PB1703 PPP1R15A GENE IS OVEREXPRESSED IN CHEMORESISTANT ACUTE MYELOID LEUKEMIA (AML)

Background: AML is the most common malignant myeloid disorder in adults. Relapses are initiated by chemoresistant leukemic cells. DNA damage and repair mechanisms influence not only the genetic predisposition to leukemia but are also very important for refractoriness to treatment. Aims: The aim of this study was to investigate the possible alterations in the gene expression profile in DNA damage signaling pathways and apoptosis in two leukemic cell lines following their exposure to chemotherapeutic agents, idarubicin and cytarabine and to confirm the results in AML patients’ cells. Methods: Kasumi‐1 and MV4‐11 AML cells were treated with either idarubicin (0.1 μM) for 6 h or cytarabine (1 μM) for 48 h. Dead cells were eliminated after exposure to the drug using the appropriate commercial kit. Gene expression profiling through PCR arrays analysis (RT 2 Profiler, Qiagen) was performed after RNA extraction from untreated, drug‐treated and chemoresistant (live) cells. Human DNA Damage Signaling pathway related genes’ expression was evaluated and analyzed through RT 2 Profiler PCR Array data analysis tool. Following our initial results, PPP1R15A gene's relative expression was evaluated by qRT‐PCR analysis with QuantiTect Primer Assays kit (Qiagen) in 12 de novo AML patients before the onset of the 7 + 3 combination chemotherapy and 17 healthy donors using the 2^‐ΔΔCt method. Results: PCR Array analysis after idarubicin treatment of Kasumi‐1 cells revealed a significant up‐regulation of genes involved in apoptosis (BBC3), cell cycle (CDKN1A, PPP1R15A), DNA damage and repair (PNP), and ATM/ATR signaling (RBBP8) while cytarabine treatment led to the up‐regulation of genes involved mostly in the DSB repair pathway i.e. HUS1,MLH1,NBN,XRCC1,XRCC2). Interestingly, significant differences in their gene expression patterns were observed between cytarabine‐treated Kasumi‐1 cells and chemoresistant ones. HUS‐1 gene (DSB) was up‐regulated in cytarabine‐treated cells and down‐regulated in chemoresistant cells, while MLH1 and NBN genes presented the opposite pattern. Most importantly, PPP1R15A gene's expression in both cytarabine and idarubicin chemoresistant MV4‐11 cells was significantly up‐regulated compared to drug treated cells. PPP1R15A gene's relative expression was significantly up‐regulated in AML patients’ cells compared to controls (median: 0.72 vs 1.72, p < 0.05) and in non‐responding to chemotherapy AML patients compared to controls (median: 0.72 vs 1.21, p < 0.05). Summary/Conclusion: Our data suggest differences in the gene expression pattern between chemoresistant cells and drug‐treated cells, indicating the significance of DNA damage and repair pathways involved in chemoresistance. Specifically, the up‐regulation of PPP1R15A gene in chemoresistant MV4‐11 cells after treatment with both agents is of great importance since this gene participates in cell cycle and its transcript levels are increased following stressful growth arrest and treatment with DNA‐damaging agents. Considering that MV4‐11 is an AML chemoresistant cell line, up‐regulation of the expression of PPP1R15A gene which facilitates recovery of cells from stress is totally compatible. These findings were further verified by qRT‐PCR demonstrating overexpression of PPP1R15A gene in AML chemoresistant patients indicating the involvement of this gene in chemoresistance mechanisms.