Monocytes polarize into pro-inflammatory macrophage-1 (M1) or alternative macrophage-2 (M2) states with distinct phenotypes and physiological functions. M2 cells promote tumour growth and metastasis whereas M1 macrophages show anti-tumour effects. We found that M2 cells were increased whereas M1 cells were decreased in bone marrow (BM) from multiple myeloma (MM) patients with progressive disease (PD) compared to those in complete remission (CR). Gene expression of Tribbles homolog 1 (TRIB1) protein kinase, an inducer of M2 polarization, was increased in BM from MM patients with PD compared to those in CR. Ruxolitinib (RUX) is an inhibitor of the Janus kinase family of protein tyrosine kinases (JAKs) and is effective for treating patients with myeloproliferative disorders. RUX markedly reduces both M2 polarization and TRIB1 gene expression in MM both in vitro and in vivo in human MM xenografts in severe combined immunodeficient mice. RUX also downregulates the expression of CXCL12, CXCR4, MUC1, and CD44 in MM cells and monocytes co-cultured with MM tumour cells; overexpression of these genes is associated with resistance of MM cells to the immunomodulatory agent lenalidomide. These results provide the rationale for evaluation of JAK inhibitors, including MM BM in combination with lenalidomide, for the treatment of MM patients.

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TNF receptor-associated factor 6 (TRAF6) has been implicated in polyubiquitin-mediated IL1R/TLR signaling through activation of kinase (IKK) to regulate the NF-κB and JNK signaling pathways. Here, TRAF6 protein was determined to be overexpressed in bone marrow mononuclear cells (BMMC) from patients with multiple myeloma. expression in BMMCs from patients with progressive disease is significantly elevated as compared with individuals in complete remission, with monoclonal gammopathy of undetermined significance, or healthy subjects. Furthermore, TRAF6 dominant-negative (TRAF6dn) peptides were constructed which specifically reduced TRAF6 signaling and activation of IKK. TRAF6 not only reduced cellular growth but also increased the apoptosis of multiple myeloma tumor cells in a concentration-dependent fashion. Because TRAF6 activates IKK through polyubiquitination, independent of its proteasome activity, a TRAF6dn peptide was combined with the proteasome inhibitors bortezomib or carfilzomib to treat multiple myeloma. Importantly, targeting of TRAF6 in the presence of proteasome inhibition enhanced anti-multiple myeloma effects and also decreased TLR/TRAF6/NF-κB-related signaling. Finally, TRAF6dn dose dependently inhibited osteoclast cell formation from CD14 monocytes, induced with and, and markedly reduced bone resorption in dentin pits. In all, these data demonstrate that blocking TRAF6 signaling has anti-multiple myeloma effects and reduces bone loss. The ability to target TRAF6 signaling and associated pathways in multiple myeloma suggests a promising new therapeutic approach. .

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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. Disclosures Chen: OncoTracker: Equity Ownership. Li:OncoTracker: Equity Ownership. Sanchez:OncoTracker: Equity Ownership. Wang:OncoTracker: Equity Ownership. Berenson:Janssen: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria; Daiichi: Speakers Bureau; Takeda: Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau.