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Introduction: Recombinant human interleukin 15 (rhIL-15) has been shown to stimulate the activation and expansion in the number of effector T lymphocytes and natural killer cells in patients. The addition of the immune checkpoint inhibitors (ICI) CTLA-4 and PD-1 has shown increased antitumor efficacy in preclinical models, and the triplet is hypothesized to enhance anti-tumor immune response by augmenting effector cell expansion, differentiation, cytotoxic activity, and immune checkpoint inhibition. Results of the lead-in safety arms of rhIL-15/ipilimumab (Ipi) or rhIL-15/nivolumab (N) doublets were previously reported (O’Sullivan et al. AACR. 2019) and we now report results for the dose escalation phase of the triplet combination (NCT03388632). Methods: This phase 1, open-label, 3+3 dose escalation trial enrolled adult patients (pts) with measurable metastatic or refractory solid tumors and ECOG performance status ≤2. Prior therapy with 2 of the 3 study agents was permitted. Pts were treated on three dose levels (DL 1-3) of rhIL-15 (0.5, 1, or 2 mcg/kg/day) administered subcutaneously on days (D)1-8 and D22-29 of a 6-week cycle for the first 4 cycles only. Nivolumab (240mg IV) was administered on D8, 22, 36 and ipilimumab (1mg/kg IV) on D8 of every cycle. Response was assessed by RECIST 1.1. Upon progression, pts could continue for response assessment utilizing iRECIST if clinically stable and without toxicity. Treatment-induced changes in circulating and tumor T cell activation, signaling, and the PD-L1 checkpoint will be assessed with validated pharmacodynamic biomarker assays. Results: A total of 17 pts were enrolled on the triplet dose escalation phase; 15 pts were evaluable for response and 6 pts had received prior treatment with a PD-1/PD-L1 inhibitor. Median time on treatment (MTT) was 12 weeks (range 3-101). A confirmed partial response (PR) was observed in a pt with intrahepatic cholangiocarcinoma (MSI-H) after 3 cycles (total time on treatment 101 weeks). Across all dose levels, 7 pts (46.7%) had stable disease (SD) that prolonged their time on treatment with MTT of 20.4 weeks; 7 pts (46.7%) experienced progressive disease (PD) with MTT of 6.4 weeks. To better profile toxicity, 5 pts were replaced due to incomplete dosing of C1. Dose limiting toxicities at DL 3 (rhIL-15 at 2 mcg/kg/day) were photosensitivity and rash. Main drug-related adverse events included grade 4 lymphopenia (n=1) and grade 3 colitis, hyperthyroidism, hyponatremia, myocarditis, neutropenia, and photosensitivity (n=1, each). No pts discontinued therapy due to toxicity events. No deaths occurred on study. Conclusions: The recommended phase 2 dose (RP2D) of rhIL-15 is 1 mcg/kg/day when administered in combination with N+Ipi. The triplet dose expansion phase with the RP2D is currently accruing and includes biomarker studies in blood and tumor biopsies to assess the tumor microenvironment. This study was funded in part by NCI Contract HHSN261200800001E. *In memorium: Dr. Thomas Waldmann7. Citation Format: Sarah J. Shin, Geraldine O'Sullivan Coyne, Howard Streicher, Naoko Takebe, Ashley Bruns, Elad Sharon, Richard Piekarz, Lamin Juwara, Larry Rubinstein, Ralph Parchment, Kristin Fino, King L. Fung, Katherine Ferry-Galow, Arjun Mittra, Abdul Rafeh Naqash, Kevin Conlon, James H. Doroshow, Alice P. Chen. Phase 1 study of recombinant interleukin 15 (rhIL-15) in combination with checkpoint inhibitors nivolumab and ipilimumab in subjects with refractory cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT147.

Background: Ataxia-telangiectasia-related (ATR) protein kinase is central to the repair of damaged DNA through the homologous recombination (HR) pathway. We conducted a phase I trial of cisplatin in combination with the ataxia-telangiectasia-related (ATR) protein kinase inhibitor berzosertib (M6620) and the poly (ADP-ribose) polymerase (PARP) 1/2 inhibitor veliparib, postulating that simultaneous PARP and ATR inhibition impairs DNA repair and induces a “BRCA null”-like phenotype which could potentiate the antitumor activity of cisplatin. We evaluated the safety, maximal tolerated dose (MTD) and preliminary anti-tumor activity of this treatment triplet. The study was conducted through the Experimental Therapeutics Clinical Trials Network (ETCTN). Methods: This open label trial used a 3+3 design. Cisplatin and berzosertib were each administered intravenously on separate, sequential days for two continuous weeks [day (D) 1 and 8; D2 and 9 respectively], together with veliparib orally twice daily during IV therapy and for 24 hours after (D1-3 and 8-10) in 21-day cycles. Prior platinum and PARP inhibitor therapy were permitted. Tumor expression of DNA damage repair (DDR) biomarkers was assessed at the MTD using validated, quantitative immunofluorescence assays. Results: Fifty-three patients (pts) enrolled, 46 pts evaluable for response. The MTD and RP2D dose is cisplatin 40 mg/m2 D1 and D8, berzosertib 210 mg/m2 D2 and D9, and veliparib 200mg BID (D1-3, D8-10). Three patients achieved a confirmed partial response (PR; 5.6%). A further 2 patients had an unconfirmed PR (breast carcinoma, -30%; HGS carcinoma, -36%). Four responders (3 PR and 1 uPR) received prior platinum. Median time on study: 4 cycles (range 1-25). Genomic data was available on four pts achieving a response: ovarian cancer (BRCA-wildtype, ATM unknown; 25 cycles), breast carcinoma (ATM mutation; 14 cycles), SCC of the tongue (Chek2 mutation; 20 cycles), and HGS carcinoma (ATM/BRIP1 mutation; 4 cycles). Twenty-six pts (56.5%) had stable disease. Thirty-five patients (66.0%) required dose reduction in at least 1 agent, most commonly veliparib (25; 47.2%). Most common grade 3 and 4 adverse events were related to myelosuppression: anemia (20; 37.7%), thrombocytopenia (17; 32.1%), leukopenia (13; 24.5%), neutropenia (12; 22.6%), and lymphopenia (11; 20.8%). Ongoing pharmacodynamic analyses of tumor biopsies collected on C1D1 (post-veliparib/cisplatin) and C1D9 (post-veliparib/cisplatin/M6620) have demonstrated a combination-induced increase in RAD51, indicative of replication stress. Conclusions: The BVP combination shows anti-tumor activity in HR-compromised tumors and M6620 further increases the DDR response elicited by combination cisplatin/veliparib. Citation Format: Geraldine O'Sullivan Coyne, Khanh T. Do, Shivaani Kummar, Naoko Takebe, Sarina Piha-Paul, Richard Piekarz, Deborah Wilsker, Brandon Miller, Katherine Ferry-Galow, Ralph Parchment, Lamin Juwara DNP, Mary Jane Ong, Arjun Mittra, James H. Doroshow, Alice P. Chen. Phase I trial of the triplet berzosertib (M6620, VX-970), veliparib and cisplatin (BVP) in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT115.

