Acute Erythroleukemia Research Articles

Marine derived macrolide bryostatin 4 inhibits the TGF-β signaling pathway against acute erythroleukemia.

Acute erythroleukemia (AEL) is a rare and highly aggressive subtype of acute myeloid leukemia (AML) with an extremely poor prognosis when treated with available drugs. Therefore, new investigational agents capable of inducing remission are urgently required. Bioinformatics analysis, western blot and qRT-PCR were used to reveal the potential biological mechanism of bryostatin 4 (B4), an antineoplastic macrolide derived from the marine bryozoan Bugula neritina. Then, in vivo experiments were conducted to evaluate the role of transforming growth factor (TGF)-β signaling in the progression of AEL. Our results revealed that the proliferation of K562 cells and TF-1 cells was significantly inhibited by B4 at IC50 values of 37 nM and 52 nM, respectively. B4 inhibited TGF-β signaling and its downstream pathway targets, particularly the phosphorylation of Smad2, Smad3, Ras, C-RAF, ERK1/2, and MEK. B4 also played an important role in cell invasion and migration in K562 cells and TF-1 cells by reducing the protein levels of the mesenchymal cell marker vimentin. Moreover, Flow cytometry and western blot analyses demonstrated that B4 induced apoptosis and initiated G0/G1 phase arrest by modulating mitochondrial dysfunction and cyclin-dependent kinase (CDK) expression. These findings indicated that B4 could inhibit the proliferation, migration, invasion, and TGF-β signaling pathways of AEL cells, thus suggesting that B4 possesses therapeutic potential as a treatment for AEL.

Germ line ERG haploinsufficiency defines a new syndrome with cytopenia and hematological malignancy predisposition

AbstractThe genomics era has facilitated the discovery of new genes that predispose individuals to bone marrow failure (BMF) and hematological malignancy (HM). We report the discovery of ETS-related gene (ERG), a novel, autosomal dominant BMF/HM predisposition gene. ERG is a highly constrained transcription factor that is critical for definitive hematopoiesis, stem cell function, and platelet maintenance. ERG colocalizes with other transcription factors, including RUNX family transcription factor 1 (RUNX1) and GATA binding protein 2 (GATA2), on promoters or enhancers of genes that orchestrate hematopoiesis. We identified a rare heterozygous ERG missense variant in 3 individuals with thrombocytopenia from 1 family and 14 additional ERG variants in unrelated individuals with BMF/HM, including 2 de novo cases and 3 truncating variants. Phenotypes associated with pathogenic germ line ERG variants included cytopenias (thrombocytopenia, neutropenia, and pancytopenia) and HMs (acute myeloid leukemia, myelodysplastic syndrome, and acute lymphoblastic leukemia) with onset before 40 years. Twenty ERG variants (19 missense and 1 truncating), including 3 missense population variants, were functionally characterized. Thirteen potentially pathogenic erythroblast transformation specific (ETS) domain missense variants displayed loss-of-function (LOF) characteristics, thereby disrupting transcriptional transactivation, DNA binding, and/or nuclear localization. Selected variants overexpressed in mouse fetal liver cells failed to drive myeloid differentiation and cytokine-independent growth in culture and to promote acute erythroleukemia when transplanted into mice, concordant with these being LOF variants. Four individuals displayed somatic genetic rescue by copy neutral loss of heterozygosity. Identification of predisposing germ line ERG variants has clinical implications for patient and family diagnoses, counseling, surveillance, and treatment strategies, including selection of bone marrow donors and cell or gene therapy.

Myelodysplastic Syndrome with Excess Blasts and Erythroid Predominance (MDS-EB-E) With NPM1 Mutation and P53 Expression

Nucleophosmin 1 (NPM1) mutated Myelodysplastic Syndrome (MDS) is a distinctly rare sub-group of myeloid neoplasm with limited available data describing its biological behavior and outcome using different therapeutic strategies. We describe a case of NPM1 mutated MDS in a middle-aged male with excess blasts and excess erythroid precursors (MDS-EB-E) morphologically mimicking Acute Erythroleukemia (AEL) with an unfavorable outcome following Hypomethylating Agent (HMA) therapy. We briefly reviewed the clinicopathological and genetic landscape data of NPM1 mutated MDS along with the role of p53 immunohistochemistry in differentiating MDS-EB-E from AEL.

