HR-associated Genes Research Articles

A novel targeted NGS panel identifies numerous homologous recombination deficiency (HRD)-associated gene mutations in addition to known BRCA mutations

Deleterious mutations in the BRCA1 and BRCA2 genes have significant therapeutic relevance in clinical settings regarding personalized therapy approaches. BRCA1 and BRCA2 play a pivotal role in homologous recombination (HR) and thus are sensitive for PARP inhibitors (PARPi). Beyond the narrow scope of evaluating only the BRCA mutation status, PARPi can be beneficial for HR deficient (HRD) patients, who harbor mutations in other HR-associated genes. In the present retrospective study, a novel targeted HRD gene panel was validated and implemented for use with FFPE tissue. Samples of patients with ovarian, breast, pancreatic and prostate cancer were included. Variants were robustly detected with various DNA input amounts and the use of test samples showed complete concordance between previously known mutations and HRD panel results. From all the 90 samples included in this cohort, TP53 was the most frequently altered gene (73%). Deleterious BRCA1/2 mutations were found in 20 (22%) of all samples. New pathogenic or likely pathogenic mutations in additional HR-associated genes were identified in 22 (24%) patients. Taken together, the present study proves the feasibility of a new HRD gene panel with reliable panel performance and offers the possibility to easily screen for resistance mutations acquired over treatment time.Mutations in HR-associated genes, besides BRCA1/2, might represent promising potential targets for synthetic lethality approaches. Thus, a substantial number of patients may benefit from expanding the scope of therapeutic agents like PARPi.

An activity-based functional test for identifying homologous recombination deficiencies across cancer types in real time

Homologous recombination (HR)-mediated DNA repair is a prerequisite for maintaining genome stability. Cancer cells displaying HR deficiency (HRD) are selectively eliminated by poly(ADP-ribose) polymerase inhibitors (PARPis). To date, sequencing of HR-associated genes and analyzing genome instability have been used as clinical predictions for PARPi therapy. However, these genetic tests cannot reflect dynamic changes in the HR status. Here, we have developed a virus- and activity-based functional assay to quantify real-time HR activity directly. Instead of focusing on a few HR-associated genes, our functional assay detects endpoint HR activity and establishes an activity threshold for identifying HRD across cancer types, validated by PARPi sensitivity and BRCA status. Notably, this fluorescence-based assay can be applied to primary ovarian cancer cells from patients to reflect their level of HRD, which is associated with survival benefits. Thus, our work provides a functional test to predict the response of primary cancer cells to PARPis.

Somatic Variants in DNA Damage Response Genes in Ovarian Cancer Patients Using Whole-exome Sequencing.

Several clinical trials have investigated homologous recombination deficiency and BRCA1/2 status to select ovarian cancer patients for treatment with poly(ADP-ribose) polymerase-inhibitors (PARPi), but less attention has been given to other DNA-damage response (DDR) pathways. Therefore, we investigated somatic single/multiple nucleotide variants and small insertions/deletions in exonic and splice-site regions of 356 DDR genes to examine whether genes other than BRCA1/2 are altered. Whole-exome sequencing data from eight high-grade serous adenocarcinoma (HGSC) and four clear cell carcinoma (oCCC) patients were analyzed. Forty-two variants (pathogenic, likely pathogenic or variants of uncertain significance) in 28 genes from DDR pathways were identified. Seven out of nine TP53 variants were previously described in The Cancer Genome Atlas Ovarian Cancer; other variants were found in 23 out of 28 unique genes, whereas no variants were reported in FAAP24, GTF2H4, POLE4, RPA3, and XRCC4. As the identified variants were not only limited to well-known TP53, BRCA1/2, and HR-associated genes, our study might contribute to the better understanding of which DDR pathways potentially influence disease progression. Moreover, they may display a potential role as biomarkers to predict platinum-based chemotherapy or PARPi treatment response or disease progression, as differences in disrupted DDR pathways were observed between patients with long and short overall survival in HGSC and oCCC groups.

