And Effective in Patients with Relapsed Or Refractory T-Cell 3 Lymphoma: Results of a Translational Phase I Dose-Escalation 4 Study

Home , Romidepsin

Author Manuscript Published OnlineFirst on November 26, 2019; DOI: 10.1158/1078-0432.CCR-19-2152 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Running Title: HDAC inhibitor and anthracycline combination, CTCL

1 Romidepsin in Combination with Liposomal Doxorubicin is Safe 2 and Effective in Patients with Relapsed or Refractory T-cell 3 Lymphoma: Results of a Translational Phase I Dose-Escalation 4 Study

5 Khoan Vu1, Chi-Heng Wu1, Chen-Yen Yang1, Aaron Zhan1, Erika Cavallone1, Wade Berry1, Pamela 6 Heeter2, Laura Pincus1, Matthew Wieduwilt3, Basem William2, Charalambos Andreadis1, Lawrence 7 Kaplan1, Frank McCormick4, Pierluigi Porcu2, Jonathan E. Brammer2*, Weiyun Z. Ai1*

8 1Division of Hematology and Blood and Marrow Transplantation, Department of Medicine, 9 University of California, San Francisco, 2Division of Hematology, James Comprehensive Cancer 10 Center, Department of Internal Medicine, The Ohio State University, Columbus, OH, 3Moores Cancer 11 Center, University of California, San Diego, 4Hellen Diller Family Cancer Center, University of 12 California, San Francisco 13 *J.E.B. and W.Z.A. contributed to the work equally 14 Correspondence: Weiyun Ai, Division of Hematology and Blood and Marrow Transplantation, University 15 of California, San Francisco, 505 Parnassus Avenue, M1286, Box 0324, San Francisco, CA 94143- 16 0324. Phone: 415-353-4061; E-mail: [email protected]. 17 18 Disclosure of Potential Conflicts of Interest: 19 K.V. owns stocks of Celgene. C.A. has received research funding and honorarium from Celgene. P.P. 20 has received research funding from Viracta, Innate Pharma, Kiowa, Daiichi, Incyte, and BeiGene, as 21 well as consulting from Innate Pharma, Viracta, and Spectrum. J.E.B. has received research funding 22 from Celgene. The remaining authors declare no competing financial interests. 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 26, 2019; DOI: 10.1158/1078-0432.CCR-19-2152 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Running Title: HDAC inhibitor and anthracycline combination, CTCL

1 Abstract 2 Purpose: The histone deacetylase (HDAC) inhibitor romidepsin and the anthracycline liposomal 3 doxorubicin (LD) have modest single-agent activity in cutaneous T-cell lymphoma (CTCL) and 4 peripheral T-cell lymphoma (PTCL). We investigated the safety and efficacy of the combination of 5 these two agents in CTCL and PTCL. 6 7 Patients and Methods: Using CTCL cell lines and primary CTCL tumor cells, we demonstrated 8 synergistic anti-tumor activity with romidepsin plus doxorubicin. We then conducted a phase I dose- 9 escalation study of the romidepsin/LD combination in relapsed/refractory CTCL and PTCL. The 10 primary objective was to determine the maximum tolerated dose (MTD) of romidepsin in combination 11 with LD at 20 mg/m2 IV, once every 28 days. 12 13 Results: Eleven CTCL and 12 PTCL patients were treated. The MTD of romidepsin was determined to 14 be 12 mg/m2. Grade 3/4 hematologic toxicities included thrombocytopenia (17%), anemia (13%), and 15 neutropenia (9%). The most frequent treatment-related non-hematologic adverse events were fatigue 16 (48%), nausea (48%), vomiting (35%), and anorexia (30%). Among 21 evaluable patients, the overall 17 response rate was 70% (1 CR, 6 PR) in CTCL and 27% (3 CR, 0 PR) in PTCL. Of the CTCL patients, 18 8 of 10 had skin response, including 6 patients (60%) achieving skin involvement < 10% of their body 19 surface area at time of best response. 20 21 Conclusions: Romidepsin plus LD demonstrated an acceptable safety profile and promising clinical 22 efficacy with deep skin responses in relapsed/refractory CTCL. Thus, this combination could be 23 considered as a bridge to skin-directed treatment or allogeneic hematopoietic cell transplantation in 24 patients with aggressive CTCL. 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

Running Title: HDAC inhibitor and anthracycline combination, CTCL

1 Translational Relevance 2 Based on in vitro studies showing that histone deacetylase (HDAC) inhibitors such as romidepsin 3 potentiate the activity of anthracyclines in both B-cell lymphoma and acute leukemia cell lines, we 4 tested the hypothesis that the combination of romidepsin and doxorubicin would have synergistic 5 effects against T-cell lymphoma in pre-clinical models and, by extension, in patients with 6 relapsed/refractory T-cell lymphoma. We found that romidepsin was synergistic with doxorubicin in 7 growth inhibition and apoptosis induction in both cutaneous T-cell lymphoma (CTCL) cell lines and 8 patient-derived primary CTCL cells. This led us to develop a phase I clinical trial testing romidepsin 9 combined with liposomal doxorubicin (LD) in patients with relapsed/refractory CTCL and peripheral T- 10 cell lymphoma (PTCL). Results from our trial show that this drug combination is highly active and well- 11 tolerated in CTCL leading to deep skin responses, consistent with our hypothesis. 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

