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 standard-of-care therapy for patients with glioblastoma, the most common brain tumor. Recently, large-scale 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 treated TCGA glioblastomas. Knockdown of MSH6 in the glioblastoma line U251 increased 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.

Figure 1

MSH6 mutation c.3166G>A (p.Val1056Met) is present in the post-treatment TCGA-02-0083 glioblastoma (right) but not in the matched pre-treatment sample (left).

Figure 2

a: The parental A172 and the TMZ-resistant subclone A172TR3 exhibit similar growth in the absence of TMZ (solid and dashed lines, square markers). Parental A172 (solid line, triangle marker) is sensitive to 100μM TMZ for seven days, whereas of A172TR3 growth (dashed line, triangle marker) is minimally affected at 100μM TMZ (p<0.01). b: The TMZ-resistant subclone TR3 (left) has a c.3656C>T MSH6 mutation, resulting in a Thr to Ile amino acid change at codon 1219, in comparison to the TMZ-sensitive, parental glioblastoma A172 line (right).

Figure 3

a: Western blot for MSH6 protein in U251 cells stably infected with five candidate shRNA lentiviral constructs show variable reduction of MSH6 expression, with Sh1 demonstrating maximal inhibition. MGMT is not expressed by any of the knockdown clones or the parental U251 cells (positive glioblastoma control SF295). b: Parental U251 and MSH6-knockdown U251-Sh1 cells exhibit similar growth in the absence of TMZ (solid and dashed lines, square markers). Whereas growth of parental U251 is significantly reduced in the presence of 100μM TMZ for seven days (solid line, closed circle), U251-Sh1 appear resistant to 100μM TMZ (dashed line, open circle) (p<0.01). Addition of 40μmol/l of O⁶-BG had no effect on TMZ cytotoxicity in both parental U251 and MSH6-knockdown U251-Sh1 cells (solid and dashed lines, triangle markers).

Figure 4

The Gli60 primary glioblastoma, which is MSH6-null secondary to nonsense mutation and loss of the remaining chromosome 2, infected with control empty lentiviral vector (Gli60-Con vector) and with MSH6-vector (Gli60-MSH6) showed differential sensitivity to TMZ after five days of drug exposure. Gli60 cells with restored MSH6 expression are significantly more sensitive to TMZ than cells infected with control vectors (p<0.01).