c-Mpl-del, a c-Mpl alternative splicing isoform, promotes AMKL progression and chemoresistance.

Acute megakaryocytic leukemia (AMKL) is a clinically heterogeneous subtype of acute myeloid leukemia characterized by unrestricted megakaryoblast proliferation and poor prognosis. Thrombopoietin receptor c-Mpl is a primary regulator of megakaryopoeisis and a potent mitogenic receptor. Aberrant c-Mpl signaling has been implicated in a myriad of myeloid proliferative disorders, some of which can lead to AMKL, however, the role of c-Mpl in AMKL progression remains largely unexplored. Here, we identified increased expression of a c-Mpl alternative splicing isoform, c-Mpl-del, in AMKL patients. We found that c-Mpl-del expression was associated with enhanced AMKL cell proliferation and chemoresistance, and decreased survival in xenografted mice, while c-Mpl-del knockdown attenuated proliferation and restored apoptosis. Interestingly, we observed that c-Mpl-del exhibits preferential utilization of phosphorylated c-Mpl-del C-terminus Y607 and biased activation of PI3K/AKT pathway, which culminated in upregulation of GATA1 and downregulation of DDIT3-related apoptotic responses conducive to AMKL chemoresistance and proliferation. Thus, this study elucidates the critical roles of c-Mpl alternative splicing in AMKL progression and drug resistance, which may have important diagnostic and therapeutic implications for leukemia accelerated by c-Mpl-del overexpression.

[Blocking PAK1 kinase activity promotes the differentiation of acute megakaryocytic leukemia cells and induces their apoptosis].

Objective: To investigate the effect of blocking P21 activated kinase 1 (PAK1) activity on the proliferation, differentiation, and apoptosis of acute megakaryocytic leukemia (AMKL) cell lines (CHRF and CMK) . Methods: Cell counts were used to detect the effects of PAK1 inhibitors (IPA-3 and G5555) on AMKL cell proliferation inhibition and colony formation, and flow cytometry was used to detect its effects on AMKL cell cycle. The effect of PAK1 inhibitor on the expression of cyclin D1 and apoptosis-related protein Cleaved caspase 3 was detected using Western blot, while interference with the protein expression level of PAK1 in AMKL cells was assessed using lentivirus-mediated shRNA transfection technology. Flow cytometry was used to detect the effects of knockdown of PAK1 kinase activity on the ability of polyploid DNA formation and cell apoptosis in AMKL cells. Results: PAK1 inhibitors inhibited the proliferation of AMKL cells in a dose-dependent manner and reduced the ability of cell colony formation, and the difference was statistically significant when compared with the control group (P<0.05) . Moreover, they also reduced the percentage of AMKL cells in S phase, and Western blot detection showed that the expression levels of phosphorylated PAK1 and cyclin D1 decreased significantly. Finally, PAK1 inhibitors induced AMKL cell apoptosis by up-regulating Cleaved caspase 3 and showed different abilities to increase the content of polyploid DNA in megakaryocytes. Only high concentrations of IPA-3 and low doses of G5555 increased the number of polyploid megakaryocytes, while knockdown of PAK1 kinase activity promoted AMKL cell differentiation and increased the apoptosis rate. Conclusion: PAK1 inhibitor significantly arrests AMKL cell growth and promotes cell apoptosis. Knocking down the expression of PAK1 promotes the formation of polyploid DNA and induces AMKL cell apoptosis. The above findings indicate that inhibiting the activity of PAK1 may control AMKL effectively.

Stepwise GATA1 and SMC3 mutations alter megakaryocyte differentiation in a Down syndrome leukemia model.

Acute megakaryoblastic leukemia of Down syndrome (DS-AMKL) is a model of clonal evolution from a preleukemic transient myeloproliferative disorder requiring both a trisomy 21 (T21) and a GATA1s mutation to a leukemia driven by additional driver mutations. We modeled the megakaryocyte differentiation defect through stepwise gene editing of GATA1s, SMC3+/-, and MPLW515K, providing 20 different T21 or disomy 21 (D21) induced pluripotent stem cell (iPSC) clones. GATA1s profoundly reshaped iPSC-derived hematopoietic architecture with gradual myeloid-to-megakaryocyte shift and megakaryocyte differentiation alteration upon addition of SMC3 and MPL mutations. Transcriptional, chromatin accessibility, and GATA1-binding data showed alteration of essential megakaryocyte differentiation genes, including NFE2 downregulation that was associated with loss of GATA1s binding and functionally involved in megakaryocyte differentiation blockage. T21 enhanced the proliferative phenotype, reproducing the cellular and molecular abnormalities of DS-AMKL. Our study provides an array of human cell-based models revealing individual contributions of different mutations to DS-AMKL differentiation blockage, a major determinant of leukemic progression.

