Chromosome Breakage Research Articles

RAD51 protects abasic sites to prevent replication fork breakage

Abasic sites are DNA lesions repaired by base excision repair. Cleavage of unrepaired abasic sites in single-stranded DNA (ssDNA) can lead to chromosomal breakage during DNA replication. How rupture of abasic DNA is prevented remains poorly understood. Here, using cryoelectron microscopy (cryo-EM), Xenopus laevis egg extracts, and human cells, we show that RAD51 nucleofilaments specifically recognize and protect abasic sites, which increase RAD51 association rate to DNA. In the absence of BRCA2 or RAD51, abasic sites accumulate as a result of DNA base methylation, oxidation, and deamination, inducing abasic ssDNA gaps that make replicating DNA fibers sensitive to APE1. RAD51 assembled on abasic DNA prevents abasic site cleavage by the MRE11-RAD50 complex, suppressing replication fork breakage triggered by an excess of abasic sites or POLθ polymerase inhibition. Our study highlights the critical role of BRCA2 and RAD51 in safeguarding against unrepaired abasic sites in DNA templates stemming from base alterations, ensuring genomic stability.

Clinical utility of relative telomere length analysis in pediatric bone marrow failure

IntroductionTelomere length related studies are limited in pediatric marrow failure cases due to difficulty in establishing population specific age related normograms. Moreover, there is paucity of data related to clinical relevance of telomere length in idiopathic aplastic anemia (IAA) and non telomere biology inherited bone marrow failure syndrome (IBMFS) cases. Methodology: Hence, in current study we investigated Relative telomere length (RTL) by RQ-PCR in 83 samples as: healthy controls (n = 44), IAA (n = 15) and IBMFS (n = 24). In addition, we performed chromosomal breakage studies and targeted NGS to screen for pathogenic variants. Results & Conclusion: Median RTL was significantly different between control vs. IBMFS (p-0.002), IAA vs. IBMFS (p-0.0075) and DC vs. non-DC IBMFS (p-0.011) but not between control vs. IAA (p-0.46). RTL analysis had clinical utility in differentiating BMF cases as 75 % (9/12) of DC had short/very short telomeres compared to only 17 % (2/12) of non-DC IBMFS, 7 % (1/15) of IAA and 7 % (3/44) of controls (p < 0.001).

HBx integration in diffuse large B-cell lymphoma inhibits Caspase-3-PARP related apoptosis

Diffuse large B-cell lymphoma (DLBCL) is the most common pathological type of non-Hodgkin lymphoma, and is closely associated with hepatitis B virus (HBV) infection status and hepatitis B X (HBx) gene integration. This project investigated the cellular biological effects and molecular mechanisms responsible for lymphomagenesis and the progression of HBx integration in DLBCL. The data showed that clinical DLBCL cells demonstrated HBx integration, and the sequencing analysis of integrated sites validated HBx integration in the constructed HBx-transfected cells. Compared with control cells, HBx-transfected cells had a significantly reduced proportion of mitochondrial membrane potential, signals of chromosomal DNA breaks, and proportion of apoptotic cells. Further studies found that this decreased apoptosis level was associated with a significant reduction of cleaved Caspase-3 and downstream poly ADP-ribose polymerase (PARP) proteins, revealing the molecular mechanisms of HBx-associated apoptosis in DLBCL. Animal experiments also demonstrated that the protein expression of cleaved Caspase-3 and PARP was prominently reduced in HBx-transfected cells from subcutaneous tumors in mice. Furthermore, the HBx-integrated cells in clinical tissues had significantly lower cleaved PARP levels than the HBx-negative samples. Therefore, HBx integration inhibits cell apoptosis through the Caspase-3-PARP pathway in DLBCL indicating a potential biomarker and therapeutic target in HBV related DLBCL.

