Combination Of Entinostat Research Articles

Triple Combination of Entinostat, a Bromodomain Inhibitor, and Cisplatin Is a Promising Treatment Option for Bladder Cancer.

Cisplatin is part of the first-line treatment of advanced urothelial carcinoma. Cisplatin resistance is a major problem but may be overcome by combination treatments such as targeting epigenetic aberrances. Here, we investigated the effect of the class I HDACi entinostat and bromodomain inhibitors (BETis) on the potency of cisplatin in two pairs of sensitive and cisplatin-resistant bladder cancer cell lines. Cisplatin-resistant J82cisR and T24 LTT were 3.8- and 24-fold more resistant to cisplatin compared to the native cell lines J82 and T24. In addition, a hybrid compound (compound 20) comprising structural features of an HDACi and a BETi was investigated. We found complete (J82cisR) or partial (T24 LTT) reversal of chemoresistance upon combination of entinostat, JQ1, and cisplatin. The same was found for the BETis JQ35 and OTX015, both in clinical trials, and for compound 20. The combinations were highly synergistic (Chou Talalay analysis) and increased caspase-mediated apoptosis accompanied by enhanced expression of p21, Bim, and FOXO1. Notably, the combinations were at least 4-fold less toxic in non-cancer cell lines HBLAK and HEK293. The triple combination of entinostat, a BETi, and cisplatin is highly synergistic, reverses cisplatin resistance, and may thus serve as a novel therapeutic approach for bladder cancer.

Entinostat, a class I selective histone deacetylase inhibitor, plus exemestane for Chinese patients with hormone receptor-positive advanced breast cancer: An overall survival update and long-term safety from the randomised, double-blind, placebo-controlled, phase 3 trial.

1050 Background: Entinostat, a synthetic benzamide derivative HDAC inhibitor, selectively targets Class I HDAC enzymes. In a randomized, double-blind, phase 3 trial, entinostat with exemestane demonstrated a statistically significant progression-free survival (PFS) benefit compared to placebo, with a median PFS of 6.32 months versus 3.72 months (HR 0.76, 95% CI 0.58-0.98, p=0.046; Binghe Xu, et al. Acta Pharm Sin B. 2023). This analysis focus on the results of overall survival (OS) and long-term safety. Methods: Patients eligible for this study had HR+/HER2– advanced breast cancer (ABC) with disease relapse or progression following at least one prior endocrine therapy (ET). They were randomly assigned in a 2:1 ratio to receive either entinostat (5mg, weekly) or placebo in combination with exemestane until disease progression or unacceptable toxicity. The primary endpoint of the study was PFS. Secondary endpoints included OS, objective response rate, clinical benefit rate and safety. Median OS was estimated using the Kaplan-Meier method and the HR and its 95% CI were calculated using the multivariate Cox proportional hazard regression model. Results: A total of 354 patients were randomized to receive either entinostat (n=235) or placebo (n=119). As of the data cutoff date (July 31, 2023), 196 (55.4%) OS events had occurred, with a median follow-up of 31.83 months. There were 127 (54.0%) deaths in the entinostat arm compared to 69 (58.0%) deaths in the placebo arm. The entinostat demonstrated improved OS compared to placebo, with a median OS of 38.39 months versus 29.18 months (HR 0.837, 95% CI 0.624-1.124, p=0.237) in the full analysis set (FAS). The results of the per-protocol analysis set (PPS) were consistent with those of the FAS, showing a median OS of 39.01 months versus 33.98 months (HR 0.796, 95% CI 0.588-1.079, p=0.142). Notably, 179 (76.2%) patients in the entinostat arm and 100 (84%) patients in the placebo arm received new anti-cancer regimens, including CDK4/6 inhibitors (14.5% vs 24.4%), chemotherapy (54.9% vs 63.9%), endocrine therapy (40.4% vs 42.9%), and herbal medicines (9.8% vs 3.4%). After adjusting for the imbalance in the use of CDK4/6 inhibitors and chemotherapy between the treatment arms, the HR for overall survival was 0.78 (95% CI 0.58-1.05, p=0.097). The updated safety results were generally consistent with the previous analysis. Conclusions: The combination of entinostat with exemestane has provided clinically meaningful OS benefit in addition to the previously observed PFS benefit over exemestane alone in patients with pre-treated, ET-resistant ABC. These findings further support entinostat as an effective and safe treatment option for the specific patient population. Clinical trial information: NCT03538171 .

Nanoencapsulation of MDM2 Inhibitor RG7388 and Class-I HDAC Inhibitor Entinostat Enhances their Therapeutic Potential Through Synergistic Antitumor Effects and Reduction of Systemic Toxicity.

Inhibitors of the p53-MDM2 interaction such as RG7388 have been developed to exploit latent tumor suppressive properties in p53 in 50% of tumors in which p53 is wild-type. However, these agents for the most part activate cell cycle arrest rather than death, and high doses in patients elicit on-target dose-limiting neutropenia. Recent work from our group indicates that combination of p53-MDM2 inhibitors with the class-I HDAC inhibitor Entinostat (which itself has dose-limiting toxicity issues) has the potential to significantly augment cell death in p53 wild-type colorectal cancer cells. We investigated whether coencapsulation of RG7388 and Entinostat within polymeric nanoparticles (NPs) could overcome efficacy and toxicity limitations of this drug combination. Combinations of RG7388 and Entinostat across a range of different molar ratios resulted in synergistic increases in cell death when delivered in both free drug and nanoencapsulated formats in all colorectal cell lines tested. Importantly, we also explored the in vivo impact of the drug combination on murine blood leukocytes, showing that the leukopenia induced by the free drugs could be significantly mitigated by nanoencapsulation. Taken together, this study demonstrates that formulating these agents within a single nanoparticle delivery platform may provide clinical utility beyond use as nonencapsulated agents.

