Translesion DNA synthesis (TLS) and homologous recombination (HR) cooperate during S-phase to safeguard replication forks integrity. Thus, the inhibition of TLS becomes a promising point of therapeutic intervention in HR-deficient cancers, where TLS impairment might trigger synthetic lethality (SL). The main limitation to test this hypothesis is the current lack of selective pharmacological inhibitors of TLS. Herein, we developed a miniaturized screening assay to identify inhibitors of PCNA ubiquitylation, a key post-translational modification required for efficient TLS activation. After screening a library of 627 kinase inhibitors, we found that targeting the pro-survival kinase AKT leads to strong impairment of PCNA ubiquitylation. Mechanistically, we found that AKT-mediated modulation of Proliferating Cell Nuclear Antigen (PCNA) ubiquitylation after UV requires the upstream activity of DNA PKcs, without affecting PCNA ubiquitylation levels in unperturbed cells. Moreover, we confirmed that persistent AKT inhibition blocks the recruitment of TLS polymerases to sites of DNA damage and impairs DNA replication forks processivity after UV irradiation, leading to increased DNA replication stress and cell death. Remarkably, when we compared the differential survival of HR-proficient vs HR-deficient cells, we found that the combination of UV irradiation and AKT inhibition leads to robust SL induction in HR-deficient cells. We link this phenotype to AKT ability to inhibit PCNA ubiquitylation, since the targeted knockdown of PCNA E3-ligase (RAD18) and a non-ubiquitylable (PCNA K164R) knock-in model recapitulate the observed SL induction. Collectively, this work identifies AKT as a novel regulator of PCNA ubiquitylation and provides the proof-of-concept of inhibiting TLS as a therapeutic approach to selectively kill HR-deficient cells submitted to replication stress.

中文翻译：

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AKT抑制削弱PCNA的泛素化并触发同源复制缺陷细胞中的复制杀伤力的合成杀伤力。

跨病变DNA合成（TLS）和同源重组（HR）在S期协同工作，以保护复制叉的完整性。因此，对TLS的抑制成为HR缺乏型癌症中治疗干预的有希望的点，其中TLS损伤可能会触发合成杀伤力（SL）。测试该假设的主要限制是目前缺乏TLS的选择性药理抑制剂。在本文中，我们开发了一种小型筛选测定法，以鉴定PCNA泛素化的抑制剂，这是有效TLS激活所需的关键翻译后修饰。在筛选了627种激酶抑制剂的文库后，我们发现靶向生存激酶AKT会导致PCNA泛素化的强烈损害。机械上，我们发现，紫外线后，AKT介导的对增殖细胞核抗原（PCNA）泛素化的调节需要DNA PKcs的上游活性，而不会影响不受干扰的细胞中PCNA泛素化的水平。此外，我们证实了持久的AKT抑制作用会阻止TLS聚合酶募集到DNA损伤部位，并削弱UV照射后DNA复制叉的合成能力，从而导致DNA复制压​​力增加和细胞死亡。值得注意的是，当我们比较HR缺陷型细胞与HR缺陷型细胞的差异存活时，我们发现UV照射和AKT抑制的组合可导致HR缺陷型细胞中强大的SL诱导。我们将此表型与AKT抑制PCNA泛素化的能力联系起来，因为有针对性的敲低PCNA E3-连接酶（RAD18）和不可泛素化（PCNA K164R）敲入模型可以概括观察到的SL诱导。总的来说，这项工作将AKT鉴定为PCNA泛素化的新型调节剂，并提供了抑制TLS的概念验证，作为选择性杀死提交复制压力的HR缺陷细胞的治疗方法。