DDDR-18. SINGLE-CELL FUNCTIONAL GENOMIC ANALYSIS OF PHARMACOLOGIC RESPONSES IN NF1 MUTANT GLIOBLASTOMA REVEALS DISTINCT MECHANISMS OF ACTION FOR MEK AND SHP2 INHIBITION

Abstract

Abstract BACKGROUND Glioblastomas harbor recurrent mutations in NF1 that activate Ras/Raf/MEK signaling, motivating pharmacologic approaches targeting downstream (MEK) or upstream (SHP2) of Ras signaling. However, the mechanisms underlying therapeutic response remain unclear. Here, we integrate genome wide CRISPRi screens and single cell sequencing in cell lines and mouse xenografts to identify functional genomic mechanisms underlying responses to the MEK inhibitor selumetinib or the SHP2 inhibitor RMC-4550. METHODS Human NF1 mutant GBM43 cells expressing CRISPRi machinery were used for CRISPRi screens with selumetinib or RMC-4550. Intracranial xenografts were established with GBM43 cells and treated with selumetinib or RMC-4550. Tumor growth kinetics were measured with bioluminescent imaging. RESULTS Genome-wide CRISPRi screens with selumetinib or RMC-4550 in NF1 mutant human GBM43 cells identified 39 genes (14 sensitivity, 25 resistance) mediating selumetinib response and 327 genes (51 sensitivity, 276 resistance) mediating RMC-4550 response. While both selumetinib and RMC-4550 hits were enriched for components of Ras/Raf/MEK, hits sensitizing RMC-4550 response were uniquely enriched for PI3K signaling and focal adhesion components while hits mediating resistance were enriched for cell cycle genes. In NF1 mutant GBM43, RMC-4550 significantly reduced tumor volume (p=0.02) while selumetinib did not. Single cell RNA-sequencing (scRNA-seq) analysis of 90,782 tumor cells from NF1 mutant GBM43 xenografts treated with selumetinib or RMC-4550 identified 12 clusters across all samples. RMC-4550, but not selumetinib, resulted in significant depletion of cells expressing an epithelial-mesenchymal transition (EMT) signature marked by expression of focal adhesion genes mediating RMC-4550 sensitivity, suggesting this signature comprises a distinct mechanism of action in response to RMC-4550 but not selumetinib. CONCLUSIONS In NF1 mutant glioblastoma in vitro and in vivo models, cellular response to both upstream (SHP2) and downstream (MEK) inhibition are mediated by Ras/Raf/MEK signaling while RMC-4550 alone uniquely regulates a focal adhesion network associated with EMT and demonstrates improved efficacy in vivo.