ALBERT

Beschreibung: Histone deacetylases (HDACs), originally discovered as histone modifiers are now proposed as important regulators of non-chromatin related processes, including the regulation of cellular pathways involved in the production of anti- and pro-inflammatory cytokines and the subsequent function of antigen-presenting cells (APCs). We have recently identified HDAC6 as a positive regulatory factor in the production of IL-10. However, the participation of this HDAC in other immune related cellular processes remains unknown. In this work we present evidence of the important role of HDAC6 in the modulation of the JAK/STAT pathway through the IL-6 regulation. We generated knockdown cell lines of HDAC6 (HDAC6KD) and non-target (NT) cells as a control in RAW264.7 murine macrophages using lentiviral shRNA. Two HADC6KD and two NT cell lines were treated with LPS or were left untreated and then analyzed by microarray. In HDAC6KD cells we found 1542 genes were down-regulated and 775 up-regulated in HDAC6KD cells. Their ontology distribution revealed significant changes in immune-related and apoptosis/cell cycle control genes. Importantly, we observed that most STAT3 and SP1 target genes were down regulated in HDAC6KD cells, suggesting the participation of HDAC6 in the regulation of these two transcription factors. Further analysis evidenced that the phosphorylation of STAT3 and the acetylation of Sp1 were diminished in HDAC6KD cells when compared against control cells. Chromatin immuneprecipitacion (CHIP) assays indicate that this particular effect of abrogation of HDAC6 involved histone modifications at the IL-6 promoter level, and more importantly, the recruitment of STAT3 and Sp1 to the IL-6 promoter was abrogated. Then, we analyzed the relevance of these findings by studying the tolerogenic JAK/STAT signaling pathway, which is known to be activated by IL-6 and critical in the final outcome of APCs in response to stimuli. Our observations included a complete abrogation in the phosphorylation of JAK2 and STAT3 proteins in HDAC6KD cells in response to LPS, which was reverted when these cells were treated with exogenous IL-6. Our final results demonstrate a critical role of HDAC6 in the modulation of IL-6 and the potential role of HDAC6 in the regulation of the JAK/STAT3 pathway. In addition HDAC6 is a regulator of SP1 and STAT3 target genes. These findings provide insight into the molecular mechanisms controlling the immunogenicity of APCs, supporting the use of HDAC6 inhibitors to enhance immune activation, and positioning HDAC6 as a potential therapeutic target. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 1039 Background Histone Deacetylases (HDACs) have divergent effects over the production of anti- and pro-inflammatory cytokines in Antigen Presenting Cells (APCs). We have previously shown that modulation of specific HDACs can alter the immunogenicity of APCs, either to an activating or tolerogenic phenotype. We recently identified HDAC6 to positively regulate IL-10 production. However, the participation of this HDAC in other immune related cellular processes remains unknown. In this work we are presenting evidence of the important role of HDAC6 in the regulation of IL-6 via activation of the JAK/STAT3 pathway. Methods Stable knockdown clones of HDAC6 (KDHDAC6) and Non-target (NT) cells were generated in RAW264.7 murine macrophages using lentiviral shRNA for HDAC6 or non-target (NT) respectively. Two KDHADC6 and two NT clones were treated with LPS or untreated and then analyzed by microarray using the Affymetrix GeneChip Mouse 430 2.0. Significantly down- or up-regulated genes were analyzed by their ontology distribution and selected genes were validated by quantitative real-time RT-PCR, ELISA, or immunoblots. Additionally, changes in the expression of these selected genes were tested in cells treated with selective HDAC6 inhibitors. Results 1542 genes were down-regulated and 775 up-regulated in KDHDAC6 cells. Their ontology distribution revealed significant changes in immune-related (632) and apoptosis/cell cycle control (47) genes. Importantly, IL-6 was one of the most highly down-regulated genes in KDHDAC6 cells. Therefore, we next analyzed the relevance of these findings by studying the tolerogenic JAK/STAT3 signaling pathway which is known to be activated by IL-6 and critical in the final outcome of APCs in response to stimuli. We observed a complete abrogation in the phosphorylation of JAK2 and STAT3 proteins in KDHDAC6 cells in response to LPS, which was reverted when these cells were treated with exogenous IL-6. Conclusions These results demonstrate the requirement of HDAC6 in the production of IL-6 in response to LPS, and therefore positions this deacetylase as an important regulator of the JAK/STAT3 pathway. These findings provide insight into the molecular mechanisms controlling the immunogenicity of APCs, supporting the use of HDAC6 inhibitors to enhance immune activation. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Cutaneous T-cell lymphoma (CTCL) includes a spectrum of non-Hodgkin’s T-cell lymphomas. While most patients with CTCL experience non-life threatening skin symptoms, those who do progress to later stages of the disease may develop serious complications. For patients with tumor stage or lymph node involvement there is a significant decline in treatment response & few treatment options are available. Current available therapeutic options include the use of histone deacetylase (HDAC) inhibitors as Romidepsin, Panobinostat and Vorinostat. However, their mechanism of action is still unknown. The role of histone deacetylases (HDACs) in cell biology, initially limited to their effects upon histones, now encompasses more complex regulatory functions that are dependent on their tissue expression, cellular compartment distribution, pathophysiological conditions and stage of cellular differentiation. Although major advances have been made in understanding the role of specific HDACs in cell proliferation and the survival of cancer cells, their individual participation in specific intracellular pathways is not completely understood. Here we present data demonstrating the changes in several immune-related pathways in two CTCL cell lines after exposure to both pan-HDAC inhibitors and selective HDAC6 inhibitors. We compared the pharmacological effects of the pan-HDAC inhibitor LBH589 (Panbinostat), the class I selective Romidepsin, and the selective HDAC6 inhibitors such as TubastatinA and NexturstatB, on two CTCL cell lines (HuT78, HuT102). Our findings thus far demonstrate the following: First, we observed a marked inhibition of proliferation capacity of both cell lines when treated with either pan-HDACi or the more selective HDAC6 inhibitors. However, the selective HDAC6 inhibitors showed less cytotoxicity. Second, we observed important changes in the expression of the co-inhibitory molecules, such as PD-L1. Given our results, we conclude that the selective targeting of HDAC6 could recapitulate the anti-tumor effects of pan-HDAC inhibitors without having the non-target cytotoxic effects often encountered when using pan-HDAC inhibitors. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Graft-versus-host disease (GVHD) remains the principal obstacle to successful outcomes in allogeneic hematopoietic stem cell transplant (HCT). Glucocorticoids are the current standard initial treatment for acute GVHD with variable complete responses rates (30% to 60%). New immunosuppressive strategies are required to improve survival and to decrease immunosuppressive toxicities. Vorinostast, a histone deacetylase inhibitor (HDACi), have shown efficacy for acute GVHD prevention in MRD HCT. Panobinostat is a potent inhibitor of deacetylases and HSP90 belonging to a structurally novel class of the cinnamic hydroxamic acid class and is one of most potent pan-HDACi. This protocol tested the safety and efficacy of Panobinostat (LBH589) as initial adjunct treatment for acute GVHD, administered within 72 hours of the first high dose glucocorticoid (methylprednisolone 0.8 mg/Kg/day IV or equivalent PO for 14 days and then taper per MD discretion). We have enrolled 19 subjects, median age 53 years (range, 34-76), male (n=12)/female (n=7), white(n=14)/hispanic(n=5); with diagnosis of CLL (n=2), MDS (n=2), Myeloma (n=1), Follicular NHL (n=1), CML(n=1), Myelofibrois (n=3), AML (n=5), MDS/CMML (n=3) or ALL(n=1). Conditioning regimens included Busulfan(BU)/fludarabine(FLU) AUC 5300 (n=10) or AUC 3500 (n=3), FLU/Melphalan (n=4) or Pentostatin/BU (n=2); and GVHD prophylaxis for MUD 8/8 (n=11) or MRD (n=5) HCT with TAC/MTX (n=6), TAC/rapamycin(n=7), TAC/MMF(n=3) and for mismatched transplants with either TAC/RAPA/ATG (n=2) or TAC/MTX/ATG (n=1). Median day of acute GVHD (n=16) onset was day + 37 post HCT (26 -109 days) with overall grade GVHD II (n=13) or III (n=6); and median day of acute symptoms in overlap GVHD patients (n=3) was day + 712 (528-981). All Patients were treated with voriconazole (n=15) or micafungin (n=4) for fungal prophylaxis. For the first four patients Panobinostat was administered intravenously (IV) weekly x 4 at 2.5MG/M2 (n=3) or 5MG/M2 IV (n=1) with all 4 achieving either CR (n=3) or PR (n=1) GVHD responses by day +15 of Panobinostat. Due to manufacturer discontinuation of IV formulation, the protocol was amended to use PO Panobinostat. Using 10mg PO TIW 3 