Homologous Recombination DNA Repair Research Articles

RAD18 O-GlcNAcylation promotes translesion DNA synthesis and homologous recombination repair

RAD18, an important ubiquitin E3 ligase, plays a dual role in translesion DNA synthesis (TLS) and homologous recombination (HR) repair. However, whether and how the regulatory mechanism of O-linked N-acetylglucosamine (O-GlcNAc) modification governing RAD18 and its function during these processes remains unknown. Here, we report that human RAD18, can undergo O-GlcNAcylation at Ser130/Ser164/Thr468, which is important for optimal RAD18 accumulation at DNA damage sites. Mechanistically, abrogation of RAD18 O-GlcNAcylation limits CDC7-dependent RAD18 Ser434 phosphorylation, which in turn significantly reduces damage-induced PCNA monoubiquitination, impairs Polη focus formation and enhances UV sensitivity. Moreover, the ubiquitin and RAD51C binding ability of RAD18 at DNA double-strand breaks (DSBs) is O-GlcNAcylation-dependent. O-GlcNAcylated RAD18 promotes the binding of RAD51 to damaged DNA during HR and decreases CPT hypersensitivity. Our findings demonstrate a novel role of RAD18 O-GlcNAcylation in TLS and HR regulation, establishing a new rationale to improve chemotherapeutic treatment.

Abstract PO2-08-11: The MAGIC study: Universal whole genome tumour and germline sequencing of newly diagnosed breast cancer identifies a high proportion of ER+ BRCA-like tumours

Abstract Background: Identification of germline mutations in hereditary breast cancer (HBC) genes can have profound benefits in the treatment of breast cancer (BC) and manging risk for patient and the family. In addition, sequencing of the tumours can reveal clinically relevant somatic features, such as homologous recombination deficiency (HRD) and mutational signatures, that can guide treatment which are not discernible by germline sequencing alone. We hypothesised that current standard of care where women diagnosed with BC are not routinely offered tumour sequencing is suboptimal as fails to exploit all therapeutic vulnerabilities which can be exploited in the treatment of BC. The MAGIC study is the first prospective trial in Australia of unselected invasive BCs in general oncology practice combining both germline and tumour sequencing. Methods: The MAGIC study included a total of 651 consecutive consented patients presenting with non-metastatic BC between June 2020 to March 2023. It included invasive BC, high-grade DCIS and pleomorphic LCIS. The sequencing was conducted in two phases; for phase one, 157 cases underwent whole genome sequencing (WGS) on both germline and matched BC DNA while for phase two, 494 cases underwent only germline whole exome sequencing. Germline variants were interrogated for pathogenic variants in BRCA1, BRCA2, PALB2, ATM, CHEK2, BARD1, BRIP1, RAD51B, RAD51C, RAD51D, MLH1, MSH2, MSH6, PMS2, CDH1, PTEN, STK11, TP53 and NTHL1. Tumour homologous recombination (HR) repair deficiency was calculated using HRDetect (Nat Med 2017;23:517). For BCs showing a high HRDetect score ( >0.75) all HBC gene promoter CpG islands were assessed for hypermethylation using the Twist NGS Methylation system. Mutational signatures were calculated from somatic mutations identified from whole genome sequenced BCs using the DeconstructSig package in R (Genome Biol. 2016;17:31) and referenced against the COSMIC v2 signature catalogue. Results: Actionable germline variants in any HBC gene were identified in 7.7% of cases (7.8% of invasive and 6.1% of in situ BC) with 5.2% having an actionable mutation in an HR pathway gene (BRCA1, BRCA2, PALB2 and RAD51C). Most cases with pathogenic variants showed bi-allelic inactivation in the tumour. All BRCA1, BRCA2 and PALB2 tumours showed high HRDetect and/or HRD scores consistent with loss of HR repair function, indicating them to be the underlying hereditary driver of the BC. One BC with a pathogenic PMS2 variant showed retention of the wild-type allele with no evidence of a mismatch repair mutational signature suggesting PMS2 was not the driver of this BC. Of the 157 BC in phase one that underwent WGS and did not have a germline pathogenic HBC gene variant, 16% (18/117 invasive and 3/13 DCIS) had an HRDetect score of >0.7 indicative of a homologous recombination repair defect and therefore biologically a “BRCA-like” tumour. Of these 21 BRCA-like cases, only 3 invasive BCs had clear somatic bi-allelic inactivation of an HBC gene that could explain the high HRDetect score (2 BRCA1 and 1 BRCA2). One of the 21 BRCA-like cases had a germline variant of unknown significance (VUS) in an HBC gene (a BRCA2 missense variant in an ER+ tumour. The tumour showed loss of the wild-type allele). Of the BRCA-like cases, 71% were ER positive which included 3 invasive lobular cancers. Conclusion: Tumour sequencing identified three times as many cases that might be eligible for HR repair defect targeted therapy than germline testing alone (16% versus 5.2%). Unlike a previous study (Ann Oncol., 2019 30:1071-1079), the majority of the BRCA-like BCs were ER+ with no evidence of a VUS in a known HBC gene suggesting these are driven by novel germline or somatic mutations in genes involved in DNA HR repair. Citation Format: Ian Campbell, Lisa Devereux, Kirsten Hogg, Luxi Lal, Michelle Sinclair, Lesley Stafford, Magnus Zethoven, Anita Skandarajah, Paul James, Geoffrey Lindeman, Bruce Mann, Dilanka De Silva. The MAGIC study: Universal whole genome tumour and germline sequencing of newly diagnosed breast cancer identifies a high proportion of ER+ BRCA-like tumours [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-08-11.

