BRCA1 Foci Formation Research Articles

DAG-Lactone Radiosensitization of Human Prostate Cancer Cells Is Mediated by ATM Down-regulation But Not Due to Abnormal DNA Repair

DAG-Lactone Radiosensitization of Human Prostate Cancer Cells Is Mediated by ATM Down-regulation But Not Due to Abnormal DNA Repair

Abstract B27: CDK inhibition impairs homologous recombination and induces PARP inhibitor sensitivity via loss of c-myc expression in TNBC

Abstract The c-myc oncogene is a “master regulator” amplified in several types of cancer including ovarian, breast, colorectal and pancreatic cancers. C-myc controls several cellular processes during tumor maintenance including the cell cycle, metabolism, and DNA damage repair while also playing an essential role in transforming normal cells during tumorigensis. C-myc amplification correlates with poor survival and a higher frequency of relapse in triple negative breast cancer (TNBC) patients. C-myc amplification promotes deregulation of the cell cycle, via transcriptional activation of G1-S-phase cell cycle and DNA replication genes. CDK inhibition has been shown to down regulate c-myc expression in cancer cells, in vitro. Therefore, we hypothesize that transient CDK inhibition downregulates c-myc expression and increases susceptibility to DNA damaging agents in TNBC cells. We observed the long-term response of several c-myc overexpressing TNBC cells to Dinaciclib, a potent CDK inhibitor (CDK 1, 2, 5, 9) in vitro. High Throughput Survival Assay (HTSA) analysis revealed that c-myc expressing TNBC cells have a potent response to CDK inhibition via Dinaciclib (IC50 10-20nM). Dinaciclib induces an S-phase arrest and downregulation of the c-myc/E2F1 pathway in both normal (MCF10A) and TNBC (MB231, SUM149, HCC1937) breast cancer cells. Loss of c-myc expression correlates with increased apoptosis in TNBC cells but not HMECs. C-myc downregulation also correlates with increased γH2AX DNA damage and downregulation of BRCA1 foci formation. The resultant impaired homologous recombination (HR) pathway in TNBC cells treated with Dinaciclib proposed an increased susceptibility to PARP inhibitor induced DNA damage. Combination Dinaciclib + PARP inhibition exhibited a Dinaciclib dose dependent synergistic growth inhibition of BRCA1 wild-type and mutant TNBC cells to PARP inhibitors. Dinaciclib also increased susceptibility of PARP inhibition in resistant BRCA1 mutant cell lines (HCC1937, SUM149). Dinaciclib + PARP Inhibitor combination therapy induced increased apoptosis, γH2AX DNA damage and decreased BRCA1, RAD51 foci formation. These results demonstrate that Dinaciclib + PARP inhibition induces growth arrest and induction of apoptosis in c-myc overexpressing cancer cells. C-myc is frequently amplified in TNBC tumors and may serve as a biomarker for CDK + PARP inhibitor combination therapy. Citation Format: Jason Patrick Carey, Khandan Keyomarsi. CDK inhibition impairs homologous recombination and induces PARP inhibitor sensitivity via loss of c-myc expression in TNBC. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B27.

MERIT40 cooperates with BRCA2 to resolve DNA interstrand cross-links.

MERIT40 is an essential component of the RAP80 ubiquitin recognition complex that targets BRCA1 to DNA damage sites. Although this complex is required for BRCA1 foci formation, its physiologic role in DNA repair has remained enigmatic, as has its relationship to canonical DNA repair mechanisms. Surprisingly, we found that Merit40(-/-) mice displayed marked hypersensitivity to DNA interstrand cross-links (ICLs) but not whole-body irradiation. MERIT40 was rapidly recruited to ICL lesions prior to FANCD2, and Merit40-null cells exhibited delayed ICL unhooking coupled with reduced end resection and homologous recombination at ICL damage. Interestingly, Merit40 mutation exacerbated ICL-induced chromosome instability in the context of concomitant Brca2 deficiency but not in conjunction with Fancd2 mutation. These findings implicate MERIT40 in the earliest stages of ICL repair and define specific functional interactions between RAP80 complex-dependent ubiquitin recognition and the Fanconi anemia (FA)-BRCA ICL repair network.

Abstract 5467: BRCA1 N-terminal-deficient proteins provide PARP inhibitor and platinum resistance

