MRN Complex Research Articles

Resection of DNA double-strand breaks activates Mre11-Rad50-Nbs1- and Rad9-Hus1-Rad1-dependent mechanisms that redundantly promote ATR checkpoint activation and end processing in Xenopus egg extracts.

Sensing and processing of DNA double-strand breaks (DSBs) are vital to genome stability. DSBs are primarily detected by the ATM checkpoint pathway, where the Mre11-Rad50-Nbs1 (MRN) complex serves as the DSB sensor. Subsequent DSB end resection activates the ATR checkpoint pathway, where replication protein A, MRN, and the Rad9-Hus1-Rad1 (9-1-1) clamp serve as the DNA structure sensors. ATR activation depends also on Topbp1, which is loaded onto DNA through multiple mechanisms. While different DNA structures elicit specific ATR-activation subpathways, the regulation and mechanisms of the ATR-activation subpathways are not fully understood. Using DNA substrates that mimic extensively resected DSBs, we show here that MRN and 9-1-1 redundantly stimulate Dna2-dependent long-range end resection and ATR activation in Xenopus egg extracts. MRN serves as the loading platform for ATM, which, in turn, stimulates Dna2- and Topbp1-loading. Nevertheless, MRN promotes Dna2-mediated end processing largely independently of ATM. 9-1-1 is dispensable for bulk Dna2 loading, and Topbp1 loading is interdependent with 9-1-1. ATR facilitates Mre11 phosphorylation and ATM dissociation. These data uncover that long-range end resection activates two redundant pathways that facilitate ATR checkpoint signaling and DNA processing in a vertebrate system.

Suppression of NBS1 Upregulates CyclinB to Induce Olaparib Sensitivity in Ovarian Cancer.

Background: Deficiencies in DNA damage repair responses promote chemotherapy sensitivity of tumor cells. The Nibrin homolog encoding gene Nijmegen Breakage Syndrome 1 (NBS1) is a crucial component of the MRE11-RAD50-NBN complex (MRN complex) and is involved in the response to DNA double-strand breaks (DSBs) repair that has emerged as an attractive strategy to overcome tumor drug resistance, but the functional relationship between NBS1 regulated DNA damage repair and cell cycle checkpoints has not been fully elucidated. Methods: In this study, lentivirus-mediated RNAi was used to construct NBS1-downregulated cells. Flow cytometry, qPCR, and immunohistochemistry were used to explore the regulatory relationship between NBS1 and CyclinB in vivo and in vitro. Results: Our findings suggest that NBS1 deficiency leads to defective homologous recombination repair. Inhibition of NBS1 expression activates CHK1 and CyclinB signaling pathways leading to cell cycle arrest and sensitizes ovarian cancer cells to Olaparib treatment in vitro and in vivo. NBS1-deficient ovarian cancer cells tend to maintain sensitivity to chemotherapeutic drugs through activation of cell cycle checkpoints. Conclusions: NBS1 may be a potential therapeutic target for epithelial ovarian cancer as it plays a role in the regulation of the DNA damage response and cell cycle checkpoints. Suppression of NBS1 upregulates CyclinB to induce Olaparib sensitivity in ovarian cancer.

DNA damage remodels the MITF interactome to increase melanoma genomic instability.

Since genome instability can drive cancer initiation and progression, cells have evolved highly effective and ubiquitous DNA damage response (DDR) programs. However, some cells (for example, in skin) are normally exposed to high levels of DNA-damaging agents. Whether such high-risk cells possess lineage-specific mechanisms that tailor DNA repair to the tissue remains largely unknown. Using melanoma as a model, we show here that the microphthalmia-associated transcription factor MITF, a lineage addition oncogene that coordinates many aspects of melanocyte and melanoma biology, plays a nontranscriptional role in shaping the DDR. On exposure to DNA-damaging agents, MITF is phosphorylated at S325, and its interactome is dramatically remodeled; most transcription cofactors dissociate, and instead MITF interacts with the MRE11-RAD50-NBS1 (MRN) complex. Consequently, cells with high MITF levels accumulate stalled replication forks and display defects in homologous recombination-mediated repair associated with impaired MRN recruitment to DNA damage. In agreement with this, high MITF levels are associated with increased single-nucleotide and copy number variant burdens in melanoma. Significantly, the SUMOylation-defective MITF-E318K melanoma predisposition mutation recapitulates the effects of DNA-PKcs-phosphorylated MITF. Our data suggest that a nontranscriptional function of a lineage-restricted transcription factor contributes to a tissue-specialized modulation of the DDR that can impact cancer initiation.