A Pilot Study of the Combination of Entinostat with Capecitabine in Advanced Breast Cancer.

Breast cancer has an unacceptably high recurrence rate when any residual disease is found following neoadjuvant treatment of high-risk disease. Based on clinical data suggesting an adjuvant role for epigenetic modifying agents in breast cancer and preclinical data suggesting synergistic activity of entinostat combined with capecitabine, we conducted a phase I, open-label study of these agents in metastatic breast cancer (MBC). Both agents have published doses for use in combination therapy, but the agents had not previously been combined with each other in a human trial. A multisite phase I dose escalation study was performed at two academic centers. Patients with pretreated, HER2-negative MBC, and measurable disease were enrolled. Dual dose escalation was performed via a Bayesian partial order continual assessment method. Dose levels ranged from entinostat 3 mg to 5 mg and capecitabine 800 mg/m2 to 1000 mg/m2. Thirteen patients with MBC and a median of 4 lines of prior therapy were enrolled across four dose level combinations. The most common toxicities were neutropenia, thrombocytopenia, and palmar-plantar dysesthesia, which were expected toxicities. No new safety signals were observed. One dose-limiting toxicity was observed, which did not exceed a prespecified toxicity rate of 25%. The median treatment duration was 2.37 months. No partial nor complete responses were observed. The study was halted early prior to entering an expansion phase, due to drug supply limitations. The tested dosing combinations of entinostat and capecitabine are likely safe in heavily pretreated metastatic breast cancer. This study's clinical investigation of entinostat in breast cancer was halted, but drug development of this agent continues outside the US. There remains a need for postoperative adjuvant drug therapy for the subpopulation of breast cancer patients with high-risk residual cancer after curative therapy. This trial is registered with NCT03473639.

Entinostat Enhances the Efficacy of Chemotherapy in Small Cell Lung Cancer Through S-phase Arrest and Decreased Base Excision Repair.

Acquired chemoresistance is a frequent event in small cell lung cancer (SCLC), one of the deadliest human malignancies. Histone deacetylase inhibitors (HDACi) have been shown to synergize with different chemotherapeutic agents including cisplatin. Accordingly, we aimed to investigate the dual targeting of HDAC inhibition and chemotherapy in SCLC. The efficacy of HDACi and chemotherapy in SCLC was investigated both in vitro and in vivo. Synergistic drug interactions were calculated based on the HSA model (Combenefit software). Results from the proteomic analysis were confirmed via ICP-MS, cell-cycle analysis, and comet assays. Single entinostat- or chemotherapy significantly reduced cell viability in human neuroendocrine SCLC cells. The combination of entinostat with either cisplatin, carboplatin, irinotecan, epirubicin, or etoposide led to strong synergy in a subset of resistant SCLC cells. Combination treatment with entinostat and cisplatin significantly decreased tumor growth in vivo. Proteomic analysis comparing the groups of SCLC cell lines with synergistic and additive response patterns indicated alterations in cell-cycle regulation and DNA damage repair. Cell-cycle analysis revealed that cells exhibiting synergistic drug responses displayed a shift from G1 to S-phase compared with cells showing additive features upon dual treatment. Comet assays demonstrated more DNA damage and decreased base excision repair in SCLC cells more responsive to combination therapy. In this study, we decipher the molecular processes behind synergistic interactions between chemotherapy and HDAC inhibition. Moreover, we report novel mechanisms to overcome drug resistance in SCLC, which may be relevant to increasing therapeutic success.

Targeting YB-1 via entinostat enhances cisplatin sensitivity of pleural mesothelioma in vitro and in vivo

Pleural mesothelioma (PM) is characterized by poor prognosis and limited therapeutic options. Y-box-binding protein 1 (YB-1) was shown to drive growth and migration of PM cells. Here, we evaluated the effect of genetic and pharmacological targeting of YB-1 on PM growth and response to cisplatin and radiation treatment. YB-1 knockdown via siRNA resulted in reduced PM cell growth, which significantly correlated with wt BAP1 and mutant NF2 and P53 status. Entinostat inhibited YB-1 deacetylation and its efficacy correlated with YB-1 knockdown-induced growth inhibition in 20 PM cell lines. Tumor growth inhibition by siRNA as well as entinostat was confirmed in mouse xenotransplant models. Furthermore, both YBX1-targeting siRNA and entinostat enhanced sensitivity to cisplatin and radiation. In particular, entinostat showed strong synergistic interactions with cisplatin which was linked to significantly increased cellular platinum uptake in all investigated cell models. Importantly, in a mouse model, the combination of cisplatin and entinostat also resulted in stronger growth inhibition than each treatment alone. Our study highlights YB-1 as an attractive target in PM and demonstrates that targeting YB-1 via entinostat is a promising approach to enhance cisplatin and radiation sensitivity.

Class I HDAC inhibition reduces DNA damage repair capacity of MYC-amplified medulloblastoma cells

PurposeMYC-driven Group 3 medulloblastoma (MB) (subtype II) is a highly aggressive childhood brain tumor. Sensitivity of MYC-driven MB to class I histone deacetylase inhibitors (HDACi) has been previously demonstrated in vitro and in vivo. In this study we characterize the transcriptional effects of class I HDACi in MYC-driven MB and explore beneficial drug combinations.MethodsMYC-amplified Group 3 MB cells (HD-MB03) were treated with class I HDACi entinostat. Changes in the gene expression profile were quantified on a microarray. Bioinformatic assessment led to the identification of pathways affected by entinostat treatment. Five drugs interfering with these pathways (olaparib, idasanutlin, ribociclib, selinexor, vinblastine) were tested for synergy with entinostat in WST-8 metabolic activity assays in a 5 × 5 combination matrix design. Synergy was validated in cell count and flow cytometry experiments. The effect of entinostat and olaparib on DNA damage was evaluated by γH2A.X quantification in immunoblotting, fluorescence microscopy and flow cytometry.ResultsEntinostat treatment changed the expression of genes involved in 22 pathways, including downregulation of DNA damage response. The PARP1 inhibitors olaparib and pamiparib showed synergy with entinostat selectively in MYC-amplified MB cells, leading to increased cell death, decreased viability and increased formation of double strand breaks, as well as increased sensitivity to additional induction of DNA damage by doxorubicin. Non-MYC-amplified MB cells and normal human fibroblasts were not susceptible to this triple treatment.ConclusionOur study identifies the combination of entinostat with olaparib as a new potential therapeutic approach for MYC-driven Group 3 MB.

