Recombination DNA Repair Pathway Research Articles

Abstract B029: Repurposing Digital PCR to Diagnose and Create a Novel Genomic Signature for Homologous Recombination Deficiency in Pancreatic Adenocarcinoma

Abstract Introduction: Germline mutations in DNA repair genes are often associated with increased risk of cancer. Mutations that affect the homologous recombination (HR) DNA repair pathway ( e.g., BRCA 1 and BRCA2) are known drivers of pancreatic adenocarcinoma. Early detection relies on identification of pathogenic mutations in patients with a family history of cancer. Next generation sequencing panels often use a select set of pathogenic variants for diagnosis, meaning that individuals with variants of uncertain significance may be missed, limiting testing of other family members for the potentially disease-causing variant. Here we describe a digital PCR assay developed to detect BRCA1 and BRCA2 variant that may eventually serve as a rapid, cost-effective alternative to sequencing that costs less than $5.00 per sample with results in 24 hours. This study highlights the need for better diagnostic tools, which will become critically important as we begin to study pathogenic mutations in more diverse populations. Methods: We have collected blood from 4 BRCA1 mutants and 5 BRCA2 mutants who have already undergone germline sequencing from commercially available assays. Clinicopathologic data has been collected as well. We designed two fluorescently labelled TaqMan probes, one that recognizes the wild-type BRCA alleles and one specific for mutant BRCA alleles. Briefly, 1 ng of genomic DNA is mixed with amplicon primers and fluorescent Taq-Man probes, then partitioned into 20,000 separate 517 picoliter wells. Quantification and detection are achieved based upon the fluorescent signal measured from each well. Results: We have successfully confirmed pathologic BRCA1 and BRCA2 mutations previously identified with next generation sequencing in 100% of samples analyzed. All three BRCA2 mutations generate a premature stop codon leading to a truncated protein and are known to be pathogenic. Two related patients have the same BRCA1 frameshift mutation that results in a premature stop codon, and one patient has a duplicated cytosine that leads to a frameshift mutation that is one of three characterized Ashkenazi Jewish founder mutations that increases cancer risk in this population . Conclusions: Here we report an innovative assay using digital PCR (dPCR) that provides a cost-effective, time-efficient method to identify MMR mutants and characterize VUS in patients and their families. Citation Format: Jennifer Brooke Valerin, Sophie Hasson, Valeria Rangel, Nicholas Pannunzio. Repurposing Digital PCR to Diagnose and Create a Novel Genomic Signature for Homologous Recombination Deficiency in Pancreatic Adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B029.

Triple Combinations of Histone Lysine Demethylase Inhibitors with PARP1 Inhibitor-Olaparib and Cisplatin Lead to Enhanced Cytotoxic Effects in Head and Neck Cancer Cells.

PARP inhibitors are used to treat cancers with a deficient homologous recombination (HR) DNA repair pathway. Interestingly, recent studies revealed that HR repair could be pharmacologically impaired by the inhibition of histone lysine demethylases (KDM). Thus, we investigated whether KDM inhibitors could sensitize head and neck cancer cells, which are usually HR proficient, to PARP inhibition or cisplatin. Therefore, we explored the effects of double combinations of KDM4-6 inhibitors (ML324, CPI-455, GSK-J4, and JIB-04) with olaparib or cisplatin, or their triple combinations with both drugs, on the level of DNA damage and apoptosis. FaDu and SCC-040 cells were treated with individual compounds and their combinations, and cell viability, apoptosis, DNA damage, and gene expression were assessed using the resazurin assay, Annexin V staining, H2A.X activation, and qPCR, respectively. Combinations of KDM inhibitors with cisplatin enhanced cytotoxic effects, unlike combinations with olaparib. Triple combinations of KDM inhibitors with cisplatin and olaparib exhibited the best cytotoxic activity, which was associated with DNA damage accumulation and altered expression of genes associated with apoptosis induction and cell cycle arrest. In conclusion, triple combinations of KDM inhibitors (especially GSK-J4 and JIB-04) with cisplatin and olaparib represent a promising strategy for head and neck cancer treatment.

