Inhibition Of Non-homologous End Joining Research Articles

Abstract CT018: TMPRSS2-ERG and RB1 as candidate predictive biomarkers for efficacy in TALAPRO-2: Phase 3 study of talazoparib (TALA) + enzalutamide (ENZA) vs placebo (PBO) + ENZA as first-line (1L) treatment in patients (pts) with metastatic castration-resistant prostate cancer (mCRPC)

Abstract Background: In TALAPRO-2 (NCT03395197), pts unselected for homologous recombination repair (HRR) gene mutations (muts) received TALA + ENZA or PBO + ENZA in 1L mCRPC. TALA + ENZA significantly improved radiographic progression-free survival (rPFS). Previous analyses focused on the association of tumor HRR gene muts with outcome; here, we examined TALA + ENZA efficacy in pts with ≥1 non-HRR gene mut with/without HRR gene muts. Methods: This is a post-hoc exploratory agnostic analysis of a TALAPRO-2 dataset of prospectively collected/retrospectively analyzed plasma ctDNA (FoundationOne®Liquid CDx). Response was assessed per RECIST v1.1. Results: Overall, 616 pts in the intent-to-treat population had non-HRR gene muts with/without HRR gene muts; 98 (16%) pts had TMPRSS2-ERG fusions. In these 616 pts, TALA + ENZA improved rPFS vs PBO + ENZA (Table). TALA + ENZA improved rPFS in pts with TMPRSS2-ERG, RB1, or MLL2 muts, and objective response rates (ORR) in pts with TMPRSS2-ERG or RB1 muts and measurable disease at baseline (the association of MLL2 with enhanced rPFS was lost without TMPRSS2-ERG, not shown). In pts with non-HRR gene muts and no HRR gene muts (n=427), TALA + ENZA showed improvement in rPFS vs PBO + ENZA. Enhanced rPFS benefit with TALA + ENZA vs PBO + ENZA was noted in pts with TMPRSS2-ERG, RB1, or MLL2 muts, but TMPRSS2-ERG ORRs were similar across treatment arms. Conclusions: TALA + ENZA improved outcomes compared with PBO + ENZA in patients with muts in specific non-HRR genes (regardless of HRR gene muts). TMPRSS2-ERG and RB1 emerged as candidate predictive biomarkers for differential efficacy favoring TALA + ENZA vs PBO + ENZA. PARP inhibitors may induce a synthetically lethal interaction with TMPRSS2-ERG-mediated inhibition of non-homologous end joining and help overcome RB1-mediated ENZA resistance. ≥1 non-HRR gene muts ≥1 non-HRR gene muts with no HRR gene muts Median rPFS, mo HR (95% CI) Median rPFS, mo HR (95% CI) Gene mutation TALA + ENZA (N=310) PBO + ENZA (N=306) TALA + ENZA (N=218) PBO + ENZA (N=209) Any non-HRR NR 19.3 0.63 (0.50–0.80) NR 22.5 0.66 (0.49–0.89) TMPRSS2-ERG, [n] 25.9 [n=43] 11.0 [n=55] 0.36 (0.20–0.64) 19.4 [n=32] 11.0 [n=44] 0.40 (0.20–0.76) MLL2, [n] 18.2 [n=20] 13.8 [n=20] 0.43 (0.19–0.98) NR [n=8] 17.2 [n=11] 0.50 (0.14–1.74) RB1, [n] 9.8 [n=14] 1.9 [n=8] 0.22 (0.07–0.73) 10.8 [n=6] 1.8 [n=3] 0.40 (0.05–2.92) ORR, % (n/N)a OR (95% CI) ORR, % (n/N)a OR (95% CI) TALA + ENZA (N=96) PBO + ENZA (N=105) TALA + ENZA (N=62) PBO + ENZA (N=75) TMPRSS2-ERG 56 (13/23) 41 (7/17) 0.54 (0.12–2.27) 53 (9/17) 47 (7/15) 0.78 (0.16–3.85) MLL2 64 (7/11) 70 (7/10) 1.33 (0.15–12.60) ND ND ND RB1 62 (5/8) 0 (0/6) 0.00 (0.00–0.75) ND ND ND CI, confidence interval; HR, hazard ratio; mo, months; ND, not displayed (combined prevalence <10 pts); NR, not reached; OR, odds ratio. aMeasurable disease at baseline Citation Format: Josep M. Piulats, Arun A. Azad, A. Douglas Laird, Nobuaki Matsubara, Karim Fizazi, Neal D. Shore, Lawrence Karsh, Glenn Liu, Andre P. Fay, Joan Carles, Robert J. Jones, Eric Voog, Stefanie Zschäbitz, Ugo De Giorgi, Steven M. Yip, Xinmeng Jasmine Mu, Xun Lin, Arne Engelsberg, Neeraj Agarwal. TMPRSS2-ERG and RB1 as candidate predictive biomarkers for efficacy in TALAPRO-2: Phase 3 study of talazoparib (TALA) + enzalutamide (ENZA) vs placebo (PBO) + ENZA as first-line (1L) treatment in patients (pts) with metastatic castration-resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT018.

