Alejandro Villagra

Histone deacetylases and the immunological network: implications in cancer and inflammation

The initiation, magnitude and duration of an immune response against antigens are a tightly regulated process involving a dynamic, orchestrated balance of pro- and anti-inflammatory pathways in immune cells. Such a delicate balance is critical for allowing efficient immune response against foreign antigens while preventing autoimmune attack against self-antigens. In recent years, much effort has been devoted to understanding immune evasion by cancer cells. Also, significant advances have been made in mechanistically understanding the role of pro- and anti-inflammatory cytokines in the regulation of immune responses against antigens, including those expressed by tumors. However, we still know very little about the regulation of inflammatory/anti-inflammatory genes in their natural setting, the chromatin substrate. Several mechanisms have been identified to influence chromatin flexibility and allow dynamic changes in gene expression. Among those, chromatin modifications induced by acetylation and deacetylation of histone tails have gained wide attention. In this study, we discuss the role of histone deacetylases in the transcriptional regulation of genes involved in the inflammatory response and how these enzymes coordinate the dynamic expression of these genes during an immune response. This emerging knowledge is opening new avenues to better understand epigenetic regulation of inflammatory responses and providing new molecular targets for either amplifying or ameliorating immune responses.

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.

Essential role of HDAC6 in the regulation of PD‐L1 in melanoma

Histone deacetylases (HDACs), originally described as histone modifiers, have more recently been demonstrated to target a variety of other proteins unrelated to the chromatin environment. In this context, our present work demonstrates that the pharmacological or genetic abrogation of HDAC6 in primary melanoma samples and cell lines, down‐regulates the expression of PD‐L1, an important co‐stimulatory molecule expressed in cancer cells, which activates the inhibitory regulatory pathway PD‐1 in T‐cells. Our data suggests that this novel mechanism of PD‐L1 regulation is mainly mediated by the influence of HDAC6 over the recruitment and activation of STAT3. Additionally, we observed that selective HDAC6 inhibitors impairs tumor growth and reduce the in vivo expression of several inhibitory check‐point molecules and other regulatory pathways involved in immunosurveillance. Most importantly, these results provide a key pre‐clinical rationale and justification to further study isotype selective HDAC6 inhibitors as potential immuno‐modulatory agents in cancer.

Tet-mediated DNA demethylation is required for SWI/SNF-dependent chromatin remodeling and histone-modifying activities that trigger expression of the Sp7 osteoblast master gene during mesenchymal lineage commitment

ABSTRACT Here we assess histone modification, chromatin remodeling, and DNA methylation processes that coordinately control the expression of the bone master transcription factor Sp7 (osterix) during mesenchymal lineage commitment in mammalian cells. We find that Sp7 gene silencing is mediated by DNA methyltransferase1/3 (DNMT1/3)-, histone deacetylase 1/2/4 (HDAC1/2/4)-, Setdb1/Suv39h1-, and Ezh1/2-containing complexes. In contrast, Sp7 gene activation involves changes in histone modifications, accompanied by decreased nucleosome enrichment and DNA demethylation mediated by SWI/SNF- and Tet1/Tet2-containing complexes, respectively. Inhibition of DNA methylation triggers changes in the histone modification profile and chromatin-remodeling events leading to Sp7 gene expression. Tet1/Tet2 silencing prevents Sp7 expression during osteoblast differentiation as it impairs DNA demethylation and alters the recruitment of histone methylase (COMPASS)-, histone demethylase (Jmjd2a/Jmjd3)-, and SWI/SNF-containing complexes to the Sp7 promoter. The dissection of these interconnected epigenetic mechanisms that govern Sp7 gene activation reveals a hierarchical process where regulatory components mediating DNA demethylation play a leading role.

Synthesis and Pharmacological Evaluation of Selective Histone Deacetylase 6 Inhibitors in Melanoma Models

Only a handful of therapies offer significant improvement in the overall survival in cases of melanoma, a cancer whose incidence has continued to rise in the past 30 years. In our effort to identify potent and isoform-selective histone deacetylase (HDAC) inhibitors as a therapeutic approach to melanoma, a series of new HDAC6 inhibitors based on the nexturastat A scaffold were prepared. The new analogues 4d, 4e, and 7b bearing added hydrophilic substituents, so as to establish additional hydrogen bonding on the rim of the HDAC6 catalytic pocket, exhibit improved potency against HDAC6 and retain selectivity over HDAC1. Compound 4d exhibits antiproliferative effects on several types of melanoma and lymphoma cells. Further studies indicates that 4d selectively increases acetylated tubulin levels in vitro and elicits an immune response through down-regulating cytokine IL-10. A preliminary in vivo efficacy study indicates that 4d possesses improved capability to inhibit melanoma tumor growth and that this effect is based on the regulation of inflammatory and immune responses.