Mapping of SINEs in the genome of Proechimys (Mammalia: Rodentia)

This study aimed to analyze the distribution of short interspersed elements (SINEs) in the chromosomes of five species of rodents of the genus Proechimys and in a variant karyotype of P. guyannensis. Molecular cytogenetic techniques were used to characterize the sequences of the B1, B4, MAR and THER SINEs, which were used as probes for hybridization in metaphase chromosomes. A wide distribution of SINEs was observed in the chromosomes of the Proechimys species examined, thus indicating differentiation of these retroelements. The signal of the B4 SINE was more evident than that of the B1 SINE, especially in P. echinothrix, P. longicaudatus, and P. cuvieri. Although the signal of the MAR SINE was more explosive than that of the THER SINE, in the species P. echinothrix, P. guyannensis (2n = 46) and P. longicaudatus, its distribution in the karyotypes was similar. The signals of these retroelements occurred at specific heterochromatic sites and were centromeric/pericentromeric and at the terminal regions in most chromosomes. This appears to be a typical distribution pattern of the SINEs and may indicate involvement with rearrangements during karyotypic diversification in Proechimys. The variation of the SINEs in the genome of Proechimys species demonstrates that these elements are distributed in a specific way in this genus and the preference for some sites, considered hotspots for chromosomal breakage, allows us to propose that these elements are related to the karyotypic evolution of Proechimys.

Causes of Chromosome Breakage and Mis-segregation Affecting Pregnancy and Newborn Health: An Insight into Developing Reproductive Health Preventive Strategies

Causes of Chromosome Breakage and Mis-segregation Affecting Pregnancy and Newborn Health: An Insight into Developing Reproductive Health Preventive Strategies

The anti-tumor effect of trifluridine via induction of aberrant mitosis is unaffected by mutations modulating p53 activity

The fluorinated thymidine analog trifluridine (FTD) is a chemotherapeutic drug commonly used to treat cancer; however, the mechanism by which FTD induces cytotoxicity is not fully understood. In addition, the effect of gain-of-function (GOF) missense mutations of the TP53 gene (encoding p53), which promote cancer progression and chemotherapeutic drug resistance, on the chemotherapeutic efficacy of FTD is unclear. Here, we revealed the mechanisms by which FTD-induced aberrant mitosis and contributed to cytotoxicity in both p53-null and p53-GOF missense mutant cells. In p53-null mutant cells, FTD-induced DNA double-stranded breaks, single-stranded DNA accumulation, and the associated DNA damage responses during the G2 phase. Nevertheless, FTD-induced DNA damage and the related responses were not sufficient to trigger strict G2/M checkpoint arrest. Thus, these features were carried over into mitosis, resulting in chromosome breaks and bridges, and subsequent cytokinesis failure. Improper mitotic exit eventually led to cell apoptosis, caused by the accumulation of extensive DNA damage and the presence of micronuclei encapsulated in the disrupted nuclear envelope. Upon FTD treatment, the behavior of the p53-GOF-missense mutant, isogenic cell lines, generated by CRISPR/Cas9 genome editing, was similar to that of p53-null mutant cells. Thus, our data suggest that FTD treatment overrode the effect on gene expression induced by p53-GOF mutants and exerted its anti-tumor activity in a manner that was independent of the p53 function.

End resection and telomere healing of DNA double-strand breaks during nematode programmed DNA elimination.

Most DNA double-strand breaks (DSBs) are harmful to genome integrity. However, some forms of DSBs are essential to biological processes, such as meiotic recombination and V(D)J recombination. DSBs are also required for programmed DNA elimination (PDE) in ciliates and nematodes. In nematodes, the DSBs are healed with telomere addition. While telomere addition sites have been well characterized, little is known regarding the DSBs that fragment nematode chromosomes. Here, we used embryos from the human and pig parasitic nematode Ascaris to characterize the DSBs. Using END-seq, we demonstrate that DSBs are introduced before mitosis, followed by extensive end resection. The resection profile is unique for each break site, and the resection generates 3'-overhangs before the addition of neotelomeres. Interestingly, telomere healing occurs much more frequently on retained DSB ends than on eliminated ends. This biased repair of the DSB ends may be due to the sequestration of the eliminated DNA into micronuclei, preventing neotelomere formation at their ends. Additional DNA breaks occur within the eliminated DNA in both Ascaris and Parascaris, ensuring chromosomal breakage and providing a fail-safe mechanism for PDE. Overall, our data indicate that telomere healing of DSBs is specific to the break sites responsible for nematode PDE.

