PARG inhibitor sensitivity correlates with accumulation of single strand DNA gaps in preclinical models of ovarian cancer
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PROVIDER: PRJNA1126286 | ENA |
REPOSITORIES: ENA
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Project description:Poly (ADP-ribose) glycohydrolase (PARG) is a dePARylating enzyme which promotes DNA repair by removal of poly (ADP-ribose) (PAR) from PARP1-parylated proteins. Loss or inhibition of PARG results in replication stress and sensitizes cancer cells to DNA damaging agents, suggesting that PARG inhibitors may provide a therapeutic option for cancer therapy. Indeed, PARG inhibitors (PARGi) are now undergoing clinical development for patients having tumors with homologous recombination deficiency (HRD), such as ovarian and breast cancer patients with germline or somatic BRCA1/2-mutations. Biomarkers of PARGi response will be required for the optimal development of these important new drugs. PARP inhibitors kill BRCA-deficient cancer cells by increasing ssDNA gaps during replication. Here, we report that, similar to PARP inhibitors, PARGi treatment can also cause an accumulation of ssGAPs in sensitive cells. PARGi exposure increased accumulation of S-Phase specific PAR, a marker for Okazaki fragment processing (OFP) defects on lagging strands, in sensitive cells but not in resistant cells. PARGi also caused accumulation of PAR at the replication fork and increased pRPA, a marker for replication stress, at the replication fork in sensitive cells. Additionally, PARGi exhibited monotherapy activity in some HR-deficient, as well as HR-proficient, patient-derived organoids of ovarian cancer, and drug sensitivity directly correlated with the accumulation of ssDNA gaps. Taken together, PARGi treatment results in toxic accumulation of PAR at replication forks resulting in ssDNA gaps due to OFP defects during replication. Regardless of the BRCA-status, the induction of ssDNA gaps in preclinical models of ovarian cancer cells correlates with PARGi sensitivity. Patient-derived organoids will be a useful model system for testing PARGi sensitivity and functional biomarkers
2024-10-31 | GSE270384 | GEO
Project description:The finished human genome-assemblies comprise several hundred un-sequenced euchromatic gaps, which may be rich in long polypurine/polypyrimidine stretches. Human chromosome 20 currently has three remaining un-sequenced gaps on its q-arm. All three gaps are within gene-dense regions, or overlap loci associated with human disorders, including one gap, which is at DLGAP4. In this study we sequenced, determined the complete sizes and assessed epigenetic landscapes of all three un-sequenced gaps on human chromosome 20 using a methodological approach involving Sanger sequencing, mate-pair paired-end high-throughput sequencing and chromatin and methylation analysis. We found histone H3K27me3 to be distributed across all three gaps in immortalized B-lymphocytes. We found five novel CpG islands in one gap to be highly hypermethylated in genomic DNA from both peripheral blood lymphocytes and human cerebellum. One of these CpG islands was differentially methylated and paternally hypermethylated. Furthermore, computational analyses predicted the presence of structured non-coding RNAs (ncRNAs) in all three chromosome 20 gaps. We verified expression for thirteen candidate ncRNAs, some of which showed tissue-specificity. Four ncRNAs expressed within the gap at DLGAP4 show elevated expression particularly in the human brain. Our data suggests that un-sequenced human genome gaps may comprise functional elements. Mate-pair paired end sequencing using genomic DNA from human translocation carriers having chromosomal rearrangments of chromosomes other than chromosome 20 and chromatin, DNA methylation analysis using human peripheral blood lymphocytes and/or human cerebellum tissue. Analysis done for three remaining human chromosome 20 un-sequenced gap regions.
2012-04-17 | E-GEOD-35405 | biostudies-arrayexpress
Project description:The finished human genome-assemblies comprise several hundred un-sequenced euchromatic gaps, which may be rich in long polypurine/polypyrimidine stretches. Human chromosome 20 currently has three remaining un-sequenced gaps on its q-arm. All three gaps are within gene-dense regions, or overlap loci associated with human disorders, including one gap, which is at DLGAP4. In this study we sequenced, determined the complete sizes and assessed epigenetic landscapes of all three un-sequenced gaps on human chromosome 20 using a methodological approach involving Sanger sequencing, mate-pair paired-end high-throughput sequencing and chromatin and methylation analysis. We found histone H3K27me3 to be distributed across all three gaps in immortalized B-lymphocytes. We found five novel CpG islands in one gap to be highly hypermethylated in genomic DNA from both peripheral blood lymphocytes and human cerebellum. One of these CpG islands was differentially methylated and paternally hypermethylated. Furthermore, computational analyses predicted the presence of structured non-coding RNAs (ncRNAs) in all three chromosome 20 gaps. We verified expression for thirteen candidate ncRNAs, some of which showed tissue-specificity. Four ncRNAs expressed within the gap at DLGAP4 show elevated expression particularly in the human brain. Our data suggests that un-sequenced human genome gaps may comprise functional elements.
2012-04-17 | GSE35405 | GEO
Project description:The published finished human genome contained 340 gaps including 250 gaps in the euchromatic region. The reasons for these gaps were not fully understood, although subsequent analysis revealed that presence of segmentally duplicated sequences were a good predictor for the presence of gaps. However, not all segmentally duplicated regions contained gaps. We made a systemic effort to close euchromatic gaps and understand the nature of gap closing sequences. Our studies clearly demonstrate that the gap closing sequences analyzed were over 2.3-fold more enriched in segmental duplications and that about 40% of the gap closing sequences were structurally variant. The structural variant nature of gap closing sequences was verified by aCGH analysis, and by paired-end-sequence and fingerprint analysis of gap spanning clones from recently available human genome fosmid libraries from eight individuals. Identification and characterization of gap closing sequences provides an effective approach for closing the remaining euchromatic gaps in the human genome. Keywords: comparative genomic hybridization
2007-12-23 | GSE9075 | GEO
Project description:In response to DNA replication stress, DNA replication checkpoint is activated to maintain fork stability, a process critical for maintenance of genome stability. However, how DNA replication checkpoint regulates replication forks remain elusive. Here we show that Rad53, a highly conserved replication checkpoint kinase, functions to couple leading and lagging strand DNA synthesis. In wild type cells under HU induced replication stress, synthesis of lagging strand, which contains ssDNA gaps, is comparable to leading strand DNA. In contrast, synthesis of lagging strand is much more than leading strand, and consequently, leading template ssDNA coated with ssDNA binding protein RPA was detected in rad53-1 mutant cells, suggesting that synthesis of leading strand and lagging strand DNA is uncoupled. Mechanistically, we show that replicative helicase MCM and leading strand DNA polymerase Pole move beyond actual DNA synthesis and that an increase in dNTP pools largely suppresses the uncoupled leading and lagging strand DNA synthesis. Our studies reveal an unexpected mechanism whereby Rad53 regulates replication fork stability.
2018-02-11 | GSE89721 | GEO
Project description:Replication gaps that persist into mitosis likely represent important threats to genome stability, but experimental identification of these gaps has proved challenging. We have developed a technique that allows us to explore the dynamics by which genome replication is completed prior to mitosis. Using this approach, we demonstrate that excessive allocation of replication resources to origins within repetitive regions, induced by SIR2 deletion, leads to persistent replication gaps and genome instability. Conversely, the weakening of replication origins in repetitive regions suppresses these gaps. Given known age- and cancer-associated changes in chromatin accessibility at repetitive sequences, we suggest that replication gaps resulting from misallocation of replication resources underlie age- and disease-associated genome instability.
