AKT inhibition impairs PCNA ubiquitylation and triggers synthetic lethality in homologous-recombination-deficient cells

SOFIA CARBAJOSA; MARÍA LAURA GUANTAY; VANESA GOTTIFREDI; ALEJANDRA GARCÍA; SABRINA MANSILLA; ISRO GLOGER; GASTÓN SORIA; FLORENCIA VILLAFAÑEZ; MARÍA FLORENCIA PANSA; KEVIN MADAUSS; JOSÉ LUIS BOCCO

San Carlos de Bariloche

Simposio; SISTAM 2018 - The fourth South American Symposium in Signal Transduction and Molecular Medicine; 2018

IUBMB

Translesion DNA Synthesis (TLS) and homologous recombination (HR) cooperate during S-phase to ensure the completion of DNA duplication in the presence of DNA damage. While TLS promotes DNA damage bypass to avoid replication stalling, HR acts as a safeguard mechanism that restores stalled/collapsed replication forks in an error-free manner. Thus, the inhibition of TLS becomes a promising point for therapeutic intervention in HR-deficient cancers, where TLS impairment might trigger synthetic lethality (SL) during S-phase. The main limitation to test this hypothesis is the current lack of selective pharmacological inhibitors of TLS. To this end, 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 a strong impairment of PCNA ubiquitylation. We confirmed that AKT inhibition also 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 evaluated the differential survival of isogenic 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 recapitulates 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.