Differential Gene Expression Profile in DNA Damage Signaling Pathways, in De Novo Acute Myeloid Leukemia Patients before Induction Chemotherapy

Introduction: Acute Myeloid Leukemia (AML) is a life-threatening myeloid malignancy with poor prognosis. Response to induction treatment determines long-term prognosis; however, mechanisms underlying response have not been thoroughly investigated. DNA damage and repair mechanisms influence not only the genetic predisposition to leukemia but are also important for refractoriness to treatment.

Aim: The aim of this study is to investigate the possible alterations in the gene expression profile of DNA damage signaling pathways in two leukemic cell lines following their exposure to chemotherapeutic agents, namely, idarubicin and cytarabine, and to verify the findings in AML patient samples at diagnosis before the onset of chemotherapy.

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 (chemo resistant) cells following chemotherapy exposure. Human DNA damage signaling pathway-related gene expression and statistical analysis was performed through the RT2 Profiler PCR Array data analysis tool. Moreover, mononuclear cells were isolated from bone marrow of 11 lymphoma patients, used as controls, and from 64 AML patients at diagnosis who subsequently received chemotherapy with the 7+3 regimen. Response to first-line chemotherapy was recorded and patients were assigned in two groups: Responders (n=53 patients) and non-Responders (n=11 patients). Total RNA was isolated (RNeasy plus Mini Kit, Qiagen), quantified and cDNA was synthesized. Relative expression of genes of interest was quantified using SYBR® Greenbased real-time RT-PCR (QuantiTect® Primer Assay, Qiagen) based on the ddCt method (Housekeeping gene GADPH). All samples were obtained from patients after informed consent and treated in accordance with the Declaration of Helsinki. Statistical analysis was performed using R Statistical Software (version 4.2.1).

Results: Five genes were found significantly [p < 0.05, Students t-test] up-regulated in live cells from either leukemic cell lines after treatment with both agents and had a FoldChange > 2. More specifically PPP1R15A, CDKN1A and GADD45G genes were up-regulated in both live cell lines, GADD45A in live MV4-11 cells and EXO1 in live Kasumi cells (Figure 1A). The aforementioned genes were selected and their expression levels were quantified in human bone marrow samples. CDKN1A and GADD45G were found down-regulated in Responders [p=0.032 and p=0.001 respectively; Welch t-test] while PPP1R15A was significantly up-regulated [over 8-fold change, p= 2.08E-09; Welch t-test] compared to controls. Likewise, GADD45G and PPP1R15A were also significantly down and up regulated respectively, in non-Responders [p=0.08 and p=1.51E-05, Welch t-test]. Interestingly, PPP1R15A presented a 1.7-fold overexpression trend in non-Responders compared to Responders [p=0.18, Welch t-test] (Figure 1B).

Conclusion: The results of our study suggest a dysregulation of the DNA damage and repair pathways in AML. Upregulation of GADD45G is known to dramatically induce apoptosis, differentiation, and growth arrest while increasing sensitivity of AML cells to chemotherapeutic drugs (Guo, Dan et al., Blood 2021). CDKN1A is involved in p53/TP53 mediated inhibition of cellular proliferation in response to DNA damage. Both were down-regulated in AML patients and are likely to play an important role in the pathogenesis of AML. PPP1R15A participates in DNA damage response facilitating the recovery of cells from stress. The up-regulation of PPP1R15A both in live cells treated with Ara-C and Cytarabine, as well as the 1.7-fold overexpression trend in non-Responders compared to Responders, supports the notion that PPP1R15A could be associated with resistance to induction treatment, which may have significant prognostic and therapeutic implications. A greater sample of AML patients both responding and not responding to chemotherapy, would be essential to delineate the role of genes involved in DNA damage and repair pathways in resistance to induction treatment.

Symeonidis:Astellas: Research Funding; Abbvie, Amgen, Astra-Zeneca, BMS, GenesisPharma, Gilead, Glaxo, Integris, Janssen, Novartis, Pfizer, Sanofi, Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Vianex: Research Funding; Roche: Research Funding; Servier SOBI: Membership on an entity's Board of Directors or advisory committees; Rafarm: Honoraria; Demo/Apopharma: Research Funding; WinMedica: Research Funding.