Functional screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair

Homology Directed Repair (HDR) enables precise genome editing, but the implementation of HDR-based therapies is hindered by limited efficiency in comparison to methods that exploit alternative DNA repair routes, such as Non-Homologous End Joining (NHEJ). In this study, we develop a functional, pooled screening platform to identify protein-based reagents that improve HDR in human hematopoietic stem and progenitor cells (HSPCs). We leverage this screening platform to explore sequence diversity at the binding interface of the NHEJ inhibitor i53 and its target, 53BP1, identifying optimized variants that enable new intermolecular bonds and robustly increase HDR. We show that these variants specifically reduce insertion-deletion outcomes without increasing off-target editing, synergize with a DNAPK inhibitor molecule, and can be applied at manufacturing scale to increase the fraction of cells bearing repaired alleles. This screening platform can enable the discovery of future gene editing reagents that improve HDR outcomes.

Concomitant inhibition of the thioredoxin system and nonhomologous DNA repair potently sensitizes Philadelphia-positive lymphoid leukemia to tyrosine kinase inhibitors.

Breakpoint cluster region-Abelson (BCR::ABL1) gene fusion is an essential oncogene in both chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) B-cell acute lymphoblastic leukemia (B-ALL). While tyrosine kinase inhibitors (TKIs) are effective in up to 95% of CML patients, 50% of Ph+ B-ALL cases do not respond to treatment or relapse. This calls for new therapeutic approaches for Ph+ B-ALL. Previous studies have shown that inhibitors of the thioredoxin (TXN) system exert antileukemic activity against B-ALL cells, particularly in combination with other drugs. Here, we present that peroxiredoxin-1 (PRDX1), one of the enzymes of the TXN system, is upregulated in Ph+ lymphoid as compared to Ph+ myeloid cells. PRDX1 knockout negatively affects the viability of Ph+ B-ALL cells and sensitizes them to TKIs. Analysis of global gene expression changes in imatinib-treated, PRDX1-deficient cells revealed that the nonhomologous end-joining (NHEJ) DNA repair is a novel vulnerability of Ph+ B-ALL cells. Accordingly, PRDX1-deficient Ph+ B-ALL cells were susceptible to NHEJ inhibitors. Finally, we demonstrated the potent efficacy of a novel combination of TKIs, TXN inhibitors, and NHEJ inhibitors against Ph+ B-ALL cell lines and primary cells, which can be further investigated as a potential therapeutic approach for the treatment of Ph+ B-ALL.