Abstract PO1-06-09: Phase 2 Trial with Safety Run-In of Gedatolisib Plus Talazoparib in Advanced Triple Negative or BRCA1/2 Positive, HER2 Negative Breast CancersBig Ten Cancer Research Consortium BTCRC-BRE18-337

Abstract Up to 2/3 of triple negative breast cancers (TNBC) have acquired defects in homologous recombination (HR) DNA repair, yet poly (ADP-ribose) polymerase inhibitor (PARPi) monotherapy has been largely ineffective in the absence of a germline BRCA 1/2 mutation (gBRCA1/2). Phosphoinositide-3-kinase (PI3K)/mTOR pathway alterations are also common in breast cancers. Preclinical data suggest that PI3K/mTOR inhibition may disrupt normal function of the HR complex and increase dependency on PARP enzymes for HR DNA repair. Thus, combining a PI3K/mTOR inhibitor with a PARPi may result in a synergistic anti-neoplastic effect. The run-in portion of this study evaluated the safety of weekly IV gedatolisib (PI3K/mTORi) and continuous daily talazoparib to determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). A 3+3 design for dose escalation explored two dose levels. The phase II study began once the MTD and R2PD were confirmed. Eligibility required age ≥ 18, 1-2 prior lines of therapy (protocol later amended to allow up to 3 lines), and advanced TNBC or advanced HER2-negative BC with gBRCA1/2 mutation. The sample size for the phase II study was determined based on a 1-sided binomial test under the null and alternative ORR of 5% vs 20% with a type I error rate of 0.1 and power level of 80%. The primary endpoint was overall response rate (ORR) in TNBC without known gBRCA1/2 with secondary endpoints of progression-free survival (PFS) and overall survival (OS). Patients with a gBRCA1/2 mutation were not included in the primary endpoint analysis. Correlative studies are planned to evaluate HR deficiency mutations, PIK3CA mutations, and other exploratory genomics. A total of 33 patients were enrolled: 14 in the safety run-in phase of the trial and 19 in the phase II study (17 TNBC, 2 with gBRCA2 mutation). In the safety run-in, 6 patients were enrolled at dose level 1 (0.75 mg talazoparib po daily and 180 mg gedatolisib IV weekly) and 8 at dose level 2 (1 mg talazoparib po daily and 180 mg gedatolisib IV weekly). 42% of the cohort developed hyperglycemia, which was mostly grade 1. There was 1 DLT of grade 3 neutropenia at dose level 1. There were 3 patients who experienced grade 4 AEs, thrombocytopenia (2) and lymphopenia (1) which were outside of the DLT window. The MTD was 1 mg of talazoparib daily and 180 mg of gedatolisib weekly, and this was selected as the RP2D. A protocol amendment was made during the phase II portion to allow for a 3 week on/1 week off schedule for gedatolisib due to emerging data showing a more favorable safety profile and enhanced antitumor activity with this dosing schedule. In the 17 patients with TNBC and no gBRCA mutation in the phase II cohort, the ORR was 12%. Best response was partial response (PR) in 2 patients (12%), stable disease (SD) in 7 patients (41%), and progressive disease (PD) in 8 patients (47%). The clinical benefit rate (ORR+SD) at 16 weeks was 23.5%. The most common adverse events (AEs) in all 33 patients were anemia (70%), fatigue (67%), oral mucositis (64%), nausea (60%), neutropenia (45%) and anorexia (45%). Of these, most were grade 1-2 other than anemia (35% grade 3), neutropenia (20% grade 3), fatigue (18% grade 3), and oral mucositis (10% grade 3). There were no grade 4 AEs in the phase II study. Median PFS was approximately 3 months and median OS was approximately 6.4 months. Although this study did not meet its primary endpoint, there were 2 TNBC patients without a gBRCA1/2 mutation who achieved a partial response to this non-chemotherapy regimen. Future biomarker testing may help elucidate these findings and possible predictors of response. Citation Format: Sneha Phadke, Kari Wisinski, Oana Danciu, Ami Shah, Menggang Yu, Yi Chen, Kathy Miller, Mark Burkard. Phase 2 Trial with Safety Run-In of Gedatolisib Plus Talazoparib in Advanced Triple Negative or BRCA1/2 Positive, HER2 Negative Breast CancersBig Ten Cancer Research Consortium BTCRC-BRE18-337 [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-06-09.

