OS07.1.A INTEGRATIVE SINGLE NUCLEI, SPATIAL, AND FUNCTIONAL GENOMIC ANALYSES REVEAL DNA-PK DRIVES GLIOBLASTOMA RADIORESISTANCE THROUGH TUMOR CELLS AND THE MYELOID MICROENVIRONMENT
Authors: Liu J , Majd N , Zou C , Mirchia K , Pak J , Foster K , Lin K , Casey-Clyde T , Seo K , Pan S , Payne E , Vasudevan H , Ozawa T , Berger M , de Groot J , Weiss W , Gilbert L , Raleigh D 
Published: October 2024

Abstract

BACKGROUND
Glioblastoma (GBM) is a heterogenous disease comprised of malignant cell states and an immunosuppressive microenvironment that underlie radiotherapy (RT) resistance. Here we integrate genome wide and single cell in vivo functional genomic screens of preclinical models with single nuclei and spatial transcriptomics of human tumors to identify therapeutic vulnerabilities driving GBM radioresistance.

MATERIAL AND METHODS
Perturb-seq of intracranial GBM models was performed using human (GBM43) or mouse (GL261, SB28) GBM cells expressing CRISPR interference (CRISPRi) machinery, with sgRNA libraries delivered to intracranial tumors using convection enhanced delivery. Genome wide dual sgRNA CRISPRi screens were performed in GBM cells (GBM43, LN18, GL261, SB28) ± RT. Functional genomic analyses across preclinical models were integrated with single nuclei and spatial transcriptomics from human GBMs. Validation of genetic phenotypes was performed using the DNA-PK inhibitor peposertib to treat GBM cells and orthotopic mouse models (SB28, GBM43, GBM6), which were analyzed using IF and single cell RNA sequencing.

RESULTS
Genome wide CRISPRi screens identified 114 radiation sensitizing targets. In vivo perturb-seq of radiation sensitizing targets reprogrammed 25 gene modules that spanned homeostatic, metabolic, and inflammatory pathways and nominated DNA-PK as a pleotropic radiation sensitizer that regulates interactions between malignant cells and the myeloid microenvironment. Genetic and pharmacologic inhibition of DNA-PK revealed radiotherapy dependent reprograming of cell states through cell intrinsic mechanisms (e.g. cell cycle, proteasome) and cell extrinsic pathways (e.g. TNFα, IFN). Peposertib sensitized GBMs to radiotherapy and extended survival in mice harboring intracranial xenografts. DNA-PK inhibition induced cGAS/STING and IFN signaling in malignant cells (n = 58,161) that were associated with pro-inflammatory polarization of infiltrating myeloid cells (n = 22,067). These alterations were synergistic with RT but were not induced by RT alone. Spatial transcriptomics of 8 matched primary-recurrent GBMs (20,793 transcriptomes) from patients treated with RT and peposertib, and single nuclei transcriptomics from 100 GBMs (76 matched primary-recurrent tumors, 4 after treatment with RT and peposertib, 356,997 nuclei) demonstrated malignant cell differentiation and enrichment of pro-inflammatory macrophages in the tumor microenvironment after inhibition of DNA-PK.

CONCLUSION
Integration of genome wide CRISPRi screens and in vivo perturb-seq in preclinical models with single nuclei and spatial transcriptomic analyses of human tumors enables discovery and mechanistic validation of therapeutic targets in GBM. These results reveal DNA-PK as a target for radiation sensitization of tumor and myeloid microenvironment cells in GBM.