Abstract Introduction: Tumors harboring BRCA1 mutations initially respond well to platinum and PARP inhibitor therapy; however, resistance invariably arises and is a major clinical problem. The BRCA1 185delAG allele is a common founder mutation located close to the protein translation start site, thought to produce a short peptide devoid of function. Experimental procedures: In this study, we utilized the SUM1315MO2 breast cancer cell line that harbors a BRCA1 185delAG mutation to study mechanisms of PARP inhibitor and platinum resistance. SUM1315MO2 cells were cultured in the presence of increasing concentrations of the PARP inhibitor rucaparib or cisplatin until rucaparib resistant (RR) and cisplatin resistant (CR) clones emerged. Results: DNA sequencing revealed that no BRCA1 gene reversion mutations were present in resistant cells. We next measured BRCA1 protein levels by Western blotting with antibodies specific for the N- and C-terminal domains of BRCA1. As a control, the wild-type BRCA1 protein expressed in MDA-MB-231 cells was detectable with both N- and C-terminal antibodies. In contrast, BRCA1 protein was undetectable in both SUM1315MO2 parental and resistant clones using the N-terminal specific antibody. However, the C-terminal specific antibody identified a more quickly migrating band of low abundance in parental cells, but with elevated expression in both RR and CR clones. We inferred that this protein was devoid of the extreme N-terminal RING domain-containing region that mediates interaction with BARD1. To investigate the functionality of the N-terminal truncated BRCA1 protein (Nt-BRCA1), we measured BRCA1 and RAD51 irradiation-induced focus formation by immunofluorescence. RR and CR clones demonstrated 2.25 - 2.75-fold increase (P = 0.024) in the number of cells with BRCA1 foci compared to parental cells. Similarly, we detected a 2 - 2.85-fold increase (P = 0.0325) in RAD51 foci formation in resistant clones compared to parental cells. Additionally, BRCA1 siRNA treated cells were 12- and 9-fold more sensitive to rucaparib compared to scrambled siRNA-treated control cells (P = 0.0002). Similarly, CR-1 cells treated with BRCA1 siRNAs were 1.56- and 1.7-fold more sensitive to cisplatin (P = 0.0346) compared to scrambled siRNA treated cells. Furthermore, N-terminal truncated BRCA1 proteins were detectable in a primary tumor from a germline BRCA1185delAG mutation carrier. Conclusions: Taken together, these results provide evidence for a novel, mutation location-dependent mechanism of PARP inhibitor and platinum resistance. Citation Format: Yifan Wang, Andrea Bernhardy, Emmanuelle Nicolas, Ryan Winters, Kathy Cai, Kelly Duncan, James Duncan, Maria Harrell, Elizabeth Swisher, Neil Johnson. BRCA1 N-terminal-deficient proteins provide PARP inhibitor and platinum resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5467. doi:10.1158/1538-7445.AM2015-5467

Abstract P6-03-09: Targeting the c-myc/E2F1 pathway in TNBC promotes a DNA damage dependent synthetic lethality

Abstract The c-myc/E2F1 pathway is highly upregulated in TNBC and dictates increased genomic instability, a higher frequency of relapse and poor overall survival. C-myc is a potent oncogene with overexpression activity that influences several pathways including metabolism, DNA replication, DNA damage, cell cycle and apoptosis. The essential role of c-myc in normal cells has made therapeutic intervention elusive. However the role of c-myc influencing cell cycle progression has demonstrated that CDK inhibition downregulates c-myc expression in TNBC breast cancer cells. We hypothesize that CDK inhibition downregulates c-myc expression resulting in an accumulation of DNA damage thus increasing the susceptibility of TNBC cells to PARP inhibition. A panel of TNBC cell lines was assessed for c-myc/E2F1 pathway expression and downregulation in the presence of CDK inhibitor Dinaciclib. An MTT-based High Throughput Survival Assay (HTSA) was utilized to assess growth inhibition response to drug treatment in TNBC cells. FACS analysis was performed on Dinaciclib treated TNBC cell lines to asses cell cycle response to CDK inhibition. DNA damage and Homologous Recombination DNA repair was assessed by γH2AX, BRCA1, RAD51 foci formation by immunofluorescence. Lentiviral knockdown of c-myc, E2F1, BRCA1 and RAD51 were performed to assess susceptibility to PARP inhibition. Combination treatment of Dinaciclib + PARP inhibitor MK4827 was assessed via combination index values calculated by Calcusyn. CDK inhibition resulted in downregulation of both c-myc and E2F1 in all TNBC cells. The pan-CDK inhibitor Dinaciclib universally inhibited proliferation in all TNBC cell lines in the presence of the drug however drug removal allowed TNBC cells to recover a normal proliferative profile. Dinaciclib induction increased γH2AX foci formation while decreasing BRCA1 and RAD51 foci formation resulting in an increase in DNA damage induced cell death that correlated with c-myc downregulation. CDK inhibition correlated with downregulation of c-myc/E2F1 target genes. Lentiviral knockdown of c-myc and not E2F1 induced susceptibility to PARP inhibitor MK4827. Combination CDK + PARP inhibitor resulted in a dose dependent synthetic lethal increase in PARP inhibitor efficacy in both BRCA1 mutant and wild-type cells. Together these results suggest that c-myc downregulation via CDK inhibition in combination with PARP inhibition may pose a novel combination therapy for TNBC patients. Citation Format: Jason P Carey, Khandan Keyomarsi. Targeting the c-myc/E2F1 pathway in TNBC promotes a DNA damage dependent synthetic lethality [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-03-09.

β-HPV 5 and 8 E6 disrupt homology dependent double strand break repair by attenuating BRCA1 and BRCA2 expression and foci formation.

Recent work has explored a putative role for the E6 protein from some β-human papillomavirus genus (β-HPVs) in the development of non-melanoma skin cancers, specifically β-HPV 5 and 8 E6. Because these viruses are not required for tumor maintenance, they are hypothesized to act as co-factors that enhance the mutagenic capacity of UV-exposure by disrupting the repair of the resulting DNA damage. Supporting this proposal, we have previously demonstrated that UV damage signaling is hindered by β-HPV 5 and 8 E6 resulting in an increase in both thymine dimers and UV-induced double strand breaks (DSBs). Here we show that β-HPV 5 and 8 E6 further disrupt the repair of these DSBs and provide a mechanism for this attenuation. By binding and destabilizing a histone acetyltransferase, p300, β-HPV 5 and 8 E6 reduce the enrichment of the transcription factor at the promoter of two genes critical to the homology dependent repair of DSBs (BRCA1 and BRCA2). The resulting diminished BRCA1/2 transcription not only leads to lower protein levels but also curtails the ability of these proteins to form repair foci at DSBs. Using a GFP-based reporter, we confirm that this reduced foci formation leads to significantly diminished homology dependent repair of DSBs. By deleting the p300 binding domain of β-HPV 8 E6, we demonstrate that the loss of robust repair is dependent on viral-mediated degradation of p300 and confirm this observation using a combination of p300 mutants that are β-HPV 8 E6 destabilization resistant and p300 knock-out cells. In conclusion, this work establishes an expanded ability of β-HPV 5 and 8 E6 to attenuate UV damage repair, thus adding further support to the hypothesis that β-HPV infections play a role in skin cancer development by increasing the oncogenic potential of UV exposure.

