Ligation of the CD44 adhesion molecule reverses blockage of differentiation in human acute myeloid leukemia.

Blockage in myeloid differentiation characterizes acute myeloid leukemia (AML); the stage of the blockage defines distinct AML subtypes (AML1/2 to AML5). Differentiation therapy in AML has recently raised interest because the survival of AML3 patients has been greatly improved using the differentiating agent retinoic acid. However, this molecule is ineffective in other AML subtypes. The CD44 surface antigen, on leukemic blasts from most AML patients, is involved in myeloid differentiation. Here, we report that ligation of CD44 with specific anti-CD44 monoclonal antibodies or with hyaluronan, its natural ligand, can reverse myeloid differentiation blockage in AML1/2 to AML5 subtypes. The differentiation of AML blasts was evidenced by the ability to produce oxidative bursts, the expression of lineage antigens and cytological modifications, all specific to normal differentiated myeloid cells. These results indicate new possibilities for the development of CD44-targeted differentiation therapy in the AML1/2 to AML5 subtypes.     

ERK2-Pyruvate Kinase Axis Permits Phorbol 12-Myristate 13-Acetate-induced Megakaryocyte Differentiation in K562 Cells

Metabolic changes that contribute to differentiation are not well understood. Overwhelming evidence shows the critical role of glycolytic enzyme pyruvate kinase (PK) in directing metabolism of proliferating cells. However, its role in metabolism of differentiating cells is unclear. Here we studied the role of PK in phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation in human leukemia K562 cells. We observed that PMA treatment decreased cancer-type anabolic metabolism but increased ATP production, along with up-regulated expression of two PK isoforms (PKM2 and PKR) in an ERK2-dependent manner. Interestingly, silencing of PK (PKM2 and PKR) inhibited PMA-induced megakaryocytic differentiation, as revealed by decreased expression of megakaryocytic differentiation marker CD61 and cell cycle behavior. Further, PMA-induced ATP production reduced greatly upon PK silencing, suggesting that PK is required for ATP synthesis. In addition to metabolic effects, PMA treatment also translocated PKM2, but not PKR, into nucleus. ERK1/2 knockdowns independently and together suggested the role of ERK2 in the up-regulation of both the isoforms of PK, proposing a role of ERK2-PK isoform axis in differentiation. Collectively, our findings unravel ERK2 guided PK-dependent metabolic changes during PMA induction, which are important in megakaryocytic differentiation.     

Control of glioma cell death and differentiation by PKM2–Oct4 interaction

Glioma stem cells are highly resistant to cell death and as such are supposed to contribute to tumor recurrence by eluding anticancer treatments. Here, we show that spheroids that contain rat neural stem cells (NSCs) or rat glioma stem cells (cancer stem cells, CSCs) express isoforms 1 and 2 of pyruvate kinase (PKM1 and PKM2); however, the expression of PKM2 is considerably higher in glioma spheroids. Silencing of PKM2 enhances both apoptosis and differentiation of rat and human glioma spheroids. We establish that PKM2 was implicated in glioma spheroid differentiation through its interaction with Oct4, a major regulator of self-renewal and differentiation in stem cells. The small molecule Dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, increases the amount of PKM2/Oct4 complexes and thus inhibited Oct4-dependent gene expression. Taken together, our results highlight a new molecular pathway through which PKM2 can manage gliomagenesis via the control of glioma stemness by Oct4.     

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.     

Molecular mechanisms that produce secondary MDS/AML by RUNX1/AML1 point mutations

RUNX1/AML1 point mutations have been identified in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients. A heterozygous germline mutation of the RUNX1 gene causes a familial platelet disorder with a predisposition to AML. RUNX1 mutations have also been detected with high frequency in minimally differentiated AML M0 subtypes and myelodysplastic/myeloproliferative neoplasms. Here we propose a new disease category of myelodysplastic neoplasms (MDN) consisting of MDS refractory anemia with excess blasts and AML with myelodysplasia-related changes, including therapy-related cases. RUNX1 mutations have been detected in about 20% of patients with "MDN". Among the MDN cases, histories of radiation exposure, therapy-related myeloid neoplasms after successful treatment for acute promyelocytic leukemia, and leukemic transformation of myeloproliferative neoplasms have been reported to have a strong association with RUNX1 mutations. The mutations occur in a normal, a receptive, or a disease-committed hematopoietic stem cell. It is suspected that the "MDN" phenotypes are defined by the RUNX1 mutations in addition to some other abnormalities.     

