Regulation Of DNA Damage Repair Research Articles

ATM-dependent activation of SIM2s regulates homologous recombination and epithelial-mesenchymal transition.

There is increasing evidence that genomic instability is a prerequisite for cancer progression. Here we show that SIM2s, a member of the bHLH/PAS family of transcription factors, regulates DNA damage repair through enhancement of homologous recombination, and prevents epithelial mesenchymal transitions (EMT) in an ATM dependent manner. Mechanistically, we found that SIM2s interacts with ATM and is stabilized through ATM-dependent phosphorylation in response to ionizing radiation (IR). Once stabilized, SIM2s interacts with BRCA1 and supports RAD51 recruitment to the site of DNA damage. Loss of SIM2s through the introduction of shSIM2 or the mutation of SIM2s at one of the predicted ATM phosphorylation sites (S115) reduces homologous recombination efficiency through disruption of RAD51 recruitment, resulting in genomic instability and induction of EMT. The EMT induced by the mutation of S115 is characterized by a decrease in E-cadherin and an induction of the basal marker, K14, resulting in increased invasion and metastasis. Together, these results identify a novel player in the DNA damage repair pathway and provides a link in DCIS progression to IDC through loss of SIM2s, increased genomic instability, EMT and metastasis.

Abstract B095: An automated tissue staining and quantitative digital image analysis pipeline for quantification of DNA damage repair at the single-cell level

Abstract Background: The repair of double-stranded breaks in DNA occurs through homologous recombination (HR) or nonhomologous enjoining (NHEJ) pathways. Germline mutations in DNA damage repair enzymes reduce efficiency of DNA damage repair (DDR) and increase cancer risk, but also the sensitivity to DNA damaging agents, such as cisplatinum and PARP1 inhibitors. Because the regulation of DDR occurs through protein-protein interactions and post-translational modifications, genomic methods are insufficient to identify cancers with defective DDR pathways. The formation of DDR protein complexes underlies strict spatial regulation. Therefore, the measurement of DDR efficiency requires in situ detection of DDR foci by visualizing the location of multiple proteins within the nucleus. This can be accomplished by multiantibody immunofluorescent staining combined with digital image analysis. The main goal of the project is to develop tissue staining assays for protein complexes involved in DDR using 5 antibodies per slide and quantitative digital image analysis pipelines to integrate data from consecutive slides in order to establish a DDR index of a patient’s prostate cancer. Methods: We developed 2 fully automated 5-plex fluorescent (RPA32, RAD51, Ku80, XRCC4, pH2AX) (53BP1, pDNA-PK, PARP1, PAR, AR) antibody protocols on the Ventana Discovery autostainer. The fluorescent assays were combined with an automated cytokeratin (CK), Ki-67, and Geminin chromogenic staining protocol on the exact same tissue. The panels were applied to a TMA of high-grade prostate cancer and for comparison to a bladder and a rectal cancer TMA slide. Slides were imaged after the fluorescent portion of the assay was completed and subsequently processed for staining with CK (yellow) and Ki-67 (teal) and Geminin (brown). Chromogenic and fluorescent images were coregistered. To integrate images from adjacent slides, a transformation matrix was applied to distort the images in order to maximize the overlap of nuclear DAPI stains. The nuclear staining of each antibody was quantified through intensity, area, and texture measurements and compared in nuclei positive and negative for pH2AX. After histogram normalization and subtraction of background and autofluorescence, the amount of colocalized pixels between antibody pairs was determined. Results: We have demonstrated feasibility of automated multiplex staining protocols that increase the reproducibility and future clinical utility of DDR assays. Specific staining of individual antibodies was identified as a punctate nuclear staining pattern in a fraction of cancer cells, and multiple parameters of the staining pattern (intensity, stained area, subnuclear distribution) were quantified using digital image analysis. DDR occurred in high-grade and low-grade prostate cancer as determined by the percentage of cancer cells that stained positive for pH2AX. pH2AX staining was also noticed in normal epithelium (weakly proliferative), stroma (nonproliferative), and inflammatory cells (proliferative). At least 5 nuclei from each compartment (tumor, normal, stroma, and inflammation) per TMA core were selected for analyses based on pH2AX positivity. Using a consistent image analysis pipeline to set staining intensity thresholds, overlapping pixels between RPA32-RAD51, Ku80-XRCC4, 53BP1-pDNAPK, and the overlap of pixels of each protein complex with AR were determined. The intracase and intercase variance of DDR complexes was compared for each cell type (cancer, normal epithelium, stroma, and inflammation) and correlated with the amount of cell proliferation. Conclusion: We demonstrate feasibility of measuring DDR complexes in single nuclei. The assay has the potential to be used in a CLIA-certified laboratory and the quantitative measurements from the multiplex DDR assays can be compared to those from other OMICS platforms, such as mutation burden and copy number alterations. Funding: Precision Medicine initiative and Translational Research Core at Cedars-Sinai Medical Center. Citation Format: Joshua Saylor, Eric Weterings, Nathan Ellis, James Hinton, Esteban Roberts, Anne Cress, Beatrice Knudsen. An automated tissue staining and quantitative digital image analysis pipeline for quantification of DNA damage repair at the single-cell level [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B095.