Targeting Loss of the Tumor Suppressor TENT5C in Multiple Myeloma

Introduction: Multiple myeloma (MM) is a genetically complex and heterogeneous neoplasm in which multiple genomic events lead to tumor development and progression. Loss of TENT5C through mutation or deletion on chromosome 1p occurs in more than 20% of newly diagnosed MM patients and is associated with a poor progression free and overall survival. Although, many studies have identified TENT5C as a tumor suppressor gene in MM, the impact of gene loss on MM physiopathology remain to be elucidated. Here we sought to identify drugs which may specifically target TENT5C-null MM cells using targeted and high-throughput drug screens (HTS). Methods: We generated CRISPR/Cas9 knockout (ko) of TENT5C in wild type (wt) U266 and KMS-11 MM cell lines and over-expressed (oe) TENT5C in MM cell lines with biallelic inactivation (H929 -/- and LP-1 -/-). Modified cell lines were compared to their original counterparts for gene expression, proliferation, and cell cycle differences. Targeted and HTS were performed to identify synthetic lethal pathways related to loss of TENT5C in MM that will result in specific cell death. Results: In TENT5C ko cells the RNA poly(A) polymerase activity was decreased at two TENT5C top targeted substrates, immunoglobulin kappa light chain and signal sequence receptor. Consistent with TENT5C tumor suppressor function, cell proliferation analysis showed that TENT5C ko cells had a higher growth rate than TENT5C wt cells. Cell cycle analysis, after nocodazole synchronization, showed TENT5C-null cells had an increased G2/M population compared to TENT5C-positive cells (U266 TENT5C ko 19.86% vs. TENT5C wt 2.6%, p<0.001; H929 TENT5C -/- 8.22% vs. TENT5C oe 1.18%, p<0.001), indicating an increase in cell cycle progression in TENT5C-null cells. Given our prior data indicating an increase in splicing abnormalities and non-homologous end joining (NHEJ) activation in TENT5C -/- patient samples, we performed growth inhibition curves using appropriate drugs. The spliceosome inhibitors pladienolide B and isoginkgetin affected TENT5C ko cells at lower concentrations than TENT5C wt cells (U266 TENT5C ko GI 50=0.58 vs. TENT5C wt GI 50=1.43 nM, U266 TENT5C ko GI 50=7.03 vs. TENT5C wt GI 50=13.72 µM, p<0.001, respectively) as did NHEJ inhibitors STL127705 and SCR7 (U266 TENT5C ko GI 50=22.5 vs. TENT5C wt GI 50=62.55 µM, U266 TENT5C ko GI 50=41.93 vs. TENT5C wt GI 50=117.38 µM, p<0.01). These results were confirmed on the other modified cells to ensure TENT5C specificity. A HTS using the LOPAC1280 library also identified several hits specifically affecting TENT5C null cells. The hits were categorized according to their targets to identify pathway enrichment. Inhibitors targeting the cell cycle (roscovitine, apigenin, SP60012) and angiogenesis (amsacrine) were selected for validation and further investigation. The differential effect observed in the HTS data was confirmed with full dose kill-curves on the same cell line (U266) and validated in another TENT5C ko cell line (KMS-11) to ensure target specificity. For example, amsacrine, an angiogenesis inhibitor, exhibited differential activity onT ENT5C ko and TENT5C wt cells (U266 TENT5C ko GI 50=5.63 µM vs. U266 TENT5C wt GI 50=9.19, p<0.001; KMS-11 TENT5C ko GI 50=9.26 µM vs. KMS-11 TENT5C wt GI 50=14.38, p<0.001). Similarly, roscovitine, a CDK inhibitor, affected TENT5C ko cells at lower concentrations than TENT5C wt cells (U266 TENT5C ko GI 50=23.33 vs. U266 TENT5C wt GI 50=34.91 µM; KMS-11 TENT5C ko GI 50=13.47 vs. KMS-11 TENT5C wt GI 50=29.33 µM, p<0.001). Additionally, cell cycle analysis showed that roscovitine induced a G 0/G 1 arrest in a dose-response manner in U266 and KMS-11 cells. Interestingly, TENT5C ko cells were more sensitive to the CDK inhibitor, and the arrested KMS-11 G 0/G 1 populations increased from 3.35 in TENT5C wt to 6.46 % in TENT5C ko and from 15.51 in TENT5C wt to 21.15 % in TENT5C ko (p<0.05), after treatment with 12.5 and 25 µM of roscovitine, respectively. The same trend was also validated in U266 TENT5C ko cells to ensure target specificity. Conclusion: Our results indicate a tumor suppressor role of TENT5C in MM, with antiproliferative properties and a susceptibility to spliceosome and NHEJ inhibitors. The HTP screen identified other targets that are currently undergoing validation leading to the identification of the molecular mechanisms underlying this function.

Impact of sequencing of androgen receptor-signaling inhibition and radiotherapy in prostate cancer: importance of homologous recombination disruption.

The synergy of combining androgen receptor-signaling inhibition (ARSI) to radiotherapy (RT) in prostate cancer has been largely attributed to non-homologous end joining (NHEJ) inhibition. However, this mechanism is unlikely to explain recently observed trial results that demonstrated the sequencing of ARSI and RT significantly impacts clinical outcomes, with adjuvant ARSI following RT yielding superior outcomes to neoadjuvant/concurrent therapy. We hypothesized this is driven by differential effects on AR-signaling and alternative DNA repair pathway engagement based on ARSI/RT sequencing. We explored the effects of ARSI sequencing with RT (neoadjuvant vs concurrent vs adjuvant) in multiple prostate cancer cell lines using androgen-deprived media and validation with the anti-androgen enzalutamide. The effects of ARSI sequencing were measured with clonogenic assays, AR-target gene transcription and translation quantification, cell cycle analysis, DNA damage and repair assays, and xenograft animal validation studies. Adjuvant ARSI after RT was significantly more effective at killing colony forming cells and decreasing the transcription and translation of downstream AR-target genes across all prostate cancer models evaluated. These results were reproduced in xenograft studies. The differential effects of ARSI sequencing were not fully explained by NHEJ inhibition alone, but by the additional disruption of homologous recombination specifically with adjuvant sequencing of ARSI. We demonstrate that altered sequencing of ARSI and RT mediates differential anti-AR-signaling and anti-cancer effects, with the greatest benefit from adjuvant ARSI following RT. These results, combined with our prior clinical findings, support the superiority of an adjuvant-based sequencing approach when using ARSI with RT.