Running Title: HDAC inhibitor and anthracycline combination, CTCL

1 Introduction 2 Cutaneous T-cell lymphoma (CTCL) is a rare non-Hodgkin lymphoma that principally involves the skin 3 and, in advanced stages, the lymph nodes, peripheral blood, and visceral organs. Mycosis fungoides 4 (MF) is the most common subtype of CTCL, comprises 50% of all CTCLs.1 Sézary syndrome (SS) is a 5 leukemic form of CTCL, characterized by diffuse erythroderma and circulating neoplastic T-cells in the 6 blood. While patients with early stage disease generally have a good prognosis, patients with relapsed 7 or advanced stage (IIB to IVB) MF or SS, have a median overall survival (OS) of 4-5 years, a result that 8 has not improved in over four decades.2–4 These patients frequently require systemic therapy; 9 however, complete response (CR) rates are low, and response durations are typically short. Thus, 10 there is an urgent need for systemic therapies that improve response rates, duration of response, and 11 OS in relapsed and advanced stage CTCL. 12 13 Peripheral T-cell lymphoma (PTCL) represents 10-15% of all non-Hodgkin lymphoma cases.5 PTCL is a 14 heterogeneous disease with over twenty subtypes with different morphologies and clinical features. 15 Clinical outcomes in PTCL, particularly in patients with relapsed/refractory disease, remain poor with a 16 median OS after relapse of 5.5 months.6 Multiple agents, including pralatrexate and romidepsin, have 17 been approved by the U.S. Food and Drug Administration for relapsed/refractory PTCL and have 18 modest response rates ranging from 20-30%.7–9 Thus, there is a critical need for novel therapies and 19 drug combinations that are highly effective and tolerable for patients with relapsed/refractory PTCL. 20 21 Histone deacetylase (HDAC) inhibitors have been shown to be clinically active in both CTCL and PTCL 22 as hyper-acetylation is a recurrent aberrancy in T-cell lymphomas.7,9–16 Romidepsin is a unique 23 bicyclic, class 1, selective HDAC inhibitor that has demonstrated efficacy in CTCL and PTCL.10,16 In a 24 phase II trial in relapsed/refractory CTCL, romidepsin demonstrated an ORR of 34%, including 6% of 25 patients with CR.16 A subsequent phase II trial of romidepsin in relapsed/refractory CTCL incorporating 26 the more stringent 2011 International Society for Cutaneous Lymphomas (ISCL) consensus response 27 criteria, the global ORR was 34% with a skin response of 38 – 41%.17 In a pivotal phase II trial in 28 relapsed/refractory PTCL, romidepsin demonstrated an overall response rate (ORR) of 25%, including 29 a 15% rate of CR or unconfirmed complete response (Cru).10 The results led to FDA approval for 30 romidepsin for the treatment of relapsed/refractory CTCL and PTCL. 31 32 Liposomal doxorubicin (LD) is an effective chemotherapeutic option for patients with CTCL. The 33 EORTC 21012 multicenter phase II study of LD 20 mg/m2 IV given every 2 weeks demonstrated a 34 global ORR of 41% (20/49) with a CR rate of 6% and a skin response of 60.5%.18 Subsequently, a 35 phase II trial of LD 20 mg/m2 every 2 weeks for 8 cycles followed by bexarotene reported an ORR of 36 41% at 16 weeks.19 LD has also been evaluated in patients with TCL, including PTCL, in combination 37 with gemcitabine with modest results.20 38 39 Romidepsin and LD have demonstrated modest effectiveness in patients with relapsed and refractory 40 CTCL and PTCL. However, there is a critical need to identify novel combinations to more effectively 41 treat these aggressive diseases. In vitro studies have shown that HDAC inhibitors such as romidepsin 42 potentiate the cytotoxic effects of anthracyclines in both B-cell lymphoma and acute myeloid leukemia 43 cell lines.21–23 However, whether the combination of an HDAC inhibitor and anthracycline has 44 augmented activity in mature T-cell lymphoma is unknown. Therefore, we hypothesized that the

Running Title: HDAC inhibitor and anthracycline combination, CTCL

1 combination of romidepsin with LD would have synergistic effects in vitro against T-cell lymphoma pre- 2 clinical models and when administered together for patients with relapsed or refractory T-cell 3 lymphoma. To test this hypothesis, we set out to determine whether the romidepsin and LD 4 combination is effective first in laboratory studies utilizing CTCL cell lines and patient-derived primary 5 CTCL cells, then in a phase I translational clinical trial. 6 7 8 Patients and methods 9 10 Drugs and cell lines 11 Romidepsin and doxorubicin used in pre-clinical studies were purchased from Selleckchem. Both 12 agents were dissolved in DMSO to provide a stock concentration of 10 mM. 13 14 Human CTCL cell lines (HH, H9, Hut78) were acquired from American Type Culture Collection (ATCC). 15 All cell lines were authenticated by STR analysis and tested for Mycoplasma by ATCC prior to receipt. 16 Experiments were performed within 6 months of receipt of cell lines. In brief, cell lines were cultured at o 17 37 C, 5% CO2, in 10% FBS of RPMI-1640 or 20% FBS of IMDM and passaged three times per week. 18 All experiments were done between 4 to 8 passages after thawing the cell lines and maintained at a cell 19 density below 106 cells/mL. Logarithmically growing cells were used for all experiments. 20 21 Primary CTCL cells (PRS-1, PRS-3) were obtained from the spleens of patient-derived CTCL xenograft 22 (PDX) murine models that we developed. The PDX models were established by injecting lymphocytes 23 obtained from either lymph nodes or blood of CTCL patients into 6- to 8-week old NSG mice (NOD.Cg- 24 Prkdcscid Il2rgtm1Wjl/SzJ). Successful engraftment of malignant cells in PDX was validated by 25 immunohistochemistry staining and T-cell receptor clonality analysis (manuscript submitted). Spleens 26 from the PDX models were homogenized in cold PBS with 0.2% FBS and filtered through a 70 µm 27 sterile cell strainer. Red blood cells were removed by ACK lysing buffer. Isolated mononuclear cells 28 were cultured in high glucose RPMI-1640 (ATCC) supplemented with 20% Human AB serum (MP 29 Biomedicals), 100 U/mL of hIL-2, 500 U/mL of hIL-7, and 1% penicillin-streptomycin. 30 31 In vitro cell proliferation assay 32 To assess growth inhibition in CTCL cell lines, 5x104/mL cells were seeded into 96-well plates and 33 cultured with a serial dilution of romidepsin and/or doxorubicin for 48 hours. Cell viability was

34 measured by the CellTiter-Glo luminescent cell viability assay (Promega), and the GI50 for each agent

35 was calculated using GraphPad Prism. GI50 was defined as the drug concentration that inhibits cell 36 growth by 50% as compared with the control. To investigate the growth inhibition activity of the 37 romidepsin/doxorubicin combination and determine the ideal range of concentrations of each drug that 38 could achieve the highest synergy, we performed combination assays using four different 39 concentrations of each agent, yielding sixteen different dose combinations. The four different

40 concentrations were selected based on the GI50 of each drug. In the combination assays, both drugs 41 were added simultaneously and cultured with seeded cells for 48 hours. Percentages of viable cells 42 were expressed relative to the viability of the untreated control cells. Each experiment was 43 independently performed at least twice with duplicates in each. The most synergistic drug 44 combinations from the combination assay were used to perform apoptosis assays.