USP14 regulates DNA damage repair by targeting RNF168-dependent ubiquitination

ABSTRACT Recent reports have made important revelations, uncovering direct regulation of DNA damage response (DDR)-associated proteins and chromatin ubiquitination (Ubn) by macroautophagy/autophagy. Here, we report a previously unexplored connection between autophagy and DDR, via a deubiquitnase (DUB), USP14. Loss of autophagy in prostate cancer cells led to unrepaired DNA double-strand breaks (DSBs) as indicated by persistent ionizing radiation (IR)-induced foci (IRIF) formation for γH2AFX, and decreased protein levels and IRIF formation for RNF168, an E3-ubiquitin ligase essential for chromatin Ubn and recruitment of critical DDR effector proteins in response to DSBs, including TP53BP1. Consistently, RNF168-associated Ubn signaling and TP53BP1 IRIF formation were reduced in autophagy-deficient cells. An activity assay identified several DUBs, including USP14, which showed higher activity in autophagy-deficient cells. Importantly, inhibiting USP14 could overcome DDR defects in autophagy-deficient cells. USP14 IRIF formation and protein stability were increased in autophagy-deficient cells. Co-immunoprecipitation and colocalization of USP14 with MAP1LC3B and the UBA-domain of SQSTM1 identified USP14 as a substrate of autophagy and SQSTM1. Additionally, USP14 directly interacted with RNF168, which depended on the MIU1 domain of RNF168. These findings identify USP14 as a novel substrate of autophagy and regulation of RNF168-dependent Ubn and TP53BP1 recruitment by USP14 as a critical link between DDR and autophagy. Given the role of Ubn signaling in non-homologous end joining (NHEJ), the major pathway for repair of IR-induced DNA damage, these findings provide unique insights into the link between autophagy, DDR-associated Ubn signaling and NHEJ DNA repair. Abbreviations: ATG7: autophagy related 7; CQ: chloroquine; DDR: DNA damage response; DUB: deubiquitinase; HR: homologous recombination; IR: ionizing radiation; IRIF: ionizing radiation-induced foci; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MIU1: motif interacting with ubiquitin; NHEJ: non homologous end-joining; PCa: prostate cancer; TP53BP1/53BP1: tumor protein p53 binding protein 1; RNF168: ring finger protein 168; SQSTM1/p62 sequestosome 1; γH2AFX/γH2AX: H2A histone family member X: phosphorylated, UBA: ubiquitin-associated; Ub: ubiquitin; Ubn: ubiquitination; USP14: ubiquitin specific peptidase 14.

Abstract 340: CTDP1 regulates FANCI activation and DNA repair

Abstract Objective: The modular BRCA1 C-terminal (BRCT) domain plays a central role in the regulation of DNA damage repair (DDR). RNA Pol II C-terminal domain phosphatase 1 (CTDP1), the only phosphatase encoding a BRCT domain, is one of the few BRCT domain-containing proteins without a formal role ascribed to DDR. Thus, the goal of this study is to characterize the DDR-specific role of CTDP1 through interrogation of the BRCT domain-protein interaction with FANCI and the repair of DNA interstrand cross-links (ICL) through the Fanconi anemia pathway. Methods: Novel CTDP1 BRCT domain-protein interactions were identified with mass spectrometry and analyzed with gene ontology enrichment. Molecular biology experiments were conducted to examine the effects of CTDP1 knockdown or overexpression on cell growth, survival, apoptosis, regulation of FANCI activation and localization, FANCD2 foci formation and ubiquitination, and homologous recombination repair. Mass spectrometry was used to determine both CTDP1-regulated FANCI phosphorylation sites and FANCI protein interactions. Results: The BRCT domain of CTDP1 was found to interact with three Fanconi anemia proteins-FANCA, FANCD2, and FANCI. This interaction remains unchanged upon mitomycin C treatment. Loss of CTDP1 induces a four-fold decrease in homologous recombination repair efficiency. CTDP1 prevents ICL-induced apoptosis. Knockdown of CTDP1 negatively affects plating efficiency and decreases the rate of cellular growth in breast cancer cell lines, but not the untransformed MCF-10A breast cell line. CTDP1 prevents sensitivity to DNA-damaging agents repaired by homologous recombination, but not UV damage or treatment with paclitaxel. CTDP1 knockdown negatively impacts FANCD2 activation measured by DNA damage-induced foci formation and monoubiquitination, which is critical for the onset of ICL repair by the ID2 complex and downstream effector proteins of the Fanconi anemia pathway. CTDP1 regulates 15 phosphorylation sites in FANCI determined by mass spectrometry profiling of cells overexpressing wild-type CTDP1 compared to phosphatase-dead D302K mutant. Paradoxically, CTDP1 phosphatase significantly increases SQ motif phosphorylation of S559 and S556, which is important for dormant origin firing and replication fork restart. This suggests that phosphorylation regulation of FANCI at non-SQ motif residues could regulate FANCI activation potential, resulting in increased phosphorylation at the SQ motif. Significance: This is the first demonstration of a formal role of CTDP1 in DDR. CTDP1 is implicated in the regulation of the specific ICL-repair Fanconi anemia pathway, and regulates breast cancer cell survival in vitro and in vivo. Delineating the mechanism by which CTDP1 regulates FANCI activation can provide the basis for preclinical studies targeting CTDP1 in breast cancer to promote sensitivity to DNA-damaging therapies. Citation Format: Kimiko L. Krieger, Wen-Feng Hu, Dragana Lagundzin, Nicholas T. Woods. CTDP1 regulates FANCI activation and DNA repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 340.

