Error-prone DNA Double-strand Break Research Articles

Non-homologous end joining shapes the genomic rearrangement landscape of chromothripsis from mitotic errors

Mitotic errors generate micronuclei entrapping mis-segregated chromosomes, which are susceptible to catastrophic fragmentation through chromothripsis. The reassembly of fragmented chromosomes by error-prone DNA double-strand break (DSB) repair generates diverse genomic rearrangements associated with human diseases. How specific repair pathways recognize and process these lesions remains poorly understood. Here we use CRISPR/Cas9 to systematically inactivate distinct DSB repair pathways and interrogate the rearrangement landscape of fragmented chromosomes. Deletion of canonical non-homologous end joining (NHEJ) components substantially reduces complex rearrangements and shifts the rearrangement landscape toward simple alterations without the characteristic patterns of chromothripsis. Following reincorporation into the nucleus, fragmented chromosomes localize within sub-nuclear micronuclei bodies (MN bodies) and undergo ligation by NHEJ within a single cell cycle. In the absence of NHEJ, chromosome fragments are rarely engaged by alternative end-joining or recombination-based mechanisms, resulting in delayed repair kinetics, persistent 53BP1-labeled MN bodies, and cell cycle arrest. Thus, we provide evidence supporting NHEJ as the exclusive DSB repair pathway generating complex rearrangements from mitotic errors.

Abstract B004: Polymerase theta (Pol θ) is essential to repair a subset of DNA breaks in HR proficient cells

Abstract DNA polymerase theta (Pol θ), functions in theta-mediated end joining (TMEJ), an error-prone DNA double strand break repair pathway. Pol θ is synthetic lethal with homologous recombination (HR) factors, and this finding has led to the development of Pol θ inhibitors and clinical trials to determine their effectiveness in HR-deficient tumors. Despite this, the biological functions of Pol q that promote genome integrity in normal cells are poorly understood. Here, we elucidate an essential role for Pol θ during mammalian interstrand crosslink (ICL) repair. We show that Pol θ is recruited downstream of ICL nucleolytic incision, BRCA2-dependent Rad51 loading, and FBXO5-mediated Rad51 ubiquitylation at sites of unsuccessful HR. Genomic scar analyses reveal that TMEJ is specifically required for repair of clustered ICLs, which are not amenable to HR-mediated repair. Thus, Pol q may be the predominant repair pathway for clustered lesions that generate two-ended double strand breaks during DNA replication. These findings suggest that Pol θ has essential roles in DNA repair in HR proficient cells, suggesting that Pol θ inhibitors may also be beneficial in the treatment of HR proficient tumors. Citation Format: Chelsea M. Smith, Dennis Simpson, Wanjuan Feng, Gaorav Gupta. Polymerase theta (Pol θ) is essential to repair a subset of DNA breaks in HR proficient cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B004.

Low CDK Activity and Enhanced Degradation by APC/CCDH1 Abolishes CtIP Activity and Alt-EJ in Quiescent Cells.

Alt-EJ is an error-prone DNA double-strand break (DSBs) repair pathway coming to the fore when first-line repair pathways, c-NHEJ and HR, are defective or fail. It is thought to benefit from DNA end-resection-a process whereby 3' single-stranded DNA-tails are generated-initiated by the CtIP/MRE11-RAD50-NBS1 (MRN) complex and extended by EXO1 or the BLM/DNA2 complex. The connection between alt-EJ and resection remains incompletely characterized. Alt-EJ depends on the cell cycle phase, is at maximum in G2-phase, substantially reduced in G1-phase and almost undetectable in quiescent, G0-phase cells. The mechanism underpinning this regulation remains uncharacterized. Here, we compare alt-EJ in G1- and G0-phase cells exposed to ionizing radiation (IR) and identify CtIP-dependent resection as the key regulator. Low levels of CtIP in G1-phase cells allow modest resection and alt-EJ, as compared to G2-phase cells. Strikingly, CtIP is undetectable in G0-phase cells owing to APC/C-mediated degradation. The suppression of CtIP degradation with bortezomib or CDH1-depletion rescues CtIP and alt-EJ in G0-phase cells. CtIP activation in G0-phase cells also requires CDK-dependent phosphorylation by any available CDK but is restricted to CDK4/6 at the early stages of the normal cell cycle. We suggest that suppression of mutagenic alt-EJ in G0-phase is a mechanism by which cells of higher eukaryotes maintain genomic stability in a large fraction of non-cycling cells in their organisms.

