Glioblastoma (GBM) is the most common and aggressive primary brain tumor, with a 95% rate of mortality at 5 years. The current standard therapeutic paradigm for GBM was established in 2005 and consists of maximum safe surgical debulking when possible, followed by concomitant fractionated radiotherapy and daily oral temzolomide (TMZ), and subsequently monthly cycles of adjuvant TMZ. Median time to disease progression on TMZ is a short 7 months, and is frequently mediated by acquired resistance by GBM cells to the alkyl-DNA adducts generated by TMZ. Specifically, the failure of the cell's DNA repair machinery to detect base mismatches leads to evasion of normal apoptotic signaling. The goal of this application is to determine whether the epigenetic agent decitabine (DAC), a DNA demethylating agent, can improve or restore base mismatch detection and repair in order to increase GBM sensitivity to TMZ. Our central hypothesis is that a subset of GBM with hypermethylated MLH1 and/or MSH6 promoters will be amenable to demethylation with DAC, increasing gene transcription and consequently mismatch repair protein activity.
In Specific Aim 1, we will use the large bank of prospectively derived GBM spheroid cell lines at our disposal to identify lines that exhibit significantly increased sensitivity or resistance to TMZ after DAC preconditioning. In the presence and absence of DAC preconditioning, these lines will be interrogated to determine the methylation status of MMR gene promoters and the corresponding transcript and protein levels. Expected downstream effects on DNA double- strand break repair levels and tumor mutational burden will be examined. Complementary to this, we will employ single cell RNA sequencing to identify other, novel tissue biomarkers of responsiveness to DAC epigenentic preconditioning, which will enable rational patient selection for clinical trials.
In Specific Aim 2, we will use an orthotopic mouse GBM xenograft model to determine the efficacy of low-dose DAC in combination with the standard TMZ regimen in improving mouse survival. Mice will be implanted with GBM cell lines demonstrated to exhibit increased TMZ sensitivity or resistance after DAC preconditioning in our in vitro studies. Following treatment, tumors will be explanted, recultured, and examined for levels of MMR protein expression and tumor mutational burden to validate pharmacodynamics observed in vitro. An innovation of this project is our unique approach of leveraging long-read third-generation bisulfite sequencing technology (SMRT-seq) to quantitate methylation levels of all CpGs in a gene promoter region simultaneously with a single set of primers. This provides improved ability, compared to short-read sequencing technology or methylation- specific PCR, to detect patterns of demethylation produced by epigenetic agents such as DAC. This work is significant because up to 30% of IDH-wildtype GBM, including the aggressive MGMT unmethylated and recurrent forms, may be responsive to an epigenetic preconditioning approach targeted at mismatch repair gene upregulation.
Glioblastoma is the most common and aggressive intrinsic brain tumor, remaining difficult to treat due to poor response rates to the standard chemotherapeutic agent, temozolomide. In the proposed research, a strategy to significantly improve responses to temozolomide using an epigenetic agent will be tested. A long term goal is the development of a clinical test that can be applied at the time of first diagnosis to identify the patients most likely to benefit from an epigenetic preconditioning approach for chemosensitization.
