RESUMEN
BACKGROUND: Glioblastoma (GBM) carries a poor prognosis, and new therapeutic strategies are necessary to improve outcomes for patients with this disease. Alkylating chemotherapies including temozolomide (TMZ) and lomustine (CCNU) are critical for treating GBM, but resistance mechanisms, including hypomethylation of O6-methylguanine-DNA methyltransferase (MGMT) promoter, undermine treatment. CRISPRoff is a programmable epigenetic memory editor that can induce stable and heritable gene silencing after transient delivery, and we hypothesize that CRISPRoff could potentiate the activity of TMZ and CCNU through long-term suppression of target genes. METHODS: We transiently delivered CRISPRoff mRNA along with sgRNAs against target genes using both electroporation and lipid nanoparticles (LNPs) into established GBM cell lines, patient-derived primary GBM cultures, and orthotopic GBM xenografts. Gene repression, specificity, and stability were measured by RT-qPCR, Western blot, bisulfite sequencing, and RNA sequencing. Sensitivity to chemotherapies was measured by cell viability dose-response, microscopy, and bioluminescence imaging. Genome-wide mapping of CCNU sensitizers was performed using CRISPRi screens. RESULTS: CRISPRoff induced complete suppression of MGMT and sensitization to TMZ that was stable for over 8 months of continuous cell propagation. GBM orthotopic tumors treated with CRISPRoff against MGMT demonstrated sensitivity to TMZ in vivo, and CRISPRoff delivery resulted in chemosensitivity in patient-derived primary GBM. Genome-wide CRISPRi screens identified combinatorial genetic vulnerabilities (BRIP1, FANCE) that were targetable by multiplexed CRISPRoff to achieve sensitization to CCNU. CONCLUSION: Transient delivery of a site-specific epigenetic memory can induce stable, complete, and multiplexed suppression of target genes for therapeutic application in GBM.
Asunto(s)
Neoplasias Encefálicas , Sistemas CRISPR-Cas , Resistencia a Antineoplásicos , Epigénesis Genética , Edición Génica , Glioblastoma , Glioblastoma/genética , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Humanos , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Ratones , Temozolomida/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Edición Génica/métodos , Lomustina/farmacología , Células Tumorales Cultivadas , Resistencia a Antineoplásicos/genética , Enzimas Reparadoras del ADN/genética , Proteínas Supresoras de Tumor/genética , Metilasas de Modificación del ADN/genética , Ratones Desnudos , Memoria EpigenéticaRESUMEN
BACKGROUND: Genetic perturbation screens with single-cell readouts have enabled rich phenotyping of gene function and regulatory networks. These approaches have been challenging in vivo, especially in adult disease models such as cancer, which include mixtures of malignant and microenvironment cells. Glioblastoma (GBM) is a fatal cancer, and methods of systematically interrogating gene function and therapeutic targets in vivo, especially in combination with standard of care treatment such as radiotherapy, are lacking. RESULTS: Here, we iteratively develop a multiplex in vivo perturb-seq CRISPRi platform for single-cell genetic screens in cancer and tumor microenvironment cells that leverages intracranial convection enhanced delivery of sgRNA libraries into mouse models of GBM. Our platform enables potent silencing of drivers of in vivo growth and tumor maintenance as well as genes that sensitize GBM to radiotherapy. We find radiotherapy rewires transcriptional responses to genetic perturbations in an in vivo-dependent manner, revealing heterogenous patterns of treatment sensitization or resistance in GBM. Furthermore, we demonstrate targeting of genes that function in the tumor microenvironment, enabling alterations of ligand-receptor interactions between immune and stromal cells following in vivo CRISPRi perturbations that can affect tumor cell phagocytosis. CONCLUSION: In sum, we demonstrate the utility of multiplexed perturb-seq for in vivo single-cell dissection of adult cancer and normal tissue biology across multiple cell types in the context of therapeutic intervention, a platform with potential for broad application.