Abstract 4378: Novel CARP-1- H2AX interaction regulates apoptosis in breast cancer cells

Abstract CCAR1/CARP-1 is a peri-nuclear phosphoprotein that regulates apoptosis signaling by breast cancer therapeutic Doxorubicin and a novel class of CARP-1 Functional Mimetic (CFM) compounds. Prior reports have established that Doxorubicin exposure causes DNA damage in cells. Phosphorylation of histone 2AX (often referred as γH2AX) protein is involved in regulating DNA damage repair and apoptosis signaling. Although, currently it is unclear whether CFMs also induce DNA damage, we found that Doxorubicin or CFM-4.16 treatments caused elevated CARP-1 and γH2AX in HBC cells. In fact, a robust nuclear or peri-nuclear co-localization of CARP-1 and γH2AX occurred in HBC cells undergoing apoptosis. Knock-down of CARP-1 abolished increase of CARP-1 and γH2AX, their co-localization, and apoptosis in CFM-4.16 or Doxorubicin-treated HBC cells. We found that CARP-1 directly binds with H2AX, and the H2AX-interacting epitope is located within CARP-1 amino acids 600 to 652. We generated HBC cells that have stable expression of myc-tagged, wild-type CARP-1 (1-1150) or CARP-1 (1-1150; Δ600-652) mutant. Further analyses revealed that CARP-1 (1-1150), but not CARP-1 (1-1150; Δ600-652) mutant, interacted with H2AX. Moreover, HBC cells expressing CARP-1 (1-1150; Δ600-652) mutant were resistant to apoptosis by Doxorubicin or CFM-4.16, and had diminished levels of γH2AX, when compared with their counterparts expressing wild-type CARP-1. We have earlier reported involvement of the Stress-Activated Protein Kinases (SAPKs) such as p38 and JNKs in apoptosis by Doxorubicin or CFMs. Since the PI3K family of serine-threonine kinases ATM/ATR, as well as JNK SAPKs, phosphorylate serine-139 of H2AX following DNA damage, our current studies are investigating whether H2AX binding with CARP-1 serves to facilitate phosphorylation of CARP-1 and H2AX proteins for transduction of apoptosis signaling. Citation Format: Sreeja C. Sekhar, Vino T. Cheriyan, Arun K. Rishi. Novel CARP-1- H2AX interaction regulates apoptosis in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4378.

Abstract 4170: The RNA helicase, DDX3, modulates DNA damage repair in Ewing sarcoma

Abstract Introduction: Ewing sarcoma (ES) is a primary bone sarcoma occurring in adolescents and young adults, accounting for 15% of childhood/adolescent cancer. We previously reported that an RNA helicase, DDX3, regulates the translation of a variety of proteins in ES cells, including those modulating DNA damage repair. Therefore, we hypothesized that inhibiting DDX3, using a small molecule called RK-33, would radio-sensitize ES cells by preventing DNA damage repair. Methods: ES cell lines (MHH-ES and TC71) were transfected with DDX3- targeted shRNA or a control, scramble shRNA. RT-PCR and western blotting confirmed knockdown of DDX3. RK-33 cytotoxicity was evaluated using cell viability assay. DNA double strand breaks (DSB) were quantified by immunofluorescent staining for γH2AX, and subcellular localization of DDX3 was determined by immunofluorescence. ES patient derived xenografts (PDX) were subcutaneously implanted in 4 cohorts of NSG mice. Results: Expression of DDX3 mRNA and protein was decreased in DDX3-knockdown cells compared with controls. To validate that DDX3 is inhibited by RK-33, we compared the cytotoxicity of this agent in control cells and in the DDX3 knockdown cell lines. Parental ES and control cells (MHH-ES, TC71, MSD-10) were more sensitive to RK-33, with IC50 values that were significantly lower than their corresponding DDX3-knockdown lines (M2C7, M1F6; 4 μM, 2.7 μM and 2.6 μM vs 42 μM and 11.6 μM, respectively, p<0.05). To determine the effect on DNA damage repair, DSB were quantified in DDX3- knockdown cells and controls, treated with or without RK-33, at various time points after irradiation. At early time points, treatment with RK-33 had no impact on the number of ES and control cells (MHH-ES, TC71, MSD-10) with >10 γH2AX foci, but after 24 hours, the average number of irradiated cells with >10 foci was significantly higher in RK-33-treated cells than in cells treated with vehicle alone (p<0.01). The time needed for full recovery of radiation-induced DSB in untreated cells was less than 6 hr, while RK-33 pre-treated cells failed to recover even after 24 hr. Irradiated DDX3-knockdown cells demonstrated impaired DNA damage repair as well. To expand our investigation into the mechanism by which DDX3 regulates DNA damage repair, we studied the subcellular localization of the enzyme in response to radiation. We found that DDX3 translocates into the nucleus and colocalizes with γH2AX in irradiated cells, suggesting a dual role, direct and indirect, in DNA damage repair. In vivo, we found that animals treated with subtherapeutic external beam radiation concurrently with RK-33 had a substantial increase in tumor response. Our results suggest that DDX3 plays a crucial role in regulating DNA damage repair dynamics in ES, both indirectly by affecting the translation of involved proteins, and directly by localizing to sites of DSB. Therefore, inhibiting DDX3 using RK-33 may represent a novel approach to radiosensitization of cancer cells. Citation Format: Marwa Afifi, Breelyn A. Wilky, Catherine Kim, Venu Raman, David Loeb. The RNA helicase, DDX3, modulates DNA damage repair in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4170.

