Defective Homologous Recombination Research Articles

C16orf72/HAPSTR1/TAPR1 functions with BRCA1/Senataxin to modulate replication-associated R-loops and confer resistance to PARP disruption

While the toxicity of PARP inhibitors to cells with defects in homologous recombination (HR) is well established, other synthetic lethal interactions with PARP1/PARP2 disruption are poorly defined. To inform on these mechanisms we conducted a genome-wide screen for genes that are synthetic lethal with PARP1/2 gene disruption and identified C16orf72/HAPSTR1/TAPR1 as a novel modulator of replication-associated R-loops. C16orf72 is critical to facilitate replication fork restart, suppress DNA damage and maintain genome stability in response to replication stress. Importantly, C16orf72 and PARP1/2 function in parallel pathways to suppress DNA:RNA hybrids that accumulate at stalled replication forks. Mechanistically, this is achieved through an interaction of C16orf72 with BRCA1 and the RNA/DNA helicase Senataxin to facilitate their recruitment to RNA:DNA hybrids and confer resistance to PARP inhibitors. Together, this identifies a C16orf72/Senataxin/BRCA1-dependent pathway to suppress replication-associated R-loop accumulation, maintain genome stability and confer resistance to PARP inhibitors.

Platinum Sensitivity in IDH1/2 Mutated Intrahepatic Cholangiocarcinoma: Not All “BRCAness” Is Created Equal

Preclinical data suggest that IDH1/2 mutations result in defective homologous recombination repair (HRR). We hypothesized that patients with IDH1/2mt intrahepatic cholangiocarcinoma (IHCC) would benefit more from 1 L platinum chemotherapy than patients with wildtype (WT) tumors. We performed a multicenter retrospective study of 81 patients with unresectable IHCC treated with 1 L platinum with a primary endpoint of clinical benefit rate (CBR). Patients with IDH1/2mt tumors had a similar CBR and objective response rate compared to those with IDH WT disease (59 versus 54%; p = 0.803), suggesting that a relationship between platinum sensitivity and HRR gene defects may be specific to tumor context.

Hypoxia-activated prodrugs of phenolic olaparib analogues for tumour-selective chemosensitisation.

Poly(ADP-ribose)polymerase inhibitors (PARPi) are used for treatment of tumours with a defect in homologous recombination (HR) repair. Combination with radio- or chemotherapy could broaden their applicability but a major hurdle is enhancement of normal tissue toxicity. Development of hypoxia-activated prodrugs (HAPs) of PARPi has potential to restrict PARP inhibition to tumours thereby avoiding off-target toxicity. We have designed and synthesised phenolic derivatives of olaparib (termed phenolaparibs) and corresponding ether-linked HAPs. Phenolaparib cytotoxicity in HR-proficient and deficient cell lines was consistent with inhibition of PARP-1. Prodrugs were deactivated relative to phenolaparibs in biochemical PARP-1 inhibition assays, and cell culture. Prodrug 7 was selectively converted to phenolaparib 4 under hypoxia and demonstrated hypoxia-selective cytotoxicity, including chemosensitisation of HR-proficient cells in combination with temozolomide. This work demonstrates the feasibility of a HAP approach to PARPi for use in combination therapies.

Heterochromatin-dependent Replication Stress: A Lesson from IDH1/2 Mutants.

Oncogenic point mutants of isocitrate dehydrogenases 1 and 2 (IDH2) generate 2-hydroxyglutarate (2HG), which inhibits lysine demethylases and increases heterochromatin. Tumor cells expressing IDH mutants are sensitive to poly(ADP) ribose polymerase (PARP) inhibitors, offering an opportunity to eliminate IDH-driven tumor cells in therapy. Expression of an oncogenic IDH1 mutant in cells leads to aberrant heterochromatin formation at DNA breaks and impairs DNA repair through homologous recombination (HR), providing a possible explanation for the PARP inhibitor sensitivity of IDH mutant cells. However, a recent study published in Molecular Cell shows that IDH mutant tumors do not display the genomic alterations associated with HR defects. Instead, IDH mutants induce heterochromatin-dependent DNA replication stress. Furthermore, PARP is activated by the replication stress induced by IDH mutants and required for suppressing the ensuing DNA damage, providing an alternative model to explain the susceptibility of IDH mutant cells to PARP inhibitors. This study presents a new example of oncogene-induced and heterochromatin-dependent replication stress, and a role of PARP in the response to the stress, extending the molecular basis for PARP-targeted therapy.

