Alternative Non-homologous End Joining Research Articles

The cGAS-Ku80 complex regulates the balance between two end joining subpathways.

As the major DNA sensor that activates the STING-TBK1 signaling cascade, cGAS is mainly present in the cytosol. A number of recent reports have indicated that cGAS also plays critical roles in the nucleus. Our previous work demonstrated for the first time that cGAS is translocated to the nucleus upon the occurrence of DNA damage and inhibits homologous recombination (HR), one of the two major pathways of DNA double strand break (DSB) repair. However, whether nuclear cGAS regulates the other DSB repair pathway, nonhomologous end joining (NHEJ), which can be further divided into the less error-prone canonical NHEJ (c-NHEJ) and more mutagenic alternative NHEJ (alt-NHEJ) subpathways, has not been characterized. Here, we demonstrated that cGAS tipped the balance of the two NHEJ subpathways toward c-NHEJ. Mechanistically, the cGAS-Ku80 complex enhanced the interaction between DNA-PKcs and the deubiquitinase USP7 to improve DNA-PKcs protein stability, thereby promoting c-NHEJ. In contrast, the cGAS-Ku80 complex suppressed alt-NHEJ by directly binding to the promoter of Polθ to suppress its transcription. Together, these findings reveal a novel function of nuclear cGAS in regulating DSB repair, suggesting that the presence of cGAS in the nucleus is also important in the maintenance of genome integrity.

The MRE11–ATM–SOG1 DNA damage signaling pathway confers rice immunity to Xanthomonas oryzae

Plants are constantly exposed to microbial pathogens in the environment. One branch of innate plant immunity is mediated by cell-membrane-localized receptors, but less is known about associations between DNA damage and plant immune responses. Here, we show that rice (Oryza sativa) mesophyll cells are prone to DNA double-stranded breaks (DSBs) in response to ZJ173, a strain of Xanthomonas oryzae pv. oryzae (Xoo). The DSB signal transducer ataxia telangiectasia mutated (ATM), but not the ATM and Rad3-related branch, confers resistance against Xoo. Mechanistically, the MRE11–ATM module phosphorylates suppressor of gamma response 1 (SOG1), which activates several phenylpropanoid pathway genes and prompts downstream phytoalexin biosynthesis during Xoo infection. Intriguingly, overexpression of the topoisomerase gene TOP6A3 causes a switch from the classic non-homologous end joining (NHEJ) pathway to the alternative NHEJ and homologous recombination pathways at Xoo-induced DSBs. The enhanced ATM signaling of the alternative NHEJ pathway strengthens the SOG1-regulated phenylpropanoid pathway and thereby boosts Xoo-induced phytoalexin biosynthesis in TOP6A3-OE1 overexpression lines. Overall, the MRE11–ATM–SOG1 pathway serves as a prime example of plant–pathogen interactions that occur via host non-specific recognition. The function of TOP6-facilitated ATM signaling in the defense response makes it a promising target for breeding of rice germplasm that exhibits resistance to bacterial blight disease without a growth penalty.

ZSCAN4 interacts with PARP1 to promote DNA repair in mouse embryonic stem cells

BackgroundIn eukaryotic cells, DNA double strand breaks (DSB) are primarily repaired by canonical non-homologous end joining (c-NHEJ), homologous recombination (HR) and alternative NHEJ (alt-NHEJ). Zinc finger and SCAN domain containing 4 (ZSCAN4), sporadically expressed in 1–5% mouse embryonic stem cells (mESCs), is known to regulate genome stability by promoting HR.ResultsHere we show that ZSCAN4 promotes DNA repair by acting with Poly (ADP-ribose) polymerase 1 (PARP1), which is a key member of the alt-NHEJ pathway. In the presence of PARP1, ZSCAN4-expressing mESCs are associated with lower extent of endogenous or chemical induced DSB comparing to ZSCAN4-negative ones. Reduced DSBs associated with ZSCAN4 are abolished by PARP1 inhibition, achieved either through small molecule inhibitor or gene knockout in mESCs. Furthermore, PARP1 binds directly to ZSCAN4, and the second ⍺-helix and the fourth zinc finger motif of ZSCAN4 are critical for this binding.ConclusionsThese data reveal that PARP1 and ZSCAN4 have a protein–protein interaction, and shed light on the molecular mechanisms by which ZSCAN4 reduces DSB in mESCs.

The RNA m5C modification in R-loops as an off switch of Alt-NHEJ

The roles of R-loops and RNA modifications in homologous recombination (HR) and other DNA double-stranded break (DSB) repair pathways remain poorly understood. Here, we find that DNA damage-induced RNA methyl-5-cytosine (m5C) modification in R-loops plays a crucial role to regulate PARP1-mediated poly ADP-ribosylation (PARylation) and the choice of DSB repair pathways at sites of R-loops. Through bisulfite sequencing, we discover that the methyltransferase TRDMT1 preferentially generates m5C after DNA damage in R-loops across the genome. In the absence of m5C, R-loops activate PARP1-mediated PARylation both in vitro and in cells. Concurrently, m5C promotes transcription-coupled HR (TC-HR) while suppressing PARP1-dependent alternative non-homologous end joining (Alt-NHEJ), favoring TC-HR over Alt-NHEJ in transcribed regions as the preferred repair pathway. Importantly, simultaneous disruption of both TC-HR and Alt-NHEJ with TRDMT1 and PARP or Polymerase θ inhibitors prevents alternative DSB repair and exhibits synergistic cytotoxic effects on cancer cells, suggesting an effective strategy to exploit genomic instability in cancer therapy.

Fisetin overcomes non-targetability of mutated KRAS induced YB-1 signaling in colorectal cancer cells and improves radiosensitivity by blocking repair of radiation-induced DNA double-strand breaks

Background and purposeKRAS is frequently mutated, and the Y-box binding protein 1 (YB-1) is overexpressed in colorectal cancer (CRC). Mutant KRAS (KRASmut) stimulates YB-1 through MAPK/RSK and PI3K/AKT, independent of epidermal growth factor receptor (EGFR). The p21-activated kinase (PAK) family is a switch-site upstream of AKT and RSK. The flavonoid compound fisetin inhibits RSK-mediated YB-1 signaling. We sought the most effective molecular targeting approach that interferes with DNA double strand break (DSB) repair and induces radiosensitivity of CRC cells, independent of KRAS mutation status. Materials and MethodsKRAS activity and KRAS mutation were analyzed by Ras-GTP assay and NGS. Effect of dual targeting of RSK and AKT (DT), the effect of fisetin as well as targeting PAK by FRAX486 and EGFR by erlotinib on YB-1 activity was tested by Western blotting after irradiation in vitro and ex vivo. Additionally, the effect of DT and FRAX486 on DSB repair pathways was tested in cells expressing reporter constructs for the DSB repair pathways by flow cytometry analysis. Residual DSBs and clonogenicity were examined by γH2AX- and clonogenic assays, respectively. ResultsErlotinib neither blocked DSB repair nor inhibited YB-1 phosphorylation under KRAS mutation condition in vitro and ex vivo. DT and FRAX486 effectively inhibited YB-1 phosphorylation independent of KRAS mutation status and diminished homologous recombination (HR) and alternative non-homologous end joining (NHEJ) repair. DT and FRAX486 inhibited DSB repair in CaCo2 but not in isogenic KRASG12V cells. Fisetin inhibited YB-1 phosphorylation, blocked DSB repair and increased radiosensitivity, independent of KRAS mutation status. ConclusionCombination of fisetin with radiotherapy may improve CRC radiation response, regardless of KRASmut status.

Dnmt3a Loss-of-Function Enhances DNA Repair in Proliferative HSCs Under Inflammatory Stress

Somatic mutations in DNMT3A can be enriched during clonal hematopoiesis by conditions with elevated type II interferon signaling. We have recently demonstrated that loss of functional Dnmt3a endows HSCs with a fitness advantage during chronic IFNg-driven inflammation. The increased fitness of Dnmt3a-mutant HSCs in these conditions can be partly attributed to the insensitivity to IFNg-primed apoptosis upon proliferative stress and delayed cell cycle activation from quiescence. However, it is unknown if these two mechanisms are related; if not, what is the underlying mechanism of inflammation-induced apoptotic resistance by Dnmt3a loss in HSCs? If the apoptotic resistance was merely due to delayed cell cycle activation from quiescence, proliferative activation should normalize the apoptotic levels of IFNg-exposed Dnmt3a-mutant HSCs. To test this, we induced HSCs into cell cycle by injecting 5-fluorouracil (5FU) into control (Vav-cre-; Dnmt3af/f) and mice lacking Dnmt3a in hematopoietic cells (3aKO, Vav-cre+; Dnmt3af/f). Kinetic analysis by BrdU labeling revealed that HSCs reached proliferation peak 8-days post-5FU treatment (>90% BrdU+). To capture the maximal number of proliferative HSCs, we injected PBS or recombinant mouse IFNg into 5FU-treated control and 3aKO mice on day 7 and 8. Phenotypic HSCs (Lin-cKit+EPCR+CD150+CD48-) were purified 2h after the second injection and analyzed for apoptosis. Cycling control HSCs were significantly more apoptotic upon IFNg exposure, while cell-cycle activated 3aKO HSCs remained insensitive to IFNg-primed apoptosis. Functional validation by colony formation assay showed IFNg-exposed cycling control HSCs formed significantly fewer colonies, while 3aKO HSCs gave rise to significantly more colonies regardless of exposure. These data suggest that Dnmt3a mutation may render HSCs resistant to IFNg-primed apoptosis via cell-cycle independent mechanisms. It has been documented that inflammation causes HSC apoptosis by inducing DNA damage through various mechanisms. Thus, the apoptotic resistance observed in Dnmt3a mutant HSCs could be due to 1) resistance to inflammation-induced DNA damage or 2) enhanced resolution of DNA damage. To test if Dnmt3a mutation renders HSCs resistant to IFNg-primed DNA damage, neutral comet assay was performed using PBS or IFNg-exposed quiescent HSCs. Interestingly, both genotypes showed increased DNA double-strand breaks (DSBs) upon IFNg exposure, suggesting that quiescent Dnmt3a-mutant HSCs are susceptible to IFNg-primed DSBs. DNA repair is one mechanism enabling DSB resolution, and is functionally active in proliferative HSCs. Thus, we performed neutral comet assay on cycling HSCs to quantify DSBs following PBS/IFNg exposure. Expectedly, IFNg significantly increased DSBs in cycling control but not in 3aKO HSCs, suggesting Dnmt3a loss may endow HSCs with augmented DNA repair. Similarly, when DNA damage was determined in control and Dnmt3a-knockout (3aKO) murine myeloblast cells (32D) 4h and 24h post-IFNg exposure, a comparable increase in DNA damage was observed 4h post-exposure in both genotypes. Interestingly, DNA damage levels were significantly reduced 24h later only in the 3aKO cells. This data paralleled HSC data, implying that Dnmt3a ablation facilitates augmented DNA repair. DSBs are repaired primarily via three universal yet distinct pathways, homologous recombination (HR), non-homologous end joining (NHEJ), and alternative NHEJ. To investigate how Dnmt3a loss facilitates enhanced DNA repair in response to IFNg exposure, we performed a DNA repair reporter assay in 32D genotypes 24h after IFNg exposure. The plasmid repair reporter assay was built on the detection of a green fluorescent protein (GFP) expression, which is otherwise disrupted by the insertion of I-SceI restriction enzyme recognition sites. After cutting with I-SceI, GFP can only be expressed after specific repair by one of the three pathways. Results showed Dnmt3a loss specifically enhanced the NHEJ pathways upon IFNg exposure, while other pathways were comparable between genotypes and treatments. In summary, our results demonstrated that Dnmt3a loss protects cell-cycle activated HSCs from inflammation-primed DSBs and apoptosis via augmented NHEJ as one mechanism. Future studies will investigate the molecular mechanisms underlying the enhanced NHEJ in Dnmt3a-mutant HSCs in response to chronic inflammation.