Inhibition of non-homologous end joining mitigates paclitaxel resistance resulting from mitotic slippage in non-small cell lung cancer

ABSTRACT Mitotic slippage, which enables cancer cells to bypass cell death by transitioning from mitosis to the G1 phase without undergoing normal cytokinesis, is one likely mechanism of paclitaxel (PTX) resistance. DNA double-strand breaks (DSBs) in the G1 phase are mainly repaired through non-homologous end joining (NHEJ). Therefore, inhibiting NHEJ could augment the PTX-induced cytotoxicity by impeding the repair of PTX-induced DSBs during the G1 phase following mitotic slippage. We aimed to evaluate the effects of NHEJ inhibition on mitotic slippage after PTX treatment in non-small cell lung cancer (NSCLC). H1299, A549, H1975, and H520 NSCLC cell lines were employed. In addition, A-196 and JQ1 were used as NHEJ inhibitors. H1299 cells were PTX-resistant and exhibited an increased frequency of mitotic slippage upon PTX treatment. NHEJ inhibitors significantly augmented the PTX-induced cytotoxicity, DSBs, and apoptosis in H1299 cells. The newly generated PTX-resistant cells were even more prone to mitotic slippage following PTX treatment and susceptible to the combined therapy. Docetaxel further demonstrated synergistic effects with the NHEJ inhibitor in PTX-resistant cells. NHEJ inhibition may overcome intrinsic or acquired PTX resistance resulting from mitotic slippage by synergistically increasing the cytotoxic effects of antimitotic drugs in NSCLC.

Efficient high-precision homology-directed repair-dependent genome editing by HDRobust.

Homology-directed repair (HDR), a method for repair of DNA double-stranded breaks can be leveraged for the precise introduction of mutations supplied by synthetic DNA donors, but remains limited by low efficiency and off-target effects. In this study, we report HDRobust, a high-precision method that, via the combined transient inhibition of nonhomologous end joining and microhomology-mediated end joining, resulted in the induction of point mutations by HDR in up to 93% (median 60%, s.e.m. 3) of chromosomes in populations of cells. We found that, using this method, insertions, deletions and rearrangements at the target site, as well as unintended changes at other genomic sites, were largely abolished. We validated this approach for 58 different target sites and showed that it allows efficient correction of pathogenic mutations in cells derived from patients suffering from anemia, sickle cell disease and thrombophilia.

Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways.

Gene editing through repair of CRISPR-Cas9-induced chromosomal breaks offers a means to correct a wide range of genetic defects. Directing repair to produce desirable outcomes by modulating DNA repair pathways holds considerable promise to increase the efficiency of genome engineering. Here, we show that inhibition of non-homologous end joining (NHEJ) or polymerase theta-mediated end joining (TMEJ) can be exploited to alter the mutational outcomes of CRISPR-Cas9. We show robust inhibition of TMEJ activity at CRISPR-Cas9-induced double-strand breaks (DSBs) using ART558, a potent polymerase theta (Polϴ) inhibitor. Using targeted sequencing, we show that ART558 suppresses the formation of microhomology-driven deletions in favor of NHEJ-specific outcomes. Conversely, NHEJ deficiency triggers the formation of large kb-sized deletions, which we show are the products of mutagenic TMEJ. Finally, we show that combined chemical inhibition of TMEJ and NHEJ increases the efficiency of homology-driven repair (HDR)-mediated precise gene editing. Our work reports a robust strategy to improve the fidelity and safety of genome engineering.

Selective utilization of non-homologous end-joining and homologous recombination for DNA repair during meiotic maturation in mouse oocytes.

DNA double-strand breaks (DSBs) are highly toxic lesions that can cause genomic instability and can be repaired by non-homologous end-joining (NHEJ) and homologous recombination (HR) pathways. Despite extensive studies about DSB repair pathways, the roles of each pathway during meiotic maturation in oocytes are not well understood. Here we show that oocytes selectively utilize NHEJ and HR to repair DSBs during meiotic maturation. Inhibition of NHEJ impaired the meiotic maturation of oocytes with DNA damage by activating the spindle assembly checkpoint (SAC) with a concomitant increase in metaphase I (MI) arrest and DNA damage levels. In contrast, oocytes with DNA damage bypassed SAC-mediated MI arrest despite the presence of fragmented DNA when HR was inhibited. Notably, this bypass of SAC arrest by HR inhibition was associated with a loss of centromere integrity and subsequent impairment of chromosome architecture. Our results demonstrate that, while NHEJ is critical for the meiotic maturation of oocytes with DNA damage, HR is essential to maintain centromere integrity against DNA damage during meiotic maturation, revealing distinct roles of NHEJ and HR during meiotic maturation in mouse oocytes.

Targeting DNA Repair with Combined Inhibition of NHEJ and MMEJ Induces Synthetic Lethality in TP53-Mutant Cancers.

