PDF(Pubmed)
Abstract：
BACKGROUND: The cell cycle is tightly regulated by checkpoints, which play a vital role in controlling its progression and timing. Cancer cells exploit the G2/M checkpoint, which serves as a resistance mechanism against genotoxic anticancer treatments, allowing for DNA repair prior to cell division. Manipulating cell cycle timing has emerged as a potential strategy to augment the effectiveness of DNA damage-based therapies.
METHODS: In this study, we conducted a forward genome-wide CRISPR/Cas9 screening with repeated exposure to the alkylating agent temozolomide (TMZ) to investigate the mechanisms underlying tumor cell survival under genotoxic stress.
RESULTS: Our findings revealed that canonical DNA repair pathways, including the Ataxia-telangiectasia mutated (ATM)/Fanconi and mismatch repair, determine cell fate under genotoxic stress. Notably, we identified the critical role of PKMYT1, in ensuring cell survival. Depletion of PKMYT1 led to overwhelming TMZ-induced cytotoxicity in cancer cells. Isobologram analysis demonstrated potent drug synergy between alkylating agents and a Myt1 kinase inhibitor, RP-6306. Mechanistically, inhibiting Myt1 forced G2/M-arrested cells into an unscheduled transition to the mitotic phase without complete resolution of DNA damage. This forced entry into mitosis, along with persistent DNA damage, resulted in severe mitotic abnormalities. Ultimately, these aberrations led to mitotic exit with substantial apoptosis. Preclinical animal studies demonstrated that the combination regimen involving TMZ and RP-6306 prolonged the overall survival of glioma-bearing mice.
CONCLUSIONS: Collectively, our findings highlight the potential of targeting cell cycle timing through Myt1 inhibition as an effective strategy to enhance the efficacy of current standard cancer therapies, potentially leading to improved disease outcomes.
METHODS: In this study, we conducted a forward genome-wide CRISPR/Cas9 screening with repeated exposure to the alkylating agent temozolomide (TMZ) to investigate the mechanisms underlying tumor cell survival under genotoxic stress.
RESULTS: Our findings revealed that canonical DNA repair pathways, including the Ataxia-telangiectasia mutated (ATM)/Fanconi and mismatch repair, determine cell fate under genotoxic stress. Notably, we identified the critical role of PKMYT1, in ensuring cell survival. Depletion of PKMYT1 led to overwhelming TMZ-induced cytotoxicity in cancer cells. Isobologram analysis demonstrated potent drug synergy between alkylating agents and a Myt1 kinase inhibitor, RP-6306. Mechanistically, inhibiting Myt1 forced G2/M-arrested cells into an unscheduled transition to the mitotic phase without complete resolution of DNA damage. This forced entry into mitosis, along with persistent DNA damage, resulted in severe mitotic abnormalities. Ultimately, these aberrations led to mitotic exit with substantial apoptosis. Preclinical animal studies demonstrated that the combination regimen involving TMZ and RP-6306 prolonged the overall survival of glioma-bearing mice.
CONCLUSIONS: Collectively, our findings highlight the potential of targeting cell cycle timing through Myt1 inhibition as an effective strategy to enhance the efficacy of current standard cancer therapies, potentially leading to improved disease outcomes.
摘要：
背景：细胞周期受到检查点的严格调节，在控制其进展和时机方面发挥着至关重要的作用。癌细胞利用G2/M检查点,作为基因毒性抗癌治疗的耐药机制，允许在细胞分裂之前进行DNA修复。操纵细胞周期时机已经成为增强基于DNA损伤的疗法的有效性的潜在策略。
方法：在本研究中，我们在反复暴露于烷化剂替莫唑胺(TMZ)的情况下进行了CRISPR/Cas9正向基因组筛查,以研究基因毒性应激下肿瘤细胞存活的潜在机制.
结果：我们的发现揭示了典型的DNA修复途径，包括ATM/Fanconi和失配修复，决定基因毒性胁迫下的细胞命运。值得注意的是,我们确定了PKMYT1在确保细胞存活方面的关键作用.PKMYT1的耗尽导致了TMZ诱导的癌细胞中的压倒性细胞毒性。等值线分析证明了烷化剂和Myt1激酶抑制剂之间的有效药物协同作用，RP-6306。机械上,抑制Myt1迫使G2/M停滞的细胞进入计划外过渡到有丝分裂期，而DNA损伤没有完全解决。这种被迫进入有丝分裂，伴随着持续的DNA损伤，导致严重的有丝分裂异常。最终,这些畸变导致有丝分裂退出，并伴有大量细胞凋亡。临床前动物研究表明，涉及TMZ和RP-6306的组合方案延长了带有神经胶质瘤的小鼠的总体生存期。
结论：总的来说，我们的研究结果强调了通过Myt1抑制靶向细胞周期定时作为增强当前标准癌症疗法疗效的有效策略的潜力。可能导致改善疾病结果。
方法：在本研究中，我们在反复暴露于烷化剂替莫唑胺(TMZ)的情况下进行了CRISPR/Cas9正向基因组筛查,以研究基因毒性应激下肿瘤细胞存活的潜在机制.
结果：我们的发现揭示了典型的DNA修复途径，包括ATM/Fanconi和失配修复，决定基因毒性胁迫下的细胞命运。值得注意的是,我们确定了PKMYT1在确保细胞存活方面的关键作用.PKMYT1的耗尽导致了TMZ诱导的癌细胞中的压倒性细胞毒性。等值线分析证明了烷化剂和Myt1激酶抑制剂之间的有效药物协同作用，RP-6306。机械上,抑制Myt1迫使G2/M停滞的细胞进入计划外过渡到有丝分裂期，而DNA损伤没有完全解决。这种被迫进入有丝分裂，伴随着持续的DNA损伤，导致严重的有丝分裂异常。最终,这些畸变导致有丝分裂退出，并伴有大量细胞凋亡。临床前动物研究表明，涉及TMZ和RP-6306的组合方案延长了带有神经胶质瘤的小鼠的总体生存期。
结论：总的来说，我们的研究结果强调了通过Myt1抑制靶向细胞周期定时作为增强当前标准癌症疗法疗效的有效策略的潜力。可能导致改善疾病结果。