Background: The base excisional repair (BER) pathway plays an important role in the development of resistance to alkylating agents. TRC102 (methoxyamine HCL) inhibits BER by binding apurinic/apyrimidinic sites, which increases temozolomide (TMZ)-induced DNA strand breaks and apoptosis. We conducted a multihistology phase I/II trial of TRC102 in combination with TMZ in patients (pts) with refractory solid tumors (NCT01851369); here we report the clinical activity of this combination in the phase II refractory colorectal carcinoma (CRC) cohort. The phase I portion of the study demonstrated anemia to be the major dose limiting toxicity of this combination; the recommended phase II doses (RP2D) for the combination were: 125 mg TRC 102 + 150 mg/m2 TMZ (PO, D1-5 of 28-day cycles). The expanded phase II trial utilized a 3-arm Simon 2-stage design based on objective responses documented during the phase I portion of the study (CRC, NSCLC, and granulosa cell ovarian cancer). Methods: Phase II pts were treated at the RP2D, except that pts with BSA < 1.6 m2 received 100 mg of TRC102. Eligibility for the CRC cohort included pts with recurrent, metastatic, histologically confirmed colorectal adenocarcinoma after ≥ 2 lines of therapy, adequate organ function, and an ECOG performance status (PS) ≤ 1. Overall response rate (ORR) was defined as a partial response (PR) or a complete response (CR), per RECIST 1.1 criteria. Results: During the phase I trial, one pt with KRAS mutant CRC experienced a sustained PR of 21 cycles (maximum 70% decrease by RECIST). This pt decided to stop treatment due to travel constraints, but at 17 months off treatment had not progressed. Based on this response, 16 pts were enrolled in the CRC cohort of the phase II trial (3 rectal, 13 colon), all heavily pretreated (median: 6 lines of prior therapy, range: 3-14). Median pt age was 64 years (range: 46-80 years); all had an ECOG PS of 1. Median time on study was 2 cycles (range: 1 to 5 cycles) including 5 pts who came off study for early clinical progression. ORR for this cohort was 6% (1 PR). Nine pts had KRAS mutant disease, including the partial responder, who also was the only pt (of the 10 tested by tumor IHC) with MGMT-negative disease. Grade 3 and 4 protocol treatment-related toxicities included thrombocytopenia (2 pts), hemolysis (1 pt), and diarrhea (1 pt). No pts discontinued the protocol because of toxicity and only 1 pt required dose reduction (thrombocytopenia). Conclusions: Despite a promising phase I result from a pt with CRC, a phase II study of combined TRC102 and TMZ treatment in patients with metastatic colon or rectal adenocarcinoma displayed an ORR of 6%. Assessing tumor MGMT status may help identify pts who could benefit from this combination and pharmacodynamic studies are underway to assess drug activity in responding and non-responding patients. Supported in part by NCI Contract HHSN261200800001E. Citation Format: Geraldine OSullivan Coyne, Cecilia Monge Bonilla, Jennifer Zlott, Naoko Takebe, Robert Meehan, Lamin Juwara, Richard Piekarz, Laurence Rubinstein, Deborah F. Wilsker, Robert J. Kinders, Ralph E. Parchment, Shahanawaz Jiwani, Shivaani Kumar, James H. Doroshow, Alice P. Chen. A Phase II trial of TRC102 (methoxyamine HCl) in combination with temozolomide in patients with relapsed metastatic colorectal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-293.

Introduction: Treatment with rhIL-15 has been reported to significantly increase levels of circulating CD8+ T cells, NK cells, and inflammatory cytokines. In vivo administration of rhIL-15 with immune checkpoints inhibitors of CTLA-4 and PD-L1 resulted in increased tumor antigen-specific CD8+ T-cells, enhanced CD8+ tumor lytic activity, and improved mouse survival. This combination is therefore expected to enhance the anti-tumor immune response in patients by promoting T-cell expansion and cytotoxic activity. The primary objective of this trial is to determine the safety, dose-limiting toxicity (DLT), and maximum tolerated dose (MTD) of rhIL-15 with nivolumab (N) and ipilimumab (Ipi) in refractory tumors. Prior to initiating treatment with all three drugs, we treated a cohort of patients (pts) with doublet combinations (rhIL-15+Ipi; rhIL-15+N) to establish safety. Here we report results for the doublets. Methods: This is an open-label, 3+3 design, phase I trial with a pharmacodynamic expansion cohort at the MTD (NCT03388632). Pts with prior immunotherapy are eligible unless they experienced a prior grade ≥ 3 immune-related toxicity. The doses for the doublets are rhIL-15 0.5 mcg/kg/day SC and N 240 mg IV or Ipi 1 mg/kg IV. rhIL-15 is given on days 1-8 & 22-29 (first 4 cycles only), followed by N on days 8, 22, & 36 or Ipi on day 8, every 6 weeks until disease progression. No dose reductions are permitted. Response is assessed by RECIST 1.1 and iRECIST after every cycle. Blood is collected at baseline and throughout the study to assess PD-L1 levels, T-cell phenotypic markers, and markers of T-cell activation/inhibition (Zap70 pY493 and SHP2 pY580). Results: Five patients were treated on the doublet arms, three with N and two with Ipi. Histologies were pancreatic, colon, rectal, cervical and thyroid cancers. The median age was 50 (range 33-70). All patients had at least 1 prior therapy (range 1-6). Two had prior immunotherapy (pembrolizumab & brachyury vaccine). There were no DLTs in the first cycle. The most common adverse events possibly related to treatment were all grade 1: injection site reaction post rhIL-15 (n=4), leukopenia (n=3), anemia (n=3), and AST/ALT increase (n=3). One pt on N experienced a grade 3 immune colitis at week 13, which resolved with steroids. Best response was stable disease in both the rhIL-15+N arm (median 18 wks, range 8-30) and rhIL-15+Ipi arm (median 14 wks, range 12-16). Conclusions: The doublet arms met safety endpoints, and the trial is currently enrolling pts for the triplet combination. In addition to the biomarker studies in blood, tumor biopsies will be collected at the MTD to assess the tumor immune microenvironment, including the proximity of T-cells to tumor cells, and the expression of intrinsic apoptosis biomarkers. Funded in part by NCI Contract HHSN261200800001E. Citation Format: Geraldine OSullivan Coyne, Sabrina S. Khan, Kevin Conlon, Howard Streicher, Ashley Bruns, Richard Piekarz, Naoko Takebe, Lamin Juwara, Ralph Parchment, Tony Navas, Kirstin Fino, King L. Fung, Elad Sharon, James H. Doroshow, Thomas A. Waldmann, Alice P. Chen. Phase I trial of recombinant human interleukin 15 (rhIL-15) in combination with nivolumab and ipilimumab in refractory cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-015.