Homologous recombination inquiry through ovarian malignancy investigations: JGOG3025 Study.

The Cancer Genome Atlas (TCGA) network has clarified that ~50% of high-grade serous ovarian cancers show homologous recombination deficiency (HRD). However, the frequency of HRD in Japanese patients with ovarian cancer remains unclear. We aimed to identify the frequency of HR-associated gene mutations in Japanese patients with ovarian cancer. The JGOG3025 study is a multicenter collaborative prospective observational study involving 65 study sites throughout Japan. We recruited 996 patients who were clinically diagnosed with ovarian cancer before surgery from March 2017 to March 2019, and 701 patients were eligible according to the criteria. We used frozen tumor tissues to extract DNA and performed next-generation sequencing for 51 targeted genes (including 29 HR-associated genes) in 701 ovarian cancers (298 high-grade serous cases, 189 clear cell cases, 135 endometrioid cases, 12 mucinous cases, 3 low-grade serous cases, and 64 others). HRD was defined as positive when at least one HR-associated gene was mutated. The frequencies of HRD and tumor BRCA1/2 mutations were 45.2% (317/701) and 18.5% (130/701), respectively, in the full analysis set. Next, we performed multivariate Cox proportional hazards regression analysis for progression-free survival (PFS) and overall survival (OS). Advanced-stage ovarian cancer patients with HRD had adjusted hazard ratios of 0.72 (95% CI, 0.55-0.94) and 0.57 (95% CI, 0.38-0.86) for PFS and OS, respectively, compared with those without HRD (p=0.016 and 0.007). Our study demonstrated that mutations in HR-associated genes were associated with prognosis. Further studies are needed to investigate the prognostic impact of each HR-associated gene in ovarian cancer.

Abstract 5458: TONSL is an immortalizing oncogene of the chromosome 8q24.3 amplicon and new therapeutic target in breast cancer

Abstract Study of genomic aberrations leading to immortalization of epithelial cells has been technically challenging due to lack of an appropriate isogenic model system. To address this technical challenge, we utilized primary breast luminal epithelial cells propagated from healthy donors of different genetic ancestry and their hTERT-immortalized counterparts to identify functional gene expression changes associated with immortalization. We identified elevated expression of TONSL (Tonsoku Like, DNA Repair Protein) as one of the earliest events during immortalization. TONSL is located on chromosome 8q24.3 and amplified in ~20% of breast cancers with significantly higher amplification in metastatic tumors. TONSL forms a complex with FACT and MMS22L1 to modulate multiple cellular pathways including DNA replication, repair through homologous recombination (HR) and functions as a post replicative histone/chromatin reader. TONSL alone immortalized primary breast epithelial cells and increased telomerase activity. While TONSL overexpression alone was insufficient for neoplastic transformation, TONSL-immortalized primary cells modified to overexpress defined oncogenes generated estrogen receptor-positive adenocarcinomas in NSG mice. Analysis of breast tumor microarray with ~500 tumors revealed poor overall and progression free survival of patients with TONSL-overexpressing tumors. TONSL increased chromatin accessibility to pro-oncogenic transcription factors including NF-κB and limited access to the tumor suppressor p53. Most importantly, TONSL overexpression resulted in significant changes in the expression of genes associated with DNA repair hubs, including upregulation of several genes in HR and Fanconi Anemia pathways. Consistent with the effects of TONSL on HR-associated genes, TONSL overexpressing primary cells exhibited upregulated DNA repair via HR. Moreover, TONSL was an essential gene for growth of TONSL-amplified breast cancer cell lines in vivo. Breast cancer cell lines with TONSL/chr8q24.3 amplification were sensitive to TONSL-FACT complex inhibitor CBL0137, both in vitro and in vivo. To our knowledge, TONSL is the only gene other than telomerase with immortalizing function and represents a new therapeutic target for breast cancer with chr8q24.3 amplification. Citation Format: Aditi S. Khatpe, Rebecca Dirks, Poornima Bhat-Nakshatri, Henry Mang, Katie Batic, Sarah Swiezy, Jacob Olson, Xi Rao, Yue Wang, Hiromi Tanaka, Sheng Liu, Jun Wan, Duojiao Chen, Yunlong Liu, Fang Fang, Sandra Althouse, Emily Hilsey, Maggie M. Granatir, Rebekah Addison, Constance J. Temm, George Sandusky, Audrey Lee-Gosselin, Kenneth Nephew Nephew, Kathy D. Miller, Harikrishna Nakshatri. TONSL is an immortalizing oncogene of the chromosome 8q24.3 amplicon and new therapeutic target in breast 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 5458.

QRT-PCR-based DNA homologous recombination-associated 4-gene score predicts pathologic complete response to platinum-based neoadjuvant chemotherapy in triple-negative breast cancer.

Cumulative evidence suggests that the addition of platinum agents as neoadjuvant chemotherapy (NACT) could improve the pathologic complete response (pCR) rate in triple-negative breast cancer (TNBC). We aimed to develop a DNA homologous recombination (HR)-associated gene expression score to predict tumor sensitivity to platinum-based NACT in TNBC. A retrospective cohort of 127 patients who were diagnosed with TNBC and received platinum-based NACT in Fudan University Shanghai Cancer Center from 2012 to 2017 was included in this study. Using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), the expression levels of eight HR-associated genes were analyzed from formalin-fixed paraffin-embedded core-needle biopsy samples obtained before NACT. A random forest model was built to estimate the weight of each gene expression level and clinicopathological factors. The training set was used to modulate parameters and select the best model. The performance of the final model was evaluated in the validation set. A 4-gene (BRCA1, XRCC5, PARP1, and RAD51) scoring system was developed. TNBC patients with a higher score had a nearly fourfold likelihood of achieving pCR to platinum-based NACT compared with patients with a lower score [odds ratio (OR) = 3.878; P < 0.001]. At the cutoff value of -2.644, the 4-gene scoring system showed high sensitivity in predicting pCR in the breast (93.0%) and pCR in the breast/axilla (91.8%), while at the cutoff value of -1.969, the 4-gene score showed high specificity for pCR in the breast (85.7%) and pCR in the breast/axilla (80.8%). The qRT-PCR-based 4-gene score has the potential to predict pCR to platinum-based NACT in TNBC.

Frequency of homologous recombination associated gene mutations in Japanese patients with ovarian cancer

<h3>Objectives:</h3> Homologous recombination deficiency (HRD) is a well-known biomarker of PARP inhibitor sensitivity. The Cancer Genome Atlas (TCGA) has clarified that about 50% of high-grade serous ovarian cancer shows HRD. However, the frequency of HRD in Japanese patients with ovarian cancer remains unclear. The aim of JGOG3025 study (NCT03159572) is to identify the frequency of HR-associated gene mutations in ovarian cancer, especially non-serous histologic types. <h3>Methods:</h3> The JGOG3025 study is a collaborative prospective observational study involving 65 study sites throughout Japan. We recruited 996 patients who were clinically diagnosed with ovarian cancer before surgery from March 2017 to March 2019, and 711 patients were eligible for JGOG3025 criteria. After central pathological review, we used frozen tumor tissues to extract DNA and performed targeted sequencing for 51 HR-associated genes in 711 ovarian cancers (298 high-grade serous, 189 clear cell, 142 endometrioid, 15 mucinous, 4 low-grade serous, and 63 others). <h3>Results:</h3> To focus on pathogenic mutations of HR-associated genes which led to HRD, we used only loss-of-function (LOF) mutations (nonsense, readthrough, and frameshift) to perform subsequent analysis. At least one HR-associated gene was mutated in 105 high-grade serous (35.2%), 21 clear cell (11.1%), 36 endometrioid (25.4%), 1 low-grade serous (25.0%) carcinomas. Although 81.8% of ovarian cancers with <i>BRCA1</i> LOF mutation were diagnosed as high-grade serous type, one case of mucinous carcinoma harbored <i>BRCA1</i> c.1961delA. On the other hand, 71.4% of ovarian cancers harboring <i>BRCA2</i> LOF mutation were high-grade serous type. Mutant allele frequencies of <i>BRCA1</i> and <i>RAD51D</i> were apparently higher than that of other HR-associated genes, suggesting allelic imbalance. <h3>Conclusions:</h3> Our findings demonstrated that mutations in HR-associated genes were detected in not only high-grade serous but also non-serous histologic type ovarian cancers. To clarify the frequency of HRD in Japanese patients with ovarian cancer, we need to assess the pathogenicity of missense mutations and investigate the correlation between HR associated gene mutations and HRD score based on copy number assay.