Abstract 19299: PPARγ Plays a Novel, Pivotal Role in DNA Damage Sensing and Repair, That is Perturbed in Pulmonary Arterial Hypertension

We previously observed that mice with deletion of peroxisome proliferator-activated receptor γ (PPARγ) in endothelial cells (EC) (EC-Pparγ-/-) do not reverse pulmonary hypertension (PH) during recovery in room air after three weeks of chronic hypoxia. Impaired PPARγ interaction with ß-catenin was linked to pulmonary arterial (PA) EC dysfunction in the pathogenesis of PH. To gain further insight into PPARγ function, important in reversing PH, we applied affinity purification-mass spectrometry in HEK293T cells to analyse the PPARγ interactome. Analyses revealed a network highly enriched in DNA damage response (DDR) proteins, including the MRE11, RAD50 and NBN (MRN) heterotrimeric complex. MRN senses damaged DNA and activates the ATM/ATR pathway essential for DNA repair. Using co-immmunoprecipitation in PAECs, we confirmed the PPARγ:MRN interaction via the PPARγ ligand binding domain. To determine the function of this novel interaction, we depleted PPARγ with siRNA and induced DNA damage with doxorubicin (DOX) or hydroxyurea (HU). PPARγ depletion impaired DDR activation after DOX/HU, with reduced levels of pATM and its substrates, pCHK1, pCHK2 and γH2AX. To test whether these abnormalities were related to reduced DNA repair, we assessed damaged DNA 24h after removal of DOX/HU using the COMET assay. PPARγ depleted PAECs showed longer comet tails reflective of unrepaired DNA. To investigate if persistent PH in EC-Pparγ-/- mice after chronic hypoxia correlated with impaired DDR, we evaluated both γH2AX (sensor) and Brca1 (repair) foci formation in the mouse lung tissues using confocal microscopy. Consistent with impaired DNA repair, Brca1 foci formation was significantly increased in ECs of EC-Pparγ-/- mice compared to the wildtype littermates. Moreover, reduced PPARγ previously described in PAECs from PH patients relative to controls was also associated with unrepaired DNA, judged by longer comet tails and by persistent γH2AX foci as assessed by confocal microscopy in tissue sections. In summary we report a novel interaction between PPARγ and the MRN that is necessary in DNA damage sensing and repair. We suggest that persistent DNA damage associated with PPARγ deficiency in PAECs could cause dysfunction or transformation related to the pathology of PH.

Abstract 2400: Dinaciclib, an inhibitor of cyclin-dependent kinases (CDKs), impairs homologous recombination, abrogates G2 checkpoint arrest and sensitizes myeloma cells to PARP inhibition

Abstract Background: Multiple myeloma (MM) is the second most common hematologic malignancy, and yet remains incurable in the majority of patients. We previously observed dysfunctional homologous recombination (HR) to mediate genomic instability and progression of MM.CDKs are key components of HR DNA repair and have been targeted by therapeutic drugs for MM. CDK1 phosphorylates BRCA1, and CDK5 (upregulated in MM and predictive of patient survival) phosphorylates ATM, modifications considered essential for checkpoint activation and HR repair. Poly(ADP-ribose)polymerase (PARP) is required for single-strand break (SSB) repair and when inhibited, SSBs accumulate and degenerate into double-strand breaks (DSBs) during DNA replication, resulting in cell death if HR is deficient. Dinaciclib (DC) is a potent inhibitor of CDK1, 2, 5 and 9, currently in clinical trials for MM. Here, we show that DC disrupts HR repair, abrogates G2 checkpoint arrest, and sensitizes MM cells to PARP inhibition. Methods: We investigated effects of DC, doxorubicin (topoisomerase II inhibitor), and veliparib (PARP inhibitor) on MM cell lines and myeloma cells from MM patients. We used confocal immunofluorescence microscopy to assess formation of γH2AX, BRCA1 and RAD51 foci, and western blotting to measure levels of phospho-BRCA1 (at Ser1497,CDK1 phosphorylation site) in order to assess the effects of DC on recruitment of HR proteins to DSBs. We measured DC effects on HR-mediated DSB repair, following transient infection with adenovirus encoding I-SceI, which forms DSBs within a DR-GFP reporter chromosomally integrated in MM.1S myeloma cells. Cell viability was determined using WST-1 and colony-formation assays. Flow cytometry after dual staining with propidium iodide (PI)/RNaseA and anti-phospho-histone H3 (Ser10) antibody was used to determine G2 checkpoint activation. In vivo consequences of DC treatment are currently being examined in a myeloma-xenograft mouse model. Results: We observed significant decreases in the percentage of cells with ≥5 BRCA1 foci (46.7% to 5.7%, p<0.001) or ≥5 RAD51 foci (26% to 4%, p<0.001), in cells treated with veliparib plus 20 nM DC, relative to veliparib alone. DC elicited ∼8-fold decrease in doxorubicin-induced phosphorylation of BRCA1 on S1497. In the DR-GFP assay of HR, 23.9% of vehicle-treated cells and 4.2% of DC-treated cells were GFP+, indicating that DC disrupts HR repair of DSBs. Cell viability following 72h treatments with veliparib ± DC, or vehicle alone, implied synergistic cytotoxicity between veliparib and DC. DC treatment significantly inhibited doxorubicin- or veliparib-induced G2/M phase cell accumulation and increased the fraction of mitotic cells in G2/M. Conclusion: DC significantly impairs HR DNA repair and sensitizes myeloma cells to PARP inhibition, and this combination may thus offer an effective treatment strategy for MM. Citation Format: David A. Alagpulinsa, Shmuel Yaccoby, Srinivas Ayyadevara, Robert J. S. Reis. Dinaciclib, an inhibitor of cyclin-dependent kinases (CDKs), impairs homologous recombination, abrogates G2 checkpoint arrest and sensitizes myeloma cells to PARP inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2400. doi:10.1158/1538-7445.AM2014-2400