Presence of a functional vitamin D receptor does not correlate with vitamin D3 phenotypic effects in myeloid differentiation

Although VDR is expressed in all the acute myeloid leukemia cell populations studied, most of these leukemias do not exhibit any phenotypic response when exposed to VD. To determine whether VD resistance is related to an altered VDR function, we performed an analysis of VDR expression, phosphorylation, DNA binding capacity and transactivation activity in several leukemic myeloid cell lines arrested at different levels of maturation. Our results indicate that VD induces a clear phenotypic effect, i.e. terminal monocytic differentiation, only in leukemic cells of M2/M3 (intermediate myeloblasts) and M5 (monoblasts) types but not in erythroid precursor cells, early leukemic myeloblasts (M0/M1 type) and promyelocytes (M3 type). VDR expression and function are evident in all the nuclear extracts obtained from the different myeloid cell lines after 12 h of VD treatment, but VD activation of monocytic differentiation is limited to a narrow differentiation window characterized by the M2 type myeloid cellular context.     

Paraptosis Cell Death Induction by the Thiamine Analog Benfotiamine in Leukemia Cells

Benfotiamine is a synthetic thiamine analogue that stimulates transketolase, a cellular enzyme essential for glucose metabolism. Currently, benfotiamine is used to treat diabetic neuropathy. We recently reported that oral benfotiamine induced a temporary but remarkable recovery from acute myeloid leukemia in an elderly patient who was ineligible for standard chemotherapy due to dementia and renal failure. In the present study we present evidences that benfotiamine possess antitumor activity against leukemia cells. In a panel of nine myeloid leukemia cell lines benfotiamine impaired the viability of HL-60, NB4, K562 and KG1 cells and also inhibited the growing of primary leukemic blasts. The antitumor activity of benfotiamine is not mediated by apoptosis, necrosis or autophagy, but rather occurs though paraptosis cell death induction. Mechanistic studies revealed that benfotiamine inhibited the activity of constitutively active ERK1/2 and concomitantly increased the phosphorylation of JNK1/2 kinase in leukemic cells. In addition, benfotiamine induced the down regulation of the cell cycle regulator CDK3 which resulted in G1 cell cycle arrest in the sensitive leukemic cells. Moreover, combination index studies showed that benfotiamine enhanced the antiproliferative activities of cytarabine against leukemia cells. These findings suggest that benfotiamine has antitumor therapeutic potential.     

Leukemic cell products down-regulate human dendritic cell differentiation

The microenvironment produced by solid tumors is inhibitory to the immune system, inducing dendritic cell (DC) alterations, but there is a paucity of information regarding haematological malignances. The aim of this study was to investigate DC differentiation under the influence of leukemic cell products. Monocytes from healthy volunteers were cultured in the presence of IL-4 and GM-CSF for the generation of immature DCs. Supernatants from leukemic cultures were added to monocyte cultures during differentiation. The lineages used were K562, a chronic myeloid leukemia, HL-60, a promyelocytic leukemia and DAUDI, originated from Burkitt lymphoma. It was observed that the expression of CD14 remained high and the CD1a was low in the presence of tumor supernatants, while non-malignant supernatants did not affect these parameters. Furthermore, IL-1β and TNF-α production by monocytes during differentiation was increased by the presence of tumor supernatants. The modifications on CD14 and CD1a expressions could be mimicked by the addition of exogenous IL-1β and partially inhibited by the neutralization of IL-1β. These results suggest that soluble products from leukemic cells interfere with DC differentiation and, in the present work, this effect could be mediated by monocyte-derived IL-1β in response to tumor supernatants.     

In vitro generation of effector cells cytotoxic to autologous targets from chronic myeloid leukemia patients in remission

Summary Peripheral blood lymphocytes (PBL) from chronic myeloid leukemia (CML) patients in remission were stimulated in vitro, in a 3-cell assay with autologous leukemic cells or autologous bone marrow (BM) cells alone, or each in combination with allogeneic PBL. The responder cells were used as effectors in a 4-h ⁵¹Cr release cytotoxicity assay using autologous targets such as leukemic cells, BM cells, phytohemagglutinin-induced lymphoblasts, and allogeneic K562 (erythroblastoid leukemic cell line) target cells. Sensitization of lymphocytes from CML patients with either autologous leukemic cells or BM cells generated cytotoxic cells (CTCs) capable of killing both the targets. These results suggested that in CML, the PBL may have been sensitized to myeloid maturation-related antigens in vivo, which, on secondary stimulation in vitro, may result in differentiation of CTCs cytotoxic to immature myeloid cells, either from autologous leukemic cells or autologous BM. The inability of PBL from patients with oral cancers to lyse autologous BM cells upon in vitro stimulation, supported this possibility. Clonogenic assays conducted to assess the colony forming potential of BM cells which had interacted with CTCs indicated that there was about 37% reduction in committed granulocyte stem cell colony formation without an appreciable change in committed granulocyte/monocyte stem cell units and clusters. Therefore, since the BM toxicity of the CTCs is not very high, these cells may have a potential clinical use in CML.     

