Analysis of E-selectin Ligands of Human Acute Leukemia Cells and their Biology in Leukemogenesis

项目摘要 本申请是根据NOSI NOT-CA-21-034提交的。产生的工作 父母补助金（U 01 CA 225730）是对马约诊所/大学U 54资助的DRSC的补充。 明尼苏达州（U 54 CA 224018），我们建议进行合作实验，利用资源 从这DRSC的所有3个项目，以提高这两个赠款的影响。多发性骨髓瘤（MM） 以及MM细胞对目前可用的组合产生耐药性的机制 方案和最近开发的免疫疗法仍然不完全理解。我们的研究记录了 骨髓间充质干细胞（BMSC）和其他骨髓/骨的非恶性细胞发挥着 在MM、白血病和实体瘤细胞对各种药物或免疫药物的耐药性中起关键作用。 效应细胞我们观察到，人MM或白血病细胞在磷酸氢钙（BCP）支架中， 具有“人源化”间充质基质区室的BM样体内系统表现出（与体外或体外系统相比， 常规皮下异种移植物）不同的基因组依赖性和对多种 治疗在亲本U 01中，我们已经认识到表面蛋白糖基化在以下方面起关键作用： 调节恶性造血细胞如何与BM小生境相互作用。此外，我们最近观察到， 通过基于CRISPR的表面蛋白糖基化调节因子的扰动可以有助于肿瘤细胞 对药理学或免疫治疗的抗性。目前的项目旨在应用我们的 BM样“人源化”支架体内模型;利用我们的资源调节表面蛋白 我们在基于CRISPR的体内研究方面的经验以及糖基化的翻译相关性， 从U 54 DRSC至（1）的MM免疫活性Vk*myc基因工程小鼠模型（GEMM） 检查具有高表面水平sLeX的MM细胞，sLeX是造血干细胞与骨髓基质细胞相互作用的关键决定因素， 具有BM血管小生境的细胞在BM小生境内表现出对体内处理更显著的抗性; 并应用基于CRISPR的方法系统地定义MM细胞的体内抗性机制 在BM环境中，（2）针对药理学试剂的组合;和（3）最近开发的免疫- 抗MM治疗。该项目将协同应用“人性化”的翻译力量， GEMM合作项目的体内模型和全面的CRISPR方法提供了关键的 深入了解骨髓环境中的MM细胞如何对药物组合产生耐药性 或免疫疗法。这些结果将告知U 54 DRSC对新方法的临床前测试， 可以预防、延迟或克服这些形式的治疗抗性。这项合作还将提供一个 蓝图，特别是关于与表面蛋白糖基化相关的治疗抗性基因， 加速母公司U 01的工作;也可用于促进其他U 54灾难应对和应对中心的工作 涉及起源于或转移到BM/骨环境的恶性肿瘤。

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.