Regulation of DNA damage repair and lipid uptake by CX3CR1 in epithelial ovarian carcinoma

Failure of currently used cytotoxic chemotherapy is one of the main reasons behind high mortality from metastatic high grade serous ovarian carcinoma. We found that high expression of a receptor for fractalkine (CX3CR1) significantly correlated with shorter survival of patients with serous ovarian carcinoma treated with cytotoxic DNA damage chemotherapies, and reduction of CX3CR1 expression resulted in sensitization to several DNA damaging modalities, including x-ray radiation and cisplatin. Here, we show that CX3CR1 plays a role in double-strand DNA break response and repair by regulating expression of RAD50 by a MYC-dependent mechanism. We demonstrate that downregulation of CX3CR1 alone and in a combination with irradiation affects peritoneal metastasis in an organ-specific manner; we show that CX3CR1 regulates lipid uptake which could control omental metastasis. This study identifies CX3CR1 as a novel potential target for sensitization of ovarian carcinoma to DNA damage therapies and reduction of peritoneal carcinomatosis.

Fanconi Anemia FANCM/FNCM-1 and FANCD2/FCD-2 Are Required for Maintaining Histone Methylation Levels and Interact with the Histone Demethylase LSD1/SPR-5 in Caenorhabditis elegans.

The histone demethylase LSD1 was originally discovered by removing methyl groups from di- and monomethylated histone H3 lysine 4 (H3K4me2/1). Several studies suggest that LSD1 plays roles in meiosis as well as in the epigenetic regulation of fertility given that, in its absence, there is evidence of a progressive accumulation of H3K4me2 and increased sterility through generations. In addition to the progressive sterility phenotype observed in the mutants, growing evidence for the importance of histone methylation in the regulation of DNA damage repair has attracted more attention to the field in recent years. However, we are still far from understanding the mechanisms by which histone methylation is involved in DNA damage repair, and only a few studies have focused on the roles of histone demethylases in germline maintenance. Here, we show that the histone demethylase LSD1/CeSPR-5 interacts with the Fanconi anemia (FA) protein FANCM/CeFNCM-1 using biochemical, cytological, and genetic analyses. LSD1/CeSPR-5 is required for replication stress-induced S phase-checkpoint activation, and its absence suppresses the embryonic lethality and larval arrest observed in fncm-1 mutants. FANCM/CeFNCM-1 relocalizes upon hydroxyurea exposure and colocalizes with FANCD2/CeFCD-2 and LSD1/CeSPR-5, suggesting coordination between this histone demethylase and FA components to resolve replication stress. Surprisingly, the FA pathway is required for H3K4me2 maintenance, regardless of the presence of replication stress. Our study reveals a connection between FA and epigenetic maintenance and therefore provides new mechanistic insight into the regulation of histone methylation in DNA repair.

Wnt signaling induces radioresistance through upregulating HMGB1 in esophageal squamous cell carcinoma

Although many articles have uncovered that Wnt signaling is involved in radioresistance, the mechanism is rarely reported. Here we generated two radioresistant cells rECA109 and rKyse150 from parental esophageal cancer cells ECA109 and Kyse150. We then found that Wnt signaling activity was higher in radioresistant cells and was further activated upon ionizing radiation (IR) exposure. In addition, radioresistant cells acquired epithelial-to-mesenchymal transition (EMT) properties and stem quality. Wnt signaling was then found to be involved in radioresistance by promoting DNA damage repair. In our present study, high-mobility group box 1 protein (HMGB1), a chromatin-associated protein, was firstly found to be transactivated by Wnt signaling and mediate Wnt-induced radioresistance. The role of HMGB1 in the regulation of DNA damage repair with the activation of DNA damage checkpoint response in response to IR was the main cause of HMGB1-induced radioresistance.

The post-translational modification, SUMOylation, and cancer (Review).