A phase I/II study to evaluate the safety, pharmacodynamics, and efficacy of entinostat in combination with atezolizumab and bevacizumab in patients with renal cell carcinoma.

4526 Background: Immunosuppressive factors such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) limit the efficacy of immunotherapies. We have previously reported that histone deacetylase (HDAC) inhibitors have antitumor effects in combination with PD-1 inhibition by inhibiting the function of Tregs and MDSCs in preclinical models. We hypothesized that HDAC inhibition may enhance or resensitize patients (pts) with renal cell carcinoma (RCC) to immunotherapy. Thus, we conducted a Phase I/II clinical study with the HDAC inhibitor entinostat in combination with the immune check point inhibitor atezolizumab and the VEGF inhibitor bevacizumab in RCC pts, either treatment naïve or previously treated with immune checkpoint inhibitors. Methods: The primary objective was to evaluate the safety, tolerability, and efficacy of this combination strategy in a Phase I/II clinical trial. This clinical study was composed of a Dose Finding Phase (Phase I) and a two-stage Phase (Phase II) portion. Due to unforeseen circumstances, enrollment was halted during Stage I of the Phase II portion. In the Phase I dose finding cohorts, according to the 3+3 standard design, pts received oral entinostat at 1 mg (dose level or dl1), 3 mg (dl 2), or 5 mg (dl 3) every 7 days, continuously, with atezolizumab at a dose of 1,200 mg/kg mg IV every three weeks in combination with bevacizumab at a dose of 15 mg/kg IV every 3 weeks. Phase II patients were split into two distinct cohorts: Cohort A (no prior treatments with PD-1 or PD-L1 inhibitors) and Cohort B (at least one prior treatment with a PD-1 or PD-L1 inhibitor for metastatic disease). Pts treated at dl3 were included in the Phase II efficacy analyses. Results: 31 pts (18 and 13 pts during Phase I and II, respectively) were enrolled. Phase I was completed with the following DLTs: hypertension (3) in dl1, encephalopathy and hyponatremia (dl2), pneumonitis and pruritis (dl3). Dl3 was established as the recommended phase II dose. The most common treatment-related toxicities were thrombocytopenia, hypophosphatemia, proteinuria, diarrhea, fatigue, and neutropenia. A total of 21 grade≥3 events were observed. Nine pts achieved a partial response in Cohort A and one patient in Cohort B with an ORR 60% (CI 32.3-83.7) and 33.3% (28.2-56.4), respectively. According to Simon’s optimal two-stage design, the ORR met the Stage I primary objective. The median PFS was 13.7 months (CI 8.7-21.3) in Cohort A and 9.8 months (CI 1.3-NE) in Cohort B. We have collected blood samples to conduct several correlative studies on peripheral blood mononuclear cells. Conclusions: The preliminary results from this study suggest that the combination of entinostat and atezolizumab plus bevacizumab is relatively well tolerated and may be active in pts with ccRCC. Clinical trial information: NCT03024437 .

A randomized, open-label, phase II study of high-dose interleukin 2 vs high-dose interleukin 2 plus entinostat in renal cell carcinoma.

e16540 Background: Immunosuppressive factors such as regulatory T cells (Tregs) limit the efficacy of immunotherapies such as high dose interleukin 2 (HD-IL2). We previously reported that the histone deacetylase (HDAC) inhibitor entinostat has antitumor effects in combination with HD-IL2 by decreasing Tregs function in a preclinical model of kidney cancer. Based on these preliminary data, we hypothesized that HDAC inhibition may enhance or resensitize patients (pts) with clear cell renal cell carcinoma (ccRCC) to HD-IL2. Our group has previously conducted a Phase II, single arm, clinical trial of entinostat in combination with HD-IL2 in ccRCC, which showed an objective response rate (ORR) of 37% and a median progression-free survival (mPFS) of 13.6 months as compared to historical data with single agent HD-IL2 of 20-25% and 4.2 months, respectively. Thus, we designed a multi-center, randomized, open label Phase II study with entinostat in combination with HD-IL2 in ccRCC patients. Methods: The primary objective was to estimate and compare PFS in pts randomized to receive either HD-IL2 plus entinostat (Arm 1) or HD-IL2 (Arm 2) and stratified by no prior therapies vs prior therapies. Due to unforeseen circumstances, enrollment halted after the first 10 patients. The treatment schedule was HD-IL2 600,000 units/kg administered IV every 8 hrs on Days 1-5 and Days 15-19 (maximum 28 doses) +/- entinostat 5 mg orally given every 2 weeks starting on Day-14, continuously. The study was originally designed to detect a 100% improvement of median PFS to 8.4 months in ARM 1. A one-sided stratified log-rank test with an overall sample size of 46 subjects (23 per group) achieves 80% power at a 0.10 significance level to detect a hazard ratio of 0.5 (median PFS of 4.2 months vs 8.4 months). Results: 10 pts (6 pts in Arm 1 and 4 pts in Arm 2) were enrolled. Among the 10 pts, only one had prior therapy (Arm 1). The most common treatment-related toxicities ( > 50%, any grade) were diarrhea, hypotension, nausea, hypomagnesemia, hypophosphatemia, pruritus, hypocalcemia and thrombocytopenia, hypophosphatemia, proteinuria, diarrhea, fatigue, and neutropenia. The most common ≥ grade 3 toxicities were hypotension (80%), hypophosphatemia (70%), and thrombocytopenia (60%). Two pts (33%: CI 4.3%-77.7%) achieved a partial response in Arm 1 and no pts in Arm 2. The median PFS was 12.4 months (CI 2.3-NE) in Arm 1 (13.3 months in pts with no prior therapies) and 2.8 months (CI 1.2-3.3) in Arm 2 (Logrank p-value 0.0167). We have collected blood samples to conduct several correlative studies including flow cytometry and gene expression analysis on peripheral blood mononuclear cells. Conclusions: The preliminary results from this study confirm that the combination of entinostat and HD-IL2 may be more active than HD-IL2 alone in pts with ccRCC. Clinical trial information: NCT03501381 .

A pilot study of the combination of entinostat with capecitabine in high-risk breast cancer after neoadjuvant therapy.

e13107 Background: Breast cancer has an unacceptably high recurrence rate when residual disease is found following neo-adjuvant treatment of high-risk disease. Based on clinical data suggesting an adjuvant role for epigenetic modifying agents in breast cancer and preclinical data suggesting synergistic activity of entinostat combined with capecitabine, we conducted a phase I, open label study of these agents in metastatic breast cancer (MBC). Both agents have published doses for use in combination therapy, but the agents had not previously been combined with each other in a human trial. Methods: A multisite phase I dose escalation study was performed at two academic centers. Patients with pretreated, HER2 negative MBC, and measurable disease were enrolled. Dual dose escalation was performed via a Bayesian partial order continual assessment method. Dose levels ranged from entinostat 3mg to 5mg and capecitabine 800mg/m2 to 1000mg/m2. Results: Thirteen patients with MBC and a median of 4 lines of prior therapy were enrolled across four dose level combinations. The most common toxicities were neutropenia, thrombocytopenia, and palmar- plantar erythrodysesthesia, which were expected toxicities. No new safety signals were observed. One dose-limiting toxicity was observed, which did not exceed a pre-specified toxicity rate of 25%. The median treatment duration was 2.37 months. No partial nor complete responses were observed. The study was halted early prior to entering an expansion phase. Conclusions: The tested dosing combinations of entinostat and capecitabine are likely safe in heavily pretreated metastatic breast cancer patients. Entinostat clinical investigation in breast cancer was halted while this study was ongoing. Clinical trial information: NCT03473639 .

Class I HDAC inhibitor entinostat synergizes with PLK1 inhibitors in MYC-amplified medulloblastoma cells

PurposeWe and others have demonstrated that MYC-amplified medulloblastoma (MB) cells are susceptible to class I histone deacetylase inhibitor (HDACi) treatment. However, single drug treatment with HDACi has shown limited clinical efficacy. We hypothesized that addition of a second compound acting synergistically with HDACi may enhance efficacy.MethodsWe used a gene expression dataset to identify PLK1 as a second target in MB cells and validated the relevance of PLK1 in MB. We measured cell metabolic activity, viability, and cycle progression in MB cells after treatment with PLK1-specific inhibitors (PLK1i). Chou–Talalay synergy calculations were used to determine the nature of class I HDACi entinostat and PLK1i interaction which was validated. Finally, the clinical potential of the combination was assessed in the in vivo experiment.ResultsMYC-amplified tumor cells are highly sensitive towards treatment with ATP-competitive PLK1i as a monotherapy. Entinostat and PLK1i in combination act synergistically in MYC-driven MB cells, exerting cytotoxic effects at clinically relevant concentrations. The downstream effect is exerted via MYC-related pathways, pointing out the potential of MYC amplification as a clinically feasible predictive biomarker for patient selection. While entinostat significantly extended survival of mice implanted with orthotopic MYC-amplified MB PDX, there was no evidence of the improvement of survival when treating the animals with the combination.ConclusionThe combination of entinostat and PLK1i showed synergistic interaction in vitro, but not in vivo. Therefore, further screening of blood–brain barrier penetrating PLK1i is warranted to determine the true potential of the combination as no on-target activity was observed after PLK1i volasertib treatment in vivo.

Abstract 3256: Entinostat enhances the efficacy of checkpoint inhibition in breast-to-lung metastases and is associated with alterations in the phenotype and function of myeloid cell populations

