Reagan, Michaela R.

Abstract Bone marrow adipocytes (BMAd) have recently been implicated in accelerating bone metastatic cancers, such as acute myelogenous leukemia and breast cancer. Importantly, bone marrow adipose tissue (BMAT) expands with aging and obesity, two key risk factors in multiple myeloma disease prevalence, suggesting that BMAds may influence and be influenced by myeloma cells in the marrow. Here, we provide evidence that reciprocal interactions and cross-regulation of myeloma cells and BMAds play a role in multiple myeloma pathogenesis and treatment response. Bone marrow biopsies from patients with multiple myeloma revealed significant loss of BMAT with myeloma cell infiltration of the marrow, whereas BMAT was restored after treatment for multiple myeloma. Myeloma cells reduced BMAT in different preclinical murine models of multiple myeloma and in vitro using myeloma cell-adipocyte cocultures. In addition, multiple myeloma cells altered adipocyte gene expression and cytokine secretory profiles, which were also associated with bioenergetic changes and induction of a senescent-like phenotype. In vivo, senescence markers were also increased in the bone marrow of tumor-burdened mice. BMAds, in turn, provided resistance to dexamethasone-induced cell-cycle arrest and apoptosis, illuminating a new possible driver of myeloma cell evolution in a drug-resistant clone. Our findings reveal that bidirectional interactions between BMAds and myeloma cells have significant implications for the pathogenesis and treatment of multiple myeloma. Targeting senescence in the BMAd or other bone marrow cells may represent a novel therapeutic approach for treatment of multiple myeloma. Significance: This study changes the foundational understanding of how cancer cells hijack the bone marrow microenvironment and demonstrates that tumor cells induce senescence and metabolic changes in adipocytes, potentially driving new therapeutic directions.

Abstract Introduction. Growing evidence suggests that immune cells that reside within the tumor microenvironment are dysregulated and functionally impaired, leading to defective anti-tumor immunity of the host. One of the major immunosuppressive mechanisms during tumor progression is expansion of regulatory immune cells. Here, we analyzed the immune cells within the bone marrow (BM) and the peripheral blood (PB) of 2 immunocompetent multiple myeloma (MM) mouse models. We next studied the role of regulatory T cells (Tregs) in MM pathogenesis. Materials and methods. To study the immune cell populations of the BM and PB, we used two immuncompetent mouse models and transplanted VK*MYC cells or 5TGM1 cells into C57BL/6 and C57BL/Kalwrij mice respectively. The immune cell populations and checkpoint receptor expressions were analyzed by CyTOF mass cytometer or flow-cytometry. Treg induction assay was performed in vitro to study the mechanism of Treg increase in the BM of myeloma injected mice. CD4+ CD25- cells were obtained from C57BL/Kalwrij mice and were co-cultured with 5TGM1 cells or B cells from C57BL/Kalwrij mice in vitro. Treg induction was compared by flow-cytometry. Transplantable VK*MYC cells were injected into "depletion of regulatory T cell" (DEREG) mice, which expresses a simian diphtheria toxin (DT) receptor-enhanced GFP fusion protein under the control of the FOXP3 gene locus, or their wild type littermates. DT injection into these mice leads to depletion of Tregs as previously described (J Exp Med. 2007; 204: 57-63). DT was given once every week for a total 3 times i.p to the DEREG mice or the littermate controls to specifically deplete Tregs and to study the role of Tregs during MM progression. Tregs (CD4+ FOXP3-GFP+ cells) were sorted from VK*MYC injected mice or non-injected DEREG mice BM using FACSAria cell sorter. Cells isolated were subjected to RNA sequencing. Gene Set Enrichment Analysis (GSEA) was performed to define differences in molecular signatures between MM-associated and normal Tregs. Results. The Treg proportion was significantly increased within the CD4+ T cells in the BM of myeloma cell injected mice from the early stage of disease compared to control mice, while in the PB, the increase was observed only at the late stages of disease progression. The effector T cell (Teff)/Treg ratio was significantly decreased in the BM at the end-stage myeloma bearing mice (P<0.01). Checkpoint related molecules (PD-1, LAG-3 and Tim-3) on Tregs and Teffs were up-regulated at the protein level in the BM of myeloma injected mice compared to control (P<0.01). These data indicate the activation of Tregs and decrease of Teff activity, leading to suppression of anti-myeloma T cell activity in the MM BM. Under in vitro co-culture conditions, 5TGM1 cells induced a significant increase in the number of Tregs from non-Treg CD4+ T cells compared to controls (P<0.01). 5TGM1 cell-induced Tregs presented with enhanced ki-67 expression, thus suggesting the ability of MM cells to induce Treg proliferation. Additionally, the trans-well co-culture experiment showed that the major mechanism for Treg induction by 5TGM1 was by direct contact with T cells rather than secreting factors. Significant increase in survival was observed in the VK*MYC injected DEREG mice under Treg depletion compared to the DEREG mice without Treg depletion and wild type littermates under DT treatment (P<0.001). The Treg depleted DEREG mice were accompanied with an increase of Teffs (P<0.01), indicating recovery of anti-myeloma T cell activity. RNA sequencing of BM Tregs from VK*MYC injected mice showed increased expression of immune checkpoint related molecules and increased Treg effector molecules (IL-10, Granzyme B) compared to BM Tregs from control mice, indicating a more functionally active phenotype of VK*MYC associated Tregs. GSEA showed an enrichment of genes involved in type-1 interferon signaling in VK*MYC associated Tregs. Conclusions. We used 2 immunocompetent MM mouse models to study the characterization and significance of Tregs in MM progression. CyTOF analysis and RNA sequencing data indicated Treg activation in the MM BM microenvironment. The Treg in vivo depletion experiment showed that Tregs have a significant role in MM progression. These data indicate that immunotherapy targeting Tregs may represent a novel therapeutic strategy for MM. Studies are ongoing to understand the roles of Tregs in human MM patients. Disclosures No relevant conflicts of interest to declare.

