The Immune Inhibitory Hematopoietic Adaptor Protein Hacs1(Sly2) Suppresses the Proinflammatory Macrophage Function Contributing to Multiple Myeloma Progression

Introduction: Hematopoietic Adaptor-Containing SH3 and SAM domains (Hacs1) is an immune-inhibitory regulator of B and dendritic cell function. Loss of Hacs1 results in a cytokine hypersensitive immune microenvironment and experimentally promotes tumor development. Loss of Hacs1 is also implicated in the development of multiple myeloma (MM) in the 5TMM1/C57L/KaLwRij murine model, and thus acts as a MM suppressor. However, the detailed mechanism of action of Hacs1 in regulating tumor development or growth remains unexplored. In addition to B and dendritic cells immune activation is also provided by tumor associate macrophages (TAMs). Here we describe the role of Hacs1 in the generation of TAMs which then contribute to inflammation and have a tumor promoting phenotype, including in multiple myeloma (MM). Methods: We generated Hacs1-/- mice in C57 as well as a NOD SCID genetic background, and herein characterize the effect on bone marrow-derived macrophages (BMDM) and consequent MM growth and therapeutic resistance. Results: Hacs1-/- BMDMs are hyper responsive to IL-4 stimulation and display an alternative activation M2 phenotype. In addition, CD11b+/Gr-1+/ CD31+/Siglec F myeloid progenitors as well as immature myeloid progenitors and eosinophils were significantly increased in Hacs1-/- mouse bone marrow. Hacs1-/- generated TAMs prolonged survival and enhanced the growth of MM cells in vitro through both contact-mediated and non-contact mediated mechanism, leading to up-regulation of Bcl-XL, Mcl-1 expression and increased resistance to melphalan, bortezomib and venetoclax in both mouse and human MM cell lines. Adoptive transfer of Hacs1-/- TAMs accelerated both 5TGM (mouse MM) and U266 (human MM) cell growth in vivo. Further analysis suggests that the function of Hacs1 on tumor-polarized macrophages may be mediated by modulation of NF-kB and Stat3 activation. As Hacs1-/- BMDMs encountered MM cells, activation of Hck, nuclear factor kappa B(NF-kB), PI3K-Akt, and IL6/Stat3 was observed, leading to increased production of survival and proinflammatory cytokines in Hacs1-/- BMDM M2 macrophages. Conclusion: Hacs1 appears to regulate MM development and anti-apoptotic chemotherapeutic resistance through up-regulation of myeloid cell proliferation and skewed development towards M2 macrophage differentiation. Hacs1 absence accomplishes this in part through regulation of the signal transducer NF-kB, SHP2 and Hck, leading to Stat3 activation. These findings support the role of Hacs1 as a regulator of the pro-myeloma inflammatory capabilities of tumor associated macrophages. Maintenance, or up-regulation of HACS1 expression in the bone marrow microenvironment and specifically in antigen presenting cell types (Dendritic, B and macrophage) could then potentially be considered a means of MM control. Keywords: Hacs1-/-, Multiple Myeloma, macrophages, bone marrow microenvironment, chemo resistance