Xrs2/NBS1 promote end-bridging activity of the MRE11-RAD50 complex

DNA double strand breaks (DSBs) can be detrimental to the cell and need to be efficiently repaired. A first step in DSB repair is to bring the free ends in close proximity to enable ligation by non-homologous end-joining (NHEJ), while the more precise, but less available, repair by homologous recombination (HR) requires close proximity of a sister chromatid. The human MRE11-RAD50-NBS1 (MRN) complex, Mre11-Rad50-Xrs2 (MRX) in yeast, is involved in both repair pathways. Here we use nanofluidic channels to study, on the single DNA molecule level, how MRN, MRX and their constituents interact with long DNA and promote DNA bridging. Nanofluidics is a suitable method to study reactions on DNA ends since no anchoring of the DNA end(s) is required. We demonstrate that NBS1 and Xrs2 play important, but differing, roles in the DNA tethering by MRN and MRX. NBS1 promotes DNA bridging by MRN consistent with tethering of a repair template. MRX shows a “synapsis-like” DNA end-bridging, stimulated by the Xrs2 subunit. Our results highlight the different ways MRN and MRX bridge DNA, and the results are in agreement with their key roles in HR and NHEJ, respectively, and contribute to the understanding of the roles of NBS1 and Xrs2 in DSB repair.

Absolute quantification of DNA damage response proteins

BackgroundDNA damage response (DDR) and repair are vital for safeguarding genetic information and ensuring the survival and accurate transmission of genetic material. DNA damage, such as DNA double-strand breaks (DSBs), triggers a response where sensor proteins recognize DSBs. Information is transmitted to kinases, initiating a sequence resulting in the activation of the DNA damage response and recruitment of other DDR and repair proteins to the DSB site in a highly orderly sequence. Research has traditionally focused on individual protein functions and their order, with limited quantitative analysis, prompting this study’s attempt at absolute quantification of DNA damage response and repair proteins and capturing changes in protein chromatin affinity after DNA damage through biochemical fractionation methods.ResultsTo assess the intracellular levels of proteins involved in DDR and repair, multiple proteins associated with different functions were quantified in EPC2-hTERT cells. H2AX had the highest intracellular abundance (1.93 × 106 molecules/cell). The components of the MRN complex were present at the comparable levels: 6.89 × 104 (MRE11), 2.17 × 104 (RAD50), and 2.35 × 104 (NBS1) molecules/cell. MDC1 was present at 1.27 × 104 molecules/cell. The intracellular levels of ATM and ATR kinases were relatively low: 555 and 4860 molecules/cell, respectively. The levels of cellular proteins involved in NHEJ (53BP1: 3.03 × 104; XRCC5: 2.62 × 104; XRCC6: 5.05 × 105 molecules/cell) were more than an order of magnitude higher than that involved in HR (RAD51: 2500 molecules/cell). Furthermore, we analyzed the dynamics of MDC1 and γH2AX proteins in response to DNA damage induced by the unstable agent neocarzinostatin (NCS). Using cell biochemical fractionation, cells were collected and analyzed at different time points after NCS exposure. Results showed that γH2AX in chromatin fraction peaked at 1 h post-exposure and gradually decreased, while MDC1 translocated from the isotonic to the hypertonic fraction, peaking at 1 hour as well. The study suggests increased MDC1 affinity for chromatin through binding to γH2AX induced by DNA damage. The γH2AX-bound MDC1 (in the hypertonic fraction) to γH2AX ratio at 1 h post-exposure was 1:56.4, with lower MDC1 levels which may attributed to competition with other proteins.ConclusionsThe approach provided quantitative insights into protein dynamics in DNA damage response.

