Tessa Knox

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.

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.

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 4854: Evaluating HDAC6 as a causal factor in metastatic breast cancer to develop immunotherapy

Histone deacetylases (HDACs), originally described as histone modifiers, have 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 immune-biology is incompletely understood. We have found that the pharmacological or genetic abrogation of a single HDAC, i.e. HDAC6, inactivates the STAT3 pathway and modulates the expression of immuno-regulatory proteins, including the down-regulation of PD-L1, PD-L2 and B7-H4, important negative regulators of immune function, often over-expressed in cancer cells; including breast cancer. HDAC6 has been also involved in a number of structural functions related to cellular motility, shape and intracellular transport through the regulation of the acetylation of numerous targets, including tubulin and cortactin. This function is strongly suggestive of HDAC6 being a key player in metastatic cancer progression. In our initial studies we observed that the selective HDAC6 inhibitor Nexturastat A is capable of reducing the tumor growth in a highly aggressive murine mammary carcinoma that mimics human triple negative breast cancer (TNBC), under both orthotopic and subcutaneous conditions of implantation. Additionally, we observed that the size and number of secondary tumor nodules in lungs were significantly diminished after the HDAC6i treatment. In order to boost the anti-tumor T-cell response, we also tested check-point inhibitors against the tumor (such as anti PD-1 and CTLA4 antibodies). While each of the standalone treatments showed a certain degree of success in reducing tumor growth and enhancing intra-tumoral IFNγ, we demonstrated that HDAC6i improves anti-tumor immune responses when combined with immune check-point blockade. Citation Format: Debarati Banik, Melissa Hadley, Jennifer Kim, Tessa Knox, Jayakumar Nair, Alan Kozikowski, Sida Shen, Charu Vyas, Ashleyn Donohue, Eduardo Sotomayor, Alejandro Villagra. Evaluating HDAC6 as a causal factor in metastatic breast cancer to develop immunotherapy [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 4854. doi:10.1158/1538-7445.AM2017-4854

Abstract LB-294:In vivoevaluation of Ames negative HDAC6 inhibitor in melanoma model

Histone deacetylases (HDAC), originally known for their ability to modify histones, have also been found to modulate a multitude of cellular processes unrelated to chromatin modification, including the regulation of cellular pathways involved in anti-tumor immune responses. Our group and others have shown that the selective inhibition of HDAC6 impairs in vitro and in vivo tumor growth in a number of cancer models. Selective HDAC6 inhibitors (HDAC6i) such as Tubastatin A have proven to be very useful in cellular models. However, due to their mutagenic characteristics (Ames positive), usage has been limited only to preclinical research and a few types of cancer. We report that the novel and selective HDAC6i SS-2-08 is negative in the Ames assay and shows promising in vivo anti-tumor activity. SS-2-08 was obtained via five synthetic steps in 20% overall yield and 99% purity. It was incubated with two strains of Salmonella typhimurium (TA98 and TA1537) in the presence and absence of mammalian microsomal enzymes (S9 mix) for Ames assessment. Murine SM1 melanoma cells were treated with SS-2-08 in vitro to evaluate the expression of HDAC6-target genes by qRT-PCR, immunoblot and flow cytometry. CellTox Green Cytotoxicity® and HDAC-Glo I/II® assays were used to evaluate viability and HDAC activity, respectively. Cellular pathway analysis was performed by Cignal™ Reporter. In vivo studies were performed with SM1 cells injected into C57/BL/6 mice. Tumors were grown until they reached 5-8 mm in diameter and then treated with vehicle control, 25 mg/kg, and 50 mg/kg of SS-2-08 three times a week. Tumor measurements were recorded twice a week. At the experimental endpoint, tissue samples were collected for analysis. SS-2-08 shows nanomolar level potency against HDAC6 and over 400-fold selectivity over HDAC1. In the Ames assay, no significant number of reverting colonies was found for either strain, thus supporting the lack of mutagenicity of SS-2-08. Preliminary in vitro work revealed that SS-2-08 has low cytotoxicity and selective HDAC inhibitory properties, an observation verified in multiple cancer cell types. The selectivity and potency of this novel compound was similar to or better than other conventional HDAC6i as demonstrated by comprehensive molecular profiling including previously characterized HDAC6 targets such as IL-10, STAT3 and PD-L1. Additionally, in vivo experiments using murine SM1 melanoma tumors showed an important reduction in tumor growth at a dose of 25 mg/kg. Acetylated tubulin was increased in the end-point tumor samples in the treatment group, indicating that SS-2-08 reached the tumor site in vivo. Finally, we observed a down regulation of PD-L1 and PD-L2 in tumor samples from the treated group previously reported for other HDAC6i. These results provide evidence for the effectiveness of this new Ames negative-HDAC6i in vivo. SS-2-08 and derivatives have potential for use as therapeutic agents to treat cancer and other diseases where the inhibition of HDAC6 could be used as a therapeutic option. Citation Format: Melissa Hadley, Sida Shen, Debarati Banik, Jennifer Kim, Jayakumar Nair, Tessa Knox, Vincent Gallub, Shannon Kirkland, Katherine B. Chiappinelli, Eduardo Sotomayor, Alan Kozikowski, Alejandro Villagra. In vivo evaluation of Ames negative HDAC6 inhibitor in melanoma model [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 LB-294. doi:10.1158/1538-7445.AM2017-LB-294

