Abstract: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood with an unmet clinical need for decades. A single oncogenic fusion gene is associated with treatment resistance and a 40 to 45% decrease in overall survival. We previously showed that expression of this PAX3:FOXO1 fusion oncogene in alveolar RMS (aRMS) mediates tolerance to chemotherapy and radiotherapy and that the class I–specific histone deacetylase (HDAC) inhibitor entinostat reduces PAX3:FOXO1 protein abundance. Here, we established the antitumor efficacy of entinostat with chemotherapy in various preclinical cell and mouse models and found that HDAC3 inhibition was the primary mechanism of entinostat-induced suppression of PAX3:FOXO1 abundance. HDAC3 inhibition by entinostat decreased the activity of the chromatin remodeling enzyme SMARCA4, which, in turn, derepressed the microRNA miR-27a. This reexpression of miR-27a led to PAX3:FOXO1 mRNA destabilization and chemotherapy sensitization in aRMS cells in culture and in vivo. Furthermore, a phase 1 clinical trial (ADVL1513) has shown that entinostat is tolerable in children with relapsed or refractory solid tumors and is planned for phase 1B cohort expansion or phase 2 clinical trials. Together, these results implicate an HDAC3–SMARCA4–miR-27a–PAX3:FOXO1 circuit as a driver of chemoresistant aRMS and suggest that targeting this pathway with entinostat may be therapeutically effective in patients.

Abstract: Adult patients (pts) diagnosed with acute lymphocytic leukemia (ALL) are known to have a poor clinical outcome as compared to children. Studies report a 2 year event free survival of 30-40% for Philadelphia chromosome negative (Ph-) patients age 〉 30 yrs and 17% for age 〉 50 yrs. In order to improve outcome for adult ALL, agents that are effective, safe and associated with a low morbidity are needed. Clofarabine, a second generation purine nucleoside analog, has clinical activity as a single agent and in combination with cytosine arabinoside (ara-C) against refractory and relapsed ALL. Clofarabine exerts its cytotoxicity through multiple mechanisms of action, with major effects via inhibition of ribonucleotide reductase (RR) and DNA polymerase-alpha, and incorporation into DNA leading to DNA damage and activation of apoptotic pathways. Histone deacetylases (HDACs) are important regulators of chromatin involved in silencing of tumor suppressor genes. HDAC inhibitors are shown to be apoptogenic in vitro for ALL cell lines and have received FDA approval for the treatment of CTCL and peripheral T cell lymphoma. Pre-treatment with entinostat has been shown to enhance the cytotoxic activity of fludarabine in leukemia cell lines in vitro (Maggio et al, Cancer Research 2004). Given the similarity of clofarabine to fludarabine, and its FDA approval for children with refractory ALL, the combination of entinostat with clofarabine was pursued. Methods A Phase I window of opportunity study using overlapping schedule of entinostat and clofarabine was used in adult pts with ALL (B precursor) or Acute Bilineage Leukemia (ABL). Pts were enrolled onto one of two arms; arm “A” received repeated cycles of entinostat-clofarabine every 21 days as long as there was evidence of response (CR, CRi, or PR following cycle 1) and pts on arm “B” received one cycle of entinostat-clofarabine prior to standard multi-agent chemotherapy. Entinostat was administered orally on day 1 and day 8 (with dose escalation from flat dosing of 4mg to 6mg to 8mg from cohort 1 to 3). Clofarabine was administered intravenously at a fixed dose for all dose cohorts at 10mg/m2 for 5 days (day 3-7). Adults 〉 40 yrs with newly diagnosed, Ph- B-lineage ALL or ABL were eligible. Additionally, adults 〉 21 yrs with relapsed and refractory, Ph- ALL or ABL were eligible. Eligibility criteria included serum creatinine 〈 2.0 mg/dl, hepatic enzymes 〈 or = 2.5 ULN and bilirubin 〈 2.0 mg/dl. WBC 〈 150,000/mm3 with no evidence for ongoing or impending leukostasis was required. Results 23 pts from 3 institutions were enrolled on this study (18 at JHH, 3 at UMD, 2 at UColorado). 