Microhomology-mediated End Joining Research Articles

Modulation of the microhomology-mediated end joining pathway suppresses large deletions and enhances homology-directed repair following CRISPR-Cas9-induced DNA breaks

BackgroundCRISPR-Cas9 genome editing often induces unintended, large genomic rearrangements, posing potential safety risks. However, there are no methods for mitigating these risks.ResultsUsing long-read individual-molecule sequencing (IDMseq), we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs, while depleting or overexpressing RPA increases or reduces LD frequency, respectively. Interestingly, small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells.ConclusionsOur findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR, suggesting new strategies for safer and more precise genome editing.

Melanoma-Derived DNA Polymerase Theta Variants Exhibit Altered DNA Polymerase Activity.

DNA polymerase θ (Pol θ or POLQ) is primarily involved in repairing double-stranded breaks in DNA through an alternative pathway known as microhomology-mediated end joining (MMEJ) or theta-mediated end joining (TMEJ). Unlike other DNA repair polymerases, Pol θ is thought to be highly error-prone yet critical for cell survival. We have identified several POLQ gene variants from human melanoma tumors that experience altered DNA polymerase activity, including a propensity for incorrect nucleotide selection and reduced polymerization rates compared to WT Pol θ. Variants are 30-fold less efficient at incorporating a nucleotide during repair and up to 70-fold less accurate at selecting the correct nucleotide opposite a templating base. This suggests that aberrant Pol θ has reduced DNA repair capabilities and may also contribute to increased mutagenesis. Moreover, the variants were identified in established tumors, suggesting that cancer cells may use mutated polymerases to promote metastasis and drug resistance.

Abstract CT169: First-in-human, phase 1/2 study of GSK4524101, an oral DNA polymerase theta inhibitor (POLQi), alone or combined with the poly(ADP-ribose) polymerase (PARP) inhibitor (PARPi) niraparib in adults with solid tumors

Abstract Background: Double-stranded DNA breaks (DSBs) in human cells are typically repaired via nonhomologous end joining (NHEJ) or homologous recombination (HR). Deficiency of HR-mediated DNA repair plays a role in the initiation and progression of many tumor types. In tumors with HR deficiency, PARP inhibition leads to generation of DSBs that cannot be effectively repaired because of the HR defect, resulting in synthetic lethality. This finding has led to the clinical development and approval of several PARPi for the treatment of various tumors including certain ovarian, breast, prostate, and pancreatic cancers that are prone to display HR deficiency (HRd). DNA polymerase theta (encoded by POLQ) mediates an alternative DNA repair mechanism, microhomology-mediated end joining (MMEJ). DNA polymerase theta is generally not detectable in normal tissues but is upregulated in many tumor types. In preclinical studies, POLQi plus PARPi treatment demonstrated superior efficacy to PARPi alone in preventing the growth of HRd tumors. To evaluate the clinical potential of combining POLQi and PARPi, this first-in-human study investigates treatment with GSK4524101, an investigational POLQi, with or without the PARPi niraparib, in patients with solid tumors. Methods: This open-label, multicenter, phase 1/2 study (NCT06077877) opened in October 2023 and comprises dose-finding (part 1, including a food-effect cohort) and dose-expansion (part 2) parts. This trial aims to assess the maximum-tolerated dose, pharmacokinetics, safety, and preliminary antitumor activity of oral GSK4524101 with or without niraparib. The primary endpoints are safety (part 1) and confirmed objective response rate (part 2). Secondary endpoints include pharmacokinetics, safety, progression-free survival (part 2), and response duration (part 2). Up to 135 patients may be enrolled. To be eligible, patients must be aged ≥18 years; have an advanced or metastatic solid tumor, an Eastern Cooperative Oncology Group performance status score of 0-2, and a life expectancy of ≥3 months; and have exhausted all standard treatment options. Individuals are ineligible if they have not recovered from chemotherapy-associated adverse events or have symptomatic uncontrolled brain or leptomeningeal metastases, a history of myelodysplastic syndrome or acute myeloid leukemia, uncontrolled hypertension, or a second malignancy that has progressed or required active treatment in the previous 2 years. The study is actively recruiting in the US and Canada; as of January 1, 2024, 1 patient has been dosed. Patients enrolled in part 1 will receive GSK4524101 alone or GSK4524101 plus niraparib; patients enrolled in part 2 will be randomized to either GSK4524101 plus niraparib or niraparib alone. Part 1 of this study is expected to complete in 2025. Citation Format: Vivek Samnotra, Veronica Moroz, Luda Shtessel, Mita Kuchimanchi, Patrick Hanafin, Malar Pannirselvam, Aishwarya Bhaskar, Andrew Liu, Haluk Yuzugullu, Minal Barve, David Sommerhalder, Timothy Yap, Michele Sanicola-Nadel. First-in-human, phase 1/2 study of GSK4524101, an oral DNA polymerase theta inhibitor (POLQi), alone or combined with the poly(ADP-ribose) polymerase (PARP) inhibitor (PARPi) niraparib in adults with solid tumors [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 CT169.

Inspiring basic and applied research in genome integrity mechanisms: Dedication to Samuel H. Wilson.