DIPG-82. RADIOSENSITIZATION WITH RAD52 INHIBITION IN DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Diffuse midline glioma (DMG) is one of the most devastating childhood cancers that limited response to radiation therapy (RT). There is a critical need for new therapeutics that enhance the radiation effect. We, therefore, tested the hypothesis that targeting RAD52 activity sensitizes the radiation response of DMG. METHODS Genome-wide CRISPR/Cas9 screening was performed to identify the potential therapeutic target. RAD52 inhibition was used by shRNA-mediated RAD52 depletion and treatment with RAD52 inhibitor, D-I03. The protein expression, cell proliferation, DNA damage marker expression, DNA repair pathway, and transcriptional alteration were analyzed by western blotting, MTS assay, colony formation assay, immunocytochemistry, DNA repair pathway assay, and RNA sequencing. Mice with DMG patient-derived xenograft (PDX) models were treated with RAD52 inhibitor alone or combination with RT. RESULTS Genome-wide CRISPR/Cas9 screening identified RAD52 as a potential therapeutic target in DMG cells. RAD52 inhibition suppressed DMG cell proliferation. Importantly, RAD52 inhibition in combination with RT further increased the radiosensitivity of DMG cells. Immunocytochemistry of DNA double-strand breaks (DSB) marker γH2AX and repair marker 53BP1 showed that RAD52 inhibition sustained DNA damage with high levels of γH2AX at 24 hours following radiation while the level of 53BPI was decreased, thereby inhibiting DNA DSB repair. Western blotting also revealed that RAD52 inhibition causes a sustained level of phosphorylated Rad50 and γH2AX in irradiated DIPG cells over 24 hours. DNA repair assay showed that RAD52 inhibition suppressed homologous recombination DNA repair pathway. RNA sequencing showed that RAD52 inhibition downregulated genes associated with the DNA repair pathway. Finally, the combination therapy of RAD52 inhibitor and RT further suppressed tumor growth and increased survival of mice bearing DMG PDXs, outperforming either monotherapy. CONCLUSIONS these results highlight RAD52 inhibition as a potential radiosensitization and provide a rationale for developing combination therapy with radiation in the treatment of DMG.

BRCA1/BARD1 ubiquitinates PCNA in unperturbed conditions to promote continuous DNA synthesis

Deficiencies in the BRCA1 tumor suppressor gene are the main cause of hereditary breast and ovarian cancer. BRCA1 is involved in the Homologous Recombination DNA repair pathway and, together with BARD1, forms a heterodimer with ubiquitin E3 activity. The relevance of the BRCA1/BARD1 ubiquitin E3 activity for tumor suppression and DNA repair remains controversial. Here, we observe that the BRCA1/BARD1 ubiquitin E3 activity is not required for Homologous Recombination or resistance to Olaparib. Using TULIP2 methodology, which enables the direct identification of E3-specific ubiquitination substrates, we identify substrates for BRCA1/BARD1. We find that PCNA is ubiquitinated by BRCA1/BARD1 in unperturbed conditions independently of RAD18. PCNA ubiquitination by BRCA1/BARD1 avoids the formation of ssDNA gaps during DNA replication and promotes continuous DNA synthesis. These results provide additional insight about the importance of BRCA1/BARD1 E3 activity in Homologous Recombination.

Abstract P69: Immune Landscape of BRCA1/2 -mutated Breast and Ovarian Cancers in an Asian Cohort