POLQ suppresses genome instability and alterations inDNA repeat tract lengths.

DNA polymerase theta (POLQ) is a principal component of the alternative non-homologous end-joining (ANHEJ) DNA repair pathway that ligates DNA double-strand breaks. Utilizing independent models of POLQ insufficiency during telomere-driven crisis, we found that POLQ–/– cells are resistant to crisis-induced growth deceleration despite sustaining inter-chromosomal telomere fusion frequencies equivalent to wild-type (WT) cells. We recorded longer telomeres in POLQ–/– than WT cells pre- and post-crisis, notwithstanding elevated total telomere erosion and fusion rates. POLQ–/– cells emerging from crisis exhibited reduced incidence of clonal gross chromosomal abnormalities in accordance with increased genetic heterogeneity. High-throughput sequencing of telomere fusion amplicons from POLQ-deficient cells revealed significantly raised frequencies of inter-chromosomal fusions with correspondingly depreciated intra-chromosomal recombinations. Long-range interactions culminating in telomere fusions with centromere alpha-satellite repeats, as well as expansions in HSAT2 and HSAT3 satellite and contractions in ribosomal DNA repeats, were detected in POLQ–/– cells. In conjunction with the expanded telomere lengths of POLQ–/– cells, these results indicate a hitherto unrealized capacity of POLQ for regulation of repeat arrays within the genome. Our findings uncover novel considerations for the efficacy of POLQ inhibitors in clinical cancer interventions, where potential genome destabilizing consequences could drive clonal evolution and resistant disease.

Switch Tandem Repeats Influence the Choice of the Alternative End-Joining Pathway in Immunoglobulin Class Switch Recombination.

Immunoglobulin class switch recombination (CSR) plays an important role in humoral imm\\une responses by changing the effector functions of antibodies. CSR occurs between highly repetitive switch (S) sequences located upstream of immunoglobulin constant gene exons. Switch sequences differ in size, the nature of their repeats, and the density of the motifs targeted by the activation-induced cytidine deaminase (AID), the enzyme that initiates CSR. CSR involves double-strand breaks (DSBs) at the universal Sµ donor region and one of the acceptor S regions. The DSBs ends are fused by the classical non-homologous end-joining (C-NHEJ) and the alternative-NHEJ (A-NHEJ) pathways. Of the two pathways, the A-NHEJ displays a bias towards longer junctional micro-homologies (MHs). The Sµ region displays features that distinguish it from other S regions, but the molecular basis of Sµ specificity is ill-understood. We used a mouse line in which the downstream Sγ3 region was put under the control of the Eµ enhancer, which regulates Sµ, and analyzed its recombination activity by CSR-HTGTS. Here, we show that provision of Eµ enhancer to Sγ3 is sufficient to confer the recombinational features of Sµ to Sγ3, including efficient AID recruitment, enhanced internal deletions and robust donor function in CSR. Moreover, junctions involving Sγ3 display a bias for longer MH irrespective of sequence homology with switch acceptor sites. The data suggest that the propensity for increased MH usage is an intrinsic property of Sγ3 sequence, and that the tandem repeats of the donor site influence the choice of the A-NHEJ.

Nonhomologous end joining as key to CRISPR/Cas-mediated plant chromosome engineering.

Although clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-mediated gene editing has revolutionized biology and plant breeding, large-scale, heritable restructuring of plant chromosomes is still in its infancy. Duplications and inversions within a chromosome, and also translocations between chromosomes, can now be achieved. Subsequently, genetic linkages can be broken or can be newly created. Also, the order of genes on a chromosome can be changed. While natural chromosomal recombination occurs by homologous recombination during meiosis, CRISPR/Cas-mediated chromosomal rearrangements can be obtained best by harnessing nonhomologous end joining (NHEJ) pathways in somatic cells. NHEJ can be subdivided into the classical (cNHEJ) and alternative NHEJ (aNHEJ) pathways, which partially operate antagonistically. The cNHEJ pathway not only protects broken DNA ends from degradation but also suppresses the joining of previously unlinked broken ends. Hence, in the absence of cNHEJ, more inversions or translocations can be obtained which can be ascribed to the unrestricted use of the aNHEJ pathway for double-strand break (DSB) repair. In contrast to inversions or translocations, short tandem duplications can be produced by paired single-strand breaks via a Cas9 nickase. Interestingly, the cNHEJ pathway is essential for these kinds of duplications, whereas aNHEJ is required for patch insertions that can also be formed during DSB repair. As chromosome engineering has not only been accomplished in the model plant Arabidopsis (Arabidopsis thaliana) but also in the crop maize (Zea mays), we expect that this technology will soon transform the breeding process.

