Exploring drug resistance mechanisms in cancer cells via CRISPR libraries

Abstract

Cancer is a leading cause of death worldwide, and while great advances have been made particularly in chemotherapy, many types of cancer still present a dismal prognosis. Glioma is the most common primary cancer of the central nervous system, and around half of the patients present with the most aggressive form of the disease, glioblastoma. Metastatic melanoma shares several of glioma's features, in particular, high aggressiveness and poor prognosis The alkylating agent temozolomide (TMZ) is the first-line drug to treat glioblastoma patients and dacarbazine (a TMZ analogue) is used to treat melanoma, but those drugs have limited success due to drug resistance. While several TMZ resistance mechanisms have been described, it has been speculated that many more remain to be uncovered. Hence, to develop glioblastoma therapies of clinical relevance, it is imperative to better understand TMZ resistance and discover novel synthetic lethal partners for TMZ combination therapies preventing tumor recurrence. In this proposal, we ultimately aim to identify and validate major TMZ resistance molecular mechanisms. For this purpose, genome-wide CRISPR-Cas9 lentiviral screen libraries for gene knockout and activation were transduced in human glioblastoma cell line. Next-generation sequencing was used to identify gRNAs that were enriched in the knockout or activation screen libraries upon TMZ treatment compared to untreated cells. Pathway analysis of gene candidates on knockout screening revealed that mismatch repair and circadian rhythm pathway were significantly enriched. Also, activation genome-wide screen library revealed that NRF2 pathway is involved on TMZ resistance. Using TGCA RNA-seq dataset of glioblastoma patients, we confirmed that expression levels of NRF2 and related genes significantly correlate with patient survival rates. Overall, our results have identified a number of genes that contribute to TMZ resistance in human glioblastoma cell. Those findings need further validation and characterization. This Young Investigator proposal focus in 2 sub-projects: analysis of the crosstalk between the transcription factor NRF2 and autophagy on TMZ resistance; use of CRISPR genomic screening libraries on melanoma cells submitted to TMZ treatment. Once validated gene candidates involved on TMZ resistance we will design and implement pre-clinical trial using chemical modulators of genes and/or pathways in combination with TMZ in glioblastoma and melanoma xenograft tumor model. There is an urgent medical need for therapeutic alternatives to treat glioblastoma and metastatic melanoma patients. If successful, this project will identify novel combination therapy leads to pursue in the clinic with TMZ. We strongly believe that the knowledge obtained from the proposed work will help design more adequate and efficient chemotherapy strategies to treat glioblastoma and melanoma patients. Also, the strategies established for in vivo and in-vivo CRISPR-Cas9 screening to identify novel combinatorial treatments will provide a blueprint for the general assessment of resistance mechanisms in various cancer types, and will truly revolutionize the current approach to drug resistance pathway identification. (AU)