Neotelomeres and telomere-spanning chromosomal arm fusions in cancer genomes revealed by long-read sequencing

Alterations in the structure and location of telomeres are pivotal in cancer genome evolution. Here, we applied both long-read and short-read genome sequencing to assess telomere repeat-containing structures in cancers and cancer cell lines. Using long-read genome sequences that span telomeric repeats, we defined four types of telomere repeat variations in cancer cells: neotelomeres where telomere addition heals chromosome breaks, chromosomal arm fusions spanning telomere repeats, fusions of neotelomeres, and peri-centromeric fusions with adjoined telomere and centromere repeats. These results provide a framework for the systematic study of telomeric repeats in cancer genomes, which could serve as a model for understanding the somatic evolution of other repetitive genomic elements.

First report of X-ray induced somatic mutation by Muller's department chair fails to support Muller's linearity hypothesis.

This paper reevaluates the first report of X-ray-induced somatic gene mutations. It was undertaken by John Patterson, Department Chair of Hermann Muller, using the same biological model, methods and equipment of Muller. Patterson reported X-ray induced mutation frequencies for X-chromosome-linked (sex-linked) recessive gene mutations in somatic cells of Drosophila melanogaster that resulted in color changes in the ommatidia of the eyes. Results were based on color changes detected in both male and female offspring irradiated while in egg, larval or pupal stages and for unirradiated controls. Patterson claimed that the observed dose response displayed linearity, with a clear implication that the linear response extended to background exposure levels of unirradiated controls. This reanalysis disputes Patterson's interpretation, showing that the dose response in the low-dose zone strongly supported a threshold model. The doses in the experiment, which were not clearly presented, were so high that it would preclude the assumption that the experiment provided any information of relevance to radiation exposures of humans at low doses, or even at high doses delivered at low-dose rates. Induced phenotypical changes that occurred at the higher doses, especially in female offspring, overwhelmingly resulted from X-ray-induced chromosome breaks instead of point mutations as initially expected by Patterson. The Patterson findings and linearity interpretations were an important contributory factor in the acceptance of the linear non-threshold (LNT) model during the formative time of concept consolidation. It is rather shocking now to see that the actual data provided no support for the LNT model.

DNA replication in early mammalian embryos is patterned, predisposing lamina-associated regions to fragility

DNA replication in differentiated cells follows a defined program, but when and how it is established during mammalian development is not known. Here we show using single-cell sequencing, that late replicating regions are established in association with the B compartment and the nuclear lamina from the first cell cycle after fertilization on both maternal and paternal genomes. Late replicating regions contain a relative paucity of active origins and few but long genes and low G/C content. In both bovine and mouse embryos, replication timing patterns are established prior to embryonic genome activation. Chromosome breaks, which form spontaneously in bovine embryos at sites concordant with human embryos, preferentially locate to late replicating regions. In mice, late replicating regions show enhanced fragility due to a sparsity of dormant origins that can be activated under conditions of replication stress. This pattern predisposes regions with long neuronal genes to fragility and genetic change prior to separation of soma and germ cell lineages. Our studies show that the segregation of early and late replicating regions is among the first layers of genome organization established after fertilization.

