Mutant forms of c-Cbl and FLT3 cooperate to enhance myeloid leukemia development
Taylor et al., pages 191–206.
Fms-like tyrosine kinase (FLT) 3 mutations, especially internal tandem duplications (ITDs), are prevalent in acute myeloid leukemia (AML). Currently, there is conjecture over the negative regulation of the aberrantly localized FLT3-ITD protein, particularly in regard to E3 ubiquitin ligases such as c-Cbl. A greater understanding of these pathways could provide insight into novel targets for the treatment of AML. Hence, in this study, Taylor et al. investigated the role of c-Cbl in the regulation of the FLT3-ITD protein by generating of a double-mutant mouse containing both a FLT3-ITD and a c-Cbl RING finger domain mutation. The combination of the two mutations severely affected embryonic development, necessitating the generation of mice repopulated with E.14 fetal liver cells. These double-mutant transplanted mice developed an aggressive myeloid leukemia, with a phenotype markedly more severe than mice with either of the single mutations. The disease was characterized by a large increase in blast and myeloid cells and short survival. Interestingly, despite the severe phenotype, the c-Cbl mutation did not enhance FLT3-ITD protein levels or the downstream activity of signal transducer and activator of transcription 5. Rather, the severe leukemia was promoted by greater activity in additional signaling pathways that involved non-FLT3 c-Cbl targets. These included enhanced c-Kit signaling that promoted elevated activity of the phosphoinositide 3-kinase pathway. This study has highlighted the importance of wild-type signaling pathways contributing to leukemia development and suggests that pharmacological manipulation of these pathways may be of therapeutic benefit to control disease progression.
Potential targeting of B-cell lymphoma 2–associated AthanoGene-1 (BAG-1) in poor responding pediatric AML patients
Aveic et al., pages 180–190.
Although huge improvements have been made in the outcome of pediatric acute myeloid leukemia (AML), a significant number of patients still relapse, confirming that standard chemotherapy is not enough. There are several factors that may cause leukemic cells to escape the effects of cytotoxic drugs, and major among them is a failure in apoptosis activation. In the current work, Aveic et al. studied the B-cell lymphoma (BCL) 2–associated AthanoGene-1 (BAG-1), which is shown in the study to be a crucial antiapoptotic protein in AML. The authors demonstrate that the BAG-1 protein is overexpressed in a subgroup of AML patients that showed poor prognosis. The chemical inhibition of BAG-1 by Thioflavin S (which has been shown to interfere with BAG1 function) or BAG-1 silencing improved leukemia cell death by enhancing leukemic cells' sensitivity to conventional drugs. In addition, the authors report synergy between Thioflavin S and conventional chemotherapeutics and between the BCL2 inhibitor ABT-737 and Thioflavin S. In addition, the article reveals that two cellular pathways behind the activation of cell apoptosis, BCL-2 and endoplasmic reticulum stress and unfolded protein response, were both altered after BAG-1 depletion. These findings suggest that BAG-1 targeting could be evaluated as an innovative treatment that may decrease leukemia chemoresistance. The authors support the development and use of specific BAG-1 inhibitors in combination with standard chemotherapy drugs to reduce relapse and improve outcome in pediatric AML patients.
Autophagy regulates hematopoietic stem and progenitor cell cycle
Cao et al., pages 229–242.
Autophagy (or autophagocytosis) is the basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components through the actions of lysosomes. The breakdown of cellular components promotes cellular survival during starvation by maintaining cellular energy levels. In this manuscript, Cao et al. explored the role of autophagy in regulating the cell cycle of mouse hematopoietic stem and progenitor cells (HSPCs) using in vivo and ex vivo experimental systems as well as genetically modified mouse models. They show that autophagy regulates the cell cycle of HSPCs in a nutrient-dependent manner. Autophagy signaling under nutrient rich conditions were not upregulated in HSPCs, and cyclin D3 maintained a high level, which promoted cell-cycle entry and cell cycling of HSPCs; however, upon nutrient stress, autophagy of early signaling was activated, which in turn triggered ubiquitinational degradation of cyclin D3 to slow down the cell-cycle entry and G1/S transition of HSPCs, maintaining a proper cell cycling rate. Therefore, autophagy signaling accelerates or decelerates the cell cycle of HSPCs by recruiting ubiquitination machinery to upregulate or downregulate cyclin D3, depending on the nutrient supply. This study nicely demonstrates that autophagy responds to the nutrient supply to adjust the cell cycle and maintain the appropriate quiescence and self-renewal of hematopoietic stem cells as well as cell cycling of HSPCs, which is essential for a functioning hematopoietic system during adult life.
