WTAP-mediated m6A methylation of PHF19 facilitates cell cycle progression by remodeling the accessible chromatin landscape in t(8;21) AML

doi:10.21203/rs.3.rs-4756131/v1

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WTAP-mediated m⁶A methylation of PHF19 facilitates cell cycle progression by remodeling the accessible chromatin landscape in t(8;21) AML

https://doi.org/10.21203/rs.3.rs-4756131/v1

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Wilms' tumor 1-associated protein (WTAP) is a key N6-methyladenosine (m⁶A) methyltransferase that is upregulated in t(8;21) acute myeloid leukemia (AML) under hypoxia inducible factor 1α-mediated transcriptional activation, promoting leukemogenesis through transcriptome-wide m⁶A modifications. However, the specific substrates and intrinsic regulatory mechanisms of WTAP are not well understood. Here, we provide evidence that PHD finger protein 19 (PHF19) overexpression is regulated by WTAP-mediated m⁶A modification and promotes cell cycle progression by altering chromatin accessibility. At the same time, high expression of PHF19 and WTAP in t(8;21) AML patients indicates a worse prognosis. Furthermore, inhibition of PHF19 expression significantly suppresses the growth of t(8;21) AML cells in both in vitro and in vivo. Mechanistically, WTAP enhances the stability of PHF19 mRNA by binding to m⁶A sites in the 3'-untranslated region, thereby upregulating PHF19 expression. Conversely, WTAP suppression reduces m⁶A modification levels on the PHF19 transcript, leading to increased instability. Knockdown of PHF19 precipitates loss of H3K27 trimethylation and enhanced chromatin accessibility, ultimately resulting in upregulated expression of genes involved in the cell cycle and DNA damage checkpoints. Therefore, WTAP/m⁶A-dependent PHF19 upregulation accelerates leukemia progression by coordinating m⁶A modification and histone methylation, establishing its status as a novel therapeutic target for t(8;21) AML.

t(8

21)

acute myeloid leukemia

PHD finger protein 19

Wilms' tumor 1-associated protein

histone methylation

RNA methylation

Acute myeloid leukemia (AML) subtype t(8;21), marked by the translocation between 8q22 and 21q22 resulting in the production of the AML1/ETO fusion protein, is one of the most common genetic abnormalities in AML and is typically linked to a positive prognosis [1, 2]. However, despite a high initial curability rate, around 40% of patients experience relapse or display refractory disease [3], revealing significant clinical and biological diversity within this AML subset. It is crucial to recognize that AML1/ETO serves as a critical initiating trigger for leukemogenesis, rather than a sole driver [4]. Epigenetic dysregulation synergistically interacts with AML1/ETO to define the leukemia cell identity and significantly contributes to the leukemogenic and maintenance processes [5].

Our previous research has shown that AML1/ETO upregulates the expression of hypoxia-inducible factor 1α (HIF1α), a pivotal transcription factor involved in cellular and systemic responses to hypoxia. Additionally, AML1/ETO functions in cooperation with HIF1α in sparking the evolutionary transformation of leukemia [6]. N6-methyladenosine (m⁶A), a prevalent and essential epigenetic modification on eukaryotic mRNAs, significantly impacts RNA splicing, nuclear export, folding, translation, and degradation [7]. As a reversible and dynamic alteration, m⁶A is governed by methyltransferases, demethylases, and m⁶A-binding proteins, which are crucial for physiological and pathological functions. The methyltransferase complex, primarily composed of methyltransferase-like 3, methyltransferase-like 14, and Wilms' tumor 1-associated protein (WTAP), facilitates m⁶A methylation [8]. WTAP serves as the crucial regulatory subunit of the methyltransferase complex recruiting it to target mRNA [9] while also playing an important role in various types of cancers [10]. Recently, our studies demonstrated the pivotal role of m⁶A in the development and progression of leukemia with t(8;21) AML [11, 12]. We found that HIF1α can upregulate the expression of m⁶A RNA methylation regulators, WTAP and YTH-domain family protein 2, by directly binding to the promoter regions of these genes, hence activating their transcription. Elevated WTAP expression alters the m⁶A landscape in the transcriptome and bolsters leukemia cell proliferation, correlating with a dismal prognosis in t(8;21) AML patients. Nonetheless, the specific substrates of WTAP and the molecular mechanisms by which WTAP-mediated m⁶A modification controls leukemia cell fate remain elusive.