Abstract PO1-20-07: Valosin-containing protein (VCP)/p97: A novel breast cancer susceptibility gene?

Abstract Introduction: Mutations in the valosin-containing protein (VCP, also known as p97) gene are associated with autosomal dominant inclusion-body myopathy with early-onset Paget disease and frontotemporal dementia. VCP is an ATPase that subserves a variety of cellular functions, prominently, organelle biogenesis and ubiquitin-dependent protein degradation. Recently, VCP has also been shown to physically interact with BRCA1 and is postulated to orchestrate the response to DNA double-strand breaks, a critical molecular prelude to homologous recombination DNA repair. Moreover, VCP is also involved in the removal of trapped poly [ADP-ribose] polymerase 1 (PARP1) from chromatin and may play a key role in the response of tumor cells to PARP inhibitors. Whether VCP acts as a breast cancer predisposition gene is currently unknown. These observations raise speculation as to whether pathologic variation in VCP may predispose to breast cancer. Case Description: A 45-year-old female presented with early-age, locally advanced invasive ductal carcinoma, grade 2, ER < 1%, PR 10%, HER2 IHC 3+, FISH ratio 5.4. The patient completed 6 cycles of neoadjuvant PTH and was surgically downstaged to ypT1bN1a at the time of her left mastectomy. She declined adjuvant radiation. She received 1 dose of trastuzumab emtansine which was discontinued due to intolerance and transitioned to complete 1 year of adjuvant trastuzumab and pertuzumab. Her medical history is significant for inclusion body myopathy with Paget disease of bone and/or frontotemporal dementia, which was diagnosed at age 36 after presenting with proximal muscle weakness. Bone scan was consistent with Paget’s disease and neurocognitive testing did not identify any abnormalities. Diagnosis was confirmed via whole genome germline sequencing which identified a pathogenic variant in VCP [c.463C >G (p.Arg155Gly)]. Germline resequencing of 155 known cancer predisposition genes did not identify any pathogenic variants. Whole exome genomic analysis of the patient’s breast cancer revealed an elevated homologous recombination score ( > 42), consistent with molecularly important role of VCP in homologous recombination breast cancer DNA repair. The patient’s family history is significant for muscular dystrophy in multiple family members on her paternal side, each of whom was deceased prior to age 60. Her paternal half-sister has both inclusion body myopathy and breast cancer, and her paternal cousin was diagnosed with breast cancer at age 46 (unknown genetic testing for each). No history of cancer on her maternal side. Discussion: This case raises the intriguing question of whether the VCP gene represents a heretofore unrecognized breast cancer predisposition gene. Further investigation into the role of VCP in tumorigenesis is warranted, and additional population studies are necessary to confirm VCP’s role in breast cancer predisposition. Given that cancers with homologous recombination deficiencies benefit from PARP inhibitors and VCP is involved in the function of the PARP1 protein, patients with mutations in the VCP gene may benefit from PARP inhibition. Citation Format: Alexis LeVee, Duveen Sturgeon, Joanne Mortimer, Kevin McDonnell. Valosin-containing protein (VCP)/p97: A novel breast cancer susceptibility gene? [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-20-07.