Germline rad 50 mutation in a case with synchronous breast and kidney cancer: a rare case

BackgroundBreast cancer has been reported to occur synchronous with ovarian, endometrial and even colon cancers. A synchronous renal cancer is rare. And its association with RAD 50 mutation is not known.Case presentationWe are reporting a 42 year old lady who was evaluated for a breast lump and was incidentally found to have a renal lesion. She underwent surgery for both and was found to have a T1c breast tumour and a renal cell carcinoma-clear cell variant. She was advised germline testing with next generation sequencing and multiplex ligation—dependent probe amplification due to synchronous tumours and age at diagnosis. It revealed a likely pathogenic variant in the RAD 50 gene.ConclusionThe RAD 50 gene is part of the MRN complex (Mre11, Rad50 and Nbs1 also known as nibrin), which is essential for DNA repair. The recommendations for follow-up and screening are not clear for patients with a pathogenic variant of the gene. This case is presented for its rarity.

Isolation and detection of DNA-protein crosslinks in mammalian cells.

DNA-protein crosslinks (DPCs) are toxic DNA lesions wherein a protein is covalently attached to DNA. If not rapidly repaired, DPCs create obstacles that disturb DNA replication, transcription and DNA damage repair, ultimately leading to genome instability. The persistence of DPCs is associated with premature ageing, cancer and neurodegeneration. In mammalian cells, the repair of DPCs mainly relies on the proteolytic activities of SPRTN and the 26S proteasome, complemented by other enzymes including TDP1/2 and the MRN complex, and many of the activities involved are essential, restricting genetic approaches. For many years, the study of DPC repair in mammalian cells was hindered by the lack of standardised assays, most notably assays that reliably quantified the proteins or proteolytic fragments covalently bound to DNA. Recent interest in the field has spurred the development of several biochemical methods for DPC analysis. Here, we critically analyse the latest techniques for DPC isolation and the benefits and drawbacks of each. We aim to assist researchers in selecting the most suitable isolation method for their experimental requirements and questions, and to facilitate the comparison of results across different laboratories using different approaches.

TDP1 suppresses chromosomal translocations and cell death induced by abortive TOP1 activity during gene transcription

DNA topoisomerase I (TOP1) removes torsional stress by transiently cutting one DNA strand. Such cuts are rejoined by TOP1 but can occasionally become abortive generating permanent protein-linked single strand breaks (SSBs). The repair of these breaks is initiated by tyrosyl-DNA phosphodiesterase 1 (TDP1), a conserved enzyme that unlinks the TOP1 peptide from the DNA break. Additionally, some of these SSBs can result in double strand breaks (DSBs) either during replication or by a poorly understood transcription-associated process. In this study, we identify these DSBs as a source of genome rearrangements, which are suppressed by TDP1. Intriguingly, we also provide a mechanistic explanation for the formation of chromosomal translocations unveiling an error-prone pathway that relies on the MRN complex and canonical non-homologous end-joining. Collectively, these data highlight the threat posed by TOP1-induced DSBs during transcription and demonstrate the importance of TDP1-dependent end-joining in protecting both gene transcription and genome stability.

Double-strand DNA break repair: molecular mechanisms and therapeutic targets.

Double-strand break (DSB), a significant DNA damage brought on by ionizing radiation, acts as an initiating signal in tumor radiotherapy, causing cancer cells death. The two primary pathways for DNA DSB repair in mammalian cells are nonhomologous end joining (NHEJ) and homologous recombination (HR), which cooperate and compete with one another to achieve effective repair. The DSB repair mechanism depends on numerous regulatory variables. DSB recognition and the recruitment of DNA repair components, for instance, depend on the MRE11-RAD50-NBS1 (MRN) complex and the Ku70/80 heterodimer/DNA-PKcs (DNA-PK) complex, whose control is crucial in determining the DSB repair pathway choice and efficiency of HR and NHEJ. In-depth elucidation on the DSB repair pathway's molecular mechanisms has greatly facilitated for creation of repair proteins or pathways-specific inhibitors to advance precise cancer therapy and boost the effectiveness of cancer radiotherapy. The architectures, roles, molecular processes, and inhibitors of significant target proteins in the DSB repair pathways are reviewed in this article. The strategy and application in cancer therapy are also discussed based on the advancement of inhibitors targeted DSB damage response and repair proteins.