SON inhibits megakaryocytic differentiation via repressing RUNX1 and the megakaryocytic gene expression program in acute megakaryoblastic leukemia.

A high incidence of acute megakaryoblastic leukemia (AMKL) in Down syndrome patients implies that chromosome 21 genes have a pivotal role in AMKL development, but the functional contribution of individual genes remains elusive. Here, we report that SON, a chromosome 21-encoded DNA- and RNA-binding protein, inhibits megakaryocytic differentiation by suppressing RUNX1 and the megakaryocytic gene expression program. As megakaryocytic progenitors differentiate, SON expression is drastically reduced, with mature megakaryocytes having the lowest levels. In contrast, AMKL cells express an aberrantly high level of SON, and knockdown of SON induced the onset of megakaryocytic differentiation in AMKL cell lines. Genome-wide transcriptome analyses revealed that SON knockdown turns on the expression of pro-megakaryocytic genes while reducing erythroid gene expression. Mechanistically, SON represses RUNX1 expression by directly binding to the proximal promoter and two enhancer regions, the known +23 kb enhancer and the novel +139 kb enhancer, at the RUNX1 locus to suppress H3K4 methylation. In addition, SON represses the expression of the AP-1 complex subunits JUN, JUNB, and FOSB which are required for late megakaryocytic gene expression. Our findings define SON as a negative regulator of RUNX1 and megakaryocytic differentiation, implicating SON overexpression in impaired differentiation during AMKL development.

High-Throughput Production of Platelet-Like Particles.

The in vitro production of platelets could provide a life-saving intervention for patients that would otherwise require donor-derived platelets. Producing large numbers of platelets in vitro from their progenitor cells, megakaryocytes, remains remarkably difficult and inefficient. Here, a human megakaryoblast leukemia cell line (MEG-01) was used to assess the maturation of megakaryocytes and to develop a new methodology for producing high numbers of platelet-like particles from mature MEG-01 cells in vitro.

Acute Megakaryoblastic Leukemia With Diffuse Periosteal Reaction of Bilateral Lower Extremities.

We report the case of a 3-year-old girl diagnosed with acute megakaryoblastic leukemia, who presented after >1 year of bilateral leg pain. At times the pain was severe enough to prevent ambulation, prompting visits to her primary care provider. However, it was not until acute respiratory failure occurred with subsequent hospitalization in the pediatric intensive care unit that severe anemia and thrombocytopenia were discovered and the diagnosis of acute myeloid leukemia was made. Bilateral lower extremity swelling was noted on admission and radiographs showed diffusely abnormal appearance of the long bones of her lower extremities with periosteal reaction and echogenic debris in the subperiosteal space, thought to represent leukemic cells. This case highlights the importance of recognizing atypical signs and symptoms of myelodysplastic syndrome progressing to acute leukemia in the context of abnormal bone pain and radiographic changes.

A case of KMT2A-SEPT9 fusion-associated acute megakaryoblastic leukemia.

Acute megakaryoblastic leukemia (AMKL) constitutes ∼5%-15% of cases of non-Down syndrome AML in children, and in the majority of cases, chimeric oncogenes resulting from recurrent gene rearrangements are identified. Based on these rearrangements, several molecular subsets have been characterized providing important prognostic information. One such subset includes a group of patients with translocations involving the KMT2A gene, which has been associated with various fusion partners in patients with AMKL. Here we report the molecular findings of a 2-yr-old girl with AMKL and t(11;17)(q23;25) found to have a KMT2A-SEPT9 fusion identified through targeted RNA sequencing. A KMT2A-SEPT9 fusion in this subset of patients has not previously been reported.

The Comparative Sensitivity of Immunohistochemical Markers of Megakaryocytic Differentiation in Acute Megakaryoblastic Leukemia.

OBJECTIVES: Immunohistochemistry (IHC) staining of core biopsy sections often plays an essential role in the diagnosis of acute megakaryoblastic leukemia (AMKL). The goal of this study was to define the relative sensitivities of commonly used stains for markers of megakaryocytic differentiation. METHODS: The sensitivities of IHC stains for CD42b, CD61, and von Willebrand factor (vWF) were compared in 32 cases of pediatric AMKL. RESULTS: The sensitivities of CD42b, CD61, and vWF were 90.6%, 78.1% and 62.5%, respectively. When CD42b and CD61 were used together, the combined sensitivity increased to 93.6%. There were no cases in which vWF was positive when both CD42b and CD61 were negative. CONCLUSIONS: CD42b can reliably be used as a solitary first-line marker for blasts of megakaryocytic lineage, whereas CD61 may be reserved for infrequent cases that are CD42b negative. There is no role for the routine use of vWF when CD42b and CD61 are available.