Abstract 4702: Caveolin-1 confers a multi-modality therapy resistance phenotype in pancreatic cancer cells

Abstract Caveolin-1 (Cav-1) is a 21kDa protein found in 50-100nm omega-shaped invaginations of the plasma membrane known as caveolae. It has been implicated in the processes of oncogenic cell transformation, tumorigenesis and metastasis. Cav-1 is upregulated in pancreatic cancer and has been shown to be correlated with poor prognosis. We hypothesized that Cav-1 promotes resistance to chemotherapy and radiation, and that downregulation may lead to sensitization to radiation and/or chemotherapy. We found that radiation and chemotherapy up-regulate Cav-1 expression, and that downregulating Cav-1 in multiple pancreatic cancer cell lines by small interfering RNA induces sensitization to chemotherapeutics such as gemcitabine and 5-fluorouracil. Furthermore, genetic knockdown causes reduction in tumor cell proliferation and colony formation. Knockdown of caveolin-1 also led to radiosensitization of tumor cells and resulted in delayed DNA repair as demonstrated by delayed pH2.AX foci resolution, increased mitotic catastrophe, as well as decreased formation of BRCA1 and DNAPK-cs foci in the nuclei of these cells. Furthermore, Cav-1 loss promoted cleavage of Caspase-9 and PARP after treatment with gemcitabine. These results indicate that knocking down Cav-1 in pancreatic cancer cells sensitizes them to radiation with delayed DNA damage repair and also to chemotherapeutics with the onset of apoptosis. In vivo, stable knockdown of Cav-1 attenuated the rate of tumor growth significantly compared to control tumors in subcutaneous xenograft pancreatic tumors. In summary we conclude that Cav-1 is responsible in promoting resistance to radiation and chemotherapy in pancreatic cancer cells and that downregulation of this protein leads to improved therapeutic response. Thus, it is possible that caveolin-1 may serve as a prognostic predictive biomarker in pancreatic cancer patients for response to radiation and chemotherapy. Citation Format: Moumita Chatterjee, Terence M. Williams. Caveolin-1 confers a multi-modality therapy resistance phenotype in pancreatic cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4702. doi:10.1158/1538-7445.AM2014-4702

Highlights of This Issue

Espin (ESPN) is a multifunctional actin-bundling protein and is an important regulator of cytoskeleton dynamics. At present, there have been no reports showing the expression and function of Espin in cancers, including melanoma. Yanagishita and colleagues reveal that Espin is strongly expressed in human melanomas as well as in RET-transgenic mice. Moreover, depletion of Espin has a dramatic impact on the metastatic activity of melanoma cells in vitro and in vivo. These findings reveal Espin not only as a novel metastatic regulator in melanoma but also as a potential biomarker for disease progression.The p53 tumor suppressor family member, p63 (TP63), plays a pivotal role in epidermal development, aging, and tumorigenesis. In an effort to understand the underlying mechanism by which p63 expression is controlled, Xu and colleagues identify the RNA-binding protein RBM24 as a novel regulator of p63 status via mRNA stability. Specifically, overexpression of RBM24 decreases, whereas knockdown of RBM24 increases, p63 expression. Mechanistically, RBM24 is able to bind to multiple regions in the 3′UTR of p63 and destabilize the p63 transcript. Together, these observations provide significant insight into the understanding of how p63 expression is regulated through a posttranscriptional mechanism.Management of epithelial ovarian cancer (EOC) that harbors wild-type BRCA remains a major challenge due to acquired chemoresistance and poor overall survival. Development of mechanism-based and/or targeted combination therapy approaches is critically needed for improved clinical outcome. Lin and colleagues demonstrate that triapine, a small molecule inhibitor of ribonucleotide reductase, sensitizes BRCA wild-type EOC cells to PARP and topoisomerase II inhibitors. Mechanistic studies demonstrate that triapine impairs homologous recombination repair (HRR) by obliterating CtIP phosphorylation, DNA double-strand break (DSB) resection, BRCA1 and Rad51 foci formation, and endonuclease-induced DSB repair. These findings provide a strong rationale for targeting HRR and using triapine to augment the efficacy of PARP and topoisomerase inhibitors for the treatment of BRCA wild-type EOC.HER2/Neu (ERBB2) is a tyrosine kinase and is a major contributor to disease progression in various types of breast cancer. Consequently, its biomarker status is fundamentally important and its activity is a key target for therapeutics. Despite initial efficacy, HER2-targeted treatment approaches eventually result in resistance. Here, Bailey and colleagues report that NF-κB activity renders HER2-positive cancers resistant to HER2-directed therapy. Interestingly, Lapatinib-resistant cells had altered NF-κB and were more sensitive to simultaneous HER2 and NF-κB inhibitor treatment than either therapy alone. Through the use of genomic analyses, a NF-κB signature was deduced that correlated with clinical outcome. Thus, this study implicates NF-κB in HER2 therapy resistance and suggests that combinatorial NF-κB and HER2 inhibition has therapeutic benefit.