Our current study elucidates that PHF19 is persistently upregulated in t(8;21) AML patients with a dismal prognosis, thereby facilitating leukemia cell proliferation. This study identifies WTAP as a regulator that enhances PHF19 expression through m⁶A-modulated mRNA stability, leading to repressive histone modifications and subsequent inactivation of cell cycle and DNA damage checkpoints, thus promoting cell cycle progression. The interplay between RNA m⁶A methylation and histone methylation highlights an epigenetic dysregulation critical in leukemogenesis, suggesting that targeted epigenetic manipulation could serve as an effective strategy for leukemia treatment.

PHF19 is overexpressed and strongly associated with an unfavorable prognosis in t(8;21) AML

We previously conducted a comprehensive analysis of m⁶A distribution and gene expression profiling in both parental Kasumi-1 cells and HIF1α-manipulated Kasumi-1 cells using m⁶A-modified RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) (GSE168778, GSE200605) [11]. By comparing differentially expressed genes with transcripts containing m⁶A sites identified by MeRIP-seq in both cell types, we identified 14 significantly differentially methylated genes that are positively correlated with HIF1α expression [11]. To further refine our candidate gene selection, mRNA expression profiles from two independent datasets underwent bioinformatics analysis (details provided below). Our analysis revealed that PHF19 exhibited the highest expression in the t(8;21) subtype of AML, and high PHF19 expression was found to have prognostic value in t(8;21) AML. Consequently, our subsequent research focused on the PHF19 gene.

We initially conducted bioinformatic analysis using data from the BeatAML database (http://www.vizome.org/aml) to investigate the expression pattern of PHF19 in AML and elucidate the relevance of PHF19 and WTAP in the t(8;21) AML subgroup. Our findings revealed a significant upregulation of PHF19 expression levels in t(8;21) AML patients compared to non-t(8;21) AML patients or healthy donors (Fig. 1A, Supplementary Fig. 1A). To validate these findings, the expression of PHF19 was examined in the GEO dataset (GSE6891) [23], revealing significantly elevated levels of PHF19 in t(8;21) AML (Fig. 1B). The prognostic implications of high PHF19 expression were further confirmed in a cohort of AML patients from the TARGET database. Notably, PHF19 expression showed a significant association with t(8;21) AML (Fig. 1C). Subsequently, t(8;21) AML patients were stratified into two groups based on their PHF19 expression levels (Fig. 1D). There were no significant differences observed in baseline clinical features between the PHF19^high and PHF19^low groups (Supplementary Table 1). While overall survival (OS) did not exhibit significant differences between the groups (Fig. 1E), it was evident that the high PHF19 expression group displayed markedly poorer event-free survival (EFS) compared to the low-expression group (Fig. 1F; p = 0.034). With a median follow-up period of 53.1 months, the estimated four-year EFS rates were found to be 57.7% (95% CI, 44.6%-68.7%) and 77.1% (95% CI, 60.6%-87.4%), respectively (Supplementary Table 2; p = 0.034). Furthermore, multivariate analysis indicated that higher expression of PHF19 was an independent risk factor for poorer EFS with marginal significance in t(8;21) AML (Supplementary Table 3; Hazard Risk = 2.04, p = 0.066). To validate the clinical significance and relevance of PHF19 and WTAP in t(8;21) AML, a separate cohort of newly diagnosed patients with t(8;21) AML from the GEO database (GSE16432) [24] were categorized into 4 groups based on their levels of WTAP and PHF19 expression (Fig. 1G). Kaplan-Meier analysis demonstrated that patients in the WTAP^high/PHF19^high group exhibited poorer OS outcomes compared to those in the WTAP^low/PHF19^low group (Fig. 1H). Furthermore, the expression of PHF19 showed positive correlation with HIF1α and WTAP expression respectively in t(8;21) AML, indicating an association between HIF1α-WTAP axis and PHF19 (Supplementary Fig. 1B,C, Fig. 1I). Overall, high expression of PHF19 was observed in t(8;21) AML, and its co-overexpression with WTAP was associated with poorer clinical outcomes, suggesting a potential oncogenic role for PHF19 in t(8;21) AML.

PHF19 knockdown inhibits leukemia cell proliferation in both in vitro and in vivo settings

We further investigated the functional roles of PHF19 in t(8;21) AML. To assess the impact of PHF19 on leukemia cell behavior, we generated t(8;21) AML cell lines Kasumi-1 and SKNO-1 with stable knockdown of PHF19 expression using lentivirus-mediated short hairpin RNA (shRNA). The knockdown efficiency was observed at both mRNA and protein levels (Fig. 2A, Supplementary Fig. 2A). Downregulation of PHF19 significantly decreased the number and size of colonies compared to control (Fig. 2B), indicating a reduction in proliferation ability of leukemia cells in vitro. Furthermore, we evaluated the effects of PHF19 knockdown on the differentiation of t(8;21) AML cells by monitoring definitive markers of differentiated cells (CD11b and CD15) using flow cytometry. We found that PHF19 knockdown increased the proportion of cells with myeloid differentiation-specific markers (Fig. 2C). Additionally, we investigated the effect of PHF19 knockdown on apoptosis using flow cytometry in SKNO-1 and Kasumi-1 cell lines, which revealed an increase in apoptotic cells following PHF19 knockdown (Fig. 2D). To determine the functional role of PHF19 on t(8;21) AML cell proliferation in vivo, nude mice were injected with control Kasumi-1 cells and PHF19 knockdown cells to construct a subcutaneous tumor model, and tumor growth was monitored. Our results showed that PHF19 knockdown significantly inhibited tumor formation, resulting in lower tumor volume in nude mice (Fig. 2E), consistent with our in vitro findings. Overall, these data provide supporting evidence for the oncogenic role of PHF19 in t(8;21) AML, motivating us to explore the molecular regulatory basis underlying high expression levels of PHF19.

PHF19 is a potential downstream target of the HIF1α-WTAP axis

Based on our previously reported transcriptome-wide m⁶A distribution feature and gene expression profiling in parental Kasumi-1 cells, as well as HIF1α-manipulated Kasumi-1 cells using MeRIP-seq and RNA-seq (GSE168778, GSE200605) [11], we identified PHF19 as a significantly differentially methylated gene positively associated with HIF1α expression. Our analysis revealed the presence of m⁶A peaks in the 3'-untranslated region (3'-UTR) of the PHF19 gene. Furthermore, we confirmed that CoCl₂-induced accumulation of HIF1α protein led to an increase in the m⁶A level of the PHF19 gene, while echinomycin-induced HIF1α inhibition reversed this effect (Fig. 3A). Additionally, it has been previously documented that HIF1α promotes the transcription of WTAP by directly binding to hypoxia response elements within the promoter region of WTAP [11]. To elucidate the molecular mechanism by which the HIF1α-WTAP axis regulates PHF19 expression, t(8;21) AML cell lines Kasumi-1 and SKNO-1 were treated with CoCL₂ or CoCL₂ + echinomycin. The activation of the HIF1α-WTAP axis induced by CoCL₂ resulted in a significant increase in PHF19 protein levels, while inhibition of the HIF1α-WTAP axis by echinomycin reversed the effect of CoCL₂ in both cell lines (Fig. 3B). This suggests that PHF19 is positively regulated by the HIF1α-WTAP axis in t(8;21) AML. Consistent with this finding, ectopic expression of HIF1α increased both PHF19 mRNA and protein levels in t(8;21) AML cell lines (Fig. 3C, Supplementary Fig. 3A). The specific regulation of PHF19 by the HIF1α-WTAP axis was further confirmed by findings showing that PHF19 mRNA and protein levels decreased upon knockdown of HIF1α using lentivirus-mediated shRNA transfection in Kasumi-1 and SKNO-1 cells (Fig. 3D, Supplementary Fig. 3B). Taken together, these results indicate that PHF19 may be a downstream target of the HIF1α-WTAP axis.

WTAP-mediated m⁶A modification increases PHF19 mRNA stability

The strong positive correlation between WTAP and PHF19 expression levels prompted us to further investigate whether WTAP enhances the mRNA m⁶A modification and expression levels of PHF19 transcripts. To this end, we conducted MeRIP-qPCR, Western blot, and real-time PCR assays to elucidate the changes in m⁶A modification, protein, and mRNA levels of PHF19 in control leukemia cells and WTAP knockdown cells. As anticipated, our MeRIP-qPCR analyses revealed a significant decrease in the m⁶A level at the 3’-UTR of PHF19 mRNA transcript in WTAP-knockdown Kasumi-1 cells (Fig. 4A). Consistent with these findings, both the expression of PHF19 mRNA and protein levels were markedly reduced upon targeted knockdown of WTAP by shRNA in Kasumi-1 and SKNO-1 cells (Fig. 4B, Supplementary Fig. 4A). Furthermore, to rule out any potential off-target effects of shRNA, we performed a rescue experiment to confirm the role of WTAP in regulating PHF19 expression. We generated stable cell lines overexpressing WTAP using lentiviruses in both Kasumi-1 and SKNO-1 cell lines. Notably, ectopic overexpression of WTAP significantly elevated PHF19 levels in both cell lines; conversely, co-transfection with WTAP shRNA targeting the 3’-UTR of the gene resulted in reduced PHF19 levels (Fig. 4C). In summary, these results strongly suggest that WTAP plays a crucial role in modulating m⁶A modification and influencing the expression of PHF19 mRNA.