Melatonin overcomes resistance to clofarabine in two leukemic cell lines by increased expression of deoxycytidine kinase
Yamanishi et al., pages 207–214.
Drug resistance remains a serious problem in leukemia therapy. In this study, Yamanishi et al. investigated the drug resistance mechanism from the viewpoint of epigenetics. Among newly developed nucleoside antimetabolites, clofarabine has broad cytotoxic activity showing therapeutic promise and is currently approved for relapsed acute lymphoblastic leukemia. To investigate the mechanisms responsible for clofarabine resistance, the authors established two clofarabine-resistant lymphoblastic leukemia cell lines from parental lines. They found that expression of deoxycytidine kinase (dCK), which phosphorylates clofarabine to exert cytotoxicity, in clofarabine-sensitive and -resistant cells, showed significant decreased expression of dCK RNA in clofarabine-resistant cells compared with sensitive cells. Interestingly, there was no difference between clofarabine-sensitive and -resistant cells in the methylation status of CpG islands of the dCK promoter and expression of MDR1, MRP1, and ABCG2. Total histone, histone H3, and histone H4 acetylation on ChIP assay were significantly decreased in resistant cells. The authors then examined the effects of melatonin in clofarabine-resistant cells. Melatonin is an indolamine that functions in the regulation of chronobiological rhythms to exert cytotoxic effects. Melatonin treatment led to increased cytotoxicity with clofarabine in resistant cells via increased acetylation, which is another histone deacetylase inhibitor. Melatonin is readily available in the clinic and may be a useful candidate for overcoming clofarabine resistance. Based on the data presented, combination therapy with cytotoxic drugs and histone deacetylase inhibitor might be useful in the clinic for enhancement of therapeutic efficacy in relapsed or refractory leukemia.
Csf1r collaborates with a C-terminal mutant of C/EBPα to develop aggressive AML
Togami et al., pages 300–308.
CCAAT-enhancer-binding protein (C/EBPα) is a transcription factor that regulates proliferation and differentiation of myeloid cells. Two types of C/EBPα mutations are found in patients with acute myeloid leukemia (AML). It has been previously demonstrated, by using either a mouse bone marrow transplantation model or a knock-in mouse, that a C-terminal mutant of C/EBPα(C/EBPα-Cm) alone induces AML and that it collaborates with an N-terminal mutant of C/EBPα(C/EBPα-Nm) in the efficient induction of AML. In this study, Togami et al. investigate the molecular mechanisms underlying C/EBPα-Cm–induced leukemogenesis. By analyzing gene expression profiles of C/EBPα-Cm– and mock-transduced c-Kit+Sca-1+Lin− cells, they identified Csf1r as a gene downregulated by C/EBPα-Cm. In addition, leukemic cells expressing C/EBPα-Cm exhibited low levels of Csf1r in mice. The authors then tested the possibility that the downregulated expression of Csf1r plays critical roles in leukemogenesis, given that Csf1r is required for myeloid differentiation. However, in contrast to their expectation, Csf1r overexpression collaborated with C/EBPα-Cm in inducing fulminant AML with leukocytosis with shorter latencies compared to those of AML induced by C/EBPα-Cm alone. These results suggest that C/EBPα-Cm–mediated downregulation of Csf1r negatively regulates the progression of myeloid malignancies involving C/EBPα-Cm, and may relate to the fact that Csfr1 is a maker of leukemic stem cell in the mouse leukemia model induced by MOZ-TIF2. Further experiments using the C/EBPα-Cm–induced leukemia model will help us understand the molecular mechanisms by which C/EBPα-Cm–induced leukemia in mice and identify novel therapeutic targets.
BIRB796 stimulates hematopoietic progenitor stem cell growth in FANCA
Svahn et al., pages 295–299.