GATA1 insufficiencies in primary myelofibrosis and other hematopoietic disorders: consequences for therapy.

INTRODUCTION: GATA1, the founding member of a family of transcription factors, plays important roles in the development of hematopoietic cells of several lineages. Although loss of GATA1 has been known to impair hematopoiesis in animal models for nearly 25 years, the link between GATA1 defects and human blood diseases has only recently been realized. Areas covered: Here the current understanding of the functions of GATA1 in normal hematopoiesis and how it is altered in disease is reviewed. GATA1 is indispensable mainly for erythroid and megakaryocyte differentiation. In erythroid cells, GATA1 regulates early stages of differentiation, and its deficiency results in apoptosis. In megakaryocytes, GATA1 controls terminal maturation and its deficiency induces proliferation. GATA1 alterations are often found in diseases involving these two lineages, such as congenital erythroid and/or megakaryocyte deficiencies, including Diamond Blackfan Anemia (DBA), and acquired neoplasms, such as acute megakaryocytic leukemia (AMKL) and the myeloproliferative neoplasms (MPNs). Expert commentary: Since the first discovery of GATA1 mutations in AMKL, the number of diseases that are associated with impaired GATA1 function has increased to include DBA and MPNs. With respect to the latter, we are only just now appreciating the link between enhanced JAK/STAT signaling, GATA1 deficiency and disease pathogenesis.

Large-scale culture of a megakaryocytic progenitor cell line with a single-use bioreactor system.

The increasing application of regenerative medicine has generated a growing demand for stem cells and their derivatives. Single-use bioreactors offer an attractive platform for stem cell expansion owing to their scalability for large-scale production and feasibility of meeting clinical-grade standards. The current work evaluated the capacity of a single-use bioreactor system (1 L working volume) for expanding Meg01 cells, a megakaryocytic (MK) progenitor cell line. Oxygen supply was provided by surface aeration to minimize foaming and orbital shaking was used to promote oxygen transfer. Oxygen transfer rates (kL a) of shaking speeds 50, 100, and 125 rpm were estimated to be 0.39, 1.12, and 10.45 h-1 , respectively. Shaking speed was a critical factor for optimizing cell growth. At 50 rpm, Meg01 cells exhibited restricted growth due to insufficient mixing. A negative effect occurred when the shaking speed was increased to 125 rpm, likely caused by high hydrodynamic shear stress. The bioreactor culture achieved the highest growth profile when shaken at 100 rpm, achieving a total expansion rate up to 5.7-fold with a total cell number of 1.2 ± 0.2 × 109 cells L-1 . In addition, cells expanded using the bioreactor system could maintain their potency to differentiate following the MK lineage, as analyzed from specific surface protein and morphological similarity with the cells grown in the conventional culturing system. Our study reports the impact of operational variables such as shaking speed for growth profile and MK differentiation potential of a progenitor cell line in a single-use bioreactor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:362-369, 2018.

Pediatric Acute Megakaryoblastic Leukemia: Multitasking Fusion Proteins and Oncogenic Cooperations.

Pediatric leukemia presents specific clinical and genetic features from adult leukemia but the underpinning mechanisms of transformation are still unclear. Acute megakaryoblastic leukemia (AMKL) is the malignant accumulation of progenitors of the megakaryocyte lineage that normally produce blood platelets. AMKL is diagnosed de novo, in patients showing a poor prognosis, or in Down syndrome (DS) patients with a better prognosis. Recent data show that de novo AMKL is primarily associated with chromosomal alterations leading to the expression of fusions between transcriptional regulators. This review highlights the most recurrent genetic events found in de novo pediatric AMKL patients and, based on recent functional analyses, proposes a mechanism of leukemogenesis common to de novo and DS-AMKL.

New hPSC-based human models to study pediatric Acute Megakaryoblastic Leukemia harboring the fusion oncogene RBM15-MKL1.

