DIPG-94. PROTEOMIC AND BIOINFORMATICS ANALYSIS OF THE SURFACEOME AND EXTRACELLULAR MATRIX OF DIFFUSE MIDLINE GLIOMAS

Abstract

Abstract INTRODUCTION Diffuse midline glioma (DMG) is a universally fatal pediatric brain cancer with a median overall survival of 9-11-months. The extracellular matrix (ECM) plays a significant role in regulating bidirectional communication between glioma cells and the niches of the tumor immune microenvironment (TIME); influencing expression of immunosuppressive cytokines and regulating immune cell infiltrations, all of which contribute to poor outcomes. DMG immunosuppression remains poorly understood but has implications for emerging immunotherapies such as CAR T cells. Using a sucrose gradient plasma membrane purification method coupled with quantitative high-resolution proteomics we have discovered a novel cancer-associated protein termed ‘Protein-J 4 (PJ-4)’. PJ-4 is an important component of the DMG-ECM that we believe contributes to immunosuppression. METHODS Expression of PJ-4 was validated by Immunoblotting, flow cytometry and immunofluorescences microscopy. CRISPR/dCas9 gene deletion and amplification of PJ-4 and its transcription factor are current being established using DMG patient-derived cell lines, and syngeneic DIPG models to help elucidate the biological significance of PJ-4 expression. RESULTS Immunofluorescences microscopy showed PJ-4’s translocation from the Golgi apparatus to the cell membrane is dependent neurosphere formation, hence the formation of an ECM. Further, PJ-4 demonstrated high affinity with pro-TGF-β and may serve to regulate its bioavailability in the TIME, suggestive of an immunomodulatory role. Immunohistochemistry analysis of our PJ-4, TGF-β and CD45 is currently underway using a constructed tissue microarray of healthy brain tissue samples and over 30 pediatric brain cancers to determine tumor specificity and correlation with immune cell infiltration. CONCLUSIONS Resistance to conventional therapies and a poor understanding of the TIME and ECM remains a significant challenge to the development of effective DMG therapies. The identification of PJ-4 as a cancer-associated constituent of the DMG-ECM may help us to develop strategies to that increase DMG response to standard-of-care radiation therapy, novel target agents and immunotherapies.