DNA Polymerase Theta Plays a Critical Role in Pancreatic Cancer Development and Metastasis.

Simple SummaryPancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers worldwide. The occurrence of oncogenic KRAS mutations is considered a signature event in PDAC, leading to genomic instability. The aim of our study was to evaluate the impact of the oncogenic KRAS G12D mutation on the activity of the error-prone alt-EJ repair mechanism, and to investigate the potential role of Polθ in the development of pancreatic cancer. We found that oncogenic KRAS increases the expression of key alt-EJ proteins in a mouse and human PDAC model. Using TLR assay, we also found increased alt-EJ activity in mouse and human cell lines upon the expression of KRAS D12D. The inactivation/impairment of alt-EJ by polymerase theta (Polθ) depletion delays the development of pancreatic cancer and prolongs the survival of experimental mice, though it does not prevent the PDAC development, which leads to full-blown PDAC with disseminated metastasis. Our studies provide a high-value target as a novel therapeutic candidate for the treatment of pancreatic and other cancers.Pancreatic ductal adenocarcinoma (PDAC), due to its genomic heterogeneity and lack of effective treatment, despite decades of intensive research, will become the second leading cause of cancer-related deaths by 2030. Step-wise acquisition of mutations, due to genomic instability, is considered to drive the development of PDAC; the KRAS mutation occurs in 95 to 100% of human PDAC, and is already detectable in early premalignant lesions designated as pancreatic intraepithelial neoplasia (PanIN). This mutation is possibly the key event leading to genomic instability and PDAC development. Our study aimed to investigate the role of the error-prone DNA double-strand breaks (DSBs) repair pathway, alt-EJ, in the presence of the KRAS G12D mutation in pancreatic cancer development. Our findings show that oncogenic KRAS contributes to increasing the expression of Polθ, Lig3, and Mre11, key components of alt-EJ in both mouse and human PDAC models. We further confirm increased catalytic activity of alt-EJ in a mouse and human model of PDAC bearing the KRAS G12D mutation. Subsequently, we focused on estimating the impact of alt-EJ inactivation by polymerase theta (Polθ) deletion on pancreatic cancer development, and survival in genetically engineered mouse models (GEMMs) and cancer patients. Here, we show that even though Polθ deficiency does not fully prevent the development of pancreatic cancer, it significantly delays the onset of PanIN formation, prolongs the overall survival of experimental mice, and correlates with the overall survival of pancreatic cancer patients in the TCGA database. Our study clearly demonstrates the role of alt-EJ in the development of PDAC, and alt-EJ may be an attractive therapeutic target for pancreatic cancer patients.

Age-related activity of Poly (ADP-Ribose) Polymerase (PARP) in men with localized prostate cancer

Mutations in DNA repair genes have been connected with familial prostate cancer and sensitivity to targeted drugs like PARP-inhibitors. Clinical use of this information is limited by the small fraction of prostate cancer risk gene carriers, variants of unknown pathogenicity and the focus on monogenic disease mechanisms. Functional assays capturing mono- and polygenic defects were shown to detect breast and ovarian cancer risk in blood-derived cells. Here, we comparatively analyzed lymphocytes from prostate cancer patients and controls applying a sensitive DNA double-strand break (DSB) repair assay and a flow cytometrybased assay measuring the activity of Poly(ADP-Ribose)-Polymerase, a target in treatment of metastatic prostate cancer. Contrary to breast and ovarian cancer patients, error-prone DNA double-strand break repair was not activated in prostate cancer patients. Yet, the activity of PARP discriminated between prostate cancer cases and controls. PARylation also correlated with the age of male probands, suggesting male-specific links between mutation-based and aging-associated DNA damage accumulation and PARP. Our work identifies prostate cancer-specific DNA repair phenotypes characterized by increased PARP activities and carboplatin-sensitivities, detected by functional testing of lymphocytes. This provides new insights for further investigation of PARP and carboplatin sensitivity as biomarkers in peripheral cells of men and prostate cancer patients.