Evaluation of DNA damage through cytogenetic approach in smokers and vapers with and without nicotine compared with control group

The use of tobacco and electronic cigarettes is harmful to health and can cause genetic damage, increasing the risk of cancer and other serious diseases. Although e-cigarettes contain fewer toxic chemicals than conventional tobacco, recent studies have shown that the vapor caused by burning produced by e-cigarettes can also be toxic and carcinogenic. Various studies have found that those exposed to tobacco and vaping have significantly higher levels of damage to their DNA in different types of cells and tissues. Evidence has accumulated that e-cigarette vaping can alter cellular functions and DNA itself, increasing the risk of cancer and aging. The present work evaluatesthe cytogenetic damage in individuals exposed to conventional cigarette vapors by burning with nicotine and vapors without nicotine, compared with a non-smoking population. The study included participants with an average age of 30 years (+/-10), with a majority of men representing 70% of the sample. The Chi-square test found no significant statistical differences between the men and women exposed (p&lt;0.05). The results of chromosomal fragility found in the four groups studied (control group, conventional smokers, vapers with nicotine and vapers without nicotine) showed breaks and gaps in one or both of the chromatids in all exposed individuals, with highly significant statistical differences (p&lt;0.001) compared to the unexposed control group. No statistically significant differences were found between the group of conventional smokers and the vapers with and without nicotine, nor between the two types of vapers (p&gt;0.05). In conclusion, cytogenetic evidence of DNA damage produced by vaping is shown in the same proportions as a normal cigarette. This will significantly impact public health, which must be considered in preventive actions. Keywords: cigarettes, vaping, chromosome breakage, DNA damage

Evaluation of DNA damage through cytogenetic approach in smokers and vapers with and without nicotine compared with control group

The use of tobacco and electronic cigarettes is harmful to health and can cause genetic damage, increasing the risk of cancer and other serious diseases. Although e-cigarettes contain fewer toxic chemicals than conventional tobacco, recent studies have shown that the vapor caused by burning produced by e-cigarettes can also be toxic and carcinogenic. Various studies have found that those exposed to tobacco and vaping have significantly higher levels of damage to their DNA in different types of cells and tissues. Evidence has accumulated that e-cigarette vaping can alter cellular functions and DNA itself, increasing the risk of cancer and aging. The present work evaluatesthe cytogenetic damage in individuals exposed to conventional cigarette vapors by burning with nicotine and vapors without nicotine, compared with a non-smoking population. The study included participants with an average age of 30 years (+/-10), with a majority of men representing 70% of the sample. The Chi-square test found no significant statistical differences between the men and women exposed (p&lt;0.05). The results of chromosomal fragility found in the four groups studied (control group, conventional smokers, vapers with nicotine and vapers without nicotine) showed breaks and gaps in one or both of the chromatids in all exposed individuals, with highly significant statistical differences (p&lt;0.001) compared to the unexposed control group. No statistically significant differences were found between the group of conventional smokers and the vapers with and without nicotine, nor between the two types of vapers (p&gt;0.05). In conclusion, cytogenetic evidence of DNA damage produced by vaping is shown in the same proportions as a normal cigarette. This will significantly impact public health, which must be considered in preventive actions. Keywords: cigarettes, vaping, chromosome breakage, DNA damage

Investigating the potential of X chromosome shredding for mouse genetic biocontrol

CRISPR-Cas9 technology has facilitated development of strategies that can potentially provide more humane and effective methods to control invasive vertebrate species, such as mice. One promising strategy is X chromosome shredding which aims to bias offspring towards males, resulting in a gradual and unsustainable decline of females. This method has been explored in insects with encouraging results. Here, we investigated this strategy in Mus musculus by targeting repeat DNA sequences on the X chromosome with the aim of inducing sufficient DNA damage to specifically eliminate X chromosome-bearing sperm during gametogenesis. We tested three different guide RNAs (gRNAs) targeting different repeats on the X chromosome, together with three male germline-specific promoters for inducing Cas9 expression at different stages of spermatogenesis. A modest bias towards mature Y-bearing sperm was detected in some transgenic males, although this did not translate into significant male-biasing of offspring. Instead, cleavage of the X chromosome during meiosis typically resulted in a spermatogenic block, manifest as small testes volume, empty tubules, low sperm concentration, and sub/infertility. Our study highlights the importance of controlling the timing of CRISPR-Cas9 activity during mammalian spermatogenesis and the sensitivity of spermatocytes to X chromosome disruption.

