Investigating the roles of Polε in genome stability and sensitization to ATR and PARP inhibitors

Primary supervisor: Roberto Bellelli, Queen Mary University of London

Secondary supervisor: David Michod, UCL

Project

Background

Genomic instability is a major hallmark of cancer and understanding its nature has provided avenues for patient-tailored therapies; the most prominent example has been the successful use of PARP inhibitors in BRCA1-BRCA2 mutant cancers [1]. We and others have recently discovered that loss of the POLE3-POLE4 subunits of DNA Polymerase Epsilon (Polε) leads to increased sensitivity to both PARP and ATR inhibitors [2, 3, 4]. In this project, by using a combination of cell biology approaches, we will dissect the mechanism behind this phenomenon, to exploit POLE3-POLE4, and downstream pathways, as novel markers of sensitivity and therapeutic targets in cancer.

Project description

The advent of PARP inhibitors has significantly changed the treatment landscape of BRCA1-BRCA2 mutant cancers [1]. Despite this, the development of resistance is becoming a challenge in the clinic. Strikingly, we have recently discovered that loss of POLE3-POLE4 leads to increased sensitivity of cancer cells to ATR, CHK1 and PARP inhibitors [4]. Understanding the mechanism behind this phenomenon will help develop new strategies for cancer treatment and overcome PARPi resistance.

By analysing the dynamic of DNA replication we have discovered that treatment with PARPi causes replicative gaps accumulation in POLE3-POLE4 KO cells [4]; what specific function of POLE3-POLE4 is required to prevent replicative DNA damage remains however unknown. To address this question we have performed in collaboration with the CRUK Functional Genomic Centre (Cambridge) a set of genome-wide CRISPR screenings in POLE4 WT and KO cells upon treatment with ATR and PARP inhibitors. These unpublished screenings have defined a set of genes whose loss rescues sensitivity of POLE4 KO cells to these compounds or further affects their viability. During this PhD project, we will investigate the main hits of the CRISPR screenings to unravel the functions of POLE3-POLE4 during unperturbed and perturbed DNA replication and identify downstream pathways that mediate sensitization to ATR and PARP inhibitors.

We will initially validate by siRNA- and CRISPR-based approaches the most relevant hits (e.g. DNA repair factors and epigenetic regulators), prioritizing draggable proteins and/or proteins for which inhibitors are already available. We will subsequently analyse proliferation and sensitivity to ATR and PARP inhibitors upon loss of POLE3-POLE4 and the validated hits. Mechanistically, we will then analyse DNA damage accumulation by confocal microscopy (γH2AX and 53BP1 foci), and the dynamic of DNA replication by DNA fiber approaches, upon loss of the identified hits, in the presence and absence of ATR and PARP inhibitors. Validated hits will be further investigated in BRCA1-BRCA2 mutant and PARP inhibitors resistant models.

In summary, this PhD project will provide a comprehensive training for students interested in genome stability and anti-cancer therapeutics.

References

1. Lord CJ, Ashworth A. PARP inhibitors: Synthetic lethality in the clinic. Science 355, 1152-1158
2. Hustedt N, Álvarez-Quilón A, McEwan A, Yuan JY, Cho T, Koob L, Hart T, Durocher D. A consensus set of genetic vulnerabilities to ATR inhibition. Open Biol. 9, 190156
3. Su D, Feng X, Colic M, Wang Y, Zhang C, Wang C, Tang M, Hart T, Chen J. CRISPR/CAS9-based DNA damage response screens reveal gene-drug interactions. DNA Repair (Amst). 87, 102803
4. Hill BR*, Ozgencil M*, Skingsley SLP, Tomlinson D, Ortueta Eizmendi C, Agnarelli A, Bellelli R. Loss of POLE3-POLE4 unleashes replicative gaps accumulation upon treatment with PARP inhibitors. Cell Reports (under review)