SUMOylation is a reversible post-translational modification which has emerged as a crucial molecular regulatory mechanism, involved in the regulation of DNA damage repair, immune responses, carcinogenesis, cell cycle progression and apoptosis. Four SUMO isoforms have been identified, which are SUMO1, SUMO2/3 and SUMO4. The small ubiquitin-like modifier (SUMO) pathway is conserved in all eukaryotes and plays pivotal roles in the regulation of gene expression, cellular signaling and the maintenance of genomic integrity. The SUMO catalytic cycle includes maturation, activation, conjugation, ligation and de-modification. The dysregulation of the SUMO system is associated with a number of diseases, particularly cancer. SUMOylation is widely involved in carcinogenesis, DNA damage response, cancer cell proliferation, metastasis and apoptosis. SUMO can be used as a potential therapeutic target for cancer. In this review, we briefly outline the basic concepts of the SUMO system and summarize the involvement of SUMO proteins in cancer cells in order to better understand the role of SUMO in human disease.

The Yeast INO80 Complex Operates as a Tunable DNA Length-Sensitive Switch to Regulate Nucleosome Sliding

The yeast INO80 chromatin remodeling complex plays essential roles in regulating DNA damage repair, replication, and promoter architecture. INO80's role in these processes is likely related to its ability to slide nucleosomes, but the underlying mechanism is poorly understood. Here we use ensemble and single-molecule enzymology to study INO80-catalyzed nucleosome sliding. We find that the rate of nucleosome sliding by INO80 increases ∼100-fold when the flanking DNA length is increased from 40 to 60bp. Furthermore, once sliding is initiated, INO80 moves the nucleosome rapidly at least 20bp without pausing to re-assess flanking DNA length, and it can change the direction of nucleosome sliding without dissociation. Finally, we show that the Nhp10 module of INO80 plays an auto-inhibitory role, tuning INO80's switch-like response to flanking DNA. Our results indicate that INO80 is a highly processive remodeling motor that is tightly regulated by both substrate cues and non-catalytic subunits.

Regulation of Apoptosis and Radiation Sensitization in Lung Cancer Cells via the Sirt1/NF-κB/Smac Pathway

Background/Aims: SirT1, a conserved NAD⁺-dependent deacetylase, has been implicated in modulating cell survival and stress responses, and it appears to play an important role in tumorigenesis and cancer resistance to chemoradiotherapy. The mechanism of SirT1 in cancer chemoradiotherapy remains to be further elucidated, which could provide potential targets for cancer therapy. Methods: We performed colony formation, immunofluorescence microscopy, flow cytometry, RNA interference, and western blotting assays to determine whether SirT1 regulates radiation sensitization and which mechanisms and/or pathways it takes in lung cancer cell lines A549 and H460. Results: Initially, the expression of SirT1 was found to be negatively correlated with radiosensitivity in lung cancer cell lines A549 and H460. RNA interference with siSirT1 against SirT1 specifically reduced SirT1 expression and induced radiosensitivity both in A549 and H460 cell lines. In contrast, the radiosensitivity was significantly reduced once SirT1 was activated by resveratrol. Immunofluorescence assay and apoptosis analysis indicated that the effect of SirT1 on the radiosensitivity observed in the A549 and H460 cell lines was mainly achieved by regulating DNA damage repair and apoptosis processes. Furthermore, the expression of SirT1 negatively modulated the expression of apoptosis-related protein NF-κB and its downstream regulator of Smac. Conclusion: Our results indicate that SirT1 regulates apoptosis and radiation sensitization in lung cancer cell lines A549 and H460 via the SirT1/NF-κB/Smac pathway.

Abstract 3807: ATM protein loss and clinical outcomes with platinum chemotherapy in patients with advanced solid tumors

Abstract Background Loss of ataxia telangiectasia mutated (ATM), a key protein regulating DNA damage repair signaling, appears to sensitize tumors to platinum chemotherapy in preclinical studies. In this study, the incidence of ATM loss in patients with advanced solid tumors and clinical outcomes with platinum chemotherapy was analyzed. Methods Archival tumor and biopsy samples of primary or metastatic sites of patients treated in a phase I trial unit were retrieved and ATM immunohistochemistry (IHC), as well as targeted next generation sequencing (NGS) was performed. Analytic validation and assay optimization of rabbit monoclonal antibody [Y170] to ATM (Abcam ab32420) was undertaken using known positive (VCaP xenograft material and GM14680 cell pellet) and negative (GM01526 cell pellet) controls. IHC slides were assessed by a trained pathologist for nuclear staining intensity of ATM and semi-quantitatively scored. ATM loss was defined as a nuclear H score of ≤10. Clinical data were collected retrospectively from case records. Duration on platinum therapy was defined as time from start of first platinum therapy to time to subsequent therapy (adjuvant platinum excluded). Results Overall, 679 samples of 587 patients were tested for ATM IHC, of which 517 samples had NGS performed; 5% of patients (n = 31) had ATM IHC loss, and 5% (n = 27) had somatic ATM mutations detected by NGS (Table 1). In 70 matched primary/metastatic or different metastatic sites, concordance of ATM IHC loss occurred in 99% (n = 69; 7 with ATM IHC loss) of samples. Of the colorectal cancer cohort (n = 223), duration on platinum therapy was significantly longer for the ATM loss group (11 vs 8 months, HR: 0.57, 95% CI: 0.35 to 0.93, p = 0.04). Discussion Loss of ATM expression was detected among 5% of this cohort of advanced solid cancers, primarily in breast and gastrointestinal tumors. Sensitivity to platinum therapy is significantly higher in ATM loss colorectal cancer. Further prospective studies with larger cohorts are required to validate these findings. ATM IHC and ATM mutation by NGS by tumor groupTumor typesTotalATM IHC loss (%)ATM mutation (%)Concomitant ATM loss and ATM mutationConcomitant TP53 mutation with ATM IHC loss or mutationTotalMisense nFrameshift nTruncating n(as % of those with ATM IHC loss)(as % of those with ATM IHC loss/mutation)Colorectal22316(7)9(4)544 (25)10 (48)Gynaecological1051(1)7(7)4125 (63)Thoracic801(1)3(4)211 (100)2 (67)Breast448(18)4(9)223 (38)4 (44)Upper GI372(5)1(3)12 (67)Hepatobiliary221(5)1(5)11 (50)Others762(3)2(3)21 (50)Total58731(5)27(5)14 (52)3 (11)10 (37)9 (29)24 (49) Citation Format: Raghav Sundar, Susana Miranda, Suzanne Carreira, Maxime Chénard-Poirier, Daniel Nava Rodrigues, Ines Figueiredo, Claudia Bertan, Wei Yuan, Desamparados Roda Perez, Ana Ferreira, Nina Tunariu, Joaquin Mateo, Johann de Bono. ATM protein loss and clinical outcomes with platinum chemotherapy in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3807. doi:10.1158/1538-7445.AM2017-3807

