KDM7A Is Targeted By MiR-155 in Acute Myeloid Leukemia and Impacts Differentiation

Acute myeloid leukemia (AML) is a heterogeneous disease that develops secondary to the acquisition of mutations that disrupt cell differentiation, proliferation and survival. MicroRNAs (miRNAs or miRs) are short non-coding RNA molecules that modulate post-transcriptional gene expression by either cleaving or repressing translation of target mRNA transcripts. Differential expression of miRNAs has been identified in AML and noted to correlate with specific disease characteristics, cytogenetic abnormalities and prognosis.

MiR-155 expression is upregulated in both adult and pediatric patients with cytogenetically normal AML (CN-AML) and correlates with adverse clinical outcomes. Specifically, we have shown that high miR-155 expression is associated with an increased incidence of induction chemotherapy failure and inferior overall and event free survival. However, how miR-155 up-regulation contributes mechanistically to adverse clinical outcomes is poorly understood.

In prior work, we correlated the expression of predicted or validated miR-155 target genes with miR-155 expression in a gene expression profiling (GEP) dataset of pediatric AML samples. We identified 22 candidates with inversely correlated expression by GEP for further validation in diagnostic bone marrow specimens from children with the highest miR-155 expression levels (n=9) vs. children with the lowest miR-155 expression levels (n=9). Although the expression of miR-155 inversely correlated with 9 target genes, only expression of the putative target KDM7A demonstrated a statistically significant difference in expression between low and high miR-155 expressing cases (p = 0.03).

KDM7A is a lysine-specific histone demethylase enzyme that may play a role in regulating differentiation by impacting transcriptional elongation. Computational software programs, i.e. TargetScan, identified two predicted miR-155 binding sites in the KDM7A 3'UTR. To evaluate whether miR-155 directly binds to the KDM7A 3'UTR, we cloned two regions of the KDM7A 3'UTR containing predicted miR-155 binding sequences into luciferase reporter vectors and then mutated the binding sites by site-directed DNA mutagenesis. We validated that both predicted binding sites in KDM7A 3'UTR were direct miR-155 targets using HEK293T cells. Next, we examined the impact of miR-155 overexpression in K562 cells, an acute leukemia cell line that express very low levels of endogenous miR-155, and can be differentiated along the erythroid lineage after hemin exposure. KDM7A RNA expression was decreased 16-fold in miR-155 versus control lentivirally transduced K562 cells as detected by qPCR. KDM7A protein expression was also decreased in miR-155 versus control expressing K562 cells as measured via Western blot. These data demonstrate that KDM7A is a previously uncharacterized target of miR-155.

Next, we explored the effect of differential KDM7A expression on cell differentiation, and cell death and apoptosis after exposure to daunorubicin chemotherapy. For this work we used GFP-labeled miR-155 and YFP-labeled KDM7A lentiviral constructs and labeled control constructs. To examine differentiation we used benzidine staining of hemin-exposed K562 cells transduced with empty control vector (ECV), miR-155, KDM7A, or both constructs. The lowest percentage of benzidine staining, consistent with limited erythroid differentiation, was seen in K562 cells with miR-155 overexpression compared to ECV (28.2% vs. 39.8% positive). This effect on blocked erythroid differentiation was fully reversed with overexpression of KDM7A in miR-155 overexpressing cells (41.4% positive). Confirming these observations, we also observed decreased benzidine staining in hemin exposed K562 cells that were transduced with KDM7A shRNA versus control (28.9% versus 42.7%). Together, these data support that KDM7A plays a role in cell differentiation that is in part controlled by miR-155 expression. Preliminary data also support that re-expression of KDM7A in miR-155 overexpressing cells promotes cell death after exposure to daunorubicin. Further work is ongoing. In conclusion, we have identified a new target of miR-155, KDM7A. Our data suggest that KDM7A plays a role in cell differentiation and that decreased KDM7A expression in AML cells that overexpress miR-155 contributes to blocked differentiation, and may also contribute to resistance to chemotherapy.