DNMT3A Harboring Leukemia-Associated Mutations Directs Sensitivity to DNA Damage at Replication Forks.
DNMT3A Harboring Leukemia-Associated Mutations Directs Sensitivity to DNA Damage at Replication Forks.
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DOI：
10.1158/1078-0432.ccr-21-2863
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发表时间：
2022-02-15
期刊：
影响因子：
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通讯作者：
Guryanova OA
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文献类型：
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作者：
Venugopal K;Feng Y;Nowialis P;Xu H;Shabashvili DE;Berntsen CM;Kaur P;Krajcik KI;Taragjini C;Zaroogian Z;Casellas Román HL;Posada LM;Gunaratne C;Li J;Dupéré-Richer D;Bennett RL;Pondugula S;Riva A;Cogle CR;Opavsky R;Law BK;Bhaduri-McIntosh S;Kubicek S;Staber PB;Licht JD;Bird JE;Guryanova OA
在急性髓性白血病（AML）中，复发性DNA甲基转移酶3A （DNMT3A）突变与化疗耐药和预后不良相关，尤其是在老年患者中。在dnmt3a突变细胞中的基因表达研究发现了与DNA损伤反应和复制叉完整性解除调控有关的特征，表明对复制应激敏感。在这里，我们测试了药物诱导的复制叉延迟是否会在DNMT3A（R882）突变的细胞中产生治疗脆弱性，例如阿糖胞苷。使用白血病细胞系、遗传小鼠模型和具有或不具有DNMT3A（mut）的等基因细胞在体外和体内评估对核苷类似物（如阿糖胞苷）的敏感性，随后分析DNA损伤和信号传导、复制重启以及治疗和药物去除后的细胞周期进展。转录组分析鉴定了DNMT3A（mut）表达解除调控的途径。我们发现，表达DNMT3A（R882）突变体的细胞对药理学诱导的复制应激的敏感性增加，s期内检查点持续激活，PARP1募集受损，DNA损伤升高，在药物去除后不完全解决，并通过有丝分裂进行。阿糖胞苷洗脱后用EdU和BrdU进行的脉冲追踪双标记实验表明，表达DNMT3A（mut）的细胞分叉塌陷率更高。RNA-seq研究支持解除调控的细胞周期进程和p53激活，以及剪接、核糖体生物发生和代谢。总之，我们的研究表明，DNMT3A突变是复制叉停止后恢复缺陷的基础，随后积累了未解决的DNA损伤，这可能具有治疗可追溯性。这些结果表明，DNMT3A除了在表观遗传控制中发挥作用外，还有助于在复制胁迫期间保持基因组的完整性。参见Viny的相关评论，
In acute myeloid leukemia (AML), recurrent DNA methyltransferase 3A (DNMT3A) mutations are associated with chemoresistance and poor prognosis, especially in advanced-age patients. Gene-expression studies in DNMT3A-mutated cells identified signatures implicated in deregulated DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here, we tested whether pharmacologically induced replication fork stalling, such as with cytarabine, creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. Leukemia cell lines, genetic mouse models, and isogenic cells with and without DNMT3A(mut) were used to evaluate sensitivity to nucleoside analogues such as cytarabine in vitro and in vivo, followed by analysis of DNA damage and signaling, replication restart, and cell-cycle progression on treatment and after drug removal. Transcriptome profiling identified pathways deregulated by DNMT3A(mut) expression. We found increased sensitivity to pharmacologically induced replication stress in cells expressing DNMT3A(R882)-mutant, with persistent intra–S-phase checkpoint activation, impaired PARP1 recruitment, and elevated DNA damage, which was incompletely resolved after drug removal and carried through mitosis. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine washout demonstrated a higher rate of fork collapse in DNMT3A(mut)-expressing cells. RNA-seq studies supported deregulated cell-cycle progression and p53 activation, along with splicing, ribosome biogenesis, and metabolism. Together, our studies show that DNMT3A mutations underlie a defect in recovery from replication fork arrest with subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during replication stress. See related commentary by Viny,