Background: Wee1 tyrosine kinase phosphorylates and inactivates cyclin dependent kinase (Cdk) 1 as part of DNA damage response (DDR) signaling, resulting in G2 cell cycle arrest to enable DNA repair. We are conducting a Phase I study of oral Wee1 inhibitor AZD1775 in patients (pts) with advanced solid tumors using twice-daily (Arm A) and once-daily (Arm B) dosing. Results for Arm A were published previously. Here, we report updated pharmacodynamic (PD), pharmacokinetic (PK), and clinical response results for Arm B. Methods: In Arm B, AZD1775 was administered once daily on days 1-5 and 8-12 of each 21-day cycle in a 3+3 design. Primary objectives were to establish safety, tolerability, PK, and MTD. Secondary objectives included determining PD effects on DDR markers in tumor tissue and circulating tumor cells (CTCs), and tumor response (RECIST 1.1). Post-treatment PD effects were established in Arm A. Given the importance of sustained molecular target control in maximizing targeted therapy efficacy, paired tumor biopsies in Arm B were obtained at baseline and day 8 (pre-dose) to assess durability of Wee1 inhibition and downstream PD effects. Immunofluorescence assays were used to measure target inhibition (pY15-Cdk1/2) and DDR markers (e.g., γH2AX). Results: Arm B included 42 pts; of the 34 evaluable for response, 6 (18%) had a partial response (PR; 4 ovarian, 2 endometrial), and 20 (59%) had stable disease (SD; 2-26 cycles, mean 7). Of 10 evaluable BRCA-deficient pts, 2 had a PR (20%) and 6 had SD (60%; mean: 7.17 cycles). AZD1775 was well-tolerated, as previously reported. Three of 11 paired biopsies had elevated baseline pY15-Cdk (8-23% nuclear area positive [NAP]) and showed persistence of target suppression following the dosing break during days 6-8. Of the 8 pts with low baseline pY15-Cdk (0-3% NAP), 5 had elevated pY15-Cdk levels at day 8 (range: 192-1094%), suggesting a target rebound effect. Of the 8 pts with elevated pY15-CdK at baseline or day 8, 4 (50%) achieved a PR. Day 8 γH2AX activation was detected for 3 pts, all who progressed within 2 cycles. Preliminary PK data revealed low plasma drug concentrations at day 8 pre-dose (55 ± 50 nM; n = 4) relative to peak levels at day 5 (1697 ± 1062 nM; n = 9). Conclusions: Daily AZD1775 exhibits antitumor activity in pts with both BRCA-proficient and -deficient ovarian and endometrial cancer. Responses amongst pts with elevated pY15-Cdk levels at baseline or day 8 provide additional clinical/PD evidence for the presumed mechanism of action of AZ1775 on the cell cycle and suggest that levels of pY15-Cdk may be an important correlate of response. In contrast, γH2AX activation on day 8 demonstrates the presence of drug-induced DNA double strand breaks that are not associated with clinical drug activity. PK data confirmed substantially reduced plasma AZD1775 concentrations at day 8. Ongoing genomic and CTC analyses may uncover additional molecular determinants of response. NCT01748825. Supported in part by NCI Contract HHSN261200800001E. Citation Format: Arjun Mittra, Geraldine O'Sullivan Coyne, Shivaani Kummar, Khanh Do, Ashley Bruns, Lamin Juwara, Richard Piekarz, Larry Rubinstein, Sheila Prindiville, Elad Sharon, Howard Streicher, Tiffaney Hsia, Ganesh Mugundu, Jiuping Ji, Deborah Wilsker, Angie B. Dull, Brandon Miller, Robert J. Kinders, Ralph Parchment, James H. Doroshow, Alice P. Chen, Naoko Takebe. DNA damage response and therapeutic activity following once-daily administration of the Wee 1 inhibitor AZD1775 (adavosertib) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT099.