Bone marrow failure, the main cause of mortality and morbidity in Fanconi anemia (FA), has been largely related to overproduction of tumor necrosis factor α (TNF-α), to which FA stem- and progenitor-cells are hypersensitive. It is known that TNF-α suppression improves the growth of patients' hematopoietic progenitor cells and, in nonhuman experimental settings, it was shown that TNF-α overproduction involves the Toll-like receptor (TLR) 4/8 and p38 mitogen-activiated protein kinase (MAPK) pathway. In this study, Svahn et al. attempted to ameliorate the FA pathway by perturbation of p38 MAPK. The work was conducted on primary human FA complementation group A (FANCA)-deficient monocytes from nine patients and shows that: (i) inhibition of p38 MAPK reduces TLR4 and 7/8-mediated TNF-α production; (ii) inhibitors of p38MAPK improve in vitro erythropoiesis; and (iii) this effect occurs on auxiliary cells (monocytes) and not directly on CD34+ cells. The two latter findings are novel and important for two reasons. The first is that translation to patients' primary cells confirms the same findings obtained on cell lines and mice. This may not always be true; FANCA mice, for example, do not have detectable developmental abnormalities. Therefore, “real ground” confirmation supports continuation of this research. The second reason is that, not only did the authors show the positive effect of inhibition of p38 MAPK on FA erythropoiesis, but they also identified the cellular target of this inhibition. This is important to direct the effect of future compounds counteracting marrow failure on a selective cell target preserving CD34+ cells, which is a severely hampered cell population in FA. At large, these findings might be also useful in other marrow failure disorders like acquired aplastic anemia, in which TNF-α is known to play an important pathogenic role.
The antimalarial drug artemisinin depletes erythrocytes
Yang et al., pages 331–341.
Artemisinin is a major antimalarial drug due to its extraordinary efficacy. However, hemolytic anemia is an evident adverse effect for which underlying mechanisms are unclear. Through a small-molecule screen in zebrafish embryos, Yang et al. found in this study that artemisinin treatments led to diminishing red blood cells but had little to no effect on hematopoietic stem cells and myeloid cells. RNA-Seq revealed that artemisinin suppressed a cluster of genes in the heme biosynthesis and globin synthesis pathways, including the gata 1 regulated genes alas2, urod, fech, and ppox. Artimisinin treatment resulted in erythroid cell apoptosis in both zebrafish and human K562 cell-differentiated red blood cells. Importantly, artemisinin suppressed the ectopic expression of erythroid genes in a zebrafish model of polycythemia vera, a blood disorder in which the bone marrow produces too many erythrocytes. This study provides not only a novel insight into the pharmacologic action of artemisinin on differentiated erythroids but also its potential utility in the intervention and therapy on polycythemia vera.
Bone marrow failure in C57BL/6 mice
Chen et al. pages 256–267.
Murine models of immune-mediated bone marrow (BM) failure have been previously developed with proven utility in the study of disease pathophysiology. However, important elements, such as inciting antigens that initiate autoimmune responses and key molecules that direct T cell lodging, remain uncharacterized. In this study, Chen et al developed a new model of immune-mediated BM failure in C57BL/6 (B6) mice through sublethal irradiation and infusion of allogeneic lymphocytes from major histocompatibility complex–mismatched Friend leukemia virus B/N (FVB) donors. Recipient animals underwent oligoclonal cytotoxic T cell expansion and activation along with significant elevations in inflammatory cytokines and chemokines, leading to severe marrow hypoplasia and fatal pancytopenia. This new model establishes a useful platform that could be extended to a large array of gene knockout and transgenic stocks readily available on the B6 strain background. As proof, B6 mice carrying the lpr mutation deficient in the apoptotic receptor Fas (B6-Fas−/−) were tested in the study. B6-Fas−/− mice showed significant attenuation in BM destruction when treated with the same levels of irradiation and allogeneic FVB lymphocyte infusion. Extension of this model to other B6-based knockout and transgenic mutants will help define the roles of individual genes and their products in the development of immune-mediated BM failure. This is needed to understand the mechanisms behind aplastic anemia and other forms of BM failure and treat patients with these devastating diseases.