The Role of the Complex USP1/WDR48 in Differentiation and Proliferation Processes in Cancer Stem Cells.

Recently, some studies identified the Basic-Helix-Loop-Helix (bHLH) transcription factor as a significant regulator for the evolution of neoplasms. The binding between bHLH proteins and DNA is restricted by heterodimerization with Inhibitors of DNA binding (ID). IDs prevent cellular differentiation, promote growth and sustain tumor development. The wide presence of stem cells in cancers suggests that genes ID are essential to cancer stem cells (CSC) progress. The enzyme Ubiquitin-specific protease 1 (USP1) is reported to deubiquitinate and stabilize IDs. Considering the action of the proteins ID, USP1 contributes to prevent differentiation mediated by bHLH and, consequently, keep CSC original characteristics. USP1 has its activity potentiated when bound to protein WD repeat-containing protein (WDR48). To identify the influence of the complex USP1/WDR48 during the CSC tumorigenesis process, and whether this complex is a possible therapeutic target. A literature search regarding the role of the complex USP1/WDR48 in inhibiting differentiation and increasing proliferation of CSC was performed, and possible selective molecule inhibitors of these deubiquitinase proteins were investigated. There is evidence that USP1/WDR48 complex promotes stem cell conservation and regulation of DNA damage repair. For this reason, inhibitors as Pimozide, GW7647, C527, SJB2-043, ML323 have been studied to inhibit USPs in cases of treatment intervention. It is consolidated in the literature the role of USP1/WDR48 during tumorigenesis. However, these studies are not enough to completely clarify the process; but certainly, the researchers are converging towards a promising direction to provide a new treatment option for cancer.

Targeting HDAC3, a new partner protein of AKT in the reversal of chemoresistance in acute myeloid leukemia via DNA damage response.

Resistance to cytotoxic chemotherapy drugs remains as the major cause of treatment failure in acute myeloid leukemia. Histone deacetylases (HDAC) are important regulators to maintain chromatin structure and control DNA damage; nevertheless, how each HDAC regulates genome stability remains unclear, especially under genome stress conditions. Here, we identified a mechanism by which HDAC3 regulates DNA damage repair and mediates resistance to chemotherapy drugs. In addition to inducing DNA damage, chemotherapy drugs trigger upregulation of HDAC3 expression in leukemia cells. Using genetic and pharmacological approaches, we show that HDAC3 contributes to chemotherapy resistance by regulating the activation of AKT, a well-documented factor in drug resistance development. HDAC3 binds to AKT and deacetylates it at the site Lys20, thereby promoting the phosphorylation of AKT. Chemotherapy drug exposure enhances the interaction between HDAC3 and AKT, resulting in decrease in AKT acetylation and increase in AKT phosphorylation. Whereas HDAC3 depletion or inhibition abrogates these responses and meanwhile sensitizes leukemia cells to chemotoxicity-induced apoptosis. Importantly, in vivo HDAC3 suppression reduces leukemia progression and sensitizes MLL-AF9+ leukemia to chemotherapy. Our findings suggest that combination therapy with HDAC3 inhibitor and genotoxic agents may constitute a successful strategy for overcoming chemotherapy resistance.

