Role In Homologous Recombination Repair Research Articles

Spectrum and characteristics of germline PALB2 pathogenic variants among patients with early-onset breast cancer in China.

e22536 Background: Partner and localizer of BRCA2 ( PALB2) plays a critical role in homologous recombination repair and recognized as a breast cancer predisposition gene. Early age at onset of breast cancer is considered a hereditary high-risk factor. However, limited information is available about early-onset breast cancer in China. The objective of this study was to investigate the spectrum and characteristics of germline PALB2 pathogenic variants among this population. Methods: A total of 1556 patients with BRCA1/2-negative early-onset breast cancer were included. Peripheral blood samples were collected and all coding regions and exon‒intron boundaries of the PALB2 genes were screened through next-generation sequencing. Results: The prevalence of germline PALB2 pathogenic variants was approximatel 0.77% in entire cohort. Eleven PALB2 pathogenic variants were identified in twelve participants, including five frameshift mutations, four nonsense mutations and one missense mutation. Except for PALB2 c.1056_1057del, which was detected twice, all other variants were detected once. In comparison to non-carriers, PALB2 carriers were more likely to be 30 years old or younger (25% vs. 14.4%), have a positive family history of breast or ovarian cancer (16.7% vs. 6.3%), exhibit metastatic lymph nodes (75% vs. 50.9%), belong to human epidermal growth factor receptor-2 (HER2) -negative and hormone receptor (HR) - positive subtype (75% vs 54.2%), and diagnosed with invasive micropapillary carcinoma (16.7% vs 3.1%). Conclusions: The prevalence of the germline PALB2 pathogenic variants is approximately 0.77% in BRCA1/2 mutation-negative early-onset breast cancer patients in China. Our findings contribute additional insights into the spectrum and characteristics of PALB2 pathogenic variants in Chinese early-onset breast cancer patients, providing valuable information for this population. Key words: PALB2, Germline mutation, Genetic sequencing, early-onset breast cancer, China. [Table: see text]

Targeting the hSSB1-INTS3 Interface: A Computational Screening Driven Approach to Identify Potential Modulators.

Human single-stranded DNA binding protein 1 (hSSB1) forms a heterotrimeric complex, known as a sensor of single-stranded DNA binding protein 1 (SOSS1), in conjunction with integrator complex subunit 3 (INTS3) and C9ORF80. This sensory protein plays an important role in homologous recombination repair of double-strand breaks in DNA to efficiently recruit other repair proteins at the damaged sites. Previous studies have identified elevated hSSB1-mediated DNA repair activities in various cancers, highlighting its potential as an anticancer target. While prior efforts have focused on inhibiting hSSB1 by targeting its DNA binding domain, this study seeks to explore the inhibition of the hSSB1 function by disrupting its interaction with the key partner protein INTS3 in the SOSS1 complex. The investigative strategy entails a molecular docking-based screening of a specific compound library against the three-dimensional structure of INTS3 at the hSSB1 binding interface. Subsequent assessments involve in vitro analyses of protein-protein interaction (PPI) disruption and cellular effects through co-immunoprecipitation and immunofluorescence assays, respectively. Moreover, the study includes an evaluation of the structural stability of ligands at the INTS3 hot-spot site using molecular dynamics simulations. The results indicate a potential in vitro disruption of the INTS3-hSSB1 interaction by three of the tested compounds obtained from the virtual screening with one impacting the recruitment of hSSB1 and INTS3 to chromatin following DNA damage. To our knowledge, our results identify the first set of drug-like compounds that functionally target INTS3-hSSB1 interaction, and this provides the basis for further biophysical investigations that should help to speed up PPI inhibitor discovery.

PTEN-mediated dephosphorylation of 53BP1 confers cellular resistance to DNA damage in cancer cells.

