BRCAness, DNA gaps, and gain and loss of PARP inhibitor-induced synthetic lethality.

Mutations in the tumor-suppressor genes BRCA1 and BRCA2 resulting in BRCA1/2 deficiency are frequently identified in breast, ovarian, prostate, pancreatic, and other cancers. Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPis) selectively kill BRCA1/2-deficient cancer cells by inducing synthetic lethality, providing an effective biomarker-guided strategy for targeted cancer therapy. However, a substantial fraction of cancer patients carrying BRCA1/2 mutations do not respond to PARPis, and most patients develop resistance to PARPis over time, highlighting a major obstacle to PARPi therapy in the clinic. Recent studies have revealed that changes of specific functional defects of BRCA1/2-deficient cells, particularly their defects in suppressing and protecting single-stranded DNA gaps, contribute to the gain or loss of PARPi-induced synthetic lethality. These findings not only shed light on the mechanism of action of PARPis, but also lead to revised models that explain how PARPis selectively kill BRCA-deficient cancer cells. Furthermore, new mechanistic principles of PARPi sensitivity and resistance have emerged from these studies, generating potentially useful guidelines for predicting the PARPi response and design therapies for overcoming PARPi resistance. In this Review, we will discuss these recent studies and put them in context with the classic views of PARPi-induced synthetic lethality, aiming to stimulate the development of new therapeutic strategies to overcome PARPi resistance and improve PARPi therapy.

Regulation, functional impact, and therapeutic targeting of APOBEC3A in cancer.

Enzymes of the apolipoprotein B mRNA editing catalytic polypeptide like (APOBEC) family are cytosine deaminases that convert cytosine to uracil in DNA and RNA. Among these proteins, APOBEC3 sub-family members, APOBEC3A (A3A) and APOBEC3B (A3B), are prominent sources of mutagenesis in cancer cells. The aberrant expression of A3A and A3B in cancer cells leads to accumulation of mutations with specific single-base substitution (SBS) signatures, characterized by C→T and C→G changes, in a number of tumor types. In addition to fueling mutagenesis, A3A and A3B, particularly A3A, induce DNA replication stress, DNA damage, and chromosomal instability through their catalytic activities, triggering a range of cellular responses. Thus, A3A/B have emerged as key drivers of genome evolution during cancer development, contributing to tumorigenesis, tumor heterogeneity, and therapeutic resistance. Yet, the expression of A3A/B in cancer cells presents a cancer vulnerability that can be exploited therapeutically. In this review, we discuss the recent studies that shed light on the mechanisms regulating A3A expression and the impact of A3A in cancer. We also review recent advances in the development of A3A inhibitors and provide perspectives on the future directions of A3A research.

A RAD18-UBC13-PALB2-RNF168 axis mediates replication fork recovery in BRCA1-deficient cancer cells.

BRCA1/2 proteins function in genome stability by promoting repair of double-stranded DNA breaks through homologous recombination and by protecting stalled replication forks from nucleolytic degradation. In BRCA1/2-deficient cancer cells, extensively degraded replication forks can be rescued through distinct fork recovery mechanisms that also promote cell survival. Here, we identified a novel pathway mediated by the E3 ubiquitin ligase RAD18, the E2-conjugating enzyme UBC13, the recombination factor PALB2, the E3 ubiquitin ligase RNF168 and PCNA ubiquitination that promotes fork recovery in BRCA1- but not BRCA2-deficient cells. We show that this pathway does not promote fork recovery by preventing replication fork reversal and degradation in BRCA1-deficient cells. We propose a mechanism whereby the RAD18-UBC13-PALB2-RNF168 axis facilitates resumption of DNA synthesis by promoting re-annealing of the complementary single-stranded template strands of the extensively degraded forks, thereby allowing re-establishment of a functional replication fork. We also provide preliminary evidence for the potential clinical relevance of this novel fork recovery pathway in BRCA1-mutated cancers, as RAD18 is over-expressed in BRCA1-deficient cancers, and RAD18 loss compromises cell viability in BRCA1-deficient cancer cells.

Unprocessed genomic uracil as a source of DNA replication stress in cancer cells.