Monoterpene indole alkaloids with anticancer activity from Tabernaemontana species

AbstractIndole alkaloids, predominantly found in plants, are a large group of natural product-derived compounds characterized by a remarkable chemical diversity associated with significant biological properties. Among these, terpenoid indole alkaloids are the largest group of secondary metabolites. Tabernaemontana species (Apocynaceae) are widely distributed in tropical and subtropical regions of the world and used in traditional medicine to treat a variety of illnesses, including tumours. These species biosynthesize large quantities of structurally complex monoterpene indole and bisindole alkaloids. Given the compelling biological properties of indole alkaloids, the phytochemical study of Tabernaemontana species has been widely addressed to identify potential anticancer compounds. Several indole alkaloids have shown antiproliferative effect due to different mechanisms, namely by inducing apoptosis or arresting cell cycle, in diverse cancer cell lines, including multidrug-resistant phenotypes. This review primarily aims to underscore the anticancer activity of indole and bisindole alkaloids isolated from several Tabernaemontana species. Moreover, our recent contributions to the field are also highlighted, focusing on the study of Tabernaemontana elegans. The anticancer evaluation, namely the reversion of P-glycoprotein-mediated multidrug resistance, of two sets of monoterpene indole alkaloid derivatives, obtained by modification of some functional groups of two major monoterpene indole alkaloids, is reviewed, as well as the results obtained for a derivative that targeted homologous recombination DNA repair defects.

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.

Antitumoral Activity of the Universal Methyl Donor S-Adenosylmethionine in Glioblastoma Cells.

Glioblastoma (GBM), the most frequent and lethal brain cancer in adults, is characterized by short survival times and high mortality rates. Due to the resistance of GBM cells to conventional therapeutic treatments, scientific interest is focusing on the search for alternative and efficient adjuvant treatments. S-Adenosylmethionine (AdoMet), the well-studied physiological methyl donor, has emerged as a promising anticancer compound and a modulator of multiple cancer-related signaling pathways. We report here for the first time that AdoMet selectively inhibited the viability and proliferation of U87MG, U343MG, and U251MG GBM cells. In these cell lines, AdoMet induced S and G2/M cell cycle arrest and apoptosis and downregulated the expression and activation of proteins involved in homologous recombination DNA repair, including RAD51, BRCA1, and Chk1. Furthermore, AdoMet was able to maintain DNA in a damaged state, as indicated by the increased γH2AX/H2AX ratio. AdoMet promoted mitotic catastrophe through inhibiting Aurora B kinase expression, phosphorylation, and localization causing GBM cells to undergo mitotic catastrophe-induced death. Finally, AdoMet inhibited DNA repair and induced cell cycle arrest, apoptosis, and mitotic catastrophe in patient-derived GBM cells. In light of these results, AdoMet could be considered a potential adjuvant in GBM therapy.

SIRT2 promotes base excision repair by transcriptionally activating OGG1 in an ATM/ATR-dependent manner.

Sirtuin 2 (SIRT2) regulates the maintenance of genome integrity by targeting pathways of DNA damage response and homologous recombination repair. However, whether and how SIRT2 promotes base excision repair (BER) remain to be determined. Here, we found that independent of its catalytic activity SIRT2 interacted with the critical glycosylase OGG1 to promote OGG1 recruitment to its own promoter upon oxidative stress, thereby enhancing OGG1 promoter activity and increasing BER efficiency. Further studies revealed that SIRT2 was phosphorylated on S46 and S53 by ATM/ATR upon oxidative stress, and SIRT2 phosphorylation enhanced the SIRT2-OGG1 interaction and mediated the stimulatory effect of SIRT2 on OGG1 promoter activity. We also characterized 37 cancer-derived SIRT2 mutants and found that 5 exhibited the loss of the stimulatory effects on OGG1 transcription. Together, our data reveal that SIRT2 acts as a tumor suppressor by promoting OGG1 transcription and increasing BER efficiency in an ATM/ATR-dependent manner.

