                                        |              |
| 7.3                                                                                                                                                      | PATIENT DISCONTINUATION                                                                                                                                          |              |
| 8.                                                                                                                                                       | STATISTICAL CONSIDERATIONS                                                                                                                                       |              |
| 8.1                                                                                                                                                      | PRIMARY OBJECTIVE                                                                                                                                                |              |
| 8.2                                                                                                                                                      | PRIMARY ENDPOINTS                                                                                                                                                |              |
| 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                                                                                                                                             |              |
| 9.                                                                                                                                                       | SAFETY MONITORING AND REPORTING ......................................................................................48                                         |              |
| PT-Phl-II v11.08.18                                                                                                                                      | Duke Cancer Institute                                                                                                                                            | Page 3 of 76 |
| CONFIDENTIAL                                                                                                                                             |                                                                                                                                                                  |              |  
| Version:  March 22, 2019   |                                                                                                                                           |                              |
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| A ADVERSE EVENTS           |                                                                                                                                           |                              |
| 9.1                        |                                                                                                                                           |                              |
| 9.2                        | SERIOUS ADVERSE EVENTS ................................................................................................................49 |                              |
| 9.3                        | A ADVERSE EVENT ASSESSMENT                                                                                                                |                              |
| 9.4                        | ADVERSE EVENT CAUSALITY                                                                                                                   |                              |
| 9.5                        | A ADVERSE REACTION EXPECTEDNESS                                                                                                           |                              |
| 9.6                        | ADDITIONAL ADVERSE EVENT CONSIDERATIONS                                                                                                   |                              |
| 9.7                        | PROCEDURES FOR ELICITING, RECORDING, AND REPORTING ADVERSE                                                                                |                              |
| EVENTS .                   |                                                                                                                                           |                              |
| 9.8                        | Quality Control and Quality Assurance                                                                                                     |                              |
| 9.9                        | Audits                                                                                                                                    |                              |
| 9.10                       | DATA Management and Processing .                                                                                                          |                              |
| 10.                        | ADMINISTRATIVE and ETHICAL CONSIDERATIONS                                                                                                 |                              |
| 10.1                       | Regulatory and Ethical Compliance                                                                                                         |                              |
| 10.2                       | DUHS Institutional Review Board and DCI Cancer Protocol Committee ..............................55                                        |                              |
| 10.3                       | Informed Consent                                                                                                                          |                              |
| 10.4                       | S STUDY DOCUMENTATION                                                                                                                     |                              |
| 10.5                       | Privacy, Confidentiality, and Data Storage                                                                                                |                              |
| DATA AND SAFETY MONITORING |                                                                                                                                           |                              |
| 10.6                       |                                                                                                                                           |                              |
| 10.7                       | Protocol Amendments                                                                                                                       |                              |
| 10.8                       | Records Retention                                                                                                                         |                              |
| ll.                        | SPONSOR AND MULTI-SITE INVESTIGATOR REQUIREMENTS ............................................57                                           |                              |
| 1.1                        | STUDY INITIATION                                                                                                                          |                              |
| 11.2                       | STUDY COMPLETION                                                                                                                          |                              |
| 11.3                       | INSTITUTIONAL REVIEW BOARD APPROVAL ............................................................................58                        |                              |
| QUALITY ASSURANCE          |                                                                                                                                           |                              |
| 11.4                       |                                                                                                                                           |                              |
| 11.5                       | DATA COLLECTION                                                                                                                           |                              |
| GENERAL CONSIDERATIONS .   |                                                                                                                                           |                              |
| 12.                        | 12.1                                                                                                                                      | Discontinuation of the Study |
| 12.2                       | Routine Study Termination                                                                                                                 |                              |
| 12.3                       | Premature Study Termination                                                                                                               |                              |
| 12.4                       | Changes to the Protocol                                                                                                                   |                              |
| 12.5                       | Protocol Deviations for Emergency or Adverse Event ...........................................................................59          |                              |
| 12.6                       | SOURCE DOCUMENTS .                                                                                                                        |                              |
| 13.                        | APPENDICES                                                                                                                                |                              |
| REFERENCES                 |                                                                                                                                           |                              |  
| PROTOCOL SYNOPSIS                         |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      |              |
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| 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 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 RATIONALE 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. 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. |              |
| OBJECTIVES - PHASE IB PT-PhI-II v11.08.18 | Duke Cancer Institute                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | Page 5 of 76 |
| CONFIDENTIAL                              |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      |              |  
| Version: March 22, 2019                             |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |              |
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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. 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) OBJECTIVES - PHASE II | 59 (may include 3 patients treated at MTD in Phase Ib, thus 56 additional patients) 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                                                                                                                                               |              |
| PATIENT ELIGIBILITY CRITERIA PT-PhI-II v11.08.18    | Duke Cancer Institute                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | Page 6 of 76 |  
PT-PhI-II v11.08.18 Duke Cancer Institute Page 6 of 76  
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| Version: March 22, 2019                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |                       |              |
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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≥9g/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 |                       |              |
| PT-PhI-II v11.08.18                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           | Duke Cancer Institute | Page 7 of 76 |
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| Version: March 22, 2019                                                                                                                                                                                                                                                                                                                                                           |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            |              |
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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                                                                                                                                                                                                                                                                                                                                                                      | ABC294640 will be administered orally twice a day (approximately 12 hours apart)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |              |
| ADMINISTRATION                                                                                                                                                                                                                                                                                                                                                                    | continuously for 28 days. A cycle will be defined as 28 days. 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.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |              |
| DEFINITION OF DOSE LIMITING TOXICITY                                                                                                                                                                                                                                                                                                                                              | 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  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.                                                                                                                                                                                                                                                   |              |
| TREATMENT REGIMENS                                                                                                                                                                                                                                                                                                                                                                | 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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PT-PhI-II v11.08.18 Duke Cancer Institute Page 8 of 76  
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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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| Screening Period:                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |                  |
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| PRESTUDY ASSESSMENTS                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | -                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           | 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 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. |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |                  |
| TREATMENT ASSESSMENTS                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | 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). 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. |                  |
| PHARMACOKINETIC ASSESSMENTS                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |                  |  
| Correlative studies                                                                                                                                                                                                                                                                                                                                                                                     |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |                                                                                                                                                                                                                                                                                  |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| -                                                                                                                                                                                                                                                                                                                                                                                                       | P                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |                                                                                                                                                                                                                                                                                  |
| 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. |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |                                                                                                                                                                                                                                                                                  |
| -                                                                                                                                                                                                                                                                                                                                                                                                       | S                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |                                                                                                                                                                                                                                                                                  |
| 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.                                                                                                                                                                 |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |                                                                                                                                                                                                                                                                                  |
| -                                                                                                                                                                                                                                                                                                                                                                                                       | E                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |                                                                                                                                                                                                                                                                                  |
| expression of sphingolipid signaling genes, SK2 mRNA level or activity, c-Myc, Mcl1 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.                                                                                                                                                                                                                                                                                                                                                                                                         |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |                                                                                                                                                                                                                                                                                  |
| CORRELATIVE  STUDY AND  PHARMACODYMANIC ASSESSMENTS                                                                                                                                                                                                                                                                                                                                                                                                         | -                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | 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 will be captured in a secure, web-based, 21 CFR Part 11 compliant system.                                                                                                                                                                                                                                                                                                                          |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |                                                                                                                                                                                                                                                                                  |
| DATA COLLECTION                                                                                                                                                                                                                                                                                                                                                                                         | 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.                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |                                                                                                                                                                                                                                                                                  |
| INVESTIGATIVE SITES                                                                                                                                                                                                                                                                                                                                                                                     | 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. |                                                                                                                                                                                                                                                                                  |
| STATISTICAL ANALYSIS                                                                                                                                                                                                                                                                                                                                                                                    | 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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |                                                                                                                                                                                                                                                                                  |
| PT-PhI-II v11.08.18                                                                                                                                                                                                                                                                                                                                                                                     | Duke Cancer Institute                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   | Page 11 of 76                                                                                                                                                                                                                                                                    |
| CONFIDENTIAL                                                                                                                                                                                                                                                                                                                                                                                            |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |                                                                                                                                                                                                                                                                                  |  
| 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. 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.   |
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| DURATION OF STUDY                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
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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                                                             |               |
| PT-PhI-II v11.08.18 | Duke Cancer Institute                                                                         | Page 12 of 76 |
| CONFIDENTIAL        |                                                                                               |               |  
| Version: March 22, 2019   |                                                        |               |
|---------------------------|--------------------------------------------------------|---------------|
| 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                |               |
| PT-PhI-II v11.08.18       | Duke Cancer Institute                                  | Page 13 of 76 |
| CONFIDENTIAL              |                                                        |               |  
| 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 yearly3and 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.
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1.2 sphingolipids sphingosine kinases and

