babam2 Antibody

What is BABAM2 and what are its primary cellular functions?

BABAM2, also known as BRE (Brain and reproductive organ-expressed protein), is a 415 amino acid protein that plays essential roles in promoting cell cycle progression and preventing cellular senescence. It binds to the intracellular juxtamembrane domain of death receptors including tumor necrosis factor receptor 1 (TNF-R1) and FAS. BABAM2 downregulates TNFα-induced activation of NFκB and may play roles in homeostasis or cellular differentiation in epithelial, neural, and germ line cells. Additionally, it inhibits components of death-inducing signaling complexes necessary for mitochondrial activation during apoptosis. BABAM2 is strongly expressed in adrenal cortex, medulla, testis, and pancreas, with weaker expression in thymus, thyroid, stomach, and small intestine .

What is the subcellular localization of BABAM2?

BABAM2 exhibits dual localization within cells. It is primarily found in both the cytoplasm and nucleus under normal physiological conditions. Importantly, during DNA damage responses, BABAM2 specifically localizes at sites of DNA damage, particularly at double-strand breaks, indicating its potential role in DNA repair mechanisms. This localization pattern is critical for researchers using immunofluorescence techniques to study BABAM2 in the context of DNA damage response pathways .

How does BABAM2 expression change during osteoclast differentiation?

Research has demonstrated that BABAM2 expression is significantly downregulated during osteoclast differentiation. Both mRNA and protein levels of BABAM2 gradually decrease over time in mouse bone marrow-derived macrophages (BMMs) and RAW264.7 mouse monocyte-macrophages undergoing osteoclastogenesis. This reduction correlates with increased expression of osteoclast differentiation marker genes such as Nfatc1, Ctsk, and Mmp9. Additionally, BABAM2 expression levels are significantly decreased in bone tissues of OVX (ovariectomized) mice compared to sham-operated mice, suggesting BABAM2 functions as a negative regulator of osteoclast formation .

What applications are BABAM2 antibodies commonly used for?

BABAM2 antibodies are primarily used in Western blotting (WB) and immunohistochemistry (IHC) applications. For Western blotting, BABAM2 antibodies are typically used at dilutions of 1:500-1:2000, while for IHC applications, dilutions range from 1:50-1:200. These antibodies have been verified in Western blots using A431 cell lysates and in IHC applications on human liver cancer and thyroid cancer tissues. BABAM2 antibodies reactive with human, mouse, and rat samples are available, making them versatile tools for comparative studies across different mammalian models .

How should I design experiments to investigate BABAM2's role in DNA damage response?

When studying BABAM2's involvement in DNA damage response, a comprehensive experimental approach should include both in vitro and in vivo components. For in vitro studies, induce DNA damage in mESCs or other relevant cell lines using either gamma irradiation (8 Gy for 150s using equipment such as Nordion Gammacell 3000) or chemical agents like doxorubicin (250 nM for 6h). Use both wild-type and BABAM2-knockout cells to compare responses. Monitor p53 and phosphorylated p53 (pp53) expressions using Western blot and immunofluorescence staining at different time points (2h, 4h, 8h post-treatment) to track the kinetics of p53 activation and degradation. To investigate direct interactions, perform co-immunoprecipitation assays using BABAM2- or p53-specific antibodies, followed by Western blot analysis. Additionally, examine ubiquitination patterns by overexpressing BABAM2 and assessing p53 ubiquitination status. Gene expression analysis of downstream targets like Atf3 should be conducted to elucidate the functional consequences of BABAM2-p53 interactions .

What are the best methodological approaches for studying BABAM2-protein interactions?

For studying BABAM2 protein interactions, co-immunoprecipitation (Co-IP) is the gold standard approach. The protocol should include: (1) Culture cells (BMMs or other relevant cell types) in 100mm dishes to 90% confluence; (2) Rinse cells once with ice-cold PBS and extract total proteins using IP Cell lysis buffer; (3) Add 1μg of primary antibody against BABAM2 or its potential interacting partner to the cell lysate and incubate overnight at 4°C with gentle rotation; (4) Use Protein A/G agarose to pull down the antibody-protein complexes; (5) Wash the complex four times with lysis buffer; (6) Separate immunoprecipitated proteins by suspending in SDS loading buffer and boiling for 5 minutes; (7) Analyze by Western blot. For more comprehensive analysis, Co-IP-MS (mass spectrometry) can be performed by digesting gel-separated proteins with trypsin and identifying peptide fragments by LC/MS/MS. This approach has successfully identified interactions between BABAM2 and proteins like p53 and Hey1, revealing mechanistic insights into BABAM2's functional roles in different cellular processes .