Regulation of Error-Prone DNA Double-Strand Break Repair and Its Impact on Genome Evolution.

Double-strand breaks are one of the most deleterious DNA lesions. Their repair via error-prone mechanisms can promote mutagenesis, loss of genetic information, and deregulation of the genome. These detrimental outcomes are significant drivers of human diseases, including many cancers. Mutagenic double-strand break repair also facilitates heritable genetic changes that drive organismal adaptation and evolution. In this review, we discuss the mechanisms of various error-prone DNA double-strand break repair processes and the cellular conditions that regulate them, with a focus on alternative end joining. We provide examples that illustrate how mutagenic double-strand break repair drives genome diversity and evolution. Finally, we discuss how error-prone break repair can be crucial to the induction and progression of diseases such as cancer.

BRCA1 mislocalization leads to aberrant DNA damage response in heterozygous ABRAXAS1 mutation carrier cells.

Whilst heterozygous germline mutations in the ABRAXAS1 gene have been associated with a hereditary predisposition to breast cancer, their effect on promoting tumourigenesis at the cellular level has not been explored. Here, we demonstrate in patient-derived cells that the Finnish ABRAXAS1 founder mutation (c.1082G > A, Arg361Gln), even in the heterozygous state leads to decreased BRCA1 protein levels as well as reduced nuclear localization and foci formation of BRCA1 and CtIP. This causes disturbances in basal BRCA1-A complex localization, which is reflected by a restraint in error-prone DNA double-strand break repair pathway usage, attenuated DNA damage response and deregulated G2-M checkpoint control. The current study clearly demonstrates how the Finnish ABRAXAS1 founder mutation acts in a dominant-negative manner on BRCA1 to promote genome destabilization in heterozygous carrier cells.

An optimized homologous recombination deficiency (HRD) algorithm ASGAD for predicting PARP inhibitor response in ovarian cancer patient.

e17077 Background: Homologous recombination deficiency (HRD) results less efficient and error-prone DNA double strand break (DSB) repair, thus causes genomic in stability and impacts on cancer susceptibility to Poly‐(ADP‐Ribose)‐Polymerase (PARP) inhibitors. Evaluating HRD level in gynecologic cancer patients is becoming far more important and influential, so far, there is no standard method to be used in clinical. Methods: Here, we optimized an HRD score algorithm, termed as ASGAD, which combines three classic factors, including loss of heterozygosity score (LOH), telomeric allelic imbalance score (TAI), large-scale state transition score (LST), along with tumor ploidy to predict PARP inhibitor response in ovarian cancer patient. Results: Traditional global optimization strategy for purity and ploidy calculation is usually sensitive to initial values and may lead to incorrect convergences. Here we designed a two-step optimization manner to avoid this problem. Firstly, a density-based clustering algorithm was applied on BAF and CN, and the genotype was assigned to the most legible cluster. Then the rectified BAF and CN was calculated and used to find the maximum likelihood genotype of each segment. The segmentation processed was also improved by applying a series of statistical test to merge similar adjacent segments. The accuracy of allele-specific copy-number detection is significantly improved vie this algorithm, deriving stable and reliable HRD scores especially on aneuploid and hyperploid tumor cases. In this study, we assessed ASGAD algorithm in almost 150 ovarian cancer patient samples, who had treated with platinum effectively. BRCA1/2 deficient was defined as either one deleterious mutation in BRCA1/2, with LOH in the wild type copy or two deleterious mutations in the same gene. The results showed that the 19/23 BRCA1/2 deficient samples are also HRD-high, giving the sensitivity of 82.61%. Besides, we identify 58 HRD-high samples with intact BRCA1/2, who might benefit from PARP inhibitors, by the ASGAD algorithm. Interestingly, we analyzed 4 HRD-high samples by using whole-exome sequencing (WES), and found other HR genes mutations in these samples, including PARP4, FANCM, MSH2, MSH6, ATR, POLD1. Conclusions: The expanded use of PARP inhibitors in HRD tumors using the ASGAD algorithm requires more validation.

