Overexpression of KMT2E Is Associated with Higher Granulocytic Differentiation Rate in Response to ATRA-Based Treatment in Acute Promyelocytic Leukemia Cell Lines

Introduction: In the clinical setting, low KMT2E expression has been associated with poor prognosis in both acute myeloid leukemia (Damm et al. J Clin Oncol. 2011 Feb 20;29(6):682-9) and acute promyelocytic leukemia (APL) (Lucena-Araujo et al. Br J Haematol. 2014 Aug;166(4):540-9). Member of the Trithorax-group, the KMT2E is implicated in the positive transcriptional control of a specific set of genes related to hematopoiesis and differentiation processes in many cell types. Functional analyses showed that the encoded protein is involved in terminal myeloid differentiation and facilitate retinoic-acid induced granulopoiesis in human promyelocytes. Using lentiviral gene transfer, we hypothesized that the forced expression of KMT2E is implicated in the ATRA-induced granulocytic differentiation in APL. Aims: To investigate the effects of overexpression of KMT2E gene in cell viability and signaling in response to ATRA plus anthracycline or arsenic trioxide (ATO) treatment in APL cell lines. Methods: NB4 and NB4-R2 (resistant to ATRA treatment) cell lines were transduced with pMEG or pMEG- KMT2E lentiviral vectors and cultured with stable selection using puromycin. Before all functional experiments, cells were tested for KMT2E gene and protein expression by qPCR and confocal microscopy, respectively. The cells transduced with KMT2E and control cells were evaluated for cell viability (MTT assay), proliferation (Ki-67 staining), cell cycle (flow cytometry), mitochondrial reactive oxygen species (ROS) production (flow cytometry) in basal conditions and under treatment with ATRA (1 μM), ATO (0.187, 0.375, 0.75, 1.5 and 3 μM), and the association of ATO (1 μM) plus ATRA (1 μM) or cytarabine (150 nM). The granulocytic differentiation in response to ATRA treatment was evaluated based on the CD11b expression (flow cytometry) plus morphological analysis (Leishman staining). The expression of the main target genes (p21, MDM2, and BMI1) encoding for proteins known to be involved in KMT2E function were evaluated by qPCR. Results: Overexpression of KMT2E was significantly associated with higher cell proliferation rates in basal or ATO and cytarabine treatment compared to vehicle treated cells (all P<.001). KMT2E overexpression significantly increase cell proliferation in NB4, but not in NB4-R2 cells (Ki-67 mean intensity of fluorescence (MFI) value - pMEG: 489±10 vs KMT2E : 1432±25; P<.001). The distribution of cells in the phases of the cell cycle was similar between groups. Using a nonlinear regression analysis, IC50 for ATO was of 2.96 and 6 μM for NB4 pMEG and KMT2E transduced cells, respectively. NB4- KMT2E cells showed lower levels of ROS generation upon ATO treatment (1.5 μM) compared to controls (ROS - MFI value - pMEG: 270.6±6 vs KMT2E : 84±11; P <.001). In the attempt to assess the role of KMT2E in granulocytic differentiation, NB4- KMT2E cells were treated with ATRA 1 μM. The cells containing an extra copy of KMT2E gene showed more differentiation rate in 72, 96 and 120 hours compared to empty vector transduced cells (all p<.05). Considering the expression of target genes of KMT2E, only p21 showed an increase in KMT2E cells treated with ATRA. Conclusion: In summary, the overexpression of KMT2E is associated with higher proliferation capacity and increased resistance to ATO treatment, associated with less ROS generation.