The levels of p53 govern the hierarchy of DNA damage tolerance pathway usage.

It is well-established that, through canonical functions in transcription and DNA repair, the tumor suppressor p53 plays a central role in safeguarding cells from the consequences of DNA damage. Recent data retrieved in tumor and stem cells demonstrated that p53 also carries out non-canonical functions when interacting with the translesion synthesis (TLS) polymerase iota (POLι) at DNA replication forks. This protein complex triggers a DNA damage tolerance (DDT) mechanism controlling the DNA replication rate. Given that the levels of p53 trigger non-binary rheostat-like functions in response to stress or during differentiation, we explore the relevance of the p53 levels for its DDT functions at the fork. We show that subtle changes in p53 levels modulate the contribution of some DDT factors including POLι, POLη, POLζ, REV1, PCNA, PRIMPOL, HLTF and ZRANB3 to the DNA replication rate. Our results suggest that the levels of p53 are central to coordinate the balance between DDT pathways including (i) fork-deceleration by the ZRANB3-mediated fork reversal factor, (ii) POLι-p53-mediated fork-slowing, (iii) POLι- and POLη-mediated TLS and (iv) PRIMPOL-mediated fork-acceleration. Collectively, our study reveals the relevance of p53 protein levels for the DDT pathway choice in replicating cells.

Impact of the interplay between stemness features, p53 and pol iota on replication pathway choices.

Using human embryonic, adult and cancer stem cells/stem cell-like cells (SCs), we demonstrate that DNA replication speed differs in SCs and their differentiated counterparts. While SCs decelerate DNA replication, differentiated cells synthesize DNA faster and accumulate DNA damage. Notably, both replication phenotypes depend on p53 and polymerase iota (POLι). By exploring protein interactions and newly synthesized DNA, we show that SCs promote complex formation of p53 and POLι at replication sites. Intriguingly, in SCs the translocase ZRANB3 is recruited to POLι and required for slow-down of DNA replication. The known role of ZRANB3 in fork reversal suggests that the p53-POLι complex mediates slow but safe bypass of replication barriers in SCs. In differentiated cells, POLι localizes more transiently to sites of DNA synthesis and no longer interacts with p53 facilitating fast POLι-dependent DNA replication. In this alternative scenario, POLι associates with the p53 target p21, which antagonizes PCNA poly-ubiquitination and, thereby potentially disfavors the recruitment of translocases. Altogether, we provide evidence for diametrically opposed DNA replication phenotypes in SCs and their differentiated counterparts putting DNA replication-based strategies in the spotlight for the creation of therapeutic opportunities targeting SCs.

Simulating Space Conditions Evokes Different DNA Damage Responses in Immature and Mature Cells of the Human Hematopoietic System.

The impact of space radiation and microgravity on DNA damage responses has been discussed controversially, largely due to the variety of model systems engaged. Here, we performed side-by-side analyses of human hematopoietic stem/progenitor cells (HSPC) and peripheral blood lymphocytes (PBL) cultivated in a 2D clinostat to simulate microgravity before, during and after photon and particle irradiation. We demonstrate that simulated microgravity (SMG) accelerates the early phase of non-homologous end joining (NHEJ)-mediated repair of simple, X-ray-induced DNA double-strand breaks (DSBs) in PBL, while repair kinetics in HSPC remained unaltered. Repair acceleration was lost with increasing LET of ion exposures, which increases the complexity of DSBs, precluding NHEJ and requiring end resection for successful repair. Such cell-type specific effect of SMG on DSB repair was dependent on the NF-кB pathway pre-activated in PBL but not HSPC. Already under unperturbed growth conditions HSPC and PBL suffered from SMG-induced replication stress associated with accumulation of single-stranded DNA and DSBs, respectively. We conclude that in PBL, SMG-induced DSBs promote repair of radiation-induced damage in an adaptive-like response. HSPC feature SMG-induced single-stranded DNA and FANCD2 foci, i.e., markers of persistent replication stress and senescence that may contribute to a premature decline of the immune system in space.

Neutrophils Alter DNA Repair Landscape to Impact Survival and Shape Distinct Therapeutic Phenotypes of Colorectal Cancer.

BACKGROUND & AIMS: Tumor-infiltrating neutrophils (polymorphonuclear neutrophils [PMNs]) are a prominent feature of colorectal cancer (CRC), where they can promote cytotoxicity or exacerbate disease outcomes. We recently showed that in acute colon injury, PMNs can increase DNA double-strand break (DSB) burden and promote genomic instability via microRNA-dependent inhibition of homologous recombination (HR) repair. In this study, we aimed to establish whether in inflamed colon, neutrophils shape the DSB-repair responses to impact CRC progression and sensitivity/resistance to DNA-repair targeted therapy. METHODS: Human sporadic CRC biopsies, The Cancer Genome Atlas gene expression analyses, tumor xenografts, and murine CRC models, as well as small-molecule inhibition of key DSB-repair factors were leveraged to investigate changes in the DSB-repair landscape and identify unique CRC responses with/without tumor infiltration by PMNs. RESULTS: We reveal that neutrophils exert a functional dualism in cancer cells, driving temporal modulation of the DNA damage landscape and resolution of DSBs. PMNs were found to promote HR deficiency in low-grade CRC by miR-155-dependent downregulation of RAD51, thus attenuating tumor growth. However, neutrophil-mediated genotoxicity due to accumulation of DSBs led to the induction of non-homologous end-joining (NHEJ), allowing for survival and growth of advanced CRC. Our findings identified a PMN-induced HR-deficient CRC phenotype, featuring low RAD51 and low Ku70 levels, rendering it susceptible to synthetic lethality induced by clinically approved PARP1 inhibitor Olaparib. We further identified a distinct PMN-induced HR-deficient CRC phenotype, featuring high Ku70 and heightened NHEJ, which can be therapeutically targeted by specific inhibition of NHEJ. CONCLUSIONS: Our work delineates 2 mechanism-based translatable therapeutic interventions in sporadic CRC.

Multiple biochemical properties of the p53 molecule contribute to activation of polymerase iota-dependent DNA damage tolerance.

We have previously reported that p53 decelerates nascent DNA elongation in complex with the translesion synthesis (TLS) polymerase ι (POLι) which triggers a homology-directed DNA damage tolerance (DDT) pathway to bypass obstacles during DNA replication. Here, we demonstrate that this DDT pathway relies on multiple p53 activities, which can be disrupted by TP53 mutations including those frequently found in cancer tissues. We show that the p53-mediated DDT pathway depends on its oligomerization domain (OD), while its regulatory C-terminus is not involved. Mutation of residues S315 and D48/D49, which abrogate p53 interactions with the DNA repair and replication proteins topoisomerase I and RPA, respectively, and residues L22/W23, which disrupt formation of p53-POLι complexes, all prevent this DDT pathway. Our results demonstrate that the p53-mediated DDT requires the formation of a DNA binding-proficient p53 tetramer, recruitment of such tetramer to RPA-coated forks and p53 complex formation with POLι. Importantly, our mutational analysis demonstrates that transcriptional transactivation is dispensable for the POLι-mediated DDT pathway, which we show protects against DNA replication damage from endogenous and exogenous sources.

DNA damage repair as a target in pancreatic cancer: state-of-the-art and future perspectives.

Complex rearrangement patterns and mitotic errors are hallmarks of most pancreatic ductal adenocarcinomas (PDAC), a disease with dismal prognosis despite some therapeutic advances in recent years. DNA double-strand breaks (DSB) bear the greatest risk of provoking genomic instability, and DNA damage repair (DDR) pathways are crucial in preserving genomic integrity following a plethora of damage types. Two major repair pathways dominate DSB repair for safeguarding the genome integrity: non-homologous end joining and homologous recombination (HR). Defective HR, but also alterations in other DDR pathways, such as BRCA1, BRCA2, ATM and PALB2, occur frequently in both inherited and sporadic PDAC. Personalised treatment of pancreatic cancer is still in its infancy and predictive biomarkers are lacking. DDR deficiency might render a PDAC vulnerable to a potential new therapeutic intervention that increases the DNA damage load beyond a tolerable threshold, as for example, induced by poly (ADP-ribose) polymerase inhibitors. The Pancreas Cancer Olaparib Ongoing (POLO) trial, in which olaparib as a maintenance treatment improved progression-free survival compared with placebo after platinum-based induction chemotherapy in patients with PDAC and germline BRCA1/2 mutations, raised great hopes of a substantially improved outcome for this patient subgroup. This review summarises the relationship between DDR and PDAC, the prevalence and characteristics of DNA repair mutations and options for the clinical management of patients with PDAC and DNA repair deficiency.

Synergistic targeting and resistance to PARP inhibition in DNA damage repair-deficient pancreatic cancer.

OBJECTIVE: ATM serine/threonine kinase (ATM) is the most frequently mutated DNA damage response gene, involved in homologous recombination (HR), in pancreatic ductal adenocarcinoma (PDAC). DESIGN: Combinational synergy screening was performed to endeavour a genotype-tailored targeted therapy. RESULTS: Synergy was found on inhibition of PARP, ATR and DNA-PKcs (PAD) leading to synthetic lethality in ATM-deficient murine and human PDAC. Mechanistically, PAD-induced PARP trapping, replication fork stalling and mitosis defects leading to P53-mediated apoptosis. Most importantly, chemical inhibition of ATM sensitises human PDAC cells toward PAD with long-term tumour control in vivo. Finally, we anticipated and elucidated PARP inhibitor resistance within the ATM-null background via whole exome sequencing. Arising cells were aneuploid, underwent epithelial-mesenchymal-transition and acquired multidrug resistance (MDR) due to upregulation of drug transporters and a bypass within the DNA repair machinery. These functional observations were mirrored in copy number variations affecting a region on chromosome 5 comprising several of the upregulated MDR genes. Using these findings, we ultimately propose alternative strategies to overcome the resistance. CONCLUSION: Analysis of the molecular susceptibilities triggered by ATM deficiency in PDAC allow elaboration of an efficient mutation-specific combinational therapeutic approach that can be also implemented in a genotype-independent manner by ATM inhibition.

BRCA1 mislocalization leads to aberrant DNA damage response in heterozygous ABRAXAS1 mutation carrier cells.

Whilst heterozygous germline mutations in the ABRAXAS1 gene have been associated with a hereditary predisposition to breast cancer, their effect on promoting tumourigenesis at the cellular level has not been explored. Here, we demonstrate in patient-derived cells that the Finnish ABRAXAS1 founder mutation (c.1082G > A, Arg361Gln), even in the heterozygous state leads to decreased BRCA1 protein levels as well as reduced nuclear localization and foci formation of BRCA1 and CtIP. This causes disturbances in basal BRCA1-A complex localization, which is reflected by a restraint in error-prone DNA double-strand break repair pathway usage, attenuated DNA damage response and deregulated G2-M checkpoint control. The current study clearly demonstrates how the Finnish ABRAXAS1 founder mutation acts in a dominant-negative manner on BRCA1 to promote genome destabilization in heterozygous carrier cells.

Therapeutic targeting of mutant p53 in pediatric acute lymphoblastic leukemia.

Alterations of the tumor suppressor gene TP53 are found in different cancers, in particular in carcinomas of adults. In pediatric acute lymphoblastic leukemia (ALL), TP53 mutations are infrequent but enriched at relapse. As in most cancers, mainly DNA-binding domain missense mutations are found, resulting in accumulation of mutant p53, poor therapy response, and inferior outcome. Different strategies to target mutant p53 have been developed including reactivation of p53's wildtype function by the small molecule APR-246. We investigated TP53 mutations in cell lines and 62 B-cell precursor ALL samples and evaluated the activity of APR-246 in TP53-mutated or wildtype ALL. We identified cases with TP53 missense mutations, high (mutant) p53 expression and insensitivity to the DNA-damaging agent doxorubicin. In TP53-mutated ALL, APR-246 induced apoptosis showing strong anti-leukemia activity. APR-246 restored mutant p53 to its wildtype conformation, leading to pathway activation with induction of transcriptional targets and re-sensitization to genotoxic therapy in vitro and in vivo In addition, induction of oxidative stress contributed to APR-246-mediated cell death. In a preclinical model of patient-derived TP53-mutant ALL, APR-246 reduced leukemia burden and synergized strongly with the genotoxic agent doxorubicin, leading to superior leukemia-free survival in vivo Thus, targeting mutant p53 by APR-246, restoring its tumor suppressive function, seems to be an effective therapeutic strategy for this high-risk group of TP53-mutant ALL.

The C-terminal domain of p53 orchestrates the interplay between non-covalent and covalent poly(ADP-ribosyl)ation of p53 by PARP1.

The post-translational modification poly(ADP-ribosyl)ation (PARylation) plays key roles in genome maintenance and transcription. Both non-covalent poly(ADP-ribose) binding and covalent PARylation control protein functions, however, it is unknown how the two modes of modification crosstalk mechanistically. Employing the tumor suppressor p53 as a model substrate, this study provides detailed insights into the interplay between non-covalent and covalent PARylation and unravels its functional significance in the regulation of p53. We reveal that the multifunctional C-terminal domain (CTD) of p53 acts as the central hub in the PARylation-dependent regulation of p53. Specifically, p53 bound to auto-PARylated PARP1 via highly specific non-covalent PAR-CTD interaction, which conveyed target specificity for its covalent PARylation by PARP1. Strikingly, fusing the p53-CTD to a protein that is normally not PARylated, renders this a target for covalent PARylation as well. Functional studies revealed that the p53-PAR interaction had substantial implications on molecular and cellular levels. Thus, PAR significantly influenced the complex p53-DNA binding properties and controlled p53 functions, with major implications on the p53-dependent interactome, transcription, and replication-associated recombination. Remarkably, this mechanism potentially also applies to other PARylation targets, since a bioinformatics analysis revealed that CTD-like regions are highly enriched in the PARylated proteome.

Feasibility of a large multi-center translational research project for newly diagnosed breast and ovarian cancer patients with affiliated biobank: the BRandO biology and outcome (BiO)-project.

INTRODUCTION: Large translational research projects may contribute to further progress in cancer treatment by exploring molecular biology, immunologic approaches and identification of new prognostic and predictive factors. Therefore, the BRandOBio-project combines a clinical registry for collection of patient and tumor characteristics with a biobank comprising tumor and liquid biopsies. In addition, sociodemographic, environmental and lifestyle factors of included patients with primary newly diagnosed breast or ovarian cancer, other rare malignant ovarian tumors or gestational trophoblastic disease are prospectively collected. METHODS: The target population includes the German "Alb-Allgäu-Bodensee Region" which constitutes the outreach area of the University Hospital Ulm with affiliated academic centers and private practices. Clinical data combined with primary tumor tissue samples and longitudinal repeatedly collected blood samples [before, 6 (in high-risk situations), 12, 36 and 60 months after treatment and at relapse] will be acquired from more than 4000 patients within the next years. Standardized questionnaires are given to patients of the University Hospital Ulm and eight selected external sites for assessing life style and cancer risk factors. Concomitantly, storage of paraffin-embedded tumor samples as well as liquid biopsy samples will allow translational research projects, for example in terms of investigating circulating DNA and germ line DNA from cell pellets. RESULTS: Starting in January 2016 at the University Hospital Ulm, 19 additional external sites started recruiting patients in March 2017. As of September 15th 2019, 2151 patients with newly diagnosed cancers could be recruited (2044 breast cancer; 107 ovarian cancer). Nearly all patients provided biological samples (tumor and liquid biopsy) and about 80% returned the standardized questionnaire. After 1 year follow-up, blood samples were available from more than 80% of the participating patients. CONCLUSIONS: The BRandO BIO study is a large prospective cohort study with integrated comprehensive biobank and evaluation of sociodemographic and life style factors of gynecological cancer patients in a well-defined geographical area in the South West of Germany. Continuous high patient recruitment and stable rates over 80% for returned questionnaires as well as for repeated blood sampling show high acceptance of the BRandO study program and confirms feasibility of the project.

DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression.

DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker-induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress.

A low-frequency haplotype spanning SLX4/FANCP constitutes a new risk locus for early-onset breast cancer (<60 years) and is associated with reduced DNA repair capacity.

Only a fraction of breast cancer (BC) cases can be yet explained by mutations in genes or genomic variants discovered in linkage, genome-wide association and sequencing studies. The known genes entailing medium or high risk for BC are strongly enriched for a function in DNA double strand repair. Thus, aiming at identifying low frequency variants conferring an intermediate risk, we here investigated 17 variants (MAF: 0.01-0.1) in 10 candidate genes involved in DNA repair or cell cycle control. In an exploration cohort of 437 cases and 1189 controls, we show the variant rs3810813 in the SLX4/FANCP gene to be significantly associated with both BC (≤60 years; OR = 2.6(1.6-3.9), p = 1.6E-05) and decreased DNA repair capacity (≤60 years; beta = 37.8(17.9-57.8), p = 5.3E-4). BC association was confirmed in a verification cohort (N = 2441). Both associations were absent from cases diagnosed >60 years and stronger the earlier the diagnosis. By imputation we show that rs3810813 tags a haplotype with 5 additional variants with the same allele frequency (R2 > 0.9), and a pattern of association very similar for both phenotypes (cases <60 years, p < 0.001, the Bonferroni threshold derived from unlinked variants in the region). In young cases (≤60 years) carrying the risk haplotype, micronucleus test results are predictive for BC (AUC > 0.9). Our findings propose a risk variant with high penetrance on the haplotype spanning SLX4/FANCP to be functionally associated to BC predisposition via decreased repair capacity and suggest this variant is carried by a fraction of these haplotypes that is enriched in early onset BC cases.

Quercetin alters the DNA damage response in human hematopoietic stem and progenitor cells via TopoII- and PI3K-dependent mechanisms synergizing in leukemogenic rearrangements.

Quercetin (Que) is an abundant flavonoid in the human diet and high-concentration food supplement with reported pro- and anti-carcinogenic activities. Topoisomerase II (TopoII) inhibition and subsequent DNA damage induction by Que was implicated in the mixed lineage leukemia gene (MLL) rearrangements that can induce infant and adult leukemias. This notion raised concerns regarding possible genotoxicities of Que in hematopoietic stem and progenitor cells (HSPCs). However, molecular targets mediating Que effects on DNA repair relevant to MLL translocations have not been defined. In this study we describe novel and potentially genotoxic Que activities in suppressing non-homologous end joining and homologous recombination pathways downstream of MLL cleavage. Using pharmacological dissection of DNA-PK, ATM and PI3K signalling we defined PI3K inhibition by Que with a concomitant decrease in the abundance of key DNA repair genes to be responsible for DNA repair inhibition. Evidence for the downstream TopoII-independent mutagenic potential of Que was obtained by documenting further increased frequencies of MLL rearrangements in human HSPCs concomitantly treated with Etoposide and Que versus single treatments. Importantly, by engaging a tissue engineered placental barrier, we have established the extent of Que transplacental transfer and hence provided the evidence for Que reaching fetal HSPCs. Thus, Que exhibits genotoxic effects in human HSPCs via different mechanisms when applied continuously and at high concentrations. In light of the demonstrated Que transfer to the fetal compartment our findings are key to understanding the mechanisms underlying infant leukemia and provide molecular markers for the development of safety values.

In vitro model for DNA double-strand break repair analysis in breast cancer reveals cell type-specific associations with age and prognosis.

Dysfunction of homologous recombination is a common denominator of changes associated with breast cancer-predisposing mutations. In our previous work, we identified a functional signature in peripheral blood lymphocytes from women who were predisposed that indicated a shift from homologous recombination to alternative, error-prone DNA double-strand break (DSB) repair pathways. To capture both hereditary and nonhereditary factors, we newly established a protocol for isolation and ex vivo analysis of epithelial cells, epithelial-mesenchymal transition cells (EMTs), and fibroblasts from breast cancer specimens (147 patients). By applying a fluorescence-based test system, we analyzed the error-prone DSB repair pathway microhomology-mediated end joining in these tumor-derived cell types and peripheral blood lymphocytes. In parallel, we investigated DNA lesion processing by quantitative immunofluorescence microscopy of histone H2AX phosphorylated on Ser139 focus after radiomimetic treatment. Our study reveals elevated histone H2AX phosphorylated on Ser139 damage removal in epithelial cells, not EMTs, and poly(ADP-ribose)polymerase inhibitor sensitivities, which suggested a DSB repair pathway shift with increasing patient age. Of interest, we found elevated microhomology-mediated end joining in EMTs, not epithelial cells, from patients who received a treatment recommendation of adjuvant chemotherapy, that is, those with high-risk tumors. Our discoveries of altered DSB repair activities in cells may serve as a method to further classify breast cancer to predict responsiveness to adjuvant chemotherapy and/or therapeutics that target DSB repair-dysfunctional tumors.-Deniz, M., Kaufmann, J., Stahl, A., Gundelach, T., Janni, W., Hoffmann, I., Keimling, M., Hampp, S., Ihle, M., Wiesmüller, L. In vitro model for DNA double-strand break repair analysis in breast cancer reveals cell type-specific associations with age and prognosis.

ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1.

The MRE11/RAD50/NBS1 (MRN) complex plays a central role as a sensor of DNA double strand breaks (DSB) and is responsible for the efficient activation of ataxia-telangiectasia mutated (ATM) kinase. Once activated ATM in turn phosphorylates RAD50 and NBS1, important for cell cycle control, DNA repair and cell survival. We report here that MRE11 is also phosphorylated by ATM at S676 and S678 in response to agents that induce DNA DSB, is dependent on the presence of NBS1, and does not affect the association of members of the complex or ATM activation. A phosphosite mutant (MRE11S676AS678A) cell line showed decreased cell survival and increased chromosomal aberrations after radiation exposure indicating a defect in DNA repair. Use of GFP-based DNA repair reporter substrates in MRE11S676AS678A cells revealed a defect in homology directed repair (HDR) but single strand annealing was not affected. More detailed investigation revealed that MRE11S676AS678A cells resected DNA ends to a greater extent at sites undergoing HDR. Furthermore, while ATM-dependent phosphorylation of Kap1 and SMC1 was normal in MRE11S676AS678A cells, there was no phosphorylation of Exonuclease 1 consistent with the defect in HDR. These results describe a novel role for ATM-dependent phosphorylation of MRE11 in limiting the extent of resection mediated through Exonuclease 1.

MCM3AP and POMP Mutations Cause a DNA-Repair and DNA-Damage-Signaling Defect in an Immunodeficient Child.