Association between RAD51, XRCC2 and XRCC3 gene polymorphisms and risk of ovarian cancer: a case control and an in silico study.

Homologous recombination (HR) is one of the important mechanisms in repairing double-strand breaks to maintain genomic integrity and DNA stability from the cytotoxic effects and mutations. Various studies have reported that single nucleotide polymorphisms (SNPs) in the HR-associated genes may have a significant association with ovarian cancer (OCa) risk but the results were inconclusive. In the present study, five polymorphisms of HR-associated genes (RAD51, XRCC2 and XRCC3) were genotyped by allelic discrimination assay in 200 OCa cases and 200 healthy individuals. The association with OCa risk was evaluated by unconditional logistic regression analyses. The results revealed that the mutant allele in both rs1801320 (CC) and rs1801321 (TT) of RAD51 gene was associated with increased risk of OCa (odds ratio [OR] 3.79, 95% confidence interval [CI] 1.21-11.78, p = 0.014 and OR 1.61, 95% CI 1.06-2.45, p = 0.025, respectively). Moreover, a significant association of TT allele (OR 4.68, 95% CI 1.27-17.15, p = 0.011) of rs3218536 of XRCC2 gene with OCa was observed. Stratified analysis results showed that patients with early menarche and stages 3 and 4 were found to be associated with rs1801321 of RAD51 gene and rs1799794 of XRCC3 gene. In silico analysis predicted that the two missense SNPs (rs3218536 and rs1799794) were found to have an impact on the protein structure, stability and function. The present study suggested that RAD51 and XRCC2 gene polymorphisms might have an impact on the OCa risk in the South Indian population. However, studies with a larger sample and on different populations are needed to support the conclusions.

Frequency and prognostic value of mutations associated with the homologous recombination DNA repair pathway in a large pan cancer cohort

PARP inhibitors have shown remarkable efficacy in the clinical management of several BRCA-mutated tumors. This approach is based on the long-standing hypothesis that PARP inhibition will impair the repair of single stranded breaks, causing synthetic lethality in tumors with loss of high-fidelity double-strand break homologous recombination. While this is now well accepted and has been the basis of several successful clinical trials, emerging evidence strongly suggests that mutation to several additional genes involved in homologous recombination may also have predictive value for PARP inhibitors. While this notion is supported by early clinical evidence, the mutation frequencies of these and other functionally related genes are largely unknown, particularly in cancers not classically associated with homologous recombination deficiency. We therefore evaluated the mutation status of 22 genes associated with the homologous recombination DNA repair pathway or PARP inhibitor sensitivity, first in a pan-cancer cohort of 55,586 patients, followed by a more focused analysis in The Cancer Genome Atlas cohort of 12,153 patients. In both groups we observed high rates of mutations in a variety of HR-associated genes largely unexplored in the setting of PARP inhibition, many of which were associated also with poor clinical outcomes. We then extended our study to determine which mutations have a known oncogenic role, as well as similar to known oncogenic mutations that may have a similar phenotype. Finally, we explored the individual cancer histologies in which these genomic alterations are most frequent. We concluded that the rates of deleterious mutations affecting genes associated with the homologous recombination pathway may be underrepresented in a wide range of human cancers, and several of these genes warrant further and more focused investigation, particularly in the setting of PARP inhibition and HR deficiency.

