PROJECT SUMMARY This application is being submitted in response to the NOSI NOT-CA-21-034. The work generated from the parent grant (U01 CA225730) is complementary to the U54 funded DRSC at Mayo Clinic/University of Minnesota (U54 CA224018) and we propose to perform collaborative experiments that utilize resources from all 3 projects of this DRSC to enhance the impact of both grants. Multiple myeloma (MM) remains incurable and the mechanisms through which MM cells develop resistance to currently available combination regimens and recently developed immunotherapies remain incompletely understood. Our studies documented that bone marrow (BM) mesenchymal stromal cells (BMSCs) and other nonmalignant cells of the BM/bone play critical roles in resistance of MM, leukemia and solid tumor cells to various pharmacological agents or immune effector cells. We observed that human MM or leukemic cells in a bicalcium phosphate (BCP) scaffold-based BM-like in vivo system with “humanized” mesenchymal stromal compartment exhibit (compared with in vitro or conventional subcutaneous xenografts) distinct genomic dependencies and decreased responses to diverse therapies. Under the parent U01, we have appreciated that surface protein glycosylation plays critical roles in regulating how malignant hematopoietic cells interact with the BM niche. Moreover, we recently observed through CRISPR-based that perturbation of surface protein glycosylation regulators can contribute to tumor cell resistance against pharmacological or immune-based treatments. This current project seeks to apply our the BM-like "humanized" scaffold in vivo model; leverage our resources on regulation of surface protein glycosylation, our experience with CRISPR-based studies in vivo and the translational relevance of the immunocompetent Vk*myc genetically engineered mouse model (GEMM) of MM from the U54 DRSC to (1) examine whether MM cells with high surface levels of sLeX, a key determinant of interaction of hematopoietic cells with the BM vascular niche, exhibit more pronounced resistance to in vivo treatments within the BM niche; and apply CRISPR-based approaches to systematically define the mechanisms of in vivo resistance of MM cells within the BM milieu (2) against combinations of pharmacological agents; and (3) recently developed immune- based anti-MM treatment. This project will synergistically apply the translational power of the “humanized” and GEMM in vivo models of the collaborating programs and comprehensive CRISPR approaches to provide key insights into how MM cells within the BM milieu develop resistance to combinations of pharmacological agents or to immune therapies. These results will inform the U54 DRSC on preclinical testing of new approaches that may prevent, delay or overcome these forms of treatment resistance. This collaboration will also provide a blueprint, especially in regard to treatment resistance genes related to surface protein glycosylation, that will accelerate the efforts of the parent U01; and may also be applied to facilitate the efforts of other U54 DRSCs that involve malignancies that originate from or metastasize to the BM/bone milieu.

PROJECT NARRATIVE Multiple myeloma (MM) remains an incurable neoplasia and the mechanisms of its resistance to currently available or recently developed therapies remain incompletely understood. This project will seek to address this void by applying translationally relevant preclinical in vivo models of MM and comprehensive CRISPR-Cas9- based approaches to systematically define how genes involved in surface protein glycosylation or other pathways can contribute to treatment resistance of MM cells has the latter interact with the supportive microenvironment of the bone marrow.