AML1/ETO Cooperates with HIF1α to Promote Leukemogenesis through DNMT3a Transactivation

The t(8;21)(q22;q22) translocation, resulting in a chimeric protein AML1/ETO (A/E), is one of the most common chromosomal abnormalities in acute myeloid leukemia (AML). It has been reported that additional mutagenic “hits” are required for A/E to be a leukemic driver, but it is unclear why most A/E-positive patients don’t carry such gene mutations. We hypothesized that unconventional and more universal events might cooperate with A/E to drive leukemogenesis. Hypoxia inducible factor 1a (HIF1a) is a transcription factor mediating the cellular response to hypoxia in malignant cells. To identify the key molecule responsible for A/E-driven leukemia, we focused on HIF1a signaling, as it is selectively activated in AML stem cells in a hypoxia-independent fashion.

Using qPCR analysis, we measured HIF1a mRNA levels in bone marrow mononuclear cells from 73 A/E-positive and 59 A/E-negative patients with newly diagnosed AML and 15 healthy donors. HIF1a was highly expressed in A/E-positive patients, as compared to A/E-negative patients and healthy controls. The expression of HIF1a was positively correlated with the expression of A/E, independent of KIT mutation. Patients were grouped into quartiles according to HIF1a expression levels (Q1-Q4, each quartile containing 25% of patients) and divided into high HIF1a (HIF1a high, Q4; n = 33) and low HIF1a (HIF1a low, Q1-Q3; n = 99). AE-positive patients with high HIF1a expression had a significantly shorter OS (median = 19.0 months vs. not reached, P = 0.015) and EFS (median = 9.0 months vs. 24.0 months, P = 0.001) compared to those with low HIF1a expression. Notably, HIF1a-associated inferior prognosis in A/E-positive patients was KIT mutation-independent. In patients carrying wild-type KIT [n = 53, about 73% (53/73) of the entire A/E-patients], those with high HIF1a levels had a shorter OS (median = 23.1 months vs. not reached, P = 0.008) and EFS (median = 9.4 months vs. not reached, P = 0.001) than those with low HIF1a levels. Multivariate analysis revealed that high HIF1a levels were independent prognostic indicators for both OS and EFS (P = 0.014, HR = 3.574 and P = 0.004, HR = 4.304, respectively) in AE-positive patients. Mechanistic studies revealed that A/E and HIF1a formed a positive regulatory circuit, in which A/E bound to HIF1a gene promoter and transactivated HIF1a or vice versa. Co-expression of AE and HIF1a in leukemia cells caused a higher cell proliferation rate in vitro and more serious leukemic status in mice. To gain insight into the molecular basis of A/E-HIF1a functional cooperation in leukemogenesis, we examined the mRNA levels of DNMT1, DNMT3a and DNMT3b expression in bone marrow samples from above-mentioned AML patients and healthy donors using qPCR. The result showed that DNMT3a, but not DNMT1 and DNMT3b, was significantly overexpressed in A/E-positive patients, as compared to A/E-negative patients and healthy donors. DNMT3a levels were positively correlated with both A/E and HIF1a levels. We therefore focused on evaluating the role of DNMT3a in A/E-positive AML. We found that DNMT3a expression was positively correlated with both A/E and HIF1a levels. As an individual event, A/E or HIF1a was enriched on DNMT3a gene promoter and synergistically increased DNMT3a transcription and enhanced global DNA methylation. Pharmacological or genetic interventions in the AE-HIF1a loop resulted in DNA hypomethylation, a reexpression of hypermethylated tumor suppressor p15^INK4band the blockage of leukemia growth.

In summary, we showed that A/E and HIF1a form a regulatory circuit and cooperatively control aggressive leukemia growth through DNMT3a transactivation and subsequent DNA hypermethylation. Thus, the HIF1a-DNMT3a nexus serves as a reliable marker, which identifies patients with a poor prognosis in an otherwise prognostically favorable AML group, and represents an innovative therapeutic target in high-risk AE-positive leukemia.