Running Title: HDAC inhibitor and anthracycline combination, CTCL

1 2 To assess growth inhibition in primary CTCL tumor cells, 0.50-1 x 106 cells were seeded into 96-well 3 plates. Cells were stimulated to grow with CD2/CD3/CD28 beads (T Cell Activation/Expansion Kit, 4 Miltenyi Biotec) for 24 – 48 hours before performing combination assays as described above. 5 6 Apoptosis assay 7 Cells from CTCL cell lines or primary tumor cells were seeded into a 96-well plate at the same cell 8 density as in the proliferation assay and treated for 48 hours (cell lines) or 72 hours (primary tumor 9 cells) with a combination of romidepsin and doxorubicin in selected concentrations (see above). Cells 10 were harvested, washed twice with cold phosphate buffered saline (PBS), and stained with propidium 11 iodide (PI) and annexin V using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences). 12 Populations of live, apoptotic and dead cells were visualized using BD Aria 3 flow cytometer (BD 13 Biosciences) and BD FACSDiva software. The data was analyzed by FlowJo software. 14 15 Clinical study design 16 This was a prospective, multi-center (2 U.S. centers), single-arm, open-label, phase 1 study 17 (#NCT01902225; https://clinicaltrials.gov/ct2/show/NCT01902225) that employed a standard “3 plus 3” 18 dose-escalation design with a dose-expansion cohort. Romidepsin was provided by Celgene 19 Corporation. The primary objective was to determine the maximum tolerated dose (MTD) of romidepsin 20 in combination with liposomal doxorubicin. The secondary objectives were complete response rate, 21 overall response rate (ORR), time to response, duration of response, progression-free survival (PFS), 22 and overall survival (OS). 23 24 For the purpose of the safety assessment and in order to minimize potential cardiotoxicity, particularly 25 in PTCL patients, the dose of LD was fixed at the standard dose of 20 mg/m2 every 4 weeks, while the 26 dose of romidepsin varied. Therefore, the MTD of the romidepsin plus LD combination was determined 27 based upon the dosage of romidepsin. Dose limiting toxicities (DLTs) were monitored during the first 28 28 days of treatment and defined as the highest dose level at which less than 33% of patients 29 experience a DLT. DLTs were defined as grade 4 or greater hematologic toxicity with the exception of 30 grade 4 neutropenia lasting less than 8 days; grade 3 thrombocytopenia lasting more than 7 days, 31 requiring dose modifications in cycle 1, or associated with bleeding; or any other grade 3 or greater 32 non-hematologic toxicity with the exception of grade 3 nausea, vomiting, anorexia, or diarrhea 33 adequately managed with supportive care. Among toxicities of special interest, grade 3 or greater left 34 ventricular systolic dysfunction or QTc prolongation was considered a DLT. 35 36 Once the MTD of romidepsin in combination with LD was determined from the dose-escalation cohort, 37 8 patients were enrolled in the dose-expansion cohort at the MTD and recommended phase 2 dose for 38 a total enrollment of up to 24 patients. This study was conducted in accordance with the International 39 Conference on Harmonization Guidelines for Good Clinical Practice and the Declaration of Helsinki. 40 The institutional review board (IRB) at each study site approved the study protocol. All patients 41 provided written informed consent. Financial support for this study was provided by Celgene 42 Corporation. 43 44 Patients

Running Title: HDAC inhibitor and anthracycline combination, CTCL

1 All patients were enrolled at the University of California, San Francisco (UCSF) and The Ohio State 2 University James Comprehensive Cancer Center (OSU-James). Eligible patients were 18 years of age 3 or older with relapsed or refractory a) CTCL (limited to stage IB-IVB MF or SS) after 1 or more prior 4 systemic therapies or more than 2 prior skin-directed therapies, or b) PTCL of any stage and subtype 5 after 1 or more prior systemic therapies. Eligible patients had an Eastern Cooperative Oncology Group 6 (ECOG) performance status < 2 with adequate hepatic, hematological, and renal function: aspartate 7 transaminase (AST) and alanine transaminase (ALT) < 3 X upper limit of normal (ULN), total bilirubin < 8 2 X ULN, absolute neutrophil count > 750/mm3, platelet count > 75,000/mm3, and creatinine clearance 9 > 30 mL/minute. Prior autologous stem cell transplant (ASCT) and prior exposure to HDAC inhibitors, 10 doxorubicin, and/or LD were permitted. Key exclusion criteria included: active central nervous system 11 or leptomeningeal lymphoma, HIV infection, hepatitis B/C infection, prior allogeneic hematopoietic cell 12 transplant, prior cumulative anthracycline exposure > 450 mg/m2 doxorubicin equivalents, and any 13 significant cardiac abnormalities including baseline QTc interval > 480 milliseconds. During the study, 14 medications leading to significant QTc prolongation or known to be strong cytochrome P450 (CYP3A4) 15 inhibitors or inducers were prohibited. All prior therapies, including radiation and chemotherapy, had to 16 be discontinued for at least one week or three half-lives, whichever was longest, prior to study drug 17 treatment. 18 19 Study treatment 20 The dose of LD was fixed at 20 mg/m2 intravenously (IV) over 1 hour on day 1 of each 28-day cycle. 21 Romidepsin was administered at 4 planned dose levels of 8, 10, 12, and 14 mg/m2 intravenously over 4 22 hours on days 1, 8, and 15, following LD. Treatment was continued until disease progression, 23 intolerance, physician discretion, patient preference, two cycles beyond best response, or up to 8 24 cycles, whichever came first. 25 26 Safety assessment 27 To assess safety, adverse event (AE) monitoring and laboratory testing were performed at all clinic 28 visits (days 1, 8, 15 of every cycle) until 30 days after the last dose of romidepsin. Toxicities were 29 reported according to the National Cancer Institute Common Terminology Criteria for Adverse Events, 30 version 4.0. All patients who received at least 1 dose of study drugs were included in the safety 31 analysis. 32 33 Efficacy and response criteria 34 Patients who had at least one formal planned response assessment were included in the efficacy 35 population. For PTCL and CTCL patients with radiologically measurable disease in nodal and/or 36 visceral disease, assessment was performed with PET/CT scans at the end of cycle 4 or within 1 week 37 after study completion, then every 6 months for 1 year, or until disease progression, death, or loss to 38 follow-up, whichever came first. For patients with CTCL, skin and blood responses were assessed 39 every other cycle, using the modified Severity Weighted Assessment Tool (mSWAT) and flow 40 cytometry, respectively.24 For CTCL patients, response was determined by the investigator utilizing the 41 International Society for Cutaneous Lymphomas consensus criteria.24 For PTCL, response was 42 determined utilizing the Lugano criteria.25 43 44 Statistical analyses

Running Title: HDAC inhibitor and anthracycline combination, CTCL

1 2 Pre-clinical studies 3 For combination assays, the interaction between romidepsin and doxorubicin was evaluated using the 4 Bliss Independence model.26 In brief, the fractional response of each drug in a combination is 5 presumably independent in the Bliss Independence model. The definition of an additive effect is that