Vagal-mAChR4 signaling promotes Friend virus complex (FV)-induced acute erythroleukemia

Erythroleukemia belongs to acute myeloid leukemia (AML) type 6 (M6), and treatment remains difficult due to the poor prognosis of the disease. Friend virus (FV) is a complex of two viruses: Friend murine leukemia virus (F-MuLV) strain along with a defective spleen focus-forming virus (SFFV), which can induce acute erythroleukemia in mice. We have previously reported that activation of vagal α7 nicotinic acetylcholine receptor (nAChR) signaling promotes HIV-1 transcription. Whether vagal muscarinic signaling mediates FV-induced erythroleukemia and the underlying mechanisms remain unclear. In this study, sham and vagotomized mice were intraperitoneally injected with FV. FV infection caused anemia in sham mice, and vagotomy reversed this change. FV infection increased erythroblasts ProE, EryA, and EryB cells in the spleen, and these changes were blocked by vagotomy. In bone marrow, FV infection reduced EryC cells in sham mice, an effect that was counteracted by vagotomy. FV infection increased choline acetyltransferase (ChAT) expression in splenic CD4+ and CD8+ T cells, and this change was reversed by vagotomy. Furthermore, the increase of EryA and EryB cells in spleen of FV-infected wild-type mice was reversed after deletion of ChAT in CD4+ T cells. In bone marrow, FV infection reduced EryB and EryC cells in sham mice, whereas lack of ChAT in CD4+ T cells did not affect this change. Activation of muscarinic acetylcholine receptor 4 (mAChR4) by clozapine N-oxide (CNO) significantly increased EryB in the spleen but decreased the EryC cell population in the bone marrow of FV-infected mice. Thus, vagal-mAChR4 signaling in the spleen and bone marrow synergistically promotes the pathogenesis of acute erythroleukemia. We uncover an unrecognized mechanism of neuromodulation in erythroleukemia.

A Novel Chalcone Derivative Regulates the Expression and Phosphorylation of ERK1/2 by Inhibiting Fli-1 Promoter Activity for Preventing the Malignant Progression of Erythroleukemia

Acute erythroleukemia is a rare form of acute myeloid leukemia recognized by its distinct phenotypic attribute of erythroblasts proliferation. In this study, in vitro experiments showed that a newly synthesized chalcone (ZH-254) inhibited cell proliferation, caused apoptosis, arrested the cell cycle in the G1 phase, and downregulated Fli-1 expression by inhibiting Fli-1 promoter activity. In vivo experiments showed that ZH-254 could effectively alleviate splenomegaly and prolong the survival of erythroleukemia mice. RT-PCR and Western blot analysis showed that ZH-254 could regulate the expression of Fli-1 target genes and G1-phase-related cell cycle proteins, including Rb, Bcl-2, Bax, ERK1/2, Gata-1, P110, SHIP-1, p-ERK1, CDK4, C-myc, Cyclin D1, Smad-3, GSK-3, and p21. Among them, the compound most significantly regulated the expression and phosphorylation of ERK1, the target gene of Fli-1 involved in regulating cell proliferation and apoptosis. Thus, ZH-254 restricts the malignancy of erythroleukemia by causing the inactivation of Fli-1 expression via suppressing its promoter activity, further regulating the expression and phosphorylation of ERK1- and G1-phase-related genes. These results reveal the critical role of Fli-1 in the growth and survival of various hematological malignancies and point to chalcone derivatives as lead compounds for the development of anti-Fli-1 drugs for the treatment of erythroleukemia with overexpression of Fli-1.

Acute Erythroleukemia (AML-M6) in one-year-old boy: a case report

Background: Acute Erythroleukemia (AML-M6) is a rare case with approximately 3-4% of all Acute Myeloid Leukemia (AML) cases. AML-M6 is less common in the pediatric population, with an incidence of <1% of all pediatric leukemia cases. This case study aims to evaluate Acute Erythroleukemia (AML-M6) in a one-year-old boy at Prof. dr. IGNG Ngoerah Denpasar. Case Presentation: A one-year-old boy with clinical manifestations of fluctuating fever for a month, paleness, bleeding gums, bruising all over his body, swelling of both eyelids and splenomegaly. Peripheral Blood Smear (PBS) examination revealed bicytopenia with moderate normochromic normocytic anemia, severe thrombocytopenia and leukocytosis. A hypercellular picture was obtained from the examination of Bone Marrow Aspiration (BMA) with an increase in erythroblast activity of 74%, proerythroblast at 50% and normal myeloblasts activity at 10%. The patient was diagnosed with Acute Erythroleukemia (AML-M6). The patient died on day 16 of treatment. Conclusion: Based on the examination results, it was concluded that the patient was diagnosed with AML-M6 according to the PBS and BMA evaluation.

Functional Evaluation of KEL as an Oncogenic Gene in the Progression of Acute Erythroleukemia.

