ALBERT

Description: Abstract 520 Histones play a critical role in transcriptional regulation, cell cycle progression and developmental events. Histone acetylation/deacetylation alters chromatin structure and affects transcription factor access to DNA. Histone deacetylase inhibitors (HDI) induce growth arrest, cellular differentiation, and apoptosis and are being pursued as anticancer drugs. Interestingly, in addition to their antitumor properties, HDI have also been shown to modulate inflammatory responses. Recently, we have found that HDAC6 is required for the production of the immunosuppressive cytokine IL-10 by APCs. Given the role of this cytokine in T-cell tolerance induction we asked whether inhibition of HDAC6 with Tubastatin A, a potent and selective HDAC6 inhibitor would influence the inflammatory status of APCs and their ability to determine activation versus tolerance of antigen-specific CD4+ T cells in vitro. First, treatment of peritoneal elicited macrophages (PEM) with increasing concentration of Tubastatin A resulted in enhanced tubulin acetylation which was accompanied by enhanced expression of co-stimulatory molecules in treated cells. In addition, Tubastatin-A treated PEM were unable to produce IL-10 and TNF-a in response to LPS stimulation. In sharp contrast, Tubastatin-A treated PEM produce higher level of the pro-inflammatory cytokines IL-12 and IL-6. Next, we evaluated the ability of Tubastatin A-treated APCs to present cognate antigen to naïve and tolerant CD4+ T-cells specific for a MHC class II restricted epitope of influenza hemagglutinin (HA). We found that treatment of PEM with Tubastatin A significantly enhanced their antigen-presenting capabilities leading to effective priming of naïve CD4+ T-cells confirmed by their increased production of IL-2 and IFN-g in response to cognate antigen. More importantly, Tubastatin-A treated APCs were able to restore the responsiveness of tolerant CD4+ T cells isolated from lymphoma bearing mice. Taken together, selective HDAC6 inhibition with Tubastatin A provides a novel therapeutic approach to induce inflammatory APCs and overcome the significant barrier that T-cell tolerance has imposed to effective lymphoma immunotherapy. Disclosures: No relevant conflicts of interest to declare.

Description: 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.

Description: Abstract 4728 HDAC11 is the newest member of the HDAC family. The physiological role of this HDAC was mainly unknown until the discovery by our group that HDAC11 regulates IL-10 gene expression in immune cells in-vitro1. To better elucidate the role of HDAC11 in lineage differentiation and hematopoiesis, we have utilized an HDAC11 promoter-driven eGFP reporter transgenic mice (TgHDAC11-eGFP) which allow us to “visualize” dynamic changes in HDAC11 gene expression/transcriptional activity in immune cell compartments in vivo. Thus far, our data indicates that in hematopoietic stem cells (CD34+/Lin−), transcriptional activation of HDAC11, indicated by eGFP expression appears to be absent. Also, no eGFP expression is seen in the common lymphoid progenitors (CLP-CD34+/CD127+/CD117low/Lin−) and/or the common myeloid progenitors (CMP-CD34+/CD127−/CD117high/Lin−). In the T-cell compartment, transcriptional activation of HDAC11 increases from CD4−/CD8− T-cells to CD4+/CD8+ T-cells to single positive CD4+ and CD8+ T-cells. The expression of eGFP then decreases from naive to effector memory, but then increases again at terminal effector memory. Expression of eGFP, in the bone marrow moderately increase transitioning from Pro-B-cells (CD3−/CD200+/CD19low/CD43high), Pre-B-cells (CD3−/CD200+/CD19int/CD43int), and Immature (CD3−/CD200+/CD19high/CD43low) respectively. Interestingly eGFP expression doubles in the B-1 (CD3−/CD19+/CD200low/−) stage of differentiation in the periphery. Remarkably, eGFP expression appears to be at its highest in the plasma cell compartment of the bone marrow. A second murine model also available to us, HDAC11 knockout mice (HDAC11KO) were also utilize to confirm these findings. When compared to wild-type mice, HDAC11KO mice have increased B-1 B-cells and decreased plasma cells. In the myeloid