doses q week x 4 weeks, we treated 2 subjects which were both discontinued from study drug due to presumed GHVD progression within 7 days of Panobinostat (after 3-4 doses). First subject had grade II GVHD (skin stage 3, gut stage 1 and liver stage 0) that progress in gut and skin; second subject with grade II GVHD (skin stage 3, gut stage 1, liver stage 1) with LFTs worsening ultimately evolving into VOD. Due to safety concerns next subjects were treated with 5 mg PO TIW 3 doses q week x 4 weeks, dose that was determined to be the maximal tolerated dose (MTD) after 6 patients completed therapy in phase I. Currently we are enrolling in phase II portion (n=7). GVHD response rate among MTD treated was complete in 85% (n=11), partial in 7.6% (n=1) or progressive in 7.6% (n=1) by day +36 after Panobinostat with majority achieving responses by day +21. Chronic GVHD at day +365 in evaluable patients (n=6) was none (n=3) or mild (n=3) and steroid was discontinued at a median of 3 months (3-6). Hematological toxicities in evaluable patients (n=13) were mild with worsening of prior thrombocytopenia (n=7/10), anemia (n=3/10) and leukopenia (n=3/10) and returned to baseline within 1-2 weeks; LFTs deterioration (n=1) within 1 week of Panobinostat in a GVHD stages 3 liver/3 skin patient; pericarditis/cardiogenic shock CTCAE 5 of unclear etiology (n=1); worsening thyroid function (n=1) and hypercholesterolemia (n=1). Preliminary correlative studies in MTD treated patients showed that CD4 and CD8 numbers remained stable during treatment. T regulatory cells numbers decreased at day +8 after Panobinostat and recovered by days +15 and +29 of treatment. Level of T regs inducing cytokines (TGFB and IL-10) increased, possibly contributing to an immune-modulatory environment. There is evidence of an increased in acetylation of histone 3 in CD4, CD8 and monocytes subsets over time. We are encouraged with tolerability of level -1 Panobinostat dose and the high GVHD response rate of 85% which may compare favorably to the historical GVHD response rate. These results suggest a potential role for Panobinostat as a tool to improve success of glucocorticoids for acute GVHD treatment. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Abstract 3724 MCL is an aggressive and incurable subtype of B-cell Non-Hodgkin's lymphomas. Although patients often respond to first-line chemotherapy plus monoclonal antibodies, relapse and decrease response to further lines of treatment eventually occurs. Manipulation of the immune system to unleash its specificity and long-lasting protective effect might provide a unique opportunity to induce more durable responses or prevent relapse in MCL. Previous studies by our group have shown that HDAC6 is required for production of the immunosuppressive cytokine, IL-10 in antigen-presenting cells (APCs) and that genetic or pharmacologic disruption of HDAC6 in these cells triggers potent antigen-specific T-cell responses. Given the role of IL-10 in suppressing the immunogenicity of B-cells, we asked therefore whether inhibition of HDAC6 with the isotype-selective inhibitor, Tubastatin-A (Tub-A) could reverse the tolerogenic properties of malignant B-cells in a murine model of MCL1. First, in vitro treatment of FC-muMCL1 cells with Tub-A resulted in increased acetylation of a-tubulin, a known HDAC6 target. Treated B-cells also displayed an enhanced expression of MHC class II and the co-stimulatory molecules B7.1, B7.2 and CD40 relative to untreated cells. Such changes resulted in more immunogenic MCL cells able to effectively activate naïve antigen-specific CD4+ T-cells and more importantly, capable of restoring the responsiveness of anergic T-cells from lymphoma-bearing mice. Second, in vivo treatment of FC-muMCL1-bearing C57BL/6 mice with Tub-A resulted in lymphoma rejection. This antitumor effect was not observed in immunodeficient (SCID) C57BL/6 mice treated with Tub-A, pointing to the immunological effects triggered by HDAC6 inhibition in B-cells as playing a dominant role in Tub-A induced anti-MCL activity. Third, mechanistically we have found that HDAC6 physically interacts with STAT3 and it is required for STAT3 phosphorylation and recruitment to the IL-10 gene promoter. Furthermore, Tub-A treated MCL cells displayed a diminished STAT3 phosphorylation and abrogation of IL-10 gene transcriptional activity. Taken together, a concerted regulatory mechanism involving HDAC6 and STAT3 influence the immunogenicity of MCL cells. Targeting HDAC6 with specific inhibitors to disrupt the tolerogenic STAT3/IL-10 axis represents a novel strategy to trigger effective anti-MCL immunity. Disclosures: Martin: Millennium Pharmaceuticals, Inc.: Speakers Bureau.