Abstract 339: Heterozygous germline mutation in ABRAXAS causes BRCA1 mislocalization and DNA damage response defects

Abstract Whilst heterozygous germline mutations in the ABRAXAS gene have been associated with hereditary breast cancer predisposition, their initial effect on promoting tumorigenesis at the cellular level has not been explored. Here, we demonstrate in patient-derived cells that the Finnish ABRAXAS founder mutation (c.1082G>A, Arg361Gln), even in the heterozygous state leads to decreased BRCA1 protein levels as well as reduced nuclear localization and foci formation of BRCA1 and CtIP. This causes disturbances in basal BRCA1-A complex localization, which is reflected by a restraint in error-prone DNA double-strand break (DSB) repair pathway usage, attenuated DNA damage response, deregulated G2-M checkpoint control and apoptosis. Most importantly, mutation carrier cells display a change in their transcriptional profile, which we attribute to the reduced nuclear levels of BRCA1. The current study clearly demonstrates how the Finnish ABRAXAS founder mutation acts in a dominant-negative manner on BRCA1 to promote genome destabilisation in heterozygous carrier cells. Citation Format: Robert Winqvist, Muthiah Bose, Juliane Sachsenweger, Niina Laurila, Ann Christin Parplys, Jonas Willmann, Leila Eshraghi, Thomas W. Dunlop, Marco Groth, Katrin Rapakko, Pentti Nieminen, Thomas W. Friedl, Lisa Heiserich, Felix Meyer, Hanna Tuppurainen, Ganapathy Raman Devarajan, Hellevi Peltoketo, Heli Nevanlinna, Katri Pylkäs, Kerstin Borgmann, Lisa Wiesmuller, Helmut Pospiech. Heterozygous germline mutation in ABRAXAS causes BRCA1 mislocalization and DNA damage response defects [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 339.

Abstract 4145: Concurrent treatment with Pim kinase inhibitor decreases alternative non-homologous end-joining repair of DNA damage induced by topoisomerase 2 inhibitors in cells with FLT3-ITD

Abstract Purpose: Internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD) is a common (30%) molecular abnormality in acute myeloid leukemia (AML), associated with a high relapse rate and short disease-free survival. The oncogenic kinase Pim-1 is upregulated downstream of FLT3-ITD and contributes to its proliferative and anti-apoptotic effects. We recently showed that Pim kinase inhibition sensitizes AML cells with FLT3-ITD to apoptosis induction by topoisomerase 2 (TOP-2) inhibitors by increasing DNA double-strand breaks (DSBs) and oxidative stress. Here, we studied whether altered DSB repair activity contributes to the increase in DNA damage. Methods: Ba/F3 cells expressing FLT3-ITD (Ba/F3-ITD) or wild-type FLT3 (Ba/F3-WT) were studied. The pan-Pim kinase inhibitor AZD1208 was provided by AstraZeneca. TOP-2 inhibitors included daunorubicin (DNR), mitoxantrone (MXR) and etoposide (VP-16). Nuclear expression of repair proteins was measured by immunoblotting. Functional changes in DSB repair activities were measured in cell lines with stable chromosomal integration of the green fluorescent protein (GFP)-based DSB repair reporters DR-GFP, EJ5-GFP and EJ2-GFP, measuring homologous recombination (HR), classical non-homologous end-joining (C-NHEJ) and alternative (Alt-) NHEJ, respectively. Results: Nuclear expression of the HR component Rad51 decreased progressively (to 40%) in Ba/F3-ITD cells after 12 hours of co-treatment with AZD1208 and DNR, compared to DNR alone. Progressive decrease (to 60%) in nuclear expression of the Alt-NHEJ component DNA ligase 3 and the C-NHEJ component Ku-70 was also seen with combination treatment. Functionally, co-treatment with AZD1208 and DNR led to a 50% decrease in Alt-NHEJ repair activity, compared to DNR alone, but no significant change in C-NHEJ or HR repair activity. Ba/F3-WT cells did not show a significant change in Alt-NHEJ, HR or C-NHEJ activity with combination treatment. Decreased Alt-NHEJ activity was also seen in Ba/F3-ITD, but not Ba/F3-WT, cells co-treated with AZD1208 and MXR or VP-16. Conclusions: While nuclear expression of multiple DNA DSB repair proteins decreased, only Alt-NHEJ repair activity decreased in FLT3-ITD cells co-treated with Pim kinase and TOP-2 inhibitors. Alt-NHEJ, a highly error-prone DNA DSB repair pathway, is upregulated in cells with FLT3-ITD and contributes to genomic instability in these cells. Decreasing Alt-NHEJ repair activity in cells treated with TOP-2 inhibitors has the potential to decrease the genomic instability that may contribute to disease progression in AML patients with FLT3-ITD. Citation Format: Kshama A. Doshi, Pratik K. Nagaria, Adriana E. Tron, Feyruz V. Rassool, Maria R. Baer. Concurrent treatment with Pim kinase inhibitor decreases alternative non-homologous end-joining repair of DNA damage induced by topoisomerase 2 inhibitors in cells with FLT3-ITD [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 4145. doi:10.1158/1538-7445.AM2017-4145

