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. 2024 Apr;30(4):435–447. doi: 10.1261/rna.079865.123

Histone lysine demethylase KDM5B facilitates proliferation and suppresses apoptosis in human acute myeloid leukemia cells through the miR-140-3p/BCL2 axis

Jiaojuan Huang 1, Shuiling Jin 2, Rongqun Guo 3, Wei Wu 4, Chengxuan Yang 5, Yali Qin 1, Qingchuan Chen 1, Ximiao He 1, Jing Qu 1,✉, Zhenhua Yang 1,✉
PMCID: PMC10946434  PMID: 38296629

Abstract

The histone lysine demethylase KDM5B is frequently up-regulated in various human cancer cells. However, its expression and functional role in human acute myeloid leukemia (AML) cells remain unclear. Here, we found that the expression level of KDM5B is high in primary human AML cells. We have demonstrated that knocking down KDM5B leads to apoptosis and impairs proliferation in primary human AML and some human AML cell lines. We further identified miR-140-3p as a downstream target gene of KDM5B. KDM5B expression was inversely correlated with the miR-140-3p level in primary human AML cells. Molecular studies showed that silencing KDM5B enhanced H3K4 trimethylation (H3K4me3) at the promoter of miR-140-3p, leading to high expression of miR-140-3p, which in turn inhibited B-cell CLL/lymphoma 2 (BCL2) expression. Finally, we demonstrate that the defective proliferation induced by KDM5B knockdown (KD) can be rescued with the miR-140-3p inhibitor or enhanced by combining KDM5B KD with a BCL2 inhibitor. Altogether, our data support the conclusion that KDM5B promotes tumorigenesis in human AML cells through the miR-140-3p/BCL2 axis. Targeting the KDM5B/miR-140-3p/BCL2 pathway may hold therapeutic promise for treating human AML.

Keywords: acute myeloid leukemia, histone H3K4 methylation, KDM5B, miR-140-3p, BCL2

INTRODUCTION

Acute myeloid leukemia (AML) is one of the most prevalent malignancies, with a high mortality rate worldwide (Rose-Inman and Kuehl 2014; Eigendorff and Hochhaus 2015; Rowe 2015). Although advances in AML treatment have significantly improved outcomes for younger patients, the prognosis for older adults, who account for most new cases, remains poor (Shah et al. 2013). Therefore, there is an urgent need to elucidate the pathogenesis of AML and identify more predictive biomarkers to improve the outcomes of AML patients.

Cancer progression is a complicated process that involves multiple critical epigenetic modifications (Rodríguez-Paredes and Esteller 2011; Timp and Feinberg 2013; Su et al. 2015; Yen et al. 2016). Several driver mutations, including gene rearrangement, have been identified in AML patients. These mutations can affect genes that regulate cell growth and differentiation, leading to the uncontrolled proliferation of immature blood cells and the onset of AML. Recent research suggests that targeting these epigenetic alterations is crucial in developing new therapies for AML. For example, drugs that inhibit histone deacetylase (HDAC), DNA methyltransferase (DNMT3A), or isocitrate dehydrogenase 2 (IDH2), which are involved in epigenetic modifications, have shown efficacy in preclinical and early clinical trials (Fennell et al. 2019). Additionally, researchers are exploring the potential of combining epigenetic-targeted therapies with other treatments like chemotherapy or immunotherapy to improve outcomes for AML patients (Fennell et al. 2019; Verma et al. 2021).

Lysine demethylase 5 (KDM5) family proteins, including KDM5A, KDM5B, KDM5C, and KDM5D, are broadly involved in a variety of biological processes, such as mitosis, cell cycle, stem cell self-renewal, and tumorigenesis (Bernstein et al. 2006). KDM5B catalyzes the demethylation of histone 3 lysine 4 (H3K4), which profoundly affects gene expression (Wang et al. 2016a; Xhabija and Kidder 2019). Recent studies have shown that KDM5B is overexpressed in various human cancers, including breast, gastric, and lung, demonstrating its oncogenic function (Flavahan et al. 2017; Li et al. 2020; Chen et al. 2022). In several leukemia studies, it has been reported that KDM5B acted as a negative regulator by altering the H3K4 trimethylation (H3K4me3) of crucial genes associated with the maintenance of leukemia stem cells (LSCs) in mixed-lineage leukemia (MLL)-rearranged AML (Wong et al. 2015; Ren et al. 2022). However, other studies showed that depletion of KDM5B inhibited proliferation and induced apoptosis in AML cells (Su et al. 2015; Shokri et al. 2018). These controversial results drove us further to explore the functional role of KDM5B in AML.

This study shows that KDM5B is highly expressed in AML clinical specimens. We then demonstrated that silencing KDM5B induces apoptosis in primary human AML cells. Moreover, our mechanism studies revealed that KDM5B promotes leukemogenesis through the miR-140-3p/BCL2 axis in AML. Our findings provide novel insights into the oncogenic role of KDM5B in AML.

RESULTS

KDM5B is overexpressed in AML patients

To examine the expression level of KDM5B in AML patients and healthy donors (HDs), we started by analyzing two published data sets (GSE114868 and GSE13159) (Kohlmann et al. 2008; Huang et al. 2019). We observed that KDM5B was highly expressed in AML patients with different types of chromosome rearrangements compared with HDs or MDS (myelodysplastic syndrome) patients (Fig. 1A,B). Early reports showed that the expression level of KDM5B was reduced in mouse LSC compared with differentiated leukemia cells (non-LSC) (Wong et al. 2015). Therefore, we also compared KDM5B expression in human LSCs (CD34+ CD38−) and non-LSCs (CD34−) from the GSE63270 data set (Jung et al. 2015). Interestingly, we found a higher KDM5B level in human LSCs than in non-LSC cells, opposite to the results obtained from the mouse MLL-AF9 leukemia model (Fig. 1C; Wong et al. 2015). To verify our analysis further, we collected mononuclear cells from the peripheral blood (PB) of 21 AML patients and 16 HDs (Supplemental Table S1) and verified the mRNA and protein expression of KDM5B. Consistent with database analysis, we found that the mRNA (monocytes and CD11b+ of peripheral blood mononuclear cells [PBMC]) and protein levels of KDM5B were significantly higher in AML patients compared with HDs (Fig. 1D,E). Collectively, these results demonstrated that KDM5B is highly expressed in primary human AML cells.

FIGURE 1.

FIGURE 1.

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KDM5B is highly expressed in AML patients. (A) Expression levels of KDM5B in HDs (n = 20) and AML patients (n = 698). Data were analyzed from the published GSE114868 data set. (B) Expression levels of KDM5B in normal (HDs, n = 76), AML patients with different types of chromosome rearrangements, and patients with MDS (from A.K to MDS, n = 48, 28, 351, 40, 37, 206, respectively). Data were analyzed from the published GSE13159 data set. (A.K) Aberrant karyotype, (N.K) normal karyotype. (C) Expression levels of KDM5B in LSC (CD34+ CD38−) and corresponding non-LSC cells (CD34−) (n = 20). Data were analyzed from the published GSE63270 data set. (D) The mRNA levels of KDM5B were examined in PBMCs from HD monocytes (n = 10) and CD11b+ (n = 6), as well as from AML patients (n = 15). The levels were determined by real-time quantitative PCR (RT-qPCR) and normalized by Actb. (E) The protein level of KDM5B in AML patients (n = 6) and HDs (n = 6) were examined by western blotting. (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001, by Student's t-test.

