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Supplementary Figure 1. Detection of MSH6 somatic mutations by cDNA and genomic DNA sequencing. A-D, cDNA sequencing (A) revealed a heterozygous T1219I mutation that was also confirmed by sequencing the genomic DNA of cell line 119 (B) and the fresh frozen tumor from patient 119 (C). The T1219I mutation was not present in genomic DNA from a blood sample of patient 119 (D) demonstrating it to be of somatic rather than germline origin.

Oral administration of AZD is compatible with TMZ chemotherapy in vivo.

AZD and TMZ administration do not significantly influence body weights of treated animals.

Purpose: The current standard of care for glioblastoma (GBM) involves a combination of surgery, radiotherapy, and temozolomide chemotherapy, but this regimen fails to achieve long-term tumor control. Resistance to temozolomide is largely mediated by expression of the DNA repair enzyme MGMT; however, emerging evidence suggests that inactivation of MSH6 and other mismatch repair proteins plays an important role in temozolomide resistance. Here, we investigate endogenous MSH6 mutations in GBM, anaplastic oligodendroglial tumor tissue, and corresponding brain tumor–initiating cell lines (BTIC).Experimental Design:MSH6 sequence and MGMT promoter methylation were determined in human tumor samples and BTICs. Sensitivity to temozolomide was evaluated in vitro using BTICs in the absence and presence of O6-benzylguanine to deplete MGMT. The influence of MGMT and MSH6 status on in vivo sensitivity to temozolomide was evaluated using intracranial BTIC xenografts.Results: We identified 11 previously unreported mutations in MSH6 in nine different glioma samples and six paired BTIC lines from adult patients. In addition, MSH6 mutations were documented in three oligodendrogliomas and two treatment-naïve gliomas, both previously unreported findings. These mutations were found to influence the sensitivity of BTICs to temozolomide both in vitro and in vivo, independent of MGMT promoter methylation status.Conclusions: These data demonstrate that endogenous MSH6 mutations may be present before alkylator therapy and occur in at least two histologic subtypes of adult glial neoplasms, with this report serving as the first to note these mutations in oligodendroglioma. These findings broaden our understanding of the clinical response to temozolomide in gliomas. Clin Cancer Res; 20(18); 4894–903. ©2014 AACR.

Molecular and genetic analysis of BTICs identifies subgroups with endogenous mutations in EGFR and PTEN.

Rapamycin partially inhibits growth of most BTICs irrespective of EGFR or PTEN mutational status.

Pharmacological inhibition of EGFR and PI3K/mTORC1/2 signaling in BTICs.

Purpose: The EGFR and PI3K/mTORC1/2 pathways are frequently altered in glioblastoma (GBM), but pharmacologic targeting of EGFR and PI3K signaling has failed to demonstrate efficacy in clinical trials. Lack of relevant models has rendered it difficult to assess whether targeting these pathways might be effective in molecularly defined subgroups of GBMs. Here, human brain tumor–initiating cell (BTIC) lines with different combinations of endogenous EGFR wild-type, EGFRvIII, and PTEN mutations were used to investigate response to the EGFR inhibitor gefitinib, mTORC1 inhibitor rapamycin, and dual mTORC1/2 inhibitor AZD8055 alone and in combination with temozolomide (TMZ)Experimental Design:In vitro growth inhibition and cell death induced by gefitinib, rapamycin, AZD8055, and TMZ or combinations in human BTICs were assessed by alamarBlue, neurosphere, and Western blotting assays. The in vivo efficacy of AZD8055 was assessed in subcutaneous and intracranial BTIC xenografts. Kaplan–Meier survival studies were performed with AZD8055 and in combination with TMZ.Results: We confirm that gefitinib and rapamycin have modest effects in most BTIC lines, but AZD8055 was highly effective at inhibiting Akt/mTORC2 activity and dramatically reduced the viability of BTICs regardless of their EGFR and PTEN mutational status. Systemic administration of AZD8055 effectively inhibited tumor growth in subcutaneous BTIC xenografts and mTORC1/2 signaling in orthotopic BTIC xenografts. AZD8055 was synergistic with the alkylating agent TMZ and significantly prolonged animal survival.Conclusion: These data suggest that dual inhibition of mTORC1/2 may be of benefit in GBM, including the subset of TMZ-resistant GBMs. Clin Cancer Res; 20(22); 5756–67. ©2014 AACR.

Gefitinib inhibits growth of EGFRvIII and PTENwt BTICs.

Supplementary Figure Legends from Dual mTORC1/2 Blockade Inhibits Glioblastoma Brain Tumor Initiating Cells In Vitro and In Vivo and Synergizes with Temozolomide to Increase Orthotopic Xenograft Survival