Background: Although PARP inhibition is effective against HR repair-deficient cancers, efficacy is limited by HR proficiency, whether present de novo or as a result of acquired resistance, prompting HR disrupting strategies to sensitize tumor cells. Inhibition of CDK1 and CDK12 compromise HR by blocking BRCA1 phosphorylation, affecting recruitment to sites of DNA damage, and by reducing HR gene expression, respectively. Dinaciclib is a pan-CDK inhibitor that inhibits both CDK1 and CDK12 at nanomolar potency. We conducted a Phase 1 study combining dinaciclib and veliparib in patients with advanced solid tumors who are not germline BRCA carriers. Methods: A 3+3 design was utilized. Veliparib was administered twice daily continuously in 28-day cycles. Dinaciclib was administered intravenously on days 8 and 22. In part 1A, escalation followed a two-dimensional schema, utilizing doses of dinaciclib between 10 - 45 mg/m2 and veliparib between 20 - 120 mg. In part 1B, veliparib was escalated between 200 mg - 400 mg with dinaciclib maintained at 30 mg/m2. PK and PD assessments were performed at baseline, after veliparib, and after the combination. Preliminary Results: Sixty-three heavily pretreated patients were enrolled in part 1A (n = 39) and 1B (n = 24). Thirty-four patients had breast or gynecologic malignancies. The MTD was 400 mg twice-daily veliparib with dinaciclib at 30 mg/m2. DLTs included G4 neutropenia > 7 days (n =1), febrile neutropenia (n = 1), mucositis (n = 1) and fatigue (n = 1). Common drug-related toxicities were neutropenia (78%), nausea (75%), fatigue (67%), electrolyte abnormalities (59%), elevated liver function tests (57%), diarrhea (52%), lymphopenia (52%), anemia (43%), dehydration (37%), anorexia (30%), vomiting (29%), hypoalbuminemia (29%), dizziness (29%), headache (22%), mucositis (18%), elevated creatinine (16%), alopecia (16%), thrombocytopenia (14%), abdominal pain (13%), insomnia (13%), and dysgeusia (11%). The median number of cycles completed was 2 (r: 1 - 10). One patient with TNBC achieved complete resolution of axillary adenopathy lasting > 8 months. Twenty-four patients (38%) had stable disease as the best response, with 9 progression-free > 4 months (TNBC, gynecologic and thymic ca). Paired tumor biopsies from one patient demonstrated reduced Ki-67 and increased gamma-H2AX staining after combination treatment compared to after veliparib alone. Conclusions: Dinaciclib administered at doses known to produce PD effects is tolerable with full dose veliparib. Anti-tumor activity is limited in non-BRCA carriers, possibly related to intermittent administration of a CDK inhibitor with known short half-life. Additional patients are being enrolled utilizing dinaciclib in more dose-intense schedules. Citation Format: Geoffrey I. Shapiro, Khanh T. Do, Sara M. Tolaney, John F. Hilton, James M. Cleary, Andrew Wolanski, Brian Beardslee, Faith Hassinger, Ketki Bhushan, Dongpo Cai, Elizabeth Downey, Solida Pruitt-Thompson, Suzanne M. Barry, Bose Kochupurakkal, Joseph Geradts, Christine Unitt, Alan D. D'Andrea, Alona Muzikansky, Richard Piekarz, L. Austin Doyle, Jeffrey Supko. Phase 1 dose-escalation study of the CDK inhibitor dinaciclib in combination with the PARP inhibitor veliparib in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT047. doi:10.1158/1538-7445.AM2017-CT047

Background: Immunosuppressive factors such as regulatory T cells (Tregs) and myeloid-derived suppressive cells (MDSCs) limit the efficacy of immunotherapies. Histone deacetylase (HDAC) inhibitors have been shown to have immunomodulatory effects. We have previously reported that the class I selective HDAC inhibitor entinostat has synergistic antitumor effects in combination with high dose interleukin-2 (IL-2) in a renal cell carcinoma model by down-regulating Foxp3 expression and function of Tregs. Thus, we have conducted a phase I/II clinical study with entinostat and high dose IL-2 in patients (pts) with metastatic clear cell renal cell carcinoma. Methods: The primary objectives were to evaluate the safety, tolerability and efficacy of this combination strategy. The main eligibility criteria were clear cell histology, no prior treatments, and being fit to receive high dose IL-2. The phase I portion consisted of two dose levels of entinostat (3 and 5 mg, PO every14 days) and a fixed standard dose of IL-2 (600,000 units/kg IV every 8 hrs, up to 14 doses). Each cycle was 85 days. To test our hypothesis, the fixed sample size for the phase II was 36 with a type I/II error of 10%. If 11 or more of the pts have a response, the hypothesis that the response rate is ? 20% is rejected. Results: Dose levels 1 and 2 were completed without DLTs and 5 mg was the recommended phase II dose for entinostat. The most common transient grade 3/4 toxicities were hypophosphatemia (16%), lymphopenia (15%), and hypocalcemia (7%). We have enrolled a total of 47 pts (44 at dose level 2), and 37 have completed one cycle (84 days) treatment. Four pts were not evaluable and were replaced. To date, the objective response is 35% (10 PR, 3 CR), the median progression-free survival is 16.1 months (6.2,27.8) and the median overall survival is 65.3 months (52.6,65.3). Decreased Tregs have been observed following treatment and lower numbers were associated with response. Analysis of monocityc MDSCs and activated antigen presenting cells will be also reported. Conclusions: The results from this clinical trial suggest that entinostat may increase the therapeutic effect of high dose IL-2 by modulating immunosuppressive cells, and represent the first example of how epigenetic agents may be rationally combined with immunotherapies. (R21CA137649; U01CA70095) Citation Format: Roberto Pili, David I. Quinn, Hans J. Hammers, J. Paul Monk, Saby George, Tanya B. Dorff, Thomas Olencki, Li Shen, Dominick Lamonica, Alan Hutson, Adrienne Groman, Susan M. Perkins, Richard Piekarz, Michael Carducci. Results from a phase I/II study with the HDAC inhibitor entinostat in combination with high-dose interleukin-2 in renal cell carcinoma patients (CTEP#7870). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT015.