Recombinant human colony stimulating factor-granulocyte/macrophage and -granulocyte, but not macrophage induce the development of a respiratory burst in primary human myeloid leukemic cells in vitro

Respiratory burst develops in myeloid blast cells if they differentiate functionally along the monocytic or granulocytic lineage. Using the nitroblue tetrazolium (NBT) assay we studied the effects of recombinant human granulocyte/macrophage colony stimulating factor (rhuGM-CSF), rhuG-CSF and rhuM-CSF on development of respiratory burst activity in primary blast cells from patients with myeloid leukemia. Assessing suspension cultures containing cells from patients with acute myeloid leukemia (AML, n = 13) or myeloid-blast crisis (myBC) of chronic myeloid leukemia (CML, n = 5) it was found that the percentage of NBT positive cells was increased by at least 20% as compared to control cultures by rhuGM-CSF in 6/17 cases, by rhuG-CSF in 7/17 cases and by rhuM-CSF in 0/16 cases, representing in 'responders' a mean increase of 267% and 270% in the absolute number of NBT positive cells by rhuGM-CSF and rhuG-CSF, respectively. Morphological examination of cultured cells from 'responders', as compared to controls, showed decreased blast cell content but generally no evidence of terminal differentiation. The demonstration of Auer rods in NBT positive cells indicates that respiratory burst developed in a leukemic clone. These findings may be of physiological, pathophysiological and clinical relevance.     

hERG Potassium Channel Blockage by Scorpion Toxin BmKKx2 Enhances Erythroid Differentiation of Human Leukemia Cells K562

Background

The hERG potassium channel can modulate the proliferation of the chronic myelogenous leukemic K562 cells, and its role in the erythroid differentiation of K562 cells still remains unclear.

Principal Findings

The hERG potassium channel blockage by a new 36-residue scorpion toxin BmKKx2, a potent hERG channel blocker with IC₅₀ of 6.7±1.7 nM, enhanced the erythroid differentiation of K562 cells. The mean values of GPA (CD235a) fluorescence intensity in the group of K562 cells pretreated by the toxin for 24 h and followed by cytosine arabinoside (Ara-C) treatment for 72 h were about 2-fold stronger than those of K562 cells induced by Ara-C alone. Such unique role of hERG potassium channel was also supported by the evidence that the effect of the toxin BmKKx2 on cell differentiation was nullified in hERG-deficient cell lines. During the K562 cell differentiation, BmKKx2 could also suppress the expression of hERG channels at both mRNA and protein levels. Besides the function of differentiation enhancement, BmKKx2 was also found to promote the differentiation-dependent apoptosis during the differentiation process of K562 cells. In addition, the blockage of hERG potassium channel by toxin BmKKx2 was able to decrease the intracellular Ca²⁺ concentration during the K562 cell differentiation, providing an insight into the mechanism of hERG potassium channel regulating this cellular process.

Conclusions/Significance

Our results revealed scorpion toxin BmKKx2 could enhance the erythroid differentiation of leukemic K562 cells via inhibiting hERG potassium channel currents. These findings would not only accelerate the functional research of hERG channel in different leukemic cells, but also present the prospects of natural scorpion toxins as anti-leukemic drugs.     