17 pts were treated on arm A and 6 pts on arm B. 6 pts were treated in each dose level with responses as follows: in dose level one (1 CR, 5 NR) and dose level two (1 CR, 1 CRi, 4NR) and dose level three (3 PR and 3 NR). The dose level 3 cohort was expanded with a total of 5 additional pts to date (1 PR, 1 SD, 3NR). Thus, the overall response rate on dose level 3 was 4PR, 1SD, 6NR. The 4 pts with CR/CRi/PR were all de novo treated elderly pts from Arm A. Notably, one pt on arm A has been in remission for over 1.5 yrs. Toxicities to date included expected but manageable grade (G) 3 and 4 cytopenias. There were G3 elevations of ALT (N=2) and AST (2) and bilirubin (1) and one bacteremia (1) and G4 cellulitis (1). Planned correlative studies are ongoing and include evaluation of acetylation of target proteins using multiparameter flow cytometry and western blot as well as methylation evaluation. Conclusions Combination therapy with entinostat-clofarabine is feasible and is well tolerated with minimal toxicity. Promising durable responses were observed in older pts that were not otherwise able to receive multi-agent induction chemotherapy upfront. This is notable given the low dose of clofarabine used in every cohort. Correlative studies evaluating protein hyperacetylation and DNA methylation in serial samples from treated pts are in progress. Disclosures: Off Label Use: This clinical study is a Phase 1 investigation and it discusses non-FDA approved doses of both clofarabine and entinostat for adults with Acute Lymphocytic Leukemia. The reason for this is that this study examines these agents in combination in a Phase 1 fashion and we started with low doses of each agent. Ordentlich:Syndax: Employment. Trepel:Syndax: Research Funding.

Abstract: Background: Axatilimab (Axa) is an IgG4 humanized monoclonal antibody with high affinity binding to CSF-1R. Axa blocks CSF1 and IL-34 binding and activation of CSF-1R signaling, a key pathway involved in the expansion and infiltration of donor-derived macrophages that mediate chronic graft-versus-host disease (cGVHD). We previously reported preliminary phase (Ph) 1 data demonstrating clinical activity and safety of Axa in patients with active cGVHD (Arora, ASH 2020). Here, we provide updated results, including Ph 2 clinical data, for doses chosen to move forward in a global, randomized pivotal study, AGAVE-201 (SNDX-6352-504). Methods: SNDX-6352-0503 is a Ph 1/2 study evaluating safety, tolerability, and efficacy of Axa in pts ≥6 years of age with active cGVHD despite ≥2 prior lines of systemic therapy. Ph 1 evaluated Axa at doses of 0.15mg/kg (n=1), 0.5mg/kg (n=1), 1mg/kg (n=3), and 3mg/kg (n=6) Q2W and 3mg/kg Q4W (n=6). The Ph 2 dose expansion evaluated Axa at 1mg/kg Q2W (n=23) with a primary objective of overall response rate (CR+PR) at 6 months. The data cutoff was 28 Jun 2021. Results: Forty pts (17 Ph 1 and 23 Ph 2) were enrolled and received at least 1 dose of axatilimab. Median age was 59 y (range, 16-73). Pts had received a median of 4 prior lines of treatment (range, 1-11), including ibrutinib (n=25), ruxolitinib (n=21), and belumosudil (n=8). Pts had a median of 4 involved organ systems at baseline (range, 1-9). At the time of the data cut, 22 pts (Ph 1, n=6; Ph 2, n=16) were continuing study treatment. Reasons for discontinuation included progression (n=5, 13%), physician decision (n=5, 13%), adverse events (AEs) (n=4, 10%; grade [Gr]) 3 periorbital edema, Gr 3 hypersensitivity reaction, Gr 4 CPK increased, Gr 5 fall (n=1 each), other (n=2, 5%), and withdrawal of consent (n=2, 5%). Thirty-eight pts were evaluable for response across Ph 1 & Ph 2. Overall response rate was 66% (n=25/38) and similar in pts previously treated with ibrutinib (n=16/24; 67%), ruxolitinib (n=13/20; 65%), and belumosudil (n=4/7; 57%). Response rates were similar for moderate severity cGVHD (60% [6/10] ) vs severe cGVHD (70% [19/27]). A 7-point improvement in the normalized Lee Symptom score was seen in 54% (n=19/35) of pts (Fig 1). Focusing on the 32 pts treated at 2 of the doses selected to move forward (1mg/kg Q2W [n=26] and 3 mg/kg Q4W [n=6] ), AEs related to Axa occurred in 66% (n=21/32) of pts with 13% (n=4/32) of pts experiencing grade ≥3 related-AEs. At the 1mg/kg Q2W dose, 62% (n=16/26) of pts experienced a related-AE with 8% (n=2/26) of pts experiencing grade ≥3 related-AEs (hypersensitivity, septic arthritis; both grade 3). Related-AEs, regardless of grade, in the 32 pts demonstrate a trend toward dose dependency (1mg/kg Q2W vs 3 mg/kg Q4W) with a higher proportion having elevations in AST (23% vs 50%), CPK (12% vs 67%), ALT (12% vs 33%), lipase (12% vs 50%), and incidence of periorbital edema (8% vs 50%) in the 3mg/kg Q4W cohort (Table 1). Transient elevated circulating enzyme levels have not been associated with hepatotoxicity or any other end-organ damage and are likely due to CSF-1R blockade on Kupffer cells, which are liver macrophages that mediate clearance of these enzymes. Importantly, the risk of infection was low, and no cases of viral reactivation were reported (cytomegalovirus, Epstein-Barr, and/or herpes simplex virus). Of the 32 pts treated at 1mg/1kg Q2W or 3mg/kg Q4W, 30 pts were considered evaluable for response (2 pts had not undergone a postbaseline assessment at the time of the data cut). A best overall response rate (CR+PR) of 70% (75% [18/24] 1mg/kg Q2W; 50% [3/6] 3mg/kg Q4W) as defined by the 2014 NIH cGVHD Consensus Criteria was observed. Responses were noted in difficult-to-treat organ manifestations, with 31% (n=4/13) experiencing a response in lung, 19% (n=5/27) in skin, and 57% (n=13/23) in joints and fascia. Median time to first response was 0.95 months (Fig 2). Conclusions: Axa is a novel agent targeting a pathway different than other cGVHD treatments. Data from this Ph 1/2 study demonstrate the safety and clinical activity of Axa in heavily pre-treated pts with active cGVHD, particularly those with fibrotic manifestations. A randomized pivotal study (AGAVE-201) has started enrolling a similar pt population, evaluating doses of 1 mg/kg Q2W and 3mg/kg Q4W, along with a lower dose of 0.3mg/kg Q2W. Figure 1 Figure 1. Disclosures Lee: Syndax: Research Funding; Takeda: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Kadmon: Research Funding; AstraZeneca: Research Funding; Incyte: Research Funding; Janssen: Other; Amgen: Research Funding. Arora: Syndax: Research Funding; Pharmacyclics: Research Funding; Kadmom: Research Funding. Defilipp: Incyte Corp.: Research Funding; Regimmune Corp.: Research Funding; Omeros, Corp.: Consultancy; Syndax Pharmaceuticals, Inc: Consultancy. Abu Zaid: Syndax: Consultancy, Research Funding; Pieris: Current equity holder in publicly-traded company; Pharamcyclic: Research Funding; Incyte: Research Funding. Di Stasi: Syndax Pharmaceutical: Honoraria, Membership on an entity's Board of Directors or advisory committees; University of Alabama at Birmingham: Current Employment. Radojcic: Syndax Pharmaceuticals: Research Funding; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Allakos: Membership on an entity's Board of Directors or advisory committees. Meyers: Nuvalent: Consultancy, Membership on an entity's Board of Directors or advisory committees; Syndax Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Patents & Royalties. Qamoos: Syndax Pharmaceuticals: Current Employment. Ordentlich: Novartis: Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months; Patrys Lmtd: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Syndax Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Twenty-eight Seven Therapeutics: Consultancy; Cymabay Therapeutics: Current equity holder in publicly-traded company; Pfizer: Current equity holder in publicly-traded company; Viking Therapeutics: Current equity holder in publicly-traded company. Quaranto: Syndax Pharmaceuticals, LLC: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Schmitt: Syndax Pharmaceuticals, LLC: Current Employment, Current holder of stock options in a privately-held company; Fractyl Laboratories Inc. (Now Fractyl Health): Ended employment in the past 24 months. Gu: Syndax: Current Employment, Current equity holder in publicly-traded company; AstraZeneca: Ended employment in the past 24 months. Pusic: Syndax: Other: Advisory Board. Kitko: Horizon: Membership on an entity's Board of Directors or advisory committees; Co-investigator on two NIH grants as part of the cGVHD consortium: Research Funding; PER: Other: PER - CME educational talks about GVHD; Vanderbilt University Medical Center: Current Employment.

