PF169 LONG‐TERM INHIBITION OF CHK1/CHK2 KINASES MODIFY ACUTE LYMPHOBLASTIC LEUKEMIA CELL LINE RESPONSE TO DNA DAMAGING AGENTS

Abstract

Background:Acute lymphoblastic leukemia (ALL) cells respond to chemotherapy, or more generally to DNA damages, by activating different DNA Damage Response (DDR) pathways. DDR‐pathways regulate cell cycle progression and DNA damages repair. Molecular and functional alterations in key DDR‐related genes drastically affect the effectiveness of DNA‐damaging treatments in cancer cells. For this reasons selective DDR‐inhibitors have been developed in order to sensitize cancer cells against conventional chemotherapy. Despite the proven efficacy of DDR‐inhibitors in cancer treatment, only few studies have highlighted the biological consequences the prolonged inhibition of DDR‐pathways in cancer cells.Aims:The aim of the study was to evaluate biological consequences of the prolonged inhibition of two crucial DDR‐related kinase, the cell cycle checkpoint kinase 1 (CHK1) and 2 (CHK2), in B‐ALL cell lines.Methods:The resistant cell lines were obtained from B‐ALL NALM‐6 cell line by prolonged incubation with a CHK1/CHK2 inhibitor, PF‐00477736. Parental cells were progressively incubated with increasing concentration of PF‐00477736 (from 100 nM to 10uM). The functional characterization of resistant cell lines was evaluated in vitro in term of response to different chemotherapy agents (doxorubicin, methotrexate and clofarabine) in term of reduction of cell viability, induction of apoptosis, cell cycle perturbation and activation of DDR pathways. The molecular characterization of resistant cell lines was evaluated in term of Copy Number Alteration (SNP‐array, Affimetrix).Results:Starting from NALM‐6 parental cell lines, we generated two resistant models, N6R‐PF4 and N6R‐PF8. N6R‐PF8 was generated form N6R‐PF4 cells after a prolong incubation with increased PF‐00477736 concentration. In term of reduction of cell viability and induction of apoptosis the resistant cell lines were not only significantly unresponsive to PF‐00477736 (increasing the IC50 up to 8‐fold) but also to different DNA damaging agents in comparison to parental NALM‐6. The cell cycle profile revealed that the resistant cell lines had a significant reduction in the percentage of cells in S phase and a significant increase of cells in G2/M phase. SNP microarray highlighted different alterations in DDR‐related genes and in particular in the ATM/CHK2 pathway. Three regions in copy number LOSS (CN = 1) containing several genes involved in cell cycle checkpoint regulation (ATM and NPAT) and in the apoptosis (BIRC2, BIRC3, CARD17, CASP1, CASP12 and CASP5) were detectable only in NALM‐6 parental cell lines and were copy number neutral in the two resistant models. Immunoblotting analysis confirmed that in the resistant cell lines the ATM/CHK2 down‐stream pathways was significant over‐expressed and activated in comparison to the parental NALM‐6. By chromosome binding analysis we identified that NALM‐6 cells were composed by a predominant clone with ATM locus CN loss and by a sub‐clone (45,X,‐Y,t(5;12)(q33;p13),t(7;19)(q11;p13)) with ATM locus in CN neutral. The treatment with PF‐00477736 progressively selected the sub‐clone, reaching 60% of clones in N6R‐PF4 and 100% of clone in N6R‐PF8.Summary/Conclusion:We showed that the higher protein expression of ATM and CHK2 affected not only the sensitivity to PF‐00477736 but also compromised the efficacy of conventional chemotherapy. In this scenario the level of expression of these two kinases seems to correlate with the sensitivity to DNA damaging agents and to PF‐00477736.