Homologous recombination (HR) repair for DNA double-strand breaks (DSBs) is critical for maintaining genome stability and conferring the resistance of tumor cells to chemotherapy. Nuclear PTEN which contains both phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and protein phosphatase plays a key role in HR repair, but the underlying mechanism remains largely elusive. We find that SUMOylated PTEN promotes HR repair but represses nonhomologous end joining (NHEJ) repair by directly dephosphorylating TP53-binding protein 1 (53BP1). During DNA damage responses (DDR), tumor suppressor ARF (p14ARF) was phosphorylated and then interacted efficiently with PTEN, thus promoting PTEN SUMOylation as an atypical SUMO E3 ligase. Interestingly, SUMOylated PTEN was subsequently recruited to the chromatin at DSB sites. This was because SUMO1 that was conjugated to PTEN was recognized and bound by the SUMO-interacting motif (SIM) of breast cancer type 1 susceptibility protein (BRCA1), which has been located to the core of 53BP1 foci on chromatin during S/G2 stage. Furthermore, these chromatin-loaded PTEN directly and specifically dephosphorylated phosphothreonine-543 (pT543) of 53BP1, resulting in the dissociation of the 53BP1 complex, which facilitated DNA end resection and ongoing HR repair. SUMOylation-site-mutated PTENK254R mice also showed decreased DNA damage repair in vivo. Blocking the PTEN SUMOylation pathway with either a SUMOylation inhibitor or a p14ARF(2-13) peptide sensitized tumor cells to chemotherapy. Our study therefore provides a new mechanistic understanding of PTEN in HR repair and clinical intervention of chemoresistant tumors.

A Highly Conserved Region in BRCA2 Suppresses the RAD51-Interaction Activity of BRC Repeats

Mammary tumors are the most prevalent type of tumors in female dogs. Breast cancer 2, early onset (BRCA2) malignant mutations are associated with tumorigenesis in humans and dogs. BRCA2 plays a pivotal role in homologous recombination repair by recruiting RAD51 recombinase to DNA damage sites to maintain genome stability. To recruit RAD51, BRCA2 must interact with RAD51 via BRC repeats, but the regulation of this interaction has been unclear. In this study, we focused on a highly conserved region (HCR) near BRC repeats. Using co-immunoprecipitation and mammalian two-hybrid assay, we found that HCR suppressed the RAD51-interaction activity of BRC repeats and that substitutions of HCR phosphorylation sites affected it. In canine tumor samples, we found ten mutations, including a novel HCR mutation (I1110M) from canine tumor samples. The effect of four HCR mutations, including I1110M, on the RAD51-interaction activity of BRC repeats was tested. One of the HCR mutations found in canine mammary tumors increased the interaction, but the two mutations found in human breast cancers decreased it. This study suggested that the HCR regulated the RAD51-interacting activity of BRC repeats through HCR phosphorylation and that mutations in HCR may be related to tumorigenesis in both dogs and humans.

The Interplay between the Cellular Response to DNA Double-Strand Breaks and Estrogen.

Cancer development is often connected to impaired DNA repair and DNA damage signaling pathways. The presence of DNA damage in cells activates DNA damage response, which is a complex cellular signaling network that includes DNA repair, activation of the cell cycle checkpoints, cellular senescence, and apoptosis. DNA double-strand breaks (DSBs) are toxic lesions that are mainly repaired by the non-homologous end joining and homologous recombination repair (HRR) pathways. Estrogen-dependent cancers, like breast and ovarian cancers, are frequently associated with mutations in genes that play a role in HRR. The female sex hormone estrogen binds and activates the estrogen receptors (ERs), ERα, ERβ and G-protein-coupled ER 1 (GPER1). ERα drives proliferation, while ERβ inhibits cell growth. Estrogen regulates the transcription, stability and activity of numerus DDR factors and DDR factors in turn modulate ERα expression, stability and transcriptional activity. Additionally, estrogen stimulates DSB formation in cells as part of its metabolism and proliferative effect. In this review, we will present an overview on the crosstalk between estrogen and the cellular response to DSBs. We will discuss how estrogen regulates DSB signaling and repair, and how DDR factors modulate the expression, stability and activity of estrogen. We will also discuss how the regulation of HRR genes by estrogen promotes the development of estrogen-dependent cancers.

