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Targeting DNA Homologous Repair Proficiency With Concomitant Topoisomerase II and c-Abl Inhibition.

ABSTRACT: Critical DNA repair pathways become deranged during cancer development. This vulnerability may be exploited with DNA-targeting chemotherapy. Topoisomerase II inhibitors induce double-strand breaks which, if not repaired, are detrimental to the cell. This repair process requires high-fidelity functional homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). If either of these pathways is defective, a compensatory pathway may rescue the cells and induce treatment resistance. Consistently, HR proficiency, either inherent or acquired during the course of the disease, enables tumor cells competent to repair the DNA damage, which is a major problem for chemotherapy in general. In this context, c-Abl is a protein tyrosine kinase that is involved in DNA damage-induced stress. We used a low-dose topoisomerase II inhibitor mitoxantrone to induce DNA damage which caused a transient cell cycle delay but allowed eventual passage through this checkpoint in most cells. We show that the percentage of HR and NHEJ efficient HeLa cells decreased more than 50% by combining c-Abl inhibitor imatinib with mitoxantrone. This inhibition of DNA repair caused more than 87% of cells in G2/M arrest and a significant increase in apoptosis. To validate the effect of the combination treatment, we tested it on commercial and patient-derived cell lines in high-grade serous ovarian cancer (HGSOC), where chemotherapy resistance correlates with HR proficiency and is a major clinical problem. Results obtained with HR-proficient and deficient HGSOC cell lines show a 50-85% increase of sensitivity by the combination treatment. Our data raise the possibility of successful targeting of treatment-resistant HR-proficient cancers.

SUBMITTER: Siddiqui A

PROVIDER: S-EPMC8488401 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Deregulation of receptor tyrosine kinase (RTK)-signaling is frequently observed in many human malignancies, making activated RTKs the promising therapeutic targets. In particular, activated RTK-signaling has a strong impact on tumor resistance to various DNA damaging agents, e.g., ionizing radiation and chemotherapeutic drugs. We showed recently that fibroblast growth factor receptor (FGFR)-signaling might be hyperactivated in imatinib (IM)-resistant gastrointestinal stromal tumors (GIST) and inhibition of this pathway sensitized tumor cells to the low doses of chemotherapeutic agents, such as topoisomerase II inhibitors. Here, we report that inhibition of FGFR-signaling in GISTs attenuates the repair of DNA double-strand breaks (DSBs), which was evidenced by the delay in ?-H2AX decline after doxorubicin (Dox)-induced DNA damage. A single-cell gel electrophoresis (Comet assay) data showed an increase of tail moment in Dox-treated GIST cells cultured in presence of BGJ398, a selective FGFR1-4 inhibitor, thereby revealing the attenuated DNA repair. By utilizing GFP-based reporter constructs to assess the efficiency of DSBs repair via homologous recombination (HR) and non-homologous end-joining (NHEJ), we found for the first time that FGFR inhibition in GISTs attenuated the homology-mediated DNA repair. Of note, FGFR inhibition/depletion did not reduce the number of BrdU and phospho-RPA foci in Dox-treated cells, suggesting that inhibition of FGFR-signaling has no impact on the processing of DSBs. In contrast, the number of Dox-induced Rad51 foci were decreased when FGFR2-mediated signaling was interrupted/inhibited by siRNA FGFR2 or BGJ398. Moreover, Rad51 and -H2AX foci were mislocalized in FGFR-inhibited GIST and the amount of Rad51 was substantially decreased in -H2AX-immunoprecipitated complexes, thereby illustrating the defect of Rad51 recombinase loading to the Dox-induced DSBs. Finally, as a result of the impaired homology-mediated DNA repair, the increased numbers of hypodiploid (i.e., apoptotic) cells were observed in FGFR2-inhibited GISTs after Dox treatment. Collectively, our data illustrates for the first time that inhibition of FGF-signaling in IM-resistant GIST interferes with the efficiency of DDR signaling and attenuates the homology-mediated DNA repair, thus providing the molecular mechanism of GIST's sensitization to DNA damaging agents, e.g., DNA-topoisomerase II inhibitors.

Project description:DNA topoisomerases are enzymes that modulate DNA topology. Among them, topoisomerase 3α is engaged in genomic maintenance acting in DNA replication termination, sister chromatid separation, and dissolution of recombination intermediates. To evaluate the role of this enzyme in Trypanosoma cruzi, the etiologic agent of Chagas disease, a topoisomerase 3α knockout parasite (TcTopo3α KO) was generated, and the parasite growth, as well as its response to several DNA damage agents, were evaluated. There was no growth alteration caused by the TcTopo3α knockout in epimastigote forms, but a higher dormancy rate was observed. TcTopo3α KO trypomastigote forms displayed reduced invasion rates in LLC-MK2 cells when compared with the wild-type lineage. Amastigote proliferation was also compromised in the TcTopo3α KO, and a higher number of dormant cells was observed. Additionally, TcTopo3α KO epimastigotes were not able to recover cell growth after gamma radiation exposure, suggesting the involvement of topoisomerase 3α in homologous recombination. These parasites were also sensitive to drugs that generate replication stress, such as cisplatin (Cis), hydroxyurea (HU), and methyl methanesulfonate (MMS). In response to HU and Cis treatments, TcTopo3α KO parasites showed a slower cell growth and was not able to efficiently repair the DNA damage induced by these genotoxic agents. The cell growth phenotype observed after MMS treatment was similar to that observed after gamma radiation, although there were fewer dormant cells after MMS exposure. TcTopo3α KO parasites showed a population with sub-G1 DNA content and strong γH2A signal 48 h after MMS treatment. So, it is possible that DNA-damaged cell proliferation due to the absence of TcTopo3α leads to cell death. Whole genome sequencing of MMS-treated parasites showed a significant reduction in the content of the multigene families DFG-1 and RHS, and also a possible erosion of the sub-telomeric region from chromosome 22, relative to non-treated knockout parasites. Southern blot experiments suggest telomere shortening, which could indicate genomic instability in TcTopo3α KO cells owing to MMS treatment. Thus, topoisomerase 3α is important for homologous recombination repair and replication stress in T. cruzi, even though all the pathways in which this enzyme participates during the replication stress response remains elusive.

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Targeting DNA Homologous Repair Proficiency With Concomitant Topoisomerase II and c-Abl Inhibition.

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