Characterization of functional transposable element enhancers in acute myeloid leukemia

Transposable elements(TEs) have been shown to have important gene regulatory functions and their alteration could lead to disease phenotypes. Acute myeloid leukemia(AML) develops as a consequence of a series of genetic changes in hematopoietic precursor cells, including mutations in epigenetic factors. Here, we set out to study the gene regulatory role of TEs in AML. We first explored the epigenetic landscape of TEs in AML patients using ATAC-seq data. We show that a large number of TEs in general, and more specifically mammalian-wide interspersed repeats(MIRs), are more enriched in AML cells than in normal blood cells. We obtained a similar finding when analyzing histone modification data in AML patients. Gene Ontology enrichment analysis showed that genes near MIRs in open chromatin regions are involved in leukemogenesis. To functionally validate their regulatory role, we selected 19 MIR regions in AML cells, and tested them for enhancer activity in an AML cell line(Kasumi-1) and a chronic myeloid leukemia(CML) cell line(K562); the results revealed several MIRs to be functional enhancers. Taken together, our results suggest that TEs are potentially involved in myeloid leukemogenesis and highlight these sequences as potential candidates harboring AML-associated variation.     

In this issue of Epigenetics

The pathogenesis of acute myeloid leukemias involves complex molecular events triggered by diverse alterations of genomic DNA. A limited number of initiating lesions, such as chromosomal translocations generating fusion genes, are constantly identified in specific forms of leukemia and are critical to leukemogenesis. Leukemia fusion proteins derived from chromosomal translocations can mediate epigenetic silencing of gene expression. Epigenetic deregulation of the DNA methylation status and of the chromatin “histone code” at specific gene sites cooperate in the pathogenesis of leukemias. The neutralization of these crucial oncogenic events can revert the leukemia phenotype. Thus, their identification and the study of their molecular and biological consequences is essential for the development of novel and specific therapeutic strategies. In this context, we recently reported a link between the differentiation block of leukemia and the epigenetic silencing of the microRNA-223 gene by the AML1/ETO oncoprotein, the product of the t(8;21) the commonest AML-associated chromosomal translocation. This finding indicates microRNAs as additional epigenetic targets for leukemogenesis and for therapeutic intervention in leukemias.     

Common signaling networks characterize leukemia-initiating cells in acute myeloid leukemia

Identification and characterization of leukemia-initiating cells (LICs) is important to understand leukemogenesis and develop novel therapies for leukemia. In this issue of Cell Stem Cell, Gibbs et?al. (2012) demonstrate that common active signaling pathways in LICs may be targeted to treat acute myeloid leukemia.     

Identification of differentially methylated markers among cytogenetic risk groups of acute myeloid leukemia

Aberrant DNA methylation is known to occur in cancer, including hematological malignancies such as acute myeloid leukemia (AML). However, less is known about whether specific methylation profiles characterize specific subcategories of AML. We examined this issue by using comprehensive high-throughput array-based relative methylation analysis (CHARM) to compare methylation profiles among patients in different AML cytogenetic risk groups. We found distinct profiles in each group, with the high-risk group showing overall increased methylation compared with low- and mid-risk groups. The differentially methylated regions (DMRs) distinguishing cytogenetic risk groups of AML were enriched in the CpG island shores. Specific risk-group associated DMRs were located near genes previously known to play a role in AML or other malignancies, such as MN1, UHRF1, HOXB3, and HOXB4, as well as TRIM71, the function of which in cancer is not well characterized. These findings were verified by quantitative bisulfite pyrosequencing and by comparison with results available at the TCGA cancer genome browser. To explore the potential biological significance of the observed methylation changes, we correlated our findings with gene expression data available through the TCGA database. The results showed that decreased methylation at HOXB3 and HOXB4 was associated with increased gene expression of both HOXB genes specific to the mid-risk AML, while increased DNA methylation at DCC distinctive to the high-risk AML was associated with increased gene expression. Our results suggest that the differential impact of cytogenetic changes on AML prognosis may, in part, be mediated by changes in methylation.     