The mutational pattern of homologous recombination (HR)-associated genes and its relevance to the immunotherapeutic response in gastric cancer.

Objective:Currently, there is an urgent need to identify immunotherapeutic biomarkers to increase the benefit of immune checkpoint inhibitors (ICIs) for patients with gastric cancer (GC). Homologous recombination deficiency (HRD) can modify the tumor immune microenvironment by increasing the presence of tumor-infiltrating lymphocytes and therefore might serve as a biomarker of immunotherapeutic response. We aimed to analyze the mutational pattern of HR-associated genes in Chinese patients with GC and its relevance to the tumor immune profile and clinical immunotherapeutic response.Methods:A panel of 543 cancer-associated genes was used to analyze genomic profiles in a cohort comprising 484 Chinese patients with GC. Correlations between HR gene mutations and tumor immunity or clinical outcomes were identified via bioinformatic analysis using 2 GC genomic datasets (TCGA and MSK-IMPACT).Results:Fifty-one of the 484 (10.54%) patients carried at least one somatic mutation in an HR gene; ATM (16/484, 3.31%) was among the most frequently mutated HR genes in the Chinese cohort. Mutations in HR genes were associated with elevated tumor mutational burden, enhanced immune activity, and microsatellite instability status. In the MSK-IMPACT cohort comprising 49 patients with stomach adenocarcinoma or gastroesophageal junction adenocarcinoma treated with ICIs, patients with HR-mut GC (n = 12) had significantly better overall survival than those with HR-wt GC (n = 37) (log-rank test, P < 0.05).Conclusions:Our data suggest that detection of somatic mutations in HR genes might aid in identifying patients who might benefit from immune checkpoint blockade therapy.

Germline and somatic mutations of homologous recombination-associated genes in Japanese ovarian cancer patients

We explored the frequency of germline and somatic mutations in homologous recombination (HR)-associated genes in major histological types of ovarian cancer. We performed targeted sequencing to assess germline and somatic mutations of 16 HR-associated genes and 4 mismatch repair (MMR) genes among 207 ovarian cancer patients (50 high-grade serous carcinomas (HGSC), 99 clear cell carcinomas (CCC), 39 endometrioid carcinomas (EC), 13 mucinous carcinomas (MC), and 6 low-grade serous carcinomas (LGSC)). Germline or somatic mutations of HR-associated genes were detected in 44% of HGSC, 28% of CCC, 23% of EC, 16% of MC, and 17% of LGSC patients. The profile of HR-associated gene mutations was remarkably different among each histological type. Germline BRCA1/2 mutations were frequently detected in HGSC and were rarely observed in CCC, EC, and MC patients. ATM somatic mutation was more frequently detected in CCC (9%) and EC patients (18%) than in HGSC patients (4%). There was a positive correlation between MMR gene mutations and HR-associated gene mutations (p = 0.0072). Our findings might be useful in selection of ovarian cancer patients that should be treated with PARP inhibitors.

Abstract B02: Co-clinical trial of olaparib in breast and ovarian patient-derived tumor xenografts (PDX) enables the identification of response biomarkers