Alterations of bases in DNA constitute a major source of genomic instability. It is believed that base alterations trigger base excision repair (BER), generating DNA repair intermediates interfering with DNA replication. Here, we show that genomic uracil, a common type of base alteration, induces DNA replication stress (RS) without being processed by BER. In the absence of uracil DNA glycosylase (UNG), genomic uracil accumulates to high levels, DNA replication forks slow down, and PrimPol-mediated repriming is enhanced, generating single-stranded gaps in nascent DNA. ATR inhibition in UNG-deficient cells blocks the repair of uracil-induced gaps, increasing replication fork collapse and cell death. Notably, a subset of cancer cells upregulates UNG2 to suppress genomic uracil and limit RS, and these cancer cells are hypersensitive to co-treatment with ATR inhibitors and drugs increasing genomic uracil. These results reveal unprocessed genomic uracil as an unexpected source of RS and a targetable vulnerability of cancer cells.

Meiotic protein SYCP2 confers resistance to DNA-damaging agents through R-loop-mediated DNA repair.

Drugs targeting the DNA damage response (DDR) are widely used in cancer therapy, but resistance to these drugs remains a major clinical challenge. Here, we show that SYCP2, a meiotic protein in the synaptonemal complex, is aberrantly and commonly expressed in breast and ovarian cancers and associated with broad resistance to DDR drugs. Mechanistically, SYCP2 enhances the repair of DNA double-strand breaks (DSBs) through transcription-coupled homologous recombination (TC-HR). SYCP2 promotes R-loop formation at DSBs and facilitates RAD51 recruitment independently of BRCA1. SYCP2 loss impairs RAD51 localization, reduces TC-HR, and renders tumors sensitive to PARP and topoisomerase I (TOP1) inhibitors. Furthermore, our studies of two clinical cohorts find that SYCP2 overexpression correlates with breast cancer resistance to antibody-conjugated TOP1 inhibitor and ovarian cancer resistance to platinum treatment. Collectively, our data suggest that SYCP2 confers cancer cell resistance to DNA-damaging agents by stimulating R-loop-mediated DSB repair, offering opportunities to improve DDR therapy.

APOBEC3A induces DNA gaps through PRIMPOL and confers gap-associated therapeutic vulnerability.

Mutation signatures associated with apolipoprotein B mRNA editing catalytic polypeptide-like 3A/B (APOBEC3A/B) cytidine deaminases are prevalent across cancers, implying their roles as mutagenic drivers during tumorigenesis and tumor evolution. APOBEC3A (A3A) expression induces DNA replication stress and increases the cellular dependency on the ataxia telangiectasia and Rad3-related (ATR) kinase for survival. Nonetheless, how A3A induces DNA replication stress remains unclear. We show that A3A induces replication stress without slowing replication forks. We find that A3A induces single-stranded DNA (ssDNA) gaps through PrimPol-mediated repriming. A3A-induced ssDNA gaps are repaired by multiple pathways involving ATR, RAD51, and translesion synthesis. Both ATR inhibition and trapping of poly(ADP-ribose) polymerase (PARP) on DNA by PARP inhibitor impair the repair of A3A-induced gaps, preferentially killing A3A-expressing cells. When used in combination, PARP and ATR inhibitors selectively kill A3A-expressing cells synergistically in a manner dependent on PrimPol-generated gaps. Thus, A3A-induced replication stress arises from PrimPol-generated ssDNA gaps, which confer a therapeutic vulnerability to gap-targeted DNA repair inhibitors.

ATR inhibition induces synthetic lethality in mismatch repair-deficient cells and augments immunotherapy.

The mismatch repair (MMR) deficiency of cancer cells drives mutagenesis and offers a useful biomarker for immunotherapy. However, many MMR-deficient (MMR-d) tumors do not respond to immunotherapy, highlighting the need for alternative approaches to target MMR-d cancer cells. Here, we show that inhibition of the ATR kinase preferentially kills MMR-d cancer cells. Mechanistically, ATR inhibitor (ATRi) imposes synthetic lethality on MMR-d cells by inducing DNA damage in a replication- and MUS81 nuclease-dependent manner. The DNA damage induced by ATRi is colocalized with both MSH2 and PCNA, suggesting that it arises from DNA structures recognized by MMR proteins during replication. In syngeneic mouse models, ATRi effectively reduces the growth of MMR-d tumors. Interestingly, the antitumor effects of ATRi are partially due to CD8+ T cells. In MMR-d cells, ATRi stimulates the accumulation of nascent DNA fragments in the cytoplasm, activating the cGAS-mediated interferon response. The combination of ATRi and anti-PD-1 antibody reduces the growth of MMR-d tumors more efficiently than ATRi or anti-PD-1 alone, showing the ability of ATRi to augment the immunotherapy of MMR-d tumors. Thus, ATRi selectively targets MMR-d tumor cells by inducing synthetic lethality and enhancing antitumor immunity, providing a promising strategy to complement and augment MMR deficiency-guided immunotherapy.

ATR promotes clearance of damaged DNA and damaged cells by rupturing micronuclei.

The human ataxia telangiectasia mutated and Rad3-related (ATR) kinase functions in the nucleus to protect genomic integrity. Micronuclei (MN) arise from genomic and chromosomal instability and cause aneuploidy and chromothripsis, but how MN are removed is poorly understood. Here, we show that ATR is active in MN and promotes their rupture in S phase by phosphorylating Lamin A/C at Ser395, which primes Ser392 for CDK1 phosphorylation and destabilizes the MN envelope. In cells harboring MN, ATR or CDK1 inhibition reduces MN rupture. Consequently, ATR inhibitor (ATRi) diminishes activation of the cytoplasmic DNA sensor cGAS and compromises cGAS-dependent autophagosome accumulation in MN and clearance of micronuclear DNA. Furthermore, ATRi reduces cGAS-mediated senescence and killing of MN-bearing cancer cells by natural killer cells. Thus, in addition to the canonical ATR signaling pathway, an ATR-CDK1-Lamin A/C axis promotes MN rupture to clear damaged DNA and cells, protecting the genome in cell populations through unexpected cell-autonomous and cell-non-autonomous mechanisms.

The RNA m5C modification in R-loops as an off switch of Alt-NHEJ.

The roles of R-loops and RNA modifications in homologous recombination (HR) and other DNA double-stranded break (DSB) repair pathways remain poorly understood. Here, we find that DNA damage-induced RNA methyl-5-cytosine (m5C) modification in R-loops plays a crucial role to regulate PARP1-mediated poly ADP-ribosylation (PARylation) and the choice of DSB repair pathways at sites of R-loops. Through bisulfite sequencing, we discover that the methyltransferase TRDMT1 preferentially generates m5C after DNA damage in R-loops across the genome. In the absence of m5C, R-loops activate PARP1-mediated PARylation both in vitro and in cells. Concurrently, m5C promotes transcription-coupled HR (TC-HR) while suppressing PARP1-dependent alternative non-homologous end joining (Alt-NHEJ), favoring TC-HR over Alt-NHEJ in transcribed regions as the preferred repair pathway. Importantly, simultaneous disruption of both TC-HR and Alt-NHEJ with TRDMT1 and PARP or Polymerase θ inhibitors prevents alternative DSB repair and exhibits synergistic cytotoxic effects on cancer cells, suggesting an effective strategy to exploit genomic instability in cancer therapy.

An ATR-PrimPol pathway confers tolerance to oncogenic KRAS-induced and heterochromatin-associated replication stress.

Activation of the KRAS oncogene is a source of replication stress, but how this stress is generated and how it is tolerated by cancer cells remain poorly understood. Here we show that induction of KRASG12V expression in untransformed cells triggers H3K27me3 and HP1-associated chromatin compaction in an RNA transcription dependent manner, resulting in replication fork slowing and cell death. Furthermore, elevated ATR expression is necessary and sufficient for tolerance of KRASG12V-induced replication stress to expand replication stress-tolerant cells (RSTCs). PrimPol is phosphorylated at Ser255, a potential Chk1 substrate site, under KRASG12V-induced replication stress and promotes repriming to maintain fork progression and cell survival in an ATR/Chk1-dependent manner. However, ssDNA gaps are generated at heterochromatin by PrimPol-dependent repriming, leading to genomic instability. These results reveal a role of ATR-PrimPol in enabling precancerous cells to survive KRAS-induced replication stress and expand clonally with accumulation of genomic instability.

ATR protects ongoing and newly assembled DNA replication forks through distinct mechanisms.

