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 (HDACi) 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 anti-tumor 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 PMN- and 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 from an immunosuppressive to 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.

Background: Transcriptional co-activators YAP/TAZ are pivotal effectors of the Hippo pathway and their dysfunction promote epithelial-to-mesenchymal transition (EMT) and malignant transformation. Results: PTPN14 interacts with Kibra and activates LATS1 (upstream negative regulator of YAP). Conclusion: PTPN14 and Kibra activate LATS1 and negatively regulate the YAP oncogenic function. Significance: Study of the YAP regulatory mechanism is crucial for understanding its role in the physiological and pathological processes.

Sunitinib is considered a first-line therapeutic option for patients with advanced clear cell renal cell carcinoma (ccRCC). Despite sunitinib clinical efficacy, eventually patients develop drug resistance and disease progression. Herein, we tested the hypothesis whether initial sunitinib resistance may be transient and could be overcome by dose increase. In selected patients initially treated with 50 mg sunitinib and presenting with minimal toxicities, sunitinib dose was escalated to 62.5 mg and/or 75 mg at the time of tumor progression. Mice bearing two different patient-derived ccRCC xenografts (PDXs) were treated 5 days/week with a dose-escalation schema (40-60-80 mg/kg sunitinib). Tumor tissues were collected prior to dose increments for immunohistochemistry analyses and drug levels. Selected intra-patient sunitinib dose escalation was safe and several patients had added progression free survival. In parallel, our preclinical results showed that PDXs, although initially responsive to sunitinib at 40 mg/kg, eventually developed resistance. When the dose was incrementally increased, again we observed tumor response to sunitinib. A resistant phenotype was associated with transient increase of tumor vasculature despite intratumor sunitinib accumulation at higher dose. In addition, we observed associated changes in the expression of the methyltransferase EZH2 and histone marks at the time of resistance. Furthermore, specific EZH2 inhibition resulted in increased in vitro anti-tumor effect of sunitinib. Overall, our results suggest that initial sunitinib-induced resistance may be overcome, in part, by increasing the dose, and highlight the potential role of epigenetic changes associated with sunitinib resistance that can represent new targets for therapeutic intervention.

A major barrier for cancer immunotherapy is the presence of suppressive cell populations in cancer patients, such as myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM), which contribute to the immunosuppressive microenvironment that promotes tumor growth and metastasis. Tasquinimod is a novel antitumor agent that is currently at an advanced stage of clinical development for treatment of castration-resistant prostate cancer. A target of tasquinimod is the inflammatory protein S100A9, which has been demonstrated to affect the accumulation and function of tumor-suppressive myeloid cells. Here, we report that tasquinimod provided a significant enhancement to the antitumor effects of two different immunotherapeutics in mouse models of cancer: a tumor vaccine (SurVaxM) for prostate cancer and a tumor-targeted superantigen (TTS) for melanoma. In the combination strategies, tasquinimod inhibited distinct MDSC populations and TAMs of the M2-polarized phenotype (CD206+). CD11b+ myeloid cells isolated from tumors of treated mice expressed lower levels of arginase-1 and higher levels of inducible nitric oxide synthase (iNOS), and were less immunosuppressive ex vivo, which translated into a significantly reduced tumor-promoting capacity in vivo when these cells were co-injected with tumor cells. Tumor-specific CD8+ T cells were increased markedly in the circulation and in tumors. Furthermore, T-cell effector functions, including cell-mediated cytotoxicity and IFNγ production, were potentiated. Taken together, these data suggest that pharmacologic targeting of suppressive myeloid cells by tasquinimod induces therapeutic benefit and provide the rationale for clinical testing of tasquinimod in combination with cancer immunotherapies.

HDAC inhibitors (HDACIs) are anticancer agents being developed in preclinical and clinical settings due to their capacity to modulate gene expression involved in cell growth, differentiation and apoptosis, through modification of both chromatin histone and nonhistone proteins. Most HDACIs in clinical development have cytotoxic or cytostatic properties and their direct inhibitory effects on tumor cells are well documented. Numerous studies have revealed that HDACIs have potent immunomodulatory activity in tumor-bearing animals and cancer patients, providing guidance to apply these agents in cancer immunotherapies. Here, we summarize recent reports addressing the effects of HDACIs on tumor cell immunogenicity, and on different components of the host immune system. In addition, we discuss the complexity of the immunomodulatory activity of these agents, which depends on the class specificity of the HDACIs, different experimental settings and the target immune cell populations.

Acquired and intrinsic resistance to receptor tyrosine kinase inhibitors (RTKi) represent a major hurdle in improving the management of clear cell renal cell carcinoma (ccRCC). Recent reports suggest that drug resistance is driven by tumor adaptation via epigenetic mechanisms that activate alternative survival pathways. The histone methyl transferase EZH2 is frequently altered in many cancers including ccRCC. To evaluate its role in ccRCC resistance to RTKi, we established and characterized a spontaneously metastatic, patient-derived xenograft (PDX) model that is intrinsically resistant to the RTKI sunitinib but not to the VEGF therapeutic antibody bevacizumab. Sunitinib maintained its anti-angiogenic and anti-metastatic activity but lost its direct anti-tumor effects due to kinome reprogramming, which resulted in suppression of pro-apoptotic and cell cycle regulatory target genes. Modulating EZH2 expression or activity suppressed phosphorylation of certain RTK, restoring the anti-tumor effects of sunitnib in models of acquired or intrinsically resistant ccRCC. Overall, our results highlight EZH2 as a rational target for therapeutic intervention in sunitinib-resistant ccRCC as well as a predictive marker for RTKi response in this disease.

Purpose Based on preclinical data suggesting that the class I selective HDAC inhibitor entinostat exerts a synergistic antitumor effect in combination with high dose interleukin 2 (IL-2) in a renal cell carcinoma model by down-regulating Foxp3 expression and function of regulatory T cells (Treg), we conducted a phase I/II clinical study with entinostat and high dose IL-2 in patients with metastatic clear cell renal cell carcinoma (ccRCC). Experimental Design Clear cell histology, no prior treatments, and being sufficiently fit to receive high dose IL-2 were the main eligibility criteria. 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). Each cycle was 85 days. The primary end point was objective response rate and toxicity. Secondary end points included progression-free survival and overall survival. Results 47 patients were enrolled. At a median follow-up of 21.9 months, the objective response rate was 37% (95% CI 22%–53%), the median progression-free survival was 13.8 months (95% CI 6.0–18.8), and the median overall survival was 65.3 months (95% CI 52.6.–65.3). The most common grade 3/4 toxicities were hypophosphatemia (16%), lymphopenia (15%), and hypocalcemia (7%), and all were transient. Decreased Treg were observed following treatment with entinostat, and lower numbers were associated with response (p=0.03). Conclusions This trial suggests a promising clinical activity for entinostat in combination with high dose Il-2 in ccRCC patients, and provides the first example of an epigenetic agent being rationally combined with immunotherapy.

These results identify TFE3/IRS-1/PI3K/AKT/mTOR as a potential dysregulated pathway in TFE3-tRCC, and suggest a therapeutic potential of vertical inhibition of this axis by using a dual PI3K/mTOR inhibitor for patients with TFE3-tRCC.