ATR-binding lncRNA ScaRNA2 promotes cancer resistance through facilitating efficient DNA end resection during homologous recombination repair

BackgroundOur previous study first showed that ATR-binding long noncoding RNA (lncRNA) is necessary for ATR function and promotes cancer resistance. However, the specific lncRNAs instrumental in ATR activation remain largely unclear, which limits our comprehensive understanding of this critical biological process.MethodsRNA immunoprecipitation (RIP) followed by RNA sequencing was employed to identify ATR-binding lncRNAs, which were further validated using RIP-qPCR assays. Immunofluorescence staining and Western blotting were applied to detect the activation of DNA damage repair factors. After the effect of scaRNA2 on cellular sensitivity to DNA-damaging reagents was determined, the effects of scaRNA2 on radiotherapy were investigated in patient-derived organoids and xenograft preclinical models. The clinical relevance of scaRNA2 was also validated in tissues isolated from rectal cancer patients.ResultsScaRNA2 was identified as the most enriched ATR-binding lncRNA and was found to be essential for homologous recombination (HR) mediated DNA damage repair. Furthermore, scaRNA2 knockdown abrogated the recruitment of ATR and its substrates in response to DNA damage. Mechanistically, scaRNA2 was observed to be necessary for Exo1-mediated DNA end resection and bridged the MRN complex to ATR activation. Knockdown of scaRNA2 effectively increased the sensitivity of cancer cells to multiple kinds of DNA damage-related chemoradiotherapy. Preclinically, knockdown of scaRNA2 improved the effects of radiotherapy on patient-derived organoids and xenograft models. Finally, an increase in scaRNA2 colocalized with ATR was also found in clinical patients who were resistant to radiotherapy.ConclusionsScaRNA2 was identified as the most abundant lncRNA bound to ATR and was demonstrated to bridge DNA end resection to ATR activation; thus, it could be applied as a potent target for combined cancer treatments with chemoradiotherapy.

The molecular details of a novel phosphorylation-dependent interaction between MRN and the SOSS complex.

The repair of double-strand DNA breaks (DSBs) by homologous recombination is crucial in the maintenance of genome integrity. While the key role of the Mre11-Rad50-Nbs1 (MRN) complex in repair is well known, hSSB1 (SOSSB and OBFC2B), one of the main components of the sensor of single-stranded DNA (SOSS) protein complex, has also been shown to rapidly localize to DSB breaks and promote repair. We have previously demonstrated that hSSB1 binds directly to Nbs1, a component of the MRN complex, in a DNA damage-independent manner. However, recruitment of the MRN complex has also been demonstrated by an interaction between Integrator Complex Subunit 3 (INTS3; also known as SOSSA), another member of the SOSS complex, and Nbs1. In this study, we utilize a combined approach of in silico, biochemical, and functional experiments to uncover the molecular details of INTS3 binding to Nbs1. We demonstrate that the forkhead-associated domain of Nbs1 interacts with INTS3 via phosphorylation-dependent binding to INTS3 at Threonine 592, with contributions from Serine 590. Based on these data, we propose a model of MRN recruitment to a DSB via INTS3.

Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice

The Mre11-Rad50-Nbs1 (MRN) complex recognizes and processes DNA double-strand breaks for homologous recombination by performing short-range removal of 5ʹ strands. Endonucleolytic processing by MRN requires a stably bound protein at the break site—a role we postulate is played by DNA-dependent protein kinase (DNA-PK) in mammals. Here we interrogate sites of MRN-dependent processing by identifying sites of CtIP association and by sequencing DNA-PK-bound DNA fragments that are products of MRN cleavage. These intermediates are generated most efficiently when DNA-PK is catalytically blocked, yielding products within 200 bp of the break site, whereas DNA-PK products in the absence of kinase inhibition show greater dispersal. Use of light-activated Cas9 to induce breaks facilitates temporal resolution of DNA-PK and Mre11 binding, showing that both complexes bind to DNA ends before release of DNA-PK-bound products. These results support a sequential model of double-strand break repair involving collaborative interactions between homologous and non-homologous repair complexes.

DNA-PK and the TRF2 iDDR inhibit MRN-initiated resection at leading-end telomeres.

Telomeres replicated by leading-strand synthesis lack the 3' overhang required for telomere protection. Surprisingly, resection of these blunt telomeres is initiated by the telomere-specific 5' exonuclease Apollo rather than the Mre11-Rad50-Nbs1 (MRN) complex, the nuclease that acts at DNA breaks. Without Apollo, leading-end telomeres undergo fusion, which, as demonstrated here, is mediated by alternative end joining. Here, we show that DNA-PK and TRF2 coordinate the repression of MRN at blunt mouse telomeres. DNA-PK represses an MRN-dependent long-range resection, while the endonuclease activity of MRN-CtIP, which could cleave DNA-PK off of blunt telomere ends, is inhibited in vitro and in vivo by the iDDR of TRF2. AlphaFold-Multimer predicts a conserved association of the iDDR with Rad50, potentially interfering with CtIP binding and MRN endonuclease activation. We propose that repression of MRN-mediated resection is a conserved aspect of telomere maintenance and represents an ancient feature of DNA-PK and the iDDR.