DNA Methylation and Histone Modifications in Autoimmunity

Abstract The immune system is a complex network that is designed to optimally communicate between its components while constantly monitoring potential external and internal insults. The regulation of the network involves intrinsic and extrinsic signals, which modulate gene expression in different cell types. These processes require selective mechanisms to control the expression of cell type-specific genes. Among them, epigenetic modifiers are key components in both normal and pathophysiological immune-system conditions. This chapter provides an overview of epigenetic mechanisms as modulators of the immune system in the context of autoimmune conditions. Although several epigenetic mechanisms have been reported, this chapter will specifically focus on DNA methyltransferases (DNMTs) and histone deacetylases (HDACs). An introduction to epigenetics, epigenetic modifiers, and the dependence of the immune system on epigenetic regulation is initially provided. The discussion then expands to the framework of autoimmune conditions, ranging from local to systemic phenomena. Further diseases with significant clinical relevance are chosen for detailed study, and in each case the immunological component that is influenced by epigenetics, with a focus towards the use of DNMT and HDAC inhibitors is discussed. The ongoing efforts that are utilizing various epigenetic approaches, both in preclinical and clinical settings, are highlighted throughout the text.

Phase I trial of histone deacetylase inhibitor panobinostat in addition to glucocorticoids for primary therapy of acute graft-versus-host disease

Glucocorticoids for primary therapy of acute GVHD have limited responses. A phase I/II trial tested 4 weeks of deacetylase inhibitor panobinostat started within 48 h of glucocorticoids (1 mg/kg/day prednisone or equivalent) as primary treatment for patients with either classic acute GVHD (n = 16) or acute GVHD overlapping with chronic (n = 6). Four patients received 2.5 mg/m2 IV three times a week (TIW). Subsequent to discontinuation of IV panobinostat, patients received oral doses (PO). Two patients treated with 10 mg TIW (PO level 1) had progressive GVHD, after which patients were treated with 5 mg TIW (PO level −1; n = 16); 31/41 adverse events were possibly related, including thrombocytopenia (n = 13), leukopenia (n = 7), hypercholesterolemia (n = 3), hypertriglyceridemia (n = 5), anemia (n = 1), fatigue (n = 1), and hepatobiliary disorder (n = 1). GVHD responses were complete (n = 12) or partial (n = 3), with 1 progression at PO level −1. T-regulatory cells increased at day 8, CD4/CD8 and monocytes exhibited enhanced H3 acetylation, and CD4 or CD8 numbers remained unchanged with a decreased interleukin 12p40 plasma level. Panobinostat in combination with prednisone is safe and warrants further testing in GVHD.

Abstract 4976: Ricolinostat, a selective HDAC6 inhibitor with immunomodulatory properties, has significant antimelanoma activity in vitro and in vivo

Melanoma is a less common but the most aggressive type of skin cancer and the rates of melanoma have been rising for the last 30 years. Immunotherapy may represent a new treatment paradigm to benefit melanoma patients. Previous studies have identified HDAC6 playing a central role in the regulating melanoma immunogenicity. We have shown that genetic knockdown (KD) of HDAC6 in murine and human melanoma cells resulted in an increased the expression of MHCI and costimulatory molecules as well as melanoma associated antigens. In vivo studies demonstrated that the growth of melanoma cells lacking HDAC6 was significantly delayed as compared to wild-type cells. This growth was further delayed when HDAC6KD melanoma bearing mice were treated with anti-PD-1 antibodies. Recently, we have shown that in vitro treatment of murine melanoma cells with Ricolinostat decreases PD-L1 expression and increases the expression of acetyl tubulin. Surprisingly, in vivo treatment of B16 melanoma bearing animals with increasing concentrations of ricolinostat resulted in a dose-dependent inhibition of melanoma growth (p In summary, our results have identified HDAC6 as a novel target for melanoma immunotherapy and point out ricolinostat as an attractive agent to add to the immuno-oncology armamentarium. Citation Format: Fengdong Cheng, Sodre Andressa, Jie Chen, Alejandro Villagra, David Woods, Jeffrey Weber, Steven Quayle, Jones Simon, Eduardo Sotomayor. Ricolinostat, a selective HDAC6 inhibitor with immunomodulatory properties, has significant antimelanoma activity in vitro and in vivo [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 4976. doi:10.1158/1538-7445.AM2017-4976