Abstract Single agent immune checkpoint inhibitors (ICIs) are ineffective against metastatic breast cancer. One mechanism of intrinsic resistance is the presence of immunosuppressive myeloid cells and dysfunctional dendritic cells in the metastatic tumor microenvironment (TME) that impede the activation of anti-tumor T cells. A comparison of single-cell RNA sequencing analyses between breast-to-lung metastases and published data from breast tumors in the syngeneic NeuN mouse model of HER2+ breast cancer revealed that despite the presence of additional immune cell populations in the lungs, myeloid cells represented the predominant immune cell population in both TMEs and demonstrated similar changes in frequency and gene expression following treatment with the histone deacetylase inhibitor, entinostat. Our work highlights for the first time an increase in the frequency of conventional dendritic cells (cDC) with entinostat treatment in breast-to-lung metastases. Gene expression analyses of breast-to-lung metastases showed that entinostat-treated myeloid derived suppressor cells (MDSCs) and tumor associated macrophages (TAMs) downregulated genes associated with immunosuppression such as S100a8, Cd84, Trem2, and Mmp9. In addition, TAMs and cDCs upregulated the expression of genes associated with antigen presentation such as H2-K1, H2-Ab1, B2m, and Cd74 and pro-inflammatory markers such as Ccl3. NeuN mice were treated with the combination of entinostat and the immune checkpoint inhibitors, anti-PD-1 and anti-CTLA-4, and breast-to-lung metastases were analyzed via flow cytometry, demonstrating an increase in the frequency of cDC1s and CD8+ T cells expressing granzyme B. Furthermore, the treatment combination of entinostat + anti-PD-1 + anti-CTLA-4 significantly increased survival in an experimental model of lung metastasis using a newly characterized model derived from the original NT2.5 HER2+ cell line, NT2.5LM. These results confirm those of our breast expansion cohort from the phase I clinical trial NCI-9844 that demonstrate a 30% overall response rate in 20 heavily pretreated patients with metastatic disease treated with entinostat + Nivolumab (anti-PD-1) + Ipilimumab (anti-CTLA-4). We highlight for the first time significant alterations within multiple myeloid derived cell types, cDCs, TAMs and MDSCs, from within a native metastatic niche, that are likely driving an entinostat mediated improved response to ICI treatment. Citation Format: Edgar Gonzalez, Adam L. MacLean, Evanthia T. Roussos Torres, Aaron Baugh, Jesse Kreger, Sofi Castanon, Valerie Narumi, Elizabeth M. Jaffee. Entinostat enhances the efficacy of checkpoint inhibition in breast-to-lung metastases and is associated with alterations in the phenotype and function of myeloid cell populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3256.

HDAC3 is critical in tumor development and therapeutic resistance in Kras-mutant non-small cell lung cancer.

HDAC3 is one of the main targets of histone deacetylase (HDAC) inhibitors in clinical development as cancer therapies, yet the in vivo role of HDAC3 in solid tumors is unknown. We identified a critical role for HDAC3 in Kras-mutant lung cancer. Using genetically engineered mouse models (GEMMs), we found that HDAC3 is required for lung tumor growth in vivo. HDAC3 was found to direct and enhance the transcription effects of the lung cancer lineage transcription factor NKX2-1 to mediate expression of a common set of target genes. We identified FGFR1 as a critical previously unidentified target of HDAC3. Leveraging this, we identified that an HDAC3-dependent transcriptional cassette becomes hyperactivated as Kras/LKB1-mutant cells develop resistance to the MEK inhibitor trametinib, and this can be reversed by treatment with the HDAC1/HDAC3 inhibitor entinostat. We found that the combination of entinostat plus trametinib treatment elicits therapeutic benefit in the Kras/LKB1 GEMM.

A phase II study to evaluate the safety, pharmacodynamics and efficacy of entinostat in combination with nivolumab plus ipilimumab in patients with renal cell carcinoma previously treated with nivolumab plus ipilimumab or nivolumab alone.

668 Background: Immunosuppressive factors such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) limit the efficacy of immunotherapies. We have previously reported that histone deacetylase (HDAC) inhibitors have antitumor effects in combination with PD-1 inhibition by inhibiting the function of Tregs and MDSCs in preclinical models. We hypothesized that HDAC inhibition may resensitize patients (pts) with renal cell carcinoma (RCC) to immunotherapy. Thus, we opened a Phase II clinical study with the HDAC inhibitor entinostat and the immune check point inhibitors nivolumab (nivo) and ipilimumab (ipi) in clear cell RCC pts previously treated with nivo + ipi or nivo alone. Methods: The primary objective was to evaluate the safety, tolerability, and efficacy of this combination strategy in a dose de-escalation, two stage, phase II clinical trial with a safety lead-in. Due to a funder decision, enrollment was halted after completion of accrual to the safety lead in group. Pts received oral entinostat at a dose of 5 mg (dose level 1) or 3 mg (dose level 2) every 7 days, continuously, with nivo at a dose of 3 mg/kg mg IV every three weeks in combination with ipi at a dose of 1 mg/kg every 3 weeks X 4 doses. Then, nivo 480 mg was continued every 4 weeks. Results: A total of 12 pts with prior nivo+ipi or nivo treatments was enrolled. Dose level 1 (5 pts enrolled) was completed with the following DLTs: thrombocytopenia, nausea and vomiting, pneumonitis. Seven pts were enrolled on dose level 2 which was established as the recommended phase II dose. The most common treatment-related toxicities were fatigue, hypophosphatemia, neutropenia and thrombocytopenia. All were transient. Most of the pts had 2 or more prior treatments. Four pts achieved a partial response, with 3 out 4 pts previously treated with nivo alone (ORR 33%; 95% CI 9.9-65.1). The median PFS was 11.1 months (CI 1.3-26.4). We collected blood samples to conduct several correlative studies including flow cytometry and gene expression analysis on peripheral blood mononuclear cells. Conclusions: The preliminary results from this study suggest that the combination of entinostat and nivo+ ipi is relatively well tolerated and may be active in pts with RCC. Clinical trial information: NCT03552380 .

27 Entinostat restores responsiveness to olaparib in ID8 TP53 null and ID8 TP53 null/BRCA2 null mouse ovarian cancer cell lines

