Screening of PCNA ubiquitylation inhibitors as novel synthetic lethality inducers in Homologous-Recombination-deficient cancer cells

GARCÍA, IRIS ALEJANDRA*; VILLAFAÑEZ, FLORENCIA*; QUIROGA, RODRIGO*; PANSA, MARÍA FLORENCIA; CARBAJOSA, SOFIA; MANSILLA, SABRINA; JACOBS, HEINZ; GOTTIFREDI, VANESA; BOCCO, JOSE LUIS; VILLARREAL, MARCOS; SORIA, GASTÓN

Egmond aan Zee

Congreso; IABCR 2019 31st International Association for Breast Cancer Research Conference; 2019

International Association for Breast Cancer Research

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. In this work we show the development of two complementary screenings assays to identify small molecules that inhibit PCNA ubiquitylation, a key post-translational modification required for efficient TLS activation. First, we setup a miniaturized WB assay with dual antibodies for ubi-PCNA and total PCNA coupled to infrared laser scanning. Second, we designed a virtual screening strategy to identify direct blockers of PCNA-ubiquitylation using molecular modeling. Herein, we present the identification of strong PCNA ubiquitylation inhibitors using both strategies. Moreover, using multiple experimental models we performed proof-of-concept experiments showing that the abolishment of PCNA ubiquitylation triggers synthetic lethality in HR-deficient cells submitted to UV irradiation and Cisplatin treatment. Collectively, these findings set the ground for developing a first-in-class type of therapeutic approach, where TLS inhibition can be achieved by the pharmacological inhibition of PCNA ubiquitylation. Given the vast incidence of HR deficiencies in multiple types of human cancers (i.e. Breast and Ovarian cancer), the pharmacological inhibition of TLS could become a niche for drug discovery as an alternative targeted therapy to treat these malignancies.