Abstract 3344: Widespread BRCA1/2-independent homologous recombination defects are caused by alterations in RNA binding proteins

Abstract Summary: Defects in homologous recombination DNA repair (HRD) both predispose to cancer development and produce therapeutic vulnerabilities, making it critical to define the spectrum of genetic events that cause HRD. However, we found that mutations in BRCA1/2 and other canonical HR genes only identified 10-20% of tumors that display genomic evidence of HRD. Using a networks-based approach, we discovered that over half of putative genes causing HRD originated outside of canonical DNA damage response genes, with a particular enrichment for RNA binding protein (RBP)-encoding genes. These putative drivers of HRD were experimentally validated, cross-validated in an independent cohort, and enriched in cancer-associated GWAS loci. Mechanistic studies indicate that some RBPs are recruited to sites of DNA damage to facilitate repair, whereas others control the expression of canonical HR genes. Overall, this study greatly expands the repertoire of known drivers of HRD, with implications for basic biology, genetic screening, and therapy stratification. Citation Format: Babita Kaundal, Daniel J. McGrail, Yang Li, Roger S. Smith, Bin Feng, Hui Dai, Limei Hu, Briana Dennehey, Sharad Awasthi, Marc L. Mendillo, Anil K. Sood, Gordon B. Mills, Shiaw-Yih Lin, S. Stephen Yi, Nidhi Sahni. Widespread BRCA1/2-independent homologous recombination defects are caused by alterations in RNA binding proteins [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 3344.

Abstract 4538: Brigatinib induces synergy with PARP inhibitors through dual inhibition of FAK and EphA2 in high-grade serous ovarian carcinoma

Abstract PARP inhibitors (PARPis) have now become a standard of care for high-grade serous ovarian cancer (HGSOC), especially for those with defects in homologous recombination (HR) DNA repair. However, the innate and acquired PARPi resistance in HGSOC poses a significant concern, necessitating the exploration of novel treatment options and strategies. Here we investigated the potential of combining brigatinib—a second-generation anaplastic lymphoma kinase (ALK) inhibitor approved for treating lung cancer—with PARPis as way to enhance PARPi activity in HGSOC. Clonogenic cell survival assays revealed that brigatinib synergizes with PARPis in HR-proficient and -deficient HGSOC cell lines. In subsequent proteomics approaches and cell-based confirmatory studies, we found that brigatnib suppressed tyrosine kinases FAK and EphA2 with reduction in downstream signaling. Follow-up pharmacologic and gene-silencing techniques demonstrated that dual FAK and EphA2 inhibition additively reduces PI3K/Akt and MAPK/ERK cell survival signaling to levels conferring PARPi sensitivity. Additionally, the efficacy of this therapeutic approach on tumor size reduction was assessed in HGSOC patient-derived xenograft (PDX) models, with combination treatment inducing tumor regression more effectively than either agent alone. Overall, our studies unveil a beneficial ALK-independent effect of brigatinib that may offer new avenues for considering its repurposing as a HGSOC therapy. Citation Format: Julie R. Duffield, Xiaonan Hou, Iman K. McKeon-Makki, Amelia M. Huehls, Benjamin W. Wilson, Anjali Prasad, Xinyan Wu, Scott H. Kaufmann, Larry M. Karnitz, John J. Weroha, Arun Kanakkanthara. Brigatinib induces synergy with PARP inhibitors through dual inhibition of FAK and EphA2 in high-grade serous ovarian carcinoma [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 4538.

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.

Abstract 1987: PARP inhibitor (PARPi) rechallenge shows differential sensitivity in PARPi olaparib-resistant BRCA1-mutated (BRCA1m) high-grade serous ovarian cancer (HGSC)