Abstract 3237: Combining PARP inhibition with the polo-like kinase 1 (PLK1) inhibitor onvansertib overcomes PARP inhibitor resistance

Abstract Background: BRCA1/2 mutant tumors cells are deficient for homologous recombination (HR)-mediated DNA repair and are particularly sensitive to PARP inhibitors (PARPi). PARPi have proved efficacy in breast, ovarian, prostate, and pancreatic cancers, particularly in HR-deficient tumors, while their activity is limited in HR-proficient tumors. However, PARPi resistance is inevitable and therapeutic resistance resulting from restoration of HR repair is a pressing clinical problem. Identifying combination treatments to sensitize tumors cells to PARPi and/or overcome PARPi resistance is critical to expand the benefit of these therapies. The Polo-like kinase 1 (PLK1), a serine threonine kinase, is a master regulator of mitosis, overexpressed in many cancers. PLK1 is also involved in the DNA damage response through the promotion of HR-mediated DNA repair and the recovery from the G2/M checkpoint. PLK1 roles in HR repair suggest that PLK1 inhibition may reverse PARPi resistance. Methods: To test the effect of PLK1 and PARP inhibitors combination, we used onvansertib, a highly selective, ATP-competitor PLK1 inhibitor currently in clinical development and the FDA-approved PARPi olaparib. The antitumor effect of the single and combined drug treatments was tested in 2 BRCA1 mutated high-grade serous ovarian cancer (HGSOC) patient derived (PDX) models resistant to olaparib. Orthotopically PDX transplanted mice were treated for 4 weeks and followed for survival. Results: The combination of onvansertib and olaparib was well tolerated and showed strong anti-tumor activity in both PDX models. The combination significantly increased mice survival in comparison to vehicle, olaparib and onvansertib, and showed that onvansertib can re-sensitize PARPi-resistant tumors to olaparib. Median survival increased by 2.7-fold and 8.1-fold respectively in the 2 PDX models in the combination group versus vehicle and the Kaplan Meyer survival curves of mice treated with the combination showed a statistically survival advantage versus control and single agent treated mice. Pharmacodynamic analyses showed an increase in mitotic, apoptotic and DNA damage markers in tumors treated with the combination versus vehicle. Conclusions: The combination of the PLK1 inhibitor onvansertib and the PARPi olaparib showed potent anti-tumor activity in olaparib-resistant BRAC1 mutant HGSOC PDX models. Additional studies are ongoing to further assess the potential of the combination in BRCA wild-type and mutant ovarian, prostate, pancreatic and breast cancer preclinical models. Citation Format: Michela Chiappa, Federica Guffanti, Alessandra Decio, Alessandro Aliverti, Francesca Ricci, Eugenio Scanziani, Federica Camin, Ilaria Craparotta, Maria Chiara Barbera, Marco Bolis, Maya Ridinger, Giovanna Damia. Combining PARP inhibition with the polo-like kinase 1 (PLK1) inhibitor onvansertib overcomes PARP inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3237.

A BRCA1 Coiled-Coil Domain Variant Disrupting PALB2 Interaction Promotes the Development of Mammary Tumors and Confers a Targetable Defect in Homologous Recombination Repair.

The BRCA1 tumor suppressor gene encodes a multidomain protein for which several functions have been described. These include a key role in homologous recombination repair (HRR) of DNA double-strand breaks, which is shared with two other high-risk hereditary breast cancer suppressors, BRCA2 and PALB2. Although both BRCA1 and BRCA2 interact with PALB2, BRCA1 missense variants affecting its PALB2-interacting coiled-coil domain are considered variants of uncertain clinical significance (VUS). Using genetically engineered mice, we show here that a BRCA1 coiled-coil domain VUS, Brca1 p.L1363P, disrupts the interaction with PALB2 and leads to embryonic lethality. Brca1 p.L1363P led to a similar acceleration in the development of Trp53-deficient mammary tumors as Brca1 loss, but the tumors showed distinct histopathologic features, with more stable DNA copy number profiles in Brca1 p.L1363P tumors. Nevertheless, Brca1 p.L1363P mammary tumors were HRR incompetent and responsive to cisplatin and PARP inhibition. Overall, these results provide the first direct evidence that a BRCA1 missense variant outside of the RING and BRCT domains increases the risk of breast cancer. SIGNIFICANCE: These findings reveal the importance of a patient-derived BRCA1 coiled-coil domain sequence variant in embryonic development, mammary tumor suppression, and therapy response.See related commentary by Mishra et al., p. 6080.