Acute erythroleukemia (AEL) is an infrequent subtype of acute myeloid leukemia (AML) with worse prognosis. Though the last decade has seen major advances in the novel features and genomic landscape in AEL, there is still a lack of specific therapeutic targets and effective treatment approaches for this disease. Here, we found a novel oncogene KEL that specifically and aberrantly expressed in patients with AEL. In this study, we demonstrated that KEL promoted cell proliferation and the downregulation of KEL reversed drug resistance in AEL cells to JQ1. Our findings suggested that KEL contributed to gain of H3K27 acetylation and promoted erythroid differentiation induced by GATA1. Additionally, GATA1 and TAL1 as cotranscription factors (TFs) modulated the expression of KEL. Maintaining cell viability and differentiation, KEL also played parts in the immune evasion of tumor cells. Our work expands the current knowledge regarding molecular mechanisms involved in cancer onset and progression, offering promising therapeutic target to broaden the treatment options.

Molecular Landscapes and Models of Acute Erythroleukemia.

Malignancies of the erythroid lineage are rare but aggressive diseases. Notably, the first insights into their biology emerged over half a century ago from avian and murine tumor viruses-induced erythroleukemia models providing the rationale for several transgenic mouse models that unraveled the transforming potential of signaling effectors and transcription factors in the erythroid lineage. More recently, genetic roadmaps have fueled efforts to establish models that are based on the epigenomic lesions observed in patients with erythroid malignancies. These models, together with often unexpected erythroid phenotypes in genetically modified mice, provided further insights into the molecular mechanisms of disease initiation and maintenance. Here, we review how the increasing knowledge of human erythroleukemia genetics combined with those from various mouse models indicate that the pathogenesis of the disease is based on the interplay between signaling mutations, impaired TP53 function, and altered chromatin organization. These alterations lead to aberrant activity of erythroid transcriptional master regulators like GATA1, indicating that erythroleukemia will most likely require combinatorial targeting for efficient therapeutic interventions.

Erythroleukemia: an Update

Acute erythroleukemia (AEL) is a rare form of acute myeloid leukemia recognized by erythroblastic proliferation. Many controversies remain around diagnosis influencing prognostic and therapeutic implications relating to this unique leukemia subset. The 2016 WHO classification includes more clear and restrictive diagnostic criteria for AEL. Primary acute erythroid leukemia is associated with complex and high-risk karyotypes including chromosomes 5q and 7q abnormalities. Mutational data shows that AEL is characterized by far lower NPM1 and FLT3-ITD mutation rates and higher mutational rates in TP53 compared with other AML subtypes. Hypomethylating agents have shown therapeutic value in AEL. In this article, we discuss the evolving diagnostic concepts of erythroleukemia, genomics, clinical outcome, and promising therapeutic targets through an appraisal of the current literature.

Acute Erythroleukemia in a Trauma Patient. A Case Report.

Introduction: Acute erythroid leukemia (AEL) is a rare type of acute myeloblastic leukemia. Acute erythroid leukemia is characterized by a predominant erythroid proliferation, there are 2 subtypes: erythroleukemia (erythroid/myeloid leukemia) and pure erythroid leukemia…
 Myelodysplastic syndromes (MDS) are a group of biologically and clinically heterogeneous clonal disorders characterized by ineffective hematopoiesis and peripheral cytopenia due to high apoptosis
 The purpose to presenting this case is how to differentiate an acute erythroleukemia (AEL) from myelodysplastic syndrome (MDS).
 Case report. A 74-year-old man came to emergency room after he falling down from the stairs and then was hospitalized with a two weeks history of severe weakness, anorexia, weight loss. He suffered of diabetes mellitus type II insulin dependent, and HTA. The physical examination showed cutaneous and conjunctival pallor, large ecchymosis in the low extremities and hepatosplenomegaly. CBC showed: pancytopenia and macrocytosis, neutrophils 60%, normoblasts 4:100 and reticulocyte count 3%.
 Conclusions: The diagnosis was Acute Erythroid Leukemia. The % of myeloblasts in bone marrow was > 20% of non-erythroid cells. Erythroleukemia characterizes from hepatomegaly this is found and in our case. Differential diagnosis is based mainly in bone marrow aspiration, immunophenotyping (flow cytometry).