Abstract Background: The majority of hereditary breast and ovarian cancers (HBOC) harbor defects in the homologous recombination (HR) DNA repair pathway, such as BRCA1 and BRCA2 mutations, which may influence the response to immune checkpoint blockade (ICB) therapy. Recent clinical trials combining poly-ADP ribose inhibitors and ICB therapy in advanced breast and ovarian cancer have shown promising results. This Asian cohort study set out to determine if immune phenotype, specifically the PD-1/PD-L1 axis, was associated with germline BRCA status in advanced breast and ovarian cancer patients and to develop novel biomarkers of HR deficiency to stratify patients for clinical management. Methods: Tissue microarrays of ovarian tumors and breast tumors were analyzed by multiplex immunohistochemistry (mIHC) to detect the expression of immune markers (CD68, CD8, PD-L1, PD-1) and HR markers (DNA polymerase theta, FANCD2, RAD51, γH2AX, phospho-ATR). The results were used to build a prediction model for BRCA using backward stepwise logistic regression analysis. In addition, we investigated immune infiltration in tumors and performed immune phenotyping on peripheral blood mononuclear cells in a subset of patients. Results: We identified potential BRCA1/2 predictors by mIHC, namely RAD51 and PD-L1 263 for ovarian tumors and FANCD2 and phospho-ATR for breast tumors. The area under the curve was 0.770 (95% confidence interval [CI]: 0.564, 0.977) in ovarian cancer patients and 0.841 (95% CI: 0.682, 0.999) in breast cancer patients. In addition, the proportion of tumor-infiltrating CD68+PD-L1+ macrophages was significantly upregulated in BRCA1-mutated tumors, while the levels of PD-1 in circulating central memory CD8+ T cells were higher in breast cancer patients with germline BRCA2 mutations, compared with non-BRCA. Conclusion: This study provides evidence of the association of the PD-1/PD-L1 axis with germline BRCA status in HBOC, which may aid clinical management. Citation Format: Siao Ting Chong, Felicia Wee, Jiang Feng Ye, Craig Joseph, Xinru Lim, Zhen Wei Neo, Justina Nadia Lee, Nur Diana Binte Ishak, Ming Ren Toh, Ee Ling Chew, Claresta Yeo, Sock Hoai Chan, Jeffrey Lim, Joe Yeong, Joanne Ngeow. Immune Landscape of BRCA1/2 -mutated Breast and Ovarian Cancers in an Asian Cohort [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P69.

Abstract 5273: A novel immunogenic cell death that exploits the cancer-specific overexpression of Rad51

Abstract Overexpression of the Homologous Recombination (HR) DNA repair pathway is often correlated with poor prognosis and metastatic disease. SyntheX developed STX100, a small, stabilized peptide that is able to disrupt the function of Rad51 - a crucial component of HR. STX100 exhibits low nanomolar affinity to the target, cell permeability, and proteolytic stability. STX100 has acute mono-agent activity that is selective towards HR-overexpressing cancer cells. Interestingly, STX100-mediated cell killing is independent of canonical cell death mechanisms (apoptosis, necroptosis, pyroptosis, ferroptosis, etc.). Rather, the elevated abundance of Rad51 in cancer cells relative to healthy tissues underlies the selective cytotoxicity resulting from an acute calcium surge upon STX100-mediated target engagement. The mechanism translates to in vivo models, where a single dose can achieve sustained survival when administered locally as a mono-therapy or in combination with an immune checkpoint blockade agent. Citation Format: Maria Soloveychik, Charly Chahwan. A novel immunogenic cell death that exploits the cancer-specific overexpression of Rad51 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5273.

BRCA1/2 alterations and reversion mutations in the area of PARP inhibitors in high grade ovarian cancer: state of the art and forthcoming challenges.

BRCA1/2 genes are part of homologous recombination (HR) DNA repair pathways in charge of error-free double-strand break (DSB) repair. Loss-of-function mutations of BRCA1/2 genes have been associated for a long time with breast and ovarian cancer hereditary syndrome. Recently, polyadenosine diphosphate-ribose polymerase inhibitors (PARPi) have revolutionized the therapeutic landscape of BRCA1/2-mutated tumors, especially of BRCA1/2 high-grade serous ovarian cancer (HGSC), taking advantage of HR deficiency through the synthetic lethality concept. However, PARPi efficiency differs among patients, and most of them will develop resistance, particularly in the relapse setting. In the current proposal, we aim to review primary and secondary resistance to PARPi in HGSC owing to BRCA1/2 alterations. Of note, as several mechanisms of primary or secondary resistance to PARPi have been described, BRCA1/2 reversion mutations that restore HR pathways are by far the most reported. First, the type and location of the BRCA1/2 primary mutation have been associated with PARPi and platinum-salt sensitivity and impact the probability of the occurrence and the type of secondary reversion mutation. Furthermore, the presence of multiple reversion mutations and the variation of allelic frequency under treatment underline the role of intratumor heterogeneity (ITH) in treatment resistance. Of note, circulating tumor DNA might help us to detect and characterize reversion mutations and ITH to finally refine the treatment strategy. Importantly, forthcoming therapeutic strategies, including combination with antiangiogenics or with targeted therapies, may help us delay and overcome PARPi resistance secondary to BRCA1/2 reversion mutations. Also, progression despite PARPi therapy does not preclude PARPi rechallenge in selected patients.

