Ruxolitinib Enhances Anti-MM Function of Cytotoxic T-Cells By Upregulating IFNγ and Gzmb

Abstract

Introduction: Multiple myeloma (MM) tumor cells evade host immunity through the interaction of checkpoint proteins on immune cells which creates an immunosuppressive milieu in the bone marrow (BM) microenvironment. The JAK/STAT signaling pathway in MM plays a pivotal role in mediating the effects of many different molecules, including interleukins, interferons (IFNs), colony-stimulating factors, growth factors, and cytokines. Our previous study demonstrated that the JAK1/2 inhibitor ruxolitinib (RUX) reverses checkpoint inhibition by downregulating PD-L1, PD-L2, CXCL12, CXCR4, MUC1, and CD44 expression in MM cells. We have reported that RUX overcomes resistance of MM patients to lenalidomide and glucocorticosteroids in an ongoing clinical trial. In this study, we further examined IFNγ and granzyme B (GZMB) expression in MM treated with RUX alone or in combination with anti-BCMA/CD3 bispecific antibodies. IFNγ is a key moderator of cell-mediated immunity that enhances anti-tumor effects of CD8 T cells and GZMB induces cancer cell death by cytotoxic T lymphocytes and natural killer cells. Method: Two fresh bone marrow (BM) aspirates from MM patients in complete remission (CR) were collected following informed consent. BM mononuclear cells (MCs) were isolated using density gradient centrifugation with Histopaque-1077 (Sigma, St. Louis) according to a standard protocol. MM tumor cells were isolated from the human myeloma xenograft LAG- λ1 after it was established in SCID mice. LAG- λ1 MM cells were co-cultured with BMMCs and treated with or without RUX alone or in combination with anti-BCMA/CD3 bispecific antibody for 48 hours. Following incubation, supernatant was collected for ELISA, and the cells were washed three times using PBS in preparation for flow cytometric analysis. Supernatants of culture medium from each experimental group were collected, and protein levels determined according to the IFN-gamma (R&D Systems™) and Granzyme B (R&D Systems™) ELISA protocols, without dilution. Each data was the average of duplicate samples. Results: IFNγ and GZMB levels were markedly increased when LAG- λ1 MM cells were co-cultured with BMMCs without any treatment. The BMMCs maintain the normal secretory activities of IFNγ and GZMB. IFNγ and GZMB levels in the culture supernatants were increased in BM treated with RUX alone. The level of IFNγ and GZMB were further increased in BMMCs co-cultured with LAG--λ1 MM cells that were treated with RUX. We further examined BMMCs co-cultured with LAG- λ1 MM cells treated with RUX in combination with anti-BCMA/CD3 bispecific antibody. The results showed that the levels of IFNγ and GZMB in the supernatants was markedly increased. We also determined the amount of CD8+ T-cells in BMMCs co-cultured with LAG--λ1 MM cells treated with or without RUX using flow cytometric analysis. The results showed that CD8+ T-cells were increased when BMMCs co-cultured with LAG- λ1 MM cells treated with 1, 5, and 15 µM. However, CD56+ cells were only slightly increased in BMMCs co-cultured with LAG- λ1 MM cells that were treated with RUX. Summary Immune checkpoint blockade therapy has become a novel therapeutic approach to treat cancer patients. We previously showed RUX reverses checkpoint inhibition by downregulating PD-L1 in MMBM. In this study, we further demonstrated RUX upregulates IFNγ and GZMB and CD8+ T-cells in MMBM. RUX may enhance anti-MM effects of immune-based therapies including anti-BCMA/CD3 bispecific antibody treatment as shown in these preclinical studies.