DNA Metabolism: From Hereditary Cancer Syndromes to Targeted Therapy
Approximately 5-10% of all breast cancers and 15-20% of all ovarian cancers arise in families carrying inherited mutations in the BRCA1 or BRCA2 tumour suppressor genes. These same genes, together with RAD51, PALB2, and the Fanconi anaemia pathway components, are also mutated in familial forms of pancreatic cancer, prostate cancer, and childhood bone marrow failure syndromes. Despite major clinical advances, patients with these hereditary cancer syndromes still face two critical challenges: why do their healthy cells tolerate decades of impaired DNA repair before a tumour emerges, and why do many of their tumours eventually resist the very therapies, such as PARP inhibitors and platinum-based chemotherapy, designed to exploit their specific genetic vulnerability? Our laboratory addresses both questions by investigating the fundamental mechanisms through which BRCA2 and RAD51 protect replicating DNA from breakage and degradation.
Every time a cell divides, billions of nucleotides must be copied with extraordinary fidelity. When this process encounters obstacles, a condition known as replication stress, BRCA2 and RAD51 safeguard the exposed nascent DNA at replication forks. When these proteins are missing or mutated, DNA replicaiton proceeds discontinuouysly accumulating gaps, unprotected forks collapse, chromosomes shatter, and the resulting genome instability fuels the transition from normal tissue to cancer.
Understanding how replication fork integrity works and how cancer cells rewire it to survive therapy is the central mission of our laboratory. Our discoveries have direct translational implications for improving the treatment of patients with BRCA-associated hereditary cancers and for identifying individuals who carry variants of uncertain significance (VUS) in DNA repair genes, whose cancer risk remains difficult to predict.
To understand how cancer-predisposing mutations disrupt DNA replication, we use a unique experimental platform that combines cell-free biochemistry with DNA electron microscopy (EM) and cryo-electron microscopy (cryo-EM). Using cell-free extracts, we can reconstitute the entire process of DNA replication in a test tube, selectively deplete cancer-relevant proteins such as BRCA2 or RAD51, and then directly visualise the resulting structural damage at replication forks. No other experimental system offers this combination of molecular control and nanometre-scale imaging.
We complement this approach with single-molecule DNA fibre assays and fluorescence microscopy in human cancer cell lines, CRISPR-engineered cellular models of hereditary cancer mutations, CRISPR-based screenings and quantitative proteomics. By moving between reconstituted biochemistry and living cells, we bridge the gap between molecular mechanism and clinical phenotype, identifying the precise molecular events that distinguish a tumor suppressor mutation carrier’s vulnerable cell from a cell that has already become malignant and therapy-resistant.
Why BRCA2 and RAD51 Loss Drives Cancer: Replication Fork Protection as a Tumour Suppressor Mechanism
For decades, the tumour suppressor function of BRCA2 was attributed solely to its role in homologous recombination, the repair of DNA double-strand breaks. Our laboratory made a paradigm-shifting discovery: BRCA2 and RAD51 have a second, independent function in protecting newly replicated DNA from nucleolytic degradation at stalled replication forks (Hashimoto et al., Nat Struct Mol Biol, 2010). We showed that when RAD51 is absent, the nuclease MRE11 attacks and degrades the nascent DNA strands, creating single-stranded DNA gaps and chromosomal instability, the very hallmarks of BRCA-mutated cancers.
This discovery, now confirmed by several laboratories worldwide, fundamentally changed our understanding of why BRCA2 mutation carriers develop cancer. It revealed that genome instability in hereditary breast and ovarian cancer does not arise only from unrepaired breaks, but also from a chronic failure to protect replicating DNA during every cell division. Critically, this replication fork protection function is recognised as a major determinant of PARP inhibitor sensitivity and resistance in the clinic: tumours that restore fork protection become resistant to olaparib and other PARP inhibitors, even when homologous recombination remains defective.