2017-02-13 | GSE90151 | GEO
Project description:Meiotic recombination is essential for proper meiotic chromosome segregation and fertility, and is initiated by programmed DNA double-strand breaks (DSBs) introduced by Spo11, a eukaryotic homolog of archaeal topoisomerase VIA. Here we report the discovery of hitherto uncharacterized Spo11-induced lesions, small gaps from 34 bp to several hundred bp, which are generated by coordinated pairs of DSBs (double DSBs or dDSBs). Isolation and genome-wide mapping of the resulting fragments with single base pair precision reveals enrichment at DSB hotspots but also a widely dispersed distribution covering the entire genome. We show that Spo11 prefers to cut at a sequence motif which promotes DNA bending, indicating that bendability of DNA contributes to cleavage site choice. Moreover, fragment lengths display a ~ (10.4n+3) bp periodicity, implying that Spo11 favours cleavage on the same face of underwound DNA. Consistently, dDSB signals overlap and correlate with topoisomerase II binding sites, which points to a role for topological stress and DNA crossings in break formation, and suggests a unified model for DSB and dDSB formation, in which Spo11 traps two DNA strands. Furthermore, gaps resulting from dDSBs, an estimated 20% of all initiation events, can account for full gene conversion events that are independent of both Msh2-dependent heteroduplex repair and MutLγ. Since non-homologous gap repair results in deletions, and ectopically re-integrated dDSB fragments result in insertions, dDSB formation represents a potential source of evolutionary diversity and pathogenic germ-line aberrations.
2021-06-09 | GSE171042 | GEO
Project description:Meiotic recombination is essential for proper meiotic chromosome segregation and fertility, and is initiated by programmed DNA double-strand breaks (DSBs) introduced by Spo11, a eukaryotic homolog of archaeal topoisomerase VIA. Here we report the discovery of hitherto uncharacterized Spo11-induced lesions, small gaps from 34 bp to several hundred bp, which are generated by coordinated pairs of DSBs (double DSBs or dDSBs). Isolation and genome-wide mapping of the resulting fragments with single base pair precision reveals enrichment at DSB hotspots but also a widely dispersed distribution covering the entire genome. We show that Spo11 prefers to cut at a sequence motif which promotes DNA bending, indicating that bendability of DNA contributes to cleavage site choice. Moreover, fragment lengths display a ~ (10.4n+3) bp periodicity, implying that Spo11 favours cleavage on the same face of underwound DNA. Consistently, dDSB signals overlap and correlate with topoisomerase II binding sites, which points to a role for topological stress and DNA crossings in break formation, and suggests a unified model for DSB and dDSB formation, in which Spo11 traps two DNA strands. Furthermore, gaps resulting from dDSBs, an estimated 20% of all initiation events, can account for full gene conversion events that are independent of both Msh2-dependent heteroduplex repair and MutLγ. Since non-homologous gap repair results in deletions, and ectopically re-integrated dDSB fragments result in insertions, dDSB formation represents a potential source of evolutionary diversity and pathogenic germ-line aberrations.
2021-06-09 | GSE169760 | GEO
Project description:GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) function as negative and positive regulators of small GTPases, respectively, and play key roles in cell fate determination. The aberrant function of GAPs and GEFs has long been implicated in cancer development but their vast number and potential redundancy have hindered comprehensive understanding of their roles. Here we performed functional genetic screens of GAPs and GEFs in primary AML specimens to uncover an unexpected and selective role for ARHGAP45 in AML. Depletion of ARHGAP45 impedes AML growth without affecting normal human CD34+ cells in vivo. Epistasis screens revealed RhoA as the major substrate of ARHGAP45 and that depletion of the Rho GTPase CDC42 synergized with ARHGAP45 deletion. Consistent with this, pharmacologic CDC42 inhibition enhanced the effect of ARHGAP45 deletion. ARHGAP45 gives rise to a minor histocompatibility antigen (termed HA-1), which can be recognized by antigen-specific T cells. CDC42 inhibition not only sensitized AML cells to ARHGAP45 inhibition but also promoted the activity of HA-1 antigen-specific T cells by upregulating ARHGAP45-derived epitope in AML cells.
2024-07-26 | GSE273164 | GEO
Project description:Pediatric Preclinical Testing Consortium (PPTC)
| PRJNA400657 | ENA