Functional Evaluation and Mechanism Study of Kel As an Oncogenic Driver in Acute Erythroleukemia

Background Acute erythroleukemia (AEL) is defined as an distinct subtype (<5%) of AML in FAB classification, characterized by predominant erythropoiesis. It is associated with a poor prognosis, with a median overall survival (OS) of 3-14 months from diagnosis. No recurrent cytogenetic abnormality is specific of AEL. Through "Oncomine" Database, we found that KEL was significantly up-regulated in AEL patients, compared with other types of AML and normal patients. It has been reported that KEL promoter exhibited a strong transcriptional erythroid activity in K562 cells, but its role in the development of AEL remains unclear. Circular RNAs (circRNAs) have been implicated in various biological processes. Here we found one circRNA transcripted from KEL gene (circ-KEL), which also had a high expression in AEL, may play a role of coordinate regulation with KEL. The present study aimed to investigate the potential modulation of KEL in the development and progression of AEL. Method In our study, we collected blood samples of 11 AEL patient, 20 other types of AML patients and 20 normal patients. qPCR and Western blot analysis were performed to examine the expression of KEL and circ-KEL. We set up the erythroid differentiation models in vitro using two AEL cells lines (K562 and HEL) , induced with 50 μM hemin from day1 to day 7. The oncogenic functions of KEL were further measured in AEL cell lines by performing RNA interfering (RNAi), immunofluorescence (IF) staining, CCK8 assay and flow cytometry. The expression levels of γ-globin and FUT1 as well as the erythroid surface markers TER119 and GPA were examined to evaluate their function in erythroid differentiation. The proliferative ability of AEL cells was determined by CCK-8 assay. AEL animal models were established using NCG mice transplanted with K562 (NC and KEL over-expression). The tumor formation rate, body weight and survival curve were investigated to evaluate the oncogenic functions of KEL in-vivo. Protein antibody microarray was used to analyze the activation of related pathways after KEL over-expression in AEL cells. Moreover, we explored the molecular mechanisms of KEL and verified the interactions among GATA1 and H3K27ac as well as the PKC/RSK2/CREB pathway by performing ChIP, RIP, dual-luciferase reporter assay and Western blotting. Result KEL was specifically higher expressed in AEL clinical samples/cells lines, consistent with the results of the database. The over-expression of KEL could up-regulate the expression of GATA1, thus promoting the erythroid differentiation of AEL cells. Moreover, protein antibody microarray results showed that the up-regulation of KEL in AEL cells leads to the activation of multiple proliferation and PDL1 related pathways. After subsequent verification, it was verified that PKC/RSK2/CREB pathway was significantly activated. Meanwhile, the expression of PDL1 was highly correlated with KEL. We found that the higher expression of KEL in AEL cells could up-regulate PDL1 expression through activating the phosphorylation of relative transcript factors on PDL1 DNA promoter. In-vivo experiment showed that KEL over-expression may accelerate the leukemia progression. In addition, we discovered that JQ1 could be used as a inhibitor of KEL, which could effectively decrease the expression of GATA1 and PDL1. A number of circRNAs have been reported to act as microRNA (miRNA) sponges, which regulate target gene expression by interacting with miRNA. Our results revealed that circ-KEL could serve as a sponge for miR-671 and up-regulate miR-617 functional target KEL, thus promote the erythroid differentiation. Conclusion Our study reveals the oncogenic role and a novel regulatory mechanism of KEL in AEL progression. For the first time, we demonstrated that KEL was specifically highly expressed in AEL cells, leading to the dysregulation of erythroid differentiation through up-regulating GATA1, and on the other hand, promoting cell proliferation via PKC/RSK2/CREB pathway. Circ-KEL which also had a high expression in AEL, may play a role of coordinate regulation with KEL. These findings may provide a promising strategy for future diagnosis, biomarker discoveries and treatment for AEL patients. Figure Disclosures No relevant conflicts of interest to declare.

NUP98-KDM5A Fusion Induces Hematopoietic Cell Proliferation and Alters Myelo-Erythropoietic Differentiation