Abstract A057: Inter-tumor heterogeneity revealed by multi-site whole genome sequencing of high grade serous ovarian cancer

Abstract A057: Inter-tumor heterogeneity revealed by multi-site whole genome sequencing of high grade serous ovarian cancer

An 19F NMR fragment-based approach for the discovery and development of BRCA2-RAD51 inhibitors to pursuit synthetic lethality in combination with PARP inhibition in pancreatic cancer

The BRCA2-RAD51 interaction remains an intriguing target for cancer drug discovery due to its vital role in DNA damage repair mechanisms, which cancer cells become particularly reliant on. Moreover, RAD51 has many synthetically lethal partners, including PARP1-2, which can be exploited to induce synthetic lethality in cancer. In this study, we established a 19F-NMR-fragment based approach to identify RAD51 binders, leading to two initial hits. A subsequent SAR program identified 46 as a low micromolar inhibitor of the BRCA2-RAD51 interaction. 46 was tested in different pancreatic cancer cell lines, to evaluate its ability to inhibit the homologous recombination DNA repair pathway, mediated by BRCA2-RAD51 and trigger synthetic lethality in combination with the PARP inhibitor talazoparib, through the induction of apoptosis. Moreover, we further analyzed the 46/talazoparib combination in 3D pancreatic cancer models. Overall, 46 showed its potential as a tool to evaluate the RAD51/PARP1-2 synthetic lethality mechanism, along with providing a prospect for further inhibitors development.

Dissection of DNA damage and repair pathways in live cells by femtosecond laser microirradiation and free-electron modeling

Understanding and predicting the outcome of the interaction of light with DNA has a significant impact on the study of DNA repair and radiotherapy. We report on a combination of femtosecond pulsed laser microirradiation at different wavelengths, quantitative imaging, and numerical modeling that yields a comprehensive picture of photon-mediated and free-electron-mediated DNA damage pathways in live cells. Laser irradiation was performed under highly standardized conditions at four wavelengths between 515 nm and 1,030 nm, enabling to study two-photon photochemical and free-electron-mediated DNA damage in situ. We quantitatively assessed cyclobutane pyrimidine dimer (CPD) and γH2AX-specific immunofluorescence signals to calibrate the damage threshold dose at these wavelengths and performed a comparative analysis of the recruitment of DNA repair factors xeroderma pigmentosum complementation group C (XPC) and Nijmegen breakage syndrome 1 (Nbs1). Our results show that two-photon-induced photochemical CPD generation dominates at 515 nm, while electron-mediated damage dominates at wavelengths ≥620 nm. The recruitment analysis revealed a cross talk between nucleotide excision and homologous recombination DNA repair pathways at 515 nm. Numerical simulations predicted electron densities and electron energy spectra, which govern the yield functions of a variety of direct electron-mediated DNA damage pathways and of indirect damage by •OH radicals resulting from laser and electron interactions with water. Combining these data with information on free electron-DNA interactions gained in artificial systems, we provide a conceptual framework for the interpretation of the wavelength dependence of laser-induced DNA damage that may guide the selection of irradiation parameters in studies and applications that require the selective induction of DNA lesions.

Final results of a pilot study of docetaxel and carboplatin for treatment of patients with mCRPC containing biallelic inactivation of genes in the homologous recombination DNA repair pathway.