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 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.
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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).  
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
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1.2.2 differential roles of sk1

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.
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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 antiinflammatory 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:
PT-PhI-II v11.08.18 Duke Cancer Institute Page 17 of 76 CONFIDENTIAL
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.
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1.3 summary of abc294640 phase

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 drugrelated 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.  
| Study ID                            | Dose                                   |                              |           |      |
|-------------------------------------|----------------------------------------|------------------------------|-----------|------|
| Patient                             | 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 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.  
  
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.  
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 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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1.4 rationale for a clinical

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 upregulates 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.
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1.5 pre-clinical efficacy of abc294640

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.
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1.5.1 sphingosine kinase 2 is

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 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 EBVImmortalized 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).  
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, RPMI8226-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).  
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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.
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1.5.2. sk2-specific shrna inhibits myeloma

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  
  
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 SK2shRNA 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

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 M.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 clls (data not shown).  
  
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).  
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 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.  
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 downregulation 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.  
  
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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1.5.5. abc294640 down-regulates ps6 and

MM originates from post-germinal-center B cells and c-Myc is a class oncogene dysregulated in postgerminal 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 upregulated 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**).
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1.5.6. abc294640 synergizes with bcl-2

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**).
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1.5.7. abc294640 induces myeloma cell

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+
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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 (OPMI, RPMI-8226, MM.1S, and U266) were treated with 30uM of ABC294640 (A) or DMSO (D) for 16 hr and whole cell Iysates were prepared and analyzed for Bcl-2 expression by Western blot. B: Combination of ABC294640 and ABT-737 led to 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.*  
  
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.  
Our pre-clinical data justify continued development of ABC294640 as a therapy for patients with myeloma.
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1.5.9. the roles of sk2

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.