Chromosomal translocations are common in acute myeloid leukemia (AML), causing gene fusions that encode oncogenic proteins. The NUP98 gene participates in chromosomal rearrangements with over 30 fusion partners and comprises 6-10% of de novo cases of pediatric AML. These fusions are associated with poor prognosis in children and adults. Among them, NUP98-KDM5A is further enriched in specific subpopulations, found in 15% of non-Down syndrome acute megakaryoblastic leukemia cases and 20% of pediatric acute erythroleukemia. Despite these associations, the direct impact of NUP98-KDM5A on the growth and differentiation of human hematopoietic cells has not been systemically studied. In this study, we transduced cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) with NUP98-KDM5A or control lentiviral vectors and examined cell proliferation and differentiation. Exposure to cytokines (SCF, TPO, IL-6, FLT3L, SR-1) selected for CD34+ cell growth, and expression of NUP98-KDM5A increased proliferation rate in liquid culture by 19.4-fold (p<0.01). In addition to a growth advantage, expression of NUP98-KDM5A led to differences in differentiation. NUP98-KDM5A expressing cells showed a 4.9-fold decrease (p<0.05) in myeloid terminal differentiation (defined as the CD33+, CD11B+ population) and 2.8-fold increase (p<0.05) in early stem cell progenitors (defined as the CD117+ population). Interestingly, NUP98-KDM5A expressing cells showed a 3.3-fold increase (p<0.01) in the erythroid population (defined by the CD235a+, CD71+ population). The increase in erythroid differentiation was confirmed by RNA sequencing, which showed an enrichment in genes involved in heme metabolism (Hallmark Heme Metabolism, FDR q-value 7.33x10-8) after only 72 hours post-transduction. Furthermore, RNA sequencing demonstrated upregulation of HOXA genes, including HOXA3, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10 and HOXA11 (p 3.36x10-2 - 1.16x10-7), which has been previously described in other high-risk leukemic fusions (including other NUP98 fusions) as a primary mechanism of stem-cell renewal. To further elucidate NUP98-KDM5A effects on differentiation, cytokine supplements known to drive myeloid differentiation (SCF, TPO, G-CSF, and GM-CSF) were added to culture media, which demonstrated the same disruption in myelo-erythropoiesis based on a 4.0-fold decrease (p<0.001) in the terminal myeloid population, a 3.2-fold increase in CD117+ (p<0.001), and 2.6-fold increase in erythroid markers (p<0.001). Furthermore, NUP98-KDM5A recapitulated this phenotype in methylcellulose colony forming assays, with increased BFU-E and reduced CFU-GM colonies compared to control (p<0.01), despite no difference in overall colony number. Taken together, these in vitro studies demonstrate increased proliferation and altered differentiation in HSPCs by NUP98-KDM5A, with the pattern of differentiation by NUP98-KDM5A paralleling the M6 (erythroleukemia) AML subtype observed in patient samples harboring this fusion, supporting the clinical observation that NUP98-KDM5A is often a monogenic driver. To evaluate the leukemic potential of NUP98-KDM5A, cells were injected into NRG-SGM3 mice with NUP98-KDM5A inducing at 12 weeks a rapidly lethal myeloid disease in vivo (97.8% human CD45+ cells vs. 2.3% in control, p<0.001). Furthermore, spleens of the NUP98-KDM5A mice were markedly enlarged, weighing on average 926.7g vs. 47.3g spleens seen in control, a 19.8-fold increase in weight (p<0.05). In conclusion, the expression of NUP98-KDM5A in HSPCs drives proliferation and alters differentiation to maintain stem cell markers while driving an erythroid phenotype in vitro. Furthermore, this fusion is sufficient to engraft in the bone marrow and spleen of NRG-SGM3 mice in vivo and cause marked splenomegaly. Taken together, these data suggest this model properly recapitulates the human disease phenotype seen in patients expressing NUP98-KDM5A and future functional assays and drug screens may provide important insights into understanding the pathophysiology and pharmacologic vulnerabilities of this fusion class. Disclosures Mullighan: Loxo Oncology: Research Funding; Amgen: Honoraria, Other: speaker, sponsored travel; Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel; AbbVie: Research Funding; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding.

Transformation Mechanisms of the Nfia-ETO2 Fusion Gene Associated with Pediatric Pure Acute Erythroleukemia