5054 Background: Inactivating mutations in genes important in homologous recombination DNA repair (HRD) are present in the tumors of ~25% of patients with metastatic castration resistant prostate cancer (mCRPC). These alterations are associated with more aggressive disease but may also predict vulnerability to DNA damaging agents. We previously reported promising interim results from a single arm, Phase 1/2 trial studying the combination of the DNA damaging agent carboplatin with docetaxel for patients with mCRPC containing HRD mutations. Here we report final results after completing accrual and two additional years of follow up. Methods: We assessed response to the combination of docetaxel 60mg/m2 and carboplatin AUC 5 IV q21 days in patients with mCRPC whose tumors had evidence of biallelic inactivation of genes involved in HRD and who had progressed on any prior treatment, including docetaxel and/or PARP inhibitor (PARPi). The primary endpoint was the proportion of patients who achieved ≥50% PSA decline from baseline (PSA50). With H0 of PSA50 of 26%, 14 patients provided 80% power to conclude H1 of PSA50 of 60% based on Simon's 2-stage design with 1-sided α = 5%. The secondary endpoints included PSA30, response duration, progression-free survival (PFS), and overall survival (OS). Results: The trial opened in Jan 2016 and completed in Sep 2021. A total of 16 patients were treated on study, including 6 who had previously received PARPi and 5 who had received docetaxel. 11/16 patients (69%) achieved PSA50. 7/9 patients (78%) with biallelic inactivation of BRCA2 (7), BRCA1 (1), or PALB2 (1) achieved PSA50. Other biallelic inactivated HRD-related genes were ATM (1), CHD1 (1), CDK12 (3), MRE11A (1), and NBN (1). Of these patients, 4/7 (57%) achieved PSA50. Notably, 3/6 patients (50%) previously treated with PARPi therapy achieved PSA50. The median PFS was 8.4 months, and the median OS was 2 years. 6/16 patients (38%) had grade 3 AEs, most commonly thrombocytopenia (25%) and anemia (13%). One patient (6%) had a grade 4 AE (thrombocytopenia). Conclusions: The addition of carboplatin to docetaxel significantly improved response rates in patients with mCRPC with biallelic inactivation in genes involved in the HRD pathway. Responses were also seen in patients who had previously received PARPi. The combination was relatively safe and well-tolerated. The encouraging activity and favorable toxicity profile observed here merits further investigation. Additional work is ongoing to further refine biomarkers predictive of treatment response and resistance. Clinical trial information: NCT02598895 .

Whole-genome sequencing based assessment of HER2 focal amplification for precision oncology of breast cancers.

609 Background: Focal oncogene amplification confers a growth advantage to tumor cells and drives cancer evolution. Herein, we aimed to explore focally amplified HER2 in breast carcinomas. Methods: We performed whole genome sequencing (WGS) and whole-transcriptome sequencing (WTS) in 787 breast cancer samples. HER2 focal amplification was defined as having a segment length less than 3Mbp and ≥4 higher copy number than the surrounding region. Results: Most of the patients were premenopausal women (n = 574, 72.9%) with a median age of 37 (range 21-76) years. We identified 57 (58.8%) and 27 (5.0%) samples with HER2 focal amplification in HER2-positive (n = 97) and even HER2-negative (n = 536) by immunohistochemistry (IHC) and in-situ hybridization (ISH) according to ASCO guideline for breast cancer, respectively. Regardless of HER2-positivity, HER2 focal amplification was significantly associated with higher HER2 RNA expression. Among 28 patients who received six cycles of docetaxel/carboplatin/trastuzumab/pertuzumab neoadjuvant therapy followed by curative surgery, 21 patients had HER2 focal amplification and 14 (66.7%) achieved pathologic complete remission (pCR). Intriguingly, all patients without HER2 focal amplification (n = 7) failed to achieve pCR. HER2 focal amplification showed enrichment of TP53 mutations (OR 0.245, q < 0.001), but was mutually exclusive with GATA3 mutations (OR 2.82, q = 0.055). The mutational spectrums suggested that the activity in APOBEC family of cytidine deaminases were responsible for the somatic single-nucleotide variants of breast cancer with HER2 focal amplification. Compared to samples without HER2 focal amplification, samples with HER2 focal amplification were less likely to have defective homologous recombination DNA repair pathway. HER focal amplifications were closely associated with various types of structural variations. Translocation partners of HER2 were widespread across the entire genome with notable exceptions on chromosome 2, and most of them were close to super-enhancers implying enhancer hijacking as one of mechanisms of HER2 overexpression. Breast cancers with or without HER2 focal amplification showed distinct RNA expression patterns. Notably, activity of genes involved in doxorubicin-resistance and AKT1 signaling were significantly associated with HER2 focal amplification. Conclusions: Our analysis demonstrates that WGS enables better classification of the HER2 amplification status in breast cancer than IHC with ISH, suggesting WGS as a sensitive tool for screening breast cancers for anti-HER2-targeted therapies.