To investigate the impact of m⁶A modification on the stability of PHF19 mRNA transcripts, we quantified the changes in PHF19 mRNA decay rates following WTAP knockdown. This was achieved by measuring mRNA abundance after transcriptional inhibition with actinomycin D treatment in both control leukemia cells and WTAP knockdown cells. Our findings revealed that PHF19 mRNA displayed increased instability in WTAP knockdown cells compared to control leukemia cells (Fig. 4D). Collectively, these data suggest that the upregulation of PHF19 levels induced by WTAP is at least partially attributed to the enhanced stability of PHF19 mRNA transcripts.

The silencing of PHF19 leads to a decrease in H3K27me3 levels

PHF19 plays a crucial role in catalyzing histone H3K27 trimethylation (H3K27me3), a repressive chromatin marker associated with epigenetic gene silencing [16]. Therefore, we aimed to investigate the impact of PHF19 on H3K27me3 levels in t(8;21) AML. The specific modulation of H3K27me3 by PHF19 was confirmed by our findings that stable knockdown of PHF19 in Kasumi-1 and SKNO-1 cells led to decreased levels of H3K27me3 (Fig. 5A). Additionally, we conducted a rescue experiment to demonstrate the role of PHF19 in regulating H3K27me3. We first established stable overexpression cell lines for PHF19 in Kasumi-1 and SKNO-1 using lentiviruses. Importantly, increased expression levels of PHF19 significantly elevated H3K27me3 levels in both cell lines, while co-transfection of PHF19 shRNA targeting the 3’-UTR of PHF19 resulted in reduced H3K27me3 levels (Fig. 5B). Consistent with these results, transfection of WTAP shRNA into Kasumi-1 and SKNO-1 cells suppressed both PHF19 and H3K27me3 levels compared to their respective controls (Fig. 5C), indicating that WTAP knockdown leads to downregulation of PHF19 and consequently a global loss of H3K27me3.

Given the evidence that the AML1/ETO-HIF1α axis promotes WTAP expression in t(8;21) AML cells [6, 11], we conducted further investigations to determine whether AML1/ETO positively regulates PHF19 expression and, consequently, H3K27me3 levels. As anticipated, overexpression of AML1/ETO led to significant increases in HIF1α, WTAP, and PHF19 levels. This was accompanied by a concurrent rise in global H3K27me3 levels in ZnSO₄-treated U937-A/E cells compared to both U937-MT cells and untreated U937-A/E cells (Supplementary Fig. 5A). These findings collectively support the notion that AML1/ETO may impact the overall level of H3K27me3 through the activation of the HIF1α-WTAP-PHF19 axis.

PHF19 regulates cell cycle and DNA damage response by modifying chromatin accessibility

Given the potential impact of altered genomic H3K27me3 levels on chromatin organization following PHF19 knockdown, we conducted chromatin accessibility analysis using assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) in control Kasumi-1 cells and PHF19 knockdown cells (Fig. 6A, Supplementary Fig. 6A). Surprisingly, our findings indicated that PHF19 knockdown led to an increase in chromatin accessibility compared to the control (Fig. 6B, Supplementary Table 7,8). Gene ontology (GO) term enrichment analysis of the genes associated with the gained peaks revealed a significant enrichment of cell cycle and DNA damage related terms in the PHF19 knockdown cells, including DNA double-strand break repair and mitotic cell cycle phase transition (Fig. 6B,C, Supplementary Table 9). Furthermore, gene set enrichment analysis (GSEA) using reactome pathways confirmed that pathways related to G1/S DNA damage checkpoints, DNA repair, cell cycle checkpoints, nucleotide excision repair, and global-genome nucleotide excision were enriched in PHF19 knockdown cells (Fig. 6D; Supplementary Table 10). Visualization of ATAC-seq tracks also demonstrated increased chromatin opening at key genes associated with DNA damage checkpoints and cell cycle phase transition following PHF19 knockdown (Supplementary Fig. 6B). These results suggest that expression of genes involved in cell cycle regulation and DNA repair may be altered upon PHF19 knockdown.

To further validate this observation, we conducted differential gene expression analysis using RNA-seq in control Kasumi-1 cells and PHF19 knockdown cells. We found that 3,043 genes were upregulated upon PHF19 knockdown (Fig. 6E, Supplementary Table 11). Additionally, the differential expression analysis of RNA-seq data exhibits many similarities with the differential analysis of ATAC-seq data. The GSEA enrichment analysis of differentially expressed genes from RNA-seq data indicated a positive correlation between PHF19 knockdown cells and gene sets related to G1/S DNA damage checkpoints, cell cycle checkpoints, and DNA repair (Fig. 6F, Supplementary Fig. 6C, Supplementary Table 12).

To investigate the role of PHF19 in regulating cell cycle progression in t(8;21) AML cells, we conducted flow cytometry analysis to examine the cell cycle distribution in PHF19 knockdown cells and control cells. Our findings were consistent with the results of cell colony-forming assays, indicating that knockdown of PHF19 led to cell cycle arrest at G1/S phase (Fig. 7A). These observations suggest that PHF19 promotes leukemia cell growth by facilitating the bypassing of G1 phase arrest through the induction of cell cycle and DNA damage checkpoints inactivation.

Targeting histone methyltransferase PRC2 inhibits t(8;21) AML cell growth

Given the pivotal role of PHF19 in the WTAP-m⁶A-PHF19 cascade, PHF19 may represent a promising new molecular target for t(8;21) AML. However, due to the current lack of suitable inhibitors against PHF19, we conducted an investigation into the cytotoxicity of polycomb repressive complex 2 (PRC2) histone methyltransferase (HMT) inhibitors as a potential therapeutic agent in t(8;21) AML. Specifically, we treated the Kasumi-1 and SKNO-1 cell lines with 9 selective HMT inhibitors respectively and assessed cell viability using the CCK-8 method. Our results demonstrated that these HMT inhibitors induced varying degrees of decrease in leukemia cell counts (Supplementary Fig. 7A), indicating a favorable responsiveness of both cell lines to the majority of tested compounds. Therefore, targeting HMT presents itself as a promising strategy for t(8;21) AML treatment.

Building upon our previous evidence demonstrating the role of the AML1/ETO-HIF1α axis in the upregulation of WTAP in t(8;21) AML [11], our current study uncovers that WTAP-mediated m⁶A RNA methylation regulates the expression and function of chromatin-modifying proteins, thereby governing leukemia cell proliferation. Specifically, the knockdown of WTAP results in the loss of m⁶A on PHF19 transcripts, leading to the downregulation of PHF19 due to increased mRNA instability. The inhibition of PHF19 causes a global reduction in H3K27me3, promoting the upregulation of cell cycle and DNA damage checkpoints, which are associated with an open chromatin state and enhanced DNA accessibility. Ultimately, this halts cell cycle progression and curtails leukemia cell growth (Fig. 7B). This study underscores the intricate interplay of epigenetic marks in remodeling the transcriptional landscape during the shifts in leukemia cell states.

PHF19, initially characterized as a partner of the PRC2 complex, emerges as a pivotal regulator in mouse embryonic stem cell self-renewal and differentiation, enhancing PRC2-driven H3K27 methylation [25–29]. Recent studies have documented overexpression of PHF19 in various cancers, correlating with aggressive tumor behaviors [30–33]. However, the biological functions of PHF19 and the regulatory mechanisms governing its expression in AML are not yet known. Here, we present evidence that WTAP overexpression mediates the abnormally high expression of PHF19 in t(8;21) AML. Our findings indicate that WTAP augments m⁶A modification in the 3’-UTR of PHF19 mRNA, thereby stabilizing PHF19 mRNA in t(8;21) AML cell lines. This research establishes a role for WTAP-mediated m⁶A modification in the regulation of PHF19 expression within the context of t(8;21) AML.

This investigation reveals that the knockdown of PHF19 triggers apoptosis and differentiation in t(8;21) AML cells, resulting in a suppression of leukemia cell growth both in vitro and in vivo. Notably, upregulation of PHF19 is linked to poorer survival rates in t(8;21) AML patients. Predominantly characterized in mouse hematopoietic precursors, PHF19 is vital for establishing hematopoietic stem cell identity and regulates the balance and differentiation of these cells [19]. Studies have more recently investigated the role of PHF19 in hematological disorders [20, 33]. PHF19 overexpression is associated with detrimental treatment outcomes in multiple myeloma, where it functions as a pivotal epigenetic regulator that fosters myeloma cell proliferation [20]. Additionally, PHF19 overexpression has been detected in several myeloid leukemia cell lines [33]. In the K562 chronic myeloid leukemia cell line, PHF19 depletion led to reduced cell proliferation and enhanced erythroid differentiation [33], underscoring its importance in promoting chronic myeloid leukemia cell growth. Our study offers further insights into the oncogenic function of PHF19 in t(8;21) AML and the regulatory mechanisms controlling its upregulation, which, to our knowledge, is the first to explore the role of m⁶A modification in modulating both the expression and function of PHF19.

We further investigated the underlying mechanism of PHF19 in controlling leukemia cell growth. Precedent research has elucidated that PHF19 functions as a ubiquitous transcriptional repressor, significantly contributing to the aggressive progression and transformation of various cancers [20, 27, 32]. Our observations align with these findings, as the upregulation of PHF19 was observed to elevate global H3K27me3 levels and establish a repressive chromatin state within t(8;21) AML cells. This regulatory mechanism facilitated leukemia cell proliferation and advancement through the cell cycle, while concurrently inhibiting apoptosis and differentiation. Mechanistically, the oncogenic effects exerted by PHF19 were contingent upon its capacity to modify chromatin accessibility. Chromatin accessibility profiling through ATAC-seq revealed a marked enhancement in ATAC-seq signals in the context of PHF19 silencing, underscoring the pivotal role of PHF19 in preserving the compacted state of chromatin in t(8;21) AML cells. The integration of chromatin accessibility data from ATAC-seq with transcriptomic profiles from RNA-seq elucidated that the activation of cell cycle and DNA damage checkpoint pathways was associated with PHF19 reduction in t(8;21) AML. This corroborates our observations of cell cycle arrest induced by PHF19 depletion in t(8;21) AML cells. Furthermore, our experiments demonstrated that PHF19 depletion potentiated apoptosis in t(8;21) AML cells. Consequently, the upregulation of PHF19 could potentially lead to the epigenetic repression of cell cycle and DNA damage checkpoint genes by altering the chromatin landscape, which in turn might promote cell cycle progression, hinder apoptosis, and stimulate cell proliferation in t(8;21) AML. The most important implication of this study is that unlike genetic changes such as chromosomal rearrangements and gene mutations, epigenetic changes are reversible. Therefore, epigenetic regulators represent a promising therapeutic target in AML. The evidence supports the notion that PHF19 acts as a tumor promoter and represents a promising molecular marker for leukemia prognosis and treatment in t(8;21) AML.

In conclusion, our discovery of the WTAP-m⁶A-PHF19 axis-driven crosstalk between m⁶A RNA methylation and histone methylation reveals a previously unrecognized regulatory mechanism for cell proliferation in t(8;21) AML. This intricate interplay not only enhances our understanding of the pathogenesis of t(8;21) AML but also unveils potential targets for therapeutic intervention. Additionally, by elucidating this novel pathway for modulating epigenetic networks to suppress leukemia development and progression, our findings provide a foundation for innovative strategies aimed at combating this devastating disease.

Cell lines and cell culture

The Kasumi-1, SKNO-1, U937-A/E and U937-MT cell lines were routinely cultivated in RPMI 1640 medium (Gibco, USA) supplemented with 50 µg/ml streptomycin, 50 IU/ml penicillin and 10% fetal calf serum (Gibco, USA). The U937-A/E cells were generated through stable transfection of HA-tagged AML1/ETO complementary DNA (cDNA) under the control of the mouse metallothionein (MT) promoter into the U937 cell line as previously reported [21]. The expression of AML1/ETO could be induced by treatment with 100 µM ZnSO₄ for 8 hours [6]. The U937-MT cells, carrying the empty vector, served as the control.