A humanized Anti-YKL-40 antibody inhibits tumor development

Drugs specifically targeting YKL-40, an over-expressed gene (CHI3L1) in various diseases remain developed. The current study is to create a humanized anti-YKL-40 neutralizing antibody and characterize its potentially therapeutic signature. We utilized in silico CDR-grafting bioinformatics to replace the complementarity determining regions (CDRs) of human IgG1 with mouse CDRs of our previously established anti-YKL-40 antibody (mAY). In fifteen candidates (VL1-3/VH1-5) of heavy and light chain variable region combination, one antibody L3H4 named Rosazumab demonstrated strong binding affinity with YKL-40 (KD = 4.645 × 10−8 M) and high homology with human IgG (80 %). In addition, we established different overlapping amino acid peptides of YKL-40 and found that Rosazumab specifically bound to residues K337, K342, and R344, the KR-rich functional domain of YKL-40. Rosazumab inhibited migration and tube formation of YKL-40-expressing tumor cells and induced tumor cell apoptosis. Mechanistically, Rosazumab induced interaction of N-cadherin with β-catenin and activation of downstream MST1/RASSF1/Histone H2B axis, leading to chromosomal DNA breakage and cell apoptosis. Treatment of xenografted tumor mice with Rosazumab twice a week for 4 weeks inhibited tumor growth and angiogenesis, but induced tumor apoptosis. Rosazumab injected in mice distributed to blood, tumor, and other multiple organs, but did not impact in function or structure of liver and kidney, indicating non-detectable toxicity in vivo. Collectively, the study is the first one to demonstrate that a humanized YKL-40 neutralizing antibody offers a valuable means to block tumor development.

Abstract PO1-16-04: Targeting PKMYT1 Kinase as a Therapeutic Strategy for Treatment of Triple Negative Breast Cancer with Low Molecular Weight Cyclin E (LMW-E) Expression

Abstract Purpose: Low molecular weight isoforms of cyclin E (LMW-E) have been implicated in various human cancers, including triple negative breast cancer (TNBC), and are associated with a poor prognosis. However, targeted therapies for TNBC based on biomarkers are currently lacking. This study aims to investigate LMW-E as a potential therapeutic target in TNBC and evaluate the efficacy of RP-6306, a selective inhibitor of the Protein Kinase, Membrane Associated Tyrosine/Threonine 1 (PKMYT1), in LMW-E-positive breast tumors. Experimental Design: Immunohistochemical (IHC) analysis was performed on pre-treatment tumor specimens from TNBC patients (n=36) to assess the correlation between LMW-E expression, CDK1 phosphorylation at Threonine 14 (pT14), and pathologic complete response to neoadjuvant chemotherapy. LMW-E inducible human mammary epithelial cells (hMEC) and breast cancer cell lines were used to investigate the regulatory effect of LMW-E on PKMYT1, the kinase responsible for CDK1 phosphorylation at T14, and the response to RP-6306, a first in-class and selective inhibitor of PKMYT1. Patient-derived xenograft (PDX) models and transgenic mouse mammary tumor virus (MMTV) models of TNBC expressing human LMW-E (hLMW-E) were also utilized to assess LMW-E as a biomarker for predicting response to RP-6306. Results. Analysis of TNBC tumor biopsies revealed a significant positive correlation between LMW-E expression and CDK1 pT14, and both biomarkers were associated with a lack of pathological complete response to neoadjuvant chemotherapy. In vitro results using LMW-E inducible hMECs and breast cancer cell lines demonstrated that LMW-E up-regulates PKMYT1 and CDK1 pT14, acting as a PKMYT1 binding protein and enhancing PKMYT1 protein stability. High LMW-E protein levels predicted a favorable response to RP-6306, resulting in the accumulation of sub-G1 and polyploid cells, decreased tolerance to replication stress, increased DNA damage, chromosomal breakage, and apoptosis. In vivo treatment of TNBC PDX models and hLMW-E transgenic tumors with RP-6306 resulted in a significant reduction in tumor volume only in mice harboring high LMW-E tumors, while low cyclin E models showed no response. Immunohistochemical analysis confirmed increased γ-H2AX and decreased CDK1-pT14 and Ki67 levels, indicating the efficacy of RP-6306 in both PDX and transgenic models. Conclusion: This study highlights the regulatory axis from LMW-E to PKMYT1 and its predictive value for pathological complete response in TNBC patients receiving neoadjuvant chemotherapy. The selective PKMYT1 kinase inhibitor RP-6306 consistently induced DNA damage and inhibited tumor growth in in vitro and in vivo pre-clinical breast tumor models. Co-expression of LMW-E and CDK1-pT14 in TNBC can be used to stratify patients whose tumors are likely to respond to RP-6306, emphasizing its therapeutic significance. Citation Format: Mi Li, Amriti Lulla, Cansu Karakas, Spiridon Tsavaschidis, Yan Wang, Tuyen Nguyun, Tuyen Bui, Gary Marshall, Kelly Hunt, Khandan Keyomarsi. Targeting PKMYT1 Kinase as a Therapeutic Strategy for Treatment of Triple Negative Breast Cancer with Low Molecular Weight Cyclin E (LMW-E) Expression [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-16-04.

