Inhibition of Histone Deacetylase 6 Effects on Interaction of Tumor and Immune Cells In Multiple Myeloma
Authors: Gorgun Gullu , Hideshima Teru , Mimura Naoya , Cirstea Diana , Santo Loredana , Hu Yiguo , Fabre Claire , Raje Noopur , Munshi Nikhil C. , Richardson Paul G , Anderson Kenneth C. 
Published: October 2019

Abstract
Abstract 2996
Histone deacetylase 6 (HDAC6), a cytoplasmic enzyme, is a member of Class IIb HDAC family and catalyses the deacetylation of histones. Acetylation of histones induces chromatin relaxation leading gene transcription; conversely, deacetylation of histones induces chromatin condensation and silencing of gene transcription. HDAC6 regulates important cellular functions including histone modifications, gene transcription, and viral infection, degradation of misfolded proteins, cell migration, and immune synapse formation. HDAC inhibitors have been developed over the last decade as anti-tumor therapeutic agents in solid tumors and hematologic malignancies including multiple myeloma (MM). Myeloma represents a model system for the development of novel drugs which impact tumor cells as well as accessory cells in the tumor microenvironment. In this study we evaluated the effects of selective HDAC6 inhibition on immune accessory cells using a novel HDAC6 small molecule inhibitor, WT-161 compared with a control HDAC inhibitor trichostatin-A (TsA). Peripheral blood (PBMC) or bone marrow mononuclear cells (BMNC) from healthy donors or MM patients were cultured in the absence or presence of WT-161 (0.25-2.5uM) for 6h to 7 days. We first determined whether WT-161 induces HDAC6 inhibition in normal PBMCs by western blot of acetylated (Lys40)-tubulin, a target for HDAC6 activity. WT-161 induced acetylation of tubulin in PBMCs as early as 6 hours of exposure. Immunophenotypic changes were determined by flow cytometric analysis. Immunophenotypic analysis of PBMCs cultured with WT-161 showed significantly increased expression of PD1 in all immune cell subpopulations including CD4T, CD8T, CD3/CD8/CD56+ NKT and NK cells as well as in CD14+ myeloid cells. Conversely, decreased expression of negative costimulatory molecules CTLA4 and PDL1 was also noted, with no significant change in the expression of positive costimulatory molecules CD28, ICOS and ICOSL. Additionally, culture of PBMCs with WT-161 significantly increased expression of effector/memory T cell marker CXCR3 and decreased expression of CXCR4 in CD4T, CD8T, NKT, NK effector cells as well as CD14+ myeloid cells. WT-161 also induced ICAM1 expression on all immune cell subpopulations. WT-161 effects on the proliferation of immune cells were next assessed by CFSE-flow cytometric analysis. CFSE stained PBMCs were stimulated with anti-CD3 ab and cultured with WT-161 for 7 days. While WT-161 triggered proliferation of CD4T and CD8T cells, proliferation of NK and NKT cells decreased and B cells were unaffected. Interestingly, CD4/CD25/Foxp3 coexpressing regulatory T cells were also significantly diminished in both PBMCs and BMNCs cultured with WT-161 for 4 days. These data suggest that HDAC6 may have an immune regulatory function, and that inhibition of HDAC6 induces changes in the immune effector cells in MM microenvironment. Ongoing analysis of effects of HDAC6 inhibition on immune cells in the tumor microenvironment will further define the role of HDAC6 in disease pathogenesis and suggest novel immune-based epigenetic-targeted therapies.

Disclosures:
Raje: AMGEN: Consultancy; Celgene: Consultancy; Novartis: Consultancy; astraZeneca: Research Funding. Munshi:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson&Johnson: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.