Vitamin C promotes anti-leukemia of DZNep in acute myeloid leukemia

The epigenetic treatment by 3-Deazaneplanocin A (DZNep), a histone methyltransferase inhibitor, shows great potential against acute myeloid leukemia (AML). However, the variant sensitivity and incomplete response to DZNep are commonly observed. Here, we reveal that vitamin C (Vc) dramatically promotes DZNep response against leukemic cells in different cell lines and primary AML samples. Vc enhances apoptosis and differentiation induced by DZNep in different AML cell lines in vitro and reduces leukemia progression in vivo. At the molecular level, Vc downregulates an enzyme of serine synthesis named D-3-phosphoglycerate dehydrogenase (PHGDH), as well as BCL2, an anti-apoptotic gene. Over-expression of PHGDH reverses the Vc-enhanced anti-leukemic effect of DZNep in AML cells. Therefore, our findings provide an effective approach to reduce the resistance against epigenetic treatment by Vc, which shows a potential improvement of their combination in AML patients.     

MicroRNA-223 Regulates Granulopoiesis but Is Not Required for HSC Maintenance in Mice

MIR233 is genetically or epigenetically silenced in a subset of acute myeloid leukemia (AML). MIR223 is normally expressed throughout myeloid differentiation and highly expressed in hematopoietic stem cells (HSCs). However, the contribution of MIR223 loss to leukemic transformation and HSC function is largely unknown. Herein, we characterize HSC function and myeloid differentiation in Mir223 deficient mice. We show that Mir223 loss results in a modest expansion of myeloid progenitors, but is not sufficient to induce a myeloproliferative disorder. Loss of Mir223 had no discernible effect on HSC quiescence, long-term repopulating activity, or self-renewal capacity. These results suggest that MIR223 loss is likely not an initiating event in AML but may cooperate with other AML associated oncogenes to induce leukemogenesis.     

The role,mechanism and potentially therapeutic application of microRNA-29 family in acute myeloid leukemia

Abnormal proliferation, apoptosis repression and differentiation blockage of hematopoietic stem/progenitor cells have been characterized to be the main reasons leading to acute myeloid leukemia (AML). Previous studies showed that miR-29a and miR-29b could function as tumor suppressors in leukemogenesis. However, a comprehensive investigation of the function and mechanism of miR-29 family in AML development and their potentiality in AML therapy still need to be elucidated. Herein, we reported that the family members, miR-29a, -29b and -29c, were commonly downregulated in peripheral blood mononuclear cells and bone marrow (BM) CD34+ cells derived from AML patients as compared with the healthy donors. Overexpression of each miR-29 member in THP1 and NB4 cells markedly inhibited cell proliferation and promoted cell apoptosis. AKT2 and CCND2 mRNAs were demonstrated to be targets of the miR-29 members, and the role of miR-29 family was attributed to the decrease of Akt2 and CCND2, two key signaling molecules. Significantly increased Akt2, CCND2 and c-Myc levels in the AML cases were detected, which were correlated with the decreased miR-29 expression in AML blasts. Furthermore, a feed-back loop comprising of c-Myc, miR-29 family and Akt2 were found in myeloid leukemogenesis. Reintroduction of each miR-29 member partially corrected abnormal cell proliferation and apoptosis repression and myeloid differentiation arrest in AML BM blasts. An intravenous injection of miR-29a, -29b and -29c in the AML model mice relieved leukemic symptoms significantly. Taken together, our finding revealed a pivotal role of miR-29 family in AML development and rescue of miR-29 family expression in AML patients could provide a new therapeutic strategy.     

CXCR4 inhibitors selectively eliminate CXCR4-expressing human acute myeloid leukemia cells in NOG mouse model

The chemokine receptor CXCR4 favors the interaction of acute myeloid leukemia (AML) cells with their niche but the extent to which it participates in pathogenesis is unclear. Here, we show that CXCR4 expression at the surface of leukemic cells allowed distinguishing CXCR4^high from CXCR4^neg/low AML patients. When high levels of CXCR4 are expressed at the surface of AML cells, blocking the receptor function with small molecule inhibitors could promote leukemic cell death and reduce NOD/Shi-scid/IL-2Rγ^null (NOG) leukemia-initiating cells (LICs). Conversely, these drugs had no efficacy when AML cells do not express CXCR4 or when they do not respond to chemokine CXC motif ligand 12 (CXCL12). Functional analysis showed a greater mobilization of leukemic cells and LICs in response to drugs, suggesting that they target the interaction between leukemic cells and their supportive bone marrow microenvironment. In addition, increased apoptosis of leukemic cells in vitro and in vivo was observed. CXCR4 expression level on AML blast cells and their migratory response to CXCL12 are therefore predictive of the response to the inhibitors and could be used as biomarkers to select patients that could potentially benefit from the drugs.     

白血病的精准基因组医学研究与转化应用

白血病是常见的血液系统恶性肿瘤,治疗主要以化疗为主,但总体治疗效果欠佳且发病的分子机制不明。因此,白血病的发病机制以及临床研究急需新的突破口。近年来,研究人员不仅发现了急性髓系白血病耐药的新靶点,揭示不同表观修饰的相互作用加速MLL白血病进展,也阐释了NK细胞白血病发病机制,发现关键表观因子在髓系肿瘤发生中的重要功能。尤其是新抑癌基因SETD2的发现,为急性髓系白血病的治疗提供了新的靶点。此外,在国际上研究人员首次将低剂量化疗方案用于治疗初诊儿童急性髓系白血病,在不影响疗效的基础上显著降低了化疗毒副作用及治疗费用。虽然基因组特征的解析加深了我们对癌症生物学分子机制的理解,但是近年来的研究表明瘤内异质性的克隆演化也是导致白血病临床治疗效果不佳的主要因素,解析不同化疗方案下白血病患者不同的克隆演化模式及其在临床预后评估中的作用是目前的研究热点之一。上述研究为急性髓系白血病诊断及治疗方法的改进提供了新的契机。     

miR-638 Regulates Differentiation and Proliferation in Leukemic Cells by Targeting Cyclin-dependent Kinase 2

MicroRNAs have been extensively studied as regulators of hematopoiesis and leukemogenesis. We identified miR-638 as a novel regulator in myeloid differentiation and proliferation of leukemic cells. We found that miR-638 was developmentally up-regulated in cells of myeloid but not lymphoid lineage. Furthermore, significant miR-638 down-regulation was observed in primary acute myeloid leukemia (AML) blasts, whereas miR-638 expression was dramatically up-regulated in primary AML blasts and leukemic cell lines undergoing forced myeloid differentiation. These observations suggest that miR-638 might play a role in myeloid differentiation, and its dysregulation may contribute to leukemogenesis. Indeed, ectopic expression of miR-638 promoted phorbol 12-myristate 13-acetate- or all-trans-retinoic acid-induced differentiation of leukemic cell lines and primary AML blasts, whereas miR-638 inhibition caused an opposite phenotype. Consistently, miR-638 overexpression induced G₁ cell cycle arrest and reduced colony formation in soft agar. Cyclin-dependent kinase 2 (CDK2) was found to be a target gene of miR-638. CDK2 inhibition phenotypically mimicked the overexpression of miR-638. Moreover, forced expression of CDK2 restored the proliferation and the colony-forming ability inhibited by miR-638. Our data suggest that miR-638 regulates proliferation and myeloid differentiation by targeting CDK2 and may serve as a novel target for leukemia therapy or marker for AML diagnosis and prognosis.     

In Vitro Transformation of Primary Human CD34+ Cells by AML Fusion Oncogenes: Early Gene Expression Profiling Reveals Possible Drug Target in AML

Different fusion oncogenes in acute myeloid leukemia (AML) have distinct clinical and laboratory features suggesting different modes of malignant transformation. Here we compare the in vitro effects of representatives of 4 major groups of AML fusion oncogenes on primary human CD34+ cells. As expected from their clinical similarities, MLL-AF9 and NUP98-HOXA9 had very similar effects in vitro. They both caused erythroid hyperplasia and a clear block in erythroid and myeloid maturation. On the other hand, AML1-ETO and PML-RARA had only modest effects on myeloid and erythroid differentiation. All oncogenes except PML-RARA caused a dramatic increase in long-term proliferation and self-renewal. Gene expression profiling revealed two distinct temporal patterns of gene deregulation. Gene deregulation by MLL-AF9 and NUP98-HOXA9 peaked 3 days after transduction. In contrast, the vast majority of gene deregulation by AML1-ETO and PML-RARA occurred within 6 hours, followed by a dramatic drop in the numbers of deregulated genes. Interestingly, the p53 inhibitor MDM2 was upregulated by AML1-ETO at 6 hours. Nutlin-3, an inhibitor of the interaction between MDM2 and p53, specifically inhibited the proliferation and self-renewal of primary human CD34+ cells transduced with AML1-ETO, suggesting that MDM2 upregulation plays a role in cell transformation by AML1-ETO. These data show that differences among AML fusion oncogenes can be recapitulated in vitro using primary human CD34+ cells and that early gene expression profiling in these cells can reveal potential drug targets in AML.     

The combination of DNA methyltransferase inhibitor therapy and immunotherapy for acute myeloid leukemia

Acute myeloid leukemia (AML), the most common form of acute leukemia in adults, is characterized by abnormal proliferation and blocked maturation and differentiation of myeloid precursor cells. AML is an aggressive cancer that progresses rapidly without treatment. Therefore, effective treatment modalities should be implemented immediately after diagnosis. The mainstay of classical AML therapy has been chemotherapy, which is not suitable for relapsing or refractory patients, especially elderly patients. Among emerging novel therapeutic approaches for AML, epigenetic therapy and immunotherapy represent two exciting therapeutic developments. This review focuses on discussion of the therapeutic considerations for AML from the perspective of combination treatment, which incorporates both DNA methyltransferase inhibitor therapy, as one of the most promising epigenetic therapies, and immune checkpoint inhibitor or dendritic cell-based vaccination treatments, as examples of immunotherapy. Both challenges and rationale in the optimization of therapeutic approaches, as well as recent clinical trial developments, along this line are summarized.     

The re-expression of the epigenetically silenced e-cadherin gene by a polyamine analogue lysine-specific demethylase-1 (LSD1) inhibitor in human acute myeloid leukemia cell lines

Aberrant epigenetic silencing of tumor suppressor genes is a common feature observed during the transformation process of many cancers, including those of hematologic origin. Histone modifications, including acetylation, phosphorylation, and methylation, collaborate with DNA CpG island methylation to regulate gene expression. The dynamic process of histone methylation is the latest of these epigenetic modifications to be described, and the identification and characterization of LSD1 as a demethylase of lysine 4 of histone H3 (H3K4) has confirmed that both the enzyme and the modified histone play important roles as regulators of gene expression. LSD1 activity contributes to the suppression of gene expression by demethylating promoter-region mono- and dimethyl-H3K4 histone marks that are associated with active gene expression. As most post-translational modifications are reversible, the enzymes involved in the modification of histones have become targets for chemotherapeutic intervention. In this study, we examined the effects of the polyamine analogue LSD1 inhibitor 2d (1,15-bis{N ⁵-[3,3-(diphenyl)propyl]-N ¹-biguanido}-4,12-diazapentadecane) in human acute myeloid leukemia (AML) cell lines. In each line studied, 2d evoked cytotoxicity and inhibited LSD1 activity, as evidenced by increases in the global levels of mono- and di-methylated H3K4 proteins. Global increases in other chromatin modifications were also observed following exposure to 2d, suggesting a broad response to this compound with respect to chromatin regulation. On a gene-specific level, treatment with 2d resulted in the re-expression of e-cadherin, a tumor suppressor gene frequently silenced by epigenetic modification in AML. Quantitative chromatin immunoprecipitation analysis of the e-cadherin promoter further confirmed that this re-expression was concurrent with changes in both active and repressive histone marks that were consistent with LSD1 inhibition. As hematologic malignancies have demonstrated promising clinical responses to agents targeting epigenetic silencing, this polyamine analogue LSD1 inhibitor presents an exciting new avenue for the development of novel therapeutic agents for the treatment of AML.     