PD34-09 EFFICACY OF PHYSICIAN'S CHOICE OF ENZALUTAMIDE OR ABIRATERONE IN THE CONTROL ARM OF PROFOUND

PD34-09 EFFICACY OF PHYSICIAN'S CHOICE OF ENZALUTAMIDE OR ABIRATERONE IN THE CONTROL ARM OF PROFOUND

Downregulation of ATM and BRCA1 Predicts Poor Outcome in Head and Neck Cancer: Implications for ATM-Targeted Therapy.

ATM and BRCA1 are DNA repair genes that play a central role in homologous recombination repair. Alterations of ATM and BRCA1 gene expression are found in cancers, some of which are correlated with treatment response and patient outcome. However, the role of ATM and BRCA1 gene expression in head and neck cancer (HNC) is not well characterized. Here, we examined the prognostic role of ATM and BRCA1 expression in two HNC cohorts with and without betel quid (BQ) exposure. The results showed that the expression of ATM and BRCA1 was downregulated in BQ-associated HNC, as the BQ ingredient arecoline could suppress the expression of both genes. Low expression of either ATM or BRCA1 was correlated with poor overall survival (OS) and was an independent prognostic factor in multivariate analysis (ATM HR: 1.895, p = 0.041; BRCA1 HR: 2.163, p = 0.040). The combination of ATM and BRCA1 expression states further improved on the prediction of OS (HR: 4.195, p = 0.001, both low vs. both high expression). Transcriptomic analysis showed that inhibition of ATM kinase by KU55933 induced apoptosis signaling and potentiated cisplatin-induced cytotoxicity. These data unveil poor prognosis in the HNC patient subgroup with low expression of ATM and BRCA1 and support the notion of ATM-targeted therapy.

Advanced Prostate Cancer: AUA/ASTRO/SUO Guideline PART II.

The summary presented herein represents Part II of the two-part series dedicated to Advanced Prostate Cancer: AUA/ASTRO/SUO Guideline discussing prognostic and treatment recommendations for patients with castration-resistant disease. Please refer to Part I for discussion of the management of patients with biochemical recurrence without metastatic disease after exhaustion of local treatment options as well as those with metastatic hormone-sensitive prostate cancer. The Advanced Prostate Cancer Panel created evidence- and consensus-based guideline statements to aid clinicians in the management of patients with advanced prostate cancer. Such statements are summarized in figure 1[Figure: see text] and detailed herein. The systematic review utilized to inform this guideline was conducted by an independent methodological consultant. A research librarian conducted searches in Ovid MEDLINE (1998 to January Week 5 2019), Cochrane Central Register of Controlled Trials (through December 2018), and Cochrane Database of Systematic Reviews (2005 through February 6, 2019). An updated search was conducted prior to publication through January 20, 2020. The methodology team supplemented searches of electronic databases with the studies included in the prior AUA review and by reviewing reference lists of relevant articles. This guideline attempts to improve a clinician's ability to treat patients diagnosed with advanced prostate cancer. Continued research and publication of high-quality evidence from future trials will be essential to improve the level of care for these patients.

Olaparib for Metastatic Castration-Resistant Prostate Cancer

BackgroundMultiple loss-of-function alterations in genes that are involved in DNA repair, including homologous recombination repair, are associated with response to poly(adenosine diphosphate–ribose) polymerase (PARP) inhibition in patients with prostate and other cancers.MethodsWe conducted a randomized, open-label, phase 3 trial evaluating the PARP inhibitor olaparib in men with metastatic castration-resistant prostate cancer who had disease progression while receiving a new hormonal agent (e.g., enzalutamide or abiraterone). All the men had a qualifying alteration in prespecified genes with a direct or indirect role in homologous recombination repair. Cohort A (245 patients) had at least one alteration in BRCA1, BRCA2, or ATM; cohort B (142 patients) had alterations in any of 12 other prespecified genes, prospectively and centrally determined from tumor tissue. Patients were randomly assigned (in a 2:1 ratio) to receive olaparib or the physician’s choice of enzalutamide or abiraterone (control). The primary end point was imaging-based progression-free survival in cohort A according to blinded independent central review.ResultsIn cohort A, imaging-based progression-free survival was significantly longer in the olaparib group than in the control group (median, 7.4 months vs. 3.6 months; hazard ratio for progression or death, 0.34; 95% confidence interval, 0.25 to 0.47; P<0.001); a significant benefit was also observed with respect to the confirmed objective response rate and the time to pain progression. The median overall survival in cohort A was 18.5 months in the olaparib group and 15.1 months in the control group; 81% of the patients in the control group who had progression crossed over to receive olaparib. A significant benefit for olaparib was also seen for imaging-based progression-free survival in the overall population (cohorts A and B). Anemia and nausea were the main toxic effects in patients who received olaparib.ConclusionsIn men with metastatic castration-resistant prostate cancer who had disease progression while receiving enzalutamide or abiraterone and who had alterations in genes with a role in homologous recombination repair, olaparib was associated with longer progression-free survival and better measures of response and patient-reported end points than either enzalutamide or abiraterone. (Funded by AstraZeneca and Merck Sharp & Dohme; PROfound ClinicalTrials.gov number, NCT02987543.)