Silencing of KDM5B-induced apoptosis and proliferation defect in primary human AML and some human AML cell lines

To investigate the functional role of KDM5B in AML, we down-regulated its expression using two pairs of short hairpin RNAs (shRNAs) and evaluated its impact on the proliferation and apoptosis of primary human AML cells. The mRNA level of KDM5B was successfully reduced by shRNAs (Fig. 2A), and silencing KDM5B inhibited proliferation (Fig. 2B) and induced apoptosis (Fig. 2C). We then repeated these experiments in several blood cancer cell lines, including MOLM-13 (FLT3-ITD mutation and expressing MLL-AF9 mRNA), MOLM-14 (a sister cell line of MOLM-13), MV-4-11 (FLT3-ITD mutation), OCI-AML3 (NPM1/DNMT3A mutations), THP-1 (MLL-AF9 rearrangement), P493 (c-Myc translocation), and K562 (BCR-ABL1 positive). Interestingly, we found that silencing KDM5B slowed proliferation and induced apoptosis in MOLM-13, MOLM-14, MV-4-11, and OCI-AML3 cells (Fig. 2D–G; Supplemental Fig. S1) but had a minor effect on THP-1, P493, or K562 cells (Supplemental Fig. S2). We also measured short-term proliferation by BrdU staining in control and KDM5B knockdown (KD) MOLM-13 cells. The results showed that BrdU incorporation was slightly inhibited by shRNA-1 but was not affected by shRNA-2, possibly due to its poor KD efficiency (Fig. 2H). In addition, the colony formation assay confirmed that the colony number was reduced by silencing KDM5B (Fig. 2I). Furthermore, lentivirus-mediated overexpression of KDM5B substantially enhanced the proliferative capacity of MOLM-13 cells (Fig. 2J). Notably, upon transplantation of KDM5B KD MOLM-13 cells into NOG mice, the size of the spleen was reduced (Fig. 2K, left), the white blood cell (WBC) count decreased (Fig. 2K, right), and their survival rate (Fig. 2L) increased compared to the controls. Altogether, these results demonstrate that KDM5B inhibits apoptosis and promotes cell proliferation in primary human AML cells and some human AML cell lines.

FIGURE 2.

FIGURE 2.

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Silencing KDM5B inhibited proliferation and induced apoptosis in primary AML cells and certain human AML cell lines. (A–C) The primary human AML cells were collected and transduced with lentivirus expressing scramble (shScr) and shRNAs. The mRNA level of KDM5B was measured by RT-qPCR and normalized by Actb (A). Proliferation was measured by Cell Counting Kit-8 (CCK-8) assay (450 nm) (B). The cell apoptosis was measured by Annexin V staining and analyzed/visualized by flow cytometry and Flowjo (C, right) Quantification from triplicates. The cells were collected from three AML patients (n = 3). The clinical information for these samples is provided in Supplemental Table S1. (D–H) MOLM-13 cells were transduced with lentivirus expressing scramble (shScr) and shRNAs. The relative mRNA and protein levels of KDM5B were measured by RT-qPCR (D) and western blotting (E), respectively. Cell apoptosis (F) was measured by Annexin V staining, and proliferation was measured by respective CCK-8 assay (G) and BrdU staining (H). (I) The colony-formation ability of KDM5B KD and control MOLM-13 cells was measured in a methylcellulose medium (STEMCELL Technologies, Cat #: M3434). (J) MOLM-13 cells were infected with lentivirus expressing GFP control and GFP-KDM5B, and the percentage of GFP+ cells was measured at indicated times. The percentage of GFP-positive cells was measured by flow cytometry, which was further analyzed by Flowjo software. (D–J) Quantification from three biological repeats. (K,L) After exposure to a nonlethal radiation dose (1 Gy), 2 × 106 MOLM-13 cells with control and KDM5B KD were transplanted into NOD mice. The spleen size/weight (K, left) and leukocyte number (K, right) were measured and recorded 2 wk after transplantation. The survival of the remaining mice was monitored (L). n = 5 for each comparison. Data represent the mean + SD (A, C, D–F, and H–K) or ±SD (B,G). (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001, by Student's t-test (A–K) and log-rank test (L).

The interferon response pathway was activated by silencing KDM5B in some human blood cancer cell lines

Since silencing KDM5B in MOLM-13 cells could recapitulate the phenomenon in primary human AML cells, this cell line was selected for our subsequent mechanistic study. To identify the downstream target gene of KDM5B, we performed mRNA-sequencing in control and KDM5B KD MOLM-13 cells. We found that a total of 5356 genes were significantly affected (P < 0.05) upon silencing KDM5B (Supplemental Table S2). Of these, we further identified 131 up-regulated and 128 down-regulated genes (fold change >2) that were significantly affected upon loss of KDM5B (Fig. 3A). KDM5B catalyzes H3K4 demethylation at the promoter of the target genes, which results in gene inactivation (Plch et al. 2019). Therefore, we focused on the up-regulated genes upon depletion of KDM5B. Consistent with recent reports that KDM5B represses the immune response in cancer cells (Wu et al. 2018; Zhang et al. 2021), gene enrichment analysis demonstrated that interferon-related signaling pathway genes, including STAT1, STAT2, IRF7, IRF9, OASL, OAS1, OAS2, IFI6, and IFIT1, were significantly activated by depletion of KDM5B (Fig. 3B). Several of the enriched genes were further validated by qPCR and western blotting (Fig. 3C,D). Primers are provided in Supplemental Table S3. In addition, we assessed the expression of the interferon response genes in various blood cancer cell lines after silencing KDM5B. We found that most of these genes, like IRF7, showed an increase in expression in MOLM-14, MV-4-11, and P493 cells, although the effect of silencing KDM5B on the proliferation and apoptosis of P493 cells was minor (Supplemental Fig. S3A–F). However, only a few genes were significantly affected by KDM5B KD in THP-1 cells (Supplemental Fig. S3G,H). Collectively, our results demonstrate that the interferon response pathway is inhibited by KDM5B, which may promote the immune evasion of specific human blood cancer cells.

FIGURE 3.

FIGURE 3.

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Silencing KDM5B activates the interferon response genes in MOLM-13 cells. (A,B) mRNA-sequencing analysis of MOLM-13 cells upon KDM5B KD. (A) Heatmap of differential expression (fold change >2) genes. (B) KEGG pathway analysis of the 131 up-regulated genes upon KDM5B KD. (C) Relative mRNA levels of indicated genes were checked by RT-qPCR and normalized to Actb. Data were generated from three biological repeats. Primers are provided in Supplemental Table S2. The P-value of significantly affected genes is marked. (D) Immunoblotting of indicated proteins in control and KDM5B KD MOLM-13 cells. (Right) Statistical analysis from three biological repeats. Data represent the mean + SD (C,D). (*) P < 0.05 and (**) P < 0.01, by Student's t-test.

Identification of miR-140-3p as a downstream target gene of KDM5B

Since the role of KDM5B in inhibiting the expression of immune response-related genes has been well investigated, we evaluated the effect of KDM5B on the expression of microRNAs (miRNAs). miRNA sequencing was performed in duplicate in control and KDM5B KD cells (Supplemental Table S4). Among the 32 known miRNAs significantly affected upon depletion of KDM5B, we selected and focused on miR-140-3p, which ranked first among 16 up-regulated known miRNAs (Fig. 4A; Supplemental Table S4). To investigate whether miR-140-3p was one of the target genes of KDM5B, we checked the expression level of miR-140-3p in several cell lines, including MOLM-13, MOLM-14, and MV-4-11, after knocking down KDM5B. The results demonstrated that the expression of miR-140-3p was significantly up-regulated by two to six folds in the KDM5B KD cells compared to the control cells (Fig. 4B–D). Alternatively, overexpression of KDM5B inhibited miR-140-3p expression (Fig. 4E). We then demonstrated that KDM5B has a minor effect on the expression of other KDM5 family genes, which rules out the possibility of a compensatory role in KDM5B KD cells (Fig. 4F). Furthermore, the results of ChIP-qPCR analysis indicated that H3K4me3 was enriched significantly at the promoter of miR-140-3p and other target genes, which showed up-regulation in KDM5B KD cells compared to control cells (Fig. 4G). Also, the CUT&Tag-qPCR assay demonstrated that the binding of KDM5B at these genes was significantly reduced in KDM5B KD cells (Fig. 4H,I). This suggests that KDM5B directly inhibits miR-140-3p expression. Altogether, our results demonstrate that miR-140-3p is a direct downstream target gene of KDM5B.

FIGURE 4.

FIGURE 4.

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Identification of miR-140-3p as a downstream target of KDM5B in human AML cells. (A) Heatmap of the differential expression of miRNAs (P < 0.05) in control and KDM5B KD MOLM-13 cells. (B–D) The expression level of miR-140-3p was determined in KDM5B KD MOLM-13 (B), MOLM-14 (C), and MV-4-11 cells (D). (E) The expression level of miR-140-3p in KDM5B overexpressed MOLM-13 cells. (F) The expression levels of indicated genes in control and KDM5B KD MOLM-13 cells. (G) Evaluation of H3K4me3 enrichment on target genes by ChIP-qPCR in control and KDM5B KD MOLM-13 cells. (H,I) Evaluation of KDM5B binding on target genes by CUT&Tag-qPCR in control and KDM5B KD MOLM-13 (H) and MOLM-14 (I) cells. Quantification from three biological repeats. Data represent the mean + SD. (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001, by Student's t-test.

The expression of miR-140-3p is negatively correlated with KDM5B in primary human AML cells

To assess the expression of miR-140-3p and its correlation with KDM5B in AML cells, we started by analyzing genetic alterations of miR-140-3p using the cBio Cancer Genomics Portal (http://www.cbioportal.org/) (Cerami et al. 2012). Interestingly, we found that miR-140-3p was frequently deleted in leukemia cells, although amplification was observed in other cancer cell types (Fig. 5A). We then analyzed the expression of miR-140-3p in HDs and AML patients from two data sets (GSE51908 and GSE209871) (Tan et al. 2014; Leoncini et al. 2022). Our analysis revealed that miR-140-3p was significantly down-regulated in AML cells compared with normal granulocytes and monocytes (GSE51908) (Fig. 5B). In the GSE209871 data set, although similar levels of miR-140-3p were observed in HDs and AML patients with relapse at diagnosis, the expression of miR-140-3p in AML patients (no relapse) was much lower than that in HDs (Fig. 5C; Leoncini et al. 2022). To verify our analysis, we collected PBMCs from HDs and AML patients and validated miR-140-3p down-regulation in AML patients by qPCR (Fig. 5D). Importantly, we further showed that the expression of miR-140-3p and KDM5B was negatively correlated in primary human AML cells (Fig. 5E). Altogether, these results demonstrate that miR-140-3p is down-regulated and negatively correlated with KDM5B expression in primary human AML cells.

FIGURE 5.

FIGURE 5.

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KDM5B negatively correlates with the expression of miR-140-3p in primary human AML. (A) Divergent alterations of the miR-140-3p in human cancers. Data were generated from cBioPortal (http://www.cbioportal.org/). (B) Expression levels of miR-140-3p in normal granulocytes (n = 13), monocytes (n = 6), and AML patients (n = 18). Data were analyzed from the published GSE51908 data set. (C) Expression levels of miR-140-3p in HDs (n = 8) and AML patients with (n = 26) or no (n = 10) relapse. Data were analyzed from the published GSE209871 data set. (D) Expression levels of miR-140-3p were measured in peripheral monocytes from HDs (n = 6) and AML patients (n = 5). (E) The expression levels of KDM5B and miR-140-3p were measured in AML patients (n = 16). Data represent the mean + SD (D). (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001, by Student's t-test (B–D).

The BCL2 3′ untranslated region (3′-UTR) is the direct target of miR-140-3p

miRNAs are short noncoding RNAs (ncRNAs) of ∼22 nt that mediate gene silencing by binding and guiding Argonaut (AGO) proteins to target sites in the 3′-UTR of mRNAs (Carthew and Sontheimer 2009). To identify the target gene of miR-140-3p, we analyzed the overlapping target mRNAs using the TargetScan, DIANA, and miRDB online analysis tools. Among a total of 36 overlapping genes (Fig. 6A; Supplemental Table S5), B-cell CLL/lymphoma 2 (BCL2), which protects leukemia cells from apoptosis, was reported to be highly expressed in leukemia (Roberts 2020), prompting us to ask whether KDM5B inhibits apoptosis through the miR-140-3p/BCL2 axis. We further found that there were two target sites (5′-CUGUGGU-3′) in the 3′ UTR of BCL2 (Fig. 6B). To confirm whether BCL2 3′ UTR is the direct target of miR-140-3p, we synthesized and transfected the mimic, inhibitor and respective negative control (NC) into 293T cells. The qPCR results showed that while the mimic could be detected by miR-140-3p-specific primers, transfection of the inhibitor significantly down-regulated the expression of miR-140-3p (Fig. 6C,D). We then cloned the full-length (WT) BCL2 3′ UTR and its mutant (MUT) into the psiCHECK reporter plasmid (Promega), generating fractions of psi-WT and psi-MUT, respectively. The dual-luciferase reporter assay was performed after transfection of these fractions with mimic and mimic NC into 293T cells. Our results showed that the luciferase activity of psi-WT was significantly decreased when cotransfected with the mimic (Fig. 6E). Moreover, a comparison of the luciferase value in psi-WT- and psi-MUT-overexpressing cells showed that the mimic could inhibit the luciferase activity of psi-WT but had a minor effect on psi-MUT (Fig. 6E), suggesting that the inhibiting function of the mimic is specifically dependent on the presence of the target sequence on psi-WT.

FIGURE 6.

FIGURE 6.

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Identification of BCL2 as a direct target of miR-140-3p. (A) Venn diagram of the miR-140-3p target genes from TargetScan, miRDB, and DIANA databases. (B) Predicted binding sites (in red) of miR-140-3p in the 3′ UTRs of BCL2. The full-length sequence of miR-140-3p (Mimic) and inhibitor was presented. (C,D) The miR-140-3p mimic (C), inhibitor (D), and NC were transfected into MOLM-13 cells, respectively. Expression of miR-140-3p was measured by RT-qPCR and quantified from three biological repeats. The sequence information of synthesized miR-140-3p mimic, inhibitor, and respective NC is provided in Supplemental Table S3. (E) The psiCHECK-2 dual-luciferase reporter plasmid harboring wild-type BCL2 3′ UTR (WT) or mutated version (both of the two binding sites were replaced with 5′-GACACCA-3′), together with miR-140-3p Mimics and NC were transfected into 293T cells, respectively. The effects of BCL2 3′-UTR WT, its mutant, and miR-140-3p Mimics on luciferase intensity were evaluated. The quantification was performed using data from two biological replicates. (F) The miR-140-3p Mimic, inhibitor, and respective NC were transfected into MOLM-13 cells, respectively. Indicated proteins were detected by western blotting. (Bottom) Quantification from three biological repeats. Data represent the mean + SD (C–F). (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001, by Student's t-test (C,D,F) and one-factor ANOVA with a post hoc t-test (E).