The CDK1 inhibitor RO3306 improves the response of BRCA-proficient breast cancer cells to PARP inhibition

Breast cancer is one of the most common malignancies in women. Approximately 15% of the patients belong to the triple-negative breast cancer (TNBC) group, and have the disadvantage of not benefiting from currently available receptor-targeted systemic therapies. Some cancers in the TNBC group harbor defects in DNA double-strand break repair by homologous recombination (HR), such as BRCA1 dysfunction, and are hypersensitive to poly (ADP-ribose) polymerase (PARP) inhibition. However, only a small fraction of the tumors are BRCA-deficient, and this restricts the therapeutic utility of the PARP inhibitor monotherapy. Cyclin-dependent kinase1 (CDK1) is necessary not only for BRCA1-mediated Sphase checkpoint activation, but also for HR, because it phosphorylates BRCA1 for the efficient formation of BRCA1 foci. In this study, we showed that the combined inhibition of CDK1 and PARP in BRCA-proficient MDA-MB-231 breast cancer cells resulted in dramatically reduced cell growth compared to PARP inhibition alone. Mechanistic investigations revealed that this sensitivity appears to be mediated by sustained DNA damage and inefficient DNA repair triggering mitochondrial-mediated apoptosis as well as autophagy. Our results suggest that CDK1 inhibition represents a plausible strategy for expanding the utility of PARP inhibitors to BRCA‑proficient breast cancers.

ATM–Dependent MiR-335 Targets CtIP and Modulates the DNA Damage Response

ATM plays a critical role in cellular responses to DNA double-strand breaks (DSBs). We describe a new ATM–mediated DSB–induced DNA damage response pathway involving microRNA (miRNA): irradiation (IR)-induced DSBs activate ATM, which leads to the downregulation of miR-335, a miRNA that targets CtIP, which is an important trigger of DNA end resection in homologous recombination repair (HRR). We demonstrate that CREB is responsible for a large portion of miR-335 expression by binding to the promoter region of miR-335. CREB binding is greatly reduced after IR, corroborating with previous studies that IR-activated ATM phosphorylates CREB to reduce its transcription activity. Overexpression of miR-335 in HeLa cells resulted in reduced CtIP levels and post-IR colony survival and BRCA1 foci formation. Further, in two patient-derived lymphoblastoid cell lines with decreased post-IR colony survival, a “radiosensitive” phenotype, we demonstrated elevated miR-335 expression, reduced CtIP levels, and reduced BRCA1 foci formation. Colony survival, BRCA1 foci, and CtIP levels were partially rescued by miRNA antisense AMO-miR-335 treatment. Taken together, these findings strongly suggest that an ATM–dependent CREB–miR-335–CtIP axis influences the selection of HRR for repair of certain DSB lesions.

Abstract 1583: Proteasome inhibition as a strategy for non-small cell lung carcinoma via inhibition of DNA double strand break repair.

Abstract Background: Each year, ∼60,000 US patients are diagnosed with locally advanced non-small cell lung carcinoma (NSCLC). Despite optimal radiation therapy (RT), chemotherapy +/- surgery, over 30% of patients fails locoregionally. The addition of concurrent chemotherapy to RT adds only ∼5% absolute survival benefit. Novel radiosensitizers may improve the therapeutic index. To identify the best potential targets, we performed whole genome RNAi screens that identified several proteasome subunits among top genes whose knockdown increased NSCLC cytotoxicity. Materials and Methods: Cytotoxicity was assessed by luminescent cell viability and clonogenic assays. Homologous recombination (HR) and non-homologous end-joining (NHEJ) were assessed using NSCLC cell lines transduced with reporter constructs that express GFP upon repair of I-SceI-induced DNA double strand breaks (DSBs). DNA damage-induced focus formation was assessed by immunofluorescence, scoring % of cells showing ≥5 foci. In vivo radiosensitization by proteasome gene knockdown was assessed using NCr nude mice injected with 1x106 NCI-H460 cells stably transfected with inducible PSMA1 shRNA. Once tumors reached ≥3 mm, knockdown was induced with doxycycline, and then one week later, RT to a dose of 20 Gy in 5 fractions was initiated. Results: Radiation and proteasome inhibition showed synergistic cytotoxicity. Irradiation of A549 cells with 1 Gy x 3 decreased clonogenic survival by 58% compared to control, while ionizing radiation (IR) plus 10 nM bortezomib decreased survival by 74% compared to bortezomib alone. Similar results were seen in NCI-H460. Proteasome inhibition via bortezomib or PSMA1 siRNA knockdown resulted in 80-90% decreased HR and NHEJ. Additionally, bortezomib or PSMA1 shRNA knockdown resulted in ≥50% decreases in BRCA1, FANCD2 or RAD51 IR-induced focus formation. Treatment of NCI-H460 xenografts with RT in the setting of PSMA1 knockdown showed marked improvements in survival; at 100 days post treatment initiation, only 20% of RT-only treated mice and 30% of doxycycline-treated mice (for PSMA1 shRNA knockdown in tumors) survived, compared to 100% following dual treatment (n = 10 per arm). There was a statistically significant difference in survival between mice with tumors treated with RT alone vs. RT + PSMA1 knockdown, with median survivals of 43 days vs. not reached, P = .0003 by the log-rank test. Conclusions: Proteasome inhibition has emerged as a promising target in NSCLC radiosensitization with evidence indicating the mechanism is through inhibition of DNA DSB repair pathways HR and NHEJ. Citation Format: Kyle R. Cron, Kaya Zhu, Deepa Kushwaha, Grace Hsieh, Dmitry Merzon, Jack Monahan, Clark C. Chen, Alan D'Andrea, David Kozono. Proteasome inhibition as a strategy for non-small cell lung carcinoma via inhibition of DNA double strand break repair. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1583. doi:10.1158/1538-7445.AM2013-1583

