Bortezomib Enhances Melphalan Response by Altering Fanconi Anemia (FA)/BRCA Pathway Expression and Function.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 840-840 ◽  
Author(s):  
Danielle N. Yarde ◽  
Lori A. Hazlehurst ◽  
Vasco A. Oliveira ◽  
Qing Chen ◽  
William S. Dalton
Keyword(s):  
Gene Expression ◽  
Dna Damage ◽  
Dna Repair ◽  
Low Dose ◽  
Dna Damage Repair ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Multiple Myeloma Cell ◽  
Damage Repair ◽  
Pre Treatment

Abstract The FA/BRCA pathway is involved in DNA damage repair and its importance in oncogenesis has only recently been implicated. Briefly, 8 FA/BRCA pathway family members facilitate the monoubiquitination of FANCD2. Upon monoubiquitination, FANCD2 translocates to the DNA repair foci where it interacts with other proteins to initiate DNA repair. Previously, we reported that the FA/BRCA pathway is upregulated in multiple myeloma cell lines selected for resistance to melphalan (Chen, et al, Blood 2005). Further, reducing FANCF in the melphalan resistant 8226/LR5 myeloma cell line partially reversed resistance, whereas overexpressing FANCF in the drug sensitive 8226/S myeloma line conferred resistance to melphalan. Others have reported, and we have also verified, that bortezomib enhances melphalan response in myeloma cells; however, the mechanism of enhanced melphalan activity in combination with bortezomib has not been reported. Based on our observation that the FA/BRCA pathway confers melphalan resistance, we hypothesized that bortezomib enhances melphalan response by targeting FA/BRCA DNA damage repair pathway genes. To investigate this hypothesis, we first analyzed FA/BRCA gene expression in 8226/S and 8226/LR5 cells treated with bortezomib, using a customized microfluidic card (to detect BRCA1, BRCA2, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, RAD51 and RAD51C) and q-PCR. Interestingly, we found that low dose (5nM) bortezomib decreased many FA/BRCA pathway genes as early as 2 hours, with maximal decreases seen at 24 hours. Specifically, 1.5- to 2.5-fold decreases in FANCA, FANCC, FANCD2, FANCE and RAD51C were seen 24 hours post bortezomib exposure. Moreover, pre-treatment of myeloma cells with low dose bortezomib followed by melphalan treatment revealed a greater than 2-fold reduction in FANCD2 gene expression levels. We also found that melphalan treatment alone enhanced FANCD2 protein expression and activation (monoubiquitination), whereas the combination treatment of bortezomib followed by melphalan decreased activation and overall expression of FANCD2 protein. Taken together, these results suggest that bortezomib enhances melphalan response in myeloma by targeting the FA/BRCA pathway. Further understanding of the role of the FA/BRCA pathway in determining melphalan response may allow for more customized and effective treatment of myeloma.

STAG2 Loss Gives Rise to Therapeutically Targetable DNA Damage Repair Defects and Altered Replication Fork Dynamics in Acute Myeloid Leukaemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1255-1255
Author(s):  
Harmony Estelle Black ◽  
Satpal Jhujh ◽  
Grant S Stewart ◽  
Kienan I Savage ◽  
Ken I Mills
Keyword(s):  
Gene Expression ◽  
Dna Damage ◽  
Dna Repair ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Replication Fork ◽  
Dna Damage Repair ◽  
Replication Forks ◽  
Damage Repair ◽  
Acute Myeloid