Chasing the backup pathway for therapeutic opportunities in cancer

BRCA mutations are usually associated with the most aggressive subtypes of cancers such as triple‐negative breast cancer and high‐grade serous ovarian cancer. Finding a drug that can address the unique molecular features in these subtypes of cancer continues to be the best hope for treatment. In tumors with impaired BRCA‐pathway, RAD52 is a candidate for targeted inhibition as it is known to take a leading role in homologous recombination repair (HRR) of DNA double‐strand breaks (DSBs) in BRCA‐deficient cancer cells [1]. Additionally, RAD52 is the main player in single‐strand annealing (SSA), which has been reported to increase activity in a number of cancers. When RAD52 joins the DSB repair complex, RPA has already bound ssDNA and the resulting protein‐protein interaction (PPI) becomes instrumental for repair to proceed. Past work showed that conformational changes offered by RPA interaction modulate RAD52 activity [2]. A combined understanding of these bimolecular studies and the value of RAD52 in tumorigenesis were motivating factors for the development of a Fluorescence‐based protein‐protein Interaction Assay (FluorIA) [3]. FluorIA provided a tool to assess domains on RPA and RAD52 important for the PPI (Figure 1). The binding affinities and thermodynamics of those interacting domains were then evaluated by methods such as surface plasmon resonance and isothermal titration calorimetry to characterize RPA:RAD52 interaction as a vital therapeutic target.FluorIA was also used as a high throughput assay to screen over 100,000 potential small molecule inhibitors (SMIs). Eleven hits were identified, three of which are FDA approved drugs (Fig. 2). Characterization experiments thus far suggest a potential impact for at least one of these three drugs on SSA repair of DSBs. Our findings may offer molecular insights into a new mechanism of action for these drugs given their reported varied sensitivity in patients carrying BRCA deficiencies. The ultimate goal is to therapeutically target the addiction of cancer cells for DNA repair. However, we anticipate that a broader understanding of the mechanisms of action for the existing cancer drugs we screened would support the effort towards personalized medicine.Support or Funding Information1) SBMB Fellowship from U.S. Department of Education GAANN (P200A120231) 2) NASA Nebraska Space Grant: Graduate fellowship 3) Nebraska Department of Health and Human services (DHHS) LB506 4) Nebraska Research Initiative 5) Fred &amp; Pamela Buffett Cancer Center Support Grant (P30CA036727) pilot project fundingCompetitive binding in FluorIA. A) Domain map of RAD52. B) SDS‐PAGE gel shown full‐length RAD52(1‐418) to the right of the molecular weight marker, RAD52(1‐303), and RAD52(1‐212). C) FluorIA reaction in which 20 pmol of EGFP tagged full‐length RAD52 is incubated with RPA alone or with a 100 pmol of unlabeled RAD52(1‐303) or (1‐212) to compete with EGFP‐RAD52 for RPA binding. Reduction in RFU signal signifies inhibition of RPA:RAD52 complex formation by the unlabeled RAD52 construct.Figure 1Inhibiti of RPA:RAD52 complex by the SMI leads. Hits are designated with the letters P or C according to the first letter of the source library name. The best IC50 are P1, 110 μM; P2, 12 μM; P3, 15 μM; C2, 120 μM and C8, 100 μM.Figure 2

PROfound: Efficacy of olaparib (ola) by prior taxane use in patients (pts) with metastatic castration-resistant prostate cancer (mCRPC) and homologous recombination repair (HRR) gene alterations.

