David M. Woods

HDAC Inhibition Upregulates PD-1 Ligands in Melanoma and Augments Immunotherapy with PD-1 Blockade.

Combining other agents with immune-based approaches can enhance treatment for melanoma. PDL-1 gene expression was increased after inhibition of histone deacetylases. Combining PD-1–blockade immunotherapy with histone deacetylase inhibition increased responses in a mouse model of melanoma. Expression of PD-1 ligands by tumors and interaction with PD-1–expressing T cells in the tumor microenvironment can result in tolerance. Therapies targeting this coinhibitory axis have proven clinically successful in the treatment of metastatic melanoma, non–small cell lung cancer, and other malignancies. Therapeutic agents targeting the epigenetic regulatory family of histone deacetylases (HDAC) have shown clinical success in the treatment of some hematologic malignancies. Beyond direct tumor cell cytotoxicity, HDAC inhibitors have also been shown to alter the immunogenicity and enhance antitumor immune responses. Here, we show that class I HDAC inhibitors upregulated the expression of PD-L1 and, to a lesser degree, PD-L2 in melanomas. Evaluation of human and murine cell lines and patient tumors treated with a variety of HDAC inhibitors in vitro displayed upregulation of these ligands. This upregulation was robust and durable, with enhanced expression lasting past 96 hours. These results were validated in vivo in a B16F10 syngeneic murine model. Mechanistically, HDAC inhibitor treatment resulted in rapid upregulation of histone acetylation of the PD-L1 gene leading to enhanced and durable gene expression. The efficacy of combining HDAC inhibition with PD-1 blockade for treatment of melanoma was also explored in a murine B16F10 model. Mice receiving combination therapy had a slower tumor progression and increased survival compared with control and single-agent treatments. These results highlight the ability of epigenetic modifiers to augment immunotherapies, providing a rationale for combining HDAC inhibitors with PD-1 blockade. Cancer Immunol Res; 3(12); 1375–85. ©2015 AACR.

Divergent roles of histone deacetylase 6 (HDAC6) and histone deacetylase 11 (HDAC11) on the transcriptional regulation of IL10 in antigen presenting cells

The anti-inflammatory cytokine IL-10 is a key modulator of immune responses. A better understanding of the regulation of this cytokine offers the possibility of tipping the balance of the immune response toward either tolerance, or enhanced immune responses. Histone deacetylases (HDACs) have been widely described as negative regulators of transcriptional regulation, and in this context, the primarily nuclear protein HDAC11 was shown to repress il-10 gene transcriptional activity in antigen-presenting cells (APCs). Here we report that another HDAC, HDAC6, primarily a cytoplasmic protein, associates with HDAC11 and modulates the expression of IL-10 as a transcriptional activator. To our knowledge, this is the first demonstration of two different HDACs being recruited to the same gene promoter to dictate divergent transcriptional responses. This dynamic interaction results in dynamic changes in the expression of IL-10 and might help to explain the intrinsic plasticity of the APC to determine T-cell activation versus T-cell tolerance.

The antimelanoma activity of the histone deacetylase inhibitor panobinostat (LBH589) is mediated by direct tumor cytotoxicity and increased tumor immunogenicity.

Melanoma is the deadliest skin cancer, and its incidence has been increasing faster than any other cancer. Although immunogenic, melanoma is not effectively cleared by host immunity. In this study, we investigate the therapeutic, antimelanoma potential of the histone deacetylase inhibitor (HDACi) panobinostat (LBH589) by assessing both its cytotoxic effects on melanoma cells as well as enhancement of immune recognition of melanoma. Utilizing murine and human melanoma cell lines, we analyzed the effects of LBH589 on proliferation and survival. In addition, we analyzed the expression of several immunologically relevant surface markers and melanoma differentiation antigens, and the ability of LBH589-treated melanoma to activate antigen-specific T cells. Finally, we assessed the in-vivo effects of LBH589 in a mouse melanoma model. Low nanomolar concentrations of LBH589 inhibit the growth of all melanoma cell lines tested, but not normal melanocytes. This inhibition is characterized by increased apoptosis as well as a G1 cell cycle arrest. In addition, LBH589 augments the expression of major histocompatibility complex and costimulatory molecules on melanoma cells leading to an increased ability to activate antigen-specific T cells. Treatment also increases expression of melanoma differentiation antigens. In vivo, LBH589 treatment of melanoma-bearing mice results in a significant increase in survival. However, in immunodeficient mice, the therapeutic effect of LBH589 is lost. Taken together, LBH589 exerts a dual effect upon melanoma cells by affecting not only growth/survival but also by increasing melanoma immunogenicity. These effects provide the framework for future evaluation of this HDAC inhibitor in melanoma treatment.