Acute Myeloid Leukaemia (AML) is a genetically heterogeneous disease of the blood and bone marrow primarily affecting older populations. Chemotherapy has been the standard of care for four decades, just 10% of elderly patients show a response and it will be curative in only a third of patients aged 18-60. Therefore, it's necessary to identify new therapeutic targets for improved treatment options. The search for novel mutations within the genomes of karyotypically normal AML patients identified Cohesin's STAG2 as a recurrently mutated gene (6%). Cohesin is a ring-shaped multimeric protein complex which has a multitude of roles including maintaining sister chromatid cohesion, DNA replication and repair, gene expression and chromatin architecture. STAG2 remains the most understudied Cohesin gene, how a mutation affects the ability of Cohesin to fulfil certain roles needs elucidation as well as how STAG2 functions as part of Cohesin to repair DNA breaks, promote faithful replication and control gene expression. Therefore, we sought to explore the consequences of loss-of-function STAG2 mutations on DNA repair and replication using a STAG2 knockout (ΔSTAG2) isogenic cell line model created using CRISPR/Cas9. Immunofluorescent staining of DNA damage repair proteins 53BP1 and RAD51 following 2Gy irradiation (IR) suggests ΔSTAG2 cells have a significant DNA repair defect. The average 53BP1 and RAD51 foci counts were significantly higher (p=0.001) in mutant cells which had three times as many foci at 24 hours after IR. In addition, 40% of ΔSTAG2 cells stain positive for 53BP1 and RAD51 respectively at 24 hours post-IR. This damage persists at 48 and 72 hours, whereas wild type (WT) damage returns to basal levels. Given that our data suggests a HR defect, we assessed the response of WT and ΔSTAG2 cells to PARP inhibitors (PARPi). Interestingly, ΔSTAG2 cells showed significantly greater sensitivity than WT cells to highly potent PARPi Talazoparib with an IC50 of 62nM. We next assayed single DNA fibres to evaluate the implications of a STAG2 mutation on the dynamics of individual replication forks. Interestingly, we observed a significantly reduced number of ongoing replication forks compared to WT cells (p=0.01) with a concomitant increase in the number of stalled forks (p=0.01) in the absence of exogeneous DNA damage. Following treatment with hydroxyurea (HU) this phenotype was exacerbated and ΔSTAG2 cells exhibit significantly more stalled replication forks than WTs (p=0.001). This indicates a role for STAG2 in maintaining replication fork stability and promoting fork restart following replication stress. Importantly, our data identifies a potential DNA damage repair deficiency that sensitises ΔSTAG2 cells to PARPi monotherapy as well as unearthing an essential role for STAG2 in replication fork progression and fork stability. This mutation could therefore be exploited therapeutically and represents a novel therapeutic target/approach for AML patients with mutated STAG2. Disclosures No relevant conflicts of interest to declare.

scholarly journals MicroRNA-146a-5p enhances radiosensitivity in hepatocellular carcinoma through replication protein A3-induced activation of the DNA repair pathway

2019 ◽  
Vol 316 (3) ◽  
pp. C299-C311 ◽  
Author(s):  
Jing Luo ◽  
Zhong-Zhou Si ◽  
Ting Li ◽  
Jie-Qun Li ◽  
Zhong-Qiang Zhang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Repair ◽  
Replication Protein ◽  
Repair Pathway ◽  
Related Factors ◽  
Damage Repair ◽  
Hcc Cells

Hepatocellular carcinoma (HCC) is known for its high mortality rate worldwide. Based on intensive studies, microRNA (miRNA) expression functions in tumor suppression. Therefore, we aimed to evaluate the contribution of miR-146a-5p to radiosensitivity in HCC through the activation of the DNA damage repair pathway by binding to replication protein A3 (RPA3). First, the limma package of R was performed to differentially analyze HCC expression chip, and regulative miRNA of RPA3 was predicted. Expression of miR-146a-5p, RPA3, and DNA damage repair pathway-related factors in tissues and cells was determined. The effects of radiotherapy on the expression of miR-146a-5p and RPA3 as well as on cell radiosensitivity, proliferation, cell cycle, and apoptosis were also assessed. The results showed that there exists a close correlation between miR-146a and the radiotherapy effect on HCC progression through regulation of RPA3 and the DNA repair pathway. The positive rate of ATM, pCHK2, and Rad51 in HCC tissues was higher when compared with that of the paracancerous tissues. SMMC-7721 and HepG2 cell proliferation were significantly inhibited following 8 Gy 6Mv dose. MiR-146a-5p restrained the expression of RPA3 and promoted the expression of relative genes associated with the DNA repair pathway. In addition, miR-146a-5p overexpression suppresses cell proliferation and enhances radiosensitivity and cell apoptosis in HCC cells. In conclusion, the present study revealed that miR-146a-5p could lead to the restriction of proliferation and the promotion of radiosensitivity and apoptosis in HCC cells through activation of DNA repair pathway and inhibition of RPA3.

scholarly journals A Novel Role for Cathepsin S as a Potential Biomarker in Triple Negative Breast Cancer

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Richard D. A. Wilkinson ◽  
Roberta E. Burden ◽  
Sara H. McDowell ◽  
Darragh G. McArt ◽  
Stephen McQuaid ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Dna Damage ◽  
Adjuvant Therapy ◽  
Dna Damage Repair ◽  
Cathepsin S ◽  
Damage Repair ◽  
Potential Biomarker ◽  
Repair Pathways ◽  
Improved Outcome