134 Background: Optimal sequencing of therapies for mCRPC is not established. In the Phase III PROfound study (NCT02987543), ola significantly prolonged radiographic progression-free survival (rPFS) vs physician’s choice of new hormonal agent (pcNHA) in pts with mCRPC and an alteration in genes with a direct or indirect role in HRR. We report exploratory subgroup analyses by prior taxane (yes vs no). Methods: Men with mCRPC that had progressed on prior NHA were randomized to ola (tablets; 300 mg bid) or pcNHA (enzalutamide or abiraterone). Pts had alterations in BRCA1, BRCA2 or ATM (Cohort A) or ≥1 of 12 other prespecified genes with a direct or indirect role in HRR (Cohort B). Stratification factors were prior taxane use and measurable disease. rPFS was assessed by blinded independent central review with RECIST v1.1 + PCWG3. Results: Subgroup analyses of rPFS and overall survival (OS) favored ola vs pcNHA irrespective of prior taxane in Cohort A, Cohorts A+B and pts with a BRCA1 and/or BRCA2 or CDK12 alteration (Table). In the ATM subgroup hazard ratio (HR) point estimates for rPFS and OS were lower in pts who had received prior taxane vs pts who had not, but 95% CIs overlapped and pt numbers were small so data should be interpreted with caution. Conclusions: The benefit of ola over pcNHA in pts with mCRPC and HRR gene alterations was generally independent of prior taxane status in the overall study population. Clinical trial information: NCT02987543. [Table: see text]

LBA8 - PROfound: Phase III study of olaparib versus enzalutamide or abiraterone for metastatic castration-resistant prostate cancer (mCRPC) with homologous recombination repair (HRR) gene alterations

LBA8 - PROfound: Phase III study of olaparib versus enzalutamide or abiraterone for metastatic castration-resistant prostate cancer (mCRPC) with homologous recombination repair (HRR) gene alterations

LBA12_PR - PROfound: Phase III study of olaparib versus enzalutamide or abiraterone for metastatic castration-resistant prostate cancer (mCRPC) with homologous recombination repair (HRR) gene alterations

LBA12_PR - PROfound: Phase III study of olaparib versus enzalutamide or abiraterone for metastatic castration-resistant prostate cancer (mCRPC) with homologous recombination repair (HRR) gene alterations

Phosphorylation of the Archaeal Holliday Junction Resolvase Hjc Inhibits Its Catalytic Activity and Facilitates DNA Repair in Sulfolobus islandicus REY15A.

Protein phosphorylation is one of the main protein post-translational modifications and regulates DNA repair in eukaryotes. Archaeal genomes encode eukaryotic-like DNA repair proteins and protein kinases (ePKs), and several proteins involved in homologous recombination repair (HRR) including Hjc, a conserved Holliday junction (HJ) resolvase in Archaea, undergo phosphorylation, indicating that phosphorylation plays important roles in HRR. Herein, we performed phosphorylation analysis of Hjc by various ePKs from Sulfolobus islandicus. It was shown that SiRe_0171, SiRe_2030, and SiRe_2056, were able to phosphorylate Hjc in vitro. These ePKs phosphorylated Hjc at different Ser/Thr residues: SiRe_0171 on S34, SiRe_2030 on both S9 and T138, and SiRe_2056 on T138. The HJ cleavage activity of the phosphorylation-mimic mutants was analyzed and the results showed that the cleavage activity of S34E was completely lost and that of S9E had greatly reduced. S. islandicus strain expressing S34E in replacement of the wild type Hjc was resistant to higher doses of DNA damaging agents. Furthermore, SiRe_0171 deletion mutant exhibited higher sensitivity to DNA damaging agents, suggesting that Hjc phosphorylation by SiRe_0171 enhanced the DNA repair capability. Our results revealed that HJ resolvase is regulated by protein phosphorylation, reminiscent of the regulation of eukaryotic HJ resolvases GEN1 and Yen1.

