Epigenetic preconditioning with decitabine sensitizes glioblastoma to temozolomide via induction of MLH1

Matthew Gallitto,Raymund L Yong,Huaien Wang,Yizhou Zhang,Isaac Wasserman,Melissa Smith,Rossana Cheng He,Julio F Inocencio,Gintaras Deikus,Maya Strahl,Robert Sebra

doi:10.1007/s11060-020-03461-4

Abstract

IntroductionTo improve the standard treatment paradigm for glioblastoma (GBM), efforts have been made to explore the efficacy of epigenetic agents as chemosensitizers. Recent data suggest possible synergy between decitabine (DAC), a DNA hypomethylating agent, and temozolomide (TMZ) in GBM, but the mechanism remains unclear. The objective of this study was to determine the effects of DAC on TMZ sensitization in a consecutively derived set of primary GBM cultures, with a focus on mismatch repair (MMR) proteins.MethodsHalf maximal inhibitory concentrations (IC50) of TMZ were calculated in eleven consecutive patient-derived GBM cell lines before and after preconditioning with DAC. MMR protein expression changes were determined by quantitative immunoblots and qPCR arrays. Single-molecule real-time (SMRT) sequencing of bisulfite (BS)-converted PCR amplicons of the MLH1 promoter was performed to determine methylation status.ResultsTMZ IC50 significantly changed in 6 of 11 GBM lines of varying MGMT promoter methylation status in response to DAC preconditioning. Knockdown of MLH1 after preconditioning reversed TMZ sensitization. SMRT-BS sequencing of the MLH1 promoter region revealed higher levels of baseline methylation at proximal CpGs in desensitized lines compared to sensitized lines.ConclusionsDAC enhances TMZ cytotoxicity in a subset of GBM cell lines, comprising lines both MGMT methylated and unmethylated tumors. This effect may be driven by levels of MLH1 via E2F1 transcription factor binding. Using unbiased long-range next-generation bisulfite-sequencing, we identified a region of the proximal MLH1 promoter with differential methylation patterns that has potential utility as a clinical biomarker for TMZ sensitization.

Highlights

To improve the standard treatment paradigm for glioblastoma (GBM), efforts have been made to explore the efficacy of epigenetic agents as chemosensitizers
We investigated whether the DAC-induced changes in mutL homolog 1 (MLH1) levels in TMZ-sensitized and desensitized GBM cell lines could be explained by CpG methylation changes at the MLH1 promoter
In vitro experiments using U251 cells demonstrated that reductions in MLH1 expression drive destabilization of its binding partner PMS2, and may be more correlated with TMZ resistance than either MSH2 or MSH6 [41, 42]

Summary

Introduction

To improve the standard treatment paradigm for glioblastoma (GBM), efforts have been made to explore the efficacy of epigenetic agents as chemosensitizers. The objective of this study was to determine the effects of DAC on TMZ sensitization in a consecutively derived set of primary GBM cultures, with a focus on mismatch repair (MMR) proteins. Results TMZ IC50 significantly changed in 6 of 11 GBM lines of varying MGMT promoter methylation status in response to DAC preconditioning. Conclusions DAC enhances TMZ cytotoxicity in a subset of GBM cell lines, comprising lines both MGMT methylated and unmethylated tumors. This effect may be driven by levels of MLH1 via E2F1 transcription factor binding. Using unbiased long-range next-generation bisulfite-sequencing, we identified a region of the proximal MLH1 promoter with differential methylation patterns that has potential utility as a clinical biomarker for TMZ sensitization. Inactivating mutations and loss of expression of MMR genes in GBM has been

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