Patricio Perez-Villarroel

Selective Histone Deacetylase 6 Inhibitors Bearing Substituted Urea Linkers Inhibit Melanoma Cell Growth

The incidence of malignant melanoma has dramatically increased in recent years thus requiring the need for improved therapeutic strategies. In our efforts to design selective histone deactylase inhibitors (HDACI), we discovered that the aryl urea 1 is a modestly potent yet nonselective inhibitor. Structure-activity relationship studies revealed that adding substituents to the nitrogen atom of the urea so as to generate compounds bearing a branched linker group results in increased potency and selectivity for HDAC6. Compound 5 g shows low nanomolar inhibitory potency against HDAC6 and a selectivity of ∼600-fold relative to the inhibition of HDAC1. These HDACIs were evaluated for their ability to inhibit the growth of B16 melanoma cells with the most potent and selective HDAC6I being found to decrease tumor cell growth. To the best of our knowledge, this work constitutes the first report of HDAC6-selective inhibitors that possess antiproliferative effects against melanoma cells.

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.

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.

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.

Essential role for histone deacetylase 11 (HDAC11) in neutrophil biology

Epigenetic changes in chromatin structure have been recently associated with the deregulated expression of critical genes in normal and malignant processes. HDAC11, the newest member of the HDAC family of enzymes, functions as a negative regulator of IL‐10 expression in APCs, as previously described by our lab. However, at the present time, its role in other hematopoietic cells, specifically in neutrophils, has not been fully explored. In this report, for the first time, we present a novel physiologic role for HDAC11 as a multifaceted regulator of neutrophils. Thus far, we have been able to demonstrate a lineage‐restricted overexpression of HDAC11 in neutrophils and committed neutrophil precursors (promyelocytes). Additionally, we show that HDAC11 appears to associate with the transcription machinery, possibly regulating the expression of inflammatory and migratory genes in neutrophils. Given the prevalence of neutrophils in the peripheral circulation and their central role in the first line of defense, our results highlight a unique and novel role for HDAC11. With the consideration of the emergence of new, selective HDAC11 inhibitors, we believe that our findings will have significant implications in a wide range of diseases spanning malignancies, autoimmunity, and inflammation.

Functional Analysis of Histone Deacetylase 11 (HDAC11).

The physiological role of histone deacetylase 11 (HDAC11), the newest member of the HDAC family, remained largely unknown until the discovery of its regulatory function in immune cells. Among them, the regulation of cytokine production by antigen-presenting cells and the modulation of the suppressive ability of myeloid-derived suppressor cells (MDSCs) (Sahakian et al. Mol Immunol 63: 579-585, 2015; Wang et al. J Immunol 186: 3986-3996, 2011; Villagra et al. Nat Immunol 10: 92-100, 2009). Our earlier data has demonstrated that HDAC11, by interacting at the chromatin level with the IL-10 promoter, downregulates il-10 transcription in both murine and human APCs in vitro and ex vivo models (Villagra et al. Nat Immunol 10: 92-100, 2009). However the role of HDAC11 in other cell types still remains unknown. Here we present several methods that can potentially be used to identify the functional role of HDAC11, assigning special attention to the evaluation of immunological parameters.

Expression and Function of Histone Deacetylase 10 (HDAC10) in B Cell Malignancies.

Histone deacetylase 10 (HDAC10) belongs to the class IIb HDAC family and its biological role remains mostly unidentified. A decreased HDAC10 expression has been reported in patients with aggressive solid tumors (Osada et al. Int J Cancer 112: 26-32, 2004; Jin et al. Int J Clin Exp Pathol 7: 5872-5879, 2014), suggesting that loss of HDAC10 expression might confer a survival advantage to malignant cells. Consequently, results from our lab suggests that overexpression of HDAC10 in aggressive mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) Z138c and MEC1 cells, respectively, resulted in a rapid induction of cell death in vitro with only 5 % of cells being alive at 48 h, cell cycle arrest, and up-regulation of co-stimulatory molecules. Here we present several standard methods to study the function of HDAC10 in B cell malignancies.

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

Abstract 4086: Histone deacetylase 6 (HDAC6) as a new target modulating the proliferation and immune-related pathways in melanoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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. This includes the deacetylation of multiple non-histone targets, such as proteins involved in cell cycle/apoptosis and immune regulation. Specifically, HDACs have garnered significant interest due to the availability of drugs that selectively inhibits HDACs. We recently identified that the pharmacological or genetic abrogation of a single HDAC, HDAC6, modifies the immunogenicity and proliferation of melanoma in both in vitro and in vivo models. Using specific HDAC6 inhibitors (HDAC6i) we observed decreased proliferation and G1 cell cycle arrest in all melanoma cell lines measured by MTS assay and flow cytometry. These results were also observed in stable HDAC6 knockdown melanoma cell lines (HDAC6KD) generated by specific lentiviral shRNA for HDAC6. In addition to the effects observed in proliferation and apoptosis after inhibiting HDAC6, we found important changes in the expression of immune-related pathways, including increased expression of MHC, co-stimulatory molecules, and specific melanoma tumor associated antigens such as gp100, MART-1, Tyrp1 and Tyrp2. Our in vitro results were further supported by in vivo tumor growth studies. We observed a delayed tumor growth of inoculated B16 melanoma cells in C57BL/6 mice treated with selective HDAC6i. A similar outcome was identified after inoculation of HDAC6KD B16 melanoma cells in C57BL/6 mice. Such an effect was reverted partially in CD4+ and CD8+ depleted C57BL/6 mice challenged with HDAC6KD cells, suggesting that the disruption of HDAC6 enhances immune system recognition of melanoma cells. This delay in tumor growth could be a reflection of their diminished proliferation and an increase in their immunogenicity leading to improved immune recognition and clearance. These studies provide critical insights into the molecular pathways that are involved in the regulatory role of HDAC6 in cell proliferation, survival, and cytokine signaling of human melanoma cells. Collectively, our data has identified HDAC6 as an attractive therapeutic target in melanoma. Citation Format: Patricio Perez-Villarroel, Maritza Lienlaf, Calvin Lee, Fengdong Cheng, David Woods, Kelly Barrios, Karrune Woan, Jorge Canales, Tessa Knox, Danay Marante, Hongwei Wang, Pedro Horna, Keiran Smalley, Esteban Celis, Ed Seto, Jeffrey S. Weber, Eduardo M. Sotomayor, Alejandro Villagra. Histone deacetylase 6 (HDAC6) as a new target modulating the proliferation and immune-related pathways in melanoma. [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 4086. doi:10.1158/1538-7445.AM2014-4086