Abstract A23: BRCA1 mutations in the BRCT domain can be removed through alternative splicing and induce PARP inhibitor resistance

Abstract Introduction: The BRCA1 protein harbors two C-terminal tandem BRCT domains. The first is encoded by BRCA1 gene sequences located toward the end of exon 16, and the second BRCT domain initiated at exon 21. BRCA1 BRCT domains bind to proteins containing a phosphorylated serine-proline-x-phenylalanine (pSPxF) motif, including Abraxas, BACH1 and CtIP. Protein–protein interactions mediated by BRCT domains regulate DNA damage repair and cell cycle checkpoint signaling. Many cancer-associated mutations located in the BRCT domains of BRCA1 result in protein structural defects, and consequently misfolding and proteasomal degradation. Experimental Procedures: In this study, we examined PARP inhibitor (PARPi) resistance mechanisms utilizing MDA-MBA-436, HCC-1395 and SNU-251 human breast cancer and ovarian cancer cell lines that harbor frameshift mutations in BRCA1 exon 20, exon 20 and exon 23, respectively; predicted to generate BRCT domain disruptions. Cells were cultured in the presence of increasing concentrations of the PARPi rucaparib until resistant clones emerged. Quantitative RT-PCR and Western blotting were used to measure BRCA1 mRNA and protein levels. Results: Despite BRCA1 mRNA being abundant in all parental cell lines, BRCA1 protein was low or undetectable. However, PARPi resistant cell lines all demonstrated elevated BRCA1 protein levels that could be detected with N- but not C-terminal specific antibodies. Notably, the gel migration and molecular weight of BRCA1 proteins were markedly lower than the mutation-induced stop codon expected size. To identify potential protein products, we carried out immunoprecipitation and mass spectrometry to analyze BRCA1 peptide sequences. BRCA1 peptides encoded by exons 2-16 were readily detected; however, no peptides encoded by exon 17-24 were present in any cell line. qRT-PCR analyses suggested that BRCA1 mRNA was subject to alternative splicing and removal of exons 16-24. To understand why PARPi selection pressure generates BRCT-less BRCA1 proteins, we ectopically overexpressed BRCA1 cDNA that harbored stop codons located in BRCT domains or prior to the first BRCT domain. BRCT mutation containing constructs had undetectable protein levels, presumably due to protein misfolding, and cells were highly PARPi sensitive. In contrast, cells expressing BRCA1 constructs with stop codons prior to the BRCT domains had robust protein expression and demonstrated residual RAD51 foci and PARPi resistance. Conclusions: Our findings indicate that alternative splicing can remove deleterious mutations that disrupt BRCT peptide folding, generating more truncated but functional proteins capable of restoring residual DNA repair and PARPi resistance. Citation Format: Yifan Wang, Andrea J. Bernhardy, Neil Johnson. BRCA1 mutations in the BRCT domain can be removed through alternative splicing and induce PARP inhibitor resistance [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A23.

Autophagy Promotes the Repair of Radiation-Induced DNA Damage in Bone Marrow Hematopoietic Cells via Enhanced STAT3 Signaling.

Autophagy protects hematopoietic cells from radiation damage in part by promoting DNA damage repair. However, the molecular mechanisms by which autophagy regulates DNA damage repair remain largely elusive. Here, we report that this radioprotective effect of autophagy depends on STAT3 signaling in murine bone marrow mononuclear cells (BM-MNCs). Specifically, we found that STAT3 activation and nuclear translocation in BM-MNCs were increased by activation of autophagy with an mTOR inhibitor and decreased by knockout of the autophagy gene Atg7. The autophagic regulation of STAT3 activation is likely mediated by induction of KAP1 degradation, because we showed that KAP1 directly interacted with STAT3 in the cytoplasm and knockdown of KAP1 increased the phosphorylation and nuclear translocation of STAT3. Subsequently, activated STAT3 transcriptionally upregulated the expression of BRCA1, which increased the ability of BM-MNCs to repair radiation-induced DNA damage. This novel finding that activation of autophagy can promote DNA damage repair in BM-MNCs via the ATG-KAP1-STAT3-BRCA1 pathway suggests that autophagy plays an important role in maintaining genomic integrity of BM-MNCs and its activation may confer protection of BM-MNCs against radiation-induced genotoxic stress.

Interactome Analysis Reveals a Novel Role for RAD6 in the Regulation of Proteasome Activity and Localization in Response to DNA Damage.

RAD6, an E2 ubiquitin-conjugating enzyme, is a key node for determining different DNA damage repair pathways, controlling both the error-prone and the error-free DNA damage repair pathways through differential regulation of the ubiquitination of the proliferating cell nuclear antigen (PCNA) protein. However, whether other pathways are involved in the RAD6-mediated regulation of DNA damage repair is still unclear. To deeply understand the molecular mechanisms of RAD6 in DNA damage repair, we performed a proteomic analysis and identified the changes of the protein-protein interaction (PPI) networks of RAD6 before and after X-ray irradiation. Furthermore, our study indicated that a proteasome-related event is likely involved in the DNA damage repair process. Moreover, we found that RAD6 promotes proteasome activity and nuclear translocation by enhancing the degradation of PSMF1 and the lamin B receptor (LBR). Therefore, we provide a novel pathway that is employed by RAD6 in response to DNA damage.

INO80 represses osmostress induced gene expression by resetting promoter proximal nucleosomes.