The DNA-dependent protease AtWSS1A suppresses persistent double strand break formation during replication.

The protease WSS1A is an important factor in the repair of DNA-protein crosslinks in plants. Here we show that the loss of WSS1A leads to a reduction of 45S rDNA repeats and chromosomal fragmentation in Arabidopsis. Moreover, in the absence of any factor of the RTR (RECQ4A/TOP3α/RMI1/2) complex, which is involved in the dissolution of DNA replication intermediates, WSS1A becomes essential for viability. If WSS1A loss is combined with loss of the classical (c) or alternative (a) nonhomologous end joining (NHEJ) pathways of double-strand break (DSB) repair, the resulting mutants show proliferation defects and enhanced chromosome fragmentation, which is especially aggravated in the absence of aNHEJ. This indicates that WSS1A is involved either in the suppression of DSB formation or in DSB repair itself. To test the latter we induced DSB by CRISPR/Cas9 at different loci in wild-type and mutant cells and analyzed their repair by deep sequencing. However, no change in the quality of the repair events and only a slight increase in their quantity was found. Thus, by removing complex DNA-protein structures, WSS1A seems to be required for the repair of replication intermediates which would otherwise be resolved into persistent DSB leading to genome instability.

Topological Analysis of γH2AX and MRE11 Clusters Detected by Localization Microscopy during X-ray-Induced DNA Double-Strand Break Repair.

Simple SummarySuper-resolution single-molecule localization microscopy (SMLM) allows the detection of cluster formation of phosphorylation sites and recruited repair proteins (e.g., MRE11) around DNA double-strand breaks induced by ionizing radiation. The spatial arrangement of these targets can be described by distance frequency distributions, which can be used as a measure for cluster size and the ratio of cluster numbers per specimen analyzed. For the analysis of the shape of clusters and their topology and similarity, persistent homology was applied and the features were compared along the DNA repair process for two different cell lines (MCF-7 breast cancer cells and CCD-1059SK human skin fibroblasts). The similarity values for components and holes were averaged for each repair time point post-irradiation so that it was possible to compare not only clusters with each other but also cell specimens of different repair periods with each other. It was shown that after X-ray irradiation, these similarity values were high for γH2AX clusters in the early repair phase, while MRE11 clusters revealed an increased similarity at later time points. DNA double-strand breaks (DSBs), known as the most severe damage in chromatin, were induced in breast cancer cells and normal skin fibroblasts by 2 Gy ionizing photon radiation. In response to DSB induction, phosphorylation of the histone variant H2AX to γH2AX was observed in the form of foci visualized by specific antibodies. By means of super-resolution single-molecule localization microscopy (SMLM), it has been recently shown in a first article about these data that these foci can be separated into clusters of about the same size (diameter ~400 nm). The number of clusters increased with the dose applied and decreased with the repair time. It has also been shown that during the repair period, antibody-labeled MRE11 clusters of about half of the γH2AX cluster diameter were formed inside several γH2AX clusters. MRE11 is part of the MRE11–RAD50–NBS1 (MRN) complex, which is known as a DNA strand resection and broken-end bridging component in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). This article is a follow-up of the former ones applying novel procedures of mathematics (topology) and similarity measurements on the data set: to obtain a measure for cluster shape and shape similarities, topological quantifications employing persistent homology were calculated and compared. In addition, based on our findings that γH2AX clusters associated with heterochromatin show a high degree of similarity independently of dose and repair time, these earlier published topological analyses and similarity calculations comparing repair foci within individual cells were extended by topological data averaging (2nd-generation heatmaps) over all cells analyzed at a given repair time point; thereby, the two dimensions (0 and 1) expressed by components and holes were studied separately. Finally, these mean value heatmaps were averaged, in addition. For γH2AX clusters, in both normal fibroblast and MCF-7 cancer cell lines, an increased similarity was found at early time points (up to 60 min) after irradiation for both components and holes of clusters. In contrast, for MRE11, the peak in similarity was found at later time points (2 h up to 48 h) after irradiation. In general, the normal fibroblasts showed quicker phosphorylation of H2AX and recruitment of MRE11 to γH2AX clusters compared to breast cancer cells and a shorter time interval of increased similarity for γH2AX clusters. γH2AX foci and randomly distributed MRE11 molecules naturally occurring in non-irradiated control cells did not show any significant topological similarity.

Somatic Mutations in Oncogenes Are in Chronic Myeloid Leukemia Acquired De Novo via Deregulated Base-Excision Repair and Alternative Non-Homologous End Joining.