This Special Issue (SI) of Environmental and Molecular Mutagenesis (EMM), entitled "Inspiring Basic and Applied Research in Genome Integrity Mechanisms," is to update the community on recent findings and advances on genome integrity mechanisms with emphasis on their importance for basic and environmental health sciences. This SI includes two research articles, one brief research communication, and four reviews that highlight cutting edge research findings and perspectives, from both established leaders and junior trainees, on DNA repair mechanisms. In particular, the authors provided an updated understanding on several distinct enzymes (e.g., DNA polymerase beta, DNA polymerase theta, DNA glycosylase NEIL2) and the associated molecular mechanisms in base excision repair, nucleotide excision repair, and microhomology-mediated end joining of double-strand breaks. In addition, genome-wide sequencing analysis or site-specific mutational signature analysis of DNA lesions from environmental mutagens (e.g., UV light and aflatoxin) provide further characterization and sequence context impact of DNA damage and mutations. This SI is dedicated to the legacy of Dr. Samuel H. Wilson from the U.S. National Institute of Environmental Health Sciences at the National Institutes of Health.

Developmental progression of DNA double-strand break repair deciphered by a single-allele resolution mutation classifier

DNA double-strand breaks (DSBs) are repaired by a hierarchically regulated network of pathways. Factors influencing the choice of particular repair pathways, however remain poorly characterized. Here we develop an Integrated Classification Pipeline (ICP) to decompose and categorize CRISPR/Cas9 generated mutations on genomic target sites in complex multicellular insects. The ICP outputs graphic rank ordered classifications of mutant alleles to visualize discriminating DSB repair fingerprints generated from different target sites and alternative inheritance patterns of CRISPR components. We uncover highly reproducible lineage-specific mutation fingerprints in individual organisms and a developmental progression wherein Microhomology-Mediated End-Joining (MMEJ) or Insertion events predominate during early rapid mitotic cell cycles, switching to distinct subsets of Non-Homologous End-Joining (NHEJ) alleles, and then to Homology-Directed Repair (HDR)-based gene conversion. These repair signatures enable marker-free tracking of specific mutations in dynamic populations, including NHEJ and HDR events within the same samples, for in-depth analysis of diverse gene editing events.

Abstract 705: miR-21-5p induces radioresistance through reinforced DNA damage response in oral squamous cell carcinoma

Abstract Cancers arising from oral cavity and oropharynx are commonly treated with radiotherapy, whereas tumor radioresistance results in poor outcome. The prerequisite for developing more effective interventions for these patients is that the molecular mechanisms leading to a radioresistant phenotype in these tumors are clear to researchers. Unfortunately, the radioresistant mechanisms remain elusive to date. We recently identified that a highly conserved and cancer-related microRNA, i.e. miR-21-5p, plays an important role in radioresistance of oral squamous cell carcinoma (OSCC). We analyzed the RNA-sequencing data of 290 tissue samples from TCGA database, and found the expression of miR-21-5p was significantly upregulated in many OSCC samples (N=271) when comparing to adjacent normal tissues (N=19). The difference was further confirmed on our freshly collected OSCC tissues from patients in operation room. We performed RNA-sequencing analysis in 11 OSCC samples and their adjacent normal tissues. Among the 2106 differentially expressed genes between OSCC and normal control groups, we found that several important DNA repair genes (RAD50 double strand break repair protein gene, RAD50; DNA Polymerase Theta, POLQ; Ataxia Telangiectasia and Rad3-related, ATR) were significantly overexpressed in OSCC samples (P<0.05), while positive correlations between miR-21-5p expression and DNA repair genes RAD51 and XRCC4 were identified in the OSCC cohort. Notably, ATR is an important DNA damage response gene, while XRCC4, RAD51, and POLQ are involved in DNA double strand break (DSB) repair through non-homologous end joining, homologous recombination, and microhomology-mediated end joining, respectively. OSCC cell lines were further recruited to study the relations of miR-21-5p with radiation-induced DSB repair. By analyzing kinetics of 53BP1 foci, a biomarker for detecting DSBs, we found that anti-miR-21-5p transfected cells showed more residual foci, indicating of unfinished repair of DSBs, at 5 hours and 18 hours post irradiation. The deficient DSB repair resulted from anti-miR-21-5p transfection were further confirmed by single cell gel electrophoresis with comet assay. Consistently, the same treatment resulted in fewer clonogenic survivors in colony formation assay, demonstrating increased radiosensitivity of OSCC cells when miR-21-5p was antagonized. This work provided novel insights about the mechanism of miR-21-5p-induced radioresistance in OSCC, which may give rise to novel therapeutic strategy for improved treatment of OSCC patients. Citation Format: Qi Liu, Weitao Hu, Xinghan Li, Yongqiang Deng, Lin Ma. miR-21-5p induces radioresistance through reinforced DNA damage response in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 705.