Modification of 3' Terminal Ends of DNA and RNA Using DNA Polymerase θ Terminal Transferase Activity.

DNA polymerase θ (Polθ) is a promiscuous enzyme that is essential for the error-prone DNA double-strand break (DSB) repair pathway called alternative end-joining (alt-EJ). During this form of DSB repair, Polθ performs terminal transferase activity at the 3' termini of resected DSBs via templated and non-templated nucleotide addition cycles. Since human Polθ is able to modify the 3' terminal ends of both DNA and RNA with a wide array of large and diverse ribonucleotide and deoxyribonucleotide analogs, its terminal transferase activity is more useful for biotechnology applications than terminal deoxynucleotidyl transferase (TdT). Here, we present in detail simple methods by which purified human Polθ is utilized to modify the 3' terminal ends of RNA and DNA for various applications in biotechnology and biomedical research.

In vitro model for DNA double-strand break repair analysis in breast cancer reveals cell type-specific associations with age and prognosis.

Dysfunction of homologous recombination is a common denominator of changes associated with breast cancer-predisposing mutations. In our previous work, we identified a functional signature in peripheral blood lymphocytes from women who were predisposed that indicated a shift from homologous recombination to alternative, error-prone DNA double-strand break (DSB) repair pathways. To capture both hereditary and nonhereditary factors, we newly established a protocol for isolation and ex vivo analysis of epithelial cells, epithelial-mesenchymal transition cells (EMTs), and fibroblasts from breast cancer specimens (147 patients). By applying a fluorescence-based test system, we analyzed the error-prone DSB repair pathway microhomology-mediated end joining in these tumor-derived cell types and peripheral blood lymphocytes. In parallel, we investigated DNA lesion processing by quantitative immunofluorescence microscopy of histone H2AX phosphorylated on Ser139 focus after radiomimetic treatment. Our study reveals elevated histone H2AX phosphorylated on Ser139 damage removal in epithelial cells, not EMTs, and poly(ADP-ribose)polymerase inhibitor sensitivities, which suggested a DSB repair pathway shift with increasing patient age. Of interest, we found elevated microhomology-mediated end joining in EMTs, not epithelial cells, from patients who received a treatment recommendation of adjuvant chemotherapy, that is, those with high-risk tumors. Our discoveries of altered DSB repair activities in cells may serve as a method to further classify breast cancer to predict responsiveness to adjuvant chemotherapy and/or therapeutics that target DSB repair-dysfunctional tumors.-Deniz, M., Kaufmann, J., Stahl, A., Gundelach, T., Janni, W., Hoffmann, I., Keimling, M., Hampp, S., Ihle, M., Wiesmüller, L. In vitro model for DNA double-strand break repair analysis in breast cancer reveals cell type-specific associations with age and prognosis.

End-processing nucleases and phosphodiesterases: An elite supporting cast for the non-homologous end joining pathway of DNA double-strand break repair.