Somatic mutations are a common molecular mechanism through which chronic myeloid leukemia (CML) cells acquire resistance to tyrosine kinase inhibitors (TKIs) therapy. While most of the mutations in the kinase domain of BCR-ABL1 can be successfully managed, the recurrent somatic mutations in other genes may be therapeutically challenging. Despite the major clinical relevance of mutation-associated resistance in CML, the mechanisms underlying mutation acquisition in TKI-treated leukemic cells are not well understood. This work demonstrated de novo acquisition of mutations on isolated single-cell sorted CML clones growing in the presence of imatinib. The acquisition of mutations was associated with the significantly increased expression of the LIG1 and PARP1 genes involved in the error-prone alternative nonhomologous end-joining pathway, leading to genomic instability, and increased expression of the UNG, FEN and POLD3 genes involved in the base-excision repair (long patch) pathway, allowing point mutagenesis. This work showed in vitro and in vivo that de novo acquisition of resistance-associated mutations in oncogenes is the prevalent method of somatic mutation development in CML under TKIs treatment.

The repair of topoisomerase 2 cleavage complexes in Arabidopsis

DNA-protein crosslinks (DPCs) and DNA double-stranded breaks (DSBs), including those produced by stalled topoisomerase 2 cleavage complexes (TOP2ccs), must be repaired to ensure genome stability. The basic mechanisms of TOP2cc repair have been characterized in other eukaryotes, but we lack information for plants. Using CRISPR/Cas-induced mutants, we show that Arabidopsis thaliana has two main TOP2cc repair pathways: one is defined by TYROSYL-DNA-PHOSPHODIESTERASE 2 (TDP2), which hydrolyzes TOP2-DNA linkages, the other by the DNA-dependent protease WSS1A (a homolog of human SPARTAN/yeast weak suppressor of smt3 [Wss1]), which also functions in DPC repair. TDP1 and TDP2 function nonredundantly in TOP1cc repair, indicating that they act specifically on their respective stalled cleavage complexes. The nuclease METHYL METHANESULFONATE AND UV-SENSITIVE PROTEIN 81 (MUS81) plays a major role in global DPC repair and a minor role in TOP2cc repair. DSBs arise as intermediates of TOP2cc repair and are repaired by classical and alternative nonhomologous end joining (NHEJ) pathways. Double-mutant analysis indicates that "clean" DNA ends caused by TDP2 hydrolysis are mainly religated by classical NHEJ, which helps avoid mutation. In contrast, the mutagenic alternative NHEJ pathway mainly processes nonligateable DNA ends. Thus, TDP2 promotes maintenance of plant genome integrity by error-free repair of TOP2cc.

Pim kinase inhibitor co-treatment decreases alternative non-homologous end-joining DNA repair and genomic instability induced by topoisomerase 2 inhibitors in cells with FLT3 internal tandem duplication.

Acute myeloid leukemia (AML) with fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) relapses with new chromosome abnormalities following chemotherapy, implicating genomic instability. Error-prone alternative non-homologous end-joining (Alt-NHEJ) DNA double-strand break (DSB) repair is upregulated in FLT3-ITD-expresssing cells, driven by c-Myc. The serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD, and inhibiting Pim increases topoisomerase 2 (TOP2) inhibitor chemotherapy drug induction of DNA DSBs and apoptosis. We hypothesized that Pim inhibition increases DNA DSBs by downregulating Alt-NHEJ, also decreasing genomic instability. Alt-NHEJ activity, measured with a green fluorescent reporter construct, increased in FLT3-ITD-transfected Ba/F3-ITD cells treated with TOP2 inhibitors, and this increase was abrogated by Pim kinase inhibitor AZD1208 co-treatment. TOP2 inhibitor and AZD1208 co-treatment downregulated cellular and nuclear expression of c-Myc and Alt-NHEJ repair pathway proteins DNA polymerase θ, DNA ligase 3 and XRCC1 in FLT3-ITD cell lines and AML patient blasts. ALT-NHEJ protein downregulation was preceded by c-Myc downregulation, inhibited by c-Myc overexpression and induced by c-Myc knockdown or inhibition. TOP2 inhibitor treatment increased chromosome breaks in metaphase spreads in FLT3-ITD-expressing cells, and AZD1208 co-treatment abrogated these increases. Thus Pim kinase inhibitor co-treatment both enhances TOP2 inhibitor cytotoxicity and decreases TOP2 inhibitor-induced genomic instability in cells with FLT3-ITD.

TET2 and DNMT3A Mutations Exert Divergent Effects on DNA Repair and Sensitivity of Leukemia Cells to PARP Inhibitors.

Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that affect the epigenetic regulation of DNA methylation. Mutations in both genes often co-occur with activating mutations in genes encoding oncogenic tyrosine kinases such as FLT3ITD, BCR-ABL1, JAK2V617F , and MPLW515L , or with mutations affecting related signaling pathways such as NRASG12D and CALRdel52 . Here, we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing these oncogenes. Malignant TET2-deficient cells displayed downregulation of BRCA1 and LIG4, resulting in reduced activity of BRCA1/2-mediated homologous recombination (HR) and DNA-PK-mediated non-homologous end-joining (D-NHEJ), respectively. TET2-deficient cells relied on PARP1-mediated alternative NHEJ (Alt-NHEJ) for protection from the toxic effects of spontaneous and drug-induced DNA double-strand breaks. Conversely, DNMT3A-deficient cells favored HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells were sensitive to PARP inhibitor (PARPi) treatment in vitro and in vivo, whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity or TET2-Wilms' tumor 1 (WT1)-binding ability was responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of WT1 mimicked the effect of TET2 mutation on DSB repair activity and sensitivity to PARPi. Collectively, these findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically. SIGNIFICANCE: TET2 and DNMT3A mutations affect distinct DNA repair mechanisms and govern the differential sensitivities of oncogenic tyrosine kinase-positive malignant hematopoietic cells to PARP inhibitors.

Concurrent Treatment with Pim Kinase Inhibitor Downregulates Alternative Non-Homologous End-Joining Repair and Decreases Genomic Instability in FLT3-ITD Cells Treated with Topoisomerase 2 Inhibitor Chemotherapy Drugs

Internal tandem duplication of fms -like tyrosine like kinase 3 (FLT3-ITD) is present in 30% of acute myeloid leukemia (AML) cases, causing constitutive FLT3 signaling. While FLT3-ITD AML patients generally achieve remission following combination chemotherapy including cytarabine and a topoisomerase (topo) 2 inhibitor, they relapse rapidly, and FLT3-ITD AML cells frequently have new structural chromosome changes at relapse. Genomic instability in FLT3-ITD AML appears to result from increased generation of reactive oxygen species, which cause DNA double-strand breaks (DSBs), and upregulated DNA DSB repair by an error-prone alternative non-homologous end-joining (Alt-NHEJ) pathway.Pim-1 is an oncogenic serine/threonine kinase that is transcriptionally upregulated downstream of FLT3-ITD, contributes directly to the proliferative and anti-apoptotic effects of FLT3-ITD, and also phosphorylates and stabilizes FLT3, creating a positive feedback loop in FLT3-ITD cells. Pim kinase inhibitors are in clinical trials. We previously showed that concurrent treatment with a Pim kinase inhibitor enhances apoptosis induction by topo 2 inhibitors in FLT3-ITD cells. Here, we tested the effect of concurrent Pim kinase inhibitor treatment on Alt-NHEJ repair of topo 2 inhibitor-induced DNA DSBs and on genomic instability.Functional changes in DSB repair activities induced by treatment with the topo 2 inhibitor daunorubicin (DNR) in the absence and presence of the pan-Pim kinase inhibitor AZD1208 were studied in Ba/F3-ITD cells, with FLT3-ITD, using stably integrated green fluorescent protein (GFP)-based DSB repair reporter constructs DR-GFP, EJ5-GFP and EJ2-GFP, measuring homologous recombination (HR), classical (C-)NHEJ and Alt-NHEJ, respectively. DNR, which induced DNA DSBs, did not induce HR or C-NHEJ repair activity, but induced a 50% increase in Alt-NHEJ activity at 36 hours. Concurrent treatment with AZD1208, which increased DNR-induced DNA DSBs more than two-fold, completely abrogated the DNR-induced increase in Alt-NHEJ repair activity. Treatment of Ba/F3-ITD cells with the topo 2 inhibitors mitoxantrone (MXR) or etoposide (VP-16) also induced Alt-NHEJ repair activity, which was abrogated by concurrent treatment with AZD1208.We next studied mechanisms underlying Pim kinase inhibitor downregulation of Alt-NHEJ. The effects of DNR, MXR or VP-16 on cellular and nuclear expression of the Alt-NHEJ proteins poly (ADP-ribose) polymerase (PARP) 1, DNA polymerase θ, DNA ligase 3 and co-factor X-ray repair cross-complementing protein 1 (XRCC1) were measured in the FLT3-ITD cell lines Ba/F3-ITD, 32D-ITD, MV4-11 and MOLM-14 in the absence and presence of AZD1208. Cellular and nuclear expression of XRCC1 and DNA ligase 3, but not PARP or DNA polymerase θ, decreased in cells co-treated with AZD1208 and topo 2 inhibitors, compared to topo 2 inhibitors alone. XRCC1 was identified as a potential direct Pim-1 substrate by global profiling using the reverse in-gel kinase assay, and was confirmed as a robust Pim substrate in a reverse in-gel kinase assay. This provides a potential mechanistic link between Pim-1 kinase and ALT NHEJ.Alt-NHEJ repair is associated with genomic instability characterized by increased chromosome deletions, insertions and translocations. We studied induction of chromosome changes by topo 2 inhibitor treatment with and without Pim kinase inhibitor in FLT3-ITD cells. Ba/F3-ITD and 32D-ITD cells were incubated with DNR and/or AZD1208, or DMSO control, for 36 hours, then prepared for cytogenetic analysis.For each treatment, 20 consecutive metaphases were analyzed for induction of chromatid breaks and exchanges. No chromosome changes were seen in either cell line with DMSO or AZD1208 treatment. In Ba/F3-ITD cells, chromosome abnormalities were seen in 17 of 20 metaphases after DNR treatment, but only 8 of 20 after combination treatment (p=0.008). In 32D-ITD cells, chromosome abnormalities were seen in 8 of 20 metaphases with DNR, and 0 metaphases with combination (p=0.003).Thus concurrent treatment with Pim kinase inhibitor abrogates induction of Alt-NHEJ repair by topo 2 inhibitor treatment in FLT3-ITD cells and decreases the genomic instability that may contribute to rapid relapse of FLT3-ITD AML after chemotherapy. The data support further preclinical and clinical testing of Pim kinase inhibitors with chemotherapy in AML with FLT3-ITD. DisclosuresTron:AstraZeneca: Employment, Equity Ownership.