Combined inhibition of NHEJ and MMEJ using two nontoxic, targeted DNA repair inhibitors can effectively induce toxic DNA damage to treat TP53-deficient cancers.

Chloroquine-Induced DNA Damage Synergizes with Nonhomologous End Joining Inhibition to Cause Ovarian Cancer Cell Cytotoxicity.

Ovarian cancer (OC) is the most lethal gynecological malignancy; therefore, more effective treatments are urgently needed. We recently reported that chloroquine (CQ) increased reactive oxygen species (ROS) in OC cell lines (OCCLs), causing DNA double-strand breaks (DSBs). Here, we analyzed whether these lesions are repaired by nonhomologous end joining (NHEJ), one of the main pathways involved in DSB repair, and if the combination of CQ with NHEJ inhibitors (NHEJi) could be effective against OC. We found that NHEJ inhibition increased the persistence of γH2AX foci after CQ-induced DNA damage, revealing an essential role of this pathway in the repair of the lesions. NHEJi decreased the proliferation of OCCLs and a strong in vitro synergistic effect on apoptosis induction was observed when combined with CQ. This effect was largely abolished by the antioxidant N-Acetyl-L-cysteine, revealing the critical role of ROS and DSB generation in CQ/NHEJi-induced lethality. We also found that the NHEJ efficiency in OCCLs was not affected by treatment with Panobinostat, a pan-histone deacetylase inhibitor that also synergizes with CQ in OCCLs by impairing homologous recombination. Accordingly, the triple combination of CQ-NHEJi-Panobinostat exerted a stronger in vitro synergistic effect. Altogether, our data suggest that the combination of these drugs could represent new therapeutic strategies against OC.

Abstract 796: Dual inhibition of NHEJ and MMEJ induces synthetic lethality in TP53 mutant cancers

Abstract DNA repair pathway inhibitors are a new class of anti-cancer drugs that are advancing in clinical trials. While inhibitors of targets in the Non-Homologous End Joining (NHEJ) DNA repair pathway, such as DNA-dependent protein kinase (DNA-PK), are available for clinical use, it remains unclear which cancers are vulnerable to these agents. In a genome-wide CRISPR knockout screen with the DNA-PK inhibitor M3814, we identify loss of POLQ, encoding polymerase theta, and other genes in the microhomology-mediated end-joining (MMEJ) pathway as key predictors of sensitivity to DNA-PK inhibition, whereas loss of TP53 conferred resistance to DNA-PK inhibition. Inhibition of DNA-PK led to increased DNA double strand break end-resection, increased expression of polymerase theta, and activation of MMEJ repair. Combined DNA-PK inhibition by M3814 and polymerase theta inhibition by novobiocin resulted in synthetic lethality mediated by the accumulation of resected DNA and apoptosis. Significantly, this drug combination efficiently killed TP53-deficient human patient-derived xenografts and the corresponding tumor organoids. Taken together, our results provide a rationale for the combination of an inhibitor of DNA-PK mediated NHEJ and an inhibitor of polymerase theta mediated MMEJ in an anti-cancer trial. If DNA-PK is inhibited, cancers develop a hyper-dependence on MMEJ and an upregulation of DNA double strand break end resection and polymerase theta expression. Similarly, P53-deficiency which confers resistance to DNA-PK inhibition, also leads to a hyper-dependence on MMEJ and an upregulation of polymerase theta expression. Thus, a combination of DNA-PK and polymerase theta inhibitors may provide a precision treatment strategy for TP53-mutant solid tumors, known to account for 50% of newly diagnosed cancers. Citation Format: Jeffrey Patterson-Fortin, Arindam Bose, Wei-Chih Tsai, Carter Grochala, Huy Nguyen, Jia Zhou, Kalindi Parmar, Jean-Bernard Lazaro, Joyce Liu, Kelsey McQueen, Geoffrey I. Shapiro, David Kozono, Alan D. D'Andrea. Dual inhibition of NHEJ and MMEJ induces synthetic lethality in TP53 mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 796.

Beta human papillomavirus 8 E6 allows colocalization of non-homologous end joining and homologous recombination repair factors.

