Inhibition Of Myeloid Derived Suppressor Cells (MDSC) In The Multiple Myeloma Bone Marrow Microenvironment

The interaction of myeloma (MM) cells with bone marrow accessory cells induces genomic, epigenomic and functional changes which promote tumor development, progression, cell adhesion mediated-drug resistance (CAM-DR), and immune suppression. As in other cancers, bidirectional interaction between MM cells and surrounding cells regulates tumor development on the one hand, while transforming the BM microenvironment into a tumor promoting and immune suppressive milieu on the other. Recent developments in targeted therapies have indicated that generation of the most effective therapeutic strategies requires not only targeting tumor or stroma cells, but also methods to overcome blockade of anti-tumor immune responses. In addition to lymphoid immune suppressor cells such as regulatory T cells (Treg), distinct populations of myeloid cells such as myeloid derived suppressor cells (MDSC) can effectively block anti-tumor immune responses, thereby representing an important obstacle for immunotherapy. While MDSC are rare or absent in healthy individuals, increased numbers of MDSC have been identified in tumor sites and peripheral circulation. We have recently assessed the presence, frequency and functional characteristics of MDSC in patients with newly diagnosed or relapsed MM compared to MM patients with response and healthy donors. We have identified an increased distinct MDSC population (CD11b⁺CD14^-HLA-DR^-/lowCD33⁺CD15⁺) with tumor promoting and immune suppressive activity in both PB and BM of MM patients. Moreover, we have shown that lenalidomide (Len) and bortezomib (Bort), either alone or in combination, do not target MDSC in MM microenvironment. Moreover, Bort-induced cytotoxicity against MM cells is abrogated in the presence of MDSCs. In solid tumors, MDSC can be targeted by treatment with the multi-targeted receptor tyrosine kinase inhibitor Sunitinib (Sun), which is therefore an effective combination agent with immunotherapy. We therefore assessed whether MDSC-mediated MM growth and immune suppression in the BM and PB can be targeted by Sun, alone or in combination with Len. We first analysed effect of Sun, alone or in combination with Len, on the tumor promoting role of MDSC versus antigen presenting cells (APC) in MM. APC (CD14⁺HLA-DR⁺), mMDSC (monocytic CD11b⁺CD14⁺HLA-DR^-/lowCD33⁺) and nMDSCs (neutrophilic CD11b⁺CD14^-HLA-DR^-/lowCD33⁺CD15⁺) were sorted by flow cytometry from MM-BM or PB and cultured with CFSE labeled MM cell lines (MM1.S, RPMI8226 and OPM1), in the absence or presence of Sun (0.5-3uM) and Len (1uM) alone or in combination. CFSE-flow analysis demonstrated that both mMDSC and nMDSC induced MM cell proliferation compared to MM cells alone (dividing cells 51%) or cultured with APC; and importantly, that Sun significantly inhibited MM cell proliferation even in the presence of MDSC (dividing cells 28%).Importantly, Sun combined with Len further enhanced MM cell cytotoxicity in the presence of MDSC. We further analysed effect of Sun on the BM stroma (BMSC)-induced MM cell growth/proliferation. Sun alone modestly inhibited BMSC-induced MM cell growth, and Len enhanced this effect. We next evaluated Sun effect on MDSC-mediated immune suppression in MM. APC, mMDSC, nMDSC were cultured with CFSE labeled autologous CD3 T cells stimulated with CD3/CD28 for 6 days, in the presence of Sun and Len alone or in combination. CFSE flow analysis demonstrated that Sun significantly reversed MDSC-induced suppression of immune effector cells (CD4 T cells, CD8 T cells and NKT cells). Finally, we determined the effect of Sun on MDSC-associated tumor promoting and immune suppressive cytokines. Flow cytometric intracellular cytokine profiling of MDSC in MM-BM and PB demonstrated that Sun increased IFNg expression, while decreasing TNFa and IL-6 expression in MDSC. Overall our data therefore show that MDSCs are increased in the MM microenvironment and play an important role in MM pathogenesis and immune suppression. They provide the rationale for clinical evaluation of Sunitinib to inhibit the tumor-promoting and immune-suppressive functions of MDSCs and improve patient outcome in MM.