The abscission checkpoint senses chromatin bridges through Top2α recruitment to DNA knots.

In response to chromatin bridges, the abscission checkpoint delays completion of cytokinesis to prevent chromosome breakage or tetraploidization. Here, we show that spontaneous or replication stress-induced chromatin bridges exhibit "knots" of catenated and overtwisted DNA next to the midbody. Topoisomerase IIα (Top2α) forms abortive Top2-DNA cleavage complexes (Top2ccs) on DNA knots; furthermore, impaired Top2α-DNA cleavage activity correlates with chromatin bridge breakage in cytokinesis. Proteasomal degradation of Top2ccs is required for Rad17 localization to Top2-generated double-strand DNA ends on DNA knots; in turn, Rad17 promotes local recruitment of the MRN complex and downstream ATM-Chk2-INCENP signaling to delay abscission and prevent chromatin breakage. In contrast, dicentric chromosomes that do not exhibit knotted DNA fail to activate the abscission checkpoint in human cells. These findings are the first to describe a mechanism by which the abscission checkpoint detects chromatin bridges, through generation of abortive Top2ccs on DNA knots, to preserve genome integrity.

The Multiple Faces of the MRN Complex: Roles in Medulloblastoma and Beyond.

Hypomorphic mutations in MRN complex genes are frequently found in cancer, supporting their role as oncosuppressors. However, unlike canonical oncosuppressors, MRN proteins are often overexpressed in tumor tissues, where they actively work to counteract DSBs induced by both oncogene-dependent RS and radio-chemotherapy. Moreover, at the same time, MRN genes are also essential genes, since the constitutive KO of each component leads to embryonic lethality. Therefore, even though it is paradoxical, MRN genes may work as oncosuppressive, oncopromoting, and essential genes. In this review, we discussed how alterations in the MRN complex impact the physiopathology of cancer, in light of our recent discoveries on the gene-dosage-dependent effect of NBS1 in Medulloblastoma. These updates aim to understand whether MRN complex can be realistically used as a prognostic/predictive marker and/or as a therapeutic target for the treatment of cancer patients in the future.

Simultaneous Nbs1 and p53 inactivation in neural progenitors triggers high-grade gliomas.

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder caused by hypomorphic mutations of NBS1. NBS1 is a member of the MRE11-RAD50-NBS1 (MRN) complex that binds to DNA double-strand breaks and activates the DNA damage response (DDR). Nbs1 inactivation in neural progenitor cells leads to microcephaly and premature death. Interestingly, p53 homozygous deletion rescues the NBS1-deficient phenotype allowing long-term survival. The objective of this work was to determine whether simultaneous inactivation of Nbs1 and p53 in neural progenitors triggered brain tumorigenesis and if so in which category this tumour could be classified. We generated a mouse model with simultaneous genetic inactivation of Nbs1 and p53 in embryonic neural stem cells and analysed the arising tumours with in-depth molecular analyses including immunohistochemistry, array comparative genomic hybridisation (aCGH), whole exome-sequencing and RNA-sequencing. NBS1/P53-deficient mice develop high-grade gliomas (HGG) arising in the olfactory bulbs and in the cortex along the rostral migratory stream. In-depth molecular analyses using immunohistochemistry, aCGH, whole exome-sequencing and RNA-sequencing revealed striking similarities to paediatric human HGG with shared features with radiation-induced gliomas (RIGs). Our findings show that concomitant inactivation of Nbs1 and p53 in mice promotes HGG with RIG features. This model could be useful for preclinical studies to improve the prognosis of these deadly tumours, but it also highlights the singularity of NBS1 among the other DNA damage response proteins in the aetiology of brain tumours.

Differential expression of meiosis and homologous recombination-related genes in the life cycle of Trypanosoma cruzi.

