Purpose: Recent advances in immunotherapy highlight the antitumor effects of immune checkpoint inhibition despite a relatively limited subset of patients receiving clinical benefit. The selective class I histone deacetylase inhibitor entinostat has been reported to have immunomodulatory activity including targeting of immune suppressor cells in the tumor microenvironment. Thus, we decided to assess whether entinostat could enhance anti–PD-1 treatment and investigate those alterations in the immunosuppressive tumor microenvironment that contribute to the combined antitumor activity. Experimental Design: We utilized syngeneic mouse models of lung (LLC) and renal cell (RENCA) carcinoma and assessed immune correlates, tumor growth, and survival following treatment with entinostat (5 or 10 mg/kg, p.o.) and a PD-1 inhibitor (10 and 20 mg/kg, s.c.). Results: Entinostat enhanced the antitumor effect of PD-1 inhibition in two syngeneic mouse tumor models by reducing tumor growth and increasing survival. Entinostat inhibited the immunosuppressive function of both polymorphonuclear (PMN)- and monocytic-myeloid derived suppressor cell (M-MDSC) populations. Analysis of MDSC response to entinostat revealed significantly reduced arginase-1, iNOS, and COX-2 levels, suggesting potential mechanisms for the altered function. We also observed significant alterations in cytokine/chemokine release in vivo with a shift toward a tumor-suppressive microenvironment. Conclusions: Our results demonstrate that entinostat enhances the antitumor effect of PD-1 targeting through functional inhibition of MDSCs and a transition away from an immune-suppressive tumor microenvironment. These data provide a mechanistic rationale for the clinical testing and potential markers of response of this novel combination in solid tumor patients. Clin Cancer Res; 23(17); 5187–201. ©2017 AACR.

Immunosuppressive factors such as regulatory T cells (Tregs) limit the efficacy of immunotherapies. Histone deacetylase (HDAC) inhibitors have been reported to have antitumor activity in different malignancies and immunomodulatory effects. Herein, we report the Tregs-targeting and immune-promoting effect of a class I specific HDAC inhibitor, entinostat, in combination with either IL-2 in a murine renal cell carcinoma (RENCA) model or a survivin-based vaccine therapy (SurVaxM) in a castration resistant prostate cancer (CR Myc-CaP) model. RENCA or CR Myc-CaP tumors were implanted orthotopically or subcutaneously, respectively. Inoculated mice were randomized into four treatment groups: vehicle, entinostat, cytokine or vaccine, and combination. Tregs in the blood were assessed by FACS analysis. Real time quantitative PCR and Western blot analysis of isolated T cell subpopulations from spleen were performed to determine Foxp3 gene and protein expression. The suppressive function of Tregs was tested by T cell proliferation assay. Low dose (5 mg/kg) entinostat reduced Foxp3 levels in Tregs and this was associated with enhanced tumor growth inhibition in combination with either IL-2 or a SurVaxM vaccine. Entinostat down-regulated Foxp3 expression transcriptionally and blocked Tregs suppressive function without affecting T effector cells (Teffs). In vitro low dose entinostat (0.5 µM) induced STAT3 acetylation and a specific inhibitor of STAT3 partially rescued entinostat-induced down-regulation of Foxp3, suggesting that STAT3 signaling is involved in Foxp3 down-regulation by entinostat. These results demonstrate a novel immunomodulatory effect of class I HDAC inhibition and provide a rationale for the clinical testing of entinostat to enhance cancer immunotherapy.

Purpose: The activity of the novel antitumor agent tasquinimod (TASQ) with S100A9 as a molecular target was investigated in men with metastatic castration-resistant prostate cancer (CRPC) and minimal symptoms. Patients and Methods: We conducted a randomized, double-blind, placebo-controlled phase II trial in men assigned (at a ratio of two to one) to either oral once-daily TASQ 0.25 mg/d escalating to 1.0 mg/d over 4 weeks or placebo. The primary end point was the proportion of patients without disease progression at 6 months, defined by Response Evaluation Criteria in Solid Tumors Group, Prostate Cancer Working Group (PCWG2), or pain criteria, excluding prostate-specific antigen. Results: Two hundred one men (134 assigned to TASQ; 67 to placebo) were evaluable, and baseline characteristics were well balanced. Six-month progression-free proportions for TASQ and placebo groups were 69% and 37%, respectively (P < .001), and median progression-free survival (PFS) was 7.6 versus 3.3 months (P = .0042). In PCWG2 CRPC clinical subgroups, PFS in months was as follows: nodal metastases, 6.1 versus 3.1; bone metastases, 8.8 versus 3.4; and visceral metastases, 6.0 versus 3.0 for patients receiving TASQ versus placebo, respectively. Bone alkaline phosphatase levels were stabilized in the TASQ group, whereas the impact on PSA kinetics was less pronounced. Adverse events (AEs) occurring more frequently in the TASQ arm included GI disorders, fatigue, musculoskeletal pains, and elevations of pancreatic and inflammatory biomarkers. Grade 3 to 4 AEs, including asymptomatic elevations of laboratory parameters, were reported in 40% of patients receiving TASQ versus 10% receiving placebo; deep vein thrombosis (4% v 0%) was more common in the TASQ arm. Conclusion: TASQ significantly slowed progression and improved PFS in patients with metastatic CRPC with an acceptable AE profile.

