Abstract 1272: <i>In vitro</i> cellular profiling of AZD5305, novel PARP1-selective inhibitor and trapper (2021)

Abstract PARP inhibitors (PARPi) have demonstrated clinical efficacy in cancers with defects in the homologous recombination repair (HRR) pathway. Recent advances in the understanding of the PARPi mechanism of action via stabilization of the PARP-DNA complex (trapping) and of the biological roles of the different PARP family members led to the development of AZD5305, a next generation, potent and selective PARP1 inhibitor and trapper. In this work, we disclose for the first time the profiling of AZD5305 in cellular models and how its activity differentiates from other PARPi. AZD5305 was able to potently inhibits overall PARylation in A549 cell line with IC50 of 3 nM, and to target PARP1 ~500 times more potently than PARP2 in A549 PARP1-KO, confirming its selectivity in cells. With our novel, sensitive and high-throughput immunofluorescence-based assay, we tested the ability of PARPi to trap PARP1 or PARP2 onto the chromatin of damaged or undamaged cells; olaparib, talazoparib and veliparib will be presented as comparative examples of non-selective PARPi. Unlike all the current clinical PARPi, AZD5305 was able to selectively induce PARP1 trapping upon treatments with concentrations as low as nanomolar (nM), whereas PARP2-trapping was not observed at any of the tested conditions. These optimal profiles of AZD5305 translated into greatly improved targeted anticancer effects in vitro compared to all other PARPi. In BRCA mutant (BRCAm) cells, treatments with AZD5305 led to antiproliferative IC50 in the single-digit nM, whilst there was no- or minimal effect in the isogenic paired BRCA wild type (BRCAwt) cells after treatments with double-digit µM concentrations. We further explored the effects of AZD5305 in genetic backgrounds “beyond BRCAm” and confirmed its superior antiproliferative and selective activity, particularly in cells isogenic for relevant genes in the HRR pathway, like PALB2 and RAD51. Screening of AZD5305 and other PARPi in larger cell lines panels revealed a differential clustering of AZD5305-treated cells between the sensitive versus the insensitive ones; this indicates that AZD5305 is also a unique instrumental tool to explore and refine selective PARP1-related activities in cancer cells, and the effects of targeting them, in different genetic backgrounds. With this goal, we are currently performing CRISPR/Cas9 screens to identify genes that, upon downregulation, cause sensitization to AZD5305. Preliminary results of these screens will be presented here. In summary, the optimal PARP1 inhibition and trapping profile of AZD5305 in cells demonstrated that AZD5305 is a next generation PARPi, with great potentials to become the best in class and deliver a markedly improved therapeutic index in the clinic. Citation Format: Giuditta Illuzzi, Lisa McWilliams, Kunzah Jamal, Alessandro Galbiati, Sabrina Bentouati, Daniel Griffiths, Elisabetta Leo. In vitro cellular profiling of AZD5305, novel PARP1-selective inhibitor and trapper [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1272.