Eva Sahakian

A Novel Chemoimmunomodulating Property of Docetaxel: Suppression of Myeloid-Derived Suppressor Cells in Tumor Bearers

Purpose: Myeloid-derived suppressor cells (MDSC) accumulate in tumor-bearing hosts and are associated with immune suppression. To date, there have only been few studies that evaluate the direct effect of chemotherapeutic agents on MDSCs. Agents that inhibit MDSCs may be useful in the treatment of patients with various cancers. Experimental Design: We investigated the in vivo effects of docetaxel on immune function in 4T1-Neu mammary tumor-bearing mice to examine if a favorable immunomodulatory effect accompanies tumor suppression. Primary focus was on the differentiation status of MDSCs and their ability to modulate T-cell responses. Results: Docetaxel administration significantly inhibited tumor growth in 4T1-Neu tumor-bearing mice and considerably decreased MDSC proportion in the spleen. The treatment also selectively increased CTL responses. Docetaxel-pretreated MDSCs cocultured with OT-II splenocytes in the presence of OVA323-339 showed OT-II–specific CD4 activation and expansion in vitro. In characterizing the phenotype of MDSCs for M1 (CCR7) and M2 [mannose receptor (CD206)] markers, MDSCs from untreated tumor bearers were primarily MR+ with few CCR7+ cells. Docetaxel treatment polarized MDSCs toward an M1-like phenotype, resulting in 40% of MDSCs expressing CCR7 in vivo and in vitro, and macrophage differentiation markers such as MHC class II, CD11c, and CD86 were upregulated. Interestingly, docetaxel induced cell death selectively in MR+ MDSCs while sparing the M1-like phenotype. Finally, inhibition of signal transducer and activator of transcription 3 may in part be responsible for the observed results. Conclusions: These findings suggest potential clinical benefit for the addition of docetaxel to current immunotherapeutic protocols. Clin Cancer Res; 16(18); 4583–94. ©2010 AACR.

Modulation of antigen-presenting cells by HDAC inhibitors: implications in autoimmunity and cancer

There is a growing body of evidence to support the use of histone deacetylase inhibitors (HDACi) in the treatment of diverse conditions from autoimmunity to cancer. In this context, HDACi have been ascribed many immunomodulatory effects, assigning novel and promising roles to these compounds. This review summarizes the current observations arising from both pre‐clinical and clinical studies in these pathological conditions. However, it is left to be explained how a single agent can have both pro‐ and anti‐inflammatory effects in either physiological or pathological conditions. This question is explored in greater detail by focusing on the effects of HDACi on antigen‐presenting cells (APCs), key regulators of immune activation. In particular, HDACi modulation of molecules involved in antigen processing and presentation, as well as co‐stimulatory and adhesion molecules, and cytokines will be discussed in the context of both professional and non‐professional APCs. Professional APCs encompass classic immune cells; however, it is increasingly evident that other somatic cells, including cancer cells, are not immunologically inert and can display functions similar to professional APCs, a challenging feature that needs to be explored as a potential therapeutic target. In this way, professional and non‐professional APCs can regulate their particular micro‐environmental niche, affecting either a pro‐ or anti‐inflammatory milieu.

Histone Deacetylase Inhibitor LAQ824 Augments Inflammatory Responses in Macrophages through Transcriptional Regulation of IL-10

APCs are important in the initiation of productive Ag-specific T cell responses and the induction of T cell anergy. The inflammatory status of the APC at the time of encounter with Ag-specific T cells plays a central role in determining such divergent T cell outcomes. A better understanding of the regulation of proinflammatory and anti-inflammatory genes in its natural setting, the chromatin substrate, might provide novel insights to overcome anergic mechanisms mediated by APCs. In this study, we show for the first time, to our knowledge, that treatment of BALB/c murine macrophages with the histone deacetylase inhibitor LAQ824 induces chromatin changes at the level of the IL-10 gene promoter that lead to enhanced recruitment of the transcriptional repressors HDAC11 and PU.1. Such an effect is associated with diminished IL-10 production and induction of inflammatory cells able of priming naive Ag-specific T cells, but more importantly, capable of restoring the responsiveness of anergized Ag-specific CD4+ T cells.