Beschreibung: Abstract 1660 Recently, we have found that HDAC6 is overexpressed in MCL cell lines and in primary human MCL cells. Knocking-down HDAC6 in MCL cells with a shRNA lentiviral system resulted in cell cycle arrest and apoptosis induction. Interestingly, MCL cells lacking HDAC6 displayed a significantly decreased STAT3 phosphorylation and abrogation of IL-10 gene transcriptional activity. ACY1215 is a novel, selective, orally bioavailable HDAC6 inhibitor. Treatment of MCL cell lines with this agent resulted in decreased cell viability and proliferation. In addition, ACY1215 inhibits IL-10 production in a dose dependent manner. Bruton tyrosine kinase (BTK) is a member of Tec family of kinases with a very distinct role in B-cell antigen receptor (BCR) signaling. The selective BTK-inhibitor PCI-32765 has shown promising pre-clinical and clinical activity in MCL. In addition to their direct anti-lymphoma effects, disruption of BTK also induces positive immunological changes such as inhibition of the immunosuppressive STAT3/IL-10 signaling pathway1. The above observations led us to determine whether the direct antitumor effects and the immunological properties of ACY1215 and PCI-32765 could be potentiated when these agents are used in combination. First, the viability of MCL cells was decreased when they were treated in vitro with either PCI-32765 or ACY1215. However, combination of these two agents resulted in a 3-fold increase in apoptosis induction, pointing to a synergistic effect of BTK and HDAC6 inhibition in MCL. The additional findings that this approach can increase the immunogenicity of MCL cells and anti-MCL immune responses has provided the proper framework for combining the selective HDAC6 inhibitor ACY1215 with BTK inhibition as a novel therapeutic strategy in MCL. Disclosures: Chen-Kiang: Bristol Myers Squibb: Consultancy; Pfizer: Research Funding. Jones:Acetylon Pharmaceuticals, Inc: Employment.

Beschreibung: Abstract 829 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 is a 131 KDa protein with preferential cytoplasmic localization where it regulates the acetylation of proteins involved in cytoskeleton, cell-cell interaction and cell migration. Emerging evidence also implicates HDAC6 in regulation of immune responses, in particular at the level of the APC/T cell immune synapse1 and in the suppressive function of regulatory T-cells2. Expanding upon these immunoregulatory properties, here we show for the first time that HDAC6 physically interacts with STAT3, a transcriptional activator of IL-10 gene expression. By co-immunoprecipitation studies and confocal studies we found that HDAC6 co-localize with STAT3 in the cytoplasm and nuclei of macrophages. Furthermore, by using several HDAC6 and STAT3 mutants we have identified that the aminoacids 503–840 of HDAC6 and the STAT3 domain comprising aminoacids 465–585 are required for this interaction. Functionally, knocking down HDAC6 in a macrophage cell line (RAW264.7-HDAC6KD) resulted in inhibition of STAT3 phosphorylation, decreased recruitment of STAT3 to the IL-10 gene promoter and abrogation of IL-10 production by these cells in response to either LPS or IL-10. Similar results were observed in dendritic cells (DCs) or macrophages isolated from HDAC6 knock-out (KO) mice. Furthermore, HDAC6KD clones or APCs from HDAC6 KO mice displayed an enhanced expression of the co-stimulatory molecule B7.2 and are better activators of antigen-specific CD4+ T-cell responses in vitro. More importantly, these APCs are able of restoring the responsiveness of anergic T-cells from lymphoma-bearing mice. Pharmacologic inhibition of HDAC6 in APCs with Tubastatin A, an isotype-selective HDAC6 inhibitor, yielded similar enhancement of APC and T-cell function in vitro. Further support for HDAC6 as an appealing target in cancer immunotherapy has been recently provided by the significant delay in tumor growth observed in either HDAC6 KO mice or in wild type mice treated with Tubastatin A. In summary, we have shown for the first time that HDAC6 interacts physically with STAT3 and such an interaction is necessary for STAT3 phosphorylation and IL-10 gene expression in APCs. Disrupting the HDAC6/STAT3/IL-10 axis in APCs with selective HDAC6 inhibitors represents a novel approach to overcome tolerogenic pathways in these cells and tip the balance towards effective antitumor immune responses. Disclosures: No relevant conflicts of interest to declare.