Plasmid construction and lentiviral cell transduction

All shRNA sequences and vectors employed for shRNA delivery are presented in Supplementary Table 4. Lentiviral expression vectors and primer sequences for gene cloning are detailed in Supplementary Table 6. Both shRNA vectors and lentiviral expression vectors were devised and synthesized by GeneChem Technologies (China). The transfection of lentivirus was carried out in accordance with the manufacturer's instructions. Leukemia cells were seeded into 6-well plates overnight prior to transfection, and the medium was replaced approximately 12–16 hours after transfection. Forty-eight hours after shRNA transfection, cells were selected with puromycin (2 µg/mL) or neomycin (400 µg/mL) for at least 24 hours before further investigations.

Drug treatment

To artificially manipulate the protein levels of HIF1α expression, cells were seeded into 6-well plates overnight and subsequently treated with 10 nM Echinomycin Streptomyces sp. (Calbiochem-Novabiochem, San Diego, CA, #330175) for 24 hours to suppress the activity of the HIF1α protein. To induce the protein expression of HIF1α, cells were treated with 100 µM CoCl₂.6H₂O (Sigma-Aldrich, #C8661-25G) for 24 hours. Cells treated with 0.1% (v/v) dimethyl sulfoxide (DMSO) served as the vehicle control. To test the drug sensitivity of PRC2 histone methyltransferase inhibitors, cells were seeded into 96-well plates and respectively treated with 9 small molecular compounds from a Chromatin Modification Compound Library (Targetmol #L8300) at a concentration of 10 µM for 24–96 hours, and then subjected to cell viability evaluation.

Western blotting and real-time quantitative reverse transcription PCR (RT-qPCR)

Detailed protocols for Western blotting, RNA extraction, cDNA preparation, and RT-qPCR can be found in the Supplementary Methods.

MeRIP assay

MeRIP assays were conducted using a Magna MeRIP™ m6A Kit (Merck #17-10499) as per the manufacturer's instructions. Total RNA was isolated from cultured cells with Ultrapure RNA Kit (DNase I) (CWBIO #CW0597S) following the manufacturer's guidelines. MeRIP was carried out using a Magna MeRIP™ m6A Kit (Merck #17-10499) as instructed. Briefly, the RNAs were chemically fragmented into ~ 100-nucleotide-long fragments by incubating at 94°C for 5 min in fragmentation buffer. The fragmentation reaction was halted with 0.5M EDTA and followed by ethanol precipitation. The fragmented RNAs were incubated for 30 minutes at room temperature with 10 µg anti-m⁶A antibody in IP buffer and then immunoprecipitated by incubation with Magna ChIP Protein A/G Magnetic Beads for 2 hours at 4℃. After washing with IP buffer, the bound RNAs were eluted from the beads with Elution Buffer (IP Buffer, N6-Methyladenosine, 5′-monophosphate sodium salt). The eluted RNA was subjected to quantitative RT-qPCR analysis after purification with RNeasy® MiniElute® Cleanup kit (QIAGEN, # 74204). The fold enrichment of m6A was calculated using the 2^−ΔΔCt method. ΔCt = Ct_IP – (Ct_input - [Input Dilution Factor]).

RNA stability assay

RNA stability assay was performed as described previously [22]. Cells with stable knockdown of WTAP were treated with actinomycin D (Sigma-Aldrich # 50-76-0) at a final concentration of 5 µg/mL for the indicated time points. Total RNA was purified using Ultrapure RNA Kit (DNase I) (CWBIO #CW0597S). The remaining mRNA was measured by RT-qPCR. The recorded value was the percentage of mRNA remaining compared with the amount before the addition of actinomycin D, normalized to the levels of GAPDH.

RNA-seq assay

Total RNA was isolated from cultured cells with TRIzol® reagent (Invitrogen #15596026). The KAPA Hyper Prep Kit (KAPA #KK8504) was employed to generate the sequencing libraries as per the manufacturer's instructions. Libraries were sequenced on the Illumina nova6000 platform (Geekgene, China) in paired-end 150 nt mode. Detailed information on the processing of RNA-seq data can be found in the Supplementary Methods.

ATAC-seq assay

ATAC-seq libraries were generated by TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme #TD502) following the manufacturer’s instructions. Briefly, for each sample, 10,000 fresh cells were lysed with pre-cooled lysis buffer and centrifuged at 500 × g for 10 min at 4°C to obtain nuclei. Nuclei were resuspended in TruePrep tagment enzyme mix and incubated for 30 min at 37°C. DNA fragments were purified with VAHTS DNA Clean Beads and subjected to PCR amplification. After size selection by VAHTS DNA Clean Beads, the samples were sequenced by the Illumina nova6000 platform (Geekgene, China) under paired-end 150 nt mode. Detailed information on the processing of ATAC-seq data can be found in the Supplementary Methods.

Colony-forming assays, flow cytometry analysis, cell viability CCK-8 assay, and animal studies

For more details on colony formation assay, cell cycle analysis, apoptosis and cell differentiation using flow cytometry, CCK-8 activity assay, and animal studies, please refer to the supplementary materials. Animal experiments were performed with the approval of the Chinese PLA General Hospital Animal Care and Use Committee and conducted according to guiding principles for research involving animals.

Statistical analysis

The data were processed using GraphPad Prism 8.0 software (GraphPad Software, Inc., La Jolla, CA, USA) and SPSS 20.0 software (IBM Corporation, Armonk, NY, USA). A threshold for statistical significance was set at a p-value of less than 0.05. Further details can be found in the Supplementary Methods.

Conflict of interest: The authors have declared that no conflict of interest exists.

DATA AVAILABILITY

The raw data generated in this study are publicly available in Gene Expression Omnibus (GEO) at GSE254526, GSE254527 and GSE254528.

AUTHOR CONTRIBUTIONS

XNG conceived ideas, designed the experiments; YQL, LLW, YLS, HSZ, YLH, and KLM performed the experiments; YQL and JL carried out statistical analysis and interpreted data; JZ and DL performed ATAC-seq and RNA-seq data processing and analysis; XNG and JL wrote the paper; JZ, CJG and DHL critically reviewed the paper. All authors vouch for the completeness and accuracy of the data and analysis.

FUNDING

The study was supported by grants from National Natural Science Foundation of China (No. 82070149, 81870109, 82122004, 82070104), National Key R&D Program of China (2020YFA0112401) and the Natural Science Foundation of Beijing Municipality (7202191).

COMPETING INTERESTS

The authors declare no competing interests.

Supplementary Information

Additional details are provided in supplementary material (available on the Oncogene Web site).

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You are reading this latest preprint version

WTAP-mediated m⁶A methylation of PHF19 facilitates cell cycle progression by remodeling the accessible chromatin landscape in t(8;21) AML

Status:

Version 1

Abstract

Figures

INTRODUCTION

RESULTS

PHF19 is overexpressed and strongly associated with an unfavorable prognosis in t(8;21) AML

PHF19 is a potential downstream target of the HIF1α-WTAP axis

WTAP-mediated m⁶A modification increases PHF19 mRNA stability

The silencing of PHF19 leads to a decrease in H3K27me3 levels

PHF19 regulates cell cycle and DNA damage response by modifying chromatin accessibility

Targeting histone methyltransferase PRC2 inhibits t(8;21) AML cell growth

DISCUSSION

Conclusion

MATERIALS AND METHODS

Cell lines and cell culture

Plasmid construction and lentiviral cell transduction

Drug treatment

Western blotting and real-time quantitative reverse transcription PCR (RT-qPCR)

MeRIP assay

RNA stability assay

RNA-seq assay

ATAC-seq assay

Colony-forming assays, flow cytometry analysis, cell viability CCK-8 assay, and animal studies

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

WTAP-mediated m6A methylation of PHF19 facilitates cell cycle progression by remodeling the accessible chromatin landscape in t(8;21) AML

Status:

Version 1

Abstract

Figures

INTRODUCTION

RESULTS

PHF19 is overexpressed and strongly associated with an unfavorable prognosis in t(8;21) AML

PHF19 is a potential downstream target of the HIF1α-WTAP axis

WTAP-mediated m6A modification increases PHF19 mRNA stability

The silencing of PHF19 leads to a decrease in H3K27me3 levels

PHF19 regulates cell cycle and DNA damage response by modifying chromatin accessibility

Targeting histone methyltransferase PRC2 inhibits t(8;21) AML cell growth

DISCUSSION

Conclusion

MATERIALS AND METHODS

Cell lines and cell culture

Plasmid construction and lentiviral cell transduction

Drug treatment

Western blotting and real-time quantitative reverse transcription PCR (RT-qPCR)

MeRIP assay

RNA stability assay

RNA-seq assay

ATAC-seq assay

Colony-forming assays, flow cytometry analysis, cell viability CCK-8 assay, and animal studies

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

WTAP-mediated m⁶A methylation of PHF19 facilitates cell cycle progression by remodeling the accessible chromatin landscape in t(8;21) AML

WTAP-mediated m⁶A modification increases PHF19 mRNA stability