In a comprehensive study to decipher the multi-layered response to the chemotherapeutic agent temozolomide (TMZ), we analyzed 427 genomes and determined mutational patterns in a collection of ~40 isogenic DNA repair-deficient human TK6 lymphoblast cell lines. We demonstrate that the spontaneous mutational background is very similar to the aging-associated mutational signature SBS40 and mainly caused by polymerase zeta-mediated translesion synthesis (TLS). MSH2-/- mismatch repair knockout in conjunction with additional repair deficiencies uncovers cryptic mutational patterns. We report how distinct mutational signatures are induced by TMZ upon sequential inactivation of DNA repair pathways, mirroring the acquisition of chemotherapy resistance by glioblastomas. The most toxic adduct induced by TMZ, O6-meG, is directly repaired by the O6-methylguanine-DNA methyltransferase (MGMT). In MGMT-/- cells, mismatch repair (MMR) leads to cell death and limits mutagenesis. MMR deficiency results in TMZ resistance, allowing the accumulation of ~10^5 C>T substitutions corresponding to signature SBS11. Under these conditions, N-alkylated bases, processed by base excision repair (BER), limit cell survival. Without BER, 3-meA is read through via error-prone TLS, causing T>A substitutions but not affecting survival. Blocking BER after abasic site formation results in large deletions and TMZ hypersensitization. Our findings reveal potential vulnerabilities of TMZ-resistant tumors.

中文翻译：

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全面的全基因组测序揭示了与衰老和替莫唑胺化疗相关的突变特征的起源

在一项旨在破译对化疗药物替莫唑胺 (TMZ) 的多层反应的综合研究中，我们分析了 427 个基因组，并确定了约 40 个等基因 DNA 修复缺陷的人 TK6 淋巴细胞系集合中的突变模式。我们证明自发突变背景与衰老相关突变特征 SBS40 非常相似，主要由聚合酶 zeta 介导的跨损伤合成 (TLS) 引起。 MSH2-/- 错配修复敲除与其他修复缺陷结合揭示了神秘的突变模式。我们报告了 TMZ 如何在 DNA 修复途径连续失活后诱导不同的突变特征，这反映了胶质母细胞瘤获得化疗耐药性。 TMZ 诱导的毒性最强的加合物 O6-meG 直接由 O6-甲基鸟嘌呤-DNA 甲基转移酶 (MGMT) 修复。在 MGMT-/- 细胞中，错配修复 (MMR) 会导致细胞死亡并限制突变。 MMR 缺陷会导致 TMZ 耐药，从而累积与特征 SBS11 相对应的~10^5 C>T 取代。在这些条件下，通过碱基切除修复 (BER) 处理的 N-烷基化碱基会限制细胞的存活。如果没有 BER，则通过容易出错的 TLS 读取 3-meA，导致 T>A 替换，但不影响生存。无碱基位点形成后阻断 BER 会导致大量缺失和 TMZ 过敏。我们的研究结果揭示了 TMZ 耐药肿瘤的潜在弱点。