Abstract 4532: Discovery of DAT-1000A, a potent Polθ inhibitor that significantly enhances anti-tumor efficacy in combination with PARP inhibitor in homologous-recombination-deficient tumors

Abstract Cancers exhibiting homologous recombination (HR) repair deficiencies due to mutations in genes like BRCA1 or BRCA2 rely on alternative DNA damage response (DDR) pathways to uphold genomic integrity. This vulnerability allows for the targeted disruption of these pathways through synthetic lethality approaches. DNA polymerase theta (Polθ, encoded by POLQ) assumes a crucial role in mending DNA double-strand breaks (DSB) via microhomology-mediated end-joining (MMEJ), one of the principal pathways for repairing DNA DSB. Notably, Polθ expression is minimal in normal tissues but becomes upregulated in HR-deficient cancer cells, positioning Polθ as a prime target for synthetic lethality in HRD cancers. Reversion mutations in HR-related genes frequently employ micro-homology deletion mechanisms. Consequently, the inhibition of Polθ holds the potential to thwart the emergence of PARPi resistance, primarily driven by BRCA reversion mutations. In this context, we introduce DAT-1000A, a novel small-molecule Polθ inhibitor exhibiting potent activity with a single-digital nanomolar IC50 value. Its efficacy in MMEJ functional assays validates its on-target cellular activity. Assessments of cell viability in HR-deficient BRCA2-KO DLD-1 cells, compared to their parental counterparts, reveal a striking synthetic lethality window exceeding 500-fold. Additionally, DAT-1000A displays synergistic anti-proliferative effects when combined with PARP inhibitors in BRCA1/2-mutated cells. In BRCA1/2-mutated xenograft models, DAT-1000A demonstrates robust and sustained tumor regression, as evidenced by the level of γH2AX, a common marker for double-strand breaks, which closely correlates with anti-tumor efficacy. Importantly, high-dose DAT-1000A treatment in mice elicits no significant abnormalities, underscoring its excellent tolerability. Collectively, DAT-1000A emerges as a novel Polθ inhibitor, offering both potent activity and a favorable safety profile, and holds promise for advancing therapeutic options in the realm of HRD cancer treatment. Citation Format: Dan Yan, Yao Zhang, Yan Zhang, Jingxi Zhang, Bin Wang, Huixian Chen, Jingxue Shi, Xiaoling Lin, Jincong Zhuo, Kevin Zhou. Discovery of DAT-1000A, a potent Polθ inhibitor that significantly enhances anti-tumor efficacy in combination with PARP inhibitor in homologous-recombination-deficient tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4532.

Abstract 4539: ZM-2311 a novel and highly potent Polθ inhibitor demonstrates synergistically anti-tumor activities in combination with different therapeutics

Abstract Homologous recombination (HR) is a high-fidelity DNA double-strand break (DSB) repair pathway, and its dysfunction leads to cell genome instability and can result in tumorigenesis. In the situation of HR deficiency, DNA polymerase theta (Polθ) mediated microhomology-mediated end joining (MMEJ) is up-regulated to serve as a backup pathway for DSB repair. Several studies have proved that the inhibition of Polθ causes synthetic lethality with HR deficiency, and Polθ emerges as a potential DNA damage repair (DDR) target for the treatment of HR deficient tumors. In addition, there is evidence showing that the depletion of Polθ improves the sensitivity of some other DDR-related targeted therapies, chemotherapy, and radiotherapy. Here we identify a novel small molecular Polθ inhibitor, ZM-2311, which inhibits Polθ activity with an IC50 of 24 nM, and strongly inhibits cellular MMEJ pathway with a single nanomolar IC50. ZM-2311 illustrates a robust potency against tumor growth in a MDA-MB-436 SHLD2-/- xenograft model, suggesting potential monotherapy application in BRCA1 mutation patients with TP53BP1/Shieldin complex deficiency. Besides monotherapy, ZM-2311 elicits strong synergetic anti-proliferation activities in combination with PARP inhibitors on BRCA2-/- DLD-1 and endogenous HR deficient tumor cells in vitro and in vivo. Tumor regression is observed even at a low dosage, indicating a high potency of ZM-2311. As reversion mutation of HR genes such as BRCA1/2 mediated by the MMEJ pathway is reported to be a mechanism of PARP inhibitor resistance, ZM-2311 is evaluated and demonstrates its potential to overcome the resistance to PARP inhibitors. Furthermore, the combination of ZM-2311 with other DDR agents, such as ATR inhibitors, induces fatal DNA damage to HR deficient cells. Considering a low risk of hematotoxicity of Polθ inhibition, ZM-2311 may provide a new combination option for DDR agents. Besides, ZM-2311 was assessed in combination with different therapies respectively which functionally induce DNA damage, such as radiotherapy and chemotherapy. ZM-2311 renders tumor cells more sensitive to radiotherapy in both HR deficient cells and proficient cells in vitro, accompanying with the upregulation of Polθ expression after radiotherapy and an enhanced phosphorylation level of H2AX in the combination treatment. Similarly, ZM-2311 also achieves a robust synergetic effect in combination with chemotherapy in cell lines with different HR statuses. These results indicate a potential application expansion of ZM-2311 to HR-proficient tumors, which is expected to bring better efficacy and reduced hematotoxicity. Taken together, ZM-2311 has proved to be a highly potent Polθ inhibitor and has promising combination potential with multiple therapy strategies. Citation Format: Feng Zhou, Lu Liu, Lei Jiang, Shuo Qian, Liting Xue, Mengying Li, Zhengtao Li, Zhen Li, Jian Li, Renhong Tang. ZM-2311 a novel and highly potent Polθ inhibitor demonstrates synergistically anti-tumor activities in combination with different therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4539.

