Abstract 2426

Acute Myeloid Leukemia (AML) is defined as rapid growth of abnormal blasts within bone marrow that invade peripheral blood. The complexity of AML is a result of the combination of cytogenetic and molecular abnormalities. In 2011, AML resulted in the highest number of deaths related to leukemia with only a 23.6% chance of five-year survival rate upon diagnosis in adults. The average age of AML patients is ∼69 years old, who are often unable to undergo induction therapy due to cytotoxicity bringing their survival rate to as low as 4–12 weeks. Developing new AML treatments is crucial for increasing survival rate and decreasing cytotoxicity. Recently, microRNA's (miR) have entered the spotlight for therapeutics in numerous diseases. By targeting 3'UTR and degrading genes, miR's are able to regulate gene expression. Within AML, miR profiling has demonstrated a global decrease in their expression implying that they could be playing a role as tumor suppressors. miR-125a previously was identified as being significantly decreased in bone marrow samples from patients in comparison to healthy controls. Though miR-125a has previously been identified as being significantly decreased in bone marrow samples from AML patients, current literature has only focused on miR-125a's role in normal hematopoiesis and not its role in AML. Therefore our focus in the present study has been to elucidate how miR-125a is suppressed in AML and attempt to define its functional role in AML. Screening of paraffin embedded bone marrow samples from AML patients in comparison to control confirmed that 42% of samples (n=34) had decreased miR-125a expression. Analysis of several human leukemic cell lines in comparison to normal primary human bone marrow CD34+ cells, identified a human acute promyelocytic leukemia, t(15; 17) NB4 cells, as having the most decreased miR-125a expression. Interestingly, miR-125a was decreased in NB4 cells in comparison to normal primary human bone marrow CD33+ cells, which is phenotypically closer to NB4 cells. Analysis of the upstream region of miR-125a indicates several CpG regions. miR-125a expression levels through RT-qPCR positively correlated to increased decitabine treatment (0–10μM), a de-methylating agent, in NB4 cells at 24 hours. Bisulfite sequencing analysis of several CpG islands upstream of miR-125a indicated that these CpG islands were methylated and partially de-methylated in response to decitabine treatment in NB4 cells. Since miR-125a was transcriptionally silenced in NB4 cells, a stable NB4 cells line transduced with mimic miR-125a or GFP (control) was generated to analyze its functional role. RT-qPCR of Erbb2's transcript level, a previously identified target of miR-125a, showed a decrease in Erbb2 in miR-125a ectopically expressed NB4 cells compared to control. Furthermore, protein expression analysis through flow cytometry illustrated decrease of Erbb2 protein expression correlating to the decreased transcript level. Total AKT1 protein analyzed through western blot (WB), a downstream Erbb2 effector, showed no significant difference between NB4 transduced with control and mimic miR-125a cells. However, phosphorylated-AKT1 (Ser473) through WB revealed a 1.5 fold decrease in NB4 cells transduced with mimic miR-125a in comparison to control. BrdU cell cycle analysis of mimic miR-125a expressing NB4 cells revealed an inhibition of cell cycle progression, causing cells to accumulate at the G0/G1 phase when compared control NB4 cells. To verify that cell cycle inhibition was due to activation of the Erbb2 pathway, Mubritrinib, a specific Erbb2 inhibitor was used. Analysis of total and phosphorylated-AKT1 verified that there was no change in total AKT1 but a 1.8 fold decrease in phosphorylated-AKT1 in Mubritinib treated NB4 cells through WB. Using a MTT cell proliferation assay, a range of Mubritinib concentrations (0–2μM) indicated a decrease of proliferation by 24 and 48 hours. Further cell proliferation analysis through BrdU showed a dramatic halt in cell cycle proliferation, losing the S-phase, in NB4 cells treated with Mubritinib. Taken together, for the first time, we show that miR-125a expression is silenced in AML due to methylation of upstream CpG islands and demonstrate that miR-125a regulates Erbb2-AKT1 pathway revealing that Mubritinib, a Erbb2-AKT inhibitor as a potential novel therapeutic agent for treating t (15; 17) translocated associated AML.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.