Ruxolitinib Reverses Checkpoint Inhibition By Downregulating PD-L1 and PD-L2 Expression on Both Tumor and Stromal Cells in Multiple Myeloma

Introduction: Multiple myeloma (MM) tumor cells evade host immunity through the interaction of PD-L1 and PD-L2 to PD-1 on T-cells. This creates an immunosuppressive milieu in the bone marrow (BM) microenvironment. The immune inhibitory proteins PD-L1 and PD-L2 are highly expressed in MM BM. Moreover, increased expression of these proteins are associated with resistance to treatment in MM. Ruxolitinib (RUX) is a JAK1/2 inhibitor that is effective for the treatment of myeloproliferative diseases. In this study, we examined PD-L1 and PD-L2 gene and protein expression in the BM of MM patients with progressive disease (PD) or in complete remission (CR). We further investigated the effects of RUX on expression of PD-L1 and PD-L2 in MMBM, and the effect of RUX in combination with anti-MM agents in vitro and in vivo.

Material and Methods: BM mononuclear cells (MCs) and serum were collected from MM patients and healthy subjects after obtaining IRB approval. Single-cell suspensions were prepared from human MM LAGκ-1A xenografts which had been grown in the mice. The cells were cultured and treated with or without RUX and then were determined by qPCR, flow cytometric analysis, ELISA, and western blot.

Results and Discussion: The results from qPCR and flow cytometric assays showed that PD-L1 and PD-L2 gene expression was markedly increased in BMMCs from MM patients with PD compared with patients in CR or with healthy controls. We further investigated the effects of RUX on PD-L1 and PD-L2 expression in primary and stromal cells from MM patients' BM samples in vitro. RUX treatment markedly reduced PD-L1and PD-L2 gene and protein expression in the MM tumor cells cultured alone or co-cultured with stromal cells in a concentration dependent pattern. We then determined whether RUX can augment the anti-MM effects of T-cells in vitro. RUX (0, 0.1, 0.5, 1, and 5 µM) increased MM cell apoptosis in the presence of IL-2 stimulated T-cells in a concentration dependent fashion, to a similar degree to anti-PD-1 (0, 0.5, 1, 5, and 10 µg/ml) or anti-PD-L1 (0, 0.5, 1, 5, and 10 µg/ml) antibody treatment. Moreover, the combination of RUX with anti-PD-1 or anti-PD-L1 antibody increased T-cell-induced MM cell apoptosis more than the agents alone. To evaluate the efficacy of drugs in vivo, severe combined immune deficient mice implanted with the human MM xenograft LAGκ-2 were treated with RUX (30mg/kg). The results showed PD-L1 expression in the xenograft was significantly decreased in RUX-treated mice compared with the untreated control group. In contrast, RUX had no effect on PD-1 expression on T-cells.

Conclusion: The PD-L1/PD-1 pathway delivers inhibitory signals that regulate both peripheral and central tolerance, and inhibit anti-tumor immune-mediated responses. This study demonstrated that the JAK inhibitor RUX downregulated PD-L1 and PD-L2 expression in both MM tumor and stromal cells. We also demonstrated that RUX alone increased T-cell-induced apoptosis of MM cells; and, moreover, the combination of RUX with anti-PD-1 and anti-PD-L1 further increased apoptosis. The results suggest that JAK inhibitors may be effective for treating MM patients through their ability to reduce expression of checkpoint proteins involved in the development of immune resistance. Thus, JAK inhibitors should help overcome the immune resistance generated by these proteins for patients with this B-cell malignancy.