IDENTIFICATION AND VALIDATION OF A NOVEL TRANSLESION DNA SYNTHESIS INHIBITOR THROUGH A WESTERN-BLOT BASED SCREENING PLATFORM

VANESA GOTTIFREDI; MARIA F. PANSA; FLORENCIA VILLAFAÑEZ; JOSÉ L BOCCO; SABRINA MANSILLA; RAMIRO GASTÓN SORIA; SOFIA CARBAJOSA; ALEJANDRA IRIS GARCÍA

Congreso; Reunión conjunta de sociedades de Biociencias; 2017

Translesion DNA synthesis (TLS) is a DNA damage tolerance processthat employs specialized polymerases to bypass DNA damageduring replication. Several lines of evidence suggest that TLSinhibition would be crucial in the homologous recombination (HR)deficient context of certain types of cancers (i.e. BRCA-/- breastand ovarian tumors). However, the limitation to explore such type oftherapeutic strategy is the lack of chemical inhibitors to target TLS.The main goal of this project is to identify specific inhibitors of TLSthat can be used as ?a proof of concept? to induce selective toxicityin BRCA-deficient cells. Our rational is that since TLS polymerasesrecruitment to sites of DNA damage is a key step for TLS success,we can indirectly monitor TLS efficiency by studying two key markers:1) The mono-ubiquitylation of PCNA and 2) the accumulationof a TLS polymerase into replication foci. Herein, we developed ascreening platform to identify inhibitors of PCNA mono-ubiquitylationthrough a Western-Blot based method.From our first screening with an open source library of kinase inhibitorsfrom GlaxoSmithKline we identified a number of hits thatinhibit mono-ubiquitylation PCNA. Here we describe the validationof these hits using commercial inhibitors, cell cycle analysis, characterizationof DNA damage markers and DNA combing assays toselect targets that impair replication processivity after UV irradiation.Moreover, with the validated hits we performed proof of concept experimentsin HR-deficient cells, unveiling a link between these pathwaysin the promotion of cell survival after UV.