Antifungal treatment by amphotericin B and 5-fluorocytosine delays the recovery of normal hematopoietic cells after intensive cytostatic therapy for acute myeloid leukemia

Abstract Using a MLL-AF9 knock-in mouse model, we discovered that consumption of a high-fat diet (HFD) accelerates the risk of developing acute myeloid leukemia (AML). This regimen increases the clusterization of FLT3 within lipid rafts on the cell surface of primitive hematopoietic cells, which overactivates this receptor as well as the downstream JAK/STAT signaling known to enhance the transformation of MLL-AF9 knock-in cells. Treatment of mice on a HFD with Quizartinib, a potent inhibitor of FLT3 phosphorylation, inhibits the JAK3/STAT3, signaling and finally antagonizes the accelerated development of AML that occurred following the HFD regimen. We can therefore conclude that, on a mouse model of AML, a HFD enforces the FLT3 signaling pathway on primitive hematopoietic cells and, in turn, improves the oncogenic transformation of MLL-AF9 knock-in cells and the leukemia initiation.

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A Critical Role of YB-1 In NPMc-Induced Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v116.21.3149.3149 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3149-3149

Author(s):

Yoko Ogawara ◽

Takuo Katsumoto ◽

Takeshi Uchiumi ◽

Kimitoshi Kohno ◽

Issay Kitabayashi

Keyword(s):

Acute Myeloid Leukemia ◽

Colony Formation ◽

Myeloid Leukemia ◽

Fetal Liver ◽

Hematopoietic Cells ◽

Wild Type ◽

Embryonic Lethal ◽

Hoxa Genes ◽

Fetal Liver Cells ◽

Acute Myeloid

Abstract Abstract 3149 Frameshift mutations in Nucleophosmin gene (NPM) are the most frequent abnormality in acute myeloid leukemia (AML), found in approximately 30% of all cases and 50% of patients with normal karyotype (NK) AML. NPM mutations result in an aberrant cytoplasmic localization of NPM protein (NPