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

Abstract

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 p15INK4band 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. Disclosures No relevant conflicts of interest to declare.

Analysis of Genomic DNA Methylation and Gene Transcription Modulation Induced by DNMT3A Deficiency in HEK293 Cells

“…In recent years, DNMT3A has been identi ed to be an ideal target for the development of personalized treatment or predict tumor prognosis [26]. This is the rst report on the effect of DNMT3A disruption in its catalytic domain on genomic DNA methylation and expression.…”

“…In recent years, DNMT3A has been intensely studied for its role in tumor prognosis or therapy [3,26]. To better reveal the functions of DNMT3A in cancer occurrence and development, in this study, we mutated HEK293 cells using the CRISPR/Cas9 technology and successfully created a DNMT3A knockout cell line, with homozygous frameshift deletion in both alleles.…”

Hypoxia-Inducible Factor1-Α (HIF1α) and Vascular Endothelial Growth Factor-A (VEGF-A) Expression in De Novo AML Patients

“…However, the contradiction in the being oncogenesis or tumor-suppressor of HIF1α has been controversial in previous studies. In this regard, a study by Gao et al, (2015) on transgenic mice concluded that inactivation of the HIF1α gene by siHIF1α inhibited tumor growth.While Velasco-Hernandez et al, (2014), by performing tests on mouse models, found results contrary to our results, which showed that HIF1α acts as a tumor suppressor and inactivation of it makes tumor progression. Given this contradiction, we decided to study the HIF1α downstream for oncogenesis, so we chose the VEGF-A gene that discussed below.…”

The RUNX1–ETO fusion protein trans‐activates c‐KIT expression by recruiting histone acetyltransferase P300 on its promoter

“…The RUNX1–ETO/RUNX1–ETO9a fusion protein comprises a conserved runt homology derived from the hematopoietic transcription factor, RUNX1, which can bind RUNX1‐binding sites . Previously, the RUNX1–ETO fusion protein was considered to be a transcription repressor through recruitment of DNA methyltransferases, histone deacetylases, or corepressors (such as miR‐193a, miR‐223, and HIF1α) . Recently, several studies reported that the RUNX1–ETO protein interacts with P300 at the docking site provided by the ETO NHR1 domain, and induces upregulation of target genes (e.g.…”

“…It has been reported that RUNX1–ETO increases c‐KIT promoter activity through directly binding to the promoter region via the RUNX1 motifs located at +655 and +674 relative to the TSS . However, RUNX1–ETO regulated downstream genes miR‐193a , DNMT3a , and SIRT1 by binding to the promoter region (−280 bp, −1890 to −1821 bp, and −546 bp realtive to the TSS) , and as is well known, promoter sequences are usually the sequence upstream of the TSS and play an important role for transcriptional regulation. Therefore, the RUNX1 binding site (−953 bp relative to the TSS) on the c‐KIT promoter may be another potential therapeutic site.…”