Beta human papillomavirus (β-HPV) are hypothesized to make DNA damage more mutagenic and potentially more carcinogenic. Double strand breaks (DSBs) are the most deleterious DNA lesion. They are typically repaired by homologous recombination (HR) or non-homologous end joining (NHEJ). HR occurs after DNA replication while NHEJ can occur at any point in the cell cycle. HR and NHEJ are not thought to occur in the same cell at the same time. HR is restricted to cells in phases of the cell cycle where homologous templates are available, while NHEJ occurs primarily during G1. β-HPV type 8 protein E6 (8E6) attenuates both repair pathways. We use a series of immunofluorescence microscopy and flow cytometry experiments to better define the impact of this attenuation. We found that 8E6 causes colocalization of HR factors (RPA70 and RAD51) with an NHEJ factor (activated DNA-PKcs or pDNA-PKcs) at persistent DSBs. 8E6 also causes RAD51 foci to form during G1. The initiation of NHEJ and HR at the same lesion could lead to antagonistic DNA end processing. Further, HR cannot be readily completed in an error-free manner during G1. Both aberrant repair events would cause deletions. To determine if these mutations were occurring, we used next generation sequencing of the 200kb surrounding a CAS9-induced DSB. 8E6 caused a 21-fold increase in deletions. Chemical and genetic inhibition of p300 as well as an 8E6 mutant that is incapable of destabilizing p300 demonstrates that 8E6 is acting via p300 destabilization. More specific chemical inhibitors of DNA repair provided mechanistic insight by mimicking 8E6-induced dysregulation of DNA repair in a virus-free system. Specifically, inhibition of NHEJ causes RAD51 foci to form in G1 and colocalization of RAD51 with pDNA-PKcs.

Precise exogenous insertion and sequence replacements in poplar by simultaneous HDR overexpression and NHEJ suppression using CRISPR-Cas9.

CRISPR-mediated genome editing has become a powerful tool for the genetic modification of biological traits. However, developing an efficient, site-specific, gene knock-in system based on homology-directed DNA repair (HDR) remains a significant challenge in plants, especially in woody species like poplar. Here, we show that simultaneous inhibition of non-homologous end joining (NHEJ) recombination cofactor XRCC4 and overexpression of HDR enhancer factors CtIP and MRE11 can improve HDR efficiency for gene knock-in. Using this approach, the BleoR gene was integrated onto the 3′ end of the MKK2 MAP kinase gene to generate a BleoR-MKK2 fusion protein. Based on fully edited nucleotides evaluated by TaqMan real-time PCR, the HDR-mediated knock-in efficiency was up to 48% when using XRCC4 silencing incorporated with a combination of CtIP and MRE11 overexpression compared with no HDR enhancement or NHEJ silencing. Furthermore, this combination of HDR enhancer overexpression and NHEJ repression also increased genome targeting efficiency and gave 7-fold fewer CRISPR-induced insertions and deletions (InDels), resulting in no functional effects on MKK2-based salt stress responses in poplar. Therefore, this approach may be useful not only in poplar and plants or crops but also in mammals for improving CRISPR-mediated gene knock-in efficiency.

Selected Articles from This Issue

Medulloblastoma is a common and deadly pediatric malignancy whose lethality is compounded by a lack of targeted therapies, partially owing to the differential biologies of the four major molecular subtypes. The most prevalent subtype, Sonic Hedgehog medulloblastoma (SHH-MB), is driven by hyperactivation of the eponymous SHH pathway within MATH-1-expressing granule cell precursors in the developing brain. Therefore, efforts to develop molecular targeted therapies for SHH-MB have focused largely on downstream targets and effectors of the SHH pathway such as the proto-oncogene SOX9, which has been shown to be specifically overexpressed in SHH-MB but whose functional role in disease progression has yet to be defined. Here, Adolphe and colleagues demonstrate that loss of SOX9 does not significantly impact the course of SHH-MB disease development and progression, indicating a dispensable role for this proto-oncogene in SHH-MB despite its elevated expression in most cases. This finding may have significant implications for ongoing preclinical and translational work aimed at identifying suitable molecular drivers of SHH-MB for therapeutic development.Lymphangioleiomyomatosis is a rare neoplastic disease of unknown origin that is nearly exclusive to women, with therapies targeting mTOR as the recognized standard of care. However, there is substantial variability in disease presentation, progression, and therap. response. Here, Espin, Baiges and colleagues perform multi-omics characterization of patient-derived lymphangioleiomyomatosis cells and tissues to identify the molecular features and potential causes of disease heterogeneity. The data suggest that differential expression of certain transcription factors—such as YB1, RUNX1, IRF1, estrogen receptor, and progesterone receptor—as well as certain immunity factors may account for some of these features. Based on the molecular features identified herein, the authors also nominate a breast mesenchymal cell line and certain pleural mesothelioma cell lines as potential surrogate models of lymphangioleiomyomatosis, expanding on the biological basis of the disease. Taken together, the data provide a high-value resource for further exploration of the molecular drivers of lymphangioleiomyomatosis progression.Glioma patients are frequently treated with temozolomide, a DNA alkylating chemotherapeutic that is known to cross the blood-brain barrier. The isocitrate dehydrogenase (IDH) R132H mutation is a common molecular feature of gliomas and is generally associated with gliomagenesis, but also with a more robust response to temozolomide therapy. However, the molecular underpinnings of this observation are not defined. In this study, Zhan, Ma, and colleagues use a heterozygous knock-in approach to model the effects of IDH R132H/wild-type allele balance on glioma cell biology. They find that the heterozygous IDH genotype in tandem with glioma driver gene alterations in p53, RB, and ATRX predisposes glioma cells to pro-senescent features. This combination of molecular features was associated with increased DNA damage markers and potentiated the effects of temozolomide in this model. However, heterozygous IDH R132H mutation also predisposed astroglial cells toward a senescence-associated secretory phenotype, which is known to exert a pro-tumorigenic effect within a tumor-permissive microenvironment. These data offer a potential mechanistic explanation for how a single mutation within IDH may provide a pro-tumorigenic stimulus while also sensitizing glioma cells to alkylating chemotherapy with temozolomide.Potentiating the effects of genotoxic interventions such as chemo- or radiotherapy requires the suppression of DNA damage response pathways, such as non-homologous end joining (NHEJ). Blocking NHEJ with chemical inhibitors is one strategy, but none have been clinically approved for this application. Building upon prior efforts to develop chemical screening strategies for NHEJ inhibitors, Du and colleagues report a miniaturized screening protocol that can be carried out in 96-well plates. By assessing an FDA-approved drug library in this format, the authors identify several potential candidates that function to inhibit NHEJ at concentrations below their cytotoxic dose. One of these drugs, ompalisib, was shown to suppress the phosphorylation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in response to chemo- or radiotherapy, a key step in the initiation of NHEJ to mediate their genotoxic effects. Overall, the authors advance a novel screening strategy to examine the effects of pharmacological interventions on DNA damage responses via NHEJ, and further nominate a candidate FDA-approved drug for development as a chemo- and radiosensitizer.

