Identification of genes that modulate Temozolomide (TMZ) response
Alvaro Curiel-García*, Seve-Ballesteros Foundation – Brain Tumour Group. BBVA Foundation-Cancer Cell Biology. Spanish National Cancer Research Center (CNIO).
Cristina Mayor*, Genomic Instability Group. Molecular Oncology programme. Spanish National Cancer Research Center (CNIO).
Oscar Fernández-Capetillo, Molecular Oncology programme. Spanish National Cancer Research Center (CNIO).
and Massimo Squatrito, Seve-Ballesteros Foundation – Brain Tumour Group. BBVA Foundation-Cancer Cell Biology. Spanish National Cancer Research Center (CNIO).
Standard of care for Glioblastoma Multiforme (GBM) includes resection of the tumor mass, followed by concurrent radiotherapy and chemotherapy with the alkylating agent Temozolomide (TMZ). Although the last decade highlighted enormous advances in treating other solid cancers, such as lung and breast, the median survival for GBM stayed nearly the same over the last 50 years, averaging 15 months from the time to diagnosis.
The resistance to TMZ represents a major obstacle to successful treatment. In order to identify genes that modulate TMZ response, we are performing a genetic screening approach using a PiggyBac transposon (PB) library in human haploid cells (HAP-1). These cells are a nearly haploid cell line in which single alelle mutations by transposon insertion, can directly cause loss-of-function phenotypes. HAP-1 cells have been transduced with a PB-transposon to generate a library of “knock-out” cells. This library was treated with a minimum lethal dose of TMZ for 2 weeks and some TMZ resistant clones were isolated. To identify the integration sites of the transposon, we analyzed these clones by Splinkerette-PCR and the remaining clones were pooled and analized by RNAseq analysis.
In a preliminary screening we have identified few candidate genes that can lead to TMZ resistance when lost. One of these genes, MSH6, has been reported to be an important modulator of TMZ response, therefore underlying the reliability of our screening approach.
We are currently validating the candidate genes in secondary analysis using the CRISPR-Cas9 system to “knock-out” genes, not only in haploid cells but also a panel of GBM cell lines. The positive hits will be further characterized and tested for TMZ modulation in a mouse model of GBM.
With these screenings we aim to find a genes that confer resistance to TMZ and identify novel biomarkers for this treatment.