Poly (adenosine diphosphate [ADP]-ribose) polymerase inhibitors (PARPi) reduce disease progression in patients diagnosed with ovarian cancer. The anticancer effects of PARPi are most effective in tumors with BRCA mutations and homologous recombination (HR) deficiency (HRD). One mechanism of PARPi resistance is through restoration of HR repair. Our group has shown that entinostat (an HDACi) potentiates the effects of olaparib (a PARPi). The objective of this study was to investigate mechanisms of intrinsic PARPi resistance by studying the combination of entinostat and olaparib in HR proficient and HR deficient mouse ovarian cancer cell lines. We used the ID8 TP53 null (HR proficient) and ID8 TP53 null/BRCA2 null (HR deficient) mouse ovarian cancer cell lines that harbor molecular features consistent with human high-grade serous ovarian cancer. To recapitulate an ongoing clinical trial using this drug combination, cells were pre-treated with 0.25 μM Entinostat or control for 24 hours, followed by 24 or 72 hours of drug treatment [0.5 μM Entinostat, 10 μM of Olaparib, the combination of both, or control]. Clonogenicity assays were used to assess colony formation and MTS assays were used to measure cell viability and proliferation. We then performed immunofluorescence (IF) staining for RAD51 (a marker for HR repair), γH2AX (a marker of DNA damage) and Ki67 (a marker of proliferation). As expected in the clonogenicity assay, there was no statistically significant difference in cell survival between the olaparib treated ID8 TP53 null (HR proficient) cells and vehicletreated controls (p=0.0993; paired t test). Alternatively, the ID8 TP53 null/BRCA2 null (HR deficient) cell line treated with olaparib alone reduced cell viability compared to control (p=0.0245; paired t test). Clonogenicity assays also demonstrated that the combination of entinostat and olaparib reduced colony formation compared to control in the ID8 TP53 null cell line (p<0.0001; one-way ANOVA). MTS assays corroborated this treatment effect (p<0.0001; two way ANOVA). Preliminary data suggests that compared to controls and each drug alone, IF staining for RAD51 and Ki67 expression was reduced in cells treated with combination therapy. Also preliminarily, there was increased number of γH2AX foci in combination treatment compared to controls and each drug alone. Entinostat restores responsiveness to olaparib in ID8 TP53 null HR proficient mouse ovarian cancer cells that molecularly resemble high-grade serous ovarian carcinoma. These results suggest this drug combination can overcome intrinsic resistance to PARPi and provide additional preclinical support for clinical investigation of this combination for the treatment of HR proficient ovarian cancer.

A Multi-Center Phase Ib Trial of the Histone Deactylase Inhibitor (HDACi) Entinostat in Combination with Anti-PD1 Antibody Pembrolizumab in Patients with Refractory/Relapsed Myelodysplastic Syndromes (RR-MDS) or Oligoblastic Acute Myeloid Leukemia (RR-AML) after Hypomethylating Agent (HMA) Failure

Introduction: Pts with MDS and HMA failure have a poor prognosis and need novel therapies. Success with anti-PD1 therapy in myeloid malignancies has been limited and resistance mechanisms are poorly understood. Preclinical data suggest that myeloid-derived suppressor cells (MDSCs) contribute to MDS progression (Chen et al. JCI 2013). and may mediate resistance to anti-PD1 therapy (Kim et al. PNAS 2014). As HDACi decrease MDSCs, synergistic anti-tumor efficacy with anti-PD-1 has been hypothesized leading to this phase Ib study (NCT02936752) of the HDACi entinostat and anti-PD1 antibody pembrolizumab in pts with MDS or oligoblastic AML and HMA failure. Methods: We conducted an investigator-initiated, NCI-Cancer Therapy Evaluation Program (NCI-CTEP)-sponsored, multicenter, dose escalation and expansion phase Ib trial. Adult (≥18 years) pts with MDS or oligoblastic AML (bone marrow [BM] blasts 20-30%) and HMA failure were enrolled. Pts had to have adequate end organ function and ECOG performance status ≤2. The dose-escalation part used 2 dose levels (DL1: entinostat 8mg PO on days 1 and 8 of each 21-day cycle; DL2: entinostat 8mg PO on days 1, 8, and 15 of each 21-day cycle) with pembrolizumab 200mg IV on day 1 of each 21-day cycle starting with cycle 2. In cycle 1, pts received only entinostat to determine its impact on MDSCs separately from pembrolizumab. Toxicities were tabulated and graded according to CTCAE-5. Dose-limiting toxicities (DLT) were defined as any of the following if deemed possibly related to trial therapy: Grade (G) 3/4 non-hematologic toxicity, any ≥G3 immune related adverse events (IrAEs) or any IrAE resulting in pembrolizumab interruption or discontinuation during the first 2 cycles of combined therapy. Hematologic AEs were considered a DLT only if they were unrelated to MDS and lasted for >42 days. The primary endpoint was safety and determination of the maximum tolerated dose. The secondary endpoint was the overall response rate defined as a composite of complete remission, partial remission, and hematologic improvement (HI) per the 2006 IWG response criteria in MDS and change in MDSCs and PD1/PDL-1 in BM. Results: Between 06/26/2017 and 02/03/2021, we enrolled 28 pts (25 MDS and 3 AML). Median age was 74 years (range [R], 57-86), all pts had received prior HMA with 5 pts (18%) having had ≥3 prior lines of therapy. Median IPSS-R at screening for MDS pts was 5.5 (R: 2-10). Baseline demographic and clinical characteristics are shown in Table 1. During dose escalation, 7 pts were enrolled in DL1 (1 pt not evaluable due to early disease progression during DLT period), and 6 pts were treated on DL2. There was 1 DLT on DL1 (G5 pneumonia/bronchoalveolar hemorrhage (BAH), while 2 pts experienced DLTs at DL2 (G3 pharyngeal mucositis and G3 anorexia). There were no hematologic DLTs during dose escalation. Thus, DL1 was expanded, and another 15 pts were enrolled at DL1. Across the entire study, pts received a median of 3 cycles of therapy (R: 1-12 cycles). The most frequent cause of treatment discontinuation was disease progression in 9 pts. Four pts died during the study, 2 due to therapy-related complications (1 from pneumonia/BAH, 1 from multi-organ failure which occurred after the DLT period in DL2). A summary of the AEs is presented in Table 2. Hematologic AEs were common. The most common non-hematologic ≥G3 AEs independent of treatment attribution were infection (32.1%), hypoxia/respiratory failure (10.7%) and dyspnea (10.7%). Potential IrAEs of any grade were reported in 5 pts including G3 pneumonitis in 2 pts. Serious AEs were reported in 14 pts; 8 of which were potentially attributed to treatment. Among the 19 pts with the first BM efficacy assessment after 3 months, no pt achieved a protocol-defined response. However, 2 pts had mCR (BM blasts decreased from a baseline of 7% and 10% to 2% and 4%, respectively) without HI. Twelve pts had stable disease, while 5 had progressive disease. With a median follow-up of 5.9 months (R: 0.5 - 59.1 months), median OS was 6.9 months (95% CI: 4.8 - 13.6 months). Correlative studies are being conducted. Conclusion: In this multi-center phase 1b investigator-initiated trial in MDS and oligoblastic AML pts after HMA failure, entinostat + pembrolizumab at DL1 was reasonably tolerated but had limited therapeutic efficacy. Ongoing correlative work will explore the disconnect between the preclinical and clinical data regarding potential synergy of entinostat + pembrolizumab. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 795: Entinostat restores responsiveness to Olaparib in ID8 TP53 null and ID8 TP53 null/BRCA2 null mouse ovarian cancer cell lines

