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HDAC6 Plays a Non-Canonical Role in the Regulation of Anti-Tumor Immune Responses, Dissemination, and Invasiveness of Breast Cancer

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HDAC6 Plays a Non-Canonical Role in the Regulation of Anti-Tumor Immune Responses, Dissemination, and Invasiveness of Breast Cancer Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/0008-5472.CAN-19-3738 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. HDAC6 plays a non-canonical role in the regulation of anti-tumor immune responses, dissemination, and invasiveness of breast cancer. Debarati Banik1, Satish Noonepalle1, Melissa Hadley1, Erica Palmer1, Maria Gracia-Hernandez1, Christian Zevallos-Delgado1, Namratta Manhas1, Hayk Simonyan1, Colin N. Young1, Anastas Popratiloff1, Katherine B. Chiappinelli1, Rohan Fernandes1, Eduardo M. Sotomayor1, and Alejandro Villagra1* 1 The George Washington University, Washington, DC Corresponding Author Contact Information: Alejandro Villagra, PhD Assistant Professor Cancer Biology Program GW Cancer Center Department of Biochemistry and Molecular Medicine School of Medicine and Health Sciences The George Washington University 800 22nd St NW, Suite 8880 | Washington, DC 20052 (202) 994 9547 | [email protected] Running Title: HDAC6 inhibitors modulate invasiveness of breast cancer Keywords: Histone Deacetylases, PD-L1, Breast Cancer, metastasis, Nexturastat A, Immunotherapy, E-Cadherin Disclosures: The authors have no financial or non-financial conflict of interest. 1 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/0008-5472.CAN-19-3738 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract: Despite the outstanding clinical results of immune checkpoint blockade (ICB) in melanoma and other cancers, clinical trials in breast cancer have reported low responses to these therapies. Current efforts are now focused on improving the treatment efficacy of ICB in breast cancer using new combination designs such as molecularly targeted agents, including histone deacetylase inhibitors (HDACi). These epigenetic drugs have been widely described as potent cytotoxic agents for cancer cells. In this work, we report new non-canonical regulatory properties of ultra-selective HDAC6i over the expression and function of epithelial-mesenchymal transition pathways and the invasiveness potential of breast cancer. These unexplored roles position HDAC6i as attractive options to potentiate ongoing immunotherapeutic approaches. These new functional activities of HDAC6i involved regulation of the E-cadherin/STAT3 axis. Pre-treatment of tumors with HDAC6i induced critical changes in the tumor microenvironment, resulting in improved effectiveness of ICB and preventing dissemination of cancer cells to secondary niches. Our results demonstrate for the first time that HDAC6i can both improve ICB antitumor immune responses and diminish the invasiveness of BC with minimal cytotoxic effects, thus departing from the cytotoxicity-centric paradigm previously assigned to HDACi. Statement of significance Ultra-selective HDAC6 inhibitors can reduce tumor growth and invasiveness of breast cancer by non-canonical mechanisms unrelated to the previously cytotoxic properties attributed to HDAC inhibitors. 2 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/0008-5472.CAN-19-3738 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction: Among breast cancer (BC) subtypes, triple-negative breast cancer (TNBC) is characterized by the lack of expression of estrogen receptor type (ER, progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) (1). This subtype presents a significant challenge to targeted therapy and is more likely to metastasize, versus other BC subtypes (2). Although the infiltration and composition of immune cells may vary across different BC subtypes (3), it has been shown that TNBC exhibits a high degree of stromal and tumor-infiltrating lymphocytes (TILs) (4), including immunosuppressive regulatory T cells (Tregs), myeloid- derived suppressor cells (MDSC), and tumor-associated macrophages (TAMs). In addition to altering the endogenous properties of tumor cells, reprogramming the inflammatory properties and cellular composition of the tumor microenvironment (TME) could be a viable choice to overcome resistance to immunotherapy, as seen in the TONIC clinical trial (5) and other studies comparing the TME between responders and non-responders to specific anticancer therapies (6). Within the realm of immunotherapy, reprogramming the T-cell activity by overcoming the co- inhibitory pathways with neutralizing antibody (Immune checkpoint blockade-mechanism) has