Mentor Name: Ranjit Bindra

Pediatric high-grade gliomas (pHGG) are among the most lethal childhood cancers. Standard-of-care radiation therapy with temozolomide (TMZ) has limited efficacy. TMZ induces cell death through deposition of O6-methylguanine (O6meG) lesions. Two critical regulators of TMZ sensitivity in adult gliomas are O6-methylguanine-DNA methyltransferase (MGMT) and the mismatch repair (MMR) pathway. MGMT is an enzyme that removes O6meG lesions from DNA, restoring guanine to its original state; methylation of the MGMT promoter CpG island can abolish MGMT expression and confer sensitivity to TMZ. In the absence of MGMT, O6meG lesions are recognized by MMR. Rather than replacing the aberrant guanine, MMR initiates futile cycling, resulting in double-strand breaks. Inactivating mutations in MMR genes, resulting in MMR deficiency (MMRd), drive resistance to TMZ by preventing such futile cycling. TMZ has exhibited limited efficacy in pHGG trials, yet MMRd and MGMT expression are rarely considered in these studies. We hypothesize that categorization of pHGG tumors based on MGMT and MMR status will better enable identification of patients who may benefit from TMZ and improve outcomes.

To test this hypothesis, we will interrogate TMZ sensitivity across pHGG cell lines with differing MGMT and MMR status to define how each influences TMZ response. In parallel, we will engineer MMRd cell lines by knocking out MSH2 using the CRISPR-Cas9 gene editing system. These models will allow us to directly test for a causal role of MMR in TMZ sensitivity. Finally, we will generate TMZ-resistant cell lines to determine whether MMR mutations occur spontaneously in pHGG cell populations chronically exposed to TMZ. In these studies, we will characterize MMR genes implicated in TMZ resistance, including MSH2, MSH6, PMS2, and MLH1. MGMT and MMR protein expression will be assessed by western blotting, and MMR mutational status by DNA sequencing. Because MMRd can occur through means other than mutation or loss of expression of core MMR genes, we will also employ an activity assay to assess MMR pathway functionality in pHGG. Specifically, we will design a reporter plasmid with an out-of-frame reporter gene directly downstream of a microsatellite region. The MMRd phenotype results in random indels that may correct this reading frame and result in reporter gene expression, while functional MMR will not. In conclusion, this work will test whether MGMT silencing and MMRd are more prevalent in pHGG than previously believed. These studies have the potential to unlock the anticancer effects of TMZ by identifying patient populations that harbor biomarkers of TMZ sensitivity or resistance.