Histone deacetylase 11: A novel epigenetic regulator of myeloid derived suppressor cell expansion and function

Myeloid-derived suppressor cells (MDSCs), a heterogeneous population of cells capable of suppressing anti-tumor T cell function in the tumor microenvironment, represent an imposing obstacle in the development of cancer immunotherapeutics. Thus, identifying elements essential to the development and perpetuation of these cells will undoubtedly improve our ability to circumvent their suppressive impact. HDAC11 has emerged as a key regulator of IL-10 gene expression in myeloid cells, suggesting that this may represent an important targetable axis through which to dampen MDSC formation. Using a murine transgenic reporter model system where eGFP expression is controlled by the HDAC11 promoter (Tg-HDAC11-eGFP), we provide evidence that HDAC11 appears to function as a negative regulator of MDSC expansion/function in vivo. MDSCs isolated from EL4 tumor-bearing Tg-HDAC11-eGFP display high expression of eGFP, indicative of HDAC11 transcriptional activation at steady state. In striking contrast, immature myeloid cells in tumor-bearing mice display a diminished eGFP expression, implying that the transition of IMC to MDSCs require a decrease in the expression of HDAC11, where we postulate that it acts as a gate-keeper of myeloid differentiation. Indeed, tumor-bearing HDAC11-knockout mice (HDAC11-KO) demonstrate a more suppressive MDSC population as compared to wild-type (WT) tumor-bearing control. Notably, the HDAC11-KO tumor-bearing mice exhibit enhanced tumor growth kinetics when compare to the WT control mice. Thus, through a better understanding of this previously unknown role of HDAC11 in MDSC expansion and function, rational development of targeted epigenetic modifiers may allow us to thwart a powerful barrier to efficacious immunotherapies.

Targeting histone deacetylase 6 mediates a dual anti-melanoma effect: Enhanced antitumor immunity and impaired cell proliferation.

The median survival for metastatic melanoma is in the realm of 8–16 months and there are few therapies that offer significant improvement in overall survival. One of the recent advances in cancer treatment focuses on epigenetic modifiers to alter the survivability and immunogenicity of cancer cells. Our group and others have previously demonstrated that pan‐HDAC inhibitors induce apoptosis, cell cycle arrest and changes in the immunogenicity of melanoma cells. Here we interrogated specific HDACs which may be responsible for this effect. We found that both genetic abrogation and pharmacologic inhibition of HDAC6 decreases in vitro proliferation and induces G1 arrest of melanoma cell lines without inducing apoptosis. Moreover, targeting this molecule led to an important upregulation in the expression of tumor associated antigens and MHC class I, suggesting a potential improvement in the immunogenicity of these cells. Of note, this anti‐melanoma activity was operative regardless of mutational status of the cells. These effects translated into a pronounced delay of in vivo melanoma tumor growth which was, at least in part, dependent on intact immunity as evidenced by the restoration of tumor growth after CD4+ and CD8+ depletion. Given our findings, we provide the initial rationale for the further development of selective HDAC6 inhibitors as potential therapeutic anti‐melanoma agents.

