Extended Data Fig. 9: MMR-deficient models of glioma, continued.

a, b, CRISPR–Cas9 MSH2 and MSH6 gene knockout in DIPG13 high-grade glioma cell line. a, Integrated genomics viewer (IGV) plots depicting MSH2 reads in between the guide RNAs in the MSH2 unedited line (sgGFP, left) and the MSH2 CRISPR knockout line (right) confirming knockout in the MSH2 edited line. b, IGV plots depicting MSH6 reads in between the guide RNAs in the MSH6 unedited line (sgGFP, left) and the MSH6 CRISPR knockout line (right) confirming knockout in the MSH6 edited line. c, Overview of in vivo temozolomide resistance study. Treatment of subcutaneous BT145 PDX-bearing animals was initiated at a volume of 100 mm³ and eight nude mice per group were randomized to 12 mg/kg/day temozolomide or vehicle for five consecutive days per 28-day cycle. Mice were treated until tumours reached a volume of 1,500 mm³, and tumours were sequenced to identify mutations and mutational signature. d, Survival of mice with BT145 xenografts (n = 8 mice per group) during treatment with vehicle (blue) or temozolomide (red). Two-sided log-rank test. e, Unique variants found in three sequenced BT145 tumours (two temozolomide-treated, and one vehicle-treated) were analysed for correlation with known mutational signatures. COSMIC Signature 11 was found in the two temozolomide-treated tumours. Mutational signatures could not be called in the vehicle-treated tumour (too few variants). After filtering of truncal variants common to all tumours, the two temozolomide-treated tumours shared only four variants, including an MSH6(T1219I) mutation and three noncoding variants. f, BT145 xenografts chronically treated with vehicle (n = 1) or temozolomide (n = 2) were removed, dissociated and cultured in serum-free medium to establish cell lines. After three passages in culture, sensitivity to temozolomide was assessed. The results of the short-term viability assays (mean ± s.e.m.) and temozolomide AUC of each cell line are depicted. g, Model of acquired hypermutation with mutational signature 11 in gliomas. Top, MMR-proficient cells repair TMZ damage and do not develop signature 11. Resistance in these cells is mediated by non-MMR pathways (for example, MGMT expression). Bottom, TMZ induces and/or selects resistant subclonal MMR-deficient cells. Further TMZ exposure produces accumulation of mutations at specific trinucleotide contexts, detected as hypermutation with signature 11.