Cathepsin S (CTSS) has previously been implicated in a number of cancer types, where it is associated with poor clinical features and outcome. To date, patient outcome in breast cancer has not been examined with respect to this protease. Here, we carried out immunohistochemical (IHC) staining of CTSS using a breast cancer tissue microarray in patients who received adjuvant therapy. We scored CTSS expression in the epithelial and stromal compartments and evaluated the association of CTSS expression with matched clinical outcome data. We observed differences in outcome based on CTSS expression, with stromal-derived CTSS expression correlating with a poor outcome and epithelial CTSS expression associated with an improved outcome. Further subtype characterisation revealed high epithelial CTSS expression in TNBC patients with improved outcome, which remained consistent across two independent TMA cohorts. Furtherin silicogene expression analysis, using both in-house and publicly available datasets, confirmed these observations and suggested high CTSS expression may also be beneficial to outcome in ER-/HER2+ cancer. Furthermore, high CTSS expression was associated with the BL1 Lehmann subgroup, which is characterised by defects in DNA damage repair pathways and correlates with improved outcome. Finally, analysis of matching IHC analysis reveals an increased M1 (tumour destructive) polarisation in macrophage in patients exhibiting high epithelial CTSS expression. In conclusion, our observations suggest epithelial CTSS expression may be prognostic of improved outcome in TNBC. Improved outcome observed with HER2+ at the gene expression level furthermore suggests CTSS may be prognostic of improved outcome in ER- cancers as a whole. Lastly, from the context of these patients receiving adjuvant therapy and as a result of its association with BL1 subgroup CTSS may be elevated in patients with defects in DNA damage repair pathways, indicating it may be predictive of tumour sensitivity to DNA damaging agents.

scholarly journals Changes in DNA Damage Repair Gene Expression and Cell Cycle Gene Expression Do Not Explain Radioresistance in Tamoxifen-Resistant Breast Cancer

2020 ◽  
Vol 28 (1) ◽  
pp. 33-40 ◽  
Author(s):  
Annemarie E. M. Post ◽  
Johan Bussink ◽  
Fred C. G. J. Sweep ◽  
Paul N. Span
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Damage Repair ◽  
Cross Resistance ◽  
Damage Repair ◽  
Breast Cancer Cohort ◽  
Tamoxifen Resistant ◽  
Repair Genes

Tamoxifen-induced radioresistance, reported in vitro, might pose a problem for patients who receive neoadjuvant tamoxifen treatment and subsequently receive radiotherapy after surgery. Previous studies suggested that DNA damage repair or cell cycle genes are involved, and could therefore be targeted to preclude the occurrence of cross-resistance. We aimed to characterize the observed cross-resistance by investigating gene expression of DNA damage repair genes and cell cycle genes in estrogen receptor-positive MCF-7 breast cancer cells that were cultured to tamoxifen resistance. RNA sequencing was performed, and expression of genes characteristic for several DNA damage repair pathways was investigated, as well as expression of genes involved in different phases of the cell cycle. The association of differentially expressed genes with outcome after radiotherapy was assessed in silico in a large breast cancer cohort. None of the DNA damage repair pathways showed differential gene expression in tamoxifen-resistant cells compared to wild-type cells. Two DNA damage repair genes were more than two times upregulated (NEIL1 and EME2), and three DNA damage repair genes were more than two times downregulated (PCNA, BRIP1, and BARD1). However, these were not associated with outcome after radiotherapy in the TCGA breast cancer cohort. Genes involved in G1, G1/S, G2, and G2/M phases were lower expressed in tamoxifen-resistant cells compared to wild-type cells. Individual genes that were more than two times upregulated (MAPK13) or downregulated (E2F2, CKS2, GINS2, PCNA, MCM5, and EIF5A2) were not associated with response to radiotherapy in the patient cohort investigated. We assessed the expression of DNA damage repair genes and cell cycle genes in tamoxifen-resistant breast cancer cells. Though several genes in both pathways were differentially expressed, these could not explain the cross-resistance for irradiation in these cells, since no association to response to radiotherapy in the TCGA breast cancer cohort was found.

ATM as a biomarker for DNA damage repair deficiency in pancreatic ductal adenocarcinoma.