Determination of Cytotoxic Impact of Heavy Metals on Plant Cells Using &lt;i.Allium cepa&lt;/i&gt; (Onion)

The environment faces significant global health challenges due to the presence of heavy metals, with harmful substances being discharged into the lithosphere, hydrosphere, and atmosphere. This study focused on assessing the cytotoxic effects of heavy metals recovered from dumpsites on the meristematic tissue of root tips of Allium cepa L. Soil samples, collected from central dumpsites containing heavy metals, underwent standard procedures. The concentrations of heavy metals in the samples were determined using the Association Official Analytical Chemist (AOAC) method. Each sample was digested with acids, neutralized with Calcium Oxide (CaO), and used to grow Allium cepa at varying concentrations, determining the LD50 of each sample. The LD50 concentrations were then employed to investigate cytotoxic effects on Allium cepa using standard techniques. Processed soil samples yielded Pb, Cr, Cd, Ni, Co, Zn, Fe, and Cu at different concentrations, many of which exceeded the WHO standard values. The mitotic index ranged from 5.34 ± 0.01 to 10.36 ± 0.00, 5.34 ± 0.00 to 13.16 ± 0.01, 3.36 ± 0.01 to 6.81 ± 0.01, 2.48 ± 0.01 to 13.16 ± 0.01, with the control at 13.32 ± 0.01. Chromosomal aberrations, including chromosome break, fragmentation, bridge, vagrant, lesion, and sticky C-mitosis, were observed. These alterations indicated that toxic substances significantly impact DNA sequence.

Two cases of cytopenia associated with multiple malformations

The first patient, a 10-year-old girl, presented with pancytopenia and recurrent epistaxis, along with a history of repeated upper respiratory infections, café-au-lait spots, and microcephaly. Genetic testing revealed compound heterozygous mutations in the DNA ligase IV (LIG4) gene, leading to a diagnosis of LIG4 syndrome. The second patient, a 6-year-old girl, was seen for persistent thrombocytopenia lasting over two years and was noted to have short stature, hyperpigmented skin, and hand malformations. She had a positive result from chromosome breakage test. She was diagnosed with Fanconi anemia complementation group A. Despite similar clinical presentations, the two children were diagnosed with different disorders, suggesting that children with hemocytopenia and malformations should not only be evaluated for hematological diseases but also be screened for other potential underlying conditions such as immune system disorders.

Assessment of genotoxicity biomarkers in gasoline station attendants due to occupational exposure.