Abstract Poly (adenosine diphosphate [ADP]-ribose) polymerase inhibitors (PARPi) reduce disease progression in patients diagnosed with ovarian cancer. The anticancer effects of PARPi are most effective in tumors with BRCA mutations and homologous recombination (HR) deficiency (HRD). One mechanism of PARPi resistance is through restoration of HR repair. Our group has shown that entinostat (an HDACi) potentiates the effects of olaparib (a PARPi). The objective of this study was to investigate mechanisms of intrinsic PARPi resistance by studying the combination of entinostat and olaparib in HR proficient and HR deficient mouse ovarian cancer cell lines. We used the ID8 TP53 null (HR proficient) and ID8 TP53 null/BRCA2 null (HR deficient) mouse ovarian cancer cell lines that harbor molecular features consistent with human high-grade serous ovarian cancer. To recapitulate an ongoing clinical trial using this drug combination, cells were pre-treated with 0.25 µM Entinostat or control for 24 hours, followed by 24 or 72 hours of drug treatment [0.5 µM Entinostat, 10 µM of Olaparib, the combination of both, or control]. Clonogenicity assays were used to assess colony formation and MTS assays were used to measure cell viability and proliferation. We then performed immunofluorescence (IF) staining for RAD51 (a marker for HR repair), γH2AX (a marker of DNA damage) and Ki67 (a marker of proliferation). As expected in the clonogenicity assay, there was no statistically significant difference in cell survival between the olaparib treated ID8 TP53 null (HR proficient) cells and vehicle-treated controls (p= 0.0993; paired t test). Alternatively, the ID8 TP53 null/BRCA2 null (HR deficient) cell line treated with olaparib alone reduced cell viability compared to control (p=0.0245; paired t test). Clonogenicity assays also demonstrated that the combination of entinostat and olaparib reduced colony formation compared to control in the ID8 TP53 null cell line (p&amp;lt;0.0001; one-way ANOVA). MTS assays corroborated this treatment effect (p&amp;lt; 0.0001; two way ANOVA). Preliminary data suggests that compared to controls and each drug alone, IF staining for RAD51 and Ki67 expression was reduced in cells treated with combination therapy. Also preliminarily, there was increased number of γH2AX foci in combination treatment compared to controls and each drug alone. Entinostat restores responsiveness to olaparib in ID8 TP53 null HR proficient mouse ovarian cancer cells that molecularly resemble high-grade serous ovarian carcinoma. These results suggest this drug combination can overcome intrinsic resistance to PARPi and provide additional preclinical support for clinical investigation of this combination for the treatment of HR proficient ovarian cancer. Citation Format: Tyler Woodard, Viju Gupta, Simona Miceska, Bisiayo Fashemi, Wendy Zhang, Dineo Khabele. Entinostat restores responsiveness to Olaparib in ID8 TP53 null and ID8 TP53 null/BRCA2 null mouse ovarian cancer cell lines [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 795.

Tumor-targeted interleukin-12 synergizes with entinostat to overcome PD-1/PD-L1 blockade-resistant tumors harboring MHC-I and APM deficiencies

BackgroundImmune checkpoint blockade (ICB) has achieved unprecedented success in treating multiple cancer types. However, clinical benefit remains modest for most patients with solid malignancies due to primary or acquired resistance....

Phase I results of the INFORM2 combination study of nivolumab and entinostat in children and adolescents: INFORM2 NivEnt.