Targeting homologous recombination deficiency in uterine leiomyosarcoma

BackgroundUterine leiomyosarcoma (uLMS) is a rare and aggressive gynaecological malignancy, with individuals with advanced uLMS having a five-year survival of < 10%. Mutations in the homologous recombination (HR) DNA repair pathway have been observed in ~ 10% of uLMS cases, with reports of some individuals benefiting from poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) therapy, which targets this DNA repair defect. In this report, we screened individuals with uLMS, accrued nationally, for mutations in the HR repair pathway and explored new approaches to therapeutic targeting.MethodsA cohort of 58 individuals with uLMS were screened for HR Deficiency (HRD) using whole genome sequencing (WGS), whole exome sequencing (WES) or NGS panel testing. Individuals identified to have HRD uLMS were offered PARPi therapy and clinical outcome details collected. Patient-derived xenografts (PDX) were generated for therapeutic targeting.ResultsAll 13 uLMS samples analysed by WGS had a dominant COSMIC mutational signature 3; 11 of these had high genome-wide loss of heterozygosity (LOH) (> 0.2) but only two samples had a CHORD score > 50%, one of which had a homozygous pathogenic alteration in an HR gene (deletion in BRCA2). A further three samples harboured homozygous HRD alterations (all deletions in BRCA2), detected by WES or panel sequencing, with 5/58 (9%) individuals having HRD uLMS. All five individuals gained access to PARPi therapy. Two of three individuals with mature clinical follow up achieved a complete response or durable partial response (PR) with the subsequent addition of platinum to PARPi upon minor progression during initial PR on PARPi. Corresponding PDX responses were most rapid, complete and sustained with the PARP1-specific PARPi, AZD5305, compared with either olaparib alone or olaparib plus cisplatin, even in a paired sample of a BRCA2-deleted PDX, derived following PARPi therapy in the patient, which had developed PARPi-resistance mutations in PRKDC, encoding DNA-PKcs.ConclusionsOur work demonstrates the value of identifying HRD for therapeutic targeting by PARPi and platinum in individuals with the aggressive rare malignancy, uLMS and suggests that individuals with HRD uLMS should be included in trials of PARP1-specific PARPi.

Clinical efficacy of PARP inhibitors in breast cancer

BRCA1 and BRCA2 are key tumor suppressor genes that are essential for the homologous recombination DNA repair pathway. Loss of function mutations in these genes result in hereditary breast and ovarian cancer syndromes, which comprise approximately 5% of cases. BRCA1/2 mutations are associated with younger age of diagnosis and increased risk of recurrences. The concept of synthetic lethality led to the development of PARP inhibitors which cause cell cytotoxicity via the inhibition of PARP1, a key DNA repair protein, in cells with germline BRCA1/2 mutations. Although still poorly understood, the most well-acknowledged proposed mechanisms of action of PARP1 inhibition include the inhibition of single strand break repair, PARP trapping, and the upregulation of non-homologous end joining. Olaparib and talazoparib are PARP inhibitors that have been approved for the management of HER2-negative breast cancer in patients with germline BRCA1/2 mutations. This review article highlights the clinical efficacy of PARP inhibitors in patients with HER2-negative breast cancer in early and advanced settings.

Abstract 875: Breast cancer bioinformatics: Untangling the roles of nucleolin and BRCA1 in dysregulated DNA repair

Abstract Proteins that enable cells to sense and repair DNA damage are essential for maintaining the stability of genomes across cell divisions; dysregulation of these processes is an established hallmark of tumorigenesis. Nucleolin (NCL), a major RNA binding protein (RBP) and a caretaker tumor suppressor protein BRCA1 (the breast &amp; ovarian cancer susceptibility gene) colocalize in breast cancer. Both NCL and BRCA1 have defined roles in homologous recombination (HR) and non-homologous end joining (NHEJ) DNA repair pathways which are often dysregulated in breast cancer. BRCA1, along with a complex molecular network of signaling proteins gets recruited to damaged replication forks to initiate the HR pathway where NCL function is also implicated. However, how NCL collaborates with BRCA1 to orchestrate the complex mechanism of DNA repair under stress conditions remains unknown. To address this gap in knowledge, we have applied in silico approaches to uncover the key players which drive the molecular interactions of NCL and BRCA1 at the damaged sites. We utilized the NIH PPI, IntAct, STRING, BioGRID, GeneMania, PrePPI, and Mentha databases to derive an overlapping interactome. In this study we present a comprehensive interactome analysis and 47 proteins identified that interact with both NCL and BRCA1. As expected, majority of these common interactors participate in DNA damage damage response along with several that are implicated in regulating the gene expression via chromatin remodeling/RNA binding or controlling cell proliferation. We classified this interactome into 5 major categories based on their GO functional annotations: chromatin remodeling, DNA damage, DNA repair, RNA-binding proteins, and cell cycle. Previously, we have successfully used computational approaches to model the RNA-binding domains (RBD) of NCL and delineate the binding interfaces between NCL-RBD and a subset of miRNA that are specifically dysregulated in breast cancer. Using the same strategy, we have modeled the full length NCL to provide a complete structural representation of the protein. Our model fills the gap in missing NCL structural data, especially the unexplored N- and C - termini that may play important roles in NCL protein-protein interactions. Our results provide predicted interaction scenarios between NCL and the subset of the overlapping interactome of NCL and BRCA1, focused on DNA repair mechanisms. These in silico models are critical to understand the protein complexity at the interface of damaged DNA to identify candidates that can be targeted in breast carcinoma. Citation Format: Nitu Farhin, Andy Lam, Anjana D. Saxena, Shaneen Singh. Breast cancer bioinformatics: Untangling the roles of nucleolin and BRCA1 in dysregulated DNA repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 875.

