Ablation of CD38 on Multiple Myeloma Cells Leads to an Aggressive Phenotype
Multiple myeloma (MM), the second most common hematological cancer in the United States, is characterized by the proliferation of malignant clonal plasma cells primarily within the bone marrow. The multifunctional cell surface glycoprotein CD38 is expressed in majority of MM cells, making it a key biomarker. Since the FDA approval of daratumumab in 2015, CD38-targeted immunotherapies have significantly advanced the treatment of MM; albeit nonuniform patient response and relapse. The role of CD38 in the complex bone microenvironment remains under investigation. This study examined the effect of CD38 on tumor cells by evaluating a CD38 knock-out (KO) clone of a human MM cell line xenografted into immunodeficient mice. CRISPR/Cas9 gene editing was used to KO CD38 expression in the wild-type (WT) MM.1S human MM cell line (MM.1S-CBR-GFP), confirmed with RNA sequencing and flow cytometry. After tumor implantation in mice, bioluminescence imaging (BLI) was employed to track tumor cells. Small animal Positron Emission Tomography (PET) was performed with the metabolic tracer, fluorodeoxyglucose ([18F]-FDG), and CD38-targeted [89Zr]-Daratumumab, respectively. Post-PET tissue biodistribution and leg bone autoradiography was performed to evaluate tracer distribution. Computed Tomography (CT), dual-energy X-ray (DEXA), and immunohistochemistry were used to evaluate changes in bone mineral density and osteolytic disease. Differences in blood composition, cytokine production, and RNA sequencing between WT and KO mice were analyzed for characterizing the new CD38 KO phenotype. BLI showed significantly increased tumor burden in the CD38 KO MM models compared to WT mice after implantation: 1-week (P=0.0011), 2-weeks (P=0.0066), 3-weeks (P=0.0046), and 4 weeks (P<0.0001). CT and DEXA imaging revealed that the KO mice had significantly decreased bone density, and similarly, planar X-ray imaging indicated severe myeloma bone disease characterized by cortical and trabecular osteolytic lesions in the femur and tibia. Bone histology further confirmed the KO tumor more aggressively invading the bone and muscle tissue. [89Zr]-Daratumumab accumulated in the spine, femur, and tibia in WT MM mice, with minimal bone uptake in the CD38 KO MM mice. [18F]-FDG PET revealed an increased metabolic activity of CD38 KO MM, with an aggressive kinetic uptake of the tracer (P < 0.001). Bilateral kidney metastases were observed in nearly all CD38 KO MM mice, while remaining undetected in WT mice. Blood analysis indicated elevated markers of disease progression and renal dysfunction, with reduced leukocyte and thrombocyte production in the CD38 KO MM xenografts. Cytokine profiling revealed altered levels of pro-inflammatory cytokines, including IL-13, IL-6, RANKL, and VEGF-a in the bone microenvironment of MM with depleted CD38. RNA sequencing showed significant transcriptional changes in CD38 KO cells, suggesting that the observed enhanced aggressiveness is predominantly fueled by the activation of key pathways that promote accelerated cell proliferation, pathways that enhance osteoclast activity, and pathways that enhance metastatic potential. Overall design: The human MM cell line, MM.1S, was obtained from Professor Katherine N. Weilbaecher in the Department of Medicine at Washington University School of Medicine (WUSM) and has been previously used to develop MM models.12, 13 The cell line was transduced with a lentivirus encoding green fluorescent protein (GFP), to enable visualization of cellular localization, and click beetle red (CBR), luciferase for bioluminescence-based tumor burden quantification and real-time monitoring of disease progression. The WT MM.1S cell line (MM.1S-WT-GFP-CBR) was genetically modified using CRISPR/Cas9 gene editing, performed by the McDonnel Genome Institute at WUSM, to create a CD38 KO clone (MM.1S-CD38 KO-GFP-CBR). The CD38 KO clone was expanded in bulk culture until sufficient cell aliquots were available for cryopreservation in liquid nitrogen. Similar to the WT MM.1S cell line, the CD38 KO clone cell line was tagged with GFP and CBR for visualization and bioluminescence analysis. RNA sequencing between WT and KO cells was performed to characterize the new CD38 KO phenotype. There were 6 RNA samples, 3 WT MM.1S cells and 3 CD38 KO cells.