2017 ◽  
Vol 35 (4_suppl) ◽  
pp. 308-308
Author(s):  
Talia Golan ◽  
Sharon Halparin ◽  
Chani Stossel ◽  
Maria Raitses-Gurevich ◽  
Dikla Atias ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Protein Expression ◽  
Dna Damage Repair ◽  
Personal History ◽  
Nuclear Staining ◽  
Repair Pathway ◽  
Damage Repair ◽  
History Of ◽  
Atm Protein

308 Background: Approximately 15% of PDAC tumors display DNA damage repair (DDR) deficiency. Germline BRCA (gBRCA) mutation serves as a robust biomarker for the DDR deficiency. A subset of patients displays a similar clinical phenotype but lack the gBRCA mutation. Identification of these BRCA-like subset of patients remains a challenge and an alternative approach may include DDR functional assays. Here we suggest loss of the ATM protein as one of the biomarkers for the identification of the DDR deficiency signature in PDAC. Methods: Patients were identified from the Sheba pancreatic cancer database based on strong family/personal history of BRCA- associated cancers or a durable response to platinum containing regimens ( ≥ 6 month) or harboring germline/somatic mutations in the DNA repair pathway (excluding gBRCA mutation). Archival FFPE blocks of primary tumors/metastatic lesions were used to explore ATM protein expression by IHC. Nuclear staining was regarded as positive. Tumor infiltrating lymphocytes served as an internal positive control. ATM loss was defined as less than10% neoplastic nuclear staining at any intensity in the presence of positive lymphocytes staining. Results: We identified 53 patients with DDR deficiency phenotype between 2014-2016 from the Sheba PDAC database (n = 250). Median age at diagnosis was 65 years (46-81) and the majority were female (62%). 47% were diagnosed at stage I/II and 53% stage IV. In the subgroup of patients with DDR deficiency phenotype, 55% displayed a family history of BRCA-associated cancers, 19% had a personal history of malignancy and23% had known mutation in DNA repair pathway. 23/53 identified subjects have been analyzed to date. We identified 52% loss of ATM in the analyzed group (n = 23). Conclusions: Loss of ATM in an unselected PDAC population is 12% (H. Kim et al, 2014). Our data demonstrate that 52% of the highly selected subgroup of PDAC patients (DDR deficiency phenotype) was found to have loss of ATM protein expression, suggesting it to be one of the biomarker for DDR signature. Identification of these patients, based on ATM protein expression profile may lead to personalized treatment options.

Retrospective analysis of patients using olaparib (O) in pancreatic cancer (PC).

2018 ◽  
Vol 36 (4_suppl) ◽  
pp. 389-389
Author(s):  
Erkut Hasan Borazanci ◽  
Carol Guarnieri ◽  
Susan Haag ◽  
Ronald Lee Korn ◽  
Courtney Edwards Snyder ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Dna Damage ◽  
Dna Repair ◽  
Retrospective Analysis ◽  
Pearson Correlation ◽  
Brca2 Mutation ◽  
Dna Damage Repair ◽  
Parp Inhibitors ◽  
Damage Repair ◽  
Repair Deficiency

389 Background: Molecular analysis has revealed four subtypes of PC giving clinicians further insight into treating this deadly disease. One subtype that was elucidated termed “unstable” is significant for the presence of DNA damage repair deficiency and can be targeted therapeutically. One such therapy, O, from the drug class poly ADP ribose polymerase (PARP) inhibitors, has already been FDA approved for individuals with BRCA mutated ovarian cancers. We performed a retrospective analysis on patients with PC treated at a single institution who have DNA damage repair deficiency mutations and have been treated with O. Methods: A chart review identified pancreatic cancer patients with DNA repair pathway mutations who were treated with O. The primary objective examined ORR in patients with PC with DNA repair mutations receiving O. Secondary objectives included tolerability, overall survival (OS), CA 19-9 change, and changes in quantitative textural analysis (QTA) on CT. Results: 11 individuals were identified, 5 carriers of a pathogenic germline (g) BRCA2 mutation, 1 carrier of a pathogenic g ATM mutation, 1 carrier of a pathogenic g BRCA1 mutation. Variants of uncertain significance (VUS) included 1 g ATM mutation, 1 g PALB2 mutation, 1 somatic (s) C11orf30 mutation, and 1 s BRCA2 mutation. Median age at diagnosis was 59, with 4 M and 7 F. No patients met criteria for familial PC and 7 had a family history consistent for breast and ovarian cancer syndrome. All individuals had metastatic PC and had progressed on at least 1 line of systemic therapy. ORR was 18%. Median time of therapy on O was 5 months (mo) (Range 1.4 to 29.567 mo) with 5 of the individuals still undergoing treatment at the time of analysis. Mean OS was 12.35 mo, 9 of the 11 individuals still alive. QTA of baseline CTs from subjects with liver (8/11) and pancreatic tumors (7/11) revealed a strong association between lesion texture and OS (Pearson correlation coefficient (PCC): hepatic mets = 0.952, p = 0.0003) and time on O (PCC: panc lesions = 0.889, p = 0.006). Conclusions: In individuals with metastatic PC with mutations involved in DNA repair, O may provide clinical benefit. QTA of individual tumors may allow for additional information that predicts outcomes to PARP inhibitors in this population.