The PI3K/mTOR Inhibitor Ompalisib Suppresses Nonhomologous End Joining and Sensitizes Cancer Cells to Radio- and Chemotherapy.

As the predominant pathway for the repair of DNA double-strand breaks (DSB), non-homologous end joining (NHEJ) attenuates the efficacy of cancer treatment which relies on the introduction of DSBs, such as radiotherapy and genotoxic drugs. Identifying novel NHEJ inhibitors is of great importance for improving the therapeutic efficiency of radio- or chemotherapy. Here we miniaturized our recently developed NHEJ detecting system into a 96-well plate-based format and interrogated an FDA approved drug library containing 1732 compounds. A collection of novel hits were considered to be potential DSB repair inhibitors at the noncytotoxic concentration. We identified omipalisib as an efficient sensitizer for DNA damage-induced cell death in vitro Furthermore, in vitro analysis uncovered the repressive effect of omipalisib on the phosphorylation of DNA-dependent protein kinase catalytic subunit induced by ionizing radiation and doxorubicin, which led to the suppression of NHEJ pathway. IMPLICATIONS: In summary, our findings suggested the possibility for repurposing these candidates as radio- or chemosensitizers, which might extend their clinical application in cancer therapy.

SCR7, an inhibitor of NHEJ can sensitize tumor cells to ionization radiation

Nonhomologous end joining (NHEJ), one of the major DNA double-strand break repair pathways, plays a significant role in cancer cell proliferation and resistance to radio and chemotherapeutic agents. Previously, we had described a small molecule inhibitor, SCR7, which inhibited NHEJ in a DNA Ligase IV dependent manner. Here, we report that SCR7 potentiates the effect of γ-radiation (IR) that induces DNA breaks as intermediates to eradicate cancer cells. Dose fractionation studies revealed that coadministration of SCR7 and IR (0.5 Gy) in mice Dalton's lymphoma (DLA) model led to a significant reduction in mice tumor cell proliferation, which was equivalent to that observed for 2 Gy dose when both solid and liquid tumor models were used. Besides, co-treatment with SCR7 and 1 Gy of IR further improved the efficacy. Notably, there was no significant change in blood parameters, kidney and liver functions upon combinatorial treatment of SCR7 and IR. Further, the co-treatment of SCR7 and IR resulted in a significant increase in unrepaired DSBs within cancer cells compared to either of the agent alone. Anatomy, histology, and other studies in tumor models confirmed the cumulative effects of both agents in activating apoptotic pathways to induce cytotoxicity by modulating DNA damage response and repair pathways. Thus, we report that SCR7 has the potential to reduce the side effects of radiotherapy by lowering its effective dose ex vivo and in mice tumor models, with implications in cancer therapy.