Pure erythroleukemia (PEL) is a very aggressive, but poorly understood form of acute myeloid leukemia characterized by malignant accumulation of erythroid progenitor cells. A novel t(1;16)(p31;q24) chromosomal translocation leading to expression of a fusion between the nuclear factor I A (NFIA) and the ETO2 transcriptional co-regulator (a.k.a. CBFA2T3 or MTG16) has been identified in pediatric patients with PEL. Based on the function of the fusion partners, we hypothesized that NFIA-ETO2 (N-E) might initiate PEL by interfering with erythroid differentiation. To investigate its function, we cloned a full-length ORF and retrovirally expressed N-E in primary mouse bone marrow (BM)- and fetal liver (FL)-derived erythroblasts (EB). N-E expression significantly increased proliferation and blocked differentiation of EB. N-E expressing BM-derived hematopoietic stem and progenitor cells (HSPC) could be plated in erythropoietin (EPO)-containing methylcellulose (MC) for up to 3 rounds. Expression of N-E deletion mutants lacking the NFIA DNA-binding, the ETO2 NHR2 or NHR4 (ΔNHR4) transcriptional repression domains were unable to block erythroid differentiation. Notably, interfering with the ETO2-NHR2 oligomerization domain by overexpressing a competing peptide overcame the N-E-mediated differentiation block. Transplantation of N-E-expressing BM-derived HSPC into irradiated syngenic mice did not induce any disease suggesting the need of genetic cooperation. As TP53 gain-of-function (GOF) mutations are molecular hallmarks of PEL, we explored functional cooperation by using a conditional TP53R248Q allele. Interestingly, the TP53 status did not affect EB in vitro proliferation or differentiation. However, N-E expression increased proliferation of TP53R248Q+ EB and resulted in the formation of abnormal round and dense colonies in MC that could be serially propagated. In addition, transplantation of N-E-expressing TP53R248Q+ EB into irradiated recipients induced a transplantable PEL-like disease after a median latency of 4 months. Symptomatic mice presented with anemia, thrombocytopenia, multi-organ tumor cell infiltration and increased white blood cell counts. To better understand the molecular mechanism, we compared the gene expression signatures before and 24 hours after induced differentiation of FL-derived EB expressing WT or the inactive ΔNHR4 N-E mutant, in presence or absence of TP53R248Q. Principal component analysis (PCA) revealed a clear separation between the transcriptomes of WT EB expressing either the active or the inactive ΔNHR4 N-E (PC1:54.7%) and by their erythroid differentiation stage (PC2:9.07%). Overall, we observed 3753 (FDR<0.05, logFC>1.5) differentially expressed genes. Many of the significantly higher expressed genes (2313/3753) were related to hematopoietic stemness (GSEAs, p<0.001). Almost 10% of the significantly lower expressed genes (92/1440) were linked to the erythroid lineage development and to erythropoietic targets of NFIA or the erythroid master regulator GATA1. Interestingly, we also found reduced expression of genes encoding for ETO2-interacting transcription factors including TAL1 and KLF1. Despite a critical role on disease progression, PCA showed only minimal changes in the N-E expression signature in presence or absence of TP53R248Q with only 12 genes differently expressed (FDR<0.05, logFC>1). These genes were previously shown to be oncogenic mediators of TP53-GOF mutations, related to metabolism and transcriptional regulation. Interestingly, the signature of differentially expressed genes in N-E transformed FL-derived EB were significantly differentially expressed in tumor cells from pediatric but not adult PEL patients (p=0.00045), indicating the pediatric origin of the fusion. Collectively, we found that the PEL-associated N-E fusion blocks erythroid differentiation, and cooperates with a TP53-GOF mutation to induce a PEL-like disease in mice that phenocopies the human disease. Mechanistically, its activity seems to correlate with repression of erythroid regulatory genes controlled by the fusion partners NFIA, ETO2, and the erythroid master regulator GATA1. Disclosures No relevant conflicts of interest to declare.

Genomic Analysis Provides Insights into Acute Erythroleukemia

Acute erythroleukemia (AEL) is a genetically distinct malignancy from AML or MDS.

Genomic subtyping and therapeutic targeting of acute erythroleukemia.

Acute erythroid leukemia (AEL) is a high-risk leukemia of poorly understood genetic basis, with controversy regarding diagnosis in the spectrum of myelodysplasia and myeloid leukemia. We compared genomic features of 159 childhood and adult AEL cases with non-AEL myeloid disorders and defined five age-related subgroups with distinct transcriptional profiles: adult, TP53 mutated; NPM1 mutated; KMT2A mutated/rearranged; adult, DDX41 mutated; and pediatric, NUP98 rearranged. Genomic features influenced outcome, with NPM1 mutations and HOXB9 overexpression being associated with a favorable prognosis and TP53, FLT3 or RB1 alterations associated with poor survival. Targetable signaling mutations were present in 45% of cases and included recurrent mutations of ALK and NTRK1, the latter of which drives erythroid leukemogenesis sensitive to TRK inhibition. This genomic landscape of AEL provides the framework for accurate diagnosis and risk stratification of this disease, and the rationale for testing targeted therapies in this high-risk leukemia.

Knockdown of Long Noncoding RNA Plasmacytoma Variant Translocation 1 with Antisense Locked Nucleic Acid GapmeRs Exerts Tumor-Suppressive Functions in Human Acute Erythroleukemia Cells Through Downregulation of C-MYC Expression.