TSP50 promotes the Warburg effect and hepatocyte proliferation via regulating PKM2 acetylation

Metabolic reprogramming is a hallmark of malignancy. Testes-specific protease 50 (TSP50), a newly identified oncogene, has been shown to play an important role in tumorigenesis. However, its role in tumor cell metabolism remains unclear. To investigate this issue, LC–MS/MS was employed to identify TSP50-binding proteins and pyruvate kinase M2 isoform (PKM2), a known key enzyme of aerobic glycolysis, was identified as a novel binding partner of TSP50. Further studies suggested that TSP50 promoted aerobic glycolysis in HCC cells by maintaining low pyruvate kinase activity of the PKM2. Mechanistically, TSP50 promoted the Warburg effect by increasing PKM2 K433 acetylation level and PKM2 acetylation site (K433R) mutation remarkably abrogated the TSP50-induced aerobic glycolysis, cell proliferation in vitro and tumor formation in vivo. Our findings indicate that TSP50-mediated low PKM2 pyruvate kinase activity is an important determinant for Warburg effect in HCC cells and provide a mechanistic link between TSP50 and tumor metabolism.Subject terms: Cancer metabolism, Oncogenes, Tumour biomarkers

Gao et al. shows that testes-specific protease 50 (TSP50) binds to PKM2 and promotes the Warburg effect by increasing PKM2 K433 acetylation level and PKM2 acetylation site (K433R) mutation remarkably abrogated the TSP50-induced aerobic glycolysis, cell proliferation in vitro and tumor formation in vivo. Our study reveals a link between an oncogene and a key enzyme in HCC glycolysis, which provides new ideas for human HCCs treatment with TSP50 as the target.     

Acetylation targets the M2 isoform of pyruvate kinase for degradation through chaperone-mediated autophagy and promotes tumor growth

Most tumor cells take up more glucose than normal cells but metabolize glucose via glycolysis even in the presence of normal levels of oxygen, a phenomenon known as the Warburg effect. Tumor cells commonly express the embryonic M2 isoform of pyruvate kinase (PKM2) that may contribute to the metabolism shift from oxidative phosphorylation to aerobic glycolysis and tumorigenesis. Here we show that PKM2 is acetylated on lysine 305 and that this acetylation is stimulated by high glucose concentration. PKM2 K305 acetylation decreases PKM2 enzyme activity and promotes its lysosomal-dependent degradation via chaperone-mediated autophagy (CMA). Acetylation increases PKM2 interaction with HSC70, a chaperone for CMA, and association with lysosomes. Ectopic expression of an acetylation mimetic K305Q mutant accumulates glycolytic intermediates and promotes cell proliferation and tumor growth. These results reveal an acetylation regulation of pyruvate kinase and the link between lysine acetylation and CMA.     

Inhibition of GATE-16 attenuates ATRA-induced neutrophil differentiation of APL cells and interferes with autophagosome formation

Autophagy is an intracellular bulk degradation process involved in cell survival upon stress induction, but also with a newly identified function in myeloid differentiation. The autophagy-related (ATG)8 protein family, including the GABARAP and LC3 subfamilies, is crucial for autophagosome biogenesis. In order to evaluate the significance of the GABARAPs in the pathogenesis of acute myeloid leukemia (AML), we compared their expression in primary AML patient samples, CD34⁺ progenitor cells and in granulocytes from healthy donors. GABARAPL1 and GABARAPL2/GATE-16, but not GABARAP, were significantly downregulated in particular AML subtypes compared to normal granulocytes. Moreover, the expression of GABARAPL1 and GATE-16 was significantly induced during ATRA-induced neutrophil differentiation of acute promyelocytic leukemia cells (APL). Lastly, knocking down GABARAPL2/GATE-16 in APL cells attenuatedneutrophil differentiation and decreased autophagic flux. In conclusion, low GABARAPL2/GATE-16 expression is associated with an immature myeloid leukemic phenotype and these proteins are necessary for neutrophil differentiation of APL cells.     

Purification of a murine leukemia inhibitory factor from Krebs ascites cells

A factor capable of inducing terminal differentiation in the murine myeloid leukemia cell line M1 has been purified to apparent homogeneity from the medium conditioned by Krebs II ascites tumor cells. The factor, termed leukemia inhibitory factor (LIF) is a single chain glycoprotein of apparent Mr 58,000 which induces differentiation and inhibits proliferation of the M1 cell line but not the WEHI-3B D+ murine myeloid leukemic cell line and has no detectable proliferative activity on normal myeloid progenitor cells. It was purified using four successive high-efficiency purification steps--anion-exchange chromatography on DEAE-Sepharose; cation-exchange chromatography on CM-Sepharose; affinity chromatography on lentil lectin-Sepharose; and reverse-phase high-performance liquid chromatography on a phenyl-silica matrix--to a specific biological activity of approximately 1.25 X 10(8) units/mg with an overall purification of 12,000-fold and a yield of 73% for the activity failing to bind to DEAE-Sepharose. Sufficient quantities of the factor (12 micrograms, 200 pmol) have been purified to allow structural and functional analysis of the molecule and comparison with other know differentiation inducers.     