Improved Therapeutic Window in BRCA-mutant Tumors with Antibody-linked Pyrrolobenzodiazepine Dimers with and without PARP Inhibition.

Pyrrolobenzodiazepine dimers (PBD) form cross-links within the minor groove of DNA causing double-strand breaks (DSB). DNA repair genes such as BRCA1 and BRCA2 play important roles in homologous recombination repair of DSB. We hypothesized that PBD-based antibody-drug conjugates (ADC) will have enhanced killing of cells in which homologous recombination processes are defective by inactivation of BRCA1 or BRCA2 genes. To support this hypothesis, we found 5T4-PBD, a PBD-dimer conjugated to anti-5T4 antibody, elicited more potent antitumor activity in tumor xenografts that carry defects in DNA repair due to BRCA mutations compared with BRCA wild-type xenografts. To delineate the role of BRCA1/2 mutations in determining sensitivity to PBD, we used siRNA knockdown and isogenic BRCA1/2 knockout models to demonstrate that BRCA deficiency markedly increased cell sensitivity to PBD-based ADCs. To understand the translational potential of treating patients with BRCA deficiency using PBD-based ADCs, we conducted a "mouse clinical trial" on 23 patient-derived xenograft (PDX) models bearing mutations in BRCA1 or BRCA2 Of these PDX models, 61% to 74% had tumor stasis or regression when treated with a single dose of 0.3 mg/kg or three fractionated doses of 0.1 mg/kg of a PBD-based ADC. Furthermore, a suboptimal dose of PBD-based ADC in combination with olaparib resulted in significantly improved antitumor effects, was not associated with myelotoxicity, and was well tolerated. In conclusion, PBD-based ADC alone or in combination with a PARP inhibitor may have improved therapeutic window in patients with cancer carrying BRCA mutations.

Using HSP90 Inhibitors to Target DNA Repair Proteins in AML

Acute myeloid leukemia (AML) is a disease of proliferative and abnormally differentiated myeloid cells(Döhner, Weisdorf, & Bloomfield, 2015) that is characterized by chromosomal aberrations and gene mutations that influence outcome(De Kouchkovsky & Abdul-Hay, 2016). Despite the approval of targeted therapies, induction therapy with cytarabine (Ara-C) and anthracycline has remained the standard of care for AML(Dombret & Gardin, 2016; Showel & Levis, 2014). Newer chemotherapeutics such as Sapacitabine have shown comparable clinical outcomes to Ara-C in older patients(Burnett et al., 2015). Ara-C is a nucleoside analog which incorporates during DNA replication and stalls replication(Liu et al., 2010) while Sapacitabine induces single stranded nicks in the DNA leading to double stranded breaks which are repaired by Homologous Recombination (HR)(Burnett et al., 2015; Lim & Jamieson, 2014). However, eventually patients develop resistance to chemotherapy leading to poor prognosis after relapse(Wu, Duan, Chen, & Chen, 2017). One aspect of this is due to upregulation of DNA damage repair proteins leading to increased repair of any DNA lesions primarily through HR. We therefore are interested in developing a way to target DNA repair to extend nucleoside analog therapy in AML.HSP90 is a molecular chaperone that is critical for the folding and proper function of multiple key proteins of various DNA repair pathways(Dote, Burgan, Camphausen, & Tofilon, 2006). Relevant to such repair, ATM and Chk1 become recruited to DNA double strand breaks to activate defined downstream signaling cascades that result in cell cycle arrest and DNA repair(Ceccaldi, Rondinelli, & D'Andrea, 2016). Of these,γ-H2AX acts a sensor of DNA damage and repair by forming foci at the sites of damaged DNA that get resolved upon successful repair(Sharma, Singh, & Almasan, 2012). Similarly, the Rad51 helicase plays a key role in HR repair and the presence of Rad51 foci can be used as a marker for successful HR(Gachechiladze, Škarda, Soltermann, & Joerger, 2017). To determine whether inhibiting HSP90 compromised DNA repair, we treated both AML cell lines OCI-AML3 and MV4-11 with HSP90 inhibitor, onalespib. OCI-AML3 cells show a time dependent decrease in DNA damage repair proteins Chk1 and Rad51 by western blotting. Furthermore, primary patient samples treated with onalespib for 24 and 36 hours also show a depletion of Chk1 and Rad51. We then used ionizing radiation (IR) to induce dsbreaks and induce signaling and repair(Ceccaldi et al., 2016). Using immunofluorescence, we found that in OCI-AML3 after exposure to IR there was a significant increase in number of Rad51 foci after 6 hours. The large number of γ-H2AX foci formed at .5 hour after IR decrease by 6 hours post-IR. This suggests that there is a successful HR repair occurring in this AML cell line. When OCI-AML3 cells were pretreated with onalespib, Rad51 foci are no longer present and γ-H2AX foci remained present at 6 hours suggesting unrepaired dsbreaks. Using the colony forming assay we see that there is a slight decrease in viability of OCI-AML3 cells exposed to onalespib however, in MV4-11 cells there is a steep decline which could be due to the differential expression of genes such as FLT3-ITD, a client protein of HSP90. Low doses of cytarabine or sapacitabine modestly decreased colony growth of OCI-AML3 and MV4-11 cells but exposing cells to onalespib for 24 hours sensitized the cells to both cytarabine and sapacitabine. Finally, we have also evaluated effects of onalespib in an in-vivo MOLM13 driven mouse model of AML and have shown that treatment with onalespib extended survival in this mouse model. DisclosuresMims:Abbvie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