Synthesis and biological evaluation of DIDS analogues as efficient inhibitors of RAD51 involved in homologous recombination

RAD51 is a pivotal protein of the homologous recombination DNA repair pathway, and is overexpressed in some cancer cells, disrupting then the efficiency of cancer-treatments. The development of RAD51 inhibitors appears as a promising solution to restore these cancer cells sensitization to radio- or chemotherapy. From a small molecule identified as a modulator of RAD51, the 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), two series of analogues with small or bulky substituents on the aromatic parts of the stilbene moiety were prepared for a structure–activity relationship study. Three compounds, the cyano analogue (12), and benzamide (23) or phenylcarbamate (29) analogues of DIDS were characterized as novel potent RAD51 inhibitors with HR inhibition in the micromolar range.

An archaeal Cas3 protein facilitates rapid recovery from DNA damage.

CRISPR-Cas systems provide heritable acquired immunity against viruses to archaea and bacteria. Cas3 is a CRISPR-associated protein that is common to all Type I systems, possesses both nuclease and helicase activities, and is responsible for degradation of invading DNA. Involvement of Cas3 in DNA repair had been suggested in the past, but then set aside when the role of CRISPR-Cas as an adaptive immune system was realized. Here we show that in the model archaeon Haloferax volcanii a cas3 deletion mutant exhibits increased resistance to DNA damaging agents compared with the wild-type strain, but its ability to recover quickly from such damage is reduced. Analysis of cas3 point mutants revealed that the helicase domain of the protein is responsible for the DNA damage sensitivity phenotype. Epistasis analysis indicated that cas3 operates with mre11 and rad50 in restraining the homologous recombination pathway of DNA repair. Mutants deleted for Cas3 or deficient in its helicase activity showed higher rates of homologous recombination, as measured in pop-in assays using non-replicating plasmids. These results demonstrate that Cas proteins act in DNA repair, in addition to their role in defense against selfish elements and are an integral part of the cellular response to DNA damage.

Pan-cancer analysis of the spectrum of homologous recombination DNA repair (HRR) pathway genes in the Indian population: A retrospective observational study

Background: Homologous recombinant repair (HRR) deficit and the associated sensitivity to poly (ADP-ribose) polymerase inhibitors (PARPi) has been well studied in breast, ovarian, prostate, and pancreatic cancers, but very little is known about it in other cancer types. Objectives: We sought to understand the spectrum of HRR mutations in various cancer types, with the goal of identifying therapeutic targets in lesser-explored cancers. Materials and Methods: In this retrospective study conducted between January 2021 and December 2022, we analyzed a cohort of 659 patients with various cancer types with mutations in 15 HRR genes using next generation sequencing, at 4baseCare Onco Solutions Pvt. Ltd., Bengaluru, Karnataka, India. Results: We identified a total of 825 gene variants, including 366 likely pathogenic/pathogenic mutations (44.4%), with BRCA1 (196 variants; 22.8%), BRCA2 (183 variants; 21.3%), and ATM (157 variants; 18.3%) being prevalent. Germline and somatic mutations were prevalent in BRCA1 (114 variants; 60.3%) and BRCA2 (46 variants; 24%), respectively. Recurrent mutations were identified in 8 genes, including BRCA1, BRCA2, BRIP1, BARD1, CDK12, CHEK2, PALB2 and RAD54L. BRCA1 and BRCA2 mutations were observed in breast, gynecologic, and musculoskeletal cancers; ATM and BRCA2 in gastrointestinal and biliary tract cancers, respiratory, and head-and-neck cancers; BRCA2 and CDK12 in genitourinary cancers. Additionally, co-occurring mutations (in genes such as BRCA1-BRIP1, ATM-BRCA2, ATM-BRIP1) and known therapeutically significant mutations were identified. Conclusions: The presence of therapeutically significant HRR mutations across a broad spectrum of cancer types in our study suggests that these mutations can possibly be targeted, especially in cancers where there is a paucity of therapeutic targets. Further, non-BRCA HRR genes, such as ATM and CDK12, could play a more prominent role than previously recognized.