We subsequently revealed the molecular architecture of this process: the fork-remodelling enzyme SMARCAL1 reverses stalled forks into “chicken-foot” structures, exposing the nascent DNA to MRE11-dependent degradation when BRCA2 is absent (Kolinjivadi et al., Mol Cell, 2017). These findings identified SMARCAL1 as a potential therapeutic target in BRCA-deficient tumours that have acquired PARP inhibitor resistance through restored fork protection.
Overcoming Drug Resistance: ssDNA Gaps as the Achilles Heel of BRCA-Deficient Cancers
The emergence of resistance to PARP inhibitors, now standard-of-care for patients with BRCA-mutated ovarian, breast, pancreatic, and prostate cancers, is a major clinical problem. Our recent work revealed an entirely new vulnerability: BRCA2-deficient cells accumulate aberrant single-stranded DNA (ssDNA) gaps on the lagging strand during replication, and the error-prone polymerase POLθ (POLQ) fills these gaps as a survival mechanism to prevent fork collapse (Mann et al., Mol Cell, 2022).
This finding has immediate clinical significance. POLθ is already the target of drugs in clinical development (e.g., novobiocin, ART558). Our work provides the mechanistic rationale for combining PARP inhibitors with POLθ inhibitors in BRCA-deficient cancers, attacking both homologous recombination and the backup gap-filling pathway simultaneously to overcome resistance. More broadly, we showed that ssDNA gap accumulation, leading to fork breakage, may be the primary toxic lesion in BRCA-deficient cells, reshaping the therapeutic logic for this entire class of hereditary cancers. Fork breakge was shown to highly frequent for the first time in BRCA-defective cells.
Why Do BRCA Carriers Accumulate DNA Damage? RAD51 as a Guardian Against Endogenous Mutagenesis
A longstanding mystery in hereditary cancer biology is how BRCA mutation carriers accumulate the additional somatic mutations needed to initiate a tumour. Our most recent discovery provides a striking answer: RAD51 directly recognises and protects abasic sites, the most common endogenous DNA lesion, arising tens of thousands of times per cell per day from spontaneous base loss, oxidative damage, and enzymatic removal of damaged bases (Hanthi et al., Mol Cell, 2024).
Using cryo-EM, we solved the structure of the RAD51 nucleofilament bound to abasic DNA, revealing how the protein’s L1 and L2 loop domains physically seal the gap left by the missing base, preventing the nuclease MRE11 from cleaving the DNA backbone, thus inhibting replication fork breakage. When BRCA2 or RAD51 are impaired, abasic sites persist and accumulate, replication forks encounter them and break, and the resulting base changes and chromosomal rearrangements drive the mutational signatures that characterise BRCA-associated breast and ovarian tumours.
Chromosomal Instability and Centromere Fragility in Cancer
Chromosomal instability (CIN), the gain or loss of whole chromosomes or large chromosomal segments, is a hallmark of the most aggressive and therapy-resistant solid tumours. Centromeres, the chromosomal regions that ensure accurate chromosome segregation during cell division, are built from highly repetitive alpha-satellite DNA that poses extraordinary challenges to the replication machinery.
We developed the first cell-free system capable of reconstituting replication of defined human centromeric sequences, and revealed that this repetitive DNA spontaneously forms DNA loop structures that suppress the ATR replication checkpoint (Aze et al., Nat Cell Biol, 2016). This checkpoint suppression may explain why centromeres are common fragile sites in cancer cells under replication stress as they fail to activate the protective signalling that would normally slow replication and prevent breakage. When centromeres break, the resulting whole-chromosome mis-segregation generates the aneuploidy that drives tumour heterogeneity and therapy resistance. Our ongoing work investigates how replication of centromeric DNA is coordinated with chromosome segregation, and how defects in this process contribute to CIN in colorectal, ovarian, and triple-negative breast cancers.
ATM Deficiency: From Ataxia Telangiectasia to Cancer Predisposition and Metabolic Vulnerability
Biallelic mutations in the ATM kinase cause Ataxia Telangiectasia (A-T), a devastating childhood syndrome characterised by neurodegeneration, immunodeficiency, and a dramatically elevated risk of leukaemia and lymphoma. Heterozygous ATM carriers, who represent approximately 1–3% of the general population, are thought to face a 2-3 fold increased risk of breast cancer, making ATM one of the most clinically important moderate-penetrance cancer susceptibility genes.