6 the total response of a combination Pcomb is equal to the sum of the two fractional responses minus

7 their product: Pcomb = Pa + Pb - PaPb. Thus, a combination will be considered synergistic if the actual

8 measured response Pcomb,m is greater than the calculated additive response Pcomb,c based on the two

9 fractional responses. The Bliss Independence Index (BI) is defined as the ratio of Pcomb,m/Pcomb,c, 10 where BI > 1 indicates synergistic, BI = 1 indicates additive, and BI < 1 indicates antagonistic activity. 11 For apoptotic assays, differences in percentage of annexin V-positive populations between single 12 agents and combinations were analyzed by an unpaired, one-tailed t test. Statistical analysis was 13 performed using Prism software. P values less than 0.05 were considered statistically significant. 14 15 Phase I clinical trial 16 The dose escalation portion of the study employed a standard “3 plus 3” design to assess safety with 17 standard dose escalation rules. If one or fewer DLTs occurred at a dose level, then enrollment 18 continued at the next dose level. If two or more DLTs occurred at the first dose level (cohort 1, 19 romidepsin 8 mg/m2), a subsequent dose level would be explored at 6 mg/m2 of romidepsin (dose level 20 -1). If two or more DLTs occurred at dose level -1, the trial would be stopped. The MTD was defined 21 as the dose level immediately prior to the dose level resulting in 2 or more DLTs. Safety and efficacy 22 were analyzed in a descriptive manner. Efficacy end points included best response, ORR, time to 23 response, duration of response, PFS, and OS. Duration of response was defined as the time from first 24 response to disease progression, initiation of new treatment with or without progression, or death from 25 any cause, with censoring at last follow-up. PFS was defined as the time from first treatment dose to 26 disease progression, initiation of new treatment with or without progression, or death from any cause, 27 with censoring at last follow-up. OS was defined as the time from first treatment dose to death from 28 any cause with censoring at last follow-up. The 95% Clopper-Pearson exact confidence intervals (CI) 29 were calculated for ORR. The Kaplan-Meier method was used to perform survival analysis. All 30 statistical analyses were performed using SAS, version 9.2 (SASA Institute Inc., Cary, NC). 31 32 33 Results 34 35 Romidepsin and doxorubicin have synergistic anti-tumor activity in CTCL cell lines and primary 36 CTCL cells 37 We performed in vitro studies in CTCL cell lines to investigate whether the combination of romidepsin 38 and doxorubicin (romi/doxo) exhibited augmented anti-tumor activity compared to each agent alone. 39 First, we verified that romidepsin and doxorubicin inhibited proliferation as single agents in all three

40 CTCL cell lines, HH, H9 and Hut78, with GI50 of 1.62, 2.21, 1.85 nM for romidepsin and 348, 121, and 41 115 nM for doxorubicin, respectively (Supplemental Figure S1A). Next, we examined the anti- 42 proliferation activity of romi/doxo. Combination assays were performed using 16 different dose 43 combinations. For growth inhibition, romi/doxo was most synergistic in H9, followed by Hut78, and not

Running Title: HDAC inhibitor and anthracycline combination, CTCL

1 synergistic in HH (Figure 1A). Importantly, romi/doxo also induced apoptosis with synergy seen in all 2 three cell lines (Figure 1C). 3 4 Next, we examined whether the synergy between romidepsin and doxorubicin can be reproduced in 5 primary CTCL cells. First, we demonstrated that romidepsin and doxorubicin inhibited proliferation of

6 CTCL cells from patient-derived CTCL xenograft murine models, PRS-1 and PRS-3, with GI50 of 1.28 7 nM and 3.21 nM for romidepsin and 140 nM and 794 nM for doxorubicin, respectively (Supplemental 8 Figure S1B). We then performed combination assays for growth inhibition in PRS-1 and PRS-3. We 9 observed that in PRS-3, romi/doxo showed synergy in all 16 dose combinations, and in PRS-1,

10 romi/doxo was synergistic when romidepsin concentration was > 1 nM (equivalent to GI22) (Figure 1B). 11 Lastly, we showed that romi/doxo synergistically induced apoptosis in both PRS-1 and PRS-3 (Figure 12 1D). 13 14 Patients 15 Twenty-four patients were enrolled with fifteen patients enrolled in the dose-escalation cohort. One 16 patient was withdrawn from the study because of worsening disease-related cytopenias before initiation 17 of study treatment. Twenty-three patients received at least one dose of study drugs. Patient 18 demographics and baseline characteristics are shown in Table 1 and supplemental Tables 1 – 2. 19 20 The CTCL cohort consisted of 11 patients: 7 with MF, one with large-cell transformation, and 3 with 21 Sézary syndrome. Four of the 8 MF patients had skin tumors (stage T3) at time of enrollment. Median 22 number of prior systemic therapies was 3 (range 1-7). Prior systemic therapies included bexarotene (n 23 = 7, 64%), HDAC inhibitor (n = 5, 45%), extracorporeal photopheresis (n = 3, 27%), methotrexate (n = 24 3, 27%), and acitretin (n = 3, 27%). 25 26 The PTCL cohort consisted of 12 patients: 5 with PTCL not otherwise specified (PTCL-NOS), 4 with 27 angioimmunoblastic T-cell lymphoma (AITL), 2 with ALK-negative anaplastic large cell lymphoma 28 (ALCL), and one with enteropathy-associated T-cell lymphoma (EATL). The median number of prior 29 therapies was 2 (range 1-3). Prior systemic therapies included CHOP or CHOP-like combinations (n = 30 10, 83%), gemcitabine-based chemotherapy (n = 7, 58%), and single-agent brentuximab vedotin (n = 2, 31 17%). Four patients (33%) had undergone prior autologous stem cell transplantation. 32 33 Dose escalation and maximum tolerated dose 34 Fifteen patients (8 CTCL, 7 PTCL) were enrolled in the dose-escalation cohort. Three DLTs occurred, 35 all in PTCL patients. One occurred at romidepsin dose level of 12mg/m2 with grade 3 36 thrombocytopenia lasting greater than 7 days, and two occurred at romidepsin dose level of 14 mg/m2 37 with grade 4 neutropenia in one and grade 3 thrombocytopenia in the other, both lasting greater than 7 38 days. The MTD of romidepsin was 12 mg/m2 in combination with LD. In the dose-expansion cohort, 8 39 patients (3 CTCL, 5 PTCL) were enrolled and treated at the MTD of romidepsin without DLTs. 40 41 Safety 42 Twenty-three patients received at least 1 dose of study drugs and were included in the safety analysis. 43 Eighteen patients discontinued treatment, and 5 patients completed all 8 cycles of treatment. Reasons 44 for treatment discontinuation were disease progression (n = 12), AEs (n = 2), switching therapy based