The conserved INO80 chromatin remodeling complex is involved in regulation of DNA damage repair, replication and transcription. It is commonly recruited to the transcription start region and contributes to the establishment of promoter-proximal nucleosomes. We find a substantial influence of INO80 on nucleosome dynamics and gene expression during stress induced transcription. Transcription induced by osmotic stress leads to genome-wide remodeling of promoter proximal nucleosomes. INO80 function is required for timely return of evicted nucleosomes to the 5΄ end of induced genes. Reduced INO80 function in Arp8-deficient cells leads to correlated prolonged transcription and nucleosome eviction. INO80 and the related complex SWR1 regulate incorporation of the H2A.Z isoform at promoter proximal nucleosomes. However, H2A.Z seems not to influence osmotic stress induced gene regulation. Furthermore, we show that high rates of transcription promote INO80 recruitment to promoter regions, suggesting a connection between active transcription and promoter proximal nucleosome remodeling. In addition, we find that absence of INO80 enhances bidirectional promoter activity at highly induced genes and expression of a number of stress induced transcripts. We suggest that INO80 has a direct repressive role via promoter proximal nucleosome remodeling to limit high levels of transcription in yeast.

Alterations of BRCA1 and PALB2 Define a Novel Class of Complex-Karyotype AML with a Very Bad Prognosis

BRCA1 is one of the most important gene associated with familial breast and ovarian cancer susceptibility and is involved in the DNA damage repair and cell cycle arrest. Alterations in BRCA1 and their consequences are well characterized in breast and ovarian tumors, while little is known about its role in Acute Myeloid Leukemia (AML).We aimed to investigate the frequency and the interplay of BRCA1 alterations and patterns of somatic mutations (SNVs) and copy number variations (CNVs) of in AML patients.We genotyped 118 AML samples at either diagnosis or relapse by Single Nucleotide Polymorphism (SNP) array (SNP6.0 and Cytoscan HD, Affymetrix) to detect CNVs. In addition, we performed Whole-Exome Sequencing (WES, 100 bp paired-end, Illumina) of 56 genotyped AML patients to detect SNVs and small indels (MuTect and Varscan 2.0). Differences in survival were assessed using Kaplan-Meier survival analysis and Long-Rank test, or Breslow when indicated.We detected BRCA1 loss in 14 out of 118 patients (12%), ranging from 1,6 Kb to the loss of the entire chromosome 17, with none of the BRCA1 loss patients having a normal karyotype. Moreover, BRCA1 losses significantly co-occurred with PALB2 and RAD50 losses. Notably, the PALB2 loss (chromosome 16) mostly involved exon 12 and was detected also in patients without the BRCA1 loss, with a frequency of 11 out of 118 cases (9%), suggesting a pivotal and previously undescribed role in AML pathogenesis. BRCA1 loss patients were enriched for copy number alterations in genes involved in the regulation of mitotic recombination, DNA repair and positive regulation of kinase activity compared with BRCA1 wild-type cases (p <0.01). To investigate which genes and biological functions cooperates with the pathways affected by structural alterations, we integrated the analysis with WES data on 56 out of 118 AML patients. We focused on the genes involved in the DNA damage repair (DDR) and we identified somatic SNVs in BRCA1, TP53 and in the Fanconi Anemia genes BRCA2 and FANCE. Analysis of co-occurrence and mutual exclusivity revealed that BRCA1 loss and DDR-related alterations (BRCA1, PALB2, BRCA2, and FANCE) were significantly associated with TP53 alterations (p=0.0001 and p=0.0003, respectively). In addition, alterations in the DDR genes were mutually exclusive with mutations in KRAS, IDH1/2, BCOR, BCORL1, NPM1 and myeloid transcription factors as RUNX1 and CEBPA. Notably, patients without alteration in the DDR genes were enriched for mutations in DNMT3A and FLT3.Finally, BRCA1 and PALB2 alterations defined group of patients with poor overall survival (OS). In particular, patients with alterations of either BRCA1 or PALB2 had a worst prognosis compared to patients without alterations in the two genes (p=0.009 and p=0.001, respectively). Patients harboring PALB2 loss had a poorer prognosis compared with patients with complex karyotype (p=0.021, Breslow test). In addition, double BRCA1 and PALB2 loss, single BRAC1 or PALB2 hits and lack of alterations in BRCA1 and PALB2 defined three different classes of risks in our AML cohort (p<0.001).In summary, we define for the first time a novel subgroup of AML patients characterized by alterations targeting the DDR pathway, and in particular the BRCA1 and PALB2 genes. Our data suggest that a signature of genes involved in the DDR and cell cycle regulation synergize and led to the uncontrolled proliferation, independently of DNA damage accumulation. Our results may help improve patient stratification and define ad hoc therapeutic strategies for this aggressive leukemia type.Acknowledgments: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), Fondazione del Monte di Bologna e Ravenna, FP7 NGS-PTL project. [Display omitted] DisclosuresGuadagnuolo:CellPly S.r.l.: Employment. Martinelli:Novartis: Speakers Bureau; Genentech: Consultancy; Roche: Consultancy, Speakers Bureau; BMS: Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; MSD: Consultancy; Pfizer: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau.