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Project description:The pleiotropic cytokine interferon-gamma (IFNγ) is known to be associated with cytostatic, anti-proliferation, and pro-apoptosis functions in cancer cells. However, cancer cells can become resistant to IFNγ, and the underlying mechanism is not fully understood. To investigate the potential IFNγ resistant mechanisms, we performed IFNγ sensitivity screens in more than 40 cancer cell lines and characterized the sensitive and resistant cell lines. By applying CRISPR screening and transcriptomic profiling in both IFNγ sensitive and resistant cells, we discovered that activation of double-strand break (DSB) repair genes could result in IFNγ resistance in cancer cells. In addition, suppression of single-strand break (SSB) repair genes increased the essentiality of DSB repair genes after IFNγ treatment. The relationship between the activation of DSB repair genes and IFNγ resistance was further confirmed in clinical tumor profiles from The Cancer Genome Atlas (TCGA) and immune checkpoint blockade (ICB) cohorts. Our study provides a comprehensive resource to elucidate IFNγ resistance in cancer and has the potential to inform combinational therapeutic strategies to overcome immunotherapy resistance.
2023-02-28 | GSE180941 | GEO
Project description:The pleiotropic cytokine interferon-gamma (IFNγ) is known to be associated with cytostatic, anti-proliferation, and pro-apoptosis functions in cancer cells. However, cancer cells can become resistant to IFNγ, and the underlying mechanism is not fully understood. To investigate the potential IFNγ resistant mechanisms, we performed IFNγ sensitivity screens in more than 40 cancer cell lines and characterized the sensitive and resistant cell lines. By applying CRISPR screening and transcriptomic profiling in both IFNγ sensitive and resistant cells, we discovered that activation of double-strand break (DSB) repair genes could result in IFNγ resistance in cancer cells. In addition, suppression of single-strand break (SSB) repair genes increased the essentiality of DSB repair genes after IFNγ treatment. The relationship between the activation of DSB repair genes and IFNγ resistance was further confirmed in clinical tumor profiles from The Cancer Genome Atlas (TCGA) and immune checkpoint blockade (ICB) cohorts. Our study provides a comprehensive resource to elucidate IFNγ resistance in cancer and has the potential to inform combinational therapeutic strategies to overcome immunotherapy resistance.
2023-02-28 | GSE180942 | GEO
Project description:Cancer Cell Resistance to IFNγ Can Occur via Enhanced Double-Strand Break Repair Pathway Activity
| PRJNA750105 | ENA
Project description:Cancer Cell Resistance to IFNγ Can Occur via Enhanced Double-Strand Break Repair Pathway Activity [CRISPR screen]
| PRJNA750249 | ENA
Project description:Cancer Cell Resistance to IFNγ Can Occur via Enhanced Double-Strand Break Repair Pathway Activity [RNA-seq]
| PRJNA750248 | ENA
Project description:Double-strand breaks (DSBs) are among the most deleterious lesions DNA can endure. Yet, DSBs are programmed at the onset of meiosis, and are required to facilitate appropriate reduction of ploidy in daughter cells. Repair of these breaks is tightly controlled to favor homologous recombination (HR)-the only repair pathway that can form crossovers. However, little is known about how the activities of alternative repair pathways are regulated at these stages. We discovered an unexpected synthetic interaction between the DSB machinery and strand-exchange proteins. Depleting the Caenorhabditis elegans DSB-promoting factors HIM-5 and DSB-2 suppresses the formation of chromosome fusions that arise in the absence of RAD-51 or other strand-exchange mediators. Our investigations reveal that nonhomologous and theta-mediated end joining (c-NHEJ and TMEJ, respectively) and single strand annealing (SSA) function redundantly to repair DSBs when HR is compromised, and that HIM-5 influences the utilization of TMEJ and SSA.
| S-EPMC6218235 | biostudies-literature
Project description:A broken DNA molecule is difficult to repair, highly mutagenic, and extremely cytotoxic. Such breaks can be repaired by homology-independent or homology-directed mechanisms. Little is known about the network that controls the repair pathway choice except that a licensing step for homology-mediated repair exists, called DNA-end resection. The choice between these two repair pathways is a key event for genomic stability maintenance, and an imbalance of the ratio is directly linked with human diseases, including cancer. Here we present novel reporters to study the balance between both repair options in human cells. In these systems, a double-strand break can be alternatively repaired by homology-independent or -dependent mechanisms, leading to the accumulation of distinct fluorescent proteins. These reporters thus allow the balance between both repair pathways to be analyzed in different experimental setups. We validated the reporters by analyzing the effect of protein downregulation of the DNA end resection and non-homologous end-joining pathways. Finally, we analyzed the role of the DNA damage response on double-strand break (DSB) repair mechanism selection. Our reporters could be used in the future to understand the roles of specific factors, whole pathways, or drugs in DSB repair pathway choice, or for genome-wide screening. Moreover, our findings can be applied to increase gene-targeting efficiency, making it a beneficial tool for a broad audience in the biological sciences.
| S-EPMC3796453 | biostudies-literature
Project description:The analysis of double-strand break (DSB) repair is complicated by the existence of several pathways utilizing a large number of genes. Moreover, many of these genes have been shown to have multiple roles in DSB repair. To address this complexity we used a repair reporter construct designed to measure multiple repair outcomes simultaneously. This approach provides estimates of the relative usage of several DSB repair pathways in the premeiotic male germline of Drosophila. We applied this system to mutations at each of 11 repair loci plus various double mutants and altered dosage genotypes. Most of the mutants were found to suppress one of the pathways with a compensating increase in one or more of the others. Perhaps surprisingly, none of the single mutants suppressed more than one pathway, but they varied widely in how the suppression was compensated. We found several cases in which two or more loci were similar in which pathway was suppressed while differing in how this suppression was compensated. Taken as a whole, the data suggest that the choice of which repair pathway is used for a given DSB occurs by a two-stage "decision circuit" in which the DSB is first placed into one of two pools from which a specific pathway is then selected.
| S-EPMC1851981 | biostudies-literature
Project description:P53-binding protein 1 (53BP1) plays critical roles in DNA double strand break (DSB) repair by promoting non-homologous end joining (NHEJ), and loss of 53BP1 abolishes PARPi sensitivity in BRCA1-deficient cells by restoring homologous recombination (HR). 53BP1 is one of the proteins initially recruited to sites of DSBs via recognition of H4K20me2 through the Tudor-UDR domain and H2AK15ub through the UDR motif. Although extensive studies have been conducted, it remains unclear how the post-translational modification of 53BP1 affects DSB repair pathway choice. Here, we identified 53BP1 as an acetylated protein and determined that acetylation of 53BP1 inhibit NHEJ and promote HR by negatively regulating 53BP1 recruitment to DSBs. Mechanistically, CBP-mediated acetylation of K1626/1628 in the UDR motif disrupted the interaction between 53BP1 and nucleosomes, subsequently blocking the recruitment of 53BP1 and its downstream factors PTIP and RIF1 to DSBs. Hyperacetylation of 53BP1, similar to depletion of 53BP1, restored PARPi resistance in BRCA1-deficient cells. Interestingly, 53BP1 acetylation was tightly regulated by HDAC2 to maintain balance between the HR and NHEJ pathways. Together, our results demonstrate that the acetylation status of 53BP1 plays a key role in its recruitment to DSBs and reveal how specific 53BP1 modification modulates the choice of DNA repair pathway.
| S-EPMC5778472 | biostudies-literature
Project description:DNA non-homologous end joining (NHEJ) and homologous recombination (HR) function to repair DNA double-strand breaks (DSBs) in G2 phase with HR preferentially repairing heterochromatin-associated DSBs (HC-DSBs). Here, we examine the regulation of repair pathway usage at two-ended DSBs in G2. We identify the speed of DSB repair as a major component influencing repair pathway usage showing that DNA damage and chromatin complexity are factors influencing DSB repair rate and pathway choice. Loss of NHEJ proteins also slows DSB repair allowing increased resection. However, expression of an autophosphorylation-defective DNA-PKcs mutant, which binds DSBs but precludes the completion of NHEJ, dramatically reduces DSB end resection at all DSBs. In contrast, loss of HR does not impair repair by NHEJ although CtIP-dependent end resection precludes NHEJ usage. We propose that NHEJ initially attempts to repair DSBs and, if rapid rejoining does not ensue, then resection occurs promoting repair by HR. Finally, we identify novel roles for ATM in regulating DSB end resection; an indirect role in promoting KAP-1-dependent chromatin relaxation and a direct role in phosphorylating and activating CtIP.
| S-EPMC3061033 | biostudies-literature