Nucleoporin 54 contributes to homologous recombination repair and post-replicative DNA integrity.

The nuclear pore complex (NPC) machinery is emerging as an important determinant in the maintenance of genome integrity and sensitivity to DNA double-strand break (DSB)-inducing agents, such as ionising radiation (IR). In this study, using a high-throughput siRNA screen, we identified the central channel NPC protein Nup54, and concomitantly its molecular partners Nup62 and Nup58, as novel factors implicated in radiosensitivity. Nup54 depletion caused an increase in cell death by mitotic catastrophe after IR, and specifically enhanced both the duration of the G2 arrest and the radiosensitivity of cells that contained replicated DNA at the time of IR exposure. Nup54-depleted cells also exhibited increased formation of chromosome aberrations arisen from replicated DNA. Interestingly, we found that Nup54 is epistatic with the homologous recombination (HR) factor Rad51. Moreover, using specific DNA damage repair reporters, we observed a decreased HR repair activity upon Nup54 knockdown. In agreement with a role in HR repair, we also demonstrated a decreased formation of HR-linked DNA synthesis foci and sister chromatid exchanges after IR in cells depleted of Nup54. Our study reveals a novel role for Nup54 in the response to IR and the maintenance of HR-mediated genome integrity.

Prognostic Value of the Expression of DNA Repair–Related Biomarkers Mediated by Alcohol in Gastric Cancer Patients

Alcohol consumption likely induces gastric carcinogenesis through deregulation of RNA polymerase (Pol) III genes and oxidative damage. Transcription factor IIB-related factor 1 (BRF1) overexpression alleviates RNA Pol III transcription inhibition through breast cancer susceptibility gene 1 (BRCA1). Myeloperoxidase (MPO) involvement in cancer is induced by alcohol-mediated oxidative damage. BRCA1/2 and MPO play key roles in DNA repair. BRCA1 and BRCA2 exert different roles in homologous recombination repair. By using human gastric cancer (GC) biopsies, we investigated the prognostic value of these proteins upon alcohol induction. In total, high expression of BRF1 (P=0.010) and positive cell infiltration of MPO (P=0.004) in tumor tissues as well as positive expression of BRCA1 (P<0.001) in para-tumor tissues were more frequent in GC patients with hazardous or harmful alcohol consumption habits. BRF1 (P=0.021), BRCA2 (P<0.001), and MPO (P=0.039) were independent prognostic factors for disease-free survival. BRCA1 (P=0.005) and BRCA2 (P<0.001) also were identified as independent prognostic factors for overall survival. Furthermore, BRCA2 was an independent unfavorable prognostic factor for disease-free survival and overall survival (P<0.001) in GC patients who underwent platinum-based adjuvant chemotherapy. BRF1, BRCA1/2, and MPO are DNA repair-related biomarkers, induced by alcohol with prognostic value in GC patients.