Identification and validation of m6A-GPI signatures as a novel prognostic model for colorectal cancer.

In order to develop an N6-methyladenosine-related gene prognostic index (m6A-GPI) that can predict the prognosis in colorectal cancer (CRC), we obtained m6A-related differentially expressed genes (DEGs) based on The Cancer Genome Atlas (TCGA) and m6Avar database, seven genes were screened by weighted gene co-expression network analysis (WGCNA) and least absolute shrinkage and selection operator (LASSO) analysis. Then, m6A-GPI was constructed based on the risk score. Survival analysis indicated that patients in the lower m6A-GPI group have more prolonged disease-free survival (DFS), and different clinical characteristic groups (tumor site and stage) also showed differential risk scores. In the analysis of the molecular characteristics, the risk score is positively associated with homologous recombination defects (HRD), copy number alterations (CNA), and the mRNA expression-based stemness index (mRNAsi). In addition, m6A-GPI also plays an essential role in tumor immune cell infiltration. The immune cell infiltration in the low m6A-GPI group is significantly higher in CRC. Moreover, we found that CIITA, one of the genes in m6A-GPI was up-regulated in CRC tissues based on real-time RT-PCR and Western blot. m6A-GPI is a promising prognostic biomarker that can be used to distinguish the prognosis of CRC patients in CRC.

Resistance mechanism and microenvironment changes after PARPi treatment: Paired analysis of ovarian cancer samples pre and post treatment.

e17586 Background: PARP inhibitors (PARPi) have been widely used in the maintenance therapy of ovarian cancer (OC) and have shown strong anti-tumor effect in tumors with homologous recombination defects (HRD). However, there is still a lack of systematic clinical data on drug resistance mechanisms and tumor microenvironment changes. Methods: 13 OC patients who were treated with PARPi at West China Second University Hospital were enrolled. PARPi treatment-naive samples (PNS, n = 11) and post-PARPi progression samples (PPS, n = 13) were acquired. Two next-generation sequencing (NGS) panels (Master panel, 563 genes for exons of DNA plus 1831 genes for RNA; HRD panel, 70,000 SNPs for HRD score evaluation, AmoyDx) were used to analyze gene variation, expression, and HRD score changes in tumors. Results: Median duration of treatment for PARPi was 17 months (9-36), and the median lines of chemotherapy before PARPi was 2 (1-3). 38.5% (5/13) patients achieved R0 resection. BRCA reversion mutations were observed in 30.0% (3/10) of the g BRCA+ PPS, including 1 splicing variant and 2 frameshift mutations. High-frequency variants also included MYC amplification (30.8%, 4/13) and ZFHX4 mutations (23.1%, 3/13); Amplification of ch11 q13 were observed in two patients (15.4%). HRD scores and tumor mutation burden (TMB) were significantly increased in PPS. RNA expression profiling showed that the expression of NGFR, CCL18, C7 and other 85 genes in PPS was up regulated. GO/KEGG analysis showed that NF-κb pathway, immune response related cell activation, cytokine - cytokine receptor interaction, B cell receptor signaling pathway, NK cytotoxicity pathways, and etc., were significantly upregulated in PPS, suggesting that PARPi may activated immune-related pathways of the tumor microenvironment (TME). Downregulated pathways in PPS include JAK-STAT signaling pathway, Wnt signaling pathway, and etc. In PPS, the level of memory B cells and neutrophils were significantly up-regulated, while the levels of immune suppression by myeloid cells were significantly down-regulated. Conclusions: Our small-scale study confirmed that BRCA reversion mutation is one of the important resistance mechanisms of PARPi, and the reversion mutation is located near the germline pathogenic mutation to counteract the reading frame change caused by the frameshift mutation. The up-regulation of B-cell receptor, NK cytotoxic cytokine receptor interaction and other related pathways suggested that PARPi may activate immune response, and the changes in the levels of memory B cells, C7 and neutrophils also confirmed the results above. Epithelial mesenchymal transition induced by CCL18 may also lead to PARPi resistance and activate the NF-κb pathway simultaneously, increasing the inflammatory characteristics of TME. These findings can provide ideas for future treatment, and larger samples are needed to verify later.

A phase II, randomized, double-blind study of the use of rucaparib vs placebo maintenance therapy in metastatic and recurrent endometrial cancer.