DNA-PKcs suppresses illegitimate chromosome rearrangements.

Two DNA repair pathways, non-homologous end joining (NHEJ) and alternative end joining (A-EJ), are involved in V(D)J recombination and chromosome translocation. Previous studies reported distinct repair mechanisms for chromosome translocation, with NHEJ involved in humans and A-EJ in mice predominantly. NHEJ depends on DNA-PKcs, a critical partner in synapsis formation and downstream component activation. While DNA-PKcs inhibition promotes chromosome translocations harboring microhomologies in mice, its synonymous effect in humans is not known. We find partial DNA-PKcs inhibition in human cells leads to increased translocations and the continued involvement of a dampened NHEJ. In contrast, complete DNA-PKcs inhibition substantially increased microhomology-mediated end joining (MMEJ), thus bridging the two different translocation mechanisms between human and mice. Similar to a previous study on Ku70 deletion, DNA-PKcs deletion in G1/G0-phase mouse progenitor B cell lines, significantly impairs V(D)J recombination and generated higher rates of translocations as a consequence of dysregulated coding and signal end joining. Genetic DNA-PKcs inhibition suppresses NHEJ entirely, with repair phenotypically resembling Ku70-deficient A-EJ. In contrast, we find DNA-PKcs necessary in generating the near-exclusive MMEJ associated with Lig4 deficiency. Our study underscores DNA-PKcs in suppressing illegitimate chromosome rearrangement while also contributing to MMEJ in both species.

Study of PARP inhibitors for breast cancer based on enhanced multiple kernel function SVR with PSO.

PARP1 is one of six enzymes required for the highly error-prone DNA repair pathway microhomology-mediated end joining (MMEJ) and needs to be inhibited when over-expressed. In order to study the PARP1 inhibitory effect of fused tetracyclic or pentacyclic dihydrodiazepinoindolone derivatives (FTPDDs) by quantitative structure-activity relationship technique, six models were established by four kinds of methods, heuristic method, gene expression programming, random forester, and support vector regression with single, double, and triple kernel function respectively. The single, double, and triple kernel functions were RBF kernel function, the integration of RBF and polynomial kernel functions, and the integration of RBF, polynomial, and linear kernel functions respectively. The problem of multi-parameter optimization introduced in the support vector regression model was solved by the particle swarm optimization algorithm. Among the models, the model established by support vector regression with triple kernel function, in which the optimal R 2 and RMSE of training set and test set were 0.9353, 0.9348 and 0.0157, 0.0288, and R2 cv of training set and test set were 0.9090 and 0.8971, shows the strongest prediction ability and robustness. The method of support vector regression with triple kernel function is a great promotion in the field of quantitative structure-activity relationship, which will contribute a lot to designing and screening new drug molecules. The information contained in the model can provide important factors that guide drug design. Based on these factors, six new FTPDDs have been designed. Using molecular docking experiments to determine the properties of new derivatives, the new drug was ultimately successfully designed.

POLQ identifies a better response subset to immunotherapy in muscle-invasive bladder cancer with high PD-L1.

Though programmed cell death-ligand 1 (PD-L1) has been used in predicting the efficacy of immune checkpoint blockade (ICB), it is insufficient as a single biomarker. As a key effector of an intrinsically mutagenic microhomology-mediated end joining (MMEJ) pathway, DNA polymerase theta (POLQ) was overexpressed in various malignancies, whose expression might have an influence on genomic stability, therefore altering the sensitivity to chemotherapy and immunotherapy. A total of 1304 patients with muscle-invasive bladder cancer (MIBC) from six independent cohorts were included in this study. The Zhongshan Hospital (ZSHS) cohort (n = 134), The Cancer Genome Atlas (TCGA) cohort (n = 391), and the Neo-cohort (n = 148) were included for the investigation of chemotherapeutic response. The IMvigor210 cohort (n = 234) and the UNC-108 cohort (n = 89) were used for the assessment of immunotherapeutic response. In addition, the relationship between POLQ and the immune microenvironment was assessed, and GSE32894 (n = 308) was used only for the evaluation of the immune microenvironment. We identified POLQhigh PD-L1high patients could benefit more from immunotherapy and platinum-based chemotherapy. Further analysis revealed that high POLQ expression was linked to chromosome instability and higher tumor mutational burden (TMB), which might elicit the production of neoantigens. Further, high POLQ expression was associated with an active tumor immune microenvironment with abundant infiltration of immune effector cells and molecules. The study demonstrated that high POLQ expression was correlated with chromosome instability and antitumor immune microenvironment in MIBC, and the combination of POLQ and PD-L1 could be used as a superior companion biomarker for predicting the efficacy of immunotherapy.