Objective: Acute erythroleukemia (AEL) is a subtype of acute myeloid leukemia (AML), with no specific treatment. Up- or downregulation of long noncoding RNAs (lncRNAs) is strongly associated with the formation and progression of many malignancies. Plasmacytoma variant translocation 1 (PVT1) is a significantly upregulated lncRNA in AML. Antisense locked nucleic acid (LNA) GapmeRs oligonucleotides are the novel tools for targeting lncRNAs. The purpose of the current study was to investigate the functional role of PVT1 antisense LNA GapmeRs on AEL cell line (KG-1). Materials and Methods: AEL cells were transfected with PVT1 antisense LNA GapmeRs at three different time points. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was accomplished to evaluate the PVT1 expression by PVT1 antisense LNA GapmeRs. The viability was evaluated by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) assay, and the apoptosis and necrosis were assessed by Annexin V/propidium iodide staining assay. The C-MYC expression level, the target gene of PVT1, was also quantified by qRT-PCR. Results: The results indicated that PVT1 inhibition could significantly decrease the viability of AEL cells, due to induction of apoptosis and necrosis, probably through the downregulation of C-MYC. Conclusions: Their findings suggest that the inhibition of lncRNA PVT1 could serve as a novel approach for controlling the proliferation of AEL cells and could open up a path for treatment of AEL.

Antiproliferative effect of upregulation of hsa-let-7c-5p in human acute erythroleukemia cells.

New achievements in the field of cancer treatment are results of recent advances in molecular medicine and gene therapy. Usage of microRNAs (miRNAs) which are small noncoding RNAs is one of the molecular research lines for the diagnosis and treatment of cancers. miRNAs have an important role in post-transcriptional regulation of the gene expression and are involved in cellular activities such as growth, differentiation, cell death and cancer development. One of the miRNAs that showed downregulation in human acute erythroleukemia is hsa-let-7c-5p. Down-regulation of hsa-let-7c-5p has been reported in in vitro studies of different cancers. In the present study, upregulation of hsa-let-7c-5p is performed in human acute erythroleukemia cell line (KG-1) using miRNA mimic. qRT-PCR, MTT assay, Annexin-V, and propidium iodide staining at different time points after miRNA mimic transfection were accomplished to assess the expression level of hsa-let-7c-5p, cell viability, apoptosis and late apoptosis. In addition, the expression level of PBX2 oncogene, a validated target gene of hsa-let-7c-5p, is evaluated by RT-qPCR to show the effectivity of this approach on erythroleukemia cancer cells. Our results can be used in translational medicine for future investigation in acute erythroleukemia and to approach treatment based on miRNA mimic therapy.

Induction of apoptosis and necrosis in human acute erythroleukemia cells by inhibition of long non-coding RNA PVT1.

Recent advances in molecular medicine have proposed new therapeutic strategies for cancer. One of the molecular research lines for the diagnosis and treatment of cancer is the use of long non-coding RNAs (LncRNAs) which are a class of non-coding RNA molecules longer than 200 base pairs in length that act as the key regulator of gene expression. Different aspects of cellular activities like cell growth, proliferation, differentiation, apoptosis and migration are regulated by lncRNAs. In various cancers, aberrant expression of lncRNAs has been reported. One of the lncRNAs that showed upregulation in human acute myeloid leukemia (AML) is lncRNA plasmacytoma variant translocation 1 (PVT1). Here, we performed blockage of lncRNA PVT1 in human acute erythroleukemia (AEL) cell line (KG1) using antisense LNA GapmeRs. Then, at different time points (24, 48 and 72 hours) after transfection, qRT‑real‑time PCR and AnnexinV/Propidium Iodide staining assay were performed. The data were processed using the ANOVA test. At all three time points, the ratio of apoptotic cells in the PVT1 antisense LNA GapmeRs treated group was higher than the other groups. The ratio of necrotic cells in the antisense LNA GapmeRs group was also higher than the other groups. These assessments show that inhibition of lncRNA PVT1 could significantly induce apoptosis and necrosis in KG1 cells. Our findings can be used in translational medicine for future investigation in acute erythroleukemia and treatment approach based on antisense therapy.