Abstract Background. Endothelial progenitor cells (EPCs) are circulating precursors with the capacity to differentiate into endothelial lineage cells through a process known as “vasculogenesis” thus contributing to vessel formation. We studied the role of endothelial progenitor cells in multiple myeloma (MM) pathogenesis. Methods. EPC levels were evaluated in peripheral blood (PB) of patients with smoldering MM (SMM), and active myeloma (MM) and in PB of healthy controls, by using flow-cytometry (CD34+VEGFR2+ cells), and by performing endothelial colony forming assays. EPC levels were studied in PB from transgenic Vk*MYC mice harboring either early MM (smoldering-like stage); or late (active MM-like stage); as well as in mice injected intravenously with either murine MM 5TGM1-turboRFP+ cells or human MM1S-GFP+/luc+ cells. Healthy syngeneic mice were used as controls. GFP-bone marrow (BM) transplantation and sub-cutaneous femur implantation were performed in recipient mice to study EPC BM mobilization (GFP+CD34+VEGFR2 cells) and EPC BM recruitment to the implanted femurs during 5TGM1 MM model progression. Transgenic ID1+/-ID3-/- mice (with a defect in EPC mobilization and differentiation ability) were injected with transplantable Vk*MYC cells and followed for survival. Wild type littermates were used as controls. Similar experiments were performed using transgenic ID1+/-ID3-/- mice transplanted with BM of wild type littermates. Therapeutic activity of DC101 anti-murine VEGFR2 Ab was evaluated in the MM1S human orthotopic xenograft model, using both early and late treatment approaches. Results. EPC levels were significantly increased in PB of MM patients, including SMM, compared to healthy controls (P<0.05). PB mononuclear cells (PBMCs) from SMM and aMM patients showed an increase ability to form both EC-CFUs (P<0.01) and ECFCs compared to those from healthy controls (P<0.05). EPC levels were significantly increased in PB of transgenic Vk*MYC mice at both early and advanced stage of MM development compared to healthy syngenic mice. In orthotopic MM models we found that levels of circulating EPCs increase early after i.v. injection of either human MM1S-GFP+/luc+ cells (1.5 and 2 times higher after 1 week and 2 weeks, respectively) or murine 5TGM1-turboRFP+ MM cells (2.5 and 3 times higher after 2 and 3 weeks, respectively) compared to naïve mice. By coupling GFP-BM transplantation and subcutaneous femur implantation, we generated a new model that allows the study of BM derived-cell trafficking from one site to another of the BM. The levels of BM-derived GFP+ EPC increased early after i.v. injection of 5TGM1-turboRFP+ cells (2 times increase after 2 weeks, P<0.01); also, the percentage of both total GFP+ cells (7.92% versus 2.635%, P<0.05) and GFP+ EPCs (0.22% versus 0.068%, P<0.05) increased in the BM of implanted femurs of tumor cell injected mice compared to controls. Survival of ID1+/-ID3-/- mice was significantly increased compared to that of wild type littermates after injection of MM Vk*MYC cells (62 versus 27 days median survival time, P=0.0005); this survival advantage was lost after wild type BM was transplanted in ID1+/-ID3-/- mice (34 versus 35 days median survival time). In human MM1S orthotopic xenograft model, treatment with anti-murine VEGFR2 DC101 Ab was able to inhibit MM progression when treatment was started early during smoldering like stages, but not when it was started after significant tumor burden (50.5, 41.5 and 40 days median survival time in DC101 early, late and untargeted IgG treated mice respectively) Conclusion. These results show that EPC mediated vasculogenesis is an important pathogenic process that occurs during the early/smoldering MM stage. Knockout mouse experiments confirm that EPCs are essential for MM tumor progression. These results indicate that anti-angiogenic agents could be effective as an early therapeutic intervention in SMM patients. Disclosures Ghobrial: Millennium/Takeda: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.