Running Title: HDAC inhibitor and anthracycline combination, CTCL

1 on patient and physician choice (n = 2), consolidation with autologous stem cell transplant (n = 1), and 2 death because of disease progression (n = 1). The 2 AEs leading to treatment discontinuation were 3 central line-associated Staphylococcus aureus (S. aureus) septicemia unrelated to the study drugs and 4 grade 3 thrombocytopenia lasting greater than 7 days despite dose reduction. 5 6 In the CTCL cohort, the treatment-related hematologic AEs were largely grade 1/2, and there were no 7 episodes of grade 3/4 neutropenia (Supplemental Table S3). In the PTCL cohort, there was a higher 8 incidence of grade 3/4 treatment-related hematologic AEs, including grade 3 neutropenia (1 patient, 9 8%), grade 4 neutropenia for > 7 days (1 patient, 8%), grade 3 thrombocytopenia (4 patients, 33%), 10 and grade 3 anemia (2 patients, 17%). The most frequently reported treatment-related non- 11 hematologic AEs that occurred in >10% of CTCL or PTCL patients are reported in Table 2. The 12 majority of AEs were grade 1/2; there were no grade 4 or 5 AEs. The most frequent AEs were fatigue 13 (48%), nausea (48%), vomiting (35%), and anorexia (30%). Three infections considered related to 14 treatment occurred in three separate patients: oropharyngeal candidiasis, urinary tract infection, and 15 cryptococcal pneumonia. Three patients developed thromboses unrelated to study drugs that required 16 therapeutic anticoagulation. No patient experienced a cardiac-related AE during the study, including 17 QTc prolongation or heart failure. 18 19 Efficacy 20 Disease response was evaluable in 21/24 (88%) of patients (10 CTCL, 11 PTCL). Three patients were 21 excluded from efficacy analysis. One patient with MF discontinued treatment prior to response 22 assessment due to S. aureus septicemia related to recent chest port placement. One patient with 23 PTCL-NOS developed grade 3 thrombocytopenia > 7 days and discontinued treatment before response 24 assessment, while another PTCL-NOS patient did not start study treatment as planned due to 25 worsening disease-related cytopenias. 26 27 The overall response rate (ORR) was 70% in the CTCL cohort and 27% in the PTCL cohort (Table 3). 28 Among the 10 CTCL patients, one patient attained a global CR and 6 patients attained global PRs. 29 Responses were observed in 2 SS patients and 5 MF patients. Median time to first response was 2 30 months, and median duration of response was 5.1 months. Median number of cycles received was 6. 31 Response by disease compartment is shown in Table 4. Eight of 10 (80%) CTCL patients had skin 32 responses (1 CRs, 7 PRs); 3 of 4 patients with skin tumors (stage T3) had skin response with no 33 residual tumor lesions at time of best response. At the time of best skin response, the median 34 reduction in mSWAT score was 88% among patients with improvement in the skin (Figure 2). Although 35 only one patient had a global CR, 6 of 10 (60%) patients had residual skin lesions < 10% of their body 36 surface area at the time of best response, all of which were either patches or plaques without tumors or 37 ulcerations. By clinical stage, responses were seen in 2/3 patients with stage IB, 2/2 patients with 38 stage IIB, and 3/6 patients with stage IVA CTCL. The 7 CTCL patients who had no prior exposure to 39 HDAC inhibitors or doxorubicin all responded to the study treatment. No responses were seen in the 3 40 CTCL patients with prior romidepsin exposure (Supplemental Table S4). Of these 3 CTCL patients with 41 prior romidepsin exposure, 2 had received single-agent romidepsin as their last treatment immediately 42 prior to study enrollment. 43

Running Title: HDAC inhibitor and anthracycline combination, CTCL

1 Three CTCL patients went on to skin-directed therapies as maintenance following initial response to the 2 combination of romidepsin plus LD. One SS patient achieved partial skin response and completed all 8 3 cycles of treatment before transitioning to extracorporeal photopheresis for maintenance without 4 progression for over 41 months. Another MF patient achieved partial remission and discontinued 5 treatment after six cycles in order to transition to skin-directed treatment. A SS patient achieved CR 6 and discontinued treatment after five cycles, per patient’s preference, before transitioning to 7 extracorporeal photopheresis for maintenance. 8 9 Among the PTCL patients, all 3 responders achieved CR: one with ALK-negative ALCL who 10 progressed at 7.7 months after start of treatment, one with AITL who proceeded to consolidative ASCT 11 and has remained in remission for over 22 months, and one with AITL who completed 8 cycles of 12 treatment and remains in remission. For patients with PTCL, the median time to first response was 3.5 13 months, median duration of response was 4.2 months, and median number of cycles received was 2. 14 15 With a median follow-up time of 17.8 months, median PFS was 6.9 months for the CTCL cohort and 2.1 16 months for the PTCL cohort (Supplemental Figure S2A-B). Median OS was not reached for the CTCL 17 cohort and was 17.5 months for the PTCL cohort (Supplemental Figure S2C-D). 18 19 20 Discussion 21 22 In this study, we took a clinical and translational approach to examine whether HDAC inhibitor 23 romidepsin can potentiate the cytotoxicity of doxorubicin. We first demonstrated the synergistic 24 interaction between romidepsin and doxorubicin in both CTCL cell lines and primary CTCL cells. Our 25 results are consistent with previously published studies, demonstrating enhanced anti-tumor activity of 26 doxorubicin when combined with an HDAC inhibitor in other malignancies, including breast cancer, soft 27 tissue sarcoma and multiple myeloma.27–29 These studies suggested several potential mechanisms 28 underlying the synergy between HDAC inhibitors and anthracyclines. Lopez et al. showed that the 29 HDAC inhibitor PCI-24781 reduced the expression of RAD51, a major mediator of DNA repair. Other 30 investigators suggested that HDAC inhibitors induced more open chromatin structure, thereby 31 augmenting the DNA accessibility to doxorubicin. 32 33 Based on our pre-clinical study, we conducted a phase I study (NCT#01902225) investigating the 34 safety of the romidepsin plus LD combination in patients with T-cell lymphoma, with preliminary data 35 evaluating the efficacy of this combination. The primary endpoint for this study was safety, with the 36 goal of determining the MTD of romidepsin in combination with LD in a heavily pre-treated population of 37 patients with CTCL or PTCL. Two DLTs, both hematologic, occurred at the 14 mg/m2 romidepsin dose; 38 therefore, the MTD of romidepsin was determined to be 12 mg/m2 when combined with 20 mg/m2 of 39 LD. It is important to note that all three DLTs were observed in PTCL patients. Additionally, there was 40 a higher rate of grade 3/4 hematologic AEs in the PTCL cohort. This is likely explained by the more 41 myelosuppressive prior treatments given to patients with relapsed/refractory PTCL as compared to 42 CTCL. Furthermore, 4 of the 12 PTCL patients had prior autologous stem cell transplant which could 43 compound this effect. In contrast, patients with CTCL had largely grade 1/2 hematologic toxicities and 44 were more tolerant to therapy overall.