Nonhomologous end joining (NHEJ) is an error-prone DNA double-strand break repair pathway that is active throughout the cell cycle. A substantial fraction of NHEJ repair events show deletions and, less often, insertions in the repair joints, suggesting an end-processing step comprising the removal of mismatched or damaged nucleotides by nucleases and other phosphodiesterases, as well as subsequent strand extension by polymerases. A wide range of nucleases, including Artemis, Metnase, APLF, Mre11, CtIP, APE1, APE2 and WRN, are biochemically competent to carry out such double-strand break end processing, and have been implicated in NHEJ by at least circumstantial evidence. Several additional DNA end-specific phosphodiesterases, including TDP1, TDP2 and aprataxin are available to resolve various non-nucleotide moieties at DSB ends. This review summarizes the biochemical specificities of these enzymes and the evidence for their participation in the NHEJ pathway.

Abstract 3629: Balance shift between error-free and error prone DNA double-strand break repair as a novel mechanism of radiosensitization by nucleoside analogs

Abstract Nucleoside analogs (NAs) represent a large group of anticancer drugs, several members of which have been shown to sensitize cells to ionizing radiation (IR). Among the lesions induced in cells by IR, the DNA double-strand break (DSB) is considered to be the most detrimental. Therefore we focused on interactions between NAs and pathways of DSB repair. We opted for post-irradiation treatment with NAs to avoid complications arising from redistribution of cells throughout the cycle prior to IR exposure. We compared the effects of 4h treatments post-IR with vidarabine (ara-A), fludarabine (F-ara-A) or gemcitabine (dFdC) on the survival of A549 lung carcinoma cells. We found that post-irradiation treatment with ara-A and F-ara-A, but not dFdC, resulted in cell radiosensitization to killing. Radiosensitization by ara-A was superior to that by F-ara-A. We employed immunofluorescence detection of IR-induced foci and I-SceI-based reporter cell lines to identify the pathways of DSB repair that are inhibited by ara-A and cause the observed radiosensitization. We found that the formation of IR-induced Rad51-foci was compromised by ara-A suggesting inhibition of homologous recombination repair (HRR). Repair of I-SceI-induced DSBs by HRR was also suppressed by ara-A. Furthermore, A549 cells deprived of Rad51 by RNAi could not be efficiently radiosensitized by ara-A. On the other hand, we could not detect impairment by ara-A of non-homologous end-joining (NHEJ), as measured by pulsed-field gel electrophoresis (PFGE) at high doses of IR (20 Gy). Notably, we observed an increase in the frequency of mutagenic NHEJ events in several I-SceI-based repair reporter systems, indicative of processing by error-prone repair pathways such as alternative/backup NHEJ (B-NHEJ). In addition, we could show that treatment with ara-A induces a dramatic increase in B-NHEJ in plateau phase Lig4-/- MEFs. Treatment with F-ara-A also inhibited HRR, but was less proficient in promoting mutagenic NHEJ. We conclude that inhibition of HRR and promotion of B-NHEJ represent two important mechanisms of radiosensitization by NAs. The strong impact of ara-A on both of these repair pathways underpins its superior radiosensitizing potential. Targeting of both HRR and B-NHEJ may provide a promising new strategy for radiosensitization. Citation Format: George Iliakis, Simon Magin. Balance shift between error-free and error prone DNA double-strand break repair as a novel mechanism of radiosensitization by nucleoside analogs. [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 3629. doi:10.1158/1538-7445.AM2015-3629

C-MYC and C-MYC-Regulated Micrornas Increase The Activity Of The Error-Prone ALT NHEJ Pathway Through Upregulation Of LIG3 and PARP1 In Tyrosine Kinase-Activated Leukemias