Tyrosine kinase inhibitors (TKI) targeting angiogenesis via inhibition of the vascular endothelial growth factor pathway have changed the medical management of metastatic renal cell carcinoma. Although treatment with TKIs has shown clinical benefit, these drugs will eventually fail patients. The potential mechanisms of resistance to TKIs are poorly understood. To address this question, we obtained an excisional biopsy of a skin metastasis from a patient with clear cell renal carcinoma who initially had a response to sunitinib and eventually progressed with therapy. Tumor pieces were grafted s.c. in athymic nude mice. Established xenografts were treated with sunitinib. Tumor size, microvascular density, and pericyte coverage were determined. Plasma as well as tissue levels for sunitinib were assessed. A tumor-derived cell line was established and assessed in vitro for potential direct antitumor effects of sunitinib. To our surprise, xenografts from the patient who progressed on sunitinib regained sensitivity to the drug. At a dose of 40 mg/kg, sunitinib caused regression of the subcutaneous tumors. Histology showed a marked reduction in microvascular density and pericyte dysfunction. More interestingly, histologic examination of the original skin metastasis revealed evidence of epithelial to mesenchymal transition, whereas the xenografts showed reversion to the clear cell phenotype. In vitro studies showed no inhibitory effect on tumor cell growth at pharmacologically relevant concentrations. In conclusion, the histologic examination in this xenograft study suggests that reversible epithelial to mesenchymal transition may be associated with acquired tumor resistance to TKIs in patients with clear cell renal carcinoma. Mol Cancer Ther; 9(6); 1525–35. ©2010 AACR.

Preclinical studies suggest that histone deacetylase (HDAC) inhibitors may restore tumour sensitivity to retinoids. The objective of this study was to determine the safety, tolerability, and the pharmacokinetic (PK)/pharmacodynamic (PD) profiles of the HDAC inhibitor entinostat in combination with 13-cis retinoic acid (CRA) in patients with solid tumours. Patients with advanced solid tumours were treated with entinostat orally once weekly and with CRA orally twice daily × 3 weeks every 4 weeks. The starting dose for entinostat was 4 mg m⁻² with a fixed dose of CRA at 1 mg kg⁻¹ per day. Entinostat dose was escalated by 1 mg m⁻² increments. Pharmacokinetic concentrations of entinostat and CRA were determined by LC/MS/MS. Western blot analysis of peripheral blood mononuclear cells and tumour samples were performed to evaluate target inhibition. A total of 19 patients were enroled. The maximum tolerated dose (MTD) was exceeded at the entinostat 5 mg m⁻² dose level (G3 hyponatremia, neutropenia, and anaemia). Fatigue (G1 or G2) was a common side effect. Entinostat exhibited substantial variability in clearance (147%) and exposure. CRA trough concentrations were consistent with prior reports. No objective responses were observed, however, prolonged stable disease occurred in patients with prostate, pancreatic, and kidney cancer. Data further showed increased tumour histone acetylation and decreased phosphorylated ERK protein expression. The combination of entinostat with CRA was reasonably well tolerated. The recommended phase II doses are entinostat 4 mg m⁻² once weekly and CRA 1 mg kg⁻¹ per day. Although no tumour responses were seen, further evaluation of this combination is warranted.

Purpose: Data from epidemiological and experimental studies suggest that dietary protein intake may play a role in inhibiting prostate and breast cancer by modulating the IGF/AKT/mTOR pathway. In this study we investigated the effects of diets with different protein content or quality on prostate and breast cancer.