Running Title: HDAC inhibitor and anthracycline combination, CTCL

1 2 Aside from the hematologic toxicities, the combination of romidepsin plus LD was well-tolerated. No 3 patients died from treatment-related AEs, and only two patients had to discontinue treatment due to 4 AEs, among which, only one was due to intolerability. Non-hematologic AEs were mostly grade 1/2 5 and consistent with the safety profile seen with single-agent romidepsin in other clinical studies. Three 6 treatment-related infections occurred, one of which was cryptococcal pneumonia. Three patients 7 developed clinically significant thromboses, though these were deemed unrelated to the study drugs. 8 Notably, there were no cardiac-related AEs such as heart failure, despite the fact that many patients 9 had prior anthracycline exposure which is a known risk factor for cardiotoxicity. 10 11 The romidepsin plus LD combination provided particularly encouraging clinical responses in both MF 12 and SS patients. In this cohort of 10 patients, the ORR was 70% (95% CI 35-93). If CTCL patients 13 with prior exposure to HDAC inhibitor or doxorubicin are excluded, the global ORR is 100%. Of the 3 14 SS patients on the study, two achieved global response, one of which was a complete response that is 15 ongoing for over 16 months. The third SS patient had stable disease in the blood and deep skin 16 response with a maximum mSWAT score reduction of 94% and has not progressed for over 41 months. 17 The combination treatment was particularly effective in the skin with a median mSWAT score reduction 18 of 88% and 6/10 patients achieving less than 10% skin involvement of their body surface area with 19 patch/plaque-only lesions at the time of best response. Three of four patients with T3 tumor lesions 20 had skin response with complete resolution of all tumors at the time of best response. The response 21 rates seen in the CTCL cohort compare favorably with response rates of 41% for single-agent LD in 22 prior studies that used similar response criteria, suggesting the hypothesis from in vitro studies that 23 romidepsin and LD have synergistic effects against malignant T-cells may apply in patients treated with 24 this combination. Specifically, the skin response rate of 80% is encouraging when compared with 25 reported response rates for single-agent romidepsin and LD. These early data suggest that this 26 combination may be suitable for rapid cytoreduction in patients with high disease burden and/or with 27 extracutaneous disease. Importantly, this regimen should be considered prior to administration of 28 single-agent romidepsin, as 100% (n = 7) of patients responded who had not received prior romidepsin 29 versus 0/3 patients who had received prior romidepsin. Based on this data, future prospective clinical 30 trials using this regimen would perhaps exclude patients who received prior romidepsin. 31 32 In contrast to the CTCL cohort, the PTCL cohort demonstrated an ORR of 27% (95% CI 6-61) which is 33 comparable to responses seen with HDAC inhibitors as a single agent in this disease.7,8,10 Additionally, 34 PFS was significantly shorter in these patients (2.1 months) than those in the CTCL cohort. It is 35 notable that the three observed responses were all CRs, two of which occurred in AITL patients. There 36 does seem to be preferential activity with HDAC inhibitors in AITL which could be explained by its high 37 frequencies of mutations in epigenetic modifiers such as TET2, DNMT3A, and IDH2 genes.30–32 These 38 results suggest that this combination is unlikely to be more efficacious than single-agent romidepsin in 39 PTCL. 40 41 In summary, we conducted a phase I clinical trial to determine the safety and preliminary efficacy of 42 patients with relapsed/refractory PTCL/CTCL treated with romidepsin and LD. In line with our pre- 43 clinical data demonstrating synergy between romidepsin and doxorubicin in CTCL cell lines and primary 44 tumor cells, romidepsin plus LD demonstrated high and rapid response rates in the CTCL cohort which

Running Title: HDAC inhibitor and anthracycline combination, CTCL

1 appeared to be better than the single-agent activities of romidepsin and LD observed in trials using 2 modern response criteria as is in this study. Romidepsin plus LD was also well-tolerated in CTCL 3 patients with mostly grade 1/2 AEs. Although our study is largely limited by the small number of 4 patients, the encouraging results in CTCL suggest that the combination of romidepsin plus LD is an 5 effective treatment option for CTCL patients with widespread thick plaques, tumors, or extracutaneous 6 involvement, who require rapid cytoreductive therapy that serves as a bridge to long-term skin-directed 7 treatment as maintenance or even allogeneic hematopoietic cell transplantation. These findings 8 provide a rationale for further investigation of romidepsin plus LD in a larger phase II clinical trial for 9 relapsed/refractory CTCL patients. 10 11 12 Acknowledgements:

13 This work was supported by UCSF Helen Diller Comprehensive Cancer Center Early Phase 14 Clinical Research Support, Celgene Inc., and generous gifts from Drs. Martin and Dorothy Spatz Family 15 Charitable Foundation, Donna L and Edward E Martins Foundation Inc and Summit Bank Foundation 16 (to W.Z.Ai) 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