How can I resolve contradictory findings in BABAM2 expression patterns across different tissues?

When facing contradictory findings regarding BABAM2 expression patterns, employ a multi-method validation approach. First, confirm antibody specificity using both positive and negative controls, including BABAM2-knockout tissues/cells and recombinant BABAM2 protein. Use at least two different antibodies targeting different epitopes to eliminate antibody-specific artifacts. Employ multiple detection methods including qPCR for mRNA expression, Western blotting for protein levels, and immunohistochemistry/immunofluorescence for tissue localization. Importantly, standardize sample preparation, fixation methods, and antigen retrieval protocols across all experiments. Consider developmental stage, physiological conditions, and disease states as variables that might affect BABAM2 expression. Single-cell analysis techniques may be necessary to resolve cell-type specific expression patterns within heterogeneous tissues. Finally, cross-validate findings using public databases like Human Protein Atlas and GTEx to establish consensus on expression patterns .

What approaches should be used to investigate the functional significance of BABAM2 in vivo?

To investigate BABAM2's functional significance in vivo, transgenic mouse models offer the most comprehensive approach. Generate BABAM2-transgenic mice (Babam2-TG) on a C57BL/6J genetic background by cloning the full-length Babam2 coding sequence into an appropriate expression vector (e.g., pPB[Exp]-CAG plasmid). Microinject the linearized plasmid into C57BL/6J oocytes and transfer them into pseudopregnant mice. Establish lines with >5-fold overexpression of Babam2 for functional studies. Design genotyping PCR primers (e.g., F: 5′- GCA GCT TTC CTC AGT CAC TTT G -3′, R: 5′- GAA TAA GGA ATG GAC AGC AGG -3′, product size 712 bp) for accurate identification of transgenic mice. For functional characterization, implement disease models such as LPS-induced bone resorption in calvarial bone, or ovariectomy (OVX) models for osteoporosis. Analyze bone mass and resorption activity using micro-CT scanning, histological analyses including TRAP staining for osteoclasts, and serum biochemical markers of bone turnover. Compare phenotypes between transgenic mice and wild-type littermates under both normal and pathological conditions to determine BABAM2's protective or pathogenic roles .

How can I optimize western blot protocols for detecting BABAM2 in different cellular compartments?

For optimal detection of BABAM2 in different cellular compartments, compartment-specific extraction is crucial. Use a sequential extraction protocol: (1) For cytoplasmic fraction, lyse cells in buffer containing 10mM HEPES (pH 7.9), 10mM KCl, 0.1mM EDTA, 0.1mM EGTA, 1mM DTT, and protease inhibitors; (2) For nuclear fraction, resuspend remaining pellet in buffer containing 20mM HEPES (pH 7.9), 400mM NaCl, 1mM EDTA, 1mM EGTA, 1mM DTT, and protease inhibitors. For chromatin-bound fraction (important for DNA damage studies), include a nuclease treatment step. Use 10% SDS-PAGE for optimal separation of BABAM2 (calculated MW: 44 kDa). Transfer to PVDF membranes at 100V for 90 minutes in cold transfer buffer containing 20% methanol. Block with 5% skim milk for 1 hour at room temperature. For primary antibody incubation, use BABAM2 antibody at 1:1000 dilution overnight at 4°C. Include compartment-specific markers (e.g., GAPDH for cytoplasm, Lamin B1 for nucleus) as controls. When analyzing DNA damage experiments, phospho-H2AX can serve as a marker for DNA damage foci. For challenging samples, increase sensitivity using enhanced chemiluminescence substrates and optimize exposure times based on signal intensity .

What are the common pitfalls when using BABAM2 antibodies for immunohistochemistry and how can they be addressed?

