Dataset Information

RNA demethylase ALKBH5 promotes tumorigenesis of t (8;21) acute myeloid leukemia via ITPA m6A modification.

ABSTRACT:

Background

Although t (8;21) is in fact considered a good risk acute myeloid leukemia (AML), only 60% of the patients live beyond 5 years after diagnosis. Studies have shown that RNA demethylase ALKBH5 promotes leukemogenesis. However, the molecular mechanism and clinical significance of ALKBH5 in t (8;21) AML have not been elucidated.

Methods

The expression of ALKBH5 was assessed in t (8;21) AML patients via qRT-PCR and western blot. The proliferative activity of these cells was examined through CCK-8 or colony-forming assays, while flow cytometry approaches were used to examine apoptotic cell rates. The in vivo role of ALKBH5 promoting leukemogenesis was assessed using t (8;21) murine model, CDX, and PDX models. RNA sequencing, m6A RNA methylation assay, RNA immunoprecipitation, and luciferase reporter assay were used to explore the molecular mechanism of ALKBH5 in t (8;21) AML.

Results

ALKBH5 is highly expressed in t (8;21) AML patients. Silencing ALKBH5 suppresses the proliferation and promotes the apoptosis of patient-derived AML cells and Kasumi-1 cells. With integrated transcriptome analysis and wet-lab confirmation, we found that ITPA is a functionally important target of ALKBH5. Mechanistically, ALKBH5 demethylates ITPA mRNA and increases its mRNA stability, leading to enhanced ITPA expression. Furthermore, transcription factor TCF15, specifically expressed in leukemia stem/initiating cells (LSCs/LICs), is responsible for the dysregulated expression of ALKBH5 in t (8;21) AML.

Conclusion

Our work uncovers a critical function for the TCF15/ALKBH5/ITPA axis and provides insights into the vital roles of m6A methylation in t (8;21) AML.

SUBMITTER: Li R

PROVIDER: S-EPMC10007764 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Publications

RNA demethylase ALKBH5 promotes tumorigenesis of t (8;21) acute myeloid leukemia via ITPA m6A modification.

Li Ran R Wu Xiaolu X Xue Kai K Feng Dandan D Li Jianyong J Li Junmin J

Biomarker research 20230310 1

<h4>Background</h4>Although t (8;21) is in fact considered a good risk acute myeloid leukemia (AML), only 60% of the patients live beyond 5 years after diagnosis. Studies have shown that RNA demethylase ALKBH5 promotes leukemogenesis. However, the molecular mechanism and clinical significance of ALKBH5 in t (8;21) AML have not been elucidated.<h4>Methods</h4>The expression of ALKBH5 was assessed in t (8;21) AML patients via qRT-PCR and western blot. The proliferative activity of these cells was ...[more]

PMID: 36899424

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Project description:BackgroundPost-ischemic angiogenesis is critical for blood flow recovery and ischemic tissue repair. N6-methyladenosine (m6A) plays essential roles in numerous biological processes. However, the impact and connected mechanism of m6A on post-ischemic angiogenesis are not fully understood.MethodsAlkB homolog 5 (ALKBH5) was screened out among several methyltransferases and demethylases involved in dynamic m6A regulation. Cardiac microvascular endothelial cells (CMECs) angiogenesis and WNT family member 5A (WNT5A) stability were analyzed upon ALKBH5 overexpression with adenovirus or knockdown with small interfering RNAs in vitro. The blood flow recovery, capillary, and small artery densities were evaluated in adeno-associated virus (AAV)-ALKBH5 overexpression or ALKBH5 knockout (KO) mice in a hind-limb ischemia model. The same experiments were conducted to explore the translational value of transient silencing of ALKBH5 with adenovirus.ResultsALKBH5 was significantly upregulated in hypoxic CMECs and led to a global decrease of m6A level. ALKBH5 overexpression further reduced m6A level in normoxic and hypoxic CMECs, impaired proliferation, migration, and tube formation only in hypoxic CMECs. Conversely, ALKBH5 knockdown preserved m6A levels and promoted angiogenic phenotypes in hypoxic but not in normoxic CMECs. Mechanistically, ALKBH5 regulated WNT5A expression through post-transcriptional mRNA modulation in an m6A-dependent manner, which decreased its stability and subsequently impeded angiogenesis in hypoxic CMECs. Furthermore, ALKBH5 overexpression hindered blood flow recovery and reduced CD31 and alpha-smooth muscle actin expression in hind-limb ischemia mice. As expected, ALKBH5-KO mice exhibited improved blood flow recovery, increased capillary, and small artery densities after hind-limb ischemia, and similar beneficial effects were observed in mice with transient adenoviral ALKBH5 gene silencing.ConclusionWe demonstrate that ALKBH5 is a negative regulator of post-ischemic angiogenesis via post-transcriptional modulation and destabilization of WNT5A mRNA in an m6A-dependent manner. Targeting ALKBH5 may be a potential therapeutic option for ischemic diseases, including peripheral artery disease.

Project description:Cardiomyocyte hypertrophy is a major outcome of pathological cardiac hypertrophy. The m6A demethylase ALKBH5 is reported to be associated with cardiovascular diseases, whereas the functional role of ALKBH5 in cardiomyocyte hypertrophy remains confused. We engineered Alkbh5 siRNA (siAlkbh5) and Alkbh5 overexpressing plasmid (Alkbh5 OE) to transfect cardiomyocytes. Subsequently, RNA immunoprecipitation (RIP)-qPCR, MeRIP-qPCR analysis and the dual-luciferase reporter assays were applied to elucidate the regulatory mechanism of ALKBH5 on cardiomyocyte hypertrophy. Our study identified ALKBH5 as a new contributor of cardiomyocyte hypertrophy. ALKBH5 showed upregulation in both phenylephrine (PE)-induced cardiomyocyte hypertrophic responses in vitro and transverse aortic constriction (TAC)/high fat diet (HFD)-induced pathological cardiac hypertrophy in vivo. Knockdown or overexpression of ALKBH5 regulated the occurrence of hypertrophic responses, including the increased cardiomyocyte surface areas and elevation of the hypertrophic marker levels, such as brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP). Mechanically, we indicated that ALKBH5 activated JAK2/STAT3 signaling pathway and mediated m6A demethylation on Stat3 mRNA, but not Jak2 mRNA, resulting in the phosphorylation and nuclear translocation of STAT3, which enhances the transcription of hypertrophic genes (e.g., Nppa) and ultimately leads to the emergence of cardiomyocytes hypertrophic growth. Our work highlights the functional role of ALKBH5 in regulating the onset of cardiomyocyte hypertrophy and provides a potential target for hypertrophic heart diseases prevention and treatment. ALKBH5 activated JAK2/STAT3 signaling pathway and mediated m6A demethylation on Stat3 mRNA, but not Jak2 mRNA, resulting in the phosphorylation and nuclear translocation of STAT3, which enhances the transcription of hypertrophic genes (e.g., Nppa) and ultimately leads to the emergence of cardiomyocytes hypertrophic growth.

Dataset Information

RNA demethylase ALKBH5 promotes tumorigenesis of t (8;21) acute myeloid leukemia via ITPA m6A modification.

Background

Methods

Results

Conclusion

Publications

RNA demethylase ALKBH5 promotes tumorigenesis of t (8;21) acute myeloid leukemia via ITPA m6A modification.

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