Running Title: HDAC inhibitor and anthracycline combination, CTCL

1 References

2 1. Willemze R, Jaffe ES, Cerroni L, et al. WHO-EORTC classifcation for cutaneous lymphomas. 3 Hematology. 2009;105(10):3768-3785. 4 2. Kim YH, Liu HL, Mraz-Gernhard S, Varghese A, Hoppe RT. Long-term outcome of 525 patients 5 with mycosis fungoides and Sezary syndrome: clinical prognostic factors and risk for disease 6 progression. Arch Dermatol. 2003;139(7):857-866. 7 3. Agar NS, Wedgeworth E, Crichton S, et al. Survival outcomes and prognostic factors in mycosis 8 fungoides/Sézary syndrome: validation of the revised International Society for Cutaneous 9 Lymphomas/European Organisation for Research and Treatment of Cancer staging proposal. J 10 Clin Oncol. 2010;28(31):4730-4739. 11 4. Talpur R, Singh L, Daulat S, et al. Long-term outcomes of 1,263 patients with mycosis fungoides 12 and Sézary syndrome from 1982 to 2009. Clin Cancer Res. 2012;18(18):5051-5060. 13 5. Moskowitz AJ, Lunning MA, Horwitz SM. How i treat the peripheral T-cell lymphomas. Blood. 14 2014;123(17):2636-2644. 15 6. Mak V, Hamm J, Chhanabhai M, et al. Survival of patients with peripheral T-cell lymphoma after 16 first relapse or progression: spectrum of disease and rare long-term survivors. J Clin Oncol. 17 2013;31(16):1970-1976. 18 7. O’Connor OA, Horwitz S, Masszi T, et al. Belinostat in Patients With Relapsed or Refractory 19 Peripheral T-Cell Lymphoma: Results of the Pivotal Phase II BELIEF (CLN-19) Study. J Clin 20 Oncol. 2015;33(23):2492-2499. 21 8. O’Connor OA, Pro B, Pinter-Brown L, et al. Pralatrexate in patients with relapsed or refractory 22 peripheral T-cell lymphoma: results from the pivotal PROPEL study. J Clin Oncol. 23 2011;29(9):1182-1189. 24 9. Coiffier B, Pro B, Prince HM, et al. Romidepsin for the treatment of relapsed/refractory peripheral 25 T-cell lymphoma: pivotal study update demonstrates durable responses. J Hematol Oncol. 26 2014;7(1):11. 27 10. Coiffier B, Pro B, Prince HM, et al. Results from a pivotal, open-label, phase II study of 28 romidepsin in relapsed or refractory peripheral T-cell lymphoma after prior systemic therapy. J 29 Clin Oncol. 2012;30(6):631-636. 30 11. Duvic M, Dummer R, Becker JC, et al. Panobinostat activity in both bexarotene-exposed and - 31 naïve patients with refractory cutaneous T-cell lymphoma: results of a phase II trial. Eur J 32 Cancer. 2013;49(2):386-394. 33 12. Duvic M, Talpur R, Ni X, et al. Phase 2 trial of oral vorinostat (suberoylanilide hydroxamic acid, 34 SAHA) for refractory cutaneous T-cell lymphoma (CTCL). Blood. 2007;109(1):31-39. 35 13. Ellis L, Pan Y, Smyth GK, et al. Histone deacetylase inhibitor panobinostat induces clinical 36 responses with associated alterations in gene expression profiles in cutaneous T-cell lymphoma. 37 Clin Cancer Res. 2008;14(14):4500-4510. 38 14. Kim EJ, Kim YH, Rook AH, et al. Clinically significant responses achieved with romidepsin 39 across disease compartments in patients with cutaneous T-cell lymphoma. Leuk Lymphoma. 40 2015;56(10):2847-2854. 41 15. Olsen EA, Kim YH, Kuzel TM, et al. Phase IIb multicenter trial of vorinostat in patients with

Running Title: HDAC inhibitor and anthracycline combination, CTCL

1 persistent, progressive, or treatment refractory cutaneous T-cell lymphoma. J Clin Oncol. 2 2007;25(21):3109-3115. 3 16. Piekarz RL, Frye R, Turner M, et al. Phase II multi-institutional trial of the histone deacetylase 4 inhibitor romidepsin as monotherapy for patients with cutaneous T-cell lymphoma. J Clin Oncol. 5 2009;27(32):5410-5417. 6 17. Kim EJ, Kim YH, Rook AH, et al. Clinically significant responses achieved with romidepsin 7 across disease compartments in patients with cutaneous T-cell lymphoma. Leuk Lymphoma. 8 2015;56(10):2847-2854. 9 18. Dummer R, Quaglino P, Becker JC, et al. Prospective international multicenter phase II trial of 10 intravenous pegylated liposomal doxorubicin monochemotherapy in patients with stage IIB, IVA, 11 or IVB advanced mycosis fungoides: final results from EORTC 21012. J Clin Oncol. 12 2012;30(33):4091-4097. 13 19. Straus DJ, Duvic M, Horwitz SM, et al. Final results of phase II trial of doxorubicin HCl liposome 14 injection followed by bexarotene in advanced cutaneous T-cell lymphoma. Ann Oncol. 15 2014;25(1):206-210. 16 20. Waller A, Lundberg J, Porter K, et al. Gemcitabine plus liposomal doxorubicin for relapsed 17 refractory T-cell lymphomas. Hematol Oncol. 2017;35:396-397. 18 21. Sanchez-Gonzalez B, Yang H, Bueso-Ramos C, et al. Antileukemia activity of the combination of 19 an anthracycline with a histone deacetylase inhibitor. Blood. 2006;108(4):1174-1182. 20 22. Kano Y, Akutsu M, Tsunoda S, et al. Cytotoxic effects of histone deacetylase inhibitor FK228 21 (depsipeptide, formally named FR901228) in combination with conventional anti- 22 leukemia/lymphoma agents against human leukemia/lymphoma cell lines. Invest New Drugs. 23 2007;25(1):31-40. 24 23. Maiso P, Colado E, Ocio EM, et al. The synergy of panobinostat plus doxorubicin in acute 25 myeloid leukemia suggests a role for HDAC inhibitors in the control of DNA repair. Leukemia. 26 2009;23(12):2265-2274. 27 24. Olsen EA, Whittaker S, Kim YH, et al. Clinical end points and response criteria in mycosis 28 fungoides and Sézary syndrome: A consensus statement of the International Society for 29 Cutaneous Lymphomas, the United States Cutaneous Lymphoma Consortium, and the 30 Cutaneous Lymphoma Task Force of the E. J Clin Oncol. 2011;29(18):2598-2607. 31 25. Cheson BD, Fisher RI, Barrington SF, et al. Recommendations for initial evaluation, staging, and 32 response assessment of hodgkin and non-hodgkin lymphoma: The Lugano classification. J Clin 33 Oncol. 2014;32(27):3059-3067. 34 26. Zhao W, Sachsenmeier K, Zhang L, Sult E, Hollingsworth RE, Yang H. A New Bliss 35 Independence Model to Analyze Drug Combination Data. J Biomol Screen. 2014;19(5):817-821. 36 27. Marchion DC, Bicaku E, Turner JG, Daud AI, Sullivan DM, Munster PN. Synergistic interaction 37 between histone deacetylase and topoisomerase II inhibitors is mediated through topoisomerase 38 IIbeta. Clin Cancer Res. 2005;11(23):8467-8475. 39 28. Lopez G, Liu J, Ren W, et al. Combining PCI-24781, a novel histone deacetylase inhibitor, with 40 chemotherapy for the treatment of soft tissue sarcoma. Clin Cancer Res. 2009;15(10):3472- 41 3483. 42 29. Cheriyath V, Kuhns MA, Kalaycio ME, Borden EC. Potentiation of apoptosis by histone

