PB1781: THERAPEUTIC POTENTIAL OF DNA DAMAGE RESPONSE IN ACUTE MYELOBLASTIC LEUKEMIA

Abstract

Background: Genome integrity is of utmost importance for cell survival and homeostasis maintenance. However, DNA integrity is threatened by endogenous and exogenous sources. DNA lesions can block genome replication and transcription, and if unrepaired or repaired incorrectly can lead to mutations and genomic instability. Hence, cells have developed a variety of mechanisms to protect genome integrity and repair damaged DNA, collectively termed the DNA damage response (DDR). These mechanisms comprise DNA damage recognition, damage tolerance, signal transduction, transcriptional regulation, cell cycle control, and DNA repair. Key DDR elements include sensor proteins that detect damage (e.g. PARP1 and 2), mediators and signaling transducers (e.g. DNA-PK) that regulate downstream targets, and effector molecules (e.g. CHK1 and 2). Increased DNA damage and altered DDR are critical features of genetic instability presumably implicated in the pathogenesis of acute myeloblastic leukemia (AML). AML is a clonal malignant disease of hematopoietic stem and/or progenitor cells characterized by a block in myeloid differentiation and increased proliferation. This neoplasia is considered the most common acute leukemia in adults and is characterized by recurrent genetic abnormalities. Recently, specific recurrent chromosomal translocations and genetic mutations typical of AML have been associated with DNA repair defects. Aims: Our goal is to assess the potential of DDR as a therapeutic target in AML, using in vitro models, in order to identify new therapeutic approaches. Methods: Five AML cell lines of different molecular and cellular subtypes were used (HEL, HL-60, K-562, LAMA-84, and NB-4). Cells were incubated in the absence and presence of increasing concentrations of two DDR inhibitors, CCT245737 (CHK1 inhibitor) and Niraparib (PARP1/2 inhibitor). Cell density and viability were assessed, for 72 hours, by trypan blue assay. Afterward, resorting to flow cytometry, the cell cycle was assessed using propidium iodide/RNAse, and the cell death was determined using Annexin V/7-AAD double staining as well as by optic microscopy (May-Grünwald-Giemsa staining). Results were statistical analyzed, considering a significance level of 95%. Results: CCT245737 reduced cell proliferation and viability in a dose-, time- and cell line-dependent manner. NB-4 was the most sensitive cell line, with an IC50 at 48 hours of 54 µM. In contrast, LAMA-84 was the most resistant cell line with an IC50 at 48 hours of 203 µM, 3.7-fold higher than NB-4. PARP1/2 inhibition also reduced cell proliferation and viability in a dose-, time- and cell line-dependent manner. Once again NB-4 was the most sensitive cell line, with an IC50 at 48 hours of 25 µM and LAMA-84 was the most resistant with an IC50 at 48 hours of 66 µM, 2.7-fold higher than NB-4. Both inhibitors induced cell death by apoptosis since an increase in the percentage of cells in late apoptosis was observed. This result is in agreement with the observed by optic microscopy. We also detected a cytostatic effect since a cell cycle arrest in the S phase was observed after treatment with both inhibitors. Summary/Conclusion: AML cell lines have different sensibilities to DDR inhibitors, particularly CCT245737 and niraparib, with the NB-4 cell line appearing to be the most sensitive to DDR inhibition (namely CHK1 and PARP1/2 inhibition), whereas LAMA-84 appears to be the most resistant to this inhibition. In conclusion, this work may help to identify new therapeutic approaches that could eventually improve AML patients’ outcomes.