CRISPR/Cas9 improves targeted knock-in efficiency in Aspergillusoryzae

Aspergillus oryzae is an important fungus in food and industrial enzyme production. In A. oryzae, targeted knock-in transformation is primarily limited to homologous recombination (HR)-based systems, in which non-homologous end-joining (NHEJ)-disruptant hosts are required. However, preparation of hosts and transformation templates for such systems is laborious, in addition to other disadvantages. In the present study, we examined alternative targeted knock-in mediated by CRISPR/Cas9, in which a microhomology-mediated end-joining (MMEJ) and single-strand annealing (SSA) repair system was employed. This approach enabled the efficient development of targeted knock-in transformants without host preparation using only a short homology template. We conclude that this new method could be applied to facilitate the transformation of A. oryzae, and will make it easier to acquire targeted knock-in transformants, especially from industrially important non-model strains.

Gross Chromosomal Rearrangement at Centromeres.

Centromeres play essential roles in the faithful segregation of chromosomes. CENP-A, the centromere-specific histone H3 variant, and heterochromatin characterized by di- or tri-methylation of histone H3 9th lysine (H3K9) are the hallmarks of centromere chromatin. Contrary to the epigenetic marks, DNA sequences underlying the centromere region of chromosomes are not well conserved through evolution. However, centromeres consist of repetitive sequences in many eukaryotes, including animals, plants, and a subset of fungi, including fission yeast. Advances in long-read sequencing techniques have uncovered the complete sequence of human centromeres containing more than thousands of alpha satellite repeats and other types of repetitive sequences. Not only tandem but also inverted repeats are present at a centromere. DNA recombination between centromere repeats can result in gross chromosomal rearrangement (GCR), such as translocation and isochromosome formation. CENP-A chromatin and heterochromatin suppress the centromeric GCR. The key player of homologous recombination, Rad51, safeguards centromere integrity through conservative noncrossover recombination between centromere repeats. In contrast to Rad51-dependent recombination, Rad52-mediated single-strand annealing (SSA) and microhomology-mediated end-joining (MMEJ) lead to centromeric GCR. This review summarizes recent findings on the role of centromere and recombination proteins in maintaining centromere integrity and discusses how GCR occurs at centromeres.

Quantitative, titratable and high-throughput reporter assays to measure DNA double strand break repair activity in cells.

Repair of DNA damage is essential for the maintenance of genome stability and cell viability. DNA double strand breaks (DSBs) constitute a toxic class of DNA lesion and multiple cellular pathways exist to mediate their repair. Robust and titratable assays of cellular DSB repair (DSBR) are important to functionally interrogate the integrity and efficiency of these mechanisms in disease models as well as in response to genetic or pharmacological perturbations. Several variants of DSBR reporters are available, however these are often limited by throughput or restricted to specific cellular models. Here, we describe the generation and validation of a suite of extrachromosomal reporter assays that can efficiently measure the major DSBR pathways of homologous recombination (HR), classical nonhomologous end joining (cNHEJ), microhomology-mediated end joining (MMEJ) and single strand annealing (SSA). We demonstrate that these assays can be adapted to a high-throughput screening format and that they are sensitive to pharmacological modulation, thus providing mechanistic and quantitative insights into compound potency, selectivity, and on-target specificity. We propose that these reporter assays can serve as tools to dissect the interplay of DSBR pathway networks in cells and will have broad implications for studies of DSBR mechanisms in basic research and drug discovery.

Insertion sequence excision is enhanced by a protein that catalyzes branch migration and promotes microhomology-mediated end joining.

Insertion sequence (IS)-excision enhancer (IEE) promotes the excision of ISs in the genome of enterohemorrhagic Escherichia coli O157. Because IEE-dependent IS excision occurs in the presence of transposase, the process of IS transposition may be involved in IS excision; however, little is understood about the molecular mechanisms of IS excision. Our in vitro analysis revealed that IEE exhibits DNA-dependent ATPase activity, which is activated by branched DNA. IEE also catalyzes the branch migration of fork-structured DNA. These results suggest that IEE remodels branched structures of the IS transposition intermediate. Sequence analysis of recombination sites in IS-excision products suggested that microhomologous sequences near the ends of the IS are involved in IS excision. IEE promoted microhomology-mediated end joining (MMEJ), in which base pairing between 6-nucleotides complementary ends of two 3'-protruding DNAs and subsequent elongation of the paired DNA strand occurred. IS-excision frequencies were significantly decreased in cells producing IEE mutants that had lost either branch migration or MMEJ activity, which suggests that these activities of IEE are required for IS excision. Based on our results, we propose a model for IS excision triggered by IEE and transposase.

DNA polymerases in precise and predictable CRISPR/Cas9-mediated chromosomal rearrangements

BackgroundRecent studies have shown that, owning to its cohesive cleavage, Cas9-mediated CRISPR gene editing outcomes at junctions of chromosomal rearrangements or DNA-fragment editing are precise and predictable; however, the underlying mechanisms are poorly understood due to lack of suitable assay system and analysis tool.ResultsHere we developed a customized computer program to take account of staggered or cohesive Cas9 cleavage and to rapidly process large volumes of junctional sequencing reads from chromosomal rearrangements or DNA-fragment editing, including DNA-fragment inversions, duplications, and deletions. We also established a sensitive assay system using HPRT1 and DCK as reporters for cell growth during DNA-fragment editing by Cas9 with dual sgRNAs and found prominent large resections or long deletions at junctions of chromosomal rearrangements. In addition, we found that knockdown of PolQ (encoding Polθ polymerase), which has a prominent role in theta-mediated end joining (TMEJ) or microhomology-mediated end joining (MMEJ), results in increased large resections but decreased small deletions. We also found that the mechanisms for generating small deletions of 1bp and >1bp during DNA-fragment editing are different with regard to their opposite dependencies on Polθ and Polλ (encoded by the PolL gene). Specifically, Polθ suppresses 1bp deletions but promotes >1bp deletions, whereas Polλ promotes 1bp deletions but suppresses >1bp deletions. Finally, we found that Polλ is the main DNA polymerase responsible for fill-in of the 5′ overhangs of staggered Cas9 cleavage ends.ConclusionsThese findings contribute to our understanding of the molecular mechanisms of CRISPR/Cas9-mediated DNA-fragment editing and have important implications for controllable, precise, and predictable gene editing.

