The JAK Inhibitor Blocks PD-L1, PD-L2 and CD44 Expression in Multiple Myeloma (MM) and Sensitizes MM Cells to Lenalidomide and Steroids

Introduction: Ruxolitinib (RUX) is an inhibitor of the Janus kinase family of protein tyrosine kinases (JAKs) that is effective for the treatment of myeloproliferative diseases. Immunomodulatory drugs including lenalidomide (LEN) and corticosteroids (dexamethasone [DEX]) have shown efficacy in the treatment of multiple myeloma (MM). We have demonstrated that the combination of the RUX, LEN and steroids shows both preclinical and clinical efficacy for the treatment of patients with relapsed and refractory MM. The immune inhibitory proteins PD-L1 and PD-L2 and CD44 are highly expressed in MM; moreover, increased expression of these proteins is associated with resistance to treatment in MM. In this study, we investigated the effects of RUX on expression of PD-L1, PD-L2 and CD44 in MM, and the effect of RUX in combination with LEN and DEX on growth of human MM in vivo.

Methods: Bone marrow mononuclear cells (BMMCs) were purified from aspirates from MM patients with progressive disease (PD) or in complete remission (CR) before and after treatment with RUX in combination with LEN and methylprednisolone (MP). The cells were stained with antibodies directed against human PD-L1, PD-L2 and CD44 and flow cytometric analysis (FCA) was performed. MM biopsies or BMMCs were analyzed using immunofluorescent analysis (IFA). Total RNA was extracted from monocytes. Quantitative PCR (qPCR) was measured for gene expression of PD-L1, PD-L2 and CD44 and the housekeeping gene HPRT1 with TaqMan technology following standard qPCR protocol. The human MM xenograft LAGκ-2 was surgically implanted into the left superficial gluteal muscle of anaesthetized naive SCID mice. LEN was administered via oral gavage daily (15 mg/kg) and DEX was administered intraperitoneally daily (1 mg/kg). RUX was given via intraperitoneal (IP) injection twice daily (1.5 mg/kg). Seven days following tumor implantation, mice were treated with vehicle alone, single agent RUX, LEN or DEX or using combination treatments.

Results: We examined PD-L1 gene expression in MM patients with PD or in CR and those with monoclonal gammopathy of undetermined significance (MGUS). The results showed that PD-L1 gene expression was markedly increased in BMMCs from MM patients with PD compared with those patients in CR or with MGUS. We determined the effects of RUX on expression of PD-L1 and CD44. The FCA data showed the percentage of PD-L1+ BMMCs was markedly reduced in MM patients treated with RUX, LEN and MP compared to those before starting treatment whereas there were decreased CD44+ cells in the MM patients after this treatment compared to those before treatment with this combination. IFA further confirmed that PD-L1+ and CD44+ BMMCs staining was markedly deceased in MM patients treated with this three drug combination compared to those without treatment. We further investigated the in vitro effects of RUX on gene expression of PD-L1, PD-L2 and CD44 in MM cell lines and primary MM cells co-cultured with THP1 monocytes. MM cells showed marked upregulation of these genes following co-culture with THP1 cells. RUX treatment markedly reduced PD-L1, PD-L2 and CD44 gene expression in the MM tumor cells co-cultured with monocytes compared with cells not treated with the JAK1/2 inhibitor. To evaluate these drugs in vivo, the human MM xenograft LAGκ-2 model was used. The mice were then treated with RUX, LEN or DEX alone, doublets or the combination of all three drugs. RUX alone produced minimal anti-MM effects whereas the doublets showed more anti-MM effects than any single agent, and the combination of all three drugs showed the most marked anti-MM effects.

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 expression of PD-L1, PD-L2 and CD44 in MM tumor cells which should help overcome the immune resistance generated by these proteins for patients with this B-cell malignancy. We also demonstrated the combination of RUX with LEN and DEX showed enhanced anti-MM efficacy in vivo. These results also 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 that frequently occurs in MM patients.