The ATR kinase safeguards genomic integrity during S phase, but how ATR protects DNA replication forks remains incompletely understood. Here, we combine four distinct assays to analyze ATR functions at ongoing and newly assembled replication forks upon replication inhibition by hydroxyurea. At ongoing forks, ATR inhibitor (ATRi) increases MRE11- and EXO1-mediated nascent DNA degradation from PrimPol-generated, single-stranded DNA (ssDNA) gaps. ATRi also exposes template ssDNA through fork uncoupling and nascent DNA degradation. Electron microscopy reveals that ATRi reduces reversed forks by increasing gap-dependent nascent DNA degradation. At new forks, ATRi triggers MRE11- and CtIP-initiated template DNA degradation by EXO1, exposing nascent ssDNA. Upon PARP inhibition, ATRi preferentially exacerbates gap-dependent nascent DNA degradation at ongoing forks in BRCA1/2-deficient cells and disrupts the restored gap protection in BRCA1-deficient, PARP-inhibitor-resistant cells. Thus, ATR protects ongoing and new forks through distinct mechanisms, providing an extended view of ATR's functions in stabilizing replication forks.

Heterochromatin-Dependent Replication Stress: A Lesson from IDH1/2 Mutants.

Oncogenic point mutants of isocitrate dehydrogenases 1 and 2 (IDH2) generate 2-hydroxyglutarate, which inhibits lysine demethylases and increases heterochromatin. Tumor cells expressing IDH mutants are sensitive to PARP inhibitors (PARPi), offering an opportunity to eliminate IDH-driven tumor cells in therapy. Expression of an oncogenic IDH1 mutant in cells leads to aberrant heterochromatin formation at DNA breaks and impairs DNA repair through homologous recombination (HR), providing a possible explanation for the PARPi sensitivity of IDH mutant cells. However, a recent study published in Molecular Cell shows that IDH mutant tumors do not display the genomic alterations associated with HR defects. Instead, IDH mutants induce heterochromatin-dependent DNA replication stress. Furthermore, PARP is activated by the replication stress induced by IDH mutants and required for suppressing the ensuing DNA damage, providing an alternative model to explain the susceptibility of IDH mutant cells to PARPis. This study presents a new example of oncogene-induced and heterochromatin-dependent replication stress, and a role of PARP in the response to the stress, extending the molecular basis for PARP-targeted therapy.

Therapy-induced APOBEC3A drives evolution of persistent cancer cells.

Acquired drug resistance to anticancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified1-4, the underlying molecular mechanisms shaping tumour evolution during treatment are incompletely understood. Genomic profiling of patient tumours has implicated apolipoprotein B messenger RNA editing catalytic polypeptide-like (APOBEC) cytidine deaminases in tumour evolution; however, their role during therapy and the development of acquired drug resistance is undefined. Here we report that lung cancer targeted therapies commonly used in the clinic can induce cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug-tolerant cancer cells persisting during therapy. Therapy-induced A3A promotes the formation of double-strand DNA breaks, increasing genomic instability in drug-tolerant persisters. Deletion of A3A reduces APOBEC mutations and structural variations in persister cells and delays the development of drug resistance. APOBEC mutational signatures are enriched in tumours from patients with lung cancer who progressed after extended responses to targeted therapies. This study shows that induction of A3A in response to targeted therapies drives evolution of drug-tolerant persister cells, suggesting that suppression of A3A expression or activity may represent a potential therapeutic strategy in the prevention or delay of acquired resistance to lung cancer targeted therapy.

A noncanonical enzymatic function of PIWIL4 maintains genomic integrity and leukemic growth in AML.

RNA-binding proteins (RBPs) form a large and diverse class of factors, many members of which are overexpressed in hematologic malignancies. RBPs participate in various processes of messenger RNA (mRNA) metabolism and prevent harmful DNA:RNA hybrids or R-loops. Here, we report that PIWIL4, a germ stem cell-associated RBP belonging to the RNase H-like superfamily, is overexpressed in patients with acute myeloid leukemia (AML) and is essential for leukemic stem cell function and AML growth, but dispensable for healthy human hematopoietic stem cells. In AML cells, PIWIL4 binds to a small number of known piwi-interacting RNA. Instead, it largely interacts with mRNA annotated to protein-coding genic regions and enhancers that are enriched for genes associated with cancer and human myeloid progenitor gene signatures. PIWIL4 depletion in AML cells downregulates the human myeloid progenitor signature and leukemia stem cell (LSC)-associated genes and upregulates DNA damage signaling. We demonstrate that PIWIL4 is an R-loop resolving enzyme that prevents R-loop accumulation on a subset of AML and LSC-associated genes and maintains their expression. It also prevents DNA damage, replication stress, and activation of the ATR pathway in AML cells. PIWIL4 depletion potentiates sensitivity to pharmacological inhibition of the ATR pathway and creates a pharmacologically actionable dependency in AML cells.