An Efficient CRISPR/Cas9 Genome Editing System for a Ganoderma lucidum Cultivated Strain by Ribonucleoprotein Method.

The CRISPR/Cas9 system has become a popular approach to genome editing. Compared with the plasmid-dependent CRISPR system, the ribonucleoprotein (RNP) complex formed by the in vitro assembly of Cas9 and single-guide RNA (sgRNA) has many advantages. However, only a few examples have been reported and the editing efficiency has been relatively low. In this study, we developed and optimized an RNP-mediated CRISPR/Cas9 genome editing system for the monokaryotic strain L1 from the Ganoderma lucidum cultivar 'Hunong No. 1'. On selective media containing 5-fluoroorotic acid (5-FOA), the targeting efficiency of the genomic editing reached 100%. The editing efficiency of the orotidine 5'-monophosphate decarboxylase gene (ura3) was greater than 35 mutants/107 protoplasts, surpassing the previously reported G. lucidum CRISPR systems. Through insertion or substitution, 35 mutants introduced new sequences of 10-569 bp near the cleavage site of ura3 in the L1 genome, and the introduced sequences of 22 mutants (62.9%) were derived from the L1 genome itself. Among the 90 mutants, 85 mutants (94.4%) repaired DNA double-strand breaks (DSBs) through non-homologous end joining (NHEJ), and five mutants (5.6%) through microhomology-mediated end joining (MMEJ). This study revealed the repair characteristics of DSBs induced by RNA-programmed nuclease Cas9. Moreover, the G. lucidum genes cyp512a3 and cyp5359n1 have been edited using this system. This study is of significant importance for the targeted breeding and synthetic metabolic regulation of G. lucidum.

Clonal Targeting of DNA Damage Response Pathways Eradicates Myeloproliferative Neoplasms

Clonal diversity of myeloproliferative neoplasms (MPN) plays a key role in poor therapeutic outcomes. MPN cells usually accumulate spontaneous DNA damage including highly toxic DNA double-strand breaks (DSBs) induced by metabolic products and replication stress. To repair numerous DSBs and survive, MPN cells activate the DNA damage response (DDR) involving the pathways that sense (ATM and ATR kinases) and repair (RAD51-mediated homologous recombination = HR, RAD52-mediated transcription associated homologous recombination = TA-HR and single strand annealing = SSA, DNA-PK -mediated non-homologous end-joining = NHEJ, PARP1/Polq-dependent microhomology-mediated end-joining = MMEJ) DSBs. Thus, DDR is a legitimate therapeutic target. Numerous MPN-driving mutations [ JAK2(V617F), TETmut, DNMT3Amut, IDH1mut] regulate DDR and affect the sensitivity of leukemia cells to DDR inhibitors (DDRi). The genetic landscape of malignant clones in a patient may be complicated since individual clones can carry multiple mutations. Thus, individual MPN clones may respond differently to DDRi depending on their mutational profile. In our experimental protocol MPN cells from individual patients were treated with various DDRi followed by single-cell targeted DNA sequencing (sctDNA-seq) to integrate patient's leukemia clonal composition with response to the inhibitors. We developed a sctDNA-seq myeloid platform interrogating up to 1394 genetic variants of 54 known leukemia driver genes, which unraveled the clonal landscape of MPN at a single-cell resolution before and after the treatment. Based on these results we designed a “clonal attack”, a patient-tailored combination of DDRi targeting all MPN clones in a patient sample. Here we show that DDRi simultaneously attacking different clones caused MPN clonal attrition in vitro. For example, phylogenetic tree analysis of MPN patient P349 sample detected linear multi-clonal architecture with 3 Lin-CD34+ clones carrying specific sets of mutations. sctDNA-seq followed by fish plot analysis revealed clonal similarities and differences in response to DDRi. The clone carrying KMT2A(L2373H) + SETBP1(H1100R) was more sensitive to ATRi than RAD52i, conversely clones with KMT2A(L2373H) alone and KMT2A(L2373H) + SETBP1(H1100R) + FLT3(R834L) were more sensitive to RAD52i than ATRi. Based on this observation, we hypothesized that simultaneous treatment with ATRi+RAD52i should result in elimination of all 3 MPN P349 clones. Remarkably, the combination of RAD52i + ATRi was >100x more effective in inhibiting clonogenic growth of Lin-CD34+ P349 cells (all clonogenic cells were eradicated). On the other hand, combination of RAD52i + ATMi, the two DDR inhibitors displaying similar pattern of clonal targeting was only 2x better than individual inhibitors. Phylogenetic tree analysis of Lin-CD34+ cells from another MPN patient P350 sample showed linear multi-clonal architecture with 4 clones. Again, sctDNA-seq followed by fish plot analysis revealed clonal similarities and differences in response to DDRi. All 4 clones displayed similar sensitivity to RAD52i and ATMi, but they responded differently to PARPi and ATRi. Clones carrying TET2(P363L) + NRAS(G12D) were more sensitive to ATRi than PARPi, whereas clones with TET2(P363L) + NRAS(G12D) + DNMT3A(W330C) and TET2(P363L) + NRAS(G12D) + DNMT3A(W330C) + IDH2(R132C) responded better to PARPi than ATRi. Importantly, the combination of PARPi + ATRi was >9x more effective in inhibiting clonogenic growth of Lin-CD34+ P349 cells, whereas RAD52i + ATMi was only 2x better than individual inhibitors. In conclusion, we postulate that sctDNA-seq combined with in vitro DDRi sensitivity testing (sctDNA-seq/DDRi) is a powerful tool to interrogate clonal sensitivity of MPN to these agents. This clonal medicine approach may become a novel therapeutic regimen to overcome clonal complexity of MPN in a cohort of patients. The “clonal attack” by DDR inhibitors shifts the paradigm of genotoxic therapies from those using non-discriminative cytotoxic drugs to those selectively attacking DDR vulnerabilities in MPN clones with minimal harm to normal cells. Since clonal heterogeneity and DNA damage are hallmarks of cancer, the “clonal attack” may be broadly applicable to the quest for cancer cure.