10034 Background: Pediatric patients with relapsed or refractory high-risk solid and CNS tumors have dismal survival. To date treatment with immune checkpoint inhibitors in this population has been disappointing. This study exploits the immune enhancing effects of entinostat on nivolumab in biomarker enriched subpopulations. The study aims to determine the pediatric recommended phase II dose (pRP2D) and to evaluate activity and safety. Methods: This is an exploratory non-randomized, open-label, multinational seamless phase I/II trial in children and adolescents with relapsed / refractory or progressive high-risk solid and CNS tumors. The phase I is divided in 2 age cohorts: 12–21 years (y) and 6–11y and follows a 3 + 3 design with two dose levels for entinostat (dose level 1: 2 mg/m2 and dose level 2: 4 mg/m2 once per week) and fixed dose nivolumab (3 mg/kg every 2 weeks). Patients entering the trial on pRP2D can seamlessly enter phase II which consists of a biomarker defined four group basket trial: high mutational load (group A), high PD-L1 mRNA expression (group B), focal MYC(N) amplification (group C), low mutational load and low PD-L1 mRNA expression and no MYC(N) amplification (group D). Results: The first patient was enrolled in May 2020 and at the time of the data cut (21-JAN-2022), 19 patients were treated. The median age at enrollment was 14 y. In the 12 – 21y cohort 15 patients were enrolled and four patients in the 6 – 11y cohort. The most frequent treatment-related AEs to date were thrombocytopenia in six (32%), nausea and vomiting both in four (21%), and neutropenia in three patients (16%). Five patients (26%) experienced grade 3/4 mostly reversible treatment-related AEs, e.g. neutropenia/leukopenia. No treatment related deaths were reported. In the 6 – 11y cohort dose escalation is ongoing. In the 12 – 21y cohort, one DLT (CTCAE grade 3 thrombocytopenia) was observed in six patients on dose level two, which was determined as the pRP2D of the combination. At the time of the data cut, 10 patients (six in arm D and four patients in which the biomarker group was not yet determined) had received at least one RECIST/RANO response evaluation by central review in phase II. One patient (17%) in arm D with metastatic relapsed renal cell carcinoma (RCC) harboring a typical PRCC-TFE3 fusion showed a PR after two cycles and finally achieved an ongoing CR. Extensive explorative analyses of immune signatures derived from INFORM RNA-Seq and WES data revealed that both the primary diagnosis and the current relapse samples harbored a remarkable high immune cell infiltration, especially CD8+ T-cells. Conclusions: The first and ongoing global INFORM2 trial has identified the pRP2D for the nivolumab and entinostat combination in the older age cohort with good tolerability. A patient with metastasized relapsed RCC experienced a CR. The role of immune infiltration as a potential predictive biomarker is currently being explored. Clinical trial information: NCT03838042.

Abstract GS1-06: A randomized control phase III trial of entinostat, a once weekly, class I selective histone deacetylase inhibitor, in combination with exemestane in patients with hormone receptor positive advanced breast cancer

Abstract Background Entinostat is a novel, potent, once weekly, orally bioavailable, class I selective histone deacetylase (HDAC) inhibitor. In a previous Phase II study, the combination of entinostat with exemestane showed significant improvement of overall survival in patients with advanced hormone receptor (HR) positive breast cancer. To verify and further confirm the benefit of HDAC inhibitor in combination with exemestane we designed a randomized, controlled trial to assess the efficacy and safety in a larger population of Chinese patients with advanced, HR positive breast cancer. Methods We carried out the randomized, double-blind, placebo-controlled, Phase III trial at 35 sites in China. Eligible patients were women (aged ≥18 years) with HR positive, human epidermal growth factor receptor-2 (HER2) negative breast cancer, whose disease had relapsed or progressed after at least one endocrine therapy (either in advanced or metastatic or adjuvant setting), and who had at least one measurable lesion, adequate organ function, ECOG performance status of 0-1, and adequate haematological and biochemical parameters. Patients were randomly assigned (2:1) via an interactive web-response system to orally take 5 mg entinostat or placebo. Both groups received oral administration of 25 mg exemestane daily. Randomization was stratified according to previous usage of CDK4/6 (yes vs no), fulvestrant (yes vs no), chemotherapy (yes vs no), and the presence of visceral metastases (yes vs no). Patients, investigators, study site staff, and the sponsor were masked to treatment assignment. The primary endpoint was Independent Radiographic Committee (IRC)-assessed progression free survival (PFS). Efficacy and safety analyses were done in all patients who received at least one dose of any study treatment. The study has reached the required number of events for final analysis of the primary endpoint. The trial is no longer enrolling patients, but follow-up for investigation of overall survival is ongoing. This study was registered with ClinicalTrials.gov with the number of NCT03538171. Results From April 16th, 2019 to May 13th, 2020, 354 patients were enrolled and randomly assigned as 235 to the entinostat group and 119 to the placebo group. IRC-assessed median PFS was 6.32 months (95% CI 5.30-9.11) in the entinostat group and 3.72 months (95% CI 1.91-5.49) in the placebo group (HR 0.74 [95% CI 0.57-0.96]; p&amp;lt;0.001). The most common Grade 3 or 4 adverse events in the entinostat group vs placebo group were neutropenia (103 [43.8%] vs 119 [0.8%] ), thrombocytopenia (20 [8.5%] vs 1 [0.8%]), and leucopenia (15 [6.4%] vs 0). Serious adverse events occurred in 28 out of 235 patients (11.9%) in the entinostat group and 11 out of 119 patients (9.2%) in the placebo group. Conclusions Entinostat and exemestane combination treatment significantly improved PFS compared with exemestane alone in patients with advanced, HR positive, HER2 negative breast cancer that progressed after previous endocrine therapy. Entinostat and exemestane combination was generally tolerated and can offer meaningful clinical benefit in these patients with unmet medical need. This phase III trial was sponsored by Taizhou EOC Pharma Co., Ltd. Citation Format: Binghe Xu, Qingyuan Zhang, Xichun Hu, Qing Li, Tao Sun, Wei Li, Quchang Ouyang, Jingfen Wang, Zhongsheng Tong, Min Yan, Huiping Li, Xiaohua Zeng, Changping Shan, Xian Wang, Xi Yan, Jian Zhang, Yue Zhang, Jiani Wang, Liang Zhang, Ying Lin, Jifeng Feng, Qianjun Chen, Jian Huang, Yongkui Lu, Hongsheng Li, Jinsheng Wu, Jing Cheng, Yanrong Hao, Cuizhi Geng, Min Lu, Yanping Li, Xi Chen, Lihua Song, Xueying Wu, Changlu Hu, Xinhong Wu, Xiaojia Wang, Yueyin Pan, Yuehong Cui, Guohua Yu, Sanyuan Sun. A randomized control phase III trial of entinostat, a once weekly, class I selective histone deacetylase inhibitor, in combination with exemestane in patients with hormone receptor positive advanced breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS1-06.