Impact of Bioflavonoid Exposure on DNA Damage Response and of Non-Homologous End Joining (NHEJ) Pathway Repair Proteins in Stem Cells

DNA double-strand breaks (DSBs) activate DNA damage response signaling pathways and multiple repair pathways depending on cell cycle stage and differentiation state. An array of recurrent, non-random chromosomal translocations are associated with hematologic malignancies, and we are interested in determining the role of specific DNA repair proteins or pathway choice in the etiology of these rearrangements. Classic non-homologous end joining (NHEJ) utilizes LIG4, XRCC4, and XLF during the final steps; LIG4 and XRCC4 deficiency is implicated in both leukemais and lymphomas but the role of XLF deficiency in leukemia etiology is not clear. Activation of alternative NHEJ (Alt-NHEJ) is largely dependent on CtIP that promotes the resection of DNA ends to uncover micro-homologies promoting the formation of large deletions and chromosomal translocations. Studies suggest that loss of efficient activity in one NHEJ repair pathway may promote use of the other more error-prone pathway. The topoisomerase II poison etoposide is a cytotoxic chemotherapeutic agent associated with therapy-related leukemias characterized by translocations involving the MLL gene at 11q23. Leukemias with MLL gene fusions have been detected in newborns indicating that these translocations can occur in utero, and additional evidence from our lab and others suggests maternal exposure to environmental topoisomerase II inhibitors such as bioflavonoids have the potential to promote these events. We are interested in understanding the crosstalk involved in bioflavonoid exposure, DNA damage response signaling, and repair pathway choice as a model system to understand the underlying mechanisms of oncogenic transformation in infant leukemias.Using established in vitro differentiation protocols, we expose embryonic stem (ES) cells and hematopoietic stem cells (HSC) derived from them to a panel of bioflavonoids alone or in combination for one hour at increasing doses ranging from 0μM-200μM based on determined LD50 values. As a control, cells are exposed to etoposide at the same doses. Following exposure, cells recovered for a period of 0-24hrs, followed by protein extraction and Western blotting. Quantitation of increases in total gH2AX confirm DNA damage, presence of Ku70 and XLF to support classical NHEJ, and CtIP to support Alt-NHEJ. Cells exposed to bioflavonoids genistein and quercetin as well as etoposide show elevated γH2AX levels by 4 hrs post-treatment compared to untreated cells. Parallel studies show dose-dependent increase in the presence of γH2AX foci in these cells. These results confirm that although bioflavonoids have pleiotropic effects, they directly promote DSBs in a short period of time post-exposure. Protein levels of Ku70 and XLF in cells exposed to genistein, quercetin, and etoposide at doses of 25, 50, 75 and 100μM are not significantly different from untreated cells. However, CtIP levels are elevated in cells exposed to quercetin compared to all other groups suggesting a specific potential for this bioflavonoid to promote Alt-NHEJ repair and genome instability.To correlate the activity of these DNA repair protein activities with downstream repair products, we use a model cell system in which parental ES cells were engineered to contain two constructs: GFP exons inserted into MLL and AF9 bcr transgene fragments. Aberrant repair resulting in chromosomal translocations are scored by GFP+ fluorescence. In both ES and derived HSC populations from them translocations can be scored following exposure to bioflavonoids and etoposide. Ongoing experiments include downregulation of XLF or CtIP by siRNA in cells prior to exposure to these compounds. Results will be presented on impact of downregulation of these two NHEJ proteins on 1) amount and persistence of γH2AX; 2) protein levels of other DNA repair pathway proteins, and 3) frequency of generating chromosomal translocations.Understanding the integrated activity and role of these proteins in preventing or promoting translocations, particularly in the context of exposure to a wide range of environmental and dietary compounds, will be critical to understanding the potential for specific isoforms or mutants to increase leukemia risk. DisclosuresNo relevant conflicts of interest to declare.