Gasoline station attendants are exposed to numerous chemicals that might have genotoxic and carcinogenic potential, such as benzene in fuel vapor and particulate matter and polycyclic aromatic hydrocarbons in vehicle exhaust emission. According to IARC, benzene and diesel particulates are Group 1 human carcinogens, and gasoline has been classified as Group 2A "possibly carcinogenic to humans." At gas stations, self-service is not implemented in Turkey; fuel-filling service is provided entirely by employees, and therefore they are exposed to those chemicals in the workplace during all working hours. Genetic monitoring of workers with occupational exposure to possible genotoxic agents allows early detection of cancer. We aimed to investigate the genotoxic damage due to exposures in gasoline station attendants in Turkey. Genotoxicity was evaluated by the Comet, chromosomal aberration, and cytokinesis-block micronucleus assays in peripheral blood lymphocytes. Gasoline station attendants (n = 53) had higher tail length, tail intensity, and tail moment values than controls (n = 61). In gasoline station attendants (n = 46), the frequencies of chromatid gaps, chromosome gaps, and total aberrations were higher compared with controls (n = 59). Increased frequencies of micronuclei and nucleoplasmic bridges were determined in gasoline station attendants (n = 47) compared with controls (n = 40). Factors such as age, duration of working, and smoking did not have any significant impact on genotoxic endpoints. Only exposure increased genotoxic damage in gasoline station attendants independently from demographic and clinical characteristics. Occupational exposure-related genotoxicity risk may increase in gasoline station attendants who are chronically exposed to gasoline and various chemicals in vehicle exhaust emissions.

Abstract LB397: Simultaneous CRISPR-Cas9 induced double strand breaks causes chromosomal catastrophe in pancreatic cancers: A parallel comparison with ionizing radiation

Abstract CRISPR-Cas9, a highly effective gene editing tool, is a molecular scissor that induces double strand breaks (DSBs) at its targeted genomic locations. Ionizing radiation (IR), in contrast, generates DSBs and other DNA lesions, making it potentially more toxic than CRISPR-Cas9 induced DSBs. Alternatively, CRISPR-Cas9 might be more toxic as the Cas9 activity would continue if the DSB were repaired to wild type, resulting in more scissions per target. We designed multitarget sgRNAs targeting 0-16 sites in the human genome and performed similar dose-response experiments with IR (0-10 Gy) over 21 days on two pancreatic cancer (PC) cell lines. Growth inhibition was measured through clonogenicity and alamarBlue assays, and γ-H2AX staining performed to calculate DSBs. Surviving colonies from sgRNA transduction underwent whole genome sequencing (WGS) and targeted deep sequencing to quantify mutations. Cell growth was assessed through cell counting and sgRNA tag survival over time. Chromosomal changes were elucidated through chromosome breakage assays and XY FISH. We found that cytotoxicity increased as a function of both IR dose and number of sgRNA target site. At a comparable number of DSBs, CRISPR-Cas9 demonstrated higher cytotoxicity (~44% growth inhibition from 5-target sgRNA, ~15 γ-H2AX foci in the PC cells) compared to IR (9% inhibition from 0.5 Gy, ~25 foci). WGS of 5-cutter treated cells also revealed ~13 CRISPR-Cas9 induced DSBs in the PC cells. &amp;gt;99% cytotoxicity was achieved with 12-target sgRNA and 8 Gy. Moreover, sequencing of surviving colonies from sgRNA-treated cells confirmed lack of widespread off-target events. Notably, no surviving colonies could be obtained from the 12- and 16-cutter treated Panc10.05 cells and 8-14 cutters treated TS0111 cells.Both CRISPR-Cas9 and IR assays demonstrated delayed cell death, and cell death began 7 days after transduction of multitarget sgRNAs. Cytogenetic analyses on cells transduced with a 14-cutter sgRNA over 21 days revealed accumulation of karyotypic abnormalities peaking at day 14, and polyploidization peaking at day 10 and decreasing by day 21. Structural variant (SV) analyses from WGS data showed that the number of SVs per colony increased and peaked in 6-cutter colonies (average = 42 SVs), then decreased in 8- and 14-cutter colonies. Finally, surviving colonies that were resistant to 5-, 8-, and 16-cutter sgRNAs were re-challenged with their original multitarget sgRNA, 12-, and 14-cutter sgRNAs. Re-treating with original sgRNA showed no growth inhibition, while &amp;gt;95% cytotoxicity was observed by re-challenging them with 12- or 14-cutter sgRNAs.In conclusion, CRISPR-Cas9 kills pancreatic cancers by inducing chromosomal catastrophe, and resistance to original set of CRISPR-Cas9 induced DSBs can be easily overcome by targeting different sites. For the same number of DSBs, CRISPR-Cas9 was surprisingly more toxic than IR. Citation Format: Selina Shiqing K. Teh, Akhil Kotwal, Eitan Halper-Stromberg, Laura Morsberger, Kirsten Bowland, Alexis Bennett, Michael Goldstein, Ying S. Zou, James R. Eshleman. Simultaneous CRISPR-Cas9 induced double strand breaks causes chromosomal catastrophe in pancreatic cancers: A parallel comparison with ionizing radiation [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 LB397.

