Combination of a Novel HDAC 6 Inhibitor ACY-241 with Anti-PD-L1 Antibody Enhances Anti-Tumor Immunity and Cytotoxicity in Multiple Myeloma

Introduction Histone deacetylases (HDACs) are attractive therapeutic targets, and selective HDAC inhibitors (HDACi), alone or in combination with other anti-cancer agents are useful treatment strategies in multiple myeloma (MM). HDACs are also involved in immune regulation in hematological malignancies. For example, blockade of HDACs upregulate immunecheckpoints such as PD-1 ligand (PD-L1). Furthermore, combination of HDACi with PD-1/PD-L1 checkpoint blockade significantly improves immunotherapy in a murine B16F10 model [Woods et al, Cancer Immunol Res. 2015; 3:1375-85]. Here we utilized an orally bioavalable HDAC6 selective inhibitor ACY-241 currently under clinical trials to examine whether combination of ACY-241 with anti-PD-L1 antibody enhances anti-MM immunity. For these studies, we utilized our previously published co-culture models of immune effector cells (plasmacytoid dendritic cells, pDCs, T cells, NK cells) and MM cells (Chauhan et al, Cancer Cell 2009, 16:309-323; Ray et al, Leukemia2015, 29:1441-1444). Specifically, in MM we have shown that: 1) PD-1 is highly expressed on T and NK cells, and both pDCs and MM cells express PD-L1; 2) MM-pDCs confer T- and NK cell immune suppression via PD-L1/PD-1 immune checkpoints and 3) blockade of PD-L1-PD-1 signaling axis by anti-PD-L1 Ab generates MM-specific CD8+ CTL activity, as well as enhances NK-cell-mediated MM cell cytolytic activity. We here examined whether combination of ACY-241 with anti-PD-L1 Ab increases anti-tumor immunity and cytotoxicity in MM.

Methods Minimally cytotoxic concentrations of ACY-241 (0.1-0.2 µM) or anti-PD-L1 Ab (5 µg/ml) were utilized to assess immune function. In T cell proliferation assay, MM patient pDCs were co-cultured with autologous T cells (pDC:T ratio; 1:10) in the presence of ACY-241, anti-PD-L1 Ab, or a combination of ACY-241 and anti-PD-L1 Ab for 5 days; proliferation was then quantified using CellTrace Violet proliferation kit for flow cytometry. CTL activity assays MM CD8+ T cells were cultured with autologous pDCs (1:10 pDC/T ratio) with ACY-241, anti-PD-L1 Ab, or both for 5 days; cells were then washed to remove drug, and pre-stained target MM cells (20:1 E/T ratio) were then added for another 2-3 days, followed by quantification of viable MM cells using FACS. NK-cell mediated cytotoxicity was assessed using flow-based CFSE-stained target lysis assays, as well as degranulation assay quantifying cell surface CD107a. Statistical parameters were calculated using GraphPad Prism.

Results. 1) Treatment of RPMI-8226, U266, and MM.1S cells with ACY-241 increases PD-L1 expression; and blockade of PD-L1 using anti-PD-L1 Ab increases the anti-MM activity of ACY-241. 2) Combination of ACY-241 and anti-PDL1 Ab enhances MM-patient pDC-induced NK cell-mediated cytolytic activities against allogenic MM cells (p < 0.008; 2-5 fold increase for combination versus anti-PD-L1 Ab alone). NK cell mediated lysis was insignificant in the absence of pDCs. 3) ACY-241, anti-PD-L1 Ab or their combination, activates patient BM-CD8+T cells in autologous pDCs/T cell co-cultures. Importantly, combination of ACY-241 and anti-PD-L1 Ab triggers a more robust allogenic and autologous MM-specific CD8+ CTL activity than anti-PD-L1 Ab alone (1.5-2.0 fold increase in combination regimen vs anti-PD-L1 Ab; p = 0.01). 4) Treatment of pDC-T cell cocultures with ACY-241 plus anti-PD-L1 Ab enhanced cytotoxic activity of CD8+ T cells against HLA-A2+ U266 cells (1.5-2 fold in combinations vs anti-PD-L1 Ab; p = 0.003). 5) In concert with above findings, ACY-241 plus anti-PD-L1 Ab increases CD107a expression in NK and CD8+T cells (CD107a+ NK: 5 fold; p = 0.003; CD107a+ CD8+T: 5 fold; p= 0.001; vs anti-PD-L1 Ab alone). Finally, 6) pDC-mediated CD8+ CTL activity was also confirmed using HLA-A2+ HCT116 colon cancer cells (~2 fold loss in viability in combination vs anti-PD-L1 Ab; p = 0.006)

Conclusions The combination of HDAC6 inhibitor ACY-241 and anti-PD-L1 Ab enhances pDC-induced T and NK cell-mediated cytolytic acitvities in MM. Our study provides the basis for combining ACY-241 with anti-PD-L1 Ab to restore immune function, and enhance MM cytotoxicity, and improve patient outcome in MM.