Abstract Background: PARPis have changed the treatment paradigm in HGSC, particularly for women with BRCA mutations. PARPi, olaparib (O), is a standard of care maintenance therapy following platinum-based chemotherapy in newly diagnosed BRCA-mutated (BRCAm) HGSC. But patients ultimately develop resistance to O. We investigated whether rechallenge with different PARPi monotherapy could cause cytotoxicity in BRCAm HGSC after becoming O-resistant. We also aimed to identify possible mechanisms of actions of PARPi’s differential sensitivity. Methods: We developed a PARPi-resistant line (UWB-OlaJR) from BRCA1m PARPi-sensitive cells (UWB1.289) by gradient exposure of O with initial lower doses to a final concentration of 20 μM over 10 months. Four PARPis including O, niraparib (N), talazoparib (T), and veliparib (V) were tested. Cell growth was assessed using 5-day XTT and 10-day clonogenic assays. DNA damage and homologous recombination (HR) repair activity were evaluated using immunofluorescence (IF) staining of γH2AX and RAD51 foci, respectively. Replication fork (RF) dynamics was measured by DNA fiber assay. All data were repeated in triplicate and analyzed using t-test or one-way ANOVA. Data were shown as mean ± SD and P < 0.05 was considered statistically significant. Results: 5-day XTT assay shows a 53.7-fold increase in resistance to O in UWB-OlaJR relative to UWB1.289 (IC50 2.2 μM vs. 118.1 μM). Cross-resistance was observed with other 3 PARPis, showing increased IC50 values compared to its parental line (V: 23.4-fold, N: 160.5-fold, T: 229.6-fold). However, 10-day clonogenic assay showed no resistance against N (1.09-fold) and T (0.51-fold) in UWB-OlaJR, indicating differential sensitivity to different PARPis by a long-term cytotoxicity measure. Surprisingly, γH2AX foci did not increase in response to PARPis in UWB-OlaJR compared to UWB1.289 (O: 2.58-fold, V: 2.33-fold; N: 2.89-fold, T: 2.58-fold; all P < 0.05), suggesting possible restored DNA repair function. All four PARPis induced replication stress in both O-sensitive and -resistant cell lines as evidenced by decreased RF speed compared to untreated group (O: 0.57- and 0.7-fold, V: 0.68- and 0.74-fold, N: 0.47- and 0.47-fold, T: 0.47- and 0.52-fold; all P < 0.001). Notably, N and T treatments reduced fork speed to a greater extent than O and V in UWB-OlaJR. However, RF protection was not affected when UWB-OlaJR treated with different PARPis compared to UWB1.289 (O: 0.89-fold, V: 0.87-fold, N: 0.71-fold, T: 0.83-fold; all P < 0.05), suggesting that differential sensitivity to different PARPis was unlikely due to modulation of RF stabilization. Conclusions: Our findings demonstrate different PARPis may induce varied levels of cytotoxicity and replication stress in O-resistant BRCA1m HGSC, possibly via modulating alternative RF dynamics besides RF protection. Citation Format: Chi-Ting Shih, Tzu-Ting Huang, Jayakumar R. Nair, Jung-Min Lee. PARP inhibitor (PARPi) rechallenge shows differential sensitivity in PARPi olaparib-resistant BRCA1-mutated (BRCA1m) high-grade serous ovarian cancer (HGSC) [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 1987.

Abstract 4447: ZC3H18: A novel regulator of NAD+ metabolism in high-grade serous ovarian cancer

Abstract ZC3H18 is a multifunctional regulator of gene expression that is lost in a subset of high-grade serous ovarian cancers (HGSOC). Our prior studies have established that the loss of ZC3H18 diminishes BRCA1 levels, leading to the disruption of homologous recombination DNA repair (HR) and heightened sensitivity of HGSOC cells to PARP inhibitors. Here we show an additional facet to ZC3H18's impact, showing that its loss also induces a reprogramming of energy metabolism in HGSOC. Our findings demonstrate that the depletion of ZC3H18 results in reduced mRNA and protein levels of the NAD+-biosynthetic enzyme, NMNAT1, in HGSOC cells. Mechanistic studies uncovered that ZC3H18 occupies the NMNAT1 promoter, facilitating NMNAT1 transcription by recruiting CDK12 onto the promoter. Consistent with this mechanism, loss of ZC3H18 led to a reduction in CDK12 levels on the NMNAT1 promoter, diminishing NMNAT1 abundance and subsequently causing a decline in cellular NAD levels. These observations unveil a previously unknown role for ZC3H18 in HGSOC, shedding light on its involvement in the regulation of energy metabolism. Importantly, our findings provide valuable insights into potential therapeutic pathways that leverage the metabolic changes linked to ZC3H18 deficiency. Citation Format: Benjamin Wilson, Natalie Wilson, Xianfeng Chen, Shuwen Zhang, Chen Wang, Larry M. Karnitz, Scott H. Kaufmann, Arun Kanakkanthara. ZC3H18: A novel regulator of NAD+ metabolism in high-grade serous ovarian cancer [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 4447.

