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Revealing Temozolomide Resistance Mechanisms via Genome-Wide CRISPR Libraries
Clarissa Ribeiro Reily Rocha1, Alexandre Reily Rocha2, Matheus Molina Silva3
1Department of Clinical and Experimental Oncology, Federal University of São Paulo (UNIFESP), São Paulo 04037-003, Brazil.
Abstract:
Glioblastoma is a severe type of brain tumor with a poor prognosis and few therapy options. Temozolomide (TMZ) is one of these options, however, with limited success, and failure is mainly due to tumor resistance. In this work, genome-wide CRISPR-Cas9 lentiviral screen libraries for gene knockout or activation were transduced in the human glioblastoma cell line, aiming to identify genes that modulate TMZ resistance. The sgRNAs enriched in both libraries in surviving cells after TMZ treatment were identified by next-generation sequencing (NGS). Pathway analyses of gene candidates on knockout screening revealed several enriched pathways, including the mismatch repair and the Sonic Hedgehog pathways. Silencing three genes ranked on the top 10 list (MSH2, PTCH2, and CLCA2) confirm cell protection from TMZ-induced death. In addition, a CRISPR activation library revealed that NRF2 and Wnt pathways are involved in TMZ resistance. Consistently, overexpression of FZD6, CTNNB1, or NRF2 genes significantly increased cell survival upon TMZ treatment. Moreover, NRF2 and related genes detected in this screen presented a robust negative correlation with glioblastoma patient survival rates. Finally, several gene candidates from knockout or activation screening are targetable by inhibitors or small molecules, and some of them have already been used in the clinic.
Insights
This study used CRISPR screening to find genes that cause resistance to temozolomide (TMZ) in glioblastoma. Researchers identified key genes and pathways, like NRF2, that could be targeted to improve brain tumor treatment.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- Glioblastoma is an aggressive brain tumor with limited treatment options and poor patient outcomes.
- Temozolomide (TMZ) is a standard chemotherapy, but its effectiveness is often compromised by intrinsic or acquired tumor resistance.
- Identifying the genetic mechanisms underlying TMZ resistance is crucial for developing more effective therapeutic strategies.
Purpose of the Study:
- To perform a genome-wide screen to identify genes that confer resistance to temozolomide (TMZ) in glioblastoma.
- To elucidate the molecular pathways involved in TMZ resistance using both gene knockout and activation screens.
- To validate identified genes and explore their correlation with patient survival and potential therapeutic targeting.
Main Methods:
- Utilized genome-wide CRISPR-Cas9 lentiviral libraries for gene knockout and activation screening in a human glioblastoma cell line.
- Employed next-generation sequencing (NGS) to identify enriched sgRNAs in cells surviving TMZ treatment.
- Validated key gene candidates through gene silencing or overexpression experiments and analyzed patient survival data.
Main Results:
- CRISPR knockout screening identified enriched pathways including mismatch repair and Sonic Hedgehog signaling.
- Silencing of MSH2, PTCH2, and CLCA2 genes confirmed their role in protecting cells from TMZ-induced death.
- CRISPR activation screening revealed the involvement of NRF2 and Wnt pathways in TMZ resistance.
- Overexpression of FZD6, CTNNB1, or NRF2 significantly enhanced glioblastoma cell survival under TMZ treatment.
- NRF2 and associated genes showed a strong negative correlation with glioblastoma patient survival.
Conclusions:
- This study successfully identified novel genetic regulators of temozolomide resistance in glioblastoma using comprehensive CRISPR screening.
- Key pathways and genes, including NRF2, MSH2, PTCH2, CLCA2, and Wnt signaling components, are implicated in modulating TMZ efficacy.
- Several identified gene candidates represent potential therapeutic targets, with some already druggable by existing clinical agents, offering new avenues for glioblastoma treatment.
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