Running Title: HDAC inhibitor and anthracycline combination, CTCL

1 deacetylase inhibitors and doxorubicin combination: cytoplasmic cathepsin B as a mediator of 2 apoptosis in multiple myeloma. Br J Cancer. 2011;104(6):957-967. 3 30. Pro B, Horwitz SM, Prince HM, et al. Romidepsin induces durable responses in patients with 4 relapsed or refractory angioimmunoblastic T-cell lymphoma. Hematol Oncol. 2017;35(4):914- 5 917. 6 31. Odejide O, Weigert O, Lane AA, et al. A targeted mutational landscape of angioimmunoblastic T- 7 cell lymphoma. Blood. 2014;123(9):1293-1296. 8 32. Fukumoto K, Nguyen TB, Chiba S, Sakata-Yanagimoto M. Review of the biologic and clinical 9 significance of genetic mutations in angioimmunoblastic T-cell lymphoma. Cancer Sci. 10 2018;109(3):490-496. 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

Running Title: HDAC inhibitor and anthracycline combination, CTCL

1 Table 1. Patient demographics and baseline characteristics CTCL PTCL All patients (n = 11) (n = 12) (n = 23) Age (y), median (range) 63 (53-79) 63 (52-83) 63 (52-83)

Sex, male, n (%) 7 (64) 6 (50) 13 (57)

ECOG performance status, n (%) 0 8 (73) 3 (25) 11 (48) 1 3 (27) 8 (67) 11 (48) 2 0 1 (8) 1 (4)

Clinical stage, n (%) Stage IB: 3 (27) Stage I: 1 (8) >Stage III: 16 (70) Stage IIB: 2 (18) Stage II: 1 (8) Stage IVA: 6 (55) Stage III: 3 (25) Stage IV: 7 (58) No. of prior systemic 3 (1-7) 2 (1-3) 2 (1-7) therapies, median (range) Prior HDAC inhibitor, n (%) 5 (45) 0 5 (22)

Prior doxorubicin-based 1 (9) 10 (83) 11 (48) therapy, n (%)

Prior ASCT, n (%) 0 4 (33) 4 (17)

2 3 4 5 6 7 8 9 10 11 12 13 14 15

Running Title: HDAC inhibitor and anthracycline combination, CTCL

1 Table 2. Treatment-related adverse events in >10% of patients Total (N = 23)

Grade 1-2, Grade 3-4, All grades, Treatment-related AE n (%) n (%) n (%) Hematologic Thrombocytopenia 11 (48) 4 (17) 15 (65) Anemia 10 (43) 3 (13) 13 (57) Neutropenia 4 (17) 2 (9) 6 (26) Non-hematologic Fatigue 10 (43) 1 (4) 11 (48) Nausea 10 (43) 1 (4) 11 (48) Vomiting 8 (35) 0 (0) 8 (35) Anorexia 7 (30) 0 (0) 7 (30) Blood alkaline phosphatase increased 4 (17) 0 (0) 4 (17) Dysgeusia 3 (13) 0 (0) 3 (13) Infection 3 (13) 0 (0) 3 (13) Hyperglycemia 3 (13) 0 (0) 3 (13) Thrombosis 3 (13) 0 (0) 3 (13) 2 3 4 5 Table 3. Summary of responses Response category CTCL (n = 10) PTCL (n = 11)

ORR (CR + PR) ORR, n (%) 7 (70) 3 (27) 95% Confidence interval (CI) (35, 93) (6, 61) Best response, n (%)* CR 1 (10) 3 (27) PR 6 (60) 0 Stable disease 2 (20) 0 Progressive disease 1 (1) 8 (73) Time to response, median months 2 (1.8-5.2) 3.5 (2.1-4.8) (range) Duration of response, median 5.1 (0.9-6.9) 4.2 (2.1-4.2) months (range) 6 *By clinical stage, responses were seen in 2/3 stage IB, 2/2 stage IIB, and 3/6 stage IVA CTCL 7 patients. 8 9 10 11

Running Title: HDAC inhibitor and anthracycline combination, CTCL

1 Table 4. Best response by compartment for all evaluable CTCL patients All evaluable CTCL Skin Lymph nodes Blood Global patients (n = 10) (n = 10) (n = 5) (n = 3) response (n = 10) ORR, n (%) 8 (80) 3 (60) 2 (67) 7 (70)

CR, n (%) 1 (10) 1 (20) 1 (33) 1 (10)

PR, n (%) 7 (70) 2 (40) 1 (33) 6 (60)

SD, n (%) 1 (10) 1 (20) 1 (33) 2 (20)

PD, n (%) 1 (10) 1 (20) 0 (0) 1 (10)

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Running Title: HDAC inhibitor and anthracycline combination, CTCL

1 Figure 1. The combination of romidepsin and doxorubicin exhibits significant synergy in growth 2 inhibition and apoptosis induction in CTCL cell lines and primary tumor cells. (A-B) Growth 3 inhibition (GI) activity of romidepsin plus doxorubicin in 16 dose combinations in CTCL cell lines and 4 primary tumor cells (top panels). The interaction between romidepsin and doxorubicin in each dose 5 combination analyzed by the Bliss Independence Index (BI). BI = 1.00 indicates additivity, BI > 1.00 6 indicates synergy (bottom panels). (C-D) Apoptosis induction of the romidepsin/doxorubicin 7 combination in CTCL cell lines and primary tumor cells represented in dot plot (top panels) and bar 8 graphs (bottom panels). For each cell line, 2 to 3 independent experiments in triplicates were 9 performed (a representative experiment is shown). Data are presented as mean ± SEM from three 10 independent experiments. *P<0.05, **P<0.01 by unpaired, one-tailed t test. 11 12 Figure 2. Maximum reduction in mSWAT score compared with baseline in CTCL patients 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Romidepsin plus Liposomal Doxorubicin is Safe and Effective in Patients with Relapsed or Refractory T-cell Lymphoma: Results of a Phase I Dose-Escalation Study

Khoan Vu, Chi-Heng Wu, Chen-Yen Yang, et al.

Clin Cancer Res Published OnlineFirst November 26, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-19-2152

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2019/11/27/1078-0432.CCR-19-2152.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/early/2019/11/26/1078-0432.CCR-19-2152. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.