Clonal Medicine Targeting DNA Damage Response Eradicates AML

Clonal diversity of acute myeloid leukemia (AML) plays a key role in poor therapeutic outcomes. AML cells usually accumulate spontaneous DNA damage including highly toxic DNA double-strand breaks (DSBs) induced by metabolic products and replication stress. To repair numerous DSBs and survive, AML cells activate the DNA damage response (DDR) involving the pathways that sense (ATM and ATR kinases) and repair (RAD51-mediated homologous recombination = HR, RAD52-mediated transcription associated homologous recombination = TA-HR and single strand annealing = SSA, DNA-PK -mediated non-homologous end-joining = NHEJ, PARP1/Polq-dependent microhomology-mediated end-joining = MMEJ) DSBs. Thus, DDR is a legitimate therapeutic target. Numerous AML-driving mutations [ FLT3(ITD), TETmut, DNMT3Amut, AML1-ETO, PML-RARa , IDH1mut, cKITmut] regulate DDR and affect the sensitivity of leukemia cells to DDR inhibitors (DDRi). The genetic landscape of malignant clones in a patient may be complicated since individual clones can carry multiple mutations. Thus, individual AML clones may respond differently to DDRi depending on their mutational profile. In our experimental protocol AML cells from individual patients were treated with various DDRi followed by single-cell targeted DNA sequencing (sctDNA-seq) to integrate patient's leukemia clonal composition with response to the inhibitors. We developed a sctDNA-seq myeloid platform interrogating up to 1394 genetic variants of 54 known leukemia driver genes, which unraveled the clonal landscape of AML at a single-cell resolution before and after the treatment. Based on these results we designed a “clonal attack”, a patient-tailored combination of DDRi targeting all AML clones in a patient sample. Here we show that DDRi simultaneously attacking different clones caused clonal attrition and eradicated the disease in vitro and in vivo. For example, in AML-MD2 sample 2 clones carrying EZH2(V679M) + TET2(L1721W) and EZH2(V679M) + TET2(L1721W) + FLT3(D835Y) were resistant/less responsive to ATRi but sensitive to PARPi, ATMi and RAD52i. Conversely, three other clones carrying EZH2(V679M) + TET2(L1721W) + RUNX1(D160Y), EZH2(V679M) + TET2(L1721W) + RUNX1(D160Y) + EZH2(E54*) and EZH2(V679M) + TET2(L1721W) + RUNX1(D160Y)EZH2(E54*) + BCOR1(R1334Tfs*32) + NRAS(G13R) were sensitive to ATRi while less responsive to PARPi, ATMi and RAD52i. Based on this observation, we hypothesized that simultaneous treatment with ATRi + PARPi or ATRi + ATMi should result in elimination of all AML-MD2 clones. In agreement with this hypothesis, in vitro treatment with these combinations was 20 - 30x more effective in eliminating clonogenic growth of Lin-CD34+ AML-MD2 cells when compared to individual inhibitors. This effect was associated with abundant accumulation of DSBs. Combinations of these inhibitors were only modestly toxic to normal hematopoietic cells. Moreover, combination of DDRi displaying similar clonal targeting specificity was only 2x better than individual inhibitors. Next, humanized NRGS immunodeficient mice bearing primary AML-MD2 xenograft were treated with vehicle, PARPi, ATRi and the combination of these drugs. We found that individual treatments reduced the number of hCD45+ AML-MD2 cells in bone marrow and spleen by approximately 2x and 3x, respectively. Remarkably, the combination of PARPi + ATRi eliminated more than 99% of leukemia cells in bone marrows of 7/10 mice and in spleens of 10/10 mice while no obvious toxicity was observed. Clonal targeting by DDR inhibitors, however, may not be applicable to all AML samples. For example, we identified AML patient samples, where clones displayed similar sensitivity pattern or were resistant to DDRi treatment. In conclusion, we postulate that sctDNA-seq combined with in vitro DDRi sensitivity testing (sctDNA-seq/DDRi) is a powerful tool to interrogate clonal sensitivity of AML to these agents. This clonal medicine approach may become a novel therapeutic regimen to overcome clonal complexity of AML in a cohort of patients. The “clonal attack” by DDR inhibitors shifts the paradigm of genotoxic therapies from those using non-discriminative cytotoxic drugs to those selectively attacking DDR vulnerabilities in AML clones with minimal harm to normal cells. Since clonal heterogeneity and DNA damage are hallmarks of cancer, the “clonal attack” may be broadly applicable to the quest for cancer cure.