CRISPR screens reveal genetic determinants of PARP inhibitor sensitivity and resistance in prostate cancer.

Prostate cancer harboring BRCA1/2 mutations are often exceptionally sensitive to PARP inhibitors. However, genomic alterations in other DNA damage response genes have not been consistently predictive of clinical response to PARP inhibition. Here, we perform genome-wide CRISPR-Cas9 knockout screens in BRCA1/2-proficient prostate cancer cells and identify previously unknown genes whose loss has a profound impact on PARP inhibitor response. Specifically, MMS22L deletion, frequently observed (up to 14%) in prostate cancer, renders cells hypersensitive to PARP inhibitors by disrupting RAD51 loading required for homologous recombination repair, although this response is TP53-dependent. Unexpectedly, loss of CHEK2 confers resistance rather than sensitivity to PARP inhibition through increased expression of BRCA2, a target of CHEK2-TP53-E2F7-mediated transcriptional repression. Combined PARP and ATR inhibition overcomes PARP inhibitor resistance caused by CHEK2 loss. Our findings may inform the use of PARP inhibitors beyond BRCA1/2-deficient tumors and support reevaluation of current biomarkers for PARP inhibition in prostate cancer.

TERRA and RAD51AP1 promote alternative lengthening of telomeres through an R- to D-loop switch.

Alternative lengthening of telomeres (ALT), a telomerase-independent process maintaining telomeres, is mediated by break-induced replication (BIR). RAD52 promotes ALT by facilitating D-loop formation, but ALT also occurs through a RAD52-independent BIR pathway. Here, we show that the telomere non-coding RNA TERRA forms dynamic telomeric R-loops and contributes to ALT activity in RAD52 knockout cells. TERRA forms R-loops in vitro and at telomeres in a RAD51AP1-dependent manner. The formation of R-loops by TERRA increases G-quadruplexes (G4s) at telomeres. G4 stabilization enhances ALT even when TERRA is depleted, suggesting that G4s act downstream of R-loops to promote BIR. In vitro, the telomeric R-loops assembled by TERRA and RAD51AP1 generate G4s, which persist after R-loop resolution and allow formation of telomeric D-loops without RAD52. Thus, the dynamic telomeric R-loops formed by TERRA and RAD51AP1 enable the RAD52-independent ALT pathway, and G4s orchestrate an R- to D-loop switch at telomeres to stimulate BIR.

Protocol to stimulate and delineate alternative lengthening of telomeres in human U2OS cells.

Alternative lengthening of telomeres (ALT) is a telomerase-independent but recombination-dependent pathway that maintains telomeres. Here, we describe a protocol to stimulate the formation of ALT-associated PML bodies (APBs) and ALT activity by tethering PML-IV to telomeres in human U2OS cells. Through immunofluorescence, in situ hybridization, and microscopy, we analyze dynamics of telomere clustering, visualize recruitment of DNA repair proteins to APBs, and measure telomere DNA synthesis during ALT. This protocol provides a unique approach to delineate the ALT pathway. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021).

Condensates induced by transcription inhibition localize active chromatin to nucleoli.

The proper function of the genome relies on spatial organization of DNA, RNA, and proteins, but how transcription contributes to the organization is unclear. Here, we show that condensates induced by transcription inhibition (CITIs) drastically alter genome spatial organization. CITIs are formed by SFPQ, NONO, FUS, and TAF15 in nucleoli upon inhibition of RNA polymerase II (RNAPII). Mechanistically, RNAPII inhibition perturbs ribosomal RNA (rRNA) processing, releases rRNA-processing factors from nucleoli, and enables SFPQ to bind rRNA. While accumulating in CITIs, SFPQ/TAF15 remain associated with active genes and tether active chromatin to nucleoli. In the presence of DNA double-strand breaks (DSBs), the altered chromatin compartmentalization induced by RNAPII inhibition increases gene fusions in CITIs and stimulates the formation of fusion oncogenes. Thus, proper RNAPII transcription and rRNA processing prevent the altered compartmentalization of active chromatin in CITIs, suppressing the generation of gene fusions from DSBs.