TPS5626 Background: There are currently no approved maintenance therapies for metastatic and recurrent endometrial cancer, which carries a dismal long-term prognosis. The PARP inhibitor drug class has demonstrated significant clinical benefit as a maintenance therapy for patients with germline/somatic BRCA mutations or homologous recombination deficiency (HRD) in ovarian cancers. However, BRCA mutations are not common in endometrial cancer. Loss of function of the tumor suppressor gene PTEN has been demonstrated in greater than 80% of endometrioid endometrial cancers 1. PTEN loss of function is well known to lead to activation of the PI3K-AKT-mTOR pathway but has also been shown to lead to defects in homologous recombination (HR) DNA repair of double strand breaks 2. Specifically, PTEN silencing leads to decreased RAD51 foci formation. Based on this mechanism, in vitro data on endometrial cancer cell lines has demonstrated sensitivity to PARP inhibitors. Notably, sensitivity of PARP inhibition in PTEN loss of function is independent of microsatellite instability (MSI) 3. Methods: This is a multi-center phase II, placebo controlled, double-blinded study of the use of rucaparib as maintenance therapy in patients with metastatic or recurrent endometrial cancer. A maximum of 138 patients will be randomized 1:1 to receive rucaparib at starting dose of 600mg BID dosing vs. placebo. Standard dose reductions of known toxicities will be followed based on FDA approved indications of rucaparib. Patients will remain on therapy until progression of disease or toxicity induced discontinuation. Eligible patients will have Stage III/IV or recurrent endometrial cancer and must have received one or two prior lines of chemotherapy. All histologic subtypes, including carcinosarcoma will be included. Treatment arms will be stratified on histology, number of lines of prior therapy, and complete vs partial response to prior line of therapy. The primary endpoint is progression free survival (PFS). Secondary endpoints include overall survival (OS), overall response rate (ORR), and toxicity. To date, 79 patients have been enrolled. 1. Mutter GL, Lin MC, Fitzgerald JT, et al. Altered PTEN expression as a diagnostic marker for the earliest endometrial precancers. J Natl Cancer Inst. 2000;92(11):924-930. 2. Shen WH, Balajee AS, Wang J, et al. Essential role for nuclear PTEN in maintaining chromosomal integrity. Cell. 2007;128(1):157-170. 3. Dedes KJ, Wetterskog D, Mendes-Pereira AM, et al. PTEN deficiency in endometrioid endometrial adenocarcinomas predicts sensitivity to PARP inhibitors. Sci Transl Med. 2010;2(53):53ra75. Clinical trial information: NCT03617679 .

The application of a multigene NGS assay in homologous recombination deficiency tracking.

e15169 Background: Several tumor types have been efficiently treated with PARP inhibitors, a class of proteins involved in DNA repair pathway, which are approved for the treatment of ovarian, breast, prostate, and pancreatic cancer. The BRCA1/2 genes are essential for homologous recombination, and tumors which carry this type of mutations appear to have a defective homologous recombination pathway as a result of the loss of the second non mutated allele in the tumor. However, both epigenetic changes and mutations in many other genes involved in the HR pathway, including Fanconi anemia genes and the ΑRID1A, ATM, ATRX, BAP1, BARD1, BLM, BRIP1, CHEK1/2, MRE11A, NBN, PALB2, RAD50, RAD51, WRN may be responsible for the HRD phenomenon. In addition to mutational status, useful for the detection of the patients that would benefit from PARPi treatment could be the presence of certain genomic scars in the tumor, such as the loss of heterozygosity (LOH). Methods: In this study 406 samples from patients with various tumor types were analyzed using a targeted NGS assay for the analysis of 513 genes associated with targeted and immuno-oncology therapies. This assay detects relevant SNVs, indels, CNVs, gene fusions, splice variants in addition to TMB and MSI simultaneously. It also measures genomic instability using sample-level LOH in addition to the analysis for 31 HR related gene alterations. Moreover, the %gLOH was also calculated by the OncoScan™ CNV Assay in 25 samples in order to compare the results obtained by these two methodologies. Results: An HR gene mutation was detected in 20.93% of the tumors analyzed, with BRCA1/2 genes being the most prevalent HR altered genes detected in 5.17% of the tumors. The majority of the non BRCA1/2 HR mutations were in ARID1A (4.18%), ATRX (2.21%) and ATM (1.97%) genes. The %gLOH was highly correlated with the presence of mutations in the BRCA1/2 genes since 76.19% (16/21) of the tumors harboring such alterations had a high %LOH value (p = 0.007). Additionally, the LOH status was highly correlated to the presence of TP53 mutations, while a negative correlation to KRAS mutations was observed. Furthermore, there was no association of %LOH and TMB value in our cohort. Lin's Concordance Correlation Coefficient was 0.93 for the 25 evaluable samples examined simultaneously by both assays, indicating an almost perfect agreement. Conclusions: A high rate of HR mutations was detected in ovarian, breast and prostate which is consistent with the PARPi approval in these tumor types. The presence of BRCA1/2 mutations was highly associated with increased LOH value whereas it did not correlate to other HR mutations. Additionally, the pancancer presence of HR gene alterations indicates that the use of such genes as biomarkers of platinum and PARPi treatments should be evaluated in a wider range of tumor types. In conclusion the addition of analysis of the gLOH seems to be useful for the detection of additional patients eligible for PARPis.