Immunodeficiency patients with DNA repair defects exhibit radiosensitivity and proneness to leukemia/lymphoma formation. Though progress has been made in identifying the underlying mutations, in most patients the genetic basis is unknown. Two de novo mutated candidate genes, MCM3AP encoding germinal center-associated nuclear protein (GANP) and POMP encoding proteasome maturation protein (POMP), were identified by whole-exome sequencing (WES) and confirmed by Sanger sequencing in a child with complex phenotype displaying immunodeficiency, genomic instability, skin changes, and myelodysplasia. GANP was previously described to promote B-cell maturation by nuclear targeting of activation-induced cytidine deaminase (AID) and to control AID-dependent hyperrecombination. POMP is required for 20S proteasome assembly and, thus, for efficient NF-κB signaling. Patient-derived cells were characterized by impaired homologous recombination, moderate radio- and cross-linker sensitivity associated with accumulation of damage, impaired DNA damage-induced NF-κB signaling, and reduced nuclear AID levels. Complementation by wild-type (WT)-GANP normalized DNA repair and WT-POMP rescued defective NF-κB signaling. In conclusion, we identified for the first time mutations in MCM3AP and POMP in an immunodeficiency patient. These mutations lead to cooperative effects on DNA recombination and damage signaling. Digenic/polygenic mutations may constitute a novel genetic basis in immunodeficiency patients with DNA repair defects.

NuMA promotes homologous recombination repair by regulating the accumulation of the ISWI ATPase SNF2h at DNA breaks.

Chromatin remodeling factors play an active role in the DNA damage response by shaping chromatin to facilitate the repair process. The spatiotemporal regulation of these factors is key to their function, yet poorly understood. We report that the structural nuclear protein NuMA accumulates at sites of DNA damage in a poly[ADP-ribose]ylation-dependent manner and functionally interacts with the ISWI ATPase SNF2h/SMARCA5, a chromatin remodeler that facilitates DNA repair. NuMA coimmunoprecipitates with SNF2h, regulates its diffusion in the nucleoplasm and controls its accumulation at DNA breaks. Consistent with NuMA enabling SNF2h function, cells with silenced NuMA exhibit reduced chromatin decompaction after DNA cleavage, lesser focal recruitment of homologous recombination repair factors, impaired DNA double-strand break repair in chromosomal (but not in episomal) contexts and increased sensitivity to DNA cross-linking agents. These findings reveal a structural basis for the orchestration of chromatin remodeling whereby a scaffold protein promotes genome maintenance by directing a remodeler to DNA breaks.

Modulation of the poly (ADP-ribose) polymerase inhibitor response and DNA recombination in breast cancer cells by drugs affecting endogenous wild-type p53.

Synthetic lethal interactions between poly (ADP-ribose) polymerase (PARP) and homologous recombination (HR) repair pathways have been exploited for the development of novel mono- and combination cancer therapies. The tumor suppressor p53 was demonstrated to exhibit indirect and direct regulatory activities in DNA repair, particularly in DNA double-strand break (DSB)-induced and replication-associated HR. In this study, we tested a potential influence of the p53 status on the response to PARP inhibition, which is known to cause replication stress. Silencing endogenous or inducibly expressing p53 we found a protective effect of p53 on PARP inhibitor (PARPi)-mediated cytotoxicities. This effect was specific for wild-type versus mutant p53 and observed in cancer but not in non-transformed cell lines. Enhanced cytotoxicities after treatment with the p53-inhibitory drug Pifithrinα further supported p53-mediated resistance to PARP inhibition. Surprisingly, we equally observed increased PARPi sensitivity in the presence of the p53-activating compound Nutlin-3. As a common denominator, both drug responses correlated with decreased HR activities: Pifithrinα downregulated spontaneous HR resulting in damage accumulation. Nutlin-3 induced a decrease of DSB-induced HR, which was accompanied by a severe drop in RAD51 protein levels. Thus, we revealed a novel link between PARPi responsiveness and p53-controlled HR activities. These data expand the concept of cell and stress type-dependent healer and killer functions of wild-type p53 in response to cancer therapeutic treatment. Our findings have implications for the individualized design of cancer therapies using PARPi and the potentially combined use of p53-modulatory drugs.