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

Abstract 4089: Histone deacetylase 6 (HDAC6) as a regulator of PD-L1 expression through STAT3 modulation in melanoma

In spite of the progress made in the understanding of the cell biology, genetics and immunology of melanoma, the outcome for patients with advanced-stage disease has remained poor with a median survival ranging from 2-16 months. Some optimism was recently provided in metastatic melanoma by the improved clinical outcomes observed in patients receiving PD-L1 blocking antibodies. A better understanding of the environmental, genetic and epigenetic factors limiting the efficacy of melanoma immunotherapy will provide appropriate partner(s) for combination with Ipilimumab or PD1/PDL1 antibodies. Among the epigenetic factors, we have found that one member of the histone deacetylase family, HDAC6, plays a critical role not only in the regulation of survival/apoptosis of melanoma cells but also in limiting their immunogenicity and recognition by immune effector cells. Particularly, we found a major role of HDAC6 as a modulator of the immunosuppresive STAT3/IL-10 pathway and down-regulation of tolerogenic PD-L1 molecules in melanoma cells. By analyzing HDAC6 knock-down melanoma cell lines (HDAC6KD) we demonstrated the inactivation of the STAT3 pathway and the subsequent down-regulation of its target genes, including the expression of PD-L1. We also observed that the PD-L1 expression and phosphorylation of STAT3 was decreased in melanoma isolated from xenograph tumor growth models after in vivo treatment with specific HDAC6 inhibitors Fortunately, there are multiple HDAC6-selective inhibitors available to mechanistically study the role of HDAC6 on these processes and provide a viable therapeutic avenue, which may minimize undesirable side effects that are characteristic of pan-HDACi such as SAHA. By building on our understanding of HDAC6 and applying these findings to novel experimental design, we hope to identify innovative therapeutic options to benefit cancer patients. Citation Format: Maritza Lienlaf, Patricio Perez-Villarroel, Calvin Lee, Fengdong Cheng, Jorge Canales, Tessa Knox, Danay Marante, Amod Sarnaik, Pedro Horna, Ed. Seto, Keiran Smalley, Jeffrey S. Weber, Eduardo M. Sotomayor, Alejandro Villagra. Histone deacetylase 6 (HDAC6) as a regulator of PD-L1 expression through STAT3 modulation 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 4089. doi:10.1158/1538-7445.AM2014-4089