Neutrophils Alter DNA Repair Landscape to Impact Survival and Shape Distinct Therapeutic Phenotypes of Colorectal Cancer

Tumor-infiltrating neutrophils (polymorphonuclear neutrophils [PMNs]) are a prominent feature of colorectal cancer (CRC), where they can promote cytotoxicity or exacerbate disease outcomes. We recently showed that in acute colon injury, PMNs can increase DNA double-strand break (DSB) burden and promote genomic instability via microRNA-dependent inhibition of homologous recombination (HR) repair. In this study, we aimed to establish whether in inflamed colon, neutrophils shape the DSB-repair responses to impact CRC progression and sensitivity/resistance to DNA-repair targeted therapy. Human sporadic CRC biopsies, The Cancer Genome Atlas gene expression analyses, tumor xenografts, and murine CRC models, as well as small-molecule inhibition of key DSB-repair factors were leveraged to investigate changes in the DSB-repair landscape and identify unique CRC responses with/without tumor infiltration by PMNs. We reveal that neutrophils exert a functional dualism in cancer cells, driving temporal modulation of the DNA damage landscape and resolution of DSBs. PMNs were found to promote HR deficiency in low-grade CRC by miR-155-dependent downregulation of RAD51, thus attenuating tumor growth. However, neutrophil-mediated genotoxicity due to accumulation of DSBs led to the induction of non-homologous end-joining (NHEJ), allowing for survival and growth of advanced CRC. Our findings identified a PMN-induced HR-deficient CRC phenotype, featuring low RAD51 and low Ku70 levels, rendering it susceptible to synthetic lethality induced by clinically approved PARP1 inhibitor Olaparib. We further identified a distinct PMN-induced HR-deficient CRC phenotype, featuring high Ku70 and heightened NHEJ, which can be therapeutically targeted by specific inhibition of NHEJ. Our work delineates 2 mechanism-based translatable therapeutic interventions in sporadic CRC.

SCR7, a potent cancer therapeutic agent and a biochemical inhibitor of nonhomologous DNA end-joining.

BackgroundDNA double‐strand breaks (DSBs) are harmful to the cell as it could lead to genomic instability and cell death when left unrepaired. Homologous recombination and nonhomologous end‐joining (NHEJ) are two major DSB repair pathways, responsible for ensuring genome integrity in mammals. There have been multiple efforts using small molecule inhibitors to target these DNA repair pathways in cancers. SCR7 is a very well‐studied anticancer molecule that blocks NHEJ by targeting one of the critical enzymes, Ligase IV.Recent findingsIn this review, we have highlighted the anticancer effects of SCR7 as a single agent and in combination with other chemotherapeutic agents and radiation. SCR7 blocked NHEJ effectively both in vitro and ex vivo. SCR7 has been used for biochemical studies like chromosomal territory resetting and in understanding the role of repair proteins in cell cycle phases. Various forms of SCR7 and its derivatives are discussed. SCR7 is also used as a potent biochemical inhibitor of NHEJ, which has found its application in improving genome editing using a CRISPR‐Cas system.ConclusionSCR7 is a potent NHEJ inhibitor with unique properties and wide applications as an anticancer agent. Most importantly, SCR7 has become a handy aid for improving genome editing across different model systems.

NEUTROPHILS ALTER DSB REPAIR PATHWAY IN INFLAMED MUCOSA TO PROMOTE COLON CARCINOGENESIS

Abstract Due to exacerbated inflammation and recurring tissue injury patients with Inflammatory Bowel Disease (IBD) are at higher risk of developing colorectal cancer (CRC). Although, PMN infiltration of the intestinal mucosa is a hallmark of IBD, and is associated with tissue injury, the contribution of PMNs to CRC and more so to IBD associated CRC is not known. We recently showed that PMNs can acutely exacerbate tissue injury and promote genomic instability by promoting miR-23a and miR-155-dependent accumulation of double-strand breaks (DSBs) and inhibition of DSB-repair by homologous recombination (HR). We now demonstrate that in chronic gut inflammation and recurring epithelial injury, as seen in IBD, via similar miRNA-dependent mechanism, PMNs alter DNA damage responses (DDR) to promote CRC progression. In murine model of Colitis-associated CRC (Azoxymethane, AOM/Dextran sodium sulfate, DSS) and human CRC xenografts, intratumoral PMNs intriguingly, functional dualism, suppressing tumor onset, but promoting tumor cell survival and growth in progressive tumors. The observed PMN effects were mediated by persistent suppression of HR-mediated DSB repair. HR suppression by PMNs, initially resulted in elevated replication stress and increased tumor cell apoptosis, however, longer term, altered DDR transcriptional profile and facilitated the upregulation of DSB repair by non-homologous end-joining (NHEJ). NHEJ upregulation enhanced CRC progression and tumor cell survival. PMN depletion, CRSPER-mediated deletion of miR-155 responsive sequence in RAD51 (preserves HR activity) or inhibition of NHEJ by small molecule inhibitors increased tumor cell death and diminished tumor development. Collectively, our data define a novel link between PMN-mediated mucosal injury and colon carcinogenesis.