FISH<Superscript>+</Superscript>CD34<Superscript>+</Superscript>CD38<Superscript>-</Superscript> cells detected in newly diagnosed acute myeloid leukemia patients can predict the clinical outcome

Background

In acute myeloid leukemia (AML), the leukemia initiating cells (LICs) or leukemia stem cells (LSCs) is found within the CD34⁺CD38^- cell compartment. The LICs subpopulation survives chemotherapy and is most probable the cause of minimal residual disease (MRD), which in turn is thought to cause relapse. The aim of this study was to determine the prognostic value of the percentage of LICs in blasts at diagnosis.

Design and methods

The percentage of LICs in the blast population was determined at diagnosis using a unique Flow-FISH analysis, which applies fluorescent in situ hybridization (FISH) analysis on flow cytometry sorted cells to distinguish LICs within the CD34⁺CD38^- cell compartment. Fourty-five AML patients with FISH-detectable cytogenetic abnormalities treated with standardized treatment program were retrospectively included in the study. Correlations with overall survival (OS), events-free survival (EFS) and cumulative incidence of relapse (CIR) were evaluated with univariate and multivariate analysis.

Results

The percentage of LICs is highly variable in patients with acute myeloid leukemia, ranged from 0.01% to 52.8% (median, 2.1%). High LIC load (≥1%) negatively affected overall survival (2-year OS: 72.57% vs. 16.75%; P?=?0.0037) and events-free survival (2-year EFS: 67.23% vs. 16.33%; P?=?0.0018), which was due to an increased cumulative incidence of relapse (2-year CIR: 56.7% vs. 18.0%; P?=?0.021). By multivariate analysis, high LIC load retained prognostic significance for OS and EFS.

Conclusions

In the present study, we established the Flow-FISH protocol as a useful method to distinguish normal and leukemic cells within the CD34⁺CD38^- cell subpopulation. The high percentage of LICs at diagnosis was significantly correlated with increased risk of poor clinical outcome.

     

Down-regulation of cyclic nucleotide phosphodiesterase PDE1A is the key event of p73 and UHRF1 deregulation in thymoquinone-induced acute lymphoblastic leukemia cell apoptosis

Thymoquinone (TQ), the active principle of Nigella sativa black seeds, has anti-proliferative properties on numerous cancer cell types. Others and we have previously reported that TQ acts as agent that triggers cell cycle arrest and apoptosis through either a p53- or p73-dependent pathway. However, the immediate targets recruited upon TQ-induced cytotoxicity have not yet been clearly identified. We therefore asked whether cyclic nucleotide phosphodiesterases (PDEs) could be involved in TQ-triggered pro-apoptotic reactivity; PDEs are regulators of intracellular levels of cyclic nucleotides and therefore can modulate cAMP and cGMP-dependent cell death pathways. Our results showed that TQ specifically repressed PDE1A expression in the acute lymphoblastic leukemia Jurkat cell line. This effect is concomitant with the previously described sequential deregulation of the expression of the tumor suppressor protein p73 and the epigenetic integrator UHRF1 (Ubiquitin-like, PHD Ring Finger 1). Interestingly, RNA-interference knock-down of PDE1A expression as well as decreased PDE1A expression induced growth inhibition of Jurkat cells, cell cycle arrest and apoptosis through an activation of p73 and a repression of UHRF1. Conversely, PDE1A re-expression counteracted the cellular pro-apoptotic effects of TQ in association with a p73 repression and UHRF1 re-expression. Altogether, our results show that TQ induced an initial down-regulation of PDE1A with a subsequent down-regulation of UHRF1 via a p73-dependent mechanism. This study further proposes that PDE1A might be involved in the epigenetic code inheritance by regulating, via p73, the epigenetic integrator UHRF1. Our findings also suggest that a forced inhibition of PDE1A expression might be a new therapeutic strategy for the management of acute lymphoblastic leukemia.