Abstract Background: Women with heterozygous germline mutations in BRCA1 or BRCA2 are predisposed to developing breast or ovarian cancers, since BRCA1/2 mutation results in impaired high-fidelity DNA repair by homologous recombination (HR) and subsequently genetic instability. In non-gBRCA TNBC, HR deficiency occurs at the somatic level, by means of BRCA1 mutation, BRCA1 epigenetic loss or mutation in other HR-associated genes. PARP1/2 inhibitors (PARPi) are active anti-cancer agents in gBRCA with advanced breast or ovarian cancer. However, not all HR-deficient tumors respond to PARP blockade, and eventually all develop acquired resistance. Here, we sought to identify PARPi response biomarkers using PDX derived from both the early disease and the metastatic setting. Methods: We developed a panel of PDX from patients harboring or not germline BRCA1/2 mutations, namely from 26 primary or advanced breast cancer and 2 high-grade serous metastatic ovarian cancer (HGSOC). The antitumor activity of the PARP1/2 inhibitor olaparib as single agent (50 mg/kg) was assessed in all models. To study the mechanisms of acquired resistance, the olaparib-sensitive PDXs were exposed to olaparib for &amp;gt;100 days, until individual tumors regrew. The tumor's capacity to repair DNA double strand breaks was estimated by quantification of the BRCA1 and RAD51 nuclear foci in the S/G2-phase of the cell cycle. We investigated the correlation between the tumor's BRCA1/RAD51 foci formation and sensitivity to olaparib, and also identified potential genetic modifiers of PARPi sensitivity by targeted sequencing. Results: Seven out of 28 PDX (25%) treated with single agent olaparib exhibited tumor regression or disease stabilization. Among the non-gBRCA PDX, BRCA1 hypermethylation or PALB2 mutation were present in olaparib-sensitive PDX. No genetic reversions in BRCA1/2 mutations were identified as the mechanism of olaparib resistance in gBRCA but BRCA1 foci formation was observed in 6 out of 10 resistant models (60%). Nuclear RAD51 foci formation correlated with PARPi resistance in twenty PDX models investigated, either with primary or acquired resistance. The duration of response was similar between gBRCA and non-gBRCA PDX. Acquired-resistance mechanisms involve restoration of HR functionality. Conclusions: Our study highlights that somatic HR-deficiency is frequent in TNBC and provides the basis of sensitivity to PARPi. In our gBRCA (n=12) and non-gBRCA (n=16) PDX, reactivation of HR functionality measured as RAD51 foci formation is a frequent event that is associated with PARPi resistance. Citation Format: Violeta Serra, Cristina Cruz, Marta Castroviejo, Sara Gutiérrez, Alba Llop, Alejandra Bruna, Beatriz Morancho, Ginevra Caratú, Ludmila Prudkin, Ana Vivancos, Paolo Nuciforo, Mark O'Connor, Jos Jonkers, Carlos Caldas, Joaquin Arribas, José Baselga, Isabel T. Rubio, Cristina Saura, Orland Díez, Judith Balmaña, Javier Cortés. Co-clinical trial of olaparib in breast and ovarian patient-derived tumor xenografts (PDX) enables the identification of response biomarkers. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B02.

The functional status of DNA repair pathways determines the sensitization effect to cisplatin in non-small cell lung cancer cells.

Cisplatin can cause a variety of DNA crosslink lesions including intra-strand and inter-strand crosslinks (ICLs), which are associated with the sensitivity of cancer cells to cisplatin. Here, we aimed to assess the contribution of the Fanconi anemia (FA), homologous recombination (HR) and nucleotide excision repair (NER) pathways to cisplatin resistance in non-small cell lung cancer (NSCLC)-derived cells. The expression of FA, HR and NER pathway-associated genes was assessed by RT-qPCR and Western blotting. siRNAs were used to knock down the expression of these genes. CCK-8 and flow cytometry assays were used to assess the viability and apoptotic rate of NSCLC-derived cells, respectively. Immunofluorescence and alkaline comet assays were used to assess the repair of ICLs. We found that acquired cisplatin-resistant NSCLC-derived A549/DR cells exhibited markedly enhanced FA and HR repair pathway capacities compared to its parental A549 cells and another independent NSCLC-derived cell line, Calu-1, which possesses a moderate innate resistance to cisplatin. siRNA-mediated silencing of the FA-associated genes FANCL and RAD18 and the HR-associated genes BRCA1 and BRCA2 significantly potentiated the sensitivity of A549/DR cells to cisplatin compared to A549 and Calu-1 cells, suggesting that the acquired cisplatin resistance in A549/DR cells may be attributed to enhanced FA and HR pathway capacities responsible for ICL repair. Although we found that expression knockdown of the NER-associated genes XPA and ERCC1 sensitized the three NSCLC-derived cell lines to cisplatin, the sensitization effect was more significant in Calu-1 cells than in A549 and A549/DR cells, implying that the innate cisplatin resistance in Calu-1 cells may result from an increased NER activity. Our results indicate that the functional status of DNA repair pathways determine the sensitivity of NSCLC cells to cisplatin. Direct targeting of the pathway that is involved in cisplatin resistance may be an effective strategy to surmount cisplatin resistance in NSCLC.