Drugging DNA Repair to Target T-ALL Cells

Despite recent progress in treatment, prognostication for T-cell acute lymphoblastic leukemia (T-ALL) patients, including those with early T-cell precursor ALL (ETP-ALL) is rather poor. Therefore, new therapies are needed to extend the complete remission time/cure and/or to treat refractory T-ALL patients. T-ALL cells accumulate potentially lethal “spontaneous” and drug-induced DNA double-strand breaks (DSBs). Thus, T-ALL cells may be “addicted” to DSB repair mechanisms to survive and targeting these pathways should sensitize them to lethal effect of DSBs. In proliferating cells DSBs are usually repaired by homologous recombination (HR) repair. HR works predominantly on broken replication forks and usually depends on BRCA1, BRCA2, PALB2, RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3), and RAD51 (BRCA pathway). However, in BRCA deficient cells displaying low expression of at least one of the proteins from the pathway, PARP1-LIG3 -mediated alternative non-homologous end-joining (Alt-NHEJ) and/or RAD52-RAD51-dependent alternative HR mechanism protect cells from lethal effect of DSBs. Therefore, PARP1 and/or RAD52 are attractive targets to eliminate BRCA-deficient leukemia cells. Although inactivating mutations in genes involved in HR are extremely rare in T-ALL, we show here that microarray gene expression profiling of T-ALLs displayed wide range of expression of mRNAs encoding the proteins in BRCA pathway suggesting that numerous leukemias from individual patients were BRCA deficient. In particular, lower expression of BRCA genes (BRCA1, BRCA2, XRCC2, RAD51B, RAD51C, RAD51) have been detected in ETP-ALLs. These data implicate malfunctioning BRCA pathway in a cohort of T-ALLs, especially in ETP-ALLs. Conversely, an average expression of PARP1 and LIG3 mRNAs seemed not to be affected, and RAD52 mRNA was even overexpressed in ETP-ALLs when compared to T-ALLs. Next, we tested the sensitivity of primary leukemia xenograft (PLX) CD3+ cells from T-ALL/ETP-ALL patients and of normal T cells to DNA repair inhibitors. All 3 samples of ETP-ALL cells were more sensitive to PARP1 inhibitor olaparib and RAD52 inhibitor 6(OH)-DL-dopa when compared to normal pan-T cells. T-ALL PLXs displayed differences in sensitivity to PARP1 and RAD52 inhibitors; two samples appeared sensitive whereas three seemed resistant. In addition, cytotoxic drug Ara-C combined with PARP1 or RAD52 inhibitors exerted stronger anti-leukemia activity when compared to individual treatments in PARP1/RAD52 inhibitor-sensitive leukemia cells. To determine if the differences in sensitivity to PARP1 and RAD52 inhibitors were associated with deficiencies in HR activity, sensitive and resistant leukemia cells as well as normal pan-T cells were transfected with the plasmid containing HR reporter cassette. HR activity was determined by restoration of a functional GFP gene and detection of GFP+ cells. T-ALL/ETP-ALL cells which were sensitive to PARP1 and RAD52 inhibitors displayed diminished HR activity in comparison to normal pan-T cells and T-ALL cells resistant to the inhibitors. Moreover, BRCA1 and RAD51 nuclear foci formation was inhibited in leukemia cells which displayed reduced HR activity and increased sensitivity to PARP1 and RAD52 inhibitors. In summary, our results suggest that majority of ETP-ALLs and a subset of T-ALLs display “BRCAness” phenotype, which predisposes them to be sensitive to PARP1 and RAD52 inhibitors. While RAD52 inhibitors await clinical development, PARP1 inhibitors such as Lynparza (olaparib), Rubraca (rucaparib) and Zeluja (niraparib) have been FDA approved to treat breast and/or ovarian cancers carrying BRCA1/2 mutations. We postulate that these inhibitors may be used to improve therapeutic effect of standard cytotoxic drugs in pre-selected patients with “BRCAness” T-ALLs/ETP-ALLs. DisclosuresMullighan:Loxo Oncology: Research Funding; Amgen: Consultancy.

Targeting the MALAT1/PARP1/LIG3 Complex Induces DNA Damage and Apoptosis in Multiple Myeloma Mouse Models

Molecular pathogenesis studies of multiple myeloma (MM) have focused on coding genes rather than long non-coding RNAs (lncRNAs). However, evidence indicates that lncRNAs are involved in the initiation and progression of almost all kinds of cancers, including MM. MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) is one of the most highly expressed conserved nuclear lncRNAs. Here, we demonstrated that MALAT1 plays important roles in MM DNA repair and cell death. We found that bone marrow plasma cells from patients with MGUS and MM express elevated levels of MALAT1 relative to those from healthy individuals. Our functional studies suggested that in MM, MALAT1 is key in regulating the DNA repair alternative non-homologous end-joining (A-NHEJ) and apoptosis pathways. Using RNA Antisense Purification-Mass Spectrum (RAP-MS), we identified MALAT1 binding proteins in MM, including PARP1 and LIG3, two key components of the A-NHEJ pathway protein complex. Degradation of the MALAT1 RNA by RNAse H using antisense gapmer DNA oligos in MM cells induced poly-ADP-ribosylation of nuclear proteins, defected the DNA repair pathway, and further induced apoptotic pathways. Anti-MALAT1 therapy combined with PARP1 inhibitor or proteasome inhibitor in MM cells showed a synergistic effect in vitro. Furthermore, using a novel single wall carbon nanotube (SWCN) conjugated with anti-MALAT1 oligos, we successfully knocked down MALAT1 RNA in cultured MM cell lines and in two xenograft mouse models. Most importantly, anti-MALAT1 therapy also could induce DNA damage and cell apoptosis in vivo, indicating that MALAT1 could serve as a potential novel therapeutic target for MM treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Creation of a new class of radiosensitizers for glioblastoma based on the mibefradil pharmacophore.

Glioblastoma (GBM) is the most common primary malignant tumor of the central nervous system with a dismal prognosis. Locoregional failure is common despite high doses of radiation therapy, which has prompted great interest in developing novel strategies to radiosensitize these cancers. Our group previously identified a calcium channel blocker (CCB), mibefradil, as a potential GBM radiosensitizer. We discovered that mibefradil selectively inhibits a key DNA repair pathway, alternative non-homologous end joining. We then initiated a phase I clinical trial that revealed promising initial efficacy of mibefradil, but further development was hampered by dose-limiting toxicities, including CCB-related cardiotoxicity, off-target hERG channel and cytochrome P450 enzymes (CYPs) interactions. Here, we show that mibefradil inhibits DNA repair independent of its CCB activity, and report a series of mibefradil analogues which lack CCB activity and demonstrate reduced hERG and CYP activity while retaining potency as DNA repair inhibitors. We present in vivo pharmacokinetic studies of the top analogues with evidence of brain penetration. We also report a targeted siRNA-based screen which suggests a possible role for mTOR and Akt in DNA repair inhibition by this class of drugs. Taken together, these data reveal a new class of mibefradil-based DNA repair inhibitors which can be further advanced into pre-clinical testing and eventually clinical trials, as potential GBM radiosensitizers.