Roles of Kruppel-associated Box (KRAB)-associated Co-repressor KAP1 Ser-473 Phosphorylation in DNA Damage Response

The Kruppel-associated box (KRAB)-associated co-repressor KAP1 is an essential nuclear co-repressor for the KRAB zinc finger protein superfamily of transcriptional factors. Ataxia telangiectasia mutated (ATM)-Chk2 and ATM- and Rad3-related (ATR)-Chk1 are two primary kinase signaling cascades activated in response to DNA damage. A growing body of evidence suggests that ATM and ATR phosphorylate KAP1 at Ser-824 in response to DNA damage and regulate KAP1-dependent chromatin condensation, DNA repair, and gene expression. Here, we show that, depending on the type of DNA damage that occurs, KAP1 Ser-473 can be phosphorylated by ATM-Chk2 or ATR-Chk1 kinases. Phosphorylation of KAP1 at Ser-473 attenuated its binding to the heterochromatin protein 1 family proteins and inhibited its transcriptional repression of KRAB-zinc finger protein (KRAB-ZFP) target genes. Moreover, KAP1 Ser-473 phosphorylation induced by DNA damage stimulated KAP1-E2F1 binding. Overexpression of heterochromatin protein 1 significantly inhibited E2F1-KAP1 binding. Elimination of KAP1 Ser-473 phosphorylation increased E2F1-targeted proapoptotic gene expression and E2F1-induced apoptosis in response to DNA damage. Furthermore, loss of phosphorylation of KAP1 Ser-473 led to less BRCA1 focus formation and slower kinetics of loss of γH2AX foci after DNA damage. KAP1 Ser-473 phosphorylation was required for efficient DNA repair and cell survival in response to DNA damage. Our studies reveal novel functions of KAP1 Ser-473 phosphorylation under stress.

Basal-like Breast cancer DNA copy number losses identify genes involved in genomic instability, response to therapy, and patient survival

Breast cancer is a heterogeneous disease with known expression-defined tumor subtypes. DNA copy number studies have suggested that tumors within gene expression subtypes share similar DNA Copy number aberrations (CNA) and that CNA can be used to further sub-divide expression classes. To gain further insights into the etiologies of the intrinsic subtypes, we classified tumors according to gene expression subtype and next identified subtype-associated CNA using a novel method called SWITCHdna, using a training set of 180 tumors and a validation set of 359 tumors. Fisher’s exact tests, Chi-square approximations, and Wilcoxon rank-sum tests were performed to evaluate differences in CNA by subtype. To assess the functional significance of loss of a specific chromosomal region, individual genes were knocked down by shRNA and drug sensitivity, and DNA repair foci assays performed. Most tumor subtypes exhibited specific CNA. The Basal-like subtype was the most distinct with common losses of the regions containing RB1, BRCA1, INPP4B, and the greatest overall genomic instability. One Basal-like subtype-associated CNA was loss of 5q11–35, which contains at least three genes important for BRCA1-dependent DNA repair (RAD17, RAD50, and RAP80); these genes were predominantly lost as a pair, or all three simultaneously. Loss of two or three of these genes was associated with significantly increased genomic instability and poor patient survival. RNAi knockdown of RAD17, or RAD17/RAD50, in immortalized human mammary epithelial cell lines caused increased sensitivity to a PARP inhibitor and carboplatin, and inhibited BRCA1 foci formation in response to DNA damage. These data suggest a possible genetic cause for genomic instability in Basal-like breast cancers and a biological rationale for the use of DNA repair inhibitor related therapeutics in this breast cancer subtype.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-011-1846-y) contains supplementary material, which is available to authorized users.

Abstract 3708: Resveratrol induces DNA damage independent of Smad4 expression in its efficacy against human head and neck carcinoma cells