Fetal Inactivation of the Nuclear Interacting SET Domain Protein 1 Impairs Terminal Erythroid Maturation and Results in Acute Erythroleukemia in Mice

The nuclear receptor binding SET domain protein 1 (NSD1) histone (H3K36) methyltransferase is target of recurrent genetic alterations in human hematological malignancies and certain solid cancers. To study its role in normal hematopoiesis, we inactivated the Nsd1 gene in the blood forming system of the mouse. Vav1-iCre mediated cleavage of floxed exon 5 resulted in almost complete loss of Nsd1 expression at the mRNA and protein level. After a latency of 7-17 weeks, all Vav1-iCre;Nsd1fl/fl mice developed signs of disease, whereas heterozygous littermates expressed normal Nsd1 levels and remained healthy. Symptomatic mice presented anemia, reticulocytosis and thrombocytopenia with presence of erythroblasts on peripheral blood smears. Diseased mice had significant splenomegaly and infiltration of erythroblasts in spleen, liver and lung. Bone marrow (BM) transplantation from symptomatic mice was able to fully propagate the disease in wild type recipients, alone or in competition with normal cells. The majority of the BM and spleen cells of diseased mice expressed modest levels of the transferrin receptor (CD71dim) and variable amounts of c-Kit and FcγR-II/III, but no TER119, CD34, B220 or Sca-1. The cells formed abnormal BFU-like dense reddish and partially hemoglobinized colonies in erythropoietin (EPO) containing methylcellulose that could be replated up to 4 rounds. In Vav1-iCre;Nsd1fl/fl fetal livers, we also observed accumulation of abnormal erythroid progenitors, deficient for Nsd1, suggesting a prenatal origin of the phenotype. RNA sequencing of lineage marker-negative, Sca1+, c-Kit+ (LSK) cells revealed aberrant regulation of genes associated with/functioning in erythroid differentiation. Indeed, in vitro differentiation of Vav1-iCre;Nsd1fl/fl erythroblasts was significantly impaired. Retroviral expression of Nsd1 was able to partially rescue the erythroid differentiation block. Interestingly, Vav1-iCre;Nsd1fl/fl erythroblasts constitutively expressed significantly increased protein levels of the erythroid master transcriptional regulator GATA1 independent whether they were expanded as immature cells or induced for terminal maturation. Impaired terminal maturation of Vav1-iCre;Nsd1fl/fl erythroblasts was associated with reduced transactivation of GATA1 positively-regulated targets (HbbA, HbbB, Gpa, Bcl2l1, p21), while expression of GATA1-repressed target genes (Gata2, c-kit, Spi1) was not affected. Strikingly, exogenous expression of Gata1 was able to overcome the differentiation block of Vav1-iCre;Nsd1fl/fl erythroblasts depending on the integrity of the N- and C-zinc fingers, as seen in colony forming assays and liquid cultures. Chromatin immunoprecipitation (ChIP) revealed that binding of GATA1 to HbbA1 locus and to a lesser extent to regulatory elements in the Gata1 promoter region (G1-HE site) was impaired in Vav1-iCre;Nsd1fl/fl erythroblasts, but restored upon overexpression of exogenous Gata1 . However, we found no significant changes of global H3K36me1 and H3K36me2 marks in wild type and Vav1-iCre;Nsd1fl/fl erythroblasts. In addition, published ChIP-seq data revealed no enrichment for H3K36me1/2 marks near critical GATA1 binding sites in HbbA1 or Gata1 suggesting that NSD1 most likely regulates GATA1 activity by direct modification or by interfering with GATA1 interacting partners necessary for efficient transactivation of critical mediators of terminal erythroid maturation. Taken together, our work unraveled NSD1 as a critical regulator of terminal erythroid differentiation. DisclosuresNo relevant conflicts of interest to declare.

MYCN contributes to the malignant characteristics of erythroleukemia through EZH2-mediated epigenetic repression of p21

MYC proto-oncogene family including c-myc and n-myc (MYCN) are critical for normal cell development and tumorigenesis. Overexpression of c-myc causes acute erythroleukemia in vivo. However, the role of MYCN in acute erythroleukemia remains poorly understood. In this study, we found that the patients with erythroleukemia showed higher expression of MYCN than normal controls. In vitro experiments, knockdown of MYCN resulted in decreased cell proliferation, elevated autonomously cell apoptosis and increased P21-mediated cell senescence. On the contrary, overexpression of MYCN obviously promoted cell proliferation, and induced erythroid differentiation block and apoptosis resistance to cytotoxic agent. Further gene microarray and functional analysis revealed that EZH2 is a target of MYCN. Knockdown of MYCN inhibited the expression of EZH2, and then activated p21 expression through removal of H3K27me3 at the p21 promoter. Overexpression of ezh2 could antagonize the p21 activation caused by MYCN knockdown. In addition, Aurora inhibitor MLN8237 inhibited the proliferation of erythroleukemia cells through repression of MYCN/EZH2 axis, whereas it minimally affected the normal hematopoietic cells. In conclusion, MYCN contributes to the malignant characteristics of erythroleukemia through EZH2-meidated epigenetic repression of p21. MYCN may serve as a therapy target for the patients with acute erythroleukemia.