Pediatric Acute Megakaryoblastic Leukemia not associated to Down Syndrome (non-DS AMKL) is a rare disease with a dismal prognosis. Around 15% of patients carry the chromosomal translocation t(1;22) that originates the fusion oncogene RBM15-MKL1, which is linked to an earlier disease onset (median of 6months of age) and arises in utero. Here we report the generation of two hPSC cell lines constitutively expressing the oncogene RBM15-MKL1, resulting in an increased expression of known RBM15-MKL1 gene targets. These cell lines represent new disease models of pediatric AMKL to study the impact of the RBM15-MKL1 oncogene on human embryonic hematopoietic development.

GATA1 Is a Sensitive and Specific Nuclear Marker for Erythroid and Megakaryocytic Lineages.

OBJECTIVES: GATA binding factor 1 (GATA1) is a transcription factor essential for erythromegakaryocytic differentiation. Given its function in lineage specification, we sought to evaluate the immunohistochemical profile of GATA1 in normal marrow and acute leukemia and assess the use of GATA1 as a specific erythromegakaryocytic immunohistochemical marker. METHODS: Immunohistochemical studies for GATA1 expression were performed on bone marrow biopsy specimens to define its role in the evaluation of acute leukemia and other hematologic disorders. RESULTS: In normal marrows, intense nuclear reactivity is seen in immature erythroid precursors and megakaryocytes. Weak to moderate nuclear positivity is seen in eosinophils and mast cells. In marrows involved by acute leukemia, blasts of pure erythroleukemia and acute megakaryoblastic leukemia exhibit intense nuclear GATA1 positivity, while blasts of acute myeloid leukemia of other categories are negative. GATA1 is also absent in the blasts of acute lymphoblastic leukemia/lymphoma and in the neoplastic cells of metastatic carcinoma and plasma cell neoplasms. CONCLUSIONS: Intense GATA1 nuclear expression is a sensitive and specific marker for cells of erythroid and megakaryocytic lineages and is an excellent marker for neoplastic cells of pure erythroleukemia and acute megakaryoblastic leukemia.

FocusHeuristics - expression-data-driven network optimization and disease gene prediction.

To identify genes contributing to disease phenotypes remains a challenge for bioinformatics. Static knowledge on biological networks is often combined with the dynamics observed in gene expression levels over disease development, to find markers for diagnostics and therapy, and also putative disease-modulatory drug targets and drugs. The basis of current methods ranges from a focus on expression-levels (Limma) to concentrating on network characteristics (PageRank, HITS/Authority Score), and both (DeMAND, Local Radiality). We present an integrative approach (the FocusHeuristics) that is thoroughly evaluated based on public expression data and molecular disease characteristics provided by DisGeNet. The FocusHeuristics combines three scores, i.e. the log fold change and another two, based on the sum and difference of log fold changes of genes/proteins linked in a network. A gene is kept when one of the scores to which it contributes is above a threshold. Our FocusHeuristics is both, a predictor for gene-disease-association and a bioinformatics method to reduce biological networks to their disease-relevant parts, by highlighting the dynamics observed in expression data. The FocusHeuristics is slightly, but significantly better than other methods by its more successful identification of disease-associated genes measured by AUC, and it delivers mechanistic explanations for its choice of genes.

CCND1-BCL2 Gene Network: A direct target of Amifostine in human acute megakaryocytic leukemia cells.

Amifostine, 2-(3-aminopropyl) aminoethyl phosphorothioate, is a broad-spectrum cytoprotective agent used to treat nuclear radiation and chemical weapon injuries. Recently, amifostine has been shown to have a profound biological influence on tumor cells. To examine the effects and mechanisms underlying the effects of amifostine on human acute megakaryocytic leukemia, we evaluated the efficacy of amifostine against Dami cells and observed a cell cycle arrest in G2 /M phase. Amifostine treatment also induced cell apoptosis of Dami cells which corresponds to formal studies. Through whole-genome microarray and bioinformatics analyses, we found that amifostine affected the gene expression of CCND1, BCL2, and CASP3 which revealed the mechanism amifostine acted on Dami cells. Thus, CCND1-BCL2 Gene Network is predicted to be a direct target of amifostine treating human acute megakaryocytic leukemia, which may provide a novel potential target for the therapy of several subtypes of human AML.

Several adaptor proteins promote intracellular localisation of the transporter MRP4/ABCC4 in platelets and haematopoietic cells.

The multidrug resistance protein 4 (MRP4/ABCC4) has been identified as an important transporter for signalling molecules including cyclic nucleotides and several lipid mediators in platelets and may thus represent a novel target to interfere with platelet function. Besides its localisation in the plasma membrane, MRP4 has been also detected in the membrane of dense granules in resting platelets. In polarised cells it is localised at the basolateral or apical plasma membrane. To date, the mechanism of MRP4 trafficking has not been elucidated; protein interactions may regulate both the localisation and function of this transporter. We approached this issue by searching for interacting proteins by in vitro binding assays, followed by immunoblotting and mass spectrometry, and by visualising their co-localisation in platelets and haematopoietic cells. We identified the PDZ domain containing scaffold proteins ezrin-binding protein 50 (EBP50/NHERF1), postsynaptic density protein 95 (PSD95), and sorting nexin 27 (SNX27), but also the adaptor protein complex 3 subunit ß3A (AP3B1) and the heat shock protein HSP90 as putative interaction partners of MRP4. The knock-down of SNX27, PSD95, and AP3B1 by siRNA in megakaryoblastic leukaemia cells led to a redistribution of MRP4 from intracellular structures to the plasma membrane. Inhibition of HSP90 led to a diminished expression and retention of MRP4 in the endoplasmic reticulum. These results indicate that MRP4 localisation and function are regulated by multiple protein interactions. Changes in the adaptor proteins can hence lead to altered localisation and function of the transporter.

Blockage of miR-92a-3p with locked nucleic acid induces apoptosis and prevents cell proliferation in human acute megakaryoblastic leukemia.

MicroRNAs (miRNAs) are non-coding RNAs involved in post-transcriptional regulation of gene expression. In many cancers, up- or downregulation of different miRNAs is reported. In acute myeloid leukemia, upregulation of miR-92a-3p was reported in human in vitro studies. We performed blockage of miR-29a-3p in human acute megakaryoblastic leukemia cell line (M-07e) by using locked nucleic acid (LNA) and cell proliferation; apoptosis and necrosis were assessed. At different time points after LNA-anti-miR92a-3p transfection, miR-92a-3p quantitation was assessed by qRT-real-time PCR, MTT assay and annexin/propidium iodide staining were performed. The data were processed using the ANOVA test. At all three time points, the expression of miR-92a-3p was lower in the LNA-anti-miR group compared with the control groups. Cell viability between LNA-Anti-miR and the control group was statistically significant. Blockage of miR-92a-3p was associated with increment of the ratio of apoptotic cells in the LNA-anti-miR group was higher than the other group. The ratio of necrotic cells in the LNA-antimiR group was higher than the other groups. These assessments indicate that miR-92a-3p blockage can decrease the viability of M-07e cells, which is mainly due to induction of apoptosis and necrosis. Our findings could open up a path to a miRNA based therapeutic approach for treatment of acute megakaryoblastic leukemia.

Inhibition of glutamate regulated calcium entry into leukemic megakaryoblasts reduces cell proliferation and supports differentiation.

Human megakaryocytes release glutamate and express glutamate-gated Ca(2+)-permeable N-methyl-D-aspartate receptors (NMDARs) that support megakaryocytic maturation. While deregulated glutamate pathways impact oncogenicity in some cancers, the role of glutamate and NMDARs in megakaryocytic malignancies remains unknown. The aim of this study was to determine if NMDARs participate in Ca(2+) responses in leukemic megakaryoblasts and if so, whether modulating NMDAR activity could influence cell growth. Three human cell lines, Meg-01, Set-2 and K-562 were used as models of leukemic megakaryoblasts. NMDAR components were examined in leukemic cells and human bone marrow, including in megakaryocytic disease. Well-established NMDAR modulators (agonists and antagonists) were employed to determine NMDAR effects on Ca(2+) flux, cell viability, proliferation and differentiation. Leukemic megakaryoblasts contained combinations of NMDAR subunits that differed from normal bone marrow and the brain. NMDAR agonists facilitated Ca(2+) entry into Meg-01 cells, amplified Ca(2+) responses to adenosine diphosphate (ADP) and promoted growth of Meg-01, Set-2 and K-562 cells. Low concentrations of NMDAR inhibitors (riluzole, memantine, MK-801 and AP5; 5-100µM) were weakly cytotoxic but mainly reduced cell numbers by suppressing proliferation. The use-dependent NMDAR inhibitor, memantine (100µM), reduced numbers and proliferation of Meg-01 cells to less than 20% of controls (IC50 20µM and 36µM, respectively). In the presence of NMDAR inhibitors cells acquired morphologic and immunophenotypic features of megakaryocytic differentiation. In conclusion, NMDARs provide a novel pathway for Ca(2+) entry into leukemic megakaryoblasts that supports cell proliferation but not differentiation. NMDAR inhibitors counteract these effects, suggesting a novel opportunity to modulate growth of leukemic megakaryoblasts.