Pb1765: differential gene expression profile in dna damage signaling pathways, in de novo acute myeloid leukemia and low-risk mds patients

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: Myeloid malignancies including Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML) are characterized by the accumulation of genetic alterations. Failure of the DNA damage response (DDR) mechanisms may by one of the underlying mechanisms for the initiation and progression of these diseases. Therefore, the investigation of the alteration of the gene expression profile of the DNA damage response genes is of particular importance. Aims: The aim of this study was to investigate the possible alterations in the gene expression profile of DNA damage response genes in patients with de novo AML and MDS as a mechanism of resistance to genotoxic stress and possibly confering resistance to treatment. Methods: Cell lines Kasumi-1 with the t(8;21) and MV4-11 (biphenotypic B-myelomonocytic leukemia) were exposed either to idarubicin (0.1μΜ) for 6h or cytarabine (1μΜ) for 48h. Dead cells were eliminated from drug-treated cells using an appropriate commercial kit. Gene expression profiling through PCR arrays analysis (RT2 Profiler, Qiagen) was performed in triplicate after RNA extraction from untreated, chemotherapy-treated and live cells following chemotherapy exposure. Human DNA damage signaling pathway-related gene expression was evaluated and analyzed through the RT2 Profiler PCR Array data analysis tool. Moreover, mononuclear cells were isolated from bone marrow of lymphoma patients, used as controls, from de novo AML patients and low-risk MDS patients. Total RNA was isolated (RNeasy plus Mini Kit, Qiagen) and quantified and cDNA was synthesized. Relative expression of genes of interest was quantified using SYBR® Green-based real-time RT-PCR (QuantiTect® Primer Assay, Qiagen). Statistics were performed using one-way Anova with significance at p<0.05. Results: The following genes were over two-fold up-regulated in live cells from leukemic cell lines after treatment with both agents: PPP1R15A, CDKN1A and GADD45G genes in both live cell lines, GADD45A in live MV4-11 cells and EXO1 in live Kasumi cells (Figure 1a). These genes were selected for relative quantification using the ΔCt method: 29 control, 20 low-risk MDS and 77 de novo AML samples were evaluated. CDKN1A and GADD45A were downregulated in MDS patients compared to controls and AML. GADD45G was down regulated in AML patients compared to controls and MDS. PPP1R15A was upregulated in AML compared to controls whereas it was down regulated in MDS. A sub-analysis of the expression of all five genes in AML, comparing responders to induction chemotherapy (n=64) with non Responders (n=13) revealed a tendency for higher PPP1R15A expression in non Responders vs Responders (p=0.17) (Figure 1b) Summary/Conclusion: The results of our study suggest a dysregulation of the DNA damage and repair pathways in AML and MDS. Upregulation of GADD45G is known to induce apoptosis, differentiation and growth arrest in response to DNA damage while increasing sensitivity of AML cells to chemotherapeutic drugs. Its downregulation in AML could be related to chemoresistance. CDKN1A and GADD45A induce apoptosis via the p53/TP53 and the p38-JNK pathway respectively. Their downregulation in MDS may represent a compensatory mechanism to increased apoptosis, characterizing low-risk MDS. PPP1R15A preserves the transcription factor eIF-2A/EIF2S1 in its active state, thereby promoting protein synthesis and facilitating recovery of cells from stress. Its upregulation in AML, which is more pronounced in non-Responders to induction chemotherapy, could be a mechanism of resistance, whereas its downregulation in MDS is compatible with increased apoptosis. Our findings may have significant prognostic and therapeutic implications.Keywords: DNA repair, DNA damage, AML, MDS