Mutation of Epigenetic Regulators at Diagnosis Is an Independent Predictor of Tyrosine Kinase Inhibitor Treatment Failure: A Report from the Residiag Study

Introduction Additional mutations at chronic myeloid leukemia (CML) diagnosis have been shown to variably affect tyrosine kinase inhibitor (TKI) response and were inconstantly detected at loss of response. Contradictory observations may have resulted from difficulties in reliably inferring CML clonal architecture from mutations quantified by NGS, BCR::ABL1 by qRT-PCR, ABL1-TK by RNA-Seq. The RESIDIAG study was designed to identify at diagnosis molecular markers predictive of TKI treatment failure and analyze the clonal dynamics using asymmetric capture sequencing (aCAP-Seq). Methods DNA samples from 60 TKI responders (median follow-up: 7.1 years) were sequenced at diagnosis by aCAP-Seq, allowing quantification and breakpoint sequencing of genomic BCR::ABL1 fusion together with 43 myeloid genes, including ABL1 with a limit of detection of ~0.1% for BCR::ABL1 and 0.5-1% for SNVs and Indels. In parallel, CML patients who experienced treatment failure (ELN 2020) were sequentially analyzed at diagnosis and at failure (n=53), at diagnosis only (n=1), and at failure only (n=3). Small deletions and BCR::ABL1 breakpoints were analyzed to search for microhomology-mediated end-joining (MMEJ) repair signatures. Mutation analysis followed strict medical standards. Results The proportion of patients receiving imatinib at diagnosis wasn't statistically different (p=0.08; Pearson's Chi-square test) between responders (70%) and the failure group (56%). At diagnosis, the number of mutations was higher (p<0.001, t-test with Welch correction) in the failure group (0.9±0.1) as compared with responders (0.18± 0.05). ASXL1, DNMT3A, and TET2 were more frequently mutated at diagnosis in the failure group (38.6%, p<0.001; 10.5%, p=0.01, and 4.3%, p=0.025 respectively; Pearson's Chi²test) as compared with responders (6.7%, 0% and 0% respectively) and the presence of a mutation in one of these genes was highly associated with a reduced failure-free survival (p<0.001, Log-ranked test, Figure 1). An MMEJ signature at BCR::ABL1 breakpoints was more frequent (p=0.014, Pearson's Chi² test) in the failure group (29.1%) than in the responder group (9.1%). and was significantly associated with the ELTS score (p=0.005, Pearson's Chi² test) but not the Sokal score (p=0.017, Pearson's Chi² test). At first failure, 69% of patients had additional mutations in ASXL1 (39%), ABL1-TK (38%), DNMT3A (18%), or TET2 (15%), while other genes were rarely (<5%) mutated. ASXL1 mutations found at diagnosis were still detectable in 18/22 patients at failure and VAF suggested that double mutant BCR::ABL1/ ASXL1 clones were driving TKI failure with or without BCR:: ABL1 tyrosine kinase domain mutation (TKD). ASXL1 mutations disappearing upon treatment were mostly at low frequency <5% at diagnosis and thus possibly not clonally related to CML. Furthermore, a stepwise selection of variables followed by multivariate analysis with the Fine-Gray model identified the age of patients (sdHR [95% C.I.] = 0.99 [0.97-1.00]; p=0.08) and the presence of a mutation in epigenetics regulators at diagnosis as factors affecting the onset of TKD mutations (sdHR = 3.01 [1.76-5.15]; p<0.001). Intriguingly, co-occurring mutations in DNMT3A and TET2 were found in five patients at TKI failure. Their allelic frequency kinetics suggested that these belonged to the same clone driving CML relapse in 4/5 CML patients, while one patient also had a TKD mutation likely involved in CML relapse. Finally, multivariate COX regression analysis identified two independent predictors of TKI failure at diagnosis: a high-risk ELTS score (p=0.001; hazard ratio=3.42 [1.62-7.22] with low-risk as baseline) and a mutation in ASXL1, DNMT3A or TET2 (p=0.002; hazard ratio=2.87 [1.49-5.51]). Conclusion Altogether these results strongly point to the contribution of epigenetic regulator mutations in the emergence of TKI failure in CML and warrant further biological studies to better understand the underlying mechanisms