KMT2E-Mediated Epigenetic Reprogramming Promotes the Sensitivity to All-Trans Retinoic Acid and Increases the Granulocytic Differentiation in AML Cells

Background: The histone-lysine N-methyltransferase 2E (KMT2E) is a member of the KMT2 family that regulates terminal myeloid differentiation and hematopoietic stem cell self-renewal. However, unlike the other members of KMT2 family, KMT2E does not exhibit the intrinsic histone methyl transferase activity, but has been shown to indirectly interact with chromatin by regulating SET7/9 expression and directing the tri-methylation state of the histone H3K4 (H3K4me3). We previously reported that the low expression of KMT2E predicts poorer prognosis in acute promyelocytic leukemia (APL) and that the overexpression of KMT2E contributed to granulocytic differentiation of APL cells in response to all-trans retinoic acid (ATRA). ATRA as a single agent is not effective in cell differentiation of non-APL acute myeloid leukemia (AML) but changes in H3K4 methylation promotes ATRA sensitivity and upregulates myeloid-differentiation-associated genes in AML cells. Therefore, to better understand the role of KMT2E in leukemogenesis and to test whether ATRA was capable to epigenetically mediate the KMT2E function in non-APL AML blasts, we investigated the effect of KMT2E overexpression in the cell viability, tumor growth and signaling response to ATRA in vitro and in vivo.

Methods: qPCR analyzes revealed that the AML cell lines OCI-AML3, U937 and THP-1 express low levels of KMT2E in relation to the APL cell line NB4 and then, they were selected for further experiments. OCIAML3, THP1, and U937 were transduced with the pMEG or the pMEG-KMT2E vector, and U937 cells were also submitted to KMT2E gene silencing by a specific shRNA. The transduced cells were subjected to ATRA (1µM) treatment to evaluate proliferation (Ki-67), clonogenicity, cell cycle and apoptosis (annexin V). For dose-response curves (MTT), cells were administrated graduated ATRA concentrations and the IC₅₀ values were calculated. ATRA-induced granulocytic differentiation was determined by immunophenotyping analysis of myeloid markers: CD11b, CD11c, CD14, CD16, CD64, and HLA-DR. Genes (SETD7/9) and protein (H3K4me3) expressions were established by qPCR and Western blot analysis respectively. 8-10 week-old NSG mice received a dorsal s.c. injection of 5 x 10⁶ U937 cells (transduced with empty/KMT2E/shKMT2E) and 7 days after initial tumor growth treatment was initiated by intraperitoneal injection of the vehicle (n=26) or ATRA (5 mg/kg, n=30) for another 7 days. Tumor volume (V) was obtained using the formula (V=W² x L x 0.52), where W represents the smaller and L the large diameter. The tumor apoptotic index was determined by TUNEL assay.

Results:KMT2E overexpression significantly increased cell proliferation and the proportion of G₂/M cells in all cell lines (all P<.01) but no differences in clonogenicity were observed. The IC₅₀ value of ATRA for U937 was of 23 µM for cells transduced with empty vector and 11 µM for pMEG-KMT2E cells, while for THP1 empty/KMT2E cells received a dose of 9.93 µM and 1.68 µM respectively. ATRA increased apoptosis in a time-dependent manner in U937 and THP1 cells (all P<.05), but not in OCIAML3 p-MEG-KMT2E cells. Furthermore, ATRA-treated U937 and THP1 cells presented G₀/G_1-cell cycle arrest (all P<.05) after 72 hours, while no effect was observed in OCI-AML3. ATRA-induced granulocytic differentiation increased the CD11b/CD14 expression in U937 and THP1-KMT2E cells, within 72 hours of treatment compared to controls (all P<.05). ATRA treatment enhanced expression of H3K4me3 in a time-dependent manner which was correlated with enhanced transcript levels of SETD7/9 (all P<.05).

In NSG mice, ATRA significantly reduced the tumor burden in U937- KMT2E cells and increased the apoptotic index (P<.01) after 7 days of treatment, albeit the high tumor burden in KMT2E cells treated with the vehicle. As a result the mean±SD of tumor volume for empty/KMT2E cells was 1296±95.6 mm³ and 445±59.6 mm³, respectively (P=.001). Finally, U937 shKMT2E indicated no difference in ATRA sensitivity and tumor volume (840±54.8 mm³ and 550±32.1 mm³ for empty/shKMT2E cells, respectively (P>.05).

Conclusion: Our results demonstrated that KMT2E increases the cellular proliferation and ATRA-induced granulocytic differentiation in non-APL AML cell lines. Furthermore, ATRA triggered epigenetic reprogramming resulting in increased levels of H3K4me3, which redirected the biological function of KMT2E.