Integrative analyses of signaling and DNA damage repair pathways in patient-derived xenograft (PDX) models from NCI’s patient-derived models repository (PDMR).

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. 3111-3111
Author(s):  
Biswajit Das ◽  
Yvonne A. Evrard ◽  
Li Chen ◽  
Rajesh Patidar ◽  
Tomas Vilimas ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Large Fraction ◽  
Dna Damage Repair ◽  
Damage Repair ◽  
Pathway Activation ◽  
Integrative Analyses ◽  
Multiple Data Sets ◽  
Repair Pathways ◽  
Pdx Models

3111 Background: Patient-derived xenografts (PDXs) are increasingly being used in translational cancer research for preclinical drug efficacy studies. The National Cancer Institute (NCI) has developed a Patient-Derived Models Repository (NCI PDMR; pdmr.cancer.gov ) of PDXs with clinical annotation, proteomics, and comprehensive genomic datasets to facilitate these studies. Here, we present an integrative genomic, transcriptomic, and proteomic analysis of critical signaling and DNA damage repair pathways in these PDX models, which represent 9 common and multiple rare tumor histologies. Methods: 304 PDX models from 294 patients were established from various solid tumor histologies from patients with primary or metastatic cancer. Whole Exome Sequencing, RNA-Seq and Reverse Phase Protein Array (RPPA) were performed on 2-9 PDXs per model across multiple passages. An integrative workflow was applied on multiple data sets to detect pathway activation. Results: We profiled 10 signaling and 5 DNA repair pathways in the PDMR dataset. We observed that: (i) a large fraction (40%) of PDX models have at least 1 targetable mutation in the RTK/RAS and/or PIK3CA pathways; (ii) 131 models (45%) have putative driver and oncogenic mutations and copy number variants (CNVs) in the WNT, TGFRb , NRF2 and NOTCH pathways. In addition, 17% of PDX models have targetable mutations in DNA damage repair pathways and 20 PDMR models have a DNA mismatch repair defect (MSI-H). We confirmed activation of the signaling pathways in a subset of PDX models by pathway enrichment analysis on gene expression data from RNASeq and phosphoprotein-specific antibody binding data from RPPA. Activation of DNA repair processes was confirmed by enrichment of relevant mutational signatures and loss of heterozygosity in these PDX models. Conclusions: Genomic analysis of NCI PDMR models revealed that a large fraction have clinically relevant somatic alterations in key signaling and DNA damage repair pathways. Further integrative analyses with matched transcriptomic and proteomic profiles confirmed pathway activation in a subset of these models, which may prioritize them for preclinical drug studies.

scholarly journals MicroRNAs in the DNA Damage/Repair Network and Cancer

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Alessandra Tessitore ◽  
Germana Cicciarelli ◽  
Filippo Del Vecchio ◽  
Agata Gaggiano ◽  
Daniela Verzella ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Repair ◽  
Transcriptional Level ◽  
Cancer Therapies ◽  
Induce Cell Cycle Arrest ◽  
Cycle Arrest ◽  
Damage Repair ◽  
Response Network ◽  
Multistep Process

Cancer is a multistep process characterized by various and different genetic lesions which cause the transformation of normal cells into tumor cells. To preserve the genomic integrity, eukaryotic cells need a complex DNA damage/repair response network of signaling pathways, involving many proteins, able to induce cell cycle arrest, apoptosis, or DNA repair. Chemotherapy and/or radiation therapy are the most commonly used therapeutic approaches to manage cancer and act mainly through the induction of DNA damage. Impairment in the DNA repair proteins, which physiologically protect cells from persistent DNA injury, can affect the efficacy of cancer therapies. Recently, increasing evidence has suggested that microRNAs take actively part in the regulation of the DNA damage/repair network. MicroRNAs are endogenous short noncoding molecules able to regulate gene expression at the post-transcriptional level. Due to their activity, microRNAs play a role in many fundamental physiological and pathological processes. In this review we report and discuss the role of microRNAs in the DNA damage/repair and cancer.

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