compartment, using TgHDAC11-eGFP mice, expression of HDAC11 transcript in myeloblasts (CD34+/CD45dim/CD117+/Lin-) appears to be absent. However the expression increases to 50% in the promyelocytes (Side Scatter high/CD45dim/+/CD34−/CD117+) and to 98% in the granulocytes specifically Neutrophils (Side scatter high/CD45dim+/CD34−/CD117−/CD14−/Ly6Gbright+). Strikingly, monocytes (dendritic cells and macrophages) showed no expression of eGFP. Taken together, HDAC11 appears to be essential for proper B-cells and T-cell differentiation. It also seems to play a critical role in differentiation of granulocytes and monocytes. Therefore it is plausible that HDAC11 might function as a regulator of hematopoietic differentiation and expansion in vivo. A better understanding of this previously unknown role of HDAC11 in hematopoiesis might lead to targeted epigenetic therapies in hematological malignancies to influence the appropriate differentiation of these cells, and possibly augmenting the efficacy of immunotherapeutic approaches against malignancies. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1363 The role of HDACs in cell biology, initially limited to their effects upon histones, encompasses now more complex regulatory functions that are dependent on their tissue expression, cellular compartment distribution and the stage of cellular differentiation. Not surprisingly, HDACs have been shown to play important roles in normal B-cell biology and, aberrant expression of these proteins has been found in some B-cell malignancies1. However, the role of specific HDACs in regulation of pro-survival and cell-cycling genes in MCL and CLL still remains poorly understood. We therefore evaluated by RT-PCR the mRNA expression of specific HDACs in MCL and CLL cell lines and in primary cells from patients with these B-cell malignancies. Our analysis revealed a unique and opposing expression of HDAC10 and HDAC11 in these malignant B-cells. While HDAC11 over-expression was frequently found in MCL and CLL cells, in particular in patients with aggressive disease, an almost complete abrogation of HDAC10 was observed in malignant B-cells as compared to normal B-cell controls. These findings led us to explore the biological consequences of manipulating HDAC11 and HDAC10 in MCL and CLL cells. First, knocking-down HDAC11 (HDAC11KD) using lentiviral shRNA resulted in downregulation of cyclin D1, Cdkn1a (p21) and bcl-2. Furthermore, HDAC11KD MCL or CLL cells displayed a slower cell proliferation relative to non-target shRNA control cells. Cell cycle analysis revealed that HDAC11KD clones are arrested in G1. Conversely, over-expression of HDAC11 in the MCL cell line Z138c or in the CLL cell line MEC1 resulted in enhanced cell survival and increased proliferative capacity. Mechanistically, we have recently found that HDAC11 over-expression is associated with increased phosphorylation of STAT3, a known survival pathway in malignant B-cells. Second, HDAC10 belongs to the class II HDAC family and its biological functions remain largely unknown. Similar to our results in aggressive MCL and CLL, a decreased HDAC10 expression has been reported in patients with aggressive solid tumors2, suggesting that loss of HDAC10 expression might confer a survival advantage to malignant cells. Indeed, over-expression of HDAC10 in Z138c and MEC1 cells resulted in a rapid induction of cell death in vitro with only 5% of cells being alive at 48 hours. Our results highlight the need for a better understanding of the expression/function of specific HDACs in MCL and CLL biology. The findings of opposing roles for HDAC11 and HDAC10 in influencing cell survival and proliferation might explain the limited efficacy of pan-HDAC inhibitors (with their indiscriminate inhibition of multiple HDACs) in these B-cell malignancies, and provide support for the development of isotype-selective inhibitors targeting HDAC11. Disclosures: Chen-Kiang: Pfizer, Inc.: Research Funding.

Description: Key Points T cells from HDAC11KO mice have increased effector functions and mediate more rapid and potent GVHD. HDAC11 associates with the Eomes and Tbet gene promoter regions in resting cells and disassociates upon activation.

Description: Abstract 519 We have previously shown that the pan-HDAC inhibitors LAQ824 and LBH589 inhibit IL-10 production in APCs, rendering these cells more inflammatory and capable of effectively priming naïve antigen-specific CD4+ T-cells and restoring the responsiveness of tolerant T-cells1. These findings led us to explore which HDAC(s) might be involved in the regulation of IL-10 gene transcription and be the putative target(s) of HDI-mediated IL-10 inhibition. To answer these questions we subjected the macrophage cell line RAW264.7 to shRNA screening using specific shRNAs to knockdown each known HDAC. We found that among all the HDACs, knocking down HDAC6 (HDAC6KD) was associated with a significant decrease in IL-10 mRNA and protein in response to LPS stimulation. Furthermore, HDAC6KD clones display an enhanced expression of the co-stimulatory molecule B7.2. Functionally, HDAC6KD cells were better activators of anti-HA (hemagglutinin-influenza) transgenic CD4+ T cells, leading to significantly enhanced production of IL-2 and IFN-g in response to cognate antigen. More importantly, anti-HA CD4+ anergic T cells isolated from animals bearing HA-expressing A20 B-cell lymphoma regained their ability to produce IL-2 and IFN-g when cultured in vitro with HDAC6KD cells. These results have been confirmed in APCs isolated from HDAC6 knock-out mice and in wild type APCs treated in vitro with isotype-selective HDAC6 inhibitors. Given that HDACs do not bind to DNA and they need to interact with transcription factors to regulate gene expression, we investigate next which transcription factor(s) HDAC6 might be associated with, to regulate IL-10 transcriptional activity. One likely candidate was Stat3, a well-known transcriptional activator of IL-10 gene expression that we have previously shown to play a central role in tolerance induction by APCs2. By co-immunoprecipitation studies we found that HDAC6 indeed interacts physically with Stat3. Of note, knocking down HDAC6 in APCs resulted in absence of Stat3 phosphorylation and decreased recruitment of Stat3 to the IL-10 gene promoter which might explain the inability of HDAC6KD cells to produce IL-10. The additional findings that IL-10 production by HDAC6KD cells was restored when these cells were transfected with a constitutively active mutant version of Stat3 (Stat3c) provides additional support for the important role of HDAC6 upon Stat3 activation. Further confirmation for a concerted regulatory mechanism involving HDAC6 and Stat3 in IL-10 gene regulation was provided by studies using CPA-7, a specific Stat3 inhibitor that disrupts Stat3 recruitment and binding to gene promoters. As expected, a complete abrogation of Stat3 recruitment to the IL-10 gene promoter was observed in CPA-7 treated APCs. Interestingly, such an effect was accompanied by a parallel decrease in HDAC6 recruitment to the IL-10 promoter and inhibition of IL-10 gene transcriptional activity. Taken together, we have shown for the first time that HDAC6 interacts physically with Stat3 and is required for its phosphorylation. Since Stat3 phosphorylation is absolutely necessary for activation of Stat3 target genes, HDAC6 inhibition is an enticing molecular approach to disrupt the Stat3/IL-10 axis and overcome tolerogenic mechanisms in APCs. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 359FN2 T-cells are an essential component of immune mediated tumor rejection. Adoptive transfer of T-cells has resulted in durable antitumor responses in some patients with hematologic malignancies. Further improvement in the efficacy of this treatment modality will require a better understanding of the regulatory checkpoint(s) limiting T-cell expansion and/or reactivity in vivo. Our group has been especially interested in the epigenetic regulatory mechanisms affecting T-cells, particularly those involving histone deacetylases (HDACs). HDACs are enzymes recruited to gene promoters where they regulate transcription through histone modifications. The role of HDACs in cell biology, initially limited to their effects upon histones, now encompasses more complex regulatory functions that are dependent on their tissue expression, subcellular compartment distribution and the stage of cellular differentiation. For instance, HDAC11, the most recently discovered member of the HDAC family, has been found to be predominantly expressed in the brain, kidney, testis as well as in T-cells. Recently, HDAC11 knock out (KO) mice have been generated by targeted deletion of floxed exon 3 of the HDAC11 gene. Although analysis of the T-cell compartment in these mice revealed no significant differences in the absolute number and/or percent of T-cells in lymphoid organs as compared to wildtype mice, striking functional differences were observed in HDAC11KO T cells. First, in response to in vitro stimulation with anti-CD3 plus anti-CD28 antibodies, HDAC11KO T-cells display an enhanced proliferation, produce significantly higher levels of the pro-inflammatory cytokines IL-2, IFN-gamma and TNF-alpha and are less susceptible to Treg-mediated suppression. Studies performed in HDAC11KO T-cells expressing a transgenic receptor (TCR) specific for ovalbumin (OTII;HDAC11KO mice) confirm that these T-cells are hyperreactive in an antigen-specific manner. In vivo, HDAC11KO T cells induced significantly more severe graft-versus-host disease (GVHD) than wildtype T-cells after allogeneic bone marrow transplantation. Enhanced GVHD mediated by HDAC11KO T cells was associated with increased levels of T-cell expansion and secreted Th1-cytokines such as IFN-gamma. Further demonstration of the intrinsic potency of HDAC11KO T-cells was provided by the finding that when the number of adoptively transferred T-cells was titrated down such that wildtype T-cells no longer induced GVHD, T-cells from HDAC11KO still potently did so. HDAC11KO T-cells are also endowed with a stronger antitumor effect given the prolonged survival observed in mice challenged with C1498 leukemic cells. Mechanistically, using ChIP analysis, we have found that HDAC11 is recruited to the T-bet promoter, which may explain, at least in part, the Th1 phenotype displayed by T-cells in the absence of the repressive effect of HDAC11. Taken together, we have unveiled for the first time that HDAC11 is a regulatory checkpoint in T-cells and represents a novel molecular target to improve the efficacy of T-cell adoptive immunotherapy for the treatment of hematologic malignancies. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2439 HDAC11 is the newest member of the HDAC family. The physiological role of this HDAC was largely unknown until the discovery by our group that HDAC11 regulates IL-10 gene expression in myeloid cells in-vitro1. To better elucidate the role of HDAC11 in these cells, we have utilized an HDAC11 promoter-driven eGFP reporter transgenic mice (TgHDAC11-eGFP) which allow us to “visualize” dynamic changes in HDAC11 gene expression /transcriptional activity in immune cells in vivo. Immature myeloid cells (IMCs) differentiate into dendritic cells, macrophages, and neutrophils and are also considered to be precursors of MDSCs in tumor-bearing hosts. Here, we show for the first time that HDAC11 plays an important role in this process. First, IMCs from the bone marrow and spleen of TgHDAC11-eGFP mice display high expression of eGFP indicative of HDAC11 transcriptional activation in these cells in the steady state. Subcutaneous injection of PANCO2 tumor cells into these mice resulted in expansion of MDSCs (identified by the expression of CD11b+/GR1+ [Ly6G and Ly6C] with variable expression of CD49d and CD115) in their lymphoid organs which was similar in magnitude to the expansion observed in tumor-bearing wild type (WT) mice. Of note, flow cytometric analysis revealed that expression of eGFP was significantly decreased in the myeloid compartment of tumor bearing TgHDAC11-eGFP mice, suggesting that the transition of IMC into MDSCs might require a decrease in HDAC11 expression. Reminiscent of our findings in the eGFP mice, studies in non-transgenic mice also demonstrated that tumor derived CD11b+ Ly6G+ MDSCs display less HDAC11 mRNA expression. Additional support for the regulatory role of HDAC11 in MDSC expansion/function has been recently provided by our studies in HDAC11KO mice, demonstrating the acquisition of a suppressive cell phenotype, by myeloid cells identical to MDSCs, in the steady state and in the absence of tumor challenge. Taken together, HDAC11 might function as a negative regulator of MDSC expansion/function in vivo. A better understanding of this previously unknown role of HDAC11 in MDSC biology might lead to targeted epigenetic therapies to influence the suppressive abilities of these cells and augment the efficacy of immunotherapeutic approaches against hematologic malignancies. 1. Villagra A, et al. Nat Immunol. 2009 Jan;10(1):92-100 Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 840 T-cells are an essential component of immune mediated tumor rejection. Adoptive transfer of T-cells results in a durable anti-tumor response in some patients with hematological malignancies. To further improve the efficacy of T-cell adoptive transfers, a better understanding of the regulatory checkpoints of these cells is needed. Here we show that HDAC11 is a negative regulator of CD8+ T-cell function, thus representing a potential target in adoptive immunotherapy. HDACs are a group of enzymes initially known for their role in deacetylating histones, thereby condensing chromatin structure and repressing gene expression. The known roles of HDACs as epigenetic regulators have recently expanded to include more complex regulatory functions including interactions with non-histone targets. HDAC11 is the most recently identified member of the HDAC family, and is highly expressed in brain, testis and T-cells. Recently, our group reported HDAC11 as a regulator of IL-10 production in antigen presenting cells. To determine the role of HDAC11 in T-cell biology, T-cells from HDAC11 knock out (HDAC11KO) mice were compared to wild-type T-cells in number, function and phenotype. HDAC11KO T-cells had no differences in absolute number or percentages of CD4+ or CD8+ lymphocytes. However CD8+ T-cells were hyper-proliferative upon CD3/CD28 stimulation and produced significantly higher levels of the pro-inflammatory, Tc1 cytokines IL-2, INF-γ, and TNF-α. However, no significant increases in the production of the Tc2 cytokines IL-4, IL-6 or IL-10 were seen. Further investigation of phenotypic differences also revealed that HDAC11KO mice have a larger percentage of central memory CD8+ T-cells. Additionally, HDAC11KO CD8+ T-cells express higher levels of the transcription factor Eomes, a known contributor to central memory cell formation as well as a controller of granzyme B and perforin production in CD8+ T-cells. This Tc1 and central memory-like phenotype translated to delayed tumor progression and survival in vivo in C1498 AML bearing mice treated with adoptively transferred HDAC11KO T-cells, as compared with wild type T-cells. Collectively, we have demonstrated HDAC11 as a negative regulator of CD8+ T-cell function, and a novel potential target to augment the efficacy of adoptive T-cell tumor immunotherapy. Disclosures: No relevant conflicts of interest to declare.