Common pitfalls in BABAM2 immunohistochemistry include high background staining, weak specific signals, and false positives/negatives. To address these issues: (1) Optimize antigen retrieval methods—compare heat-induced epitope retrieval using citrate buffer (pH 6.0) versus EDTA buffer (pH 9.0); (2) Test multiple antibody dilutions between 1:50-1:200 to determine optimal concentration; (3) Implement stringent blocking procedures using 3-5% normal serum matching the species of secondary antibody plus 1% BSA; (4) Include isotype control antibodies at matching concentrations to identify non-specific binding; (5) Validate specificity using BABAM2-knockdown tissues or absorption controls with recombinant BABAM2; (6) For tissues with high endogenous peroxidase activity, extend H₂O₂ quenching steps (3% H₂O₂ for 15-20 minutes); (7) Test different detection systems (ABC method versus polymer-based systems) for optimal signal-to-noise ratio; (8) Include parallel phosphate buffer controls to distinguish between specific antibody binding and buffer effects; (9) Standardize fixation times across samples to ensure consistent epitope preservation; (10) When analyzing IHC results, incorporate digital image analysis with positive cell counting rather than subjective scoring to improve reproducibility .

How should I troubleshoot failed co-immunoprecipitation experiments when studying BABAM2 interactions?

When troubleshooting failed co-immunoprecipitation experiments for BABAM2 interactions, systematically evaluate each step of the protocol. First, confirm protein expression of both BABAM2 and the target interacting protein in input samples via Western blot. Verify antibody quality and binding capability by performing simple immunoprecipitation of each protein individually before attempting co-IP. Consider crosslinking proteins prior to lysis using cell-permeable crosslinkers like DSP (dithiobis(succinimidyl propionate)) to stabilize transient interactions. Optimize lysis conditions by testing different buffers—for nuclear interactions, use higher salt concentrations (300-400mM NaCl) followed by dilution before immunoprecipitation; for cytoplasmic interactions, use milder detergents like 0.5% NP-40. For weak interactions, increase input material (scale up from 100mm to 150mm dishes) and reduce washing stringency. Importantly, some interactions (like BABAM2-p53) may be induced or enhanced following cellular stress—consider treating cells with gamma irradiation or doxorubicin before harvesting. For detecting ubiquitination-related interactions, add deubiquitinase inhibitors like N-ethylmaleimide to lysis buffers. Finally, reverse the antibody used for pull-down (e.g., if BABAM2 antibody failed, try the interacting partner's antibody) as epitope accessibility may differ in protein complexes .

What controls should be included when studying BABAM2 expression changes during cellular differentiation?

When studying BABAM2 expression changes during cellular differentiation processes such as osteoclastogenesis, a comprehensive set of controls is essential. Include temporal controls by collecting samples at multiple time points (day 0, 1, 3, 5, 7) to establish expression kinetics. Implement positive controls by monitoring established differentiation markers—for osteoclastogenesis, include Nfatc1, Ctsk, and Mmp9 as positive indicators of differentiation progress. Include housekeeping genes/proteins that remain stable during differentiation (GAPDH, β-actin, and α-tubulin) as loading/reference controls, but validate their stability during your specific differentiation model. Incorporate biological relevance controls by comparing in vitro findings with in vivo models (such as bone tissues from OVX versus sham-operated mice). For specificity validation, include BABAM2-knockdown and BABAM2-overexpression conditions alongside normal differentiation. Monitor both mRNA (via qPCR) and protein levels (via Western blot) to distinguish between transcriptional and post-transcriptional regulation. Include pathway activity controls by monitoring signaling pathways potentially regulating BABAM2 (NFκB, MAPK, p53) during differentiation. Finally, implement methodological controls including no-RT controls for qPCR and secondary-antibody-only controls for Western blots to exclude technical artifacts .

How can BABAM2 research contribute to understanding DNA damage repair mechanisms?

BABAM2 research significantly contributes to understanding DNA damage repair mechanisms through several key pathways. BABAM2 localizes at sites of DNA damage, particularly at double-strand breaks, suggesting direct involvement in DNA repair processes. Studies have shown that BABAM2 interacts with p53, a master regulator of cellular response to DNA damage, and promotes its ubiquitination. In BABAM2-deficient mouse embryonic stem cells (mESCs), p53 and phosphorylated p53 expressions are significantly stronger and more prolonged after gamma irradiation compared to wild-type cells. This prolonged p53 activation in BABAM2-deficient cells significantly induces expression of Atf3, a stress-response gene, following DNA damage. By regulating p53 stability and activity, BABAM2 appears to modulate cellular responses to genotoxic stress, potentially affecting decisions between cell cycle arrest, DNA repair, and apoptosis. Future research should focus on identifying specific DNA repair pathways (homologous recombination, non-homologous end joining) that require BABAM2 function, and characterizing the composition of BABAM2-containing repair complexes at DNA damage sites using techniques such as ChIP-seq and proximity-dependent labeling .

