CCL2 Promotes Macrophages-Associated Chemoresistance Via MCPIP1 Dual Catalytic Activities in Multiple Myeloma

Background: Despite with the introduction of novel chemotherapy agents to multiple myeloma (MM), chemoresistance remains the major problem in clinical management. The interaction of MM cells with different cell components in the tumor microenvironment is important for tumor growth and chemoresistance, in which macrophages (Mφs) represent the prominent components. Mφs, who provide a favorable microenvironment for MM cells, protect them from drug-induced apoptosis. Mφs have great plasticity and can differentiate into different functional states according to microenvironmental signals. We previously found that the chemokine CCL2 is overexpressed in MM patients and CCL2 promoted Mφs'infiltration in the MM-BM microenvironment. The present study aims to further investigate the clinical significance of CCL2 and elucidate the mechanism how it participates in Mφs-mediated MM chemoresistance.

Methods: Clinical significance of CCL2 were investigated by immunohistochemistry and Elisa. Flow cytometry, quantitative real-time PCR and Western blotting were conducted to evaluated the apoptosis of MM cells and Mφs' polarization. RNA sequencing was performed to determine the key molecular induced by CCL2 in Mφs. Genetically modified cells (e.g., exhibiting siRNA knockdown, generating deletion mutants through lentiviruses) were employed to evaluate the functional significance of MCPIP1. Proteome Profiler Human phosphor-kinase antibody array was used to determine the changes of protein phosphorylation in Mφs caused by CCL2. MM xenograft models were used to evaluated the role of CCL2 and MCPIP1 in vivo.

Results: We found that CCL2 expression is tightly associated with MM patients' treatment status. When newly diagnosed patients received 4 courses of PCD combined therapy, CCL2 expression in their BM significantly decreased. MM cell lines and PBMCs barely expressed CCL2, while Mφs highly expressed CCL2. Mφs interacted with MM cells further upregulated their expression of CCL2. These increased CCL2 in the MM' BM microenvironment had little effect on MM cells' proliferation and their drug responses, but CCL2-treated Mφs were more effective at protecting MM cells from bortezomib- and melphalan- induced apoptosis. CCL2 could also effectively polarize Mφs toward M2-like Mφs, which possess a stronger ability to protect MM cells from chemotherapy drug-induced apoptosis. RNA sequencing revealed that CCL2 significantly induced the immunosuppressive molecular MCP-1-induced protein (MCPIP1) in Mφs and the result was verified both in mRNA and protein level. MM cells cocultured with MCPIP1-knock down Mφs were more sensitive to drug-induced apoptosis than those cocultured with control Mφs. Mφs transfected with D141N mutant MCPIP1 lost their protective effects because the mutation resulted in impaired RNase and DUB activities of MCPIP1. Mφs isolated from NSG mice' tumor masses showed MCPIP1-knock down Mφs displayed a more M1-like phenotype and in vitro experiments showed MCPIP1-knock down Mφs were more difficult to be polarized toward M2-like Mφs by MM cells. CCL2 significantly activated JAK2/STAT3 pathway in Mφs and inhibition of STAT3 hindered CCL2-induced MCPIP1 protein expression.

Conclusion: Our study showed that increased CCL2 in MM' BM microenvironment polarizes Mφs towards M2-like phenotype and promotes Mφs to protect MM cells from chemotherapy drug-induced apoptosis. Mechanistically, CCL2 activated JAK2/STAT3 pathway to induce the expression of MCPIP1. MCPIP1, the critical negative regulator of inflammation, mediated Mφs 'polarization and protection effect via its dual catalytic activities.

Key words: Multiple Myeloma, Macrophages-Associated Chemoresistance, Polarization, CCL2, MCPIP1