Trypanosoma cruzi has a complex life cycle consisting of four morphological and distinct biological stages. Although some authors suggest that T. cruzi primarily follows clonal reproduction, recent genomic and transcriptomic studies indicate an unorthodox capacity for recombination. We aimed to estimate the differential gene expression of 10 meiosis/homologous recombination-related genes during the T. cruzi life cycle, including epimastigotes, under two different types of stress (oxidative stressand pH changes). We performed RT-qPCR tests using novel-designed primers to estimate the differential gene expression (∆Ct and ∆∆Ct) of nine genes (SPO11, HAP2, RAD50, MRN complex, BRCA2, DMC1, MND1, and RPA1) and RAD51, which was previously reported. Our results show basal expression of all genes during the life cycle, indicating their hypothetical role in several cellular processes but with specific signatures of differential gene expression during the life cycle (HAP2, RPA, RAD50, BRCA2, MND1, and DMC1) and oxidative stress (RPA, MRE11, NBS1, BRCA2, MND1, and RAD51). Additionally, we found that the MRN complex has an independent level of expression in T. cruzi, with profiles of MRE11 and NBS1 upregulated in some stages. Recent studies on other trypanosomatids have highlighted the influence of HAP2 and RPA in recombination and hybridization. If T. cruzi uses the same repertoire of genes, our findings could suggest that metacyclogenesis may be the putative step that the parasite uses to undergo recombination. Likewise, our study reveals the differential profiles of genes expressed in response to oxidative and pH stress. Further studies are necessary to confirm our findings and understand the recombination mechanism in T. cruzi.

The DNA Damage Sensor MRE11 Regulates Efficient Replication of the Autonomous Parvovirus Minute Virus of Mice.

Parvoviruses are single-stranded DNA viruses that utilize host proteins to vigorously replicate in the nuclei of host cells, leading to cell cycle arrest. The autonomous parvovirus, minute virus of mice (MVM), forms viral replication centers in the nucleus which are adjacent to cellular DNA damage response (DDR) sites, many of which are fragile genomic regions prone to undergoing DDR during the S phase. Since the cellular DDR machinery has evolved to transcriptionally suppress the host epigenome to maintain genomic fidelity, the successful expression and replication of MVM genomes at these cellular sites suggest that MVM interacts with DDR machinery distinctly. Here, we show that efficient replication of MVM requires binding of the host DNA repair protein MRE11 in a manner that is independent of the MRE11-RAD50-NBS1 (MRN) complex. MRE11 binds to the replicating MVM genome at the P4 promoter, remaining distinct from RAD50 and NBS1, which associate with cellular DNA break sites to generate DDR signals in the host genome. Ectopic expression of wild-type MRE11 in CRISPR knockout cells rescues virus replication, revealing a dependence on MRE11 for efficient MVM replication. Our findings suggest a new model utilized by autonomous parvoviruses to usurp local DDR proteins that are crucial for viral pathogenesis and distinct from those of dependoparvoviruses, like adeno-associated virus (AAV), which require a coinfected helper virus to inactivate the local host DDR. IMPORTANCE The cellular DNA damage response (DDR) machinery protects the host genome from the deleterious consequences of DNA breaks and recognizes invading viral pathogens. DNA viruses that replicate in the nucleus have evolved distinct strategies to evade or usurp these DDR proteins. We have discovered that the autonomous parvovirus, MVM, which is used to target cancer cells as an oncolytic agent, depends on the initial DDR sensor protein MRE11 to express and replicate efficiently in host cells. Our studies reveal that the host DDR interacts with replicating MVM molecules in ways that are distinct from viral genomes being recognized as simple broken DNA molecules. These findings suggest that autonomous parvoviruses have evolved distinct mechanisms to usurp DDR proteins, which can be used to design potent DDR-dependent oncolytic agents.