What is the potential significance of BABAM2 in bone-related disorders and therapies?

BABAM2 demonstrates significant potential as a therapeutic target for bone-related disorders, particularly those characterized by excessive osteoclast activity such as osteoporosis. Research has established BABAM2 as an essential negative regulator of osteoclast formation and bone resorption. Knockdown of BABAM2 significantly accelerates osteoclast formation and activity, while BABAM2 overexpression blocks these processes. In vivo studies using BABAM2-transgenic mice show increased bone mass and significantly downregulated bone resorption activity compared to wild-type littermates. Crucially, BABAM2-transgenic mice demonstrate protection against LPS-induced bone resorption activation, resulting in reduced calvarial bone lesions. Mechanistically, BABAM2 interacts with Hey1 to inhibit Nfatc1 transcription, a master regulator of osteoclastogenesis. These findings suggest that therapeutic approaches enhancing BABAM2 expression or activity could potentially inhibit excessive osteoclast formation in conditions like osteoporosis, Paget's disease, and metastatic bone disease. Development of small molecule enhancers of BABAM2 activity or gene therapy approaches to increase BABAM2 expression in bone tissue represent promising avenues for novel anti-resorptive therapies with potentially fewer side effects than current treatments targeting osteoclast function .

How does BABAM2 function in pluripotency and stem cell maintenance?

BABAM2 plays critical roles in pluripotency and stem cell maintenance through multiple mechanisms. In mouse embryonic stem cells (mESCs), BABAM2 is essential for promoting cell cycle progression and preventing cellular senescence. BABAM2-deficient mESCs show impaired proliferation compared to wild-type cells in standard proliferation assays. Additionally, BABAM2 appears to regulate the DNA damage response in stem cells through its interaction with p53. Following DNA damage induced by gamma irradiation, BABAM2-deficient mESCs exhibit prolonged and enhanced p53 activation, which inhibits NANOG expression—a core pluripotency factor. This suggests BABAM2 helps maintain pluripotency under stress conditions by limiting p53-mediated suppression of stemness factors. The interaction between BABAM2 and p53 appears to promote p53 ubiquitination, potentially marking it for degradation and thereby regulating its activity levels in stem cells. This regulatory mechanism may be crucial for balancing the competing demands of genomic integrity maintenance and pluripotency preservation in stem cell populations. Future research should investigate how BABAM2 interacts with other pluripotency network components and whether its expression or activity changes during differentiation of various stem cell types .

What methodological advances would enhance BABAM2 research in cancer biology?

Advancing BABAM2 research in cancer biology requires several methodological innovations. Development of highly specific monoclonal antibodies against different BABAM2 epitopes would enable more precise detection of BABAM2 isoforms and post-translational modifications across cancer types. Implementation of CRISPR-Cas9 gene editing to create isogenic cancer cell lines with BABAM2 knockout, knockdown, or overexpression would provide cleaner experimental systems for functional studies. Single-cell proteomics approaches would help characterize BABAM2 expression heterogeneity within tumors and identify cancer cell subpopulations with altered BABAM2 activity. Establishment of patient-derived organoid models preserving BABAM2 expression patterns would enable more physiologically relevant drug screening. Development of small-molecule modulators (inhibitors or activators) of BABAM2 would facilitate pharmacological studies. Creation of conditional BABAM2 knockout mouse models in specific cancer backgrounds would allow temporal control of BABAM2 expression during tumor development. Implementation of spatial transcriptomics and proteomics would reveal BABAM2 distribution within tumor microenvironments. Development of liquid biopsy approaches to detect circulating BABAM2 or its fragments could provide non-invasive biomarkers. Generation of comprehensive protein interaction networks using BioID or APEX proximity labeling would map BABAM2's cancer-specific interactome. Finally, computational integration of multi-omics data would identify cancer-specific BABAM2 signatures with potential diagnostic or prognostic value .

What are the key technical specifications to consider when selecting a BABAM2 antibody?

