Immunomodulatory Effects of HDACi in Combination with Checkpoint Blockade and Lenalidomide in the Immunosuppressive Multiple Myeloma Bone Marrow Microenvironment

Background: The interaction of myeloma (MM) cells with bone marrow (BM) 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 effective 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) and regulatory T cells (Treg) act as tumor promoters and suppressors of effector immune response, and therefore represent a significant barrier to current anti-tumor therapeutic strategies. We have recently shown that WT161, a novel small molecule inhibitor of HDAC6, alone or in combination with proteasome inhibitors induces MM cytotoxicity even in the bortezomib resistant MM cells. To target the MM BM immune suppressive microenvironment, we here have assessed the immunomodulatory effects of a novel triple-combination strategy using HDACi with checkpoint blockade and lenalidomide (Len) in MM BM ex vivo.

Methods: Differential effects of Class I and Class II HDACi including pan-HDAC inhibitor Trichostatin A (TsA, 5-100nM), HDAC1 selective inhibitor Romidepsin (Rom, 1.5-15nM), HDAC3 inhibitor BG45 (10-40uM), HDAC6 inhibitor Tubastatin A HCl (TubA) and WT161 (0.5-10uM) alone or in combination with anti-PD1 (20ug/ml) and Len (0.5-1uM) were evaluated in MM-BM. HDACi direct effect on MM (MM1.S, H929, and RPMI8226) cell growth was measured by MTT. In the autologous coculture settings, BMMC from patients with newly diagnosed (ND)-MM and relapsed (R)-MM were cultured with HDACi alone or in combination with anti-PD1 and Len, with or without drug-wash-out for 1-5 days. Effector cell activation, proliferation, and cytotoxic activity was measured by flow cytometry analysis. Triple-combination effects on MDSC-mediated immune suppression was determined in cultures of autologous T cells with CD11b⁺CD14⁺HLA-DR^- mMDSC, CD11b⁺CD14⁺HLA-DR^-CD33⁺CD15⁺nMDSC, and CD11b⁺CD14⁺HLA-DR⁺APCs.

Results: Healthy PBMC and MM cell line cultures demonstrated a dose-dependent toxicity and inhibition of proliferation in both CD4 and CD8 T cells induced by Rom; in contrast, TubA, WT161, and BG45 significantly induced T cell proliferation and cytotoxic activity even after wash out of HDACi. Additionally, HDAC6 inhibitors TubA and WT161 markedly increased expression of T cell exhaustion antigens TIM3 and LAG3, and these effects were partially reversed by drug washout. Moreover, triple-combination of HDACi with PD-1 blockade and Len significantly induced CD25+CD69+ activated T and NK cell mediated cytotoxicity of CD138+ MM cells. Interestingly, in the autologous co-cultures of MM-BMMC, TubA, BG45 and Rom induced CD25+CD69+ activated T cells, and the PD-1 blockade and Len further enhanced T cell activation. However, HDAC inhibitors, with the exception of HDAC3 inhibitor BG45, expanded immunosuppressive mMDSC populations within MM-BM myeloid compartment. Specifically, WT161 induced differentiation of myeloid cells by shifting CD14^-HLA-DR^-CD33⁺CD15⁺ nMDSCs to CD14⁺HLA-DR^- mMDSCs and CD14⁺HLA-DR⁺APCs. Moreover, Rom significantly increased frequency of immunosuppressive IFNα producing plasmacytoid dendritic cells (pDCs), while decreasing Tregs, in MM-BM. Importantly, WT161 and TubA in combination with PD-1 blockade and Len reversed MDSC-mediated immunosuppressive activity, as measured by T cell proliferation. Finally, inhibition of HDAC6 induced MM cell cytotoxicity, and addition of PD-1 blockade and Len further enhanced TubA- and WT161 induced anti-MM immune responses.

Conclusion: These data suggest that distinct classes of HDACi may induce differential immunomodulatory effects in the MM BM. Inhibition of HDAC6 induces changes in immune suppressor cells leading to enhanced anti-MM immune response, and addition of PD-1 blockade with Len further enhances HDAC6 inhibition-induced effector cell mediated anti-MM immune response. Ongoing analysis of triple combination of HDAC6 inhibition with checkpoint blockade and lenalidomide in the MM BM microenvironment will further define the role of HDAC6 in disease pathogenesis, and suggest novel immune-based epigenetic and targeted combination therapies for MM.