E2a/Pbx1 induces the rapid proliferation of stem cell factor-dependent murine pro-T cells that cause acute T-lymphoid or myeloid leukemias in mice

Oncoprotein E2a/Pbx1 is produced by the t(1;19) chromosomal translocation of human pre-B acute lymphoblastic leukemia. E2a/Pbx1 blocks differentiation of primary myeloid progenitors but, paradoxically, induces apoptosis in established pre-B-cell lines, and no transforming function of E2a/Pbx1 has been reported in cultured lymphoid progenitors. Here, we demonstrate that E2a/Pbx1 induces immortal proliferation of stem cell factor (SCF)-dependent pro-T thymocytes by a mechanism dependent upon both its transactivation and DNA-binding functions. E2a-Pbx1 cooperated with cytokines or activated signaling oncoproteins to induce cell division, as inactivation of conditional E2a/Pbx1 in either factor-dependent pro-T cells or pro-T cells made factor independent by expression of Bcr/Abl resulted in pro-T-cell quiescence, while reactivation of E2a/Pbx1 restored cell division. Infusion of E2a/Pbx1 pro-T cells in mice caused T lymphoblastic leukemia and, unexpectedly, acute myeloid leukemia. The acute lymphoblastic leukemia did not evidence further maturation, suggesting that E2a/Pbx1 establishes an early block in pro-T-cell development that cannot be overcome by marrow or thymic microenvironments. In an E2a/Pbx1 pro-T thymocyte clone that induced only pro-T acute lymphoblastic leukemia, coexpression of Bcr/Abl expanded its leukemic phenotype to include acute myeloid leukemia, suggesting that unique functions of cooperating signaling oncoproteins can influence the lymphoid versus myeloid character of E2a/Pbx1 leukemia and may cooperate with E2a/Pbx1 to dictate the pre-B-cell phenotype of human leukemia containing t(1;19).     

Differentiation in mature T lymphoid leukemia cells is unstable and reversible to myeloid cells, without the involvement of a common stem cell.

Mechanisms for the unstable and reversible differentiation of a mature CD3+ CD7+TCR-alpha/beta+ T lymphoid leukemia into the myeloid lineage were investigated. Inasmuch as productive rearrangement of the TCR-alpha is a late determinant of T cell differentiation, the TCR-alpha rearrangement was sequenced to determine the state of differentiation of the leukemic multipotent cell. An identical productive rearrangement of J alpha C to a novel V alpha region was found in the myeloid and T lymphoid leukemic cells. Thus, a "terminally" differentiated T lymphoid leukemic cell after productively rearranging TCR-alpha and -beta continues to display potential for multilineage differentiation. Therefore, multilineage potential is due to an unstable and reversible differentiation in a mature T lymphocyte as opposed to differentiation of an uncommitted common T and myeloid precursor cell.     

Preventing pyruvate kinase muscle expression in Chinese hamster ovary cells curbs lactogenic behavior by altering glycolysis,gating pyruvate generation,and increasing pyruvate flux into the TCA cycle

Deletion of the pyruvate kinase muscle (PKM) gene, which is involved in conversion of phosphoenolpyruvate to pyruvate, has been shown to curb lactogenic behavior in Chinese hamster ovary (CHO) cells. This study describes the generation of pyruvate kinase muscle isoforms 1 and 2 knockout (PKM-KO) and pyruvate kinase muscle isoform-1 knockout (PKM1-KO) CHO host cells to understand metabolic shifts that reduce lactate secretion in these cells. Glucose and amino acids uptake levels in wild-type (WT), PKM-KO, and PKM1-KO stable cell lines, expressing two different antibodies, were analyzed in 14-day fed-batch production assays using different vessels. PKM-KO and PKM1-KO cells consumed more glucose per cell, altered amino acids metabolism, had higher flux of pyruvate into the tricarboxylic acid (TCA) cycle, and as previously shown reduced lactate secretion levels compared with the WT cells. Additionally, both PKM-KO and PKM1-KO cells had higher specific productivity and lower cell growth rates compared with the WT cells. Our findings suggest that rewiring the flux of pyruvate to the TCA cycle by deletion of PKM or PKM1 reduced cell growth and increased specific productivity in CHO cells. Overall, PKM1-KO cells had similar product quality and comparable or better titers relative to the WT cells, hence, targeted deletion of this isoform for curbing lactogenic behavior in CHO cells is suggested.