When selecting a BABAM2 antibody for research applications, several technical specifications must be carefully evaluated. First, confirm the antibody's reactivity across species relevant to your research (human, mouse, rat) as cross-reactivity varies between products. Verify the antibody's validated applications—polyclonal BABAM2 antibodies are typically suitable for Western blotting (optimal dilution 1:500-1:2000) and immunohistochemistry (optimal dilution 1:50-1:200). Consider the clonality—polyclonal antibodies offer broader epitope recognition but potentially higher batch-to-batch variability compared to monoclonals. Identify the immunogen used to generate the antibody (e.g., recombinant protein of human BRE) and ensure it matches your experimental needs. Confirm the antibody's isotype (typically IgG) and host species (commonly rabbit) to plan appropriate secondary antibody selection and avoid cross-reactivity in multi-labeling experiments. Check the calculated molecular weight detection (44 kDa for BABAM2) to ensure proper band identification. For subcellular studies, verify the antibody's ability to detect BABAM2 in relevant compartments (cytoplasm, nucleus, DNA damage sites). Review storage requirements (typically -20°C, valid for 12 months) and shipping conditions to maintain antibody integrity. Finally, examine the validated sample types (e.g., A431 cells for WB, human liver cancer and thyroid cancer tissues for IHC) to ensure compatibility with your experimental system .

How should researchers validate BABAM2 antibody specificity for their experimental systems?

A comprehensive validation strategy for BABAM2 antibodies involves multiple complementary approaches. Begin with positive and negative control lysates—use samples with known BABAM2 expression and BABAM2 knockout/knockdown samples to confirm specific detection. Perform peptide competition assays by pre-incubating the antibody with excess immunizing peptide/protein before application; this should abolish specific signals. Compare results using multiple antibodies targeting different BABAM2 epitopes; concordant results increase confidence in specificity. Verify the molecular weight of detected bands (44 kDa for BABAM2) and investigate any unexpected bands. For antibodies intended for immunoprecipitation, perform reverse IP validation by detecting BABAM2 in immunoprecipitates pulled down using antibodies against known BABAM2-interacting partners (e.g., p53, Hey1). For immunohistochemistry applications, compare staining patterns with published BABAM2 expression data and perform parallel staining with antibodies against proteins known to colocalize with BABAM2. Use orthogonal detection methods by correlating protein detection with mRNA expression (RT-qPCR). For studies investigating BABAM2 in DNA damage responses, confirm appropriate relocalization to damage sites after induction treatments (gamma irradiation or doxorubicin). Finally, when using new lots of the same antibody, perform side-by-side comparisons with previous lots to ensure consistent performance .

What parameters should be optimized when using BABAM2 antibodies for quantitative analysis?

For accurate quantitative analysis using BABAM2 antibodies, multiple parameters must be optimized. Establish the antibody's linear detection range by creating a standard curve using known quantities of recombinant BABAM2 protein or lysates with varying BABAM2 expression levels. Optimize protein loading amounts to ensure signals fall within this linear range—typically 10-30μg total protein for Western blots. Determine optimal primary antibody concentration through titration experiments (testing dilutions from 1:500-1:2000 for Western blot and 1:50-1:200 for IHC) to maximize specific signal while minimizing background. Select appropriate detection methods—for low-abundance BABAM2 detection, enhanced chemiluminescence or fluorescent secondary antibodies offer greater sensitivity than colorimetric methods. Include multiple reference/housekeeping proteins (e.g., GAPDH, β-actin, and α-tubulin) and verify their stability under your experimental conditions. For Western blots, implement technical replicates (minimum triplicate) and biological replicates (samples from different sources) to assess reproducibility. Standardize all protocol parameters (incubation times, temperatures, buffer compositions, washing steps) for consistent results across experiments. Use image acquisition settings that avoid pixel saturation—for chemiluminescence, capture multiple exposure times to ensure optimal signal. When quantifying bands/signals, define consistent measurement regions and apply appropriate background subtraction methods. Finally, validate quantitative findings using complementary techniques such as ELISA or quantitative mass spectrometry when possible .

How might BABAM2's interaction with p53 be exploited in cancer treatment strategies?