Background: Endocrine therapy resistance in advanced breast cancer remains a significant clinical problem that may be overcome with use of histone deacetylase (HDAC) inhibitors such as entinostat. The ENCORE 301 randomized phase II study reported an improvement in progression-free (PFS) and overall survival (OS) with the addition of entinostat to the steroidal aromatase inhibitor (AI) exemestane in patients with advanced hormone receptor (HR)-positive, HER2-negative breast cancer. Protein lysine acetylation in peripheral blood mononuclear cells (PBMCs) was associated with prolonged PFS in the entinostat arm. Methods: E2112 is a multicenter randomized double-blind, placebo-controlled phase III study that enrolled men or women with advanced HR-positive, HER2-negative breast cancer whose disease had progressed on a non-steroidal AI in the adjuvant or metastatic setting (NCT02115282). Study participants were also required to have an ECOG performance status 0-1 with measurable or non-measurable (limited to 20% of the study population) disease. One prior chemotherapy for metastatic disease and prior treatment with fulvestrant and a CDK4/6 inhibitor was permitted but not required. Participants received exemestane 25mg po daily and entinostat (EE)/placebo (EP) 5mg po every week. Primary endpoints were PFS (central review) and OS. One-sided type 1 error 0.025 was split between two hypothesis tests: 0.001 for PFS test and 0.024 for OS. PFS tested in the first 360 pts, 88.5% power to detect 42% reduction in the hazard of PFS failure (median PFS, 4.1 to 7.1 months); OS tested in all 600 pts, 80% power to detect 25% reduction in the hazard of death (median OS, 22 to 29.3 months). Secondary endpoints included safety, objective response rate (ORR), and changes in protein lysine acetylation status in PBMCs (CD3+ T cells, CD14+ monocytes, CD19+ B cells, pan-leukocyte marker CD45+ cells, CD56+ NK cells) between C1D1 and C1D15 (integrated biomarker). Results: A total of 608 participants were randomized between March 2014 and October 2018 (305 EE, 303 EP), 98% enrolled in USA. Characteristics were well balanced between the arms. Median age was 63 years (range, 29-91), 99% female, 95% postmenopausal, 80% white and 15% black. A majority (84%) had disease resistant to AI in the metastatic setting at study entry, 78% had measurable disease and 60% visceral disease. Prior treatments included chemotherapy (60%), fulvestrant (30%), CDK4/6 inhibitor (35%), everolimus (3%). Median prior lines of chemotherapy was 1 (range, 0-4) and endocrine therapy was 2 (range, 1-7); in adjuvant/metastatic setting. Grade 3/4 adverse events in EE arm included neutrophil count decreased (20%), hypophosphatemia (14%), anemia (8%), white blood cell decreased (6%), fatigue (4%), diarrhea (4%), and platelet count decreased (3%). At final analysis, median PFS was 3.3 months (EE) versus 3.1 months (EP) (HR=0.87, 95% CI: 0.67, 1.13, p=0.30). Median OS was 23.4 months (EE) versus 21.7 months (EP) (HR=0.99, 95% CI: 0.82, 1.21, p=0.94). ORR was 4.6% (EE) and 4.3% (EP). The median fold change in lysine acetylation in PBMCs was approximately 1.5 in EE arm, and 1 in EP arm. Participants on EE had significantly higher increase in lysine acetylation by C1D15 than patients on EP (397 paired samples available for analysis, p Conclusion: The combination of exemestane and entinostat did not improve survival in AI resistant advanced HR-positive, HER2-negative breast cancer. Pharmacodynamic analysis confirmed target inhibition in entinostat-treated patients. Citation Format: Roisin M Connolly, Fengmin Zhao, Kathy D Miller, Min-Jung Lee, Richard L Piekarz, Karen L Smith, Ursa Brown-Glaberman, Jennifer S Winn, Bryan A Faller, Adedayo A Onitilo, Mark E Burkard, George T Budd, Ellis G Levine, Melanie E Royce, Peter A Kaufman, Alexandra Thomas, Jane B Trepel, Antonio C Wolff, Joseph A Sparano. E2112: Randomized phase 3 trial of endocrine therapy plus entinostat/placebo in patients with hormone receptor-positive advanced breast cancer. A trial of the ECOG-ACRIN cancer research group [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr GS4-02.

Background: Base excision repair (BER), one of the pathways of DNA damage repair, has been implicated in chemoresistance. TRC102 acts through a novel mechanism to inhibit BER and cause DNA strand breaks, potentiating the antitumor activity of TMZ in preclinical models. We conducted a phase I trial of TRC102 in combination with TMZ to determine the safety, tolerability, and maximum tolerated dose of the combination; to evaluate the pharmacokinetics (PK) of each agent alone and in combination; and to assess DNA damage response (percent nuclear area of γH2AX foci) in circulating tumor cells (CTCs). Methods: Eligible pts were required to have refractory advanced solid tumors that had progressed following standard therapy; ≥ 18 yrs of age; ECOG PS 0-2; and adequate organ function. TRC102 and TMZ were administered orally once daily, D1-5 of q28d cycles; Starting dose level (DL 1) was TRC102 25 mg and TMZ 125mg/m2. Accrual to DL6 (TRC102 125 mg; TMZ 150 mg/m2) is ongoing. CTCs were obtained during C1 and on C2D1. Blood samples for PK analysis were obtained during C1. Results: Twenty pts have been enrolled to date; median age 59 yrs (range 45-78 yrs); median # of prior therapies: 3.5 (1-9); Dx: GI (6), H&N (4), breast (3), GYN (3), lung (2), soft tissue sarcoma (2). Fourteen pts are evaluable for response; 2 partial responses by RECIST have been observed to date (≥ 6 cycles; NSCLC and granulosa cell tumor of the ovary). Grade 3/4 toxicities (#pts): neutropenia (2), thrombocytopenia (1), lymphopenia (1), anemia (1), leucopenia (1), hypophosphatemia (1). PK in combination was similar to single agent PK reported for both drugs, with no evidence of a PK interaction. TRC102 levels required for preclinical activity (50ng/mL) were achieved at DL1. T1/2 of TRC102 was 26 hr. CTC analysis is ongoing. Conclusions: Combination of TRC102 with TMZ is well tolerated and clinical activity was observed with 2 partial responses to date. MTD has not been reached; accrual is ongoing. Paired tumor biopsies to assess for evidence of DNA damage response and apoptosis are planned at the MTD in the expansion phase. Citation Format: Woondong Jeong, Khanh Do, Alice Chen, Jennifer Zlott, Lamin Juwara, Yvonne Horneffer, Robert Kinders, Lihua Wang, Priya Balasubramanian, Larry Anderson, Elad Sharon, Howard Streicher, Richard Piekarz, Barbara Conley, Jerry Collins, James H. Doroshow, Shivaani Kummar. A phase I trial of oral TRC102 (methoxyamine HCl) in combination with temozolomide (TMZ) in patients with relapsed solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT316. doi:10.1158/1538-7445.AM2015-CT316