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.

A ribosomal gene panel predicting a novel synthetic lethality in non-BRCAness tumors

Poly (ADP-ribose) polymerase (PARP) inhibitors are one of the most exciting classes of targeted therapy agents for cancers with homologous recombination (HR) deficiency. However, many patients without apparent HR defects also respond well to PARP inhibitors/cisplatin. The biomarker responsible for this mechanism remains unclear. Here, we identified a set of ribosomal genes that predict response to PARP inhibitors/cisplatin in HR-proficient patients. PARP inhibitor/cisplatin selectively eliminates cells with high expression of the eight genes in the identified panel via DNA damage (ATM) signaling-induced pro-apoptotic ribosomal stress, which along with ATM signaling-induced pro-survival HR repair constitutes a new model to balance the cell fate in response to DNA damage. Therefore, the combined examination of the gene panel along with HR status would allow for more precise predictions of clinical response to PARP inhibitor/cisplatin. The gene panel as an independent biomarker was validated by multiple published clinical datasets, as well as by an ovarian cancer organoids library we established. More importantly, its predictive value was further verified in a cohort of PARP inhibitor-treated ovarian cancer patients with both RNA-seq and WGS data. Furthermore, we identified several marketed drugs capable of upregulating the expression of the genes in the panel without causing HR deficiency in PARP inhibitor/cisplatin-resistant cell lines. These drugs enhance PARP inhibitor/cisplatin sensitivity in both intrinsically resistant organoids and cell lines with acquired resistance. Together, our study identifies a marker gene panel for HR-proficient patients and reveals a broader application of PARP inhibitor/cisplatin in cancer therapy.

Multi-omics analysis reveals distinct non-reversion mechanisms of PARPi resistance in BRCA1- versus BRCA2-deficient mammary tumors

BRCA1 and BRCA2 both function in DNA double-strand break repair by homologous recombination (HR). Due to their HR defect, BRCA1/2-deficient cancers are sensitive to poly(ADP-ribose) polymerase inhibitors (PARPis), but they eventually acquire resistance. Preclinical studies yielded several PARPi resistance mechanismsthat do not involve BRCA1/2 reactivation, but their relevance in the clinic remains elusive. To investigate which BRCA1/2-independent mechanisms drive spontaneous resistance invivo, we combine molecular profiling with functional analysis of HR of matched PARPi-naive and PARPi-resistant mouse mammary tumors harboring large intragenic deletions that prevent reactivation of BRCA1/2. We observe restoration of HR in 62% of PARPi-resistant BRCA1-deficient tumors but none in the PARPi-resistant BRCA2-deficient tumors. Moreover, we find that 53BP1 loss is the prevalent resistance mechanism in HR-proficient BRCA1-deficient tumors, whereas resistance in BRCA2-deficient tumors is mainly induced by PARG loss. Furthermore, combined multi-omics analysis identifies additional genes and pathways potentially involved in modulating PARPi response.

Abstract CT158: BrUOG360: A phase Ib/II study of copanlisib combined with rucaparib in patients with metastatic castration-resistant prostate cancer (mCRPC)