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.

Induction of clusterin by AKT--role in cytoprotection against docetaxel in prostate tumor cells.

Clusterin (CLU), in its cytoplasmic form, is abundant in many advanced cancers and has been established to be cytoprotective against chemotherapeutic agents including docetaxel. However, little is known of the mechanism of its induction. Here, we provide evidence that AKT plays a critical role in upregulating cytoplasmic/secretory sCLU, which is responsible for docetaxel resistance. Western blot analysis indicated that docetaxel-resistant sublines derived from DU145 and PC3 prostate tumor cell lines displayed a markedly increased phospho-AKT level closely accompanied by heightened sCLU expression when compared with parental cells. To examine if AKT has a role in sCLU expression, AKT blockade was done by treatment with a specific inhibitor, API-2, or dominant-negative AKT transduction before analysis of sCLU gene expression. Loss of AKT function resulted in loss of sCLU and was accompanied by chemosensitization to docetaxel and increased cell death via a caspase-3–dependent pathway. To confirm that AKT affected resistance to docetaxel through sCLU and not through other mediators, tumor cells were first transfected with full-length CLU for overexpression and then treated with the AKT inhibitor API-2. We found that once sCLU was overexpressed, API-2 could not chemosensitize the tumor cells to docetaxel. Thus, the chemoresistance to docetaxel is mediated by sCLU and it can be induced by AKT. Lastly, AKT was found to mediate sCLU induction via signal transducer and activator of transcription 1 activation, which we have earlier shown to drive sCLU gene expression. These results identify a previously unrecognized pathway linking AKT to cytoprotection by sCLU in tumor cells. Mol Cancer Ther; 9(6); 1831–41. ©2010 AACR.

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.

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 inxa0vivo 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.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.

Stat3 Inhibition Augments the Immunogenicity of B-cell Lymphoma Cells, Leading to Effective Antitumor Immunity

Mantle cell lymphoma (MCL) is an aggressive and incurable subtype of B-cell non-Hodgkin lymphomas. Although patients often respond initially to first-line treatment with chemotherapy plus monoclonal antibodies, relapse and decreased response to further lines of treatment eventually occurs. Harnessing the immune system to elicit its exquisite specificity and long-lasting protection might provide sustained MCL immunity that could potentially eradicate residual malignant cells responsible for disease relapse. Here, we show that genetic or pharmacologic disruption of Stat3 in malignant B cells augments their immunogenicity leading to better activation of antigen-specific CD4(+) T cells and restoration of responsiveness of tolerized T cells. In addition, treatment of MCL-bearing mice with a specific Stat3 inhibitor resulted in decreased Stat3 phosphorylation in malignant B cells and anti-lymphoma immunity in vivo. Our findings therefore indicate that Stat3 inhibition may represent a therapeutic strategy to overcome tolerance to tumor antigens and elicit a strong immunity against MCL and other B-cell malignancies.

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 48u2009h of glucocorticoids (1u2009mg/kg/day prednisone or equivalent) as primary treatment for patients with either classic acute GVHD (nu2009=u200916) or acute GVHD overlapping with chronic (nu2009=u20096). Four patients received 2.5u2009mg/m2 IV three times a week (TIW). Subsequent to discontinuation of IV panobinostat, patients received oral doses (PO). Two patients treated with 10u2009mg TIW (PO level 1) had progressive GVHD, after which patients were treated with 5u2009mg TIW (PO level −1; nu2009=u200916); 31/41 adverse events were possibly related, including thrombocytopenia (nu2009=u200913), leukopenia (nu2009=u20097), hypercholesterolemia (nu2009=u20093), hypertriglyceridemia (nu2009=u20095), anemia (nu2009=u20091), fatigue (nu2009=u20091), and hepatobiliary disorder (nu2009=u20091). GVHD responses were complete (nu2009=u200912) or partial (nu2009=u20093), 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.