Background: Epigenetic alterations in the genome, including abnormal DNA methylation and histone hypoacetylation, initiate and promote cancerous changes. A potential mechanism of resistance to endocrine therapy in breast cancer involves changes in gene expression secondary to epigenetic modifications, which might be modulated with the use of histone deacetylase (HDAC) inhibitors such as entinostat. Results from ENCORE 301, a phase II study evaluating the addition of entinostat to the steroidal aromatase inhibitor (AI) exemestane in patients with hormone receptor (HR)-positive advanced breast cancer who had experienced disease progression on a non-steroidal AI (NSAI), showed a significant improvement in progression-free survival (PFS), the primary endpoint, and overall survival (OS), an exploratory endpoint. Based on the OS results, entinostat has been designated a Breakthrough Therapy by the FDA when used in combination with exemestane in HR-positive advanced breast cancer. Trial design: E2112 is a multicenter randomized double-blind placebo-controlled phase III study (NCT02115282) enrolling patients with advanced HR-positive, HER2-negative breast cancer who have experienced disease progression on a NSAI (n=600). Patients will receive exemestane 25mg po daily and entinostat/placebo 5mg po every week (28 day cycle). Staging every 12 weeks. Eligibility Criteria: Postmenopausal women and men (≥ 18 years), ECOG performance status 0-1, locally advanced/metastatic invasive adenocarcinoma of the breast: ER/PR-positive, HER2-negative, measurable or non-measurable (20% cap) disease. Disease progression after NSAI use in the metastatic setting OR relapse while on or within ≤ 12 months of end of adjuvant NSAI therapy. Prior CDK inhibitor or everolimus permitted, but not exemestane or fulvestrant. One prior chemo permitted in metastatic setting. Specific Aims: Both PFS (defined by central review) and OS are primary endpoints, and the study is designed to show an improvement in either PFS or OS. Secondary endpoints include: Safety and tolerability, objective response rate, changes in lysine acetylation status in peripheral blood mononuclear cells, patient-reported symptom burden and treatment toxicities, adherence. Statistical Methods: One-sided type 1 error 0.025 split between two hypotheses tests: 0.001 for PFS test and 0.024 for OS. PFS is tested in the first 360 pts, 88.5% power to detect 42% reduction in the hazard of PFS failure (median PFS 4.1 to 7.1 months); OS is tested in all 600 pts, 80% power to detect 25% reduction in the hazard of death (median OS 22 to 29.3 months) Present and Targeted Accrual: This study was activated in March 2014 and accrual is anticipated to complete in 40 months. Contact Person: Dr. Roisin Connolly, Email: rconnol2@jhmi.edu Funding: ECOG-ACRIN Cancer Research Group (ECOG-ACRIN), Cancer Therapy Evaluation Program (CTEP) at the National Cancer Institute, and Syndax Pharmaceuticals. Citation Format: Roisin M Connolly, Fengmin Zhao, Kathy D Miller, Amye J Tevaarwerk, Lynne I Wagner, Min-Jung Lee, Judy Murray, Robert Gray, Richard L Piekarz, Jo Anne A Zujewski, Joseph A Sparano. E2112: A randomized phase III trial of endocrine therapy plus entinostat/placebo in patients with hormone receptor-positive advanced breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr OT2-1-06.

In ongoing efforts to increase the solid tumor efficacy of the histone deacetylase inhibitor romidepsin, we characterized romidepsin-resistant T-cell lymphoma cell lines that were found to have activation of the mitogen-activated protein kinase (MAPK) pathway, leading to subsequent phosphorylation and degradation of the proapoptotic protein Bim. This led us to examine the combination of romidepsin with MAPK pathway inhibitors in cell lines harboring the V600E BRAF mutation that leads to constitutive activation of the MAPK pathway. To more closely simulate clinical administration of romidepsin, 11 V600E BRAF positive cell lines (8 melanomas and 3 colorectal cancers) were exposed to 25 ng/ml romidepsin in the presence or absence of the MAPK pathway inhibitors AZD6244 (selumetinib, 250 nM), PD0325901 (250 nM) or PLX4032 (vemurafenib, 1 µM) for 6 h, after which the cells were placed in romidepsin-free medium containing the inhibitors and incubated for an additional 42 h. Apoptosis was subsequently measured by Annexin V and propidium iodide staining. Of the 11 cell lines, 10 exhibited significantly higher annexin staining after short-term romidepsin treatment alone (control, median annexin value 7.1%±3.8% vs. treated, median annexin value 34.7%±17.8%, p Citation Format: Susan E. Bates, Victoria Luchenko, Agnes Basseville, Julian Bahr, Arup R. Chakraborty, Andrew McDonald, Alexandra Zimmer, Richard L. Piekarz, Robert W. Robey. Short-term romidepsin treatment combined with MAPK pathway inhibition results in decreased mitochondrial hexokinase 2, increased mitochondrial Bim and apoptosis in BRAF mutant cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5536. doi:10.1158/1538-7445.AM2014-5536

Introduction: Histone deacetylases (HDAC) are frequently deregulated in human cancers and their inhibition allows re-expression of silenced genes. Belinostat is an HDAC inhibitor with in vitro and in vivo activity in multiple malignancies, currently in phase II trials. We report the pharmacokinetics of belinostat and metabolites in patients enrolled in a phase I liver dysfunction trial currently being conducted and led by the NCI. Methods: Patients enrolled into the study were assigned to varying cohorts depending on their level of hepatic dysfunction (normal=N, mild=H1, moderate=H2, and severe=H3). All patients were given a 30 minute infusion at 400 mg/m2 on cycle 1 day -7. Heparinized plasma samples were collected prior to infusion, 15 and 25 min after start, and 5, 10, 15, 30, 60, 90, 120, 240, 360, 480 and 1440 minutes after infusion. Patients from the NCIDTC had samples collected and these were quantitated by a previously validated assay for belinostat, belinostat-glucuronide, methyl-belinostat, M21, M24, and M26. Pharmacokinetic parameters were derived non-compartmentally with PK Solutions. Metabolic ratios were calculated as a measure of metabolic fate. Results: 15 patients (N=2, H1=10, H2=2, H3=1) had useable pharmacokinetic data. Observed belinostat Cmax (N=21.1±4.9, H1=55.8±78.0, H2=23.0±7.2, H3=23.1 µg/mL) and belinostat AUC0-inf (N=806±294, H1=1308±996, H2=702±82, H3=780 µg/mL•min) did not reveal any trends with dysfunction cohort. Metabolic AUC0-inf ratios of belinostat to the various metabolites also did not appear to change with dysfunction cohort. Conclusion: Liver function does not appear to affect the pharmacokinetics or metabolic fate of belinostat. Analysis of additional subjects is ongoing. The results of this study will facilitate optimal dosing for patients with liver dysfunction. Citation Format: Brian Kiesel, Robert Parise, Yan Lin, Deborah Allen, Guru Reddy, Shanta Chawla, Richard Piekarz, Percy Ivy, Shivaani Kumar, Jan H. Beumer. Pharmacokinetic analysis of the HDAC inhibitor belinostat (PXD-101) and metabolites in patients with hepatic dysfunction. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT207. doi:10.1158/1538-7445.AM2014-CT207

Inhibition of histone deacetylase (HDAC) enzymes represents a promising therapeutic approach in clinical oncology, as aberrant gene expression and alterations in histone acetylation due to HDACs have been implicated in tumor development and progression. Even though several histone deacetylase inhibitors (HDIs) are currently in clinical trials, so far only the HDIs romidepsin and vorinostat have been approved by the U.S. Food and Drug Administration for the treatment of cutaneous T-cell lymphoma (CTCL). During clinical trials with romidepsin in CTCL, disease progression was noted in some patients who initially responded to therapy, while the disease in other patients did not respond to therapy suggesting that both de novo and acquired resistance to romidepsin were observed. To identify molecular determinants of resistance, we selected HuT78 CTCL cells with romidepsin in the presence of inhibitors of P-glycoprotein (Pgp) to prevent upregulation of Pgp as a mechanism of resistance. Resistant sublines were approximately 250- to 385-fold resistant to romidepsin; the Pgp inhibitor tariquidar did not significantly reverse resistance. The sublines also exhibited resistance to apoptosis following treatment with the HDIs apicidin, belinostat, entinostat, panobinostat, and vorinostat. A custom gene-expression array detected elevated expression of insulin receptor (INSR) in romidepsin resistant cells compared to parental cells. Immunoblot analysis of downstream effectors of the IR pathway demonstrated a 4- to 8-fold increase in mitogen-activated protein kinase (MAPK) kinase (MEK) phosphorylation. Even though resistant cells did not respond to 48 h treatment with inhibitors of the insulin receptor, they exhibited exquisite sensitivity to treatment with as little as 1 nM of the MEK inhibitor PD0325901. Sensitivity to MEK inhibition in resistant cells was associated with restoration of the pro-apoptotic protein Bim. Combined treatment of romidepsin with MEK inhibitors also significantly yielded greater apoptosis in resistant cells compared to romidepsin and MEK inhibitor treatment alone. Gene expression analysis of circulating tumor samples obtained from patients with CTCL enrolled on the NCI 1312 Phase II romidepsin study suggested interaction of romidepsin with the MAPK pathway, indicated by altered expression of genes demonstrated to be under its control. These findings implicate activation of MEK as a resistance mechanism to romidepsin, and suggest combination of romidepsin with MEK inhibitors in clinical trials. Citation Format: Arup R. Chakraborty, Rob Robey, Zhirong Zhan, Victoria Luchenko, Michael Gottesman, Nathan Collie, Jean-Pierre Gillet, Richard Piekarz, Andrew Kossenkov, Louise Showe, Susan Bates. Resistance to the histone deacetylase inhibitor romidepsin is associated with degradation of Bim following MAPK pathway activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3399. doi:10.1158/1538-7445.AM2013-3399

Background: In preclinical breast cancer models, combination epigenetic therapy with a DNA methyltransferase inhibitor (DNMTI) and a histone deacetylase inhibitor (HDACI) yield superior estrogen receptor (ER) re-expression and greater restoration of tamoxifen responsiveness than either agent alone. We conducted a multicenter phase II clinical trial to evaluate the DNMTI 5-azacitidine (5-AZA) and the HDACI entinostat in women with advanced breast cancer. Methods: Women with advanced HER2-negative, either triple-negative (TN; ER/progesterone receptor [PR]/HER2-negative) or hormone-resistant breast cancer received 5-AZA 40 mg/m2 (SQ, days 1-5, 8-10) and entinostat 7 mg (PO, days 3,10) every 28 days. Primary endpoint: objective response rate (ORR) in each group. Secondary endpoints: safety, tolerability, survival, clinical benefit rate. Exploratory endpoints: pharmacokinetics, pharmacogenetics, change in candidate gene re-expression/methylation in circulating DNA and mandatory tumor samples. Patients are offered ongoing study therapy at progression with addition of hormonal therapy (optional continuation phase). Sample size: Simon two-stage design with interim analysis after 13 patients per cohort (1st stage). If ≥1 response, accrual will continue for total of 27 per cohort (2nd stage). Null hypothesis: ORR at most 5% against alternative hypothesis that is at least 20% with type I error 4% and power 90%. Preclinical TN/ ER-positive xenograft studies assessing 5-AZA impact were also performed. Results: Thirteen evaluable patients were enrolled in 1st stage of TN cohort. Median age was 47 (31-67), median prior chemotherapies 3 (1-5), 77% white/33% black, 77% visceral disease. Median cycles received 2 (1-4). Therapy was well tolerated, most common grade 3/4 treatment related adverse events leucopenia and neutropenia (23% each). No responses observed following 1st stage and this cohort was closed. Median 1.5 additional cycles (optional continuation phase) received by 4 patients with no responses to date. Exposure to 5-AZA (Cmax=1134±1670ng/mL; AUCINF=939±724 ng*h/mL) was slightly higher than previous studies, entinostat (Cmin=0.78±0.65ng/mL) was similar. Hormone-resistant cohort proceeded to 2nd stage as 1 partial response observed. Final results will be reported once accrual complete. Ongoing preclinical studies suggest that ER-positive is more sensitive than TN breast cancer to 5-AZA. Conclusion: Combination epigenetic therapy with agents, dose and schedule described was well tolerated but not associated with clinical activity in advanced TN breast cancer. Correlative analyses will be presented at meeting. Promising preclinical findings suggest epigenetic therapy may be efficacious in ER-positive breast cancer. Citation Format: Roisin M. Connolly, Rachel C. Jankowitz, Cynthia A. Zahnow, Zhe Zhang, Michelle A. Rudek, Stacie C. Jeter, Shannon Slater, Penny Powers, Antonio C. Wolff, John Fetting, Adam M. Brufsky, Richard Piekarz, Nita Ahuja, George Somlo, Augustin Garcia, Steven Baylin, Nancy E. Davidson, Vered Stearns. A phase 2 study investigating the safety, efficacy and surrogate biomarkers of response of 5-azacitidine (5-AZA) andentinostat (MS-275) in patients with triple-negative advanced breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4666. doi:10.1158/1538-7445.AM2013-4666

Solid tumor trials with histone deacetylase inhibitors (HDIs) have been largely disappointing. We recently characterized a romidepsin-resistant T-cell lymphoma cell line that was found to have activation of the mitogen-activated protein kinase (MAPK) pathway, leading to subsequent phosphorylation and degradation of the proapoptotic protein Bim, suggesting that activation of this pathway may also confer resistance to romidepsin and other HDIs. The V600E BRAF mutation has been found in approximately 60% of melanomas and approximately 15% of colon cancers and leads to constitutive activation of the MAPK pathway. We thus hypothesized that combined treatment with romidepsin and BRAF or mitogen-activated protein kinase kinase (MEK) inhibitors may be effective in cancers that harbor the V600E BRAF mutation. To more closely simulate clinical administration of romidepsin, 11 V600E BRAF mutant cell lines (8 melanomas and 3 colorectal cancers) were exposed to 25 ng/ml romidepsin for 6 h after which romidepsin was removed, cells were incubated in fresh medium for an additional 42 h and subsequently apoptosis was measured by Annexin V and propidium iodide staining. Of the 11 cell lines, 10 exhibited significantly higher annexin staining after short-term romidepsin treatment (control 7.1% ± 3.8% vs. treated 34.7% ± 17.8%, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4709. doi:1538-7445.AM2012-4709

Elevated expression of phosphorylated mitogen activated protein kinase kinase (MEK) as a mechanism of resistance to the histone deacetylase inhibitor romidepsin in HUT 78 cutaneous T-cell lymphoma cells. Histone deacetylase inhibitors (HDIs) have shown promise in the treatment of T-cell lymphomas including cutaneous and peripheral T-cell lymphomas. However, resistance to romidepsin limits its activity in some patients. A detailed understanding of the mechanisms of resistance to HDIs may lead to strategies designed to increase clinical efficacy. To study mechanisms of resistance to the HDI romidepsin, the HUT78 cutaneous T-cell lymphoma cell line was exposed to increasing concentrations of romidepsin in the presence of the P-glycoprotein (P-gp) inhibitors verapamil or valspodar (PSC-833) to prevent the emergence of P-gp, a known resistance mechanism. The DpVp35 and DpVp50 sublines are maintained in 35 ng/ml and 50 ng/ml romidepsin, respectively, in the presence of 5 µg/ml verapamil while DpP75 is maintained in 75 ng/ml romidepsin and 3 µg/ml valspodar. In 4-day cytotoxicity assays, the sublines are approximately 55-fold resistant to romidepsin and are not cross resistant to the HDIs belinostat, panobinostat or vorinostat. Low but detectable levels of P-gp do not explain the resistance. We used a custom drug resistance gene expression array and found increased expression of insulin receptor (IR) in the resistant cells that was confirmed by immunoblot analysis. Elevated expression of phosphorylated mitogen activated protein kinase kinase (MEK), a downstream effector of the IR pathway, was also observed in the resistant cells compared to the parental cells. Interestingly, resistant cells were found to be exquisitely sensitive to MEK inhibition, as significant apoptosis was observed after 48 h in the presence of 5 nM of the MEK inhibitor PD0325901 and 10 nM of the MEK inhibitor AZD 6244 as measured by flow cytometry with annexin V and by immunoblot examining poly (ADP-ribose) polymerase (PARP) cleavage. No significant apoptosis was observed in parental cells at concentrations up to 500 nM. Resistant cells were not, however, sensitive to extracellular related kinase (ERK) inhibition or phosphatidylinositol 3-kinase (PI3K) inhibition in as determined by annexin V assay. In summary, we hypothesize that activated MEK can mediate resistance to romidepsin, but may also lead to collateral sensitivity to MEK inhibitors. The emerging role of activated MEK as a mechanism of resistance to romidepsin suggests combination of romidepsin with MEK inhibitors in future clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2624. doi:10.1158/1538-7445.AM2011-2624

Purpose: P-glycoprotein (Pgp) antagonists have been difficult to develop because of complex pharmacokinetic interactions and a failure to demonstrate meaningful results. However, data highlighting Pgp-mediated drug efflux continues to be reported. A growing body of evidence suggests that sestamibi uptake in lung cancer correlates with disease response to chemotherapy and has been proposed as a pre-selection technique to identify tumors that will respond to chemotherapy. We report the results of a clinical trial using tariquidar (XR9576), a potent Pgp antagonist, in combination with docetaxel. Experimental Design: Patients first received either 40 mg/m2 of docetaxel alone or 40 mg/m2 docetaxel administered in combination with 150 mg tariquidar. In the first cycle, the pharmacokinetics of docetaxel were monitored after the day 1 and day 8 doses with or without tariquidar. 99mTc-sestamibi scanning and rhodamine efflux from CD56+ mononuclear cells were performed to establish whether tariquidar modulates Pgp. In subsequent cycles, 75 mg/m2 of docetaxel was administered with 150mg of tariquidar every three weeks. Results: Forty-eight patients were enrolled onto the trial. Twenty-nine percent of the patients (14/48) had previously treated metastatic non-small cell lung cancer (NSCLC). Three partial responses were seen in the NSCLC cohort, measuring 40%, 57% and 67% reduction in tumor size by RECIST. Two patients remained on study for 7 and 24 months, respectively. Non-hematologic grade 3/4 toxicities in 235 cycles included fatigue (6%), nausea (4%), and diarrhea (1%). Tariquidar blocked Pgp-mediated rhodamine efflux from CD56+ cells and reduced 99mTc-sestamibi clearance from the liver. Sestamibi results were available in 32 of the 48 patients, and an increased area under the curve for the liver was noted, ranging from 5.8% to 251% over the pre-tariquidar scan. A 12% to 25% increase in sestamibi uptake was noted in 8 of 10 patients with lung cancer with visible lesions. A pharmacokinetic analysis evaluating potential interaction between tariquidar and docetaxel will be presented; previous reports suggest minimal interaction with anticancer agents. Conclusions: Tariquidar is a Pgp antagonist, without significant side effects and much less pharmacokinetic interaction than previous Pgp antagonists. Surrogate studies show increased retention of the Pgp substrate sestamibi following tariquidar in imagable lung cancers. Although the percentage increase was less than 25%, we have previously noted that quantitation by planar sestamibi imaging tends to underestimate actual change in accumulation. While response in this trial was designed as an exploratory endpoint only, three responses among 14 in a heavily pretreated population with NSCLC are somewhat surprising and suggest that a follow-up phase II study may be worthwhile. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3527.