Emerging strategies: PARP inhibitors in combination with immune checkpoint blockade in BRCA1 and BRCA2 mutation-associated and triple-negative breast cancer.

Poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitor monotherapy in germline BRCA1 and BRCA2 mutation-associated metastatic breast cancer is a well-tolerated and an effective therapeutic strategy, however, the durability of response can be limited. Checkpoint inhibitors targeting the PD-1/PD-L1 axis as monotherapy in metastatic triple-negative breast cancer (mTNBC) have a limited role due to low response rates, but are capable of long, durable responses. Combination PARP inhibition with checkpoint blockade is an emerging area of investigation with potential synergy to produce robust responses with durability. Mechanistically, PARP inhibition activates the stimulator of interferon gene (STING) pathway to promote dendritic cell and T lymphocyte recruitment, increases tumor neoantigens, and upregulates PD-L1 expression to increase the immunogenicity of the tumor and thereby potentially enhance responses to immunotherapy. Several clinical trials have reported early results on PARP inhibitor and PD-1/PD-L1 checkpoint inhibitor combinations. All studies have shown safety and tolerability of this combination regimen. In advanced breast cancer associated with a germline BRCA1 or BRCA2 mutation, response rates have been high and similar to what is observed with PARP inhibitor monotherapy. Additional follow-up is needed to see if combination with a checkpoint inhibitor can lead to a clinically meaningful extension of durability of response in patients with germline mutations in BRCA1 and BRCA2. In unselected mTNBC in the 1st-3rd line setting, response rates of combined PARP inhibitor and PD-1/PD-L1 inhibitors have ranged from 18-21%, with higher rates of response among those with alterations in homologous recombination DNA repair pathway genes. Multiple ongoing studies will report additional data on combinations of PARP inhibitors and checkpoint blockade in the future and this combination strategy remains an active area of investigation.

Targeting Krebs-cycle-deficient renal cell carcinoma with Poly ADP-ribose polymerase inhibitors and low-dose alkylating chemotherapy.

Loss-of-function mutations in genes encoding the Krebs cycle enzymes Fumarate Hydratase (FH) and Succinate Dehydrogenase (SDH) induce accumulation of fumarate and succinate, respectively and predispose patients to hereditary cancer syndromes including the development of aggressive renal cell carcinoma (RCC). Fumarate and succinate competitively inhibit αKG-dependent dioxygenases, including Lysine-specific demethylase 4A/B (KDM4A/B), leading to suppression of the homologous recombination (HR) DNA repair pathway. In this study, we have developed new syngeneic Fh1- and Sdhb-deficient murine models of RCC, which demonstrate the expected accumulation of fumarate and succinate, alterations in the transcriptomic and methylation profile, and an increase in unresolved DNA double-strand breaks (DSBs). The efficacy of poly ADP-ribose polymerase inhibitors (PARPis) and temozolomide (TMZ), alone and in combination, was evaluated both in vitro and in vivo. Combination treatment with PARPi and TMZ results in marked in vitro cytotoxicity in Fh1- and Sdhb-deficient cells. In vivo, treatment with standard dosing of the PARP inhibitor BGB-290 and low-dose TMZ significantly inhibits tumor growth without a significant increase in toxicity. These findings provide the basis for a novel therapeutic strategy exploiting HR deficiency in FH and SDH-deficient RCC with combined PARP inhibition and low-dose alkylating chemotherapy.