Abstract Background: Molecular alterations in genes regulating homologous recombination (HR) confer sensitivity of mCRPC to poly ADP-ribose polymerase inhibitors (PARPi). Increased activation of thePI3K-AKT-mTOR pathway in mCRPC contributes to tumor progression and represents an important therapeutic target. Preclinical studies have shown that PI3K inhibitors (PI3Ki) impair HR and sensitize cancer cells to PARPi even in the absence of HR gene mutations. We hypothesize that dual PI3K and PARP inhibition may improve clinical outcomes of mCRPC. We describe preliminary results of a phase Ib/II study investigating safety of the combination of copanlisib (pan-class I PI3Ki) and rucaparib (PARP-1, -2 and -3 inhibitor). Methods: Enrollment criteria included progressive mCRPC, prior androgen inhibitors (abiraterone, enzalutamide, and/or apalutamide); prior taxane chemotherapy was allowed. HR defects (HRD) was not required for the phase Ib but was an inclusion criterion for the Phase II component of the trial. The phase I followed a standard 3+3 escalation design. Dose level (DL) 1 consisted of rucaparib 400 mg PO BID continuously and copanlisib 45 mg IV days 1, 8 and 15 every 28 days; in DL -1 the copanlisib was reduced to 45 mg days 1 and 15 only. Adverse events (AE) were graded by CTCAE v5.0. The primary aim of the phase I was to establish the MTD and the recommended phase II dose (RP2D) of copanlisib in combination with rucaparib. Results: Thirteen patients were enrolled with a median age of 64 (55-78) and a median baseline PSA of 11.7 ng/mL (0.015-1939.2). Nine patients (69%) received prior chemotherapy (docetaxel [6], cabazitaxel [3]). Seven patients had HRD (54%) BRCA1 (1), BRCA2 (4), CDK12 (1), and FANCA (1). Treatment-related AEs included grade 2 (G2) leukopenia (46%), G2/G3 anemia (31%), G2/G3 rash (31%). Two dose-limiting toxicities (DLTs) were observed in 5 patients treated in DL 1: G3 rash and G3 AST/ALT elevation attributed to both drugs. Six patients were treated at DL -1 without DLTs; this was identified as the RP2D. Two additional patients have been treated at this dose in Phase II of the study. By RECIST 1.1 criteria, one patient (8%) achieved a partial response (BRCA1 mutation) and three (23%) achieved stable disease resulting in a clinical benefit rate of 30.7%. Median duration of treatment was 12 weeks. One patient harboring a PALB2 VUS mutation remained on treatment for 21.7 months. Of 13 evaluable patients, there were 2 confirmed PSA responses (15%) (one patient without an HRD and one with a BRCA1 mutation). Conclusions: The combination of rucaparib and copanlisib is well tolerated. The RP2D was rucaparib 400mg BID with copanlisib 45mg (D1, D15; 28-day cycle) with signal of efficacy in patients with and without HRD. Clinical trial information: NCT04253262. Citation Format: Matthew J. Hadfield, Andre De Souza, Dragan Golijanin, Roxanne Wood, Adam Olszewski, Sheldon L. Holder, Wafik S. El-Deiry, Rahul Aggarwal, Anthony Mega, Benedito A. Carneiro. BrUOG360: A phase Ib/II study of copanlisib combined with rucaparib in patients with metastatic castration-resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT158.

Targeted therapy of advanced parathyroid carcinoma guided by genomic and transcriptomic profiling.

Parathyroid carcinoma (PC) is an ultra-rare malignancy with a high risk of recurrence after surgery. Tumour-directed systemic treatments for PC are not established. We used whole-genome and RNA sequencing in four patients with advanced PC to identify molecular alterations that could guide clinical management. In two cases, the genomic and transcriptomic profiles provided targets for experimental therapies that resulted in biochemical response and prolonged disease stabilization: (a) immune checkpoint inhibition with pembrolizumab based on high tumour mutational burden and a single-base substitution signature associated with APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) overactivation; (b) multi-receptor tyrosine kinase inhibition with lenvatinib due to overexpression of FGFR1 (Fibroblast Growth Factor Receptor 1) and RET (Ret Proto-Oncogene) and, (c)later in the course of the disease, PARP (Poly(ADP-Ribose) Polymerase) inhibition with olaparib prompted by signs of defective homologous recombination DNA repair. In addition, our data provided new insights into the molecular landscape of PC with respect to the genome-wide footprints of specific mutational processes and pathogenic germline alterations. These data underscore the potential of comprehensive molecular analyses to improve care for patients with ultra-rare cancers based on insight into disease biology.

Identification of natural killer cell associated subtyping and gene signature to predict prognosis and drug sensitivity of lung adenocarcinoma.