Abstract 1673: KRAS dependency, a gene editing approach

Abstract The RAS family genes encode small GTP-binding cytoplasmic proteins that are important signaling molecules. They regulate cell growth, survival and differentiation by coupling receptor activation to downstream effector pathways. Activating mutations of oncogenic RAS pathway genes are frequently detected in human cancers. The role of KRAS in tumor formation is not questionable. It’s implication in tumor maintenance is less well validated. The KRAS dependency is a key question to answer before to develop KRAS inhibitor. Targeted genome editing using CRISPR/Cas9 is a relatively new, revolutionary technology allowing for efficient and directed alterations of the genome. CRISPR is a genome-editing platform that makes use of the bacterially-derived endonuclease Cas9 to introduce DNA double-strand breaks at precise locations in the genome using complementary guide RNAs. These double strand breaks can be repaired by homologous recombination DNA repair mechanisms thank to a donor DNA template. By this way mutations can be introduced at desired loci, DNA fragment can be deleted or introduce at the desired location. We aimed at investigating the KRAS dependence and we applied the CRISPR-Cas9 technology to engineer isogenic cancer cells harboring a “conditional KRAS gene knockout" by exchanging the KRAS endogenous promoter. This promoter replacement approach can be used also to validate new targets. Citation Format: Christophe Marcireau, Fréderic Lacroix, Dietmar Hoffmann, May Cindhuchao, Loreley Calvet, Yvette Ruffin, Laurent Debussche. KRAS dependency, a gene editing approach [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 1673.

PRMT5-mediated homologous recombination repair is essential to maintain genomic integrity of neural progenitor cells

Maintaining genomic stability is a prerequisite for proliferating NPCs to ensure genetic fidelity. Though histone arginine methylation has been shown to play important roles in safeguarding genomic stability, the underlying mechanism during brain development is not fully understood. Protein arginine N-methyltransferase 5 (PRMT5) is a type II protein arginine methyltransferase that plays a role in transcriptional regulation. Here, we identify PRMT5 as a key regulator of DNA repair in response to double-strand breaks (DSBs) during NPC proliferation. Prmt5F/F; Emx1-Cre (cKO-Emx1) mice show a distinctive microcephaly phenotype, with partial loss of the dorsal medial cerebral cortex and complete loss of the corpus callosum and hippocampus. This phenotype is resulted from DSBs accumulation in the medial dorsal cortex followed by cell apoptosis. Both RNA sequencing and in vitro DNA repair analyses reveal that PRMT5 is required for DNA homologous recombination (HR) repair. PRMT5 specifically catalyzes H3R2me2s in proliferating NPCs in the developing mouse brain to enhance HR-related gene expression during DNA repair. Finally, overexpression of BRCA1 significantly rescues DSBs accumulation and cell apoptosis in PRMT5-deficient NSCs. Taken together, our results show that PRMT5 maintains genomic stability by regulating histone arginine methylation in proliferating NPCs.

A novel SIK2 inhibitor SIC-19 exhibits synthetic lethality with PARP inhibitors in ovarian cancer

PurposeOvarian cancer patients with HR proficiency (HRP) have had limited benefits from PARP inhibitor treatment, highlighting the need for improved therapeutic strategies. In this study, we developed a novel SIK2 inhibitor, SIC-19, and investigated its potential to enhance the sensitivity and expand the clinical utility of PARP inhibitors in ovarian cancer. MethodsThe SIK2 protein was modeled using a Molecular Operating Environment (MOE), and the most favorable model was selected based on a GBVI/WSA dG scoring function. The Chembridge Compound Library was screened, and the top 20 candidate compounds were tested for their interaction with SIK2 and downstream substrates, AKT-pS473 and MYLK-pS343. SIC-19 emerged as the most promising drug candidate and was further evaluated using multiple assays. ResultsSIC-19 exhibited selective and potent inhibition of SIK2, leading to its degradation through the ubiquitination pathway. The IC50 of SIC-19 correlated inversely with endogenous SIK2 expression in ovarian cancer cell lines. Treatment with SIC-19 significantly inhibited cancer cell growth and sensitized cells to PARP inhibitors in vitro, as well as in ovarian cancer organoids and xenograft models. Mechanistically, SIK2 knockdown and SIC-19 treatment reduced RAD50 phosphorylation at Ser635, prevented nuclear translocation of RAD50, disrupted nuclear filament assembly, and impaired DNA homologous recombination repair, ultimately inducing apoptosis. These findings highlight the crucial role of SIK2 in the DNA HR repair pathway and demonstrate the significant PARP inhibitor sensitization achieved by SIC-19 in ovarian cancer. ConclusionsSIC-19, a novel SIK2 inhibitor, effectively inhibits tumor cell growth in ovarian cancer by interfering with RAD50-mediated DNA HR repair. Furthermore, SIC-19 enhances the efficacy of PARP inhibitors, providing a promising therapeutic strategy to improve outcomes for ovarian cancer patients.

Germline Mutations of Holliday Junction Resolvase Genes in Multiple Primary Malignancies Involving Lung Cancer Lead to PARP Inhibitor Sensitization.

The incidence of multiple primary malignancies (MPM) involving lung cancer has increased in recent decades. There is an urgent need to clarify the genetic profile of such patients and explore more efficacious therapy for them. Peripheral blood samples from MPM involving patients with lung cancer were assessed by whole-exome sequencing (WES), and the identified variants were referenced for pathogenicity using the public available database. Pathway enrichment analysis of mutated genes was performed to identify the most relevant pathway. Next, the effects of mutations in relevant pathway on function and response to targeted drugs were verified by in vitro and in vivo experiments. Germline exomes of 71 patients diagnosed with MPM involving lung cancer were sequenced. Pathway enrichment analysis shows that the homologous recombination repair (HRR) pathway has the strongest correlation. Moreover, HRR genes, especially key Holliday junction resolvases (HJR) genes (GEN1, BLM, SXL4, and RMI1), were most frequently mutated, unlike the status in the samples from patients with lung cancer only. Next, we identified a total of seven mutations in HJR genes led to homologous recombination DNA repair deficiency and rendered lung cancer cells sensitive to PARP inhibitor treatment, both in vitro and in vivo. This is the first study to map the profile of germline mutations in patients with MPM involving lung cancer. This study may shed light on early prevention and novel targeted therapies for MPM involving patients with lung cancer with HJR mutations.

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

Genetic Basis of Breast and Ovarian Cancer: Approaches and Lessons Learnt from Three Decades of Inherited Predisposition Testing.

Germline variants occurring in BRCA1 and BRCA2 give rise to hereditary breast and ovarian cancer (HBOC) syndrome, predisposing to breast, ovarian, fallopian tube, and peritoneal cancers marked by elevated incidences of genomic aberrations that correspond to poor prognoses. These genes are in fact involved in genetic integrity, particularly in the process of homologous recombination (HR) DNA repair, a high-fidelity repair system for mending DNA double-strand breaks. In addition to its implication in HBOC pathogenesis, the impairment of HR has become a prime target for therapeutic intervention utilizing poly (ADP-ribose) polymerase (PARP) inhibitors. In the present review, we introduce the molecular roles of HR orchestrated by BRCA1 and BRCA2 within the framework of sensitivity to PARP inhibitors. We examine the genetic architecture underneath breast and ovarian cancer ranging from high- and mid- to low-penetrant predisposing genes and taking into account both germline and somatic variations. Finally, we consider higher levels of complexity of the genomic landscape such as polygenic risk scores and other approaches aiming to optimize therapeutic and preventive strategies for breast and ovarian cancer.

Bazedoxifene analogs as potential WDHD1 degraders and antitumor agents: Synthesis, evaluation and molecular dynamics simulation studies.

DNA repair is strongly associated with tumor resistance to radiotherapy and chemotherapy. WD repeat and HMG-box DNA binding protein 1 (WDHD1) is a key adaptor for homologous recombination repair of DNA, and its overexpression is relevant to the poor prognosis of many tumor patients. We previously have identified and validated bazedoxifene (BZA), which had 60% inhibitory rate on WDHD1 in MCF7 cells at 10 μM, from the Food and Drug Administration-approvedcompound library. Here, we initially established the binding model of BZA, synthesized and evaluated eight BZA analogs. Further, we detailed the use of molecular dynamics simulations to provide insights into the basis for activity against WDHD1. This binding mode will be instructive for the development of new WDHD1 degraders.