We are discovering that ATM plays a previously unrecognised role in cellular metabolism beyond its canonical DNA damage signalling function. Our recent findings show that ATM-deficient cells exhibit widespread metabolic reprogramming including glycogen accumulation, impaired mitochondrial respiration, and premature senescence. These metabolic defects may explain the multi-organ pathology of A-T and, critically, they reveal metabolic vulnerabilities that could be targeted pharmacologically, both in A-T patients and in the growing number of ATM-mutated cancers (including up to 10% of mantle cell lymphomas, prostate cancers, and pancreatic cancers) now being identified through clinical genome sequencing.
DNA Hypomethylation, Embryonic Gene Reactivation, and Attenuation of DNA repair Dependency in Cancer
Global DNA hypomethylation and DNA replication stress are a hallmark of virtually all cancers. We proposed that the resulting de-repression of embryonic and trophoblast gene programmes, which normally drive tissue invasion, angiogenesis, and immune evasion at the placental-maternal interface, triggered by DNA replication stress in hypomethylated stemm cells (Atashpaz et al Elife 2020), provides a unifying mechanism through which genetically diverse tumours acquire stereotypical malignant behaviours (Costanzo, et al Open Biol, 2018). We discovered that a key mediator of this switch is the chromatin remodeller SSRP1-SPT16: by evicting linker histone H1, SSRP1-SPT16 converts somatic chromatin to an embryonic-like state with dramatically increased replication origin density and accelerated cell cycles (Falbo et al., Nat Commun, 2020). SSRP1 is overexpressed in multiple aggressive cancers, where it may recapitulate this embryonic replication programme.
Critically, the high origin density of embryonic-like chromatin shortens inter-fork distances, reducing the stretches of nascent DNA that require RAD51/BRCA2-mediated protection. Cancer cells that reactivate this programme may therefore attenuate their dependency on DNA repair and fork protection, a potential mechanism of therapy resistance operating through epigenetic reprogramming rather than genetic BRCA reversion. We are testing whether inhibitors of replication origin assembly that selectively target SSRP1-overexpressing cells can resensitise resistant triple-negative breast, high-grade serous ovarian, and metastatic pancreatic cancers to PARP inhibitors and platinum chemotherapy.
ERC Starting Grant (2007-2012): Mechanisms of genome stability in vertebrates. 1.5 Million Euros.
ERC Consolidator Grant (2014-2019): Molecular mechanisms of replication fork protection. 2.5 Million Euros.
Giovanni Armenise-Harvard Career Development Award (2013): DNA metabolism and genome stability.
Lister Institute Prize for Preventive Medicine (2006): Research excellence award.
AIRC Investigator Grants (2014-2029): Consecutive grants supporting DNA replication and repair research in cancer.
Fondo Italiano per la Scienza FIS3 (2026): 1.9 Million Euros.
Photogallery
EM image of replication fork isolated from cells defective for BRCA2 showing a ssDNA gap (arrow) at fork junction (left). A typical reversed fork induced by replicaiton fork stalling (right) (Kolinjivadi et al, Molecular Cell 2017)
EM image of replication fork isolated from cells defective for RAD51 showing ssDNA gaps behid forks (white arrow) at fork junction (black arrow) (Hashimoto et al, NSBM 2010)
Immunofluoresce of H2AX foci in BRCA2-/- cancer cells that were untreated (left columns) or treated with APE1 inhibitor (right panels) (Hanthi et al, Mol Cell 2024)
CryoEM images of RAD51 nucleofilaments bound to single stranded (ss)DNA or double stranded ds(DNA)
CryoEM images of RAD51 nucleofilaments bound to single stranded (ss)DNA containing abasic sites (red) (Hanthi et al, Mol Cell 2024)
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