Abstract P4-07-04: PARP1/2 inhibition in a subset of triple negative breast cancer (TNBC) patient-derived tumor xenografts (PDX) identifies predictive biomarkers of response

Abstract Background: BRCA1/2 mutation carriers (gBRCA) have a higher risk of breast or ovarian cancer, since BRCA1/2 mutation results in impaired high-fidelity DNA repair by homologous recombination (HR) and subsequently genetic instability. In non-gBRCA TNBC, HR deficiency occurs at the somatic level, by means of BRCA1 mutation, BRCA1 epigenetic loss or mutation in other HR-associated genes. Because PARP1/2 inhibitors (PARPi) are well-tolerated and active anti-cancer agents in the advanced setting of gBRCA tumors, we sought to expand their applicability by identifying response biomarkers in TNBC. Methods: We have assessed the antitumor response of the PARP1/2 inhibitor olaparib as single agent in a panel of 12 primary and advanced TNBC PDX models. On PDXs exhibiting primary sensitivity to olaparib, we have developed models of acquired resistance by continuous exposure to the drug and identifying progression on treatment. We have characterized the models through targeted sequencing and the analysis of the hypermethylation and expression levels of BRCA1 transcript to find potential correlates of drug-sensitivity. Results: Three out of 12 PDXs (25%) treated with single agent olaparib, exhibit tumor regression or disease stabilization. BRCA1 is hypermethylated in two of these PARPi-sensitive TNBC PDX models and is associated with loss of BRCA1 mRNA expression. The third PARPi-sensitive TNBC PDX harbors a frameshift, heterozygous PALB2 mutation, which is no longer detected in the acquired resistance PDX model. Acquired resistance in the hypermethylated PDXs is under study as well as the duration of response compared to gBRCA PDX models. Conclusions: Our study highlights that somatic HR-deficiency is frequent in TNBC and provides the basis of sensitivity to PARPi. Citation Format: Serra V, Cruz C, Bruna A, Ibrahim YH, Vivancos A, Vivancos A, Nuciforo P, Bellet M, Gómez P, Pérez JM, Saura C, Vidal M, Serres X, Rueda OM, Peg V, Caldas C, O'Connor MJ, Baselga J, Cortés J. PARP1/2 inhibition in a subset of triple negative breast cancer (TNBC) patient-derived tumor xenografts (PDX) identifies predictive biomarkers of response. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-07-04.

Synthetic lethal targeting of oncogenic transcription factors in acute leukemia by PARP inhibitors.

Acute myeloid leukemia (AML) is mostly driven by oncogenic transcription factors, which have been classically viewed as intractable targets using small-molecule inhibitor approaches. Here we demonstrate that AML driven by repressive transcription factors, including AML1-ETO (encoded by the fusion oncogene RUNX1-RUNX1T1) and PML-RARα fusion oncoproteins (encoded by PML-RARA) are extremely sensitive to poly (ADP-ribose) polymerase (PARP) inhibition, in part owing to their suppressed expression of key homologous recombination (HR)-associated genes and their compromised DNA-damage response (DDR). In contrast, leukemia driven by mixed-lineage leukemia (MLL, encoded by KMT2A) fusions with dominant transactivation ability is proficient in DDR and insensitive to PARP inhibition. Intriguingly, genetic or pharmacological inhibition of an MLL downstream target, HOXA9, which activates expression of various HR-associated genes, impairs DDR and sensitizes MLL leukemia to PARP inhibitors (PARPis). Conversely, HOXA9 overexpression confers PARPi resistance to AML1-ETO and PML-RARα transformed cells. Together, these studies describe a potential utility of PARPi-induced synthetic lethality for leukemia treatment and reveal a novel molecular mechanism governing PARPi sensitivity in AML.

