Each round of duplication, mammalian cells experience replication fork arrest. To ensure the continuation of DNA replication, cells evolved multiple different and often redundant mechanisms for the handling and recovery of perturbed DNA replication forks. 

We use cell models, genetic engeneering and proteomics to identify alternative mechanisms of fork recovery that are used when the function of one or more of them is disabled or inhibited. Once identified, we functionally dissect them and investigate the correlation of each one with genome integrity and chemosensitivity of cancer cells. 

We are focusing on two crucial proteins involved in the handling of perturbed replication forks: the WRN exo/helicase, and the RAD52 protein. Additionally, we will extend the scope to other conditions. 

The MUS81 endonuclease is a protein involved in the processing of stalled forks, when they cannot be protected from degradation as occurs in BRCA1/2-deficient cells or in checkpoint-defective conditions. MUS81 is an attractive therapeutic target and a clinically relevant biomarkers for breast cancer. Indeed, MUS81 is often overexpressed in breast as well as ovarian cancer. 

Deregulated activation of MUS81 in S-phase, which can be modelled expressing the constitutively-active S87D-MUS81 allele, results in resistance to the PARP1 inhibitor, Olaparib.

Using cell biology, biochemistry and bioinformatics, we want to determine the molecular mechanisms underlying the resistance to Olaparib, and whether they confer concomitant sensitivity to other targeted approaches. 

The human RAD52 protein is involved in a back-up pathway of replication fork recovery in human cells. Despite RAD52 is often deregulated in human cancers, and its loss is synthetic lethal with that of BRCA genes, the precise role of RAD52 during replication stress is still largely undefined. We showed that, in normal cells, loss of RAD52 triggers extensive degradation of nascent strand by the MRE11 endonuclease, independently from DSB formation and DNA repair. 

We undertake analysis of replication dynamics, fork recovery and nuclear dynamics of fork remodelling factors to provide: 

1. Functional characterization of the human RAD52-dependent replication fork protection mechanisms;

2. Analysis of the RAD52-MUS81 cooperative functions;

3. Functional characterization of the effect of cancer-related mutations of RAD52 on replication fork protection, and impact of RAD52 for genome instability of cancer;

4. Identification of replication fork recovery mechanisms used in the absence of RAD52.

We hypothesize that RAD52 carries out at least two distinct functions at perturbed replication forks: a) protection of perturbed replication forks, which is an entirely novel RAD52 function, and b) recovery of collapsed replication forks. These functions may differently affect genome instability and drug sensitivity of cancer vs. normal cells.

We contributed to unveil a crucial role of the WRN exo/helicase in the recovery from perturbed replication, and identify its crosstalk with the replication checkpoint. 

Using cell models expressing different catalytic and regulatory WRN mutants, we want to determine the function of WRN at perturbed replication forks to identify its role during fork remodeling and regression. Specifically, we want to determine:

1. The function of WRN exonuclese at unperturbed and perturbed replication forks; 

2. The alternative pathways activated when WRN is deregulated;

3. The contribution of WRN-dependent replication fork processing to checkpoint activation at perturbed forks;

4. The functional characterization of the WRN-RPA complex.

Our data will contribute to the understanding of the fork remodeling mechanisms, which are relevant for basic cancer research. Moreover, as WRN inhibitors become to be of interest, and WRN expression can be found deregulated in human cancers, our proposal is expected to provide mechanistic foundations for combined treatments in anticancer therapy, especially in CRC.

The human genome can adopt several non-canonical conformations, including G-quadruplexes (G4s). G4s are not randomly distributed throughout the genome and play regulatory roles in crucial cellular processes such as DNA replication, transcription, recombination, and gene expression. In eukaryotic cells, the persistence of G4s can contribute to replication stress, emphasizing the importance of removing these structures for accurate DNA replication and maintaining genome integrity.  The mechanisms by which these non-canonical DNA structures accumulate and are resolved or removed in cells are not fully understood.

We demonstrate that the Werner helicase-interacting protein 1 (WRNIP1), a fork protective factors, is necessary to prevent the pathological persistence of G4s.

The aims of this study are:

• Analyzing the role of WRNIP1 in preventing G4 accumulation during replication stress;

• Characterizing domains of WRNIP1 required to counteract the pathological persistence of G4s;

• Identifying the mechanisms in which WRNIP1 participates in and contrubutes to the removal of G4s.

We hypothesize that WRNIP1 plays a role in stabilizing replication forks stalled at G4s and collaborates with other factors to removed them, aiming  to mitigate G4/R-loop-mediated transcription-replication conflicts and ultimately protect against genomic instability.

The aim of this study is to define a potential role of WRNIP1 in the molecular mechanisms giving rise to chemoresistance in tumours, particularly those with BRCAness. 

Since WRNIP1 binds RAD51 at perturbed forks, we hypothesize that loss of this WRNIP1 function underlies PARPi sensitivity, impairing RAD51 stabilization and, possibly, gap suppression. 

Given that WRNIP1 is often overexpressed in breast and ovarian cancers, we propose that WRNIP1 might contribute to induce resistance to PARPi in cancer cells, bypassing the need for BRCA2 to stabilise RAD51 at perturbed forks.

To do that, we sake to investigate:

• The mechanisms underlying increased sensitivity to PARPi in WRNIP1-deficient cells;

• A possible role of WRNIP1 in inducing chemoresistance in BRCA2-deficient or mutant cells;

• Analysis of the relevance of WRNIP1 levels for the onset of resistance to PARPi in cancer cells.

Clarify the role of WRNIP1 as a new factor modulating chemosensitivity in BRCAness cancers is pivotal for understanding the intricate molecular pathways influencing the response to chemotherapy in these specific cancer types.

We expect to pave the way for a rational evaluation of WRNIP1 status as a therapy biomarker and for further mechanistic studies aimed at evaluating WRNIP1 as a therapeutic target for use in combination with PARPi and a WRNIP1 inhibitor.