Constitutively activated tyrosine kinases (TK) BCR-ABL1 and FLT3/ITD not only increase cell survival and proliferation, but also increase levels of endogenous DNA damage and activity of an error-prone DNA double-strand break (DSB) repair pathway. This genomic instability leads to acquisition of genomic alterations that can result in disease progression and/or resistance to therapy. We have previously demonstrated that, in TK-activated leukemias, activity of the classic non homologous end-joining (C-NHEJ) pathway that repairs DSBs is decreased, and, as a consequence, an alternative, highly error-prone form of NHEJ (ALT NHEJ) predominates, evidenced by increased expression of DNA ligase IIIα (LIG3) and PARP1 (components of ALT NHEJ), increased frequency of large DNA deletions, and repair using DNA microhomologies. In this study, we sought to elucidate the role of a key downstream target of TKs, c-MYC, in upregulating LIG3 and PARP1 expression and consequently increasing ALT NHEJ and genomic instability. We demonstrated that MYC increases the expression of LIG3 and PARP1 through two mechanisms: 1) Increased binding to the promoters of LIG3 and PARP1, leading to increased transcription, and 2) Repression of microRNAs (miRs) that putatively regulate LIG3 and PARP1.Chemical and siRNA-mediated knockdown of MYC in MO7e-BCR/ABL and FLT3/ITD(+) MOLM14 cells results in significant reduction (p<0.05) in LIG3 and PARP1 mRNA and protein compared to controls. Chromatin immunoprecipitation assays revealed MYC binding to the promoters of LIG3 and PARP1 in AML (MOLM14) and CML (K562 and MO7e-BCR/ABL) cell lines. Additionally, transfection of PARP1 and LIG3 promoter-luciferase constructs into TK-activated (32D-FLT3/ITD, MO7e-BCR/ABL) cells showed significantly (p<0.01) increased LIG3 and PARP1 promoter activity compared to parental controls (32D, MO7e). Moreover, knockdown of MYC in 32D-FLT3/ITD and MO7e-BCR/ABL cells resulted in a significant reduction of promoter activity in luciferase assays (p<0.05). Conversely, overexpression of c-MYC in 293T cells caused an increase (p<0.05) in LIG3 and PARP1 promoter activity.We next determined whether MYC-repressed miRs that have predicted binding sites in the 3'-UTR and coding regions of LIG3 and PARP1 are involved in regulating expression of LIG3 and PARP1. We found that there was a significant inverse correlation between LIG3 expression and miR-22, miR-23a, and miR-150 (Pearson's r ≤ -0.3, p<0.05). Similarly, there was a significant inverse correlation between PARP1 expression and miR-22, miR-23a, miR-27a, and miR-150 (Pearson r ≤ -0.3, p<0.05). Over-expression of miR-22 in the CML cell line K562 decreased both LIG3 and PARP1 protein levels by 52% and 63% respectively. Similar results were seen upon over-expression of miR-34a (59% and 45%) and miR-150 (46% and 62%) for LIG3 and PARP1. This indicates that MYC-regulated miRs may function coordinately to regulate NHEJ repair. Importantly, our functional NHEJ assays demonstrate an overall significant (p<0.05) reduction in the average size of deletions at the sites of DSB repair when MYC is knocked down, indicating a reduction in ALT NHEJ activity.To determine whether increased expression of LIG3 and PARP1 correlated with MYC expression in primary leukemia samples, we examined mRNA levels from bone marrow of 21 CML patients (12 chronic phase, 1 accelerated, 7 blast crisis, and 1 unknown). Twelve patients were resistant to Imatinib, 7 were responsive, and 2 undetermined. There was a strong positive correlation between levels of MYC and PARP1 (Pearson's r= 0.75, p=0.001), as well as MYC and LIG3 (Pearson's r =0.45, p=0.03). While there was no correlation between levels of gene expression and disease phase, we found that the majority of samples with elevated levels of MYC, LIG3 and PARP1 were from Imatinib-resistant patients (64%), compared to samples from Imatinib-sensitive patients (36%) (p=0.03). Additionally, 2 patient samples with TKI-resistant T315I mutation in BCR-ABL1 exhibited elevated levels of MYC, LIG3 and PARP1. Thus, increased MYC expression, and repression of miRs 22, 150 and 34a augment expression of LIG3 and PARP1, generating DSB repair errors that may lead to resistance to TKI therapy. Altered expression of MYC, LIG3, PARP1 and miRs 22, 150 and 34a may be biomarkers for those patients likely to become resistant to TKI therapy. Disclosures:No relevant conflicts of interest to declare.