Introduction: This research explored the immune characteristics of natural killer (NK) cells in lung adenocarcinoma (LUAD) and their predictive role on patient survival and immunotherapy response. Material and methods: Molecular subtyping of LUAD samples was performed by evaluating NK cell-associated pathways and genes in The Cancer Genome Atlas (TCGA) dataset using consistent clustering. 12 programmed cell death (PCD) patterns were acquired from previous study. Riskscore prognostic models were constructed using Least absolute shrinkage and selection operator (Lasso) and Cox regression. The model stability was validated in Gene Expression Omnibus database (GEO). Results: We classified LUAD into three different molecular subgroups based on NK cell-related genes, with the worst prognosis in C1 patients and the optimal in C3. Homologous Recombination Defects, purity and ploidy, TMB, LOH, Aneuploidy Score, were the most high-expressed in C1 and the least expressed in C3. ImmuneScore was the highest in C3 type, suggesting greater immune infiltration in C3 subtype. C1 subtypes had higher TIDE scores, indicating that C1 subtypes may benefit less from immunotherapy. Generally, C3 subtype presented highest PCD patterns scores. With four genes, ANLN, FAM83A, RHOV and PARP15, we constructed a LUAD risk prediction model with significant differences in immune cell composition, cell cycle related pathways between the two risk groups. Samples in C1 and high group were more sensitive to chemotherapy drug. The score of PCD were differences in high- and low-groups. Finally, we combined Riskscore and clinical features to improve the performance of the prediction model, and the calibration curve and decision curve verified that the great robustness of the model. Conclusion: We identified three stable molecular subtypes of LUAD and constructed a prognostic model based on NK cell-related genes, maybe have a greater potential for application in predicting immunotherapy response and patient prognosis.

Abstract 6114: Race-associated base excision repair defects alter the DNA damage landscape in prostate cancer

Abstract Prostate cancer is the most diagnosed cancer among men in the United States. African American men are diagnosed with and succumb to prostate cancer at higher rates than other demographic groups. However, the biological drivers contributing to poorer outcomes in African American men compared to European American men with prostate cancer are still poorly defined. One understudied area is DNA repair defects, which drive genomic instability in cancers. Defects in homologous recombination are often at the forefront of repair defects examined and exploited for therapeutic intervention. However, defects in other DNA repair pathways may contribute to poor disease outcomes and chemoresistance. We demonstrated that defects in the base excision repair (BER) pathway contribute to chemoresistance and alter growth in breast cancer. Others have also shown chemoresistant effects from dysregulated BER protein expression in gastric, colorectal, and ovarian cancers. Examination of the TCGA database show dysregulation of key BER protein like X-ray cross complementing protein 1 (XRCC1) and DNA polymerase β (POLβ) are common across several cancers. Therefore, we decided to explore DNA damage and repair defects in prostate cancer to explore if altered DNA repair contributes to poorer outcomes in African American men. To measure DNA repair defects in formalin-fixed paraffin-embedded (FFPE) tissues, we employed a novel assay, Repair Assisted Damage Detection (RADD), which measures DNA lesions within the tissues. RADD fluorescently tags DNA lesions for quantification and is compatible with immunohistochemistry detection of DNA repair or other proteins of interest. Using the tissue microarrays, we used RADD to discover that prostate tumors from African American patients have more uracil and pyrimidine lesions, elevated uracil DNA glycosylase (UNG) levels, and reduced XRCC1 levels than European American tumors, which indicate defects in the BER pathway. In addition, these men had higher uracil monophosphate (UMP) and lower expression of folate cycle metabolites, suggesting that metabolic rewiring may also contribute to the dysregulation of BER. Defects in the BER pathway may offer new therapeutic opportunities for African American prostate cancer patients. These defects may be targeted by therapeutics which pressure the BER pathways. This work demonstrates that functional detection of DNA repair defects through RADD offers new insight into race-specific DNA repair defects offering new molecular targets or therapeutic strategies to reduce racial disparities in prostate cancer. Citation Format: Kaveri Goel, Kimiko Krieger, Manoj Sonavane, Arun Sreekumar, Natalie R. Gassman. Race-associated base excision repair defects alter the DNA damage landscape in prostate cancer [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 6114.