Abstract Resveratrol is a non-toxic natural product present in grape skin, red wine, peanuts etc. Extensive research in almost last two-decades has established that resveratrol possesses a broad range of health benefits including anti-cancer, anti-oxidant, anti aging and cardio protective properties; however, its efficacy and associated mechanisms is not examined in head and neck squamous cell carcinomas (HNSCC), which is the sixth most common human malignancy largely associated with tobacco and alcohol use. Molecular mechanistic studies have established that frequent alterations in Smad4 signaling pathway is common in HNSCC due to frequent mutations in TGF-β type II receptor. Smad4 loss has also been shown to cause genomic instability in mice and to induce HNSCC, suggesting that the agents are needed to control HNSCC that could target both Smad4 dependent and independent pathways. Accordingly, here we evaluated the efficacy of resveratrol against a panel of human HNSCC cell lines, namely FaDu (homologous deletion of Smad4), Cal27 (harbors nonsense mutation in Smad4), and Det562 (wild type Smad4). Resveratrol treatment at 5-50µM concentrations for 24 hrs significantly suppressed cell proliferation and induced DNA damage in FaDu and Cal27 cell lines; however, Det562 cells were more resistant to such treatments even at comparatively higher dose (100 µM). Cal27 cells transfected to Smad4 (Cal27-Smad4) showed similar resveratrol effects as parent line (Cal27) indicating that a lack of resveratrol effect in Det562 cells was independent of Smad4 status in these cells. Same resveratrol treatments also did not induce DNA damage in normal human epidermal keratinocytes and fibroblasts, showing its selective efficacy only in HNSCC cells. In other studies, resveratrol also caused a strong S phase arrest and induced apoptotic death of FaDu and Cal27 cells together with induction in Brca1 and H2AX nuclear foci formation. These finding for the first time established the efficacy of resveratrol in inducing DNA damage mediated cell death, which was independent of Smad4 expression in HNSCC. Further in an in vivo study, resveratrol (50 mg/kg body weight) treatment for 30 days, inhibited the tumor volume (48%; P<.01) and tumor weight (62%; p<.01) in ectopically growing human FaDu tumor xenografts in athymic nude mice. H2AX (ser 139) and cleaved caspase 3 expression was strongly increased in tumor xenograft from resveratrol-treated mice compared to controls. Together, these results clearly establish the anti-proliferative, DNA damaging and pro-apoptotic effects of resveratrol in HNSCC cells independent of Smad4 status, both in vitro and in vivo. Based on these findings, more studies are needed in future to evaluate both anti-cancer and chemopreventive efficacy of resveratrol in relevant pre-clinical models to establish its potential usefulness against human head and neck cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3708. doi:10.1158/1538-7445.AM2011-3708

Abstract 2979: Combined cdk1 and PARP inhibition results in tumor regression in a KrasG12D p53L/L murine lung adenocarcinoma model

Abstract Kras is one of the most commonly mutated oncogenes in non-small cell lung cancer (NSCLC), and targeted therapies are not available for this lung cancer subset. Consequently, there is an unmet need for improved therapies for patients with NSCLCs harboring Kras mutation. We previously demonstrated that cdk1 phosphorylates BRCA1, and is essential for efficient BRCA1 focus formation and activation of the S phase checkpoint in response to DNA damage in RAS mutant NSCLC cell lines (Johnson et al., Mol Cell 2009; 35: 327-39). PARP inhibition has been shown to selectively kill cancer cells that are unable to perform homologous recombination (HR)-mediated DNA repair. Furthermore, PARP inhibitors have provided clinical responses in patients with BRCA1 and BRCA2 mutations. We hypothesized that small molecule mediated cdk1 inhibition would disrupt BRCA1 function and sensitize BRCA-proficient, RAS mutant NSCLC cells to PARP inhibitors. Using a gene conversion assay to directly measure HR, in which GFP expression indicates the occurrence of HR repair, we demonstrated that the small molecule cdk1 inhibitors RO-3306 and AG024322 reduced GFP expression 7.7-fold (p = 0.013) and 3.4 fold (p = 0.016), respectively, compared to vehicle. In colony forming assays, RO-3306 and AG024322 reduced the LC50 value of the PARP inhibitor AG014699 6.6-fold and 18.7-fold in Nras mutant, TP53-deleted NCI-H1299 cells. Similar results were observed with Kras mutant A549 cells. In contrast to transformed cells, non-transformed Retinal Pigment Epithelial (RPE) cells were less sensitive to combined RO-3306 and AG014699 treatment. Similarly, when NCI-H1299 xenograft-bearing mice were treated with both AG024322 and AG014699, tumor growth was significantly delayed compared to vehicle exposure or treatment with either compound alone. We further assessed the therapeutic efficacy of combined AG024322 and AG014699 treatment in the KrasG12Dp53L/L murine lung adenocarcinoma model. Mice treated with vehicle, AG024322 or AG014699 alone all demonstrated tumor growth by MRI. In contrast, 87.5% (14/16) treated with combined AG024322 and AG014699 had up to 70% reduction in tumor volume. Furthermore, combined AG024322 and AG014699 treatment resulted in a marked reduction in Ki67 staining and increased TUNEL staining in residual tumor compared to vehicle or individual treatments. No toxicity or damage to normal mouse tissues and organs was found after combination treatment by pathologic assessment. This approach provides the potential to extend well-tolerated PARP inhibition to treatment for BRCA-proficient Kras mutant NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2979. doi:10.1158/1538-7445.AM2011-2979

Abstract 1406: Involvement of DNA repair pathways in DAG-lactone radiosensitization of human LNCaP cells