A Novel Role for Histone Deacetylase 6 in the Regulation of the Tolerogenic STAT3/IL-10 Pathway in APCs

APCs are critical in T cell activation and in the induction of T cell tolerance. Epigenetic modifications of specific genes in the APC play a key role in this process, and among them histone deacetylases (HDACs) have emerged as key participants. HDAC6, one of the members of this family of enzymes, has been shown to be involved in regulation of inflammatory and immune responses. In this study, to our knowledge we show for the first time that genetic or pharmacologic disruption of HDAC6 in macrophages and dendritic cells results in diminished production of the immunosuppressive cytokine IL-10 and induction of inflammatory APCs that effectively activate Ag-specific naive T cells and restore the responsiveness of anergic CD4+ T cells. Mechanistically, we have found that HDAC6 forms a previously unknown molecular complex with STAT3, association that was detected in both the cytoplasmic and nuclear compartments of the APC. By using HDAC6 recombinant mutants we identified the domain comprising amino acids 503–840 as being required for HDAC6 interaction with STAT3. Furthermore, by re–chromatin immunoprecipitation we confirmed that HDAC6 and STAT3 are both recruited to the same DNA sequence within the Il10 gene promoter. Of note, disruption of this complex by knocking down HDAC6 resulted in decreased STAT3 phosphorylation—but no changes in STAT3 acetylation—as well as diminished recruitment of STAT3 to the Il10 gene promoter region. The additional demonstration that a selective HDAC6 inhibitor disrupts this STAT3/IL-10 tolerogenic axis points to HDAC6 as a novel molecular target in APCs to overcome immune tolerance and tips the balance toward T cell immunity.

Abstract 1338: Novel implications of histone deacetylase 6 selective inhibition in melanoma T-cell immunotherapy

Adoptive T-cell therapy is an effective treatment for metastatic melanoma, achieving objective clinical responses in half of patients undergoing tumor infiltration lymphocyte (TIL) therapy. Epigenetic modifications are key players in regulating gene expression. Modulation of histone deacetylases (HDACs) has received attention for its implications in altering gene regulation. While HDAC pan-inhibition directly affects tumor growth, the immune system may be negatively impacted. Novel HDAC-selective inhibitors can ameliorate these undesirable effects. HDAC6 is unique in containing two catalytic domains, allowing for the development of isotype-selective inhibitors. Here a novel role of HDAC6 in T-cell immunity with important implications for adoptive cell therapy was explored. Initially a delay in B16 melanoma growth was observed in HDAC6KO mice (p Citation Format: Andressa Sodre Laino, David M. Woods, Amod Sarnaik, Esteban Celis, Jeffrey Weber, Eduardo M. Sotomayor. Novel implications of histone deacetylase 6 selective inhibition in melanoma T-cell immunotherapy. [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 1338. doi:10.1158/1538-7445.AM2015-1338

Abstract 638: Epigenetic reprograming of immune cells through selective inhibition of HDAC6 reduces suppressive phenotypes and augments anti-tumor properties of T-cells

Alteration of the epigenetic landscape of immune cells can modify the pattern of gene expression, thus resulting in phenotypic and functional changes. Small molecule inhibitors targeting epigenetic modifiers, such as histone deacetylases (HDACs), have been shown to reduce tumor growth. Besides promoting direct anti-tumor effects, HDAC inhibitors also target immune cells and alter their gene regulation. Here, we demonstrate that the HDAC6 selective inhibitors ACY-241 and ACY-1215 (ricolinostat) decrease the function of myeloid derived suppressor cells (MDSC) and T regulatory (Treg) cells, maintain an effector phenotype by CD8+ T cells, and do not reduce viability of immune cells. First, peripheral blood mononuclear cells derived from melanoma patients were treated with ACY-241, and the phenotype of MDSCs was assessed. Expression of the suppressive molecules ARG1 (p Citation Format: Andressa L. Sodre, David M. Woods, Amod Sarnaik, Brian C. Betts, Steven Quayle, Simon Jones, Jeffrey Weber. Epigenetic reprograming of immune cells through selective inhibition of HDAC6 reduces suppressive phenotypes and augments anti-tumor properties of T-cells [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 638. doi:10.1158/1538-7445.AM2017-638

Abstract A074: Selective Inhibition of HDAC6 augments T-cell central memory and enhances anti-tumor functions: Implications in TIL therapy