Abstract 7598: The novel eIF4A inhibitor potently synergizes with BCL2 inhibitor in high grade B-cell lymphoma with MYC and BCL2 rearrangements through regulating UPR

Abstract Double hit lymphoma(DHL) is defined to be a chromosomal break with MYC gene combined with BCL2 or BCL6. MYC is an oncogene which proliferation the cells and BCL2 inhibit cell death. These combinations could be a cause of highly aggressive clinical features and poor survival of DLBCL. The WHO-HAEM5 renames the entity DLBCL/high-grade B-cell lymphoma with MYC and BCL2 rearrangements. Despite the dismal prognosis, no widely accepted standard treatment exists to the management of DHL. Therefore, new therapeutic approaches for these patients are urgently needed. Previously, we looked at the eIf4A inhibition was shown to downregulate MYC expression in lymphoma cell lines. Venetoclax, a highly selective inhibitor of BCL2, shows clinical activity for patients with DLBCL although there are concerns about increasing toxicity. Our hypothesis in the following: If eIF4A inhibitor preferentially inhibit the translation of oncogenes such as cMyc and venetoclax inhibit the BCL2 in DHL, then the combination of eIF4A inhibitor and BCL2 inhibitor will be highly effective for DHL and potentially safe for animals and humans. We tested the anti-lymphoma effect of CR-1-31-B, in combination with Venetoclax in lymphoma cell lines using flow cytometry. We performed cell apoptosis assays in increasing concentrations of Venetoclax, CR-1-31-B and a combination of the two drugs at 48 hours. To better understand molecular mechanisms underlying the observed synergistic effect in DLBCL-DHL, RNA-seq profiling was performed on cells treated with single drugs and their combinations, respectively. Venetoclax and CR1-31-B exhibited concentration and time dependent cytotoxicity in lymphoma cell lines at low nanomolar concentrations. Overall, the combination of eIF4A inhibitor and BCL2 inhibitor showed synergy in all kinds of DLBCL cell lines. Especially DHL cell lines were more sensitive to the drug combination and showed enhanced synergy compared to the non-DHL cell lines(p&amp;lt;0.0001). We analyzed RNA-seq profiling of cells treated with single drugs and their combinations and found that the transcriptional signature of the synergistic combination was unique relative to that of either constituent monotherapy. Gene set enrichment analysis was performed in the DHL and non-DHL group. The analysis demonstrates that known Unfolded protein response(UPR) part and metabolism, are enriched in DHL groups. This suggests that the UPR and metabolism pathway are closely related to the combination effect in DHL. These findings suggest that the combination of eIF4A inhibitor and BCL2 inhibitor have potent pharmacological activity in DHL lines, it was closely related with UPR. These combination is promising and novel treatment for high-grade B-cell lymphoma with MYC and BCL2 rearrangements. Further pre-clinical investigations of this combination for these patients are needed. Citation Format: So-Young Seol, Chung Hyun Park, Soo Jeong Kim, Haerim Chung, Hyunsoo Cho, Jin Seok Kim, Yu Ri Kim. The novel eIF4A inhibitor potently synergizes with BCL2 inhibitor in high grade B-cell lymphoma with MYC and BCL2 rearrangements through regulating UPR [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 7598.