Capsicum annuum basic transcription factor 3 (CaBtf3) regulates transcription of pathogenesis-related genes during hypersensitive response upon Tobacco mosaic virus infection

Hypersensitive response (HR) cell death upon plant virus infection is an excellent plant strategy for inhibiting viral movement and obtaining systemic acquired resistance (SAR) against further infection. Various host factors are involved in these HR processes, either directly as viral resistance proteins or indirectly. We characterized a gene encoding the CaBtf3 [β-nascent polypeptide-associated complex (NAC) subunit] of NAC from the hot pepper plant. NAC contacts nascent polypeptides to prevent aggregation and degradation of newly synthesized proteins by controlling cotranslational protein folding. CaBtf3 protein fused to green fluorescent protein predominantly localized to the nucleus. Silencing phenotype of CaBtf3 upon the Tobacco mosaic virus (TMV)-P0 inoculation exhibited reduced HR cell death and decreased expression of some HR-associated genes, but increased TMV coat protein levels compared with TRV2 control plants. Furthermore, silencing of NbBtf3, a highly homologous gene of CaBtf3, also led to the reduced Bax- and Pto-mediated cell death. The results indicate that CaBtf3 might be involved in HR cell death and could function as a transcription factor in the nucleus by transcriptional regulation of HR-related gene expression.

Hypoxia down-regulates DNA double strand break repair gene expression in prostate cancer cells

Background and purpose Intratumoral hypoxia has been correlated with poor clinical outcome in prostate cancer. Prostate cancer cells can be genetically unstable and have altered DNA repair. We, therefore, hypothesized that the expression of DNA double-strand break (DNA-dsb) repair genes in normal and malignant prostate cultures can be altered under hypoxic conditions. Methods and materials The expression of homologous recombination (HR) and non-homologous recombination (NHEJ) genes following gas hypoxia (0.2%) or exposure to HIF1α-inducing agent, CoCl 2 (100 μM), was determined for normal diploid fibroblasts (GM05757) and the pre-malignant and malignant prostate cell lines, BPH-1, 22RV-1, DU145 and PC3. RNA and protein levels were determined using RT-PCR and Western blotting. Additionally, p53 genotype and function, the level of hypoxia-induced apoptosis, and cell cycle distribution, were determined to correlate to changes in DNA-dsb gene expression. Results Induction of hypoxia was confirmed using HIF1α and VEGF expression in gas- and CoCl 2-treated cultures. Hypoxia (48–72 h of 0.2% O 2) decreased RNA expression of a number of HR-related genes (e.g. Rad51, Rad52, Rad54, BRCA1, BRCA2) in both normal and malignant cultures. Similar decreases in RNA pertaining to the NHEJ-related genes (e.g. Ku70, DNA-PKcs, DNA Ligase IV, Xrcc4) were observed. In selected cases, hypoxia-mediated decreases in RNA expression led to decreased DNA-dsb protein expression. CoCl 2-treated cultures did not show decreased DNA-dsb protein expression. The ability of hypoxia to down-regulate Rad51 and other HR-associated genes under hypoxia was not correlated to c-Abl or c-Myc gene expression, p53 genotype or function, propensity for hypoxia-mediated apoptosis, or specific changes in cell cycle distribution. Conclusions Hypoxia can down-regulate expression of DNA-dsb repair genes in both normal and cancer cells. If associated with a functional decrease in DNA-dsb repair, this observation could provide a potential basis for the observed genetic instability within tumor cells exposed to hypoxia.