Abstract We previously demonstrated that pre-treatment of human androgen-dependent prostate cancer cell line LNCaP with either 12-0-tetradecanoylphorbol 13-acetate (TPA, a known protein kinase C activator), or diacylglycerol-lactone (DAG-lactone) radiosensitized these cells. This effect was mediated by down-regulation of ATM protein levels, thus de-repressing the enzyme ceramide synthase (CerS), ceramide generation and apoptosis. Here we assessed whether ATM down-regulation affected DNA repair pathways. LNCaP cells were pretreated for 16 hr with 10μM DAG-lactone and irradiated with 20 or 40Gy. Pre-treatment with DAG-lactone significantly enhanced apoptosis at 48 hours from 1.1% to 13.9% (20Gy) and 19.9% (40Gy) respectively. γ-H2AX and BRCA1 foci formation at 1 and 8 hours post-radiation were scored. Immunofluorescent staining for γ-H2AX demonstrated a dose-dependent increase in γ-H2AX foci formation at 20Gy and 40Gy for both 1hr and 8 hr time points. Our data also shows that 40Gy alone produces the same amount of DNA double strand breaks (dsbs; as measured by γ-H2AX at 8 hr) as 20Gy+DAG-lactone, yet 40Gy alone does not induce apoptosis in LNCaP cells, while 20Gy+DAG-lactone does, indicating that ATM down-regulation is necessary for the apoptotic response. Similarly there was a dose-dependent response in the generation of BRCA1 foci at 20Gy and 40 Gy at 8hr post-radiation. There was no difference in the BRCA1 foci between pre-treatment with DAG-lactone either at 20Gy or at 40 Gy at both 1 hr or at 8 hr post-radiation. While apoptotic degradation is associated with γ-H2AX phosphorylation, in LNCaP cells these events would be expected at 24-48 hr. Therefore the foci observed here are unlikely to be associated with apoptotic DNA degradation and most likely result from DNA dsbs. Our recent data showed that unrepaired DNA dsbs trigger post-transcriptional activation of CerS via a mechanism that remains unknown. Accordingly MRE 11, ATM or DNA-PKcs (SCID) deficiencies sensitize mice towards CerS-mediated apoptosis in crypt stem cells clonogens. Although these studies indicate that HR is active, further experiments need to be carried out to explore whether non-homologous end-joining (NHEJ) is or is not functional in LNCaP treated with DAG-lactone and radiation. Hence, the trigger for CerS activation still needs to be established. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1406.

Abstract 3882: Compromised Cdk1 activity sensitizes BRCA-proficient cancer cells to inhibition of Poly(ADP-ribose) polymerase

Abstract Poly(ADP-ribose) polymerase (PARP)-1 is a sensor of DNA nicks that signals the presence of DNA damage and facilitates repair. When PARP-1 is inhibited, single stranded DNA breaks (SSBs) degenerate to more lethal, double stranded DNA breaks (DSBs). BRCA1 and BRCA2 tumor suppressor proteins mediate the repair of DSBs by homologous recombination (HR). When these proteins are dysfunctional or mutated, cells become HR defective and are hypersensitive to PARP inhibition. However, BRCA-deficient tumors represent only a small fraction of adult cancers, potentially restricting the clinical utility of PARP inhibitor monotherapy. We previously showed that cdk1 phosphorylates BRCA1, an event essential for efficient BRCA1 focus formation and S phase checkpoint control in response to DNA damage (Johnson et al., Mol Cell 2009; 35: 327-39). Here, we show that shRNA-mediated depletion of cdk1 in the non-small cell lung cancer (NSCLC) cell line - NCI-H1299 compromises formation of BRCA1 foci, and consequently Rad51 foci, in response to treatment with the PARP inhibitor AG14361. PARP inhibitor treatment also resulted in increased numbers of chromosome breaks and G2/M accumulation in cdk1 depleted cells, but not in cells with normal cdk1 levels or cdk2 depleted control cells. Cdk1 depleted NCI-H1299 cells were 74-fold (p=0.004) more sensitive than cells with normal cdk1 levels to AG14361 treatment measured by colony formation (LC50 85 nM and 6300 nM, respectively). Similar results were obtained with A549 cells. In addition, cdk1 depleted NCI-H1299 cells demonstrated a substantial delay in tumor xenograft growth with PARP inhibitor treatment compared to cells expressing normal cdk1 levels. The effects of cdk1 depletion were reproduced by small molecule-mediated cdk1 inhibition. The cdk1 inhibitor RO3306 sensitized a range of BRCA1 proficient cancer cell types to PARP inhibitor treatment. In contrast, RO3306 did not sensitize the non-transformed Retinal Pigmented Epithelial (RPE) cells to PARP inhibition. Because RO3306 has poor pharmacokinetic properties, we combined cdk1 and PARP inhibition in vivo with a clinically relevant cdk1 inhibitor, AG024322. In colony forming assays, NCI-H1299 cells were 10-fold more sensitive to treatment with the PARP inhibitor AG014699 when co-treated with 50 nM AG024322. Similarly, when NCI-H1299 xenograft-bearing mice were treated with both compounds, tumor growth was significantly delayed compared to vehicle exposure or treatment with either compound alone. Because reduced cdk1 activity impairs BRCA1 function and HR repair, cdk1 inhibition represents a plausible strategy for expanding the therapeutic utility of PARP inhibitors to the BRCA-proficient cancer population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3882.

AKT1 represses gene conversion induced by different genotoxic stresses and induces supernumerary centrosomes and aneuploidy in hamster ovary cells

The oncogenic kinase AKT1 is frequently overexpressed or activated in sporadic breast and ovarian cancers. In human breast tumors, we have previously shown that AKT1 represses homologous recombination (HR) induced by one double-strand break (DSB). To further analyze the impact of AKT1 on HR, we ectopically expressed wild-type or mutant forms of AKT1 in a hamster ovary cell line containing an intrachromosomal substrate for monitoring HR. In this cell line, AKT1 repressed HR induced by different genotoxic stresses including ionizing radiation, UV-C and one single DSB introduced into the intrachromosomal substrate. Consistently, AKT1 disrupted RAD51 foci formation, showing that AKT1 specifically affects gene conversion. Concomitantly, AKT1 represses both BRCA1 foci formation and HR stimulation resulting from BRCA1 overexpression, showing that AKT1 affects BRCA1-mediated HR functions, also in another species (hamster) and in another type of cell tissue (ovary cells). Finally, consistent with the HR defects, active AKT1 expression induces supernumerary centrosomes and aneuploidy. In addition to its impact on cell proliferation and apoptosis, the present data propose a novel oncogenic function for AKT1, by producing genomic instability as a consequence of HR repression.