Abstract 6603: BRCA1 promoter methylation in sporadic breast cancer patients detected by liquid biopsy

Abstract Background: BRCA1 promoter methylation (PM) is an early initiating event in cancer, occurring in 3 to 65.2% of all breast tumors, and 30 to 65% of triple negative tumors. BRCA1 PM has been associated with defective homologous recombination repair (HRR), early onset of breast and ovarian cancer, and improved clinical response to adjuvant chemotherapy. Historically, there has been no diagnostic assay that comprehensively evaluates both BRCA1 PM and genomic alterations in cell-free circulating tumor DNA (ctDNA). Here, we describe the novel detection of BRCA1 PM and genomic alterations in a cohort of patients with breast cancer using GuardantINFINITY, a liquid biopsy assay interrogating 800+ genes and genome-wide methylation detection. Method: We assessed for BRCA1 PM in ctDNA from 274 patients with late-stage breast cancer. Genomic sequencing of 800+ genes and PM profiling of 398 genes was performed by GuardantINFINITY. The positive calling threshold for PM was established by comparing cell-free DNA derived from patients with cancer and cancer-free donors. The limit of detection (LoD) was determined through in silico and experimental titrations of ctDNA from clinical samples and cell lines with known gene PM into the plasma of cancer-free donors. Results: Among the 274 patients with advanced breast cancer, 8 (2.9%) had germline pathogenic mutations in BRCA1, BRCA2, or ATM. BRCA1 PM was detected in 11/274 (4.0%) patients at the predefined threshold of >99% specificity. BRCA1 PM detection in this cohort was 8.9% (8/90) when excluding samples with low tumor shedding (<1% epigenomic tumor fraction in cfDNA). Among the 11 patients with BRCA1 PM detected in ctDNA, one had a co-occurring somatic BRCA1 nonsense variant (p.S361*); none of the remaining patients with BRCA1 PM had another HRR-related mutation detected in cfDNA. Among patients without BRCA1 PM detected, pathogenic somatic alterations were detected in BRCA2, ATM, and CHEK2 in 25 (9.4%) patients. In silico simulations using clinical samples with BRCA1 PM indicated an LoD of 0.0408%. BRCA1 PM was not detected in 3210 individual and mixed cancer-free clinical samples, indicating a high specificity for BRCA1 PM calls. Conclusion: GuardantINFINITY, a plasma-based diagnostic assay, detected both BRCA1 PM and genomic alterations in this unspecified advanced breast cancer cohort. The BRCA1 PM detection rates of 4.0-8.9% are consistent with values previously reported in the literature. As BRCA1 PM has important prognostic and therapeutic implications for the management of breast (as well as ovarian) cancers, additional studies are warranted to further describe the PM patterns across breast cancer subtypes and how these patterns both influence and are influenced by disease evolution and therapeutic response. Liquid biopsy thus serves as a suitable method to noninvasively identify and monitor changes in both genomics and epigenomics. Citation Format: Jennifer Yen, Sai Chen, Colby Jenkins, Brooke Overstreet, Yu Fu, Jun Zhao, Tingting Jiang, Leylah Drusbosky, Stephen Pettitt, Michael Dorschner, Lauren Lawrence, Han-Yu Chuang, Andrew Tutt. BRCA1 promoter methylation in sporadic breast cancer patients detected by liquid biopsy [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 6603.

Farrerol directly activates the deubiqutinase UCHL3 to promote DNA repair and reprogramming when mediated by somatic cell nuclear transfer

Farrerol, a natural flavanone, promotes homologous recombination (HR) repair to improve genome-editing efficiency, but the specific protein that farrerol directly targets to regulate HR repair and the underlying molecular mechanisms have not been determined. Here, we find that the deubiquitinase UCHL3 is the direct target of farrerol. Mechanistically, farrerol enhanced the deubiquitinase activity of UCHL3 to promote RAD51 deubiquitination, thereby improving HR repair. Importantly, we find that embryos of somatic cell nuclear transfer (SCNT) exhibited defective HR repair, increased genomic instability and aneuploidy, and that the farrerol treatment post nuclear transfer enhances HR repair, restores transcriptional and epigenetic network, and promotes SCNT embryo development. Ablating UCHL3 significantly attenuates farrerol-mediated stimulation in HR and SCNT embryo development. In summary, we identify farrerol as an activator of the deubiquitinase UCHL3, highlighted the importance of HR and epigenetic changes in SCNT reprogramming and provide a feasible method to promote SCNT efficiency.

N-terminal acetyltransferase NatB regulates Rad51-dependent repair of double-strand breaks in Saccharomyces cerevisiae.