Abstract 4055: Enhancing anti-PD-1 immune blockade in melanoma through selective inhibition of histone deacetylase 6

Histone deacetylases (HDACs), were originally described in a limited context as histone modifiers. New evidence has demonstrated that HDACs are also involved in a diverse range of cellular processes that are not restricted to the chromatin environment, such as the regulation of the cell cycle/apoptosis and, more recently, a modulator of immune response. However, much remains unknown about the mechanism of action of HDACs and their roles in the immune-biology of cancer. The non-specific nature of pan-HDAC inhibitors results in a narrow therapeutic window of use, limiting the dose and duration due to toxicity. Our group has focused in one specific HDAC, HDAC6, and shown that both the genetic abrogation and pharmacological inhibition of this HDAC modulates the expression of a variety of immune-regulatory proteins in the tumor microenvironment, including PD-L1, PD-L2, MHC class I, B7-H4 and TRAIL-R1. In particular, we have previously demonstrated that both pharmacological inhibition and/or genetic abrogation of HDAC6 plays a critical role in the immune check point blockade by down-regulating the expression of PD-L1 and other check-point modulators such as PD-L2, B7-H4, etc. Moreover, we have also observed that in vivo inhibition of HDAC6 reduces tumor growth in B16 and SM1 murine melanoma models within syngeneic immunocompetent hosts. Additionally, we have found that the combination of low doses of the HDAC6i Nexturastat A and checkpoint immune blockade, including anti-PD-1 and anti-CTLA4, results in an important improvement in anti-tumor immune responses as evidenced by the reduction of tumor growth when compared to treatment with individual stand-alone agents. In these studies we also evidenced an increased production of IFNγ and IL-2 in the stand-alone check-point inhibitor treatments, which leads to an upregulation of PD-L1 and PD-L2. Similar levels of IFNγ and IL-2 were found in the combination groups. However, the expression level of PD-L1 and PD-L2 were comparable to the non-treated group. Taking all together, we have found that HDAC6i could be used as a potential adjuvant in ongoing therapeutic options involving immune check-point blockade. Citation Format: Tessa Knox, Eva Sahakian, Jayakumar Nair, Jennifer Kim, Debarati Banik, Melissa Hadley, John Powers, Fengdong Cheng, Sida Shen, Javier Pinilla, Jeffrey Weber, Alan Kozikowski, Eduardo Sotomayor, Alejandro Villagra. Enhancing anti-PD-1 immune blockade in melanoma through selective inhibition of histone deacetylase 6 [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 4055. doi:10.1158/1538-7445.AM2017-4055

Abstract 2331: HDAC6, new role as master regulator of PD-L1 and immune-related pathways

Histone deacetylases (HDACs), originally described as histone modifiers, have more recently been demonstrated to modify a variety of other proteins involved in diverse cellular processes unrelated to the chromatin environment, including the modulation of proteins related to cell cycle/apoptosis and immune regulation. In contrast to the well-documented effects of HDAC inhibitors (HDACi) in the control of cell cycle and apoptosis, their role in immunobiology is still not completely understood, and the reported immunological outcomes when using HDACi are heterogeneous. Our group recently reported that the pharmacological or genetic abrogation of a single HDAC, HDAC6, modulates the expression of immuno-regulatory proteins, including PD-L1, PD-L2, MHC class I, B7-H4 and TRAIL-R1. We primarily focused in PD-L1, which is an important negative regulator of T-cell function and often over-expressed in cancer cells. In a mechanistic point of view, we have found that the pharmacological inhibition and genetic abrogation of HDAC6 inactivates the STAT3 pathway, impairs the nuclear translocation and the recruitment of STAT3 to the PD-L1 promoter and subsequently down-regulates the expression of PD-L1. Moreover, the in vivo abrogation of HDAC6 reduces tumor growth in melanoma models, effect that is enhanced in the presence of the immune check-point blocking antibodies anti-PD-1 and anti-CTLA4. These results provide a key pre-clinical rationale and justification to further study isotype selective HDAC6 inhibitors as potential immunomodulatory agents in cancer. Citation Format: Tessa Knox, Maritza Lienlaf, Patricio Perez, Mibel Pabon, Calvin Lee, Fengdong Cheng, Eva Sahakian, John Powers, Susan Deng, Smalley Keiran, Alan Kozikowski, Javier Pinilla, Amod Sarnaik, Ed Seto, Jeffrey Weber, Eduardo Sotomayor, Alejandro Villagra. HDAC6, new role as master regulator of PD-L1 and immune-related pathways. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2331.