The interaction between BABAM2 and p53 presents a promising target for cancer treatment strategies, especially for tumors with wild-type p53. Research has demonstrated that BABAM2 promotes p53 ubiquitination, likely contributing to p53 degradation and activity regulation. In BABAM2-deficient cells, p53 and phosphorylated p53 levels remain elevated for prolonged periods following DNA damage, suggesting BABAM2 inhibition could enhance p53-mediated responses to genotoxic therapies. Potential therapeutic approaches include: (1) Development of small molecule inhibitors targeting the BABAM2-p53 interaction interface to stabilize p53 in cancer cells; (2) Combination therapies pairing BABAM2 inhibition with DNA-damaging chemotherapeutics or radiation to amplify p53-dependent apoptosis; (3) Synthetic lethality approaches targeting BABAM2 in cancers with specific genetic backgrounds that make them vulnerable to p53 pathway activation; (4) Development of proteolysis-targeting chimeras (PROTACs) that could selectively degrade BABAM2 in cancer cells; (5) Gene therapy approaches to downregulate BABAM2 expression in tumors with wild-type p53. Importantly, therapeutic strategies would need to account for BABAM2's tissue-specific expressions and functions, potentially focusing on tumors derived from tissues where BABAM2 is highly expressed, such as adrenal, testicular, or pancreatic cancers. Further research characterizing the precise mechanisms and structural basis of BABAM2-p53 interaction would facilitate development of these targeted approaches .

What is the potential relationship between BABAM2 and cellular senescence pathways?

BABAM2 appears to function as a critical regulator of cellular senescence pathways. Research indicates that BABAM2 plays an essential role in preventing cellular senescence, as evidenced by its involvement in promoting cell cycle progression. The relationship between BABAM2 and senescence likely involves multiple interconnected mechanisms. First, BABAM2's regulation of p53 activity through promoting ubiquitination could directly impact senescence, as p53 is a major driver of senescence programs. In BABAM2-deficient cells, the observed prolonged and enhanced p53 activation following DNA damage could potentially trigger senescence rather than apoptosis in certain cellular contexts. Second, BABAM2's localization at DNA damage sites suggests involvement in DNA repair processes; inefficient DNA repair is a known trigger for senescence. Third, BABAM2's interaction with death receptors (TNF-R1, FAS) and modulation of NFκB signaling could influence inflammatory components of senescence, particularly the senescence-associated secretory phenotype (SASP). Future research should investigate: (1) Changes in BABAM2 expression during replicative and stress-induced senescence; (2) The senescence phenotype in BABAM2-knockdown/knockout cellular models; (3) BABAM2's interaction with other senescence regulators beyond p53, such as p16INK4a and Rb; (4) The potential role of BABAM2 in regulating SASP factor expression; (5) BABAM2's contribution to senescence in aging tissues, particularly those where it is highly expressed. Understanding these relationships could identify BABAM2 as a potential target for senolytic therapies or interventions aimed at modulating age-related pathologies .

What are the implications of BABAM2's role in the BRCA1-A complex for DNA repair studies?

BABAM2's role as a member of the BRCA1-A complex has significant implications for DNA repair studies, particularly in the context of homologous recombination and breast cancer biology. As indicated by its full name (BRISC and BRCA1-A complex member 2), BABAM2 functions within complexes critical for DNA damage response. Future studies should investigate: (1) How BABAM2 specifically contributes to BRCA1-A complex assembly, stability, and recruitment to DNA damage sites; (2) Whether BABAM2 deficiency impacts homologous recombination efficiency similarly to BRCA1 deficiency; (3) The potential synthetic lethality relationships between BABAM2 deficiency and PARP inhibitors or other DNA repair-targeting therapies; (4) Whether BABAM2 mutations or expression changes occur in BRCA1-wild-type breast cancers with homologous recombination deficiency phenotypes; (5) How BABAM2 might function as a regulator of BRCA1 activity through protein stability control, similar to its role in p53 regulation; (6) The interaction between BABAM2 and other components of the DNA damage response network beyond BRCA1 and p53. Methodologically, researchers should employ techniques like CRISPR-Cas9 screen approaches to identify genetic dependencies involving BABAM2 in DNA repair-deficient backgrounds, proximity-based labeling methods to map BABAM2's protein interaction network at DNA damage sites, and high-resolution microscopy to visualize BABAM2 dynamics during DNA damage response in real-time. Understanding these relationships could identify new therapeutic vulnerabilities in cancers with specific DNA repair defects .