Abstract: 130 Background: Primary and acquired resistance to endocrine therapy in advanced breast cancer presents a significant barrier to maximizing the clinical effectiveness of these agents. We recently reported in a randomized, placebo controlled phase II study that entinostat (ENT), a class 1 selective HDAC inhibitor, combined with exemestane extended PFS and OS in postmenopausal women with ER+ positive breast cancer that had progressed on a prior nonsteroidal aromatase inhibitor. Likewise, everolimus (EVE), a mTORi, combined with exemestane or tamoxifen (TAM) also significantly extended PFS in a similar patient population. In order to determine the potential benefit of combining ENT with EVE plus hormone therapy we have piloted a series of xenograft studies in primary human tumor models of TAM sensitivity and resistance. Methods: Athymic nude mice bearing xenografts of tissue derived directly from patient tumors (MaCa4049 – TAM sensitive; MaCa3366/TAM – TAM resistant) were treated with A) vehicle B) 2 mg/kg EVE C) 2 mg/kg EVE + 10 mg/kg TAM D) 15 mg/kg ENT E) 2 mg/kg EVE + 10 mg/kg TAM + 15 mg/kg ENT. Tumor samples taken at the end of the study were cryoconserved for analysis of gene and protein expression and protein phosphorylation. Results: Growth of MaCa4049 tumors was inhibited in all treatment groups relative to vehicle in two independent studies conducted in this tumor model. Groups B, C and D demonstrated a similar level of activity while maximal inhibition was observed in Group E. All treatments were generally well tolerated with weight loss similar in groups C and E. Treatment of MaCa3366/TAM tumors is ongoing with similar levels of tumor growth inhibition in all groups observed relative to vehicle at week 7. Analysis of tumor samples from completed studies will be presented to provide insight into mechanism of action for the enhanced activity observed with the triple combination. Conclusions: Preclinical data demonstrating enhanced activity of entinostat combined with everolimus and hormone therapy provides the rationale for clinical investigation of this novel therapeutic approach to targeting hormone therapy resistance.

Abstract: Extracts of the resin of the guggul tree ( Commiphora mukul ) lower LDL (low-density lipoprotein) cholesterol levels in humans. The plant sterol guggulsterone [4,17(20)-pregnadiene-3,16-dione] is the active agent in this extract. We show that guggulsterone is a highly efficacious antagonist of the farnesoid X receptor (FXR), a nuclear hormone receptor that is activated by bile acids. Guggulsterone treatment decreases hepatic cholesterol in wild-type mice fed a high-cholesterol diet but is not effective in FXR-null mice. Thus, we propose that inhibition of FXR activation is the basis for the cholesterol-lowering activity of guggulsterone. Other natural products with specific biologic effects may modulate the activity of FXR or other relatively promiscuous nuclear hormone receptors.

Abstract: Purpose: Inflammatory breast cancer (IBC), diagnosed clinically, and triple-negative breast cancer (TNBC), diagnosed by molecular receptor status, are the two most aggressive forms of breast cancer, and both lack effective targeted therapies. We previously demonstrated involvement of histone deacetylase (HDAC) inhibitor entinostat in regulating apoptosis in IBC and TNBC cells; here, we aimed to identify novel combination therapy candidates. Experimental Design: Potential therapeutic targets were identified by mRNA expression profiling of TNBC and IBC cells treated with entinostat. Drug action and synergism were assessed by in vitro proliferation assays, tumor growth in vivo, and proteomic analyses. Gain/loss-of-expression studies were utilized to functionally validate the role of identified targets in sensitivity of TNBC and IBC cells to combination therapy. Results: Entinostat induced activity of the oncogenic ERK pathway and expression of proapoptotic NOXA. These are known to stabilize and degrade, respectively, MCL1, an antiapoptotic Bcl-2 protein. In breast cancer patients, high-MCL1/low-NOXA tumor expression correlated significantly with poor survival outcomes. Combination treatment of entinostat with MEK inhibitor pimasertib reduced the growth of TNBC and IBC cells in vitro and inhibited tumor growth in vivo. The synergistic action of combination therapy was observed in TNBC and IBC cell lines in which NOXA expression was induced following entinostat treatment. The therapeutic activity depended on induction of mitochondrial cell death pathways initiated by NOXA-mediated MCL1 degradation. Conclusions: Our preclinical findings provide a rationale for the clinical testing of combination HDAC and MEK pathway inhibition for TNBC and IBC that exhibit elevated baseline tumor MCL1 expression. Clin Cancer Res; 23(16); 4780–92. ©2017 AACR.