Immunomodulatory Effects of Histone Deacetylase 6 Inhibition in Suppressor Immune Cells in Multiple Myeloma
Authors: Gorgun Gullu , Hideshima Teru , Raje Noopur S. , Mimura Naoya , Bradner James E. , Cirstea Diana , Santo Loredana , Tai Yu-Tzu , Sellitto Michael , Hu Yiguo , Fabre Claire , Ohguchi Hiroto , Minami Jiro , Richardson Paul G. , Munshi Nikhil C. , Anderson Kenneth C. 
Published: October 2019

Abstract
Abstract 128
The interaction of myeloma (MM) cells with bone marrow accessory cells and/or the extracellular matrix induces genomic, epigenomic and functional changes which promote tumor development, progression, cell adhesion mediated-drug resistance (CAM-DR), and immune suppression. To develop the most efficient anti-MM treatment strategy and prevent tumor escape from immune recognition, both enhancing anti-MM effector immune response and overcoming MM-induced immune suppression is essential. Suppressive immune cells including myeloid derived suppressor cells (MDSC), regulatory T cells (Treg) and IL-17 secreting Th (Th17) cells act as tumor promoters and suppressors of effector immune response, and therefore represent a significant barrier to current anti-tumor therapeutic strategies. Since, we and others have reported increased numbers of Treg and Th17 cells in MM, we here assessed MDSCs in both peripheral blood (PBMC) and bone marrow (BMMC) of patients with MM compared to healthy donors. Phenotypic analysis by flow cytometry showed a significant increase in CD14−CD11b+HLA-DRlowCD15+ MDSCs in both PBMC and BMMC from MM patients compared to healthy donors (p<0.01). Furthermore, coculture of MM cell lines with healthy PBMCs for 6 days demonstrated that MM cells significantly induce MDSC differentiation in healthy PBMCs (p<0.03). Recent studies have demonstrated that histone deacytlase 6 (HDAC6) is an important regulator of monocyte/macrophage-mediated immune response. We therefore next analysed the immunomodulatory effects of WT-161, a novel small molecule inhibitor of HDAC6, alone or in combination with lenalidomide (len) and bortezomib (bort), on suppressive immune cells in the MMBM microenvironment. To keep cell-cell interaction intact reflective of the MMBM microenvironment, PBMCs or BMMCs from MM patients were cultured in the absence or presence of WT-161 (0.5–5uM), len (1–10uM), and/or bort (2–5nM), and individual cell populations were analysed by flow cytometry. Phenotypic characterization of suppressive immune cells showed a significant decrease in both CD4+CD25+Foxp3+ Treg cells and MDSCs in MM-PBMCs and MM-BMMCs cultured with WT-161, alone or in combination with len or bort (p<0.01); however, there was no change in the expression of Th17 cells. To determine the functional mechanism of immune suppression, MDSC and Treg cells were isolated by magnetic-Ab sorting and cultured for 6 days with autologous T cells (TCR/IL-2 stimulated), with or without WT-161, len and bort, alone or in combination. T cell proliferation (by 3H-thymidine assay) was significantly inhibited in the presence of MDSCs, whereas WT-161 notably reversed MDSC-mediated T cell suppression. In contrast, len and bort did not show any significant effect. Intracellular reactive oxygen species (ROS, an MDSC-derived metabolic immune inhibitory molecule) expression was significantly decreased in MDSCs from MM cultured with WT-161, alone or together with len and bort (p<0.05). Additionally, WT-161 also reversed Treg-mediated T cell suppression as well as len. Cytokine profiling by intracellular flow cytometric analysis demonstrated that WT-161 significantly decreased IL-6 and GM-CSFR expression in MDSCs, whereas it induced IFNγ and IL-12 production in effector CD4T, CD8T and NKT cells. Finally, unstimulated or IL-2 prestimulated (36h) PBMCs or NK cells were cultured with MM cell lines (MM1.S, RPMI8226), in the absence or presence of WT-161 alone or with len and bort (4h), and anti-MM cytotoxic activity was determined by Cr51-release cytotoxicity assay. While len (48% killing) and WT-161 (39% killing) induced CTL-mediated cytotoxicity, WT-161 (53% killing) and len (56% killing) induced more potent NK cell-mediated anti-MM cytotoxicity. These data suggest that HDAC6 may have an immune regulatory function, and that inhibition of HDAC6 induces changes in suppressor immune cells leading to enhanced anti-MM immune response in MM microenvironment. Ongoing analysis of the 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:
Hideshima: Acetylon: Consultancy. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding. Bradner:Acetylon: Scientific Founder. Richardson:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. 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; Onyx: Consultancy, Membership on an entity's Board of Directors or advisory committees. Anderson:Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees.