Characterization of DNA repair systems in acute myeloblastic leukemia: implications in therapy response

• Autor(a) principal: Figueiredo, Diana Filipa
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/35911
• topic: Acute myeloblastic leukemia; Genomic instability; DNA damage; DNA damage repair; Therapy response

Acute myeloblastic leukemia (AML) comprises a heterogeneous group of hematological malignancies that are characterized by the increased proliferation and accumulation of immature myeloid cells. The initiation and progression of malignant neoplasms arises from genomic instability. DNA damage and defects associated with its repair are the main drivers of this instability. Furthermore, altered DNA damage repair (DDR) pathways can also modulate chemotherapy response. Thus, understanding the mechanisms of DNA damage and repair underlying AML is essential to comprehend its progression and response to therapy. As such, the aim of this work was to characterize DNA damage and DNA damage repair in AML through the use of in vitro models, and to evaluate its impact on therapy response. To achieve this goal, seven AML cell lines (LAMA-84, K562, HEL, KG-1, HL-60, THP-1, and NB-4), and a normal lymphocyte cell line (IMC) were used. Chromosome damage levels were evaluated by the cytokinesis-block micronucleus (CBMN) assay. Double-strand breaks (DSBs) were quantified through the fast halo assay and by flow cytometry-based quantification of γH2AX. 8-hydroxy-2' -deoxyguanosine (8-OHdG) levels present in cell culture medium were quantified through the use of a competitive ELISA kit. To evaluate DNA repair kinetics, levels of chromosome damage (CBMN assay) and of DSBs (cytometry-based γH2AX quantification) were assessed immediately after exposure to hydrogen peroxide (H₂O₂), 1h or 4h, and 24h after exposure. Drug sensitivity data of the different neoplastic cell lines was retrieved from the Genomics of Drug Sensitivity in Cancer platform. Results showed that LAMA-84 cells have the highest percentage of chromosome damage (8.84%) while IMC cells have the lowest (2.52%). Additionally, LAMA-84 cells showed a higher frequency of nucleoplasmic bridges, KG-1 and IMC cells showed a higher percentage of micronucleus, and the remaining cell lines presented a higher frequency of nuclear buds. Levels of γH2AX indicated that HEL cells had the highest prevalence of DSBs (MFI = 66), while IMC cells had the lowest (MFI = 19). NB-4 cells had the highest 8-OHdG levels (8.3 ng/mL) and K562 cells the lowest (2.4 ng/mL). After exposure to H₂O₂, cells were able to revert the induced chromosome damage after 24h, with exception of KG-1 and THP-1 cells. Following exposure to this genotoxic agent, cells were also able to repair the induced DSBs, with exception of HEL cells. The combined analysis of the retrieved drug sensitivity data and the previously determined damage parameters showed that baseline DNA damage levels represent accurate biomarkers that distinguish resistance from sensitivity to conventional therapeutic agents and DDR inhibitors. Taken together these results suggest that the evaluation of background DNA damage in AML may allow to predict cell sensitivity to several drugs and could thus represent a possible treatment response biomarker.