MSH6 Mutations Arise in Glioblastomas during Temozolomide Therapy and Mediate Temozolomide Resistance

Abstract

Purpose: Over the past few years, the alkylating agent temozolomide has become the standardof-care therapy for patients with glioblastoma, the most common brain tumor. Recently, largescale cancer genome sequencing efforts have identified a hypermutation phenotype and inactivating MSH6 mismatch repair gene mutations in recurrent, post-temozolomide glioblastomas, particularly those growing more rapidly during temozolomide treatment. This study aimed to clarify the timing and role of MSH6 mutations in mediating glioblastoma temozolomide resistance. Experimental Design: MSH6 sequence and microsatellite instability (MSI) status were determined in matched prechemotherapy and postchemotherapy glioblastomas identified by The Cancer Genome Atlas (TCGA) as having posttreatment MSH6 mutations. Temozolomide-resistant lines were derived in vitro through selective growth under temozolomide, and the MSH6 gene was sequenced in resistant clones. The role of MSH6 inactivation in mediating resistance was explored using lentiviral short hairpin RNA knockdown and MSH6 reconstitution. Results: MSH6 mutations were confirmed in posttreatment TCGA glioblastomas but absent in matched pretreatment tumors. The posttreatment hypermutation phenotype displayed a signature bias toward CpC transitions and was not associated with MSI. In vitro modeling through exposure of an MSH6 wild-type glioblastoma line to temozolomide resulted in resistant clones; one clone showed an MSH6 mutation,Thr 1219 Ile, that had been independently noted in two treatedTCGA glioblastomas. Knockdown of MSH6 in the glioblastoma line U251increased resistance to temozolomide cytotoxicity and reconstitution restored cytotoxicity in MSH6-null glioma cells. Conclusions: MSH6 mutations are selected in glioblastomas during temozolomide therapy both in vitro and in vivo and are causally associated with temozolomide resistance.

Promoter methylation and expression of MGMT and the DNA mismatch repair genes MLH1, MSH2, MSH6 and PMS2 in paired primary and recurrent glioblastomas

“…13 Additional in vivo and in vitro studies provided evidence that MSH6 mutations are selected for during TMZ treatment and may thus be causally associated with TMZ resistance and glioblastoma relapse. 12 Other authors confirmed MSH6 alterations, 14 while high-level microsatellite instability was absent in recurrent glioblastomas. 12,14 Here, we report on a translational study performed within the framework of the German Glioma Network (GGN).…”

“…13 A follow-up study confirmed the presence of MSH6 mutations in post-treatment TCGA glioblastomas but not in the matched pretreatment tumors. 12 Interestingly, recurrent glioblastomas do not display high-level microsatellite instability despite the presence of mutations in MSH6 and other MMR genes. 12,14 We compared the expression of MLH1, MSH2, MSH6 and PMS2 proteins in primary and recurrent glioblastoma tissues and in line with previous studies 11 detected lower expression of MSH6 in a substantial fraction of recurrent when compared to primary glioblastomas.…”

“…Other authors determined the mRNA expression levels of MSH2, MLH1, PMS1, PMS2 and GTBPT in gliomas and found lower expression relative to normal lymphocytes in variable subsets of patients. 31 Thus, reduced expression of one or more MMR genes is frequent in glioblastoma and may contribute to the development of therapy resistance and tumor recurrence, albeit typical microsatellite instability is rare 12,14,32 and mostly restricted to tumors from a small subset of young patients and/or patients with Turcot syndrome. 33 Interestingly, we found that reduced expression of MMR proteins is not restricted to recurrent tumors previously treated with TMZ but may also be seen in patients treated with radiotherapy only.…”

MGMT promoter methylation in malignant gliomas: ready for personalized medicine?

“…50 Selection for loss of mismatch repair proteins, such as MSH6, could be involved in a minority of patients. [51][52][53] In vivo evidence for the direct involvement of MGMT in the response of glioblastoma to alkylating agents has been provided by The Cancer Genome Atlas. 54 The value of temozolomide in the setting of recurrent glioblastoma, including its relationship with MGMT promoter methylation status, must now to be determined in patients who have already been exposed to temozolomide in the first-line setting, 55,56 as is being pursued in the DIRECTOR trial (http://clinicaltrials.gov, NCT00941460).…”

Patient-derived xenografts, the cancer stem cell paradigm, and cancer pathobiology in the 21st century

“…Moreover, residual tumors generally recur as more aggressive, refractory, and lethal; likely due to the inability of chemotherapy to eliminate CSC, and resulting in additional mutations accumulated in these cells during exposure to genotoxic drug regimens. 45 Genetically engineered mouse models (GEMMs) are also popular models through which tumor biology is studied. Unfortunately, these tumor models also have their intrinsic shortcomings.…”