Two Mechanisms Produce Mutation Hotspots at DNA Breaks in Escherichia coli

SummaryMutation hotspots and showers occur across phylogeny and profoundly influence genome evolution, yet the mechanisms that produce hotspots remain obscure. We report that DNA double-strand breaks (DSBs) provoke mutation hotspots via stress-induced mutation in Escherichia coli. With tet reporters placed 2 kb to 2 Mb (half the genome) away from an I-SceI site, RpoS/DinB-dependent mutations occur maximally within the first 2 kb and decrease logarithmically to ∼60 kb. A weak mutation tail extends to 1 Mb. Hotspotting occurs independently of I-site/tet-reporter-pair position in the genome, upstream and downstream in the replication path. RecD, which allows RecBCD DSB-exonuclease activity, is required for strong local but not long-distance hotspotting, indicating that double-strand resection and gap-filling synthesis underlie local hotspotting, and newly illuminating DSB resection in vivo. Hotspotting near DSBs opens the possibility that specific genomic regions could be targeted for mutagenesis, and could also promote concerted evolution (coincident mutations) within genes/gene clusters, an important issue in the evolution of protein functions.

Mutability and importance of a hypermutable cell subpopulation that produces stress-induced mutants in Escherichia coli.

In bacterial, yeast, and human cells, stress-induced mutation mechanisms are induced in growth-limiting environments and produce non-adaptive and adaptive mutations. These mechanisms may accelerate evolution specifically when cells are maladapted to their environments, i.e., when they are are stressed. One mechanism of stress-induced mutagenesis in Escherichia coli occurs by error-prone DNA double-strand break (DSB) repair. This mechanism was linked previously to a differentiated subpopulation of cells with a transiently elevated mutation rate, a hypermutable cell subpopulation (HMS). The HMS could be important, producing essentially all stress-induced mutants. Alternatively, the HMS was proposed to produce only a minority of stress-induced mutants, i.e., it was proposed to be peripheral. We characterize three aspects of the HMS. First, using improved mutation-detection methods, we estimate the number of mutations per genome of HMS-derived cells and find that it is compatible with fitness after the HMS state. This implies that these mutants are not necessarily an evolutionary dead end, and could contribute to adaptive evolution. Second, we show that stress-induced Lac+ mutants, with and without evidence of descent from the HMS, have similar Lac+ mutation sequences. This provides evidence that HMS-descended and most stress-induced mutants form via a common mechanism. Third, mutation-stimulating DSBs introduced via I-SceI endonuclease in vivo do not promote Lac+ mutation independently of the HMS. This and the previous finding support the hypothesis that the HMS underlies most stress-induced mutants, not just a minority of them, i.e., it is important. We consider a model in which HMS differentiation is controlled by stress responses. Differentiation of an HMS potentially limits the risks of mutagenesis in cell clones.

A Switch from High-Fidelity to Error-Prone DNA Double-Strand Break Repair Underlies Stress-Induced Mutation

Special mechanisms of mutation are induced in microbes under growth-limiting stress causing genetic instability, including occasional adaptive mutations that may speed evolution. Both the mutation mechanisms and their control by stress have remained elusive. We provide evidence that the molecular basis for stress-induced mutagenesis in an E. coli model is error-prone DNA double-strand break repair (DSBR). I-SceI-endonuclease-induced DSBs strongly activate stress-induced mutations near the DSB, but not globally. The same proteins are required as for cells without induced DSBs: DSBR proteins, DinB-error-prone polymerase, and the RpoS starvation-stress-response regulator. Mutation is promoted by homology between cut and uncut DNA molecules, supporting a homology-mediated DSBR mechanism. DSBs also promote gene amplification. Finally, DSBs activate mutation only during stationary phase/starvation but will during exponential growth if RpoS is expressed. Our findings reveal an RpoS-controlled switch from high-fidelity to mutagenic DSBR under stress. This limits genetic instability both in time and to localized genome regions, potentially important evolutionary strategies.