Histone deacetylases (HDACs) are a family of epigenetic modulators responsible for regulating gene expression. As key players in orchestrating both tumor and immune responses, HDACs have received attention for their possible role in cancer immunotherapy. While pan-HDAC inhibitors directly impact tumor growth, their broad targeting can be detrimental to the immune system. In this sense, HDAC selective inhibition becomes attractive as a strategy to minimize off-target effects. Here a novel role of targeting HDAC6 in T-cells with potential implications for melanoma immunotherapy is explored. Adoptive T-cell therapy is an effective treatment for metastatic melanoma, reaching objective clinical responses in half of patients undergoing tumor infiltration lymphocyte (TIL) therapy. However, lack of persistence of reactive T-cells is a major reason why patients fail to sustain long-term responses to treatment. The presence of T-cell memory populations is associated with prolonged responses to TIL. Herein we have shown that HDAC6-selective inhibition by ricolinostat increased the central memory phenotype of human CD4 and CD8 T-cells (p Citation Format: Andressa Sodre Laino, David M. Woods, Amod Sarnaik, Jeffrey Weber, Eduardo M. Sotomayor. Selective Inhibition of HDAC6 augments T-cell central memory and enhances anti-tumor functions: Implications in TIL therapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A074.

Abstract 4090: Inhibition of class I histone deacetylases promotes robust and durable enhancement of PDL1 expression in melanoma: Rationale for combination therapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Histone deacetylase inhibitors (HDACi) have shown remarkable anti-tumor activity, leading to FDA approval of two HDACi for the treatment of CTCL and several others currently at various stages of clinical development for the treatment of both solid and hematological malignancies. Previous work from our lab has shown that treatment with HDACi results in increased expression of pro-inflammatory promoting surface markers on melanoma cells, promoting enhanced T-cell activation. Recent clinical trial data has shown that blockade of the PD1/PDL1 interaction is effective in the treatment of melanoma, renal cell and non-small cell lung cancer. Importantly, responses to PD1 blocking antibodies were preferentially seen in patients with tumors expressing PDL1. Here we report that HDACi targeting class I HDACs, but not class II, augments expression of PDL1 in melanoma cells. Two murine and five human melanoma cell lines were treated for up to 72 hours with DMSO, LBH589 (pan-HDACi), MS275 (class I inhibitor), MGCD0103 (class I inhibitor), an HDAC6 specific inhibitor, or a class IIa inhibitor. Using flow cytometry, dose dependent, increases in PDL1 expression were found in the LBH589, MS275 and MGCD0103 treated groups, but not in those receiving HDAC6i or class IIa inhibitor, relative to DMSO. Increased expression was noted as early as 24 hours after treatment and peaked at 72 to 96 hours post-treatment. As IFN-γ is known to upregulate the expression of PDL1 in both normal and transformed cells, we evaluated whether these results were associated with induction of IFN-γ expression by the melanoma cells. However, no detectable levels of IFN-γ were seen in either non-treated, class I HDACi, or class II HDACi-treated cells. Melanoma cells treated with HDACi in addition to IFN-γ have enhanced expression of PDL1 relative to either treatment alone. To further gain insight into the specific HDAC regulating the expression of PDL1, preliminary experiments utilizing knockdowns (KD) of individual class I HDACs were performed. In all KD melanoma cells no increase in PDL1 expression was seen, suggesting that the increased expression of PDL1 is dependent on inhibition of multiple class I HDACs. Supporting this conclusion, treatment of class I HDAC-KDs with HDACi recapitulates the increased PDL1 expression seen with WT melanoma. Finally, in preliminary in vivo experiments combining treatment of melanoma bearing mice with anti-PDL1 antibodies, mice receiving the combination treatment had a survival advantage over those receiving PDL1 blocking antibodies or HDACi alone. These results provide a strong rationale for the evaluation of combination therapies utilizing PDL1 or PD1 blocking antibodies in combination with HDACi. Furthermore, these results support the need for further development and investigation of iso-specific HDACi in order to obtain more directed therapeutic efficacy. Citation Format: David M. Woods, Andressa L. Sodre, Eva Sahakian, John Powers, Maritza Lienlaf-Moreno, Patricio Perez-Villarroel, Alejandro Villagra, Javier Pinilla-Ibarz, Eduardo Sotomayor. Inhibition of class I histone deacetylases promotes robust and durable enhancement of PDL1 expression in melanoma: Rationale for combination therapy. [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 4090. doi:10.1158/1538-7445.AM2014-4090