Abstract 3996: A novel 13-basepair deletion in CSPG4/NG2 abrogates protein expression, glioblastoma proliferation and invasion in vitro and in vivo in mice

Abstract

Abstract Introduction: Glioblastoma (GBM) is the most frequent treatment resistant and poor prognosis primary brain tumor. Age-adjusted incidence among Caucasians is approx. twice that of Africans. Genetic changes in ethnicity may represent druggable targets for development of new treatments for GBM. Chondroitin sulfate proteoglycan 4 (CSPG4) with sequence homology to neuron-glial-2 (NG2), henceforth CSPG4/NG2, is a transmembrane proteoglycan that is upregulated in GBM, resulting in leaky neovasculature and high cell proliferation, and is independently prognostic for poor survival. Mechanisms regulating CSPG4/NG2 expression remain to be elucidated. Experiments: Tumor-derived DNA whole exome sequences (WES) from mixed American GBM patients (n=300) collected into the TCGA database and blood-derived DNA WES from controls (n = 2504) in the 1000 Genome database were analyzed for mutations in the CSPG4 gene by GATK-3.5. We used prime editing (PE) to introduce a 13-base pair (bp) deletion through design of PE guide RNA (n=27), screening GBM cells (n=14) for CSPG4/NG2 expression, transfecting and expanding PE cells, confirming editing using Sanger sequencing. We functionally characterized the mutation in PE cells and transplanted in orthotopic PDX mouse models compared to wild type (WT) and Cas9 control cells. Western blotting, BrdU proliferation assays, clonogenic- and wound healing assays were used to investigate function. Gelatin zymography assessed CSPG4/NG2 matrix metalloproteinase (MMP) cross-talk during invasion on mixed extracellular matrix- or collagen IV- coated transwells. Results: A novel 13bp frameshift deletion in exon 3 of the CSPG4 gene was identified from WES of GBM samples deposited in TCGA. The mutation was cancer specific (p<0.001). Comparison of frequency in various ethnicities revealed the mutation to be more frequent among African American GBM patients (p=0.019). We generated 3 clones heterozygous for the CSPG4/NG2 deletion in patient derived GBMs. A homozygous deletion was lethal to the cells. The 13bp deletion reduced CSPG4/NG2 expression by approx. 60%, slowed tumor cell proliferation compared to both WT (p<0.001) and Cas9 cells (p<0.0001). PE cells had significantly reduced colony formation (p<0.001), scratch wound healing (p<0.001), had a weaker invasive phenotype when challenged with collagen IV (p<0.001), and reduced cleavage of collagen I by MMP2 that was corroborated by RNAseq gene expression. Reduced growth of PE cells in vivo was verified by multiplex immunohistochemistry on ex vivo brain sections and identified biomarkers for differential tumor development. Tumor growth was reduced in vivo in mice. Conclusions: We identified a novel 13bp deletion in CSPG4/NG2 with phenotypic effect that could be used as a future molecular target in GBM treatment, and present the first successful application of PE in human GBM cells. Citation Format: Victoria Smith Arnesen, Susina Suntharalingam, Żaneta Matuszek, Shahin Sarowar, Stian Knappskog, Stein Atle Lie, David R. Liu, Mohummad Aminur Rahman, Martha Chekenya. A novel 13-basepair deletion in CSPG4/NG2 abrogates protein expression, glioblastoma proliferation and invasion in vitro and in vivo in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3996.