SYST-26 INVESTIGATING THE FUNCTIONAL ROLE OF GPNMB IN GLIOBLASTOMA AND THE TUMOR IMMUNE MICROENVIRONMENT AND ITS TARGETED ELIMINATION USING CAR-T CELLS

Abstract INTRODUCTION Glycoprotein nonmetastatic melanoma protein B (GPNMB) is active in the extracellular matrix of glioblastoma and presents a promising immunotherapy target for both tumor cells and immunosuppressive macrophages. METHODS Immunohistochemistry was performed on patient derived xenograft (PDX) brains and tissue samples of 16 patient-matched primary/recurrent GBMs and 23 normal organ tissues. Whole cell proteomics was performed on 43 matched primary/recurrent GBM samples. CRISPR/Cas9 was used to eliminate expression in GBM lines and proliferation and mouse survival times measured. GPNMB knockout clones generated in GL261 were engrafted in immunocompetent mice to examine single cell transcriptomes using sciRNA sequencing at endpoint. A second-generation CAR-T was developed to target GPNMB-expressing populations, and efficacy was interrogated using in vitro assays and GBM PDX models. RESULTS GPNMB was absent in most normal tissues but detected in residual PDX tumors treated orthotopically with CD133 CAR-Ts. Tissue microarrays and whole cell proteomics showed GPNMB upregulation in recurrent GBMs compared to primary (p=0.0349 vs. p=0.0033) while absent in normal tissues. Single cell sequencing data of patient GBMs revealed GPNMB was also highly expressed in tumor-associated macrophages. Eliminating GPNMB in GBM cell lines decreased proliferation (P<0.001) and prolonged survival times in all mouse models (P<0.01). GPNMB knockout clones displayed downregulation of hallmark signaling pathways of GBM such as PDGFR, TGF-beta, Integrins and Stats, and decreased innate/adaptive immune activation. CAR-T cytotoxicity and activation was observed in vitro and in vivo resulting in decreased tumor burden (P<0.001) and increased survival times (P<0.001). CONCLUSIONS We show GPNMB influences tumor-intrinsic biology of GBM and is also active in macrophages in the recurrent GBM immune microenvironment. By targeting GPNMB alongside CD133, combinatorial therapeutic regimens could target both the cancer stem cell hierarchy and its supportive niche. Administration of both CAR-T cell therapies to humanized mice engrafted with patient-derived GBMs is underway.