taken up a prime role (7). Mechanisms underlying resistance to immune checkpoint blockade (ICB) are still not fully understood. Although several studies have highlighted acquired resistance mechanisms in T cells (8), primary and adaptive pathways in other components of the TME have not been explored in detail. Modalities regulating the epithelial-mesenchymal transition (EMT) have been proposed as promising targets to overcome ICB resistance (9). Specifically, recent studies indicate that cancer patients refractory to the established ICB treatment, such as anti- programmed cell death protein 1 (PD-1 therapy), have altered expression of genes involved in EMT and cell adhesion, including down-regulation of E-Cadherin (E-Cad) (10). Importantly, TAMs are negative regulators of E-Cad expression in cancer cells (11). However, it is not clear how decreased E-Cad leads to ICB resistance. Even less is known about whether a pharmacologic approach could restore E-Cad expression and thus sensitize tumors to PD-1 treatment. Epigenetic modulators have been explored as a potential class of molecules to overcome the ICB resistance pathways, due to their ability to affect multiple cellular processes (12). Histone deacetylases (HDACs), a family of 11 zinc-dependent iso-enzymes described initially as histone modifiers, modify various proteins involved in diverse cellular processes unrelated to the chromatin environment. The role of specific HDACs in cell proliferation and survival have been extensively studied (13). However, their involvement in the regulation of immune-related pathways is not entirely understood (14). Pan-HDAC inhibitors (HDACis) have been used as anticancer agents in clinical settings. But critical limitations, including low activity in solid tumors and cardiac toxicity (15), have limited their use in combination therapies. For example, vorinostat is a pan-HDACi that activates multiple apoptotic pathways in both cancer and normal cells, thus limiting its use for extended periods (16). The non-selective nature of current HDACis is likely a significant cause of HDACi-associated clinical toxicities. On the other hand, ricolinostat, which targets HDAC6 and class I-HDACs, showed relatively lower levels of adverse effects, well-tolerability, and increased efficacy in initial clinical trial, combined with anticancer drug lenalidomide and steroid drug dexamethasone (17). It remains underexplored whether selective HDACis could achieve equal efficacy with reduced side-effects than pan- HDACi. The findings from phase 1b trial with ricolinostat provide additional rationales to investigate HDAC-specific inhibitors featuring selectivity and lower toxicity. We used a highly specific HDAC6i, Nexturastat A (NextA), which has been used in multiple in vitro and in vivo models (18-21). NextA was screened against all 11 isozymes. Within the similar Class 1 and Class 4 isozymes, NextA displayed low micromolar activity compared to the low nanomolar activity against HDAC6 as well as high levels of selective inhibition against members of the related Class 2 HDAC isozymes, reaching >1000-fold selectivity in some cases. 3 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/0008-5472.CAN-19-3738 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. The functional specificity of NextA was confirmed by its ability to induce hyperacetylation of α- tubulin, a hallmark of HDAC6 inhibition (21). This next-generation HDAC6i has minimal cytotoxic effects, and mainly affects immune-related functions in tumor and immune cells (18). Examples include regulation of macrophage-phenotype and tumoral expression of immunosuppressive molecules, e.g., PD-L1, PD-L2, B7-H3, and B7-H4 (22). Also, HDAC6 regulates tubulin and cortactin, which are important modulators of cellular shape, motility, and cytoskeletal structure (23), making it a critical factor in metastasis. Overexpression of HDAC6 increased chemotactic cell motility, whereas selective inhibition of HDAC6 leading to tubulin-hyperacetylation, inhibited the invasion and motility of fibroblasts (24). Moreover, HDAC6 is associated with anchorage- independent growth in BC, such as patient-derived adenocarcinoma SKBR3 and hormonal receptor-positive MCF7 (25). Given the central role of HDAC6 in determining tumoral characteristics, we tested the hypothesis that pharmacological and genetic inhibition of HDAC6 confers benefits at multiple levels, such as primary and metastatic tumor growth. Our results indicated that NextA potentiates the anti-tumor effect of ICB by inducing noticeable changes within TME, including diminished infiltration of immunosuppressive
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