Homologous recombination (HR) is a highly accurate mechanism for repairing DNA double-strand breaks (DSBs) that arise from various genotoxic insults and blocked replication forks. Defects in HR and unscheduled HR can interfere with other cellular processes such as DNA replication and chromosome segregation, leading to genome instability and cell death. Therefore, the HR process has to be tightly controlled. Protein N-terminal acetylation is one of the most common modifications in eukaryotic organisms. Studies in budding yeast implicate a role for NatB acetyltransferase in HR repair, but precisely how this modification regulates HR repair and genome integrity is unknown. In this study, we show that cells lacking NatB, a dimeric complex composed of Nat3 and Mdm2, are sensitive to the DNA alkylating agent methyl methanesulfonate (MMS), and that overexpression of Rad51 suppresses the MMS sensitivity of nat3Δ cells. Nat3-deficient cells have increased levels of Rad52-yellow fluorescent protein foci and fail to repair DSBs after release from MMS exposure. We also found that Nat3 is required for HR-dependent gene conversion and gene targeting. Importantly, we observed that nat3Δ mutation partially suppressed MMS sensitivity in srs2Δ cells and the synthetic sickness of srs2Δ sgs1Δ cells. Altogether, our results indicate that NatB functions upstream of Srs2 to activate the Rad51-dependent HR pathway for DSB repair.

866 Preclinical Study of Pamiparib, a CNS Penetrant PARP Inhibitor, in IDH1/2 Mutated Gliomas

INTRODUCTION: Our group demonstrated IDH1/2-mutated gliomas harbor intrinsic homologous recombination (HR) defects mediated by 2-HG, conferring sensitivity to PARP-inhibitors. We studied pamiparib, a CNS-penetrant PARP-inhibitor with potent PARP-trapping ability, in combination with TMZ and radiation in IDH1/2-mutated gliomas and its pharmacokinetics to demonstrate CNS penetrance. METHODS: We performed DNA-repair functional studies with in-vitro short- and long-term viability assays, and in-vivo studies utilizing an intracranial rat glioma model with bioluminescence imaging. Pharmacokinetics was measured with LCMS/MS from harvested tumor, normal brain, and blood. RESULTS: In-vitro viability assays in paired isogenic IDH1-wildtype and mutant U87 cells showed the IDH1 mutation conferred several-fold enhanced sensitivity to TMZ, as expected. Differential TMZ sensitivity was less pronounced in HCT116 cells, as expected given higher MGMT expression. ELISA confirmed PARylation inhibition by pamiparib at the nanomolar range in a dose-dependent manner. Enhanced pamiparib sensitivity was seen in IDH-mutant U87 and HCT116 cells compared to wildtype. Pamiparib and TMZ demonstrated synergistic interactions in U87 cells, dependent upon IDH1 mutation status. In HCT116 cells, TMZ resistance was overcome with pamiparib in wildtype and mutant cells but notably at lower doses in mutant cells. Synergy was observed with pamiparib and radiation in U87-mutant, compared to wildtype cells. Pharmacokinetics demonstrated favorable CNS penetration with tumor:plasma ratios (>0.20) with 3 mg/kg and 6 mg/kg doses of pamiparib, orally administered BID to rats harboring RG2-cell intracranial tumors. These levels persisted between 2- and 8-hours after last dosing. Pamiparib selectively penetrated tumors over normal brain with normal brain:plasma ratios <0.20. CONCLUSIONS: Pamiparib may selectively target IDH1/2-mutated gliomas and act synergistically with TMZ and radiation to exploit intrinsic HR defects. Pharmacokinetics suggest favorable CNS penetration after BID oral administration.

TRABID overexpression enables synthetic lethality to PARP inhibitor via prolonging 53BP1 retention at double-strand breaks

53BP1 promotes nonhomologous end joining (NHEJ) over homologous recombination (HR) repair by mediating inactivation of DNA end resection. Ubiquitination plays an important role in regulating dissociation of 53BP1 from DNA double-strand breaks (DSBs). However, how this process is regulated remains poorly understood. Here, we demonstrate that TRABID deubiquitinase binds to 53BP1 at endogenous level and regulates 53BP1 retention at DSB sites. TRABID deubiquitinates K29-linked polyubiquitination of 53BP1 mediated by E3 ubiquitin ligase SPOP and prevents 53BP1 dissociation from DSBs, consequently inducing HR defects and chromosomal instability. Prostate cancer cells with TRABID overexpression exhibit a high sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors. Our work shows that TRABID facilitates NHEJ repair over HR during DNA repair by inducing prolonged 53BP1 retention at DSB sites, suggesting that TRABID overexpression may predict HR deficiency and the potential therapeutic use of PARP inhibitors in prostate cancer.