To assess the impact of miR-140-3p on BCL2 expression in cells, we transfected the mimic, inhibitor, and respective NC into MOLM-13 cells. Western blotting results showed that the BCL2 protein level was decreased by mimic transfection. In contrast, the transfection of the inhibitor reversed this trend (Fig. 6F). Collectively, these results demonstrate that the BCL2 3′ UTR is the direct target of miR-140-3p.

Inhibition of miR-140-3p alleviates KDM5B silencing-induced proliferation reduction

To address whether inhibition of miR-140-3p can alleviate KDM5B silencing-induced proliferation reduction in AML cells, we initially verified that the expression level of BCL2 was reduced upon silencing KDM5B in MOLM-13 (Fig. 7A) and other cell lines (Supplemental Fig. S4A–C). Moreover, overexpression of KDM5B led to increased BCL2 expression (Fig 7B). We then transfected the miR-140-3p inhibitor and NC into control and KDM5B KD cells. We found that the inhibitor could rescue the expression of BCL2 in KDM5B KD MOLM-13 and MOLM-14 cells (Fig. 7C; Supplemental Fig. S4D), suggesting that KDM5B KD-induced down-regulation of BCL2 expression was mediated by miR-140-3p. To address whether miR-140-3p/BCL2 is the main target pathway responsible for the reduction in proliferation upon silencing KDM5B, the proliferation rate of MOLM-13 (as shown in Fig. 7C) and MOLM-14 cells (Supplemental Fig. S4E) was measured by CCK-8 assay. Consistent with the expression level of BCL2, enforced expression of the miRNA inhibitor promoted cell proliferation (Fig. 7D; Supplemental Fig. S4E). Notably, the miRNA inhibitor, but not NC, could significantly alleviate KDM5B KD-induced proliferation dysfunction (Fig. 7D; Supplemental Fig. S4E), highlighting the anti-oncogenic role of miR-140-3p.

FIGURE 7.

FIGURE 7.

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The miR-140-3p inhibitor can alleviate KDM5B silencing-induced proliferation reduction. (A,B) The level of BCL2 was assessed in MOLM-13 cells with KDM5B silencing (A) or KDM5B overexpression (B). (C) miR-140-3p inhibitor and NC were transfected into control (shScr) and KDM5B KD MOLM-13 cells, respectively. BCL2 expression was measured by RT-qPCR (top) and western blotting (bottom). (D) CCK8 assay to measure the proliferation of the cells treated in (C). (E,F) MOLM-13 cells were treated with vehicle (DMSO) and increasing doses of PBIT. The mRNA levels of miR-140-3p (E) and BCL2 (F) were measured by RT-qPCR, and the cell apoptosis (G) was measured by Annexin V staining, respectively. Quantification from two (C) and three (A–C, E–G) biological repeats. (H) A model for the role of KDM5B in promoting leukemogenesis through miR-140-3p/BCL2 axis in AML cells. A high level of KDM5B inhibits the generation of miR-140-3p, which turns off its attenuation effect on BCL2, subsequently promoting the tumorigenesis of AML. Data represent the mean + SD (A–C, E–G) or ±SD (D). (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001, by Student's t-test (A,B,E–G) and one-factor ANOVA with a post hoc t-test (C,D).

To investigate the therapeutic potential of targeting KDM5B in AML, we evaluated the impact of two commercial KDM5B inhibitors, PBIT and GSK467, on the expression of miR-140-3p and BCL2, as well as on cell proliferation and apoptosis. MOLM-13 cells were more sensitive to PBIT than GSK467 (Supplemental Fig. S5A,B). Therefore, PBIT was chosen for subsequent assays. We observed that PBIT increased miR-140-3p expression (Fig. 7E), reduced BCL2 expression (Fig. 7F), and induced cell apoptosis (Fig. 7G) in a dose-dependent manner in MOLM-13 cells. However, the inhibitor PBIT had a minor effect on the apoptosis of normal PBMCs (Supplemental Fig. S5C). In addition, studies have shown that BCL2 is involved in regulating cell senescence (Gorgoulis et al. 2019; Martin et al. 2023) and activating caspase-9 (Marsden et al. 2002). Our experiments showed that silencing KDM5B had a minimal effect on cell senescence (Supplemental Fig. S6A), but promoted the cleavage of caspase-9 (Supplemental Fig. S6B) in MOLM-13 cells. Interestingly, combining KDM5B silencing with Venetoclax inhibitor resulted in a synergistic effect leading to an increase in caspase-9 cleavage and defective cell proliferation (Supplemental Fig. S6B,C). These findings demonstrate that KDM5B promotes BCL2 expression and proliferation of some types of AML cells by suppressing miR-140-3p.

DISCUSSION

KDM5B, a histone demethylase identified decades ago, is responsible for demethylating H3K4me2/3 (Xiang et al. 2007). Since H3K4 methylation is highly associated with gene activation, KDM5B has been considered a repressor of gene transcription. Later, KDM5B was reported to be highly up-regulated in breast cancer cells (Lu et al. 1999). Since then, the oncogenic function of KDM5B has been uncovered in multiple human cancers, including ovarian cancer, prostate cancer, bladder cancer, lung cancer, colorectal cancer, gastric cancer, malignant melanoma, and hepatocellular carcinoma (Xiang et al. 2007; Xhabija and Kidder 2019; He et al. 2022). However, the current understanding of the role of KDM5B in AML tumorigenesis is controversial. Wong and his colleague showed that depletion of KDM5B in a mouse MLL-AF9 model and several human AML cell lines promoted cell proliferation, suggesting that KDM5B may serve as a tumor suppressor in AML (Wong et al. 2015). In contrast, others have shown that depletion of KDM5B induces cell apoptosis in AML cell lines, which indicates that KDM5B may play an oncogenic role in AML (Su et al. 2015; Shokri et al. 2018). In this study, we started with analyzing KDM5B expression in primary human AML cells from Gene Expression Omnibus (GEO) data sets, followed by qPCR and western blot validation. These analyses confirmed a higher expression level of KDM5B in AML patients than in healthy controls. In our subsequent studies, we discovered that KDM5B plays a significant role in preventing apoptosis and promoting proliferation in primary human AML cells. We observed this effect in MOLM-13, MOLM-14, and MV-4-11 cells, which all have an FLT3-ITD mutation, but not in THP-1, K562, and P493 cell lines. Our discovery implies that the function of KDM5B in AML cells may depend on specific mutations. Therefore, KDM5B could be a potential therapeutic target for FLT3-ITD-positive AML patients. To further explore the oncogenic role of KDM5B in human AML, future works, such as transplantation experiments involving primary human AML cells with specific mutation xenografts, are needed.

miRNAs are a class of single-stranded noncoding RNAs that directly bind to the 3′ UTRs of target genes to suppress gene expression at the posttranscriptional level (Sontheimer 2005; Wang et al. 2016b; Gabra and Salmena 2017). Increasing evidence has demonstrated that miRNAs play a crucial role in tumorigenesis (Ding et al. 2018; Huang et al. 2018; Wang et al. 2018). In this study, we identified that miR-140-3p was the downstream target gene of KDM5B. Functional analyses showed that miR-140-3p significantly inhibited the proliferation of AML cells by targeting and silencing BCL2. Since BCL2 is a well-established oncogene that can promote tumorigenesis by protecting tumor cells from apoptosis, our studies imply that KDM5B may promote AML tumorigenesis through BCL2 modulation.

The innate immune system regulates the interferon gene family, which is crucial in the immune system response. Although studies have shown that interferon treatment benefits AML patients (Zhang et al. 2021, 2023), myeloid malignancies, like other types of cancer, have ways to manipulate and evade the immune system (Tettamanti et al. 2022). Studies have demonstrated that KDM5B plays a role in immune evasion by inhibiting interferon responses (Wu et al. 2018; Zhang et al. 2021). Our research has shown that when KDM5B is suppressed, the genes involved in the interferon-signaling pathway get activated in MOLM-13, MOLM-14, and P493 cells. This indicates that KDM5B has a role in inhibiting immune responses in specific blood cancer cells. Therefore, a combination of targeting KDM5B and interferon therapy may have a significant impact on the immunotherapy of some blood cancers that have specific mutations.

In conclusion, our study provides evidence that KDM5B is overexpressed in human AML. The high level of KDM5B directly inhibits the expression of miR-140-3p, leading to up-regulated expression of BCL2, which in turn promotes the proliferation of AML cells (Fig. 7H). Our studies uncover a novel oncogenic role of KDM5B. Targeting the KDM5B/miR-140-3p/BCL2 axis may offer therapeutic potential in treating certain types of human AML.

MATERIALS AND METHODS

Cell culture and antibodies

HEK293T (293T) cells were maintained in Dulbecco's modified Eagle medium (Invitrogen) containing 10% fetal bovine serum (FBS). The cell lines MOLM-13, MOLM-14, MV4-11, THP-1, P493, and K562 were incubated with RPMI 1640 medium, and the OCI-AML3 cell line was incubated with Iscove's modified Dulbecco's medium (IMDM, Invitrogen), all of which were supplemented with 20% FBS (Thermo Scientific). Stem Cell Bank, Chinese Academy of Sciences, kindly provided the above cell lines. If needed, primary human AML cells and fresh isolated PBMCs were cultured in StemSpan serum-free expansion medium (STEMCELL Technologies, Cat #: 09600). The KDM5B inhibitors PBIT (Cat #: HY-101451) and GSK467 (Cat #: HY-116761), and BCL2 inhibitor Venetoclax (Cat #: HY-15531) were obtained from MedChemExpress. Antibodies against KDM5B (Cat #: 3273) and caspase-9 (Cat #: 9504) were purchased from Cell Signaling Technology (CST). Antibodies against STAT1 (Cat #: 66545-1-Ig), STAT2 (Cat #: 16674-1-AP), IRF7 (Cat #: 22392-1-AP), and BCL-2 (Cat #: 12789-1-AP) were obtained from Proteintech. Antibody against β-actin (Cat #: ABl1010) was purchased from Abbkine.

Clinical sample collection

PBMCs anticoagulated with EDTA were obtained from AML patients and HDs from The First Affiliated Hospital of Zhengzhou University (ZZU). This study was approved by the Research and Clinical Trial ethics committee, The First Affiliated Hospital of ZZU (no. 2023-KY-0813). A total of 47 PBMC samples obtained from 24 untreated AML patients and 23 healthy controls were enrolled in this study. The clinical information of these samples is provided in Supplemental Table S1. Per the manufacturer's instructions, PBMCs were isolated from whole blood using density gradient centrifugation. CD11b+ myeloid cells were isolated from PBMCs using microbeads (Cat #: 130-097-142, Miltenyi Biotec), if required. AML patients were clinically diagnosed based on the French–American–British (FAB) classification criteria.

Lentivirus packaging and infection

pLKO.1-based lentivirus plasmids expressing scramble and KDM5B shRNAs were constructed as we recently described (Qu and Yang 2021; Liu et al. 2023). Viral particles were produced in 293T cells following the recommended protocols (Addgene) and further concentrated by PGE6000 when necessary. Cells were infected with viruses in the presence of 8 μg/mL polybrene (Sigma). Stable cell lines were obtained by puromycin (2 μg/mL) screening for 2–3 d. If needed, the primary human AML cells were grown in StemSpan serum-free expansion medium (STEMCELL Technologies, Cat #: 09600). Concentrated lentivirus was added to the cells along with 4 μg/mL polybrene (Sigma) and incubated. After 24 h, the old medium was replaced with the fresh expansion medium containing 1 μg/mL puromycin. The cells were selected by puromycin for 2 d and then used for different assays.

Real-time quantitative PCR (RT-qPCR) and RNA sequencing

Total RNA was extracted with TRIzol reagent (TaKaRa), and cDNA was obtained by reverse transcription using the PrimeScript RT Reagent Kit with gDNA Eraser (Clontech). The qPCR was conducted with TB Green Premix Ex Taq (Clontech) on a Quantagene q225 Real-Time PCR System (Kubo Technology). Relative mRNA levels of indicated genes were normalized to Actb. RNA sequencing was conducted and analyzed as we recently described (Liu et al. 2023). The Hairpin-it miRNAs quantification and U6 calibration RT-qPCR kit were used to measure miR-140-3p levels (GenePharma).

Transfection of the antagomir and Mimic of miR-140-3p

The chemically modified antagomir (Inhibitor) complementary to miR-140-3p was designed to inhibit endogenous miR-140-3p expression. The Inhibitor, Mimic of miR-140-3p, and their NCs were obtained from GenePharma. The detailed sequences are provided in Supplemental Table S3. According to the manufacturer's instructions, cells were transfected with the Mimic/Inhibitor and their NC using Lipofectamine 2000 (Invitrogen).

Cell Counting Kit-8 (CCK-8) and methylcellulose colony formation assays

The cells were resuspended in RPMI 1640 medium and seeded in 96-well plates. CCK-8 (Abbkine) reagent was added to each well and incubated for an extra 2 h before analysis. The absorbance was measured at 450 nm with a Thermo Scientific microplate reader. The colony-forming capacity of cells was measured using the methylcellulose medium (Cat #: M3434, STEMCELL Technologies) according to the manufacturer's instructions. In brief, 600 cells were seeded in 3.5 mm plates with 1.1 mL of MethoCult M3434 medium and cultured at 37°C for 10–14 d. Colonies consisting of 40 or more cells were counted and recorded.

Cell cycle and apoptosis analysis

Cell cycle and apoptosis analyses were performed as previously described (Yang et al. 2019). Briefly, the BrdU-FITC Flow Kit (BD, Cat #: 559619) and Annexin V-FITC Apoptosis Detection Kit (BD, Cat #: 556547) were used to evaluate the cell cycle and apoptosis, respectively, by following the manufacturer's instructions. Stained cells were analyzed by flow cytometer (BD Biosciences), and the acquired data were analyzed using FlowJo.

Western blotting

Total protein was extracted using RIPA lysis buffer (Sigma). The lysates were then boiled for 10 min after adding 4× loading buffer. Individual protein samples were then separated by SDS-PAGE. Then, the SDS-PAGE gel band was transferred to a nitrocellulose membrane. After adding the indicated primary antibodies, the membranes were incubated overnight at 4°C. The membranes were washed with PBS three times. Subsequently, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (Abcam) antibody for 1 h at room temperature. Actb was used as an internal control. The expression values were analyzed with ImageJ software.

Senescence staining

The senescence staining was performed using the β-Galactosidase Staining Kit according to the manufacturer's instructions (Cat #: G1580, Solarbio Life Sciences).

Chromatin immunoprecipitation (ChIP) assay

The ChIP assay for H3K4me3 was carried out as previously described (Jiang et al. 2011; Liu et al. 2023). Briefly, ∼1 × 107 cells were cross-linked by 1% formaldehyde, lysed, and sonicated to obtain chromatin fragments in a size range between 200 and 700 bp. Solubilized chromatin was diluted in ChIP dilution buffer (1:10, Upstate) and incubated with antibody overnight at 4°C. Protein A sepharose resin (Sigma) was used to capture the antibody–chromatin complex and then washed with low salt, LiCl, and TE (pH 8.0) wash buffers (Upstate). Enriched chromatin fragments were eluted at 65°C for 10 min, subjected to reversal of crosslinking at 65°C for 5 h, and purified by proteinase K (1 mg/mL) digestion, phenol–chloroform extraction, and ethanol precipitation. Purified ChIP DNA was then subjected to qPCR analysis.

CUT&Tag-qPCR

Endogenous KDM5B CUT&Tag and its qPCR were performed as described (Li et al. 2021). The Novoprotein NovoNGS CUT-Tag 3.0 High-sensitivity Kit (for Illumina) (Cat #: N259-YH01) was used for library construction. Briefly, 1 × 105 cells were incubated with 10 μL CoA magnetic beads for 10–15 min at room temperature. The mixture was then incubated with KDM5B primary antibody (1:50 dilution) for 2 h at room temperature, followed by incubation with goat anti-rabbit IgG H&L (1:200 dilution) for 1 h at room temperature. After washing, the beads were incubated with ChiTag RpAG-Transposome for 1 h at room temperature and then incubated with 40 μL Tagmentation buffer for 1 h at 37°C. DNA fragments were recovered and amplified by PCR using the primer library in the kit. Finally, the purified DNA was diluted six times and was used for qPCR. The binding site at the Actb gene locus was amplified and used as an internal reference. Primer sequences are listed in Supplemental Table S3.

Luciferase reporter assay

Fragments of the BCL-2 3′ UTR containing the putative wild-type or mutant miR-140-3p binding sites were amplified, cloned, and inserted into the psiCHECK-2 luciferase reporter plasmid (Promega), according to the manufacturer's instructions. The constructs were cotransfected into 293T cells with the empty vehicle, miR-140-3p mimic (50 nM), or mimic NC (50 nM) using Lipofectamine 2000 (Invitrogen). The Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System Kit (Promega) 48 h after transfection. At least three triplicates with two independent experiments were performed for each treatment.

Xenotransplantation of MOLM-13 cells in NOG mice

The 8–10 wk-old female NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt mice (Strain no.: T001475, GemPharmatech) were bred and maintained at the Laboratory Animal Center (Huazhong University of Science and Technology). All mice were treated humanely and under the approval of the Animal Care and Ethics Committee at Huazhong University of Science and Technology. The mice received sublethal irradiation (1 Gy) and were then transplanted with 2 × 106 control and KDM5B KD MOLM-13 cells via the tail vein (n = 10). The mice were euthanized for analysis when the control mice developed leukemia syndrome (weight loss and enlarged spleen), and the rest were monitored for survival.

Statistical analysis

Unless indicated in the figure legends, the unpaired two-tailed Student's t-test was used to calculate P-values and evaluate the statistical significance of the differences between the indicated samples. The data displayed are the mean ± standard deviation (SD). The P-value is presented as *P < 0.05, **P < 0.01, ***P < 0.001 (n.s., no significance), respectively. Four-group comparisons were analyzed by one-factor or two-factor ANOVA. If the ANOVA was overall significant, a post hoc Student's t-test was used for pairwise comparison.

DATA DEPOSITION

The original RNA sequencing data are available from the corresponding author on request. All other data generated or analyzed in this study are included in this paper.

SUPPLEMENTAL MATERIAL

Supplemental material is available for this article.

ACKNOWLEDGMENTS

We are very grateful to Wei Jie (Tsinghua University) for providing the full-length cDNA of KDM5B, Lingqiang Zhu (Huazhong University of Science and Technology) for providing psiCHECK reporter plasmid, Innovation and Research Center (School of Basic Medicine, Huazhong University of Science and Technology) for providing flow cytometry equipment, and the Laboratory Animal Center (Huazhong University of Science and Technology) for providing quality care for mice. This work was supported by the National Natural Science Foundation of China (82070171, Z.Y.; 82222002, J.Q.; 82000164, S.J.), the Department of Science and Technology of Hubei Province (2021CFA051, Z.Y.), and a startup fund from Huazhong University of Science and Technology (Z.Y.). This study was approved by the Research and Clinical Trial Ethics Committee, The First Affiliated Hospital of Zheng Zhou University (no. 2023-KY-0813).

Author contributions: J.Q. and Z.Y. conceived and supervised the study. J.H. performed most of the experiments. S.J., R.G., W.W., C.Y., and Y.Q. performed some experiments, including RT-qPCR, western blotting, and isolating primary blood cells. Q.C. analyzed some GSE data sets. X.H. provided some instructive suggestions for data analysis. Z.Y. and J.H. analyzed the data. Z.Y. wrote the paper.

Footnotes

MEET THE FIRST AUTHOR

Jiaojuan Huang.

Jiaojuan Huang

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Meet the First Author(s) is an editorial feature within RNA, in which the first author(s) of research-based papers in each issue have the opportunity to introduce themselves and their work to readers of RNA and the RNA research community. Jiaojuan Huang is the first author of this paper, “Histone lysine demethylase KDM5B facilitates proliferation and suppresses apoptosis in human acute myeloid leukemia cells through the miR-140-3p/BCL2 axis.” Jiaojuan obtained her B.S. from Anhui Medical University and M.S. from Southwest Jiaotong University. She is currently pursuing her PhD in the Department of Genetics, Tongji Medical College, Huazhong University of Science and Technology, under the guidance of Dr. Zhenhua Yang. In this experiment, the mechanism of KDM5B in human acute myeloid leukemia (AML) was studied in detail. The study provides valuable information for KDM5B in leukemia research, which enriches our understanding of the mechanism of leukemia refractory and provides a theoretical basis for designing personalized treatment programs.

What are the major results described in your paper and how do they impact this branch of the field?

Our study presents the first evidence that KDM5B is highly expressed in AML patients. We then conducted a knockdown experiment and found that the reduction of KDM5B leads to apoptosis of cells. Interestingly, we observed that this apoptosis mainly occurred in cell lines with FLT-ITD mutation, and the effect of KDM5B in other AML cell lines was not significant. The expression and function of KDM5B in AML have been a topic of controversy, but our study has clarified its role in AML. The different efficacy of KDM5B in different cell lines is an important consideration for clinical precision treatment of human AML.

What led you to study RNA or this aspect of RNA science?

In the last two decades, the discovery of numerous noncoding RNAs and their functions have highlighted the crucial role of noncoding RNA genes in regulating and expressing genetic information. Initially, we overlooked miRNA during our experiment, resulting in several detours while exploring the mechanism of KDM5B in AML. However, our perspective shifted toward miRNA, and we discovered its regulatory role in the process. This research experience sparked my interest in RNA and motivated me to explore additional RNA functions in the future.

During the course of these experiments, were there any surprising results or particular difficulties that altered your thinking and subsequent focus?

After conducting the CUT-TAG experiment, I extracted DNA, amplified it, and ran the gel. However, I did not see any bands, which was frustrating. I was unsure if the protocol went wrong, or if the antibody was not suitable for the experiment. The antibody used in the CUT-TAG experiment was not indicated as appropriate, which added to my confusion. Eventually, we increased the DNA template content and the number of amplification cycles, exceeding the maximum number of cycles given in the manual. This led to a successful outcome, which was a pleasant surprise. This experience taught me that it is important to not always follow instructions blindly, and to experiment appropriately. Despite the many failures in scientific research, it brings a sense of happiness that cannot be found elsewhere.

What are some of the landmark moments that provoked your interest in science or your development as a scientist?

During my master's degree studies, I discovered a lot of interesting things through experiments. At that time, I fell in love with research, so I chose to continue my PhD study after completing the M.S. Although there are many difficulties encountered during experiments, the surprises that experiments bring far outweigh the difficulties. Sometimes, when I think that my experimental research can contribute even a little bit to the development of human medicine, I feel that all the hard work is worth it. In short, medical research gives me a sense of fulfillment, so I love it.

If you were able to give one piece of advice to your younger self, what would that be?

First, time is precious, and especially when we're young, we have to prioritize and complete what we can. Second, we need to have the courage to explore and practice what we want, allowing ourselves more opportunities to experience trial and error, and overcome any obstacles that come our way. Finally, we mustn't be afraid of losing, as what we have lost will eventually come back to us in another form. As long as we maintain a good character and remain brave and confident, we will ultimately achieve our goals.

REFERENCES

  1. Bernstein BE, Mikkelsen TS, Xie X, Kamal M, Huebert DJ, Cuff J, Fry B, Meissner A, Wernig M, Plath K, et al. 2006. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125: 315–326. 10.1016/j.cell.2006.02.041 [DOI] [PubMed] [Google Scholar]
  2. Carthew RW, Sontheimer EJ. 2009. Origins and mechanisms of miRNAs and siRNAs. Cell 136: 642–655. 10.1016/j.cell.2009.01.035 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, Jacobsen A, Byrne CJ, Heuer ML, Larsson E, et al. 2012. The cBio Cancer Genomics Portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov 2: 401–404. 10.1158/2159-8290.CD-12-0095 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen B, Chen H, Lu S, Zhu X, Que Y, Zhang Y, Huang J, Zhang L, Zhang Y, Sun F, et al. 2022. KDM5B promotes tumorigenesis of Ewing sarcoma via FBXW7/CCNE1 axis. Cell Death Dis 13: 354. 10.1038/s41419-022-04800-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ding Q, Wang Q, Ren Y, Zhu HQ, Huang Z. 2018. MicroRNA-126 attenuates cell apoptosis by targeting TRAF7 in acute myeloid leukemia cells. Biochem Cell Biol 96: 840–846. 10.1139/bcb-2018-0017 [DOI] [PubMed] [Google Scholar]
  6. Eigendorff E, Hochhaus A. 2015. [Acute leukemia in adults]. Pathologe 36: 503–517, quiz 518–519 (in German). 10.1007/s00292-015-0087-y [DOI] [PubMed] [Google Scholar]
  7. Fennell KA, Bell CC, Dawson MA. 2019. Epigenetic therapies in acute myeloid leukemia: Where to from here? Blood 134: 1891–1901. 10.1182/blood.2019003262 [DOI] [PubMed] [Google Scholar]
  8. Flavahan WA, Gaskell E, Bernstein BE. 2017. Epigenetic plasticity and the hallmarks of cancer. Science 357: eaal2380. 10.1126/science.aal2380 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gabra MM, Salmena L. 2017. microRNAs and acute myeloid leukemia chemoresistance: a mechanistic overview. Front Oncol 7: 255. 10.3389/fonc.2017.00255 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gorgoulis V, Adams PD, Alimonti A, Bennett DC, Bischof O, Bishop C, Campisi J, Collado M, Evangelou K, Ferbeyre G, et al. 2019. Cellular senescence: defining a path forward. Cell 179: 813–827. 10.1016/j.cell.2019.10.005 [DOI] [PubMed] [Google Scholar]
  11. He R, Xhabija B, Gopi LK, Kurup JT, Xu Z, Liu Z, Kidder BL. 2022. H3K4 demethylase KDM5B regulates cancer cell identity and epigenetic plasticity. Oncogene 41: 2958–2972. 10.1038/s41388-022-02311-z [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Huang Y, Zou Y, Lin L, Ma X, Chen H. 2018. Identification of serum miR-34a as a potential biomarker in acute myeloid leukemia. Cancer Biomark 22: 799–805. 10.3233/CBM-181381 [DOI] [PubMed] [Google Scholar]
  13. Huang HH, Chen FY, Chou WC, Hou HA, Ko BS, Lin CT, Tang JL, Li CC, Yao M, Tsay W, et al. 2019. Long non-coding RNA HOXB-AS3 promotes myeloid cell proliferation and its higher expression is an adverse prognostic marker in patients with acute myeloid leukemia and myelodysplastic syndrome. BMC Cancer 19: 617. 10.1186/s12885-019-5822-y [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jiang H, Shukla A, Wang X, Chen WY, Bernstein BE, Roeder RG. 2011. Role for Dpy-30 in ES cell-fate specification by regulation of H3K4 methylation within bivalent domains. Cell 144: 513–525. 10.1016/j.cell.2011.01.020 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jung N, Dai B, Gentles AJ, Majeti R, Feinberg AP. 2015. An LSC epigenetic signature is largely mutation independent and implicates the HOXA cluster in AML pathogenesis. Nat Commun 6: 8489. 10.1038/ncomms9489 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kohlmann A, Kipps TJ, Rassenti LZ, Downing JR, Shurtleff SA, Mills KI, Gilkes AF, Hofmann WK, Basso G, Dell'orto MC, et al. 2008. An international standardization programme towards the application of gene expression profiling in routine leukaemia diagnostics: the Microarray Innovations in LEukemia study prephase. Br J Haematol 142: 802–807. 10.1111/j.1365-2141.2008.07261.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Leoncini PP, Vitullo P, Reddel S, Tocco V, Paganelli V, Stocchi F, Mariggiò E, Massa M, Nigita G, Veneziano D, et al. 2022. MicroRNA profiling of paediatric AML with FLT-ITD or MLL-rearrangements: expression signatures and in vitro modulation of miR-221-3p and miR-222-3p with BRD4/HATs inhibitors. Oncol Rep 48: 221. 10.3892/or.2022.8436 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Li G, Kanagasabai T, Lu W, Zou MR, Zhang SM, Celada SI, Izban MG, Liu Q, Lu T, Ballard BR, et al. 2020. KDM5B is essential for the hyperactivation of PI3K/AKT signaling in prostate tumorigenesis. Cancer Res 80: 4633–4643. 10.1158/0008-5472.CAN-20-0505 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Li C, Wang H, Yin Z, Fang P, Xiao R, Xiang Y, Wang W, Li Q, Huang B, Huang J, et al. 2021. Ligand-induced native G-quadruplex stabilization impairs transcription initiation. Genome Res 31: 1546–1560. 10.1101/gr.275431.121 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Liu Z, Hu W, Qin Y, Sun L, Jing L, Lu M, Li Y, Qu J, Yang Z. 2023. Isl1 promotes gene transcription through physical interaction with Set1/Mll complexes. Eur J Cell Biol 102: 151295. 10.1016/j.ejcb.2023.151295 [DOI] [PubMed] [Google Scholar]
  21. Lu PJ, Sundquist K, Baeckstrom D, Poulsom R, Hanby A, Meier-Ewert S, Jones T, Mitchell M, Pitha-Rowe P, Freemont P, et al. 1999. A novel gene (PLU-1) containing highly conserved putative DNA/chromatin binding motifs is specifically up-regulated in breast cancer. J Biol Chem 274: 15633–15645. 10.1074/jbc.274.22.15633 [DOI] [PubMed] [Google Scholar]
  22. Marsden VS, O'Connor L, O'Reilly LA, Silke J, Metcalf D, Ekert PG, Huang DC, Cecconi F, Kuida K, Tomaselli KJ, et al. 2002. Apoptosis initiated by Bcl-2-regulated caspase activation independently of the cytochrome c/Apaf-1/caspase-9 apoptosome. Nature 419: 634–637. 10.1038/nature01101 [DOI] [PubMed] [Google Scholar]
  23. Martin N, Popgeorgiev N, Ichim G, Bernard D. 2023. BCL-2 proteins in senescence: beyond a simple target for senolysis? Nat Rev Mol Cell Biol 24: 517–518. 10.1038/s41580-023-00594-y [DOI] [PubMed] [Google Scholar]
  24. Plch J, Hrabeta J, Eckschlager T. 2019. KDM5 demethylases and their role in cancer cell chemoresistance. Int J Cancer 144: 221–231. 10.1002/ijc.31881 [DOI] [PubMed] [Google Scholar]
  25. Qu J, Yang Z. 2021. Protocol to produce high-titer retrovirus for transduction of mouse bone marrow cells. STAR Protoc 2: 100459. 10.1016/j.xpro.2021.100459 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ren Z, Kim A, Huang YT, Pi WC, Gong W, Yu X, Qi J, Jin J, Cai L, Roeder RG, et al. 2022. A PRC2-Kdm5b axis sustains tumorigenicity of acute myeloid leukemia. Proc Natl Acad Sci 119: e2122940119. 10.1073/pnas.2122940119 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Roberts AW. 2020. Therapeutic development and current uses of BCL-2 inhibition. Hematology Am Soc Hematol Educ Program 2020: 1–9. 10.1182/hematology.2020000154 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rodríguez-Paredes M, Esteller M. 2011. Cancer epigenetics reaches mainstream oncology. Nat Med 17: 330–339. 10.1038/nm.2305 [DOI] [PubMed] [Google Scholar]
  29. Rose-Inman H, Kuehl D. 2014. Acute leukemia. Emerg Med Clin N Am 32: 579–596. 10.1016/j.emc.2014.04.004 [DOI] [PubMed] [Google Scholar]
  30. Rowe JM. 2015. Reasons for optimism in the therapy of acute leukemia. Best Pract Res Clin Haematol 28: 69–72. 10.1016/j.beha.2015.10.002 [DOI] [PubMed] [Google Scholar]
  31. Shah A, Andersson TM, Rachet B, Björkholm M, Lambert PC. 2013. Survival and cure of acute myeloid leukaemia in England, 1971–2006: a population-based study. Br J Haematol 162: 509–516. 10.1111/bjh.12425 [DOI] [PubMed] [Google Scholar]
  32. Shokri G, Doudi S, Fathi-Roudsari M, Kouhkan F, Sanati MH. 2018. Targeting histone demethylases KDM5A and KDM5B in AML cancer cells: a comparative view. Leuk Res 68: 105–111. 10.1016/j.leukres.2018.02.003 [DOI] [PubMed] [Google Scholar]
  33. Sontheimer EJ. 2005. Assembly and function of RNA silencing complexes. Nat Rev Mol Cell Biol 6: 127–138. 10.1038/nrm1568 [DOI] [PubMed] [Google Scholar]
  34. Su H, Ma X, Huang Y, Han H, Zou Y, Huang W. 2015. JARID1B deletion induced apoptosis in Jeko-1 and HL-60 cell lines. Int J Clin Exp Pathol 8: 171–183. [PMC free article] [PubMed] [Google Scholar]
  35. Tan YS, Kim M, Kingsbury TJ, Civin CI, Cheng WC. 2014. Regulation of RAB5C is important for the growth inhibitory effects of MiR-509 in human precursor-B acute lymphoblastic leukemia. PLoS ONE 9: e111777. 10.1371/journal.pone.0111777 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tettamanti S, Pievani A, Biondi A, Dotti G, Serafini M. 2022. Catch me if you can: how AML and its niche escape immunotherapy. Leukemia 36: 13–22. 10.1038/s41375-021-01350-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Timp W, Feinberg AP. 2013. Cancer as a dysregulated epigenome allowing cellular growth advantage at the expense of the host. Nat Rev Cancer 13: 497–510. 10.1038/nrc3486 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Verma S, Dhanda H, Singh A, Rishi B, Tanwar P, Chaudhry S, Siraj F, Misra A. 2021. Systematic review of epigenetic targets in acute myeloid leukemia. Am J Blood Res 11: 458–471. [PMC free article] [PubMed] [Google Scholar]
  39. Wang D, Han S, Peng R, Jiao C, Wang X, Yang X, Yang R, Li X. 2016a. Depletion of histone demethylase KDM5B inhibits cell proliferation of hepatocellular carcinoma by regulation of cell cycle checkpoint proteins p15 and p27. J Exp Clin Cancer Res 35: 37. 10.1186/s13046-016-0311-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wang J, Chen J, Sen S. 2016b. MicroRNA as biomarkers and diagnostics. J Cell Physiol 231: 25–30. 10.1002/jcp.25056 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wang Z, Luo H, Fang Z, Fan Y, Liu X, Zhang Y, Rui S, Chen Y, Hong L, Gao J, et al. 2018. MiR-204 acts as a potential therapeutic target in acute myeloid leukemia by increasing BIRC6-mediated apoptosis. BMB Rep 51: 444–449. 10.5483/BMBRep.2018.51.9.036 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wong SH, Goode DL, Iwasaki M, Wei MC, Kuo HP, Zhu L, Schneidawind D, Duque-Afonso J, Weng Z, Cleary ML. 2015. The H3K4-methyl epigenome regulates leukemia stem cell oncogenic potential. Cancer Cell 28: 198–209. 10.1016/j.ccell.2015.06.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wu L, Cao J, Cai WL, Lang SM, Horton JR, Jansen DJ, Liu ZZ, Chen JF, Zhang M, Mott BT, et al. 2018. KDM5 histone demethylases repress immune response via suppression of STING. PLoS Biol 16: e2006134. 10.1371/journal.pbio.2006134 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Xhabija B, Kidder BL. 2019. KDM5B is a master regulator of the H3K4-methylome in stem cells, development and cancer. Semin Cancer Biol 57: 79–85. 10.1016/j.semcancer.2018.11.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Xiang Y, Zhu Z, Han G, Ye X, Xu B, Peng Z, Ma Y, Yu Y, Lin H, Chen AP, et al. 2007. JARID1B is a histone H3 lysine 4 demethylase up-regulated in prostate cancer. Proc Natl Acad Sci 104: 19226–19231. 10.1073/pnas.0700735104 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yang Z, Shah K, Khodadadi-Jamayran A, Jiang H. 2019. Control of hematopoietic stem and progenitor cell function through epigenetic regulation of energy metabolism and genome integrity. Stem Cell Rep 13: 61–75. 10.1016/j.stemcr.2019.05.023 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yen CY, Huang HW, Shu CW, Hou MF, Yuan SS, Wang HR, Chang YT, Farooqi AA, Tang JY, Chang HW. 2016. DNA methylation, histone acetylation and methylation of epigenetic modifications as a therapeutic approach for cancers. Cancer Lett 373: 185–192. 10.1016/j.canlet.2016.01.036 [DOI] [PubMed] [Google Scholar]
  48. Zhang SM, Cai WL, Liu X, Thakral D, Luo J, Chan LH, Mcgeary MK, Song E, Blenman KRM, Micevic G, et al. 2021. KDM5B promotes immune evasion by recruiting SETDB1 to silence retroelements. Nature 598: 682–687. 10.1038/s41586-021-03994-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zhang Y, Gao Y, Jiang Y, Ding Y, Chen H, Xiang Y, Zhan Z, Liu X. 2023. Histone demethylase KDM5B licenses macrophage-mediated inflammatory responses by repressing Nfkbia transcription. Cell Death Differ 30: 1279–1292. 10.1038/s41418-023-01136-x [DOI] [PMC free article] [PubMed] [Google Scholar]

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