Title: Hacs1 is a myeloma immunosuppressor driving pro-tumorigenic myelopoiesis and myeloid cell differentiation through MS4A3/IL3/GM-CSF signaling

Abstract Introduction: The bone marrow (BM) microenvironment supports survival of the malignant multiple myeloma (MM) cells and is composed of cellular components that foster MM development and progression by suppression of immune responses. Despite major progresses in understanding the MM biology and pathophysiology, it is still incurable. Myeloid cells are well-described to suppress antitumor immunity (Barry et al., 2023; De Sanctis et al., 2023). However, the molecular mechanisms to achieve immunosuppressive myeloid cell states are unknown. Cytokine IL-4 is the primary driver of tumor infiltrating monocyte-derived macrophage (MO-MAC) phenotype as reported by LaMarche that deletion of IL-4Rα alone within early myeloid progenitors in BM reduced tumor burden; while deletion in downstream mature myeloid cells had no effect (LaMarche et al., 2024). Mechanistically, IL-4 derived from BM basophils and eosinophils acted on granulocyte-monocyte progenitors (GMPs) to transcriptionally program the development of immunosuppressive tumor-promoting myeloid cell.Hematopoietic Adaptor-Containing SH3 and SAM domains (HACS1) maps to a region on chromosome 21 that is frequently disrupted in hematopoietic malignancies, suggesting its involvement in these diseases (Claudio et al., 2001). Previously, we showed that Hacs1 is involved in MM development through changes of its microenvironment. It regulates IL4 signaling in B cell, T cell, macrophages and dendritic cells and affects their function (Wang et al., 2010). Hacs1-/- bone marrow derived macrophages (BMDMs) are hyper-responsive to IL-4 stimulation and display an alternative activation M2 phenotype. We found CD11b+/Gr-1+/ CD31+/Siglec F myeloid progenitors as well as immature myeloid progenitors and eosinophils were significantly increased in Hacs1-/- mouse bone marrow. Method: In the current study, we investigated the role of Hacs1 in MM immunosuppressive microenvironment utilizing Hacs1-/-mouse BM-myeloid derived suppressor cells (MDSCs) as well as MM tumor-bearing mice to study MM progression and therapeutic resistance. Results: Our results showed Hacs1-/-mouse bone marrow in MM tumor-bearing mice has upregulated PD-L1 expression in MDSCs, suppressing T cell function. Overexpression of Hacs1 or stimulation Hacs1 reduces MDSC accumulation, resulting in elevated T cell activity and reduced tumor load. Our data suggest that Hacs1 plays a key role in regulation of the immunosuppressive microenvironment in MM. Furthermore, myeloid cells from Hacs1-/-mouse BM have increased expression of MS4A3 gene, a key gene regulating cell differentiation to monocytic/granulocytic-myeloid-derived suppressor cells (M-MDSCs and G-MDSCs, respectively) (Liu et al., 2019). At the mechanistic level, MS4A3 enhances IL-3 and GM-CSF signaling in Hacs1-/-BM CD34+ cells, compared to Hacs1+/+cells.Conclusion: These data suggest that Hacs1 negatively regulate IL-4 signalling axis in bone marrow and drives pro-tumorigenic myelopoiesis and myeloid cell differentiation through MS4A3/IL3/GM-CSF signaling. Hacs1 is a key player in cellular signaling within the immune system with roles in immune regulation, cancer development, and potential serves as a MM therapeutic target. Keywords: Hacs1-/-, Multiple Myeloma, myeloid derived suppressor cells (MDSCs), bone marrow microenvironment, chemo resistance