Abstract CT188: A phase Ib study of sapacitabine (sapa) and olaparib (ola) in patients (pts) with BRCA1/2-mutated (BRCA1/2m) metastatic breast cancer (MBC)

Abstract Background: PARP inhibitors (PARPi) are superior to chemotherapy in pts with BRCA1/2m MBC, with median PFS (mPFS) of 7 months with single-agent ola. Sapa is an oral nucleoside analog; the active metabolite, CNDAC, generates single-strand DNA breaks that are converted to double-strand breaks (DSB) during subsequent replication, resulting in cell death. CNDAC-induced DSB repair is dependent on homologous recombination. Sapa combined with ola may augment DNA damage compared to ola alone in BRCA1/2m tumors. Methods: We conducted a phase Ib study of sapa/ola in pts with BRCA1/2m HER2-negative MBC who were not previously PARPi-exposed. Primary objective was to determine the RP2D of sapa with ola. Secondary objectives included safety, tolerability and mPFS. RAD51 staining was performed on archival tissue. Serial blood samples were collected for circulating tumor DNA (ctDNA) analysis using low-pass WGS with targeted sequencing via the DDR Discovery Panel (Resolution Bioscience). Results: 10 women (median age 46) were enrolled before the study was terminated for hematologic toxicity. Germline BRCA1 and BRCA2 mutations were present in 3 and 7 pts, respectively. 4 pts had received prior platinum. All pts initially received standard dose ola (300 mg twice daily) with 100 mg (n = 3), 150 mg (n = 4), or 200 mg (n = 3) sapa once daily on days 1-5 and 8-12 of an every 28-day cycle. The RP2D was not determined. Hematologic AEs were common, including anemia (80%), neutropenia (80%), and thrombocytopenia (40%), with 1 DLT of G4 neutropenia lasting more than 7 days. The ORR was 50% (95% CI: 18.7% - 81.3%) with mPFS of 9.7 months (95% CI: 8.02 - NA). 3 pts had clinical benefit of 15+ months, exceeding expectations with olaparib alone, including 2 with PR, and 2 who remained on trial for 36+ months. 3 patients had confirmed PRs of short duration. Of the remaining patients, one withdrew consent early and the other 3 had non-CR/non-PD or SD of 4, 7 and 9 months. At progression, 2 pts had evidence of BRCA reversion mutation associated with an MMEJ signature; ctDNA also demonstrated MRE11 and PTEN amplifications in 1 of these pts, suggesting multiple resistance mechanisms. 3 pts had no evidence of reversion but had acquired putative non-reversion mechanisms of resistance, including changes suggesting enhanced MRN complex function, FANCD2 and ATR amplifications and PIK3CA mutation. The majority of tumors (6/10) were RAD51-negative. Other tumors with RAD51 foci demonstrated replication stress evidenced by high levels of pKAP1 and pRPA, suggesting benefit associated with sapa. Conclusions: Sapa/ola produces high rates of hematologic toxicity. However, the ORR of 50%, mPFS of 9.7 months, and durability of 3+ years in 2 pts suggest possible combinatorial benefit. Further exploration with different sapa schedules or with substitution of a PARP1-selective inhibitor to decrease hematologic toxicity is warranted. Citation Format: Filipa Lynce, Noah Graham, Bose S. Kochupurakkal, Huy Nguyen, Philip D. Poorvu, Victoria Attaya, Raechel Davis, Kavita Garg, Lee P. Lim, Mark Li, Cloud P. Paweletz, Nabihah Tayob, Geoffrey I. Shapiro, Sara M. Tolaney. A phase Ib study of sapacitabine (sapa) and olaparib (ola) in patients (pts) with BRCA1/2-mutated (BRCA1/2m) metastatic breast cancer (MBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT188.

Importance of Germline and Somatic Alterations in Human MRE11, RAD50, and NBN Genes Coding for MRN Complex

The MRE11, RAD50, and NBN genes encode for the nuclear MRN protein complex, which senses the DNA double strand breaks and initiates the DNA repair. The MRN complex also participates in the activation of ATM kinase, which coordinates DNA repair with the p53-dependent cell cycle checkpoint arrest. Carriers of homozygous germline pathogenic variants in the MRN complex genes or compound heterozygotes develop phenotypically distinct rare autosomal recessive syndromes characterized by chromosomal instability and neurological symptoms. Heterozygous germline alterations in the MRN complex genes have been associated with a poorly-specified predisposition to various cancer types. Somatic alterations in the MRN complex genes may represent valuable predictive and prognostic biomarkers in cancer patients. MRN complex genes have been targeted in several next-generation sequencing panels for cancer and neurological disorders, but interpretation of the identified alterations is challenging due to the complexity of MRN complex function in the DNA damage response. In this review, we outline the structural characteristics of the MRE11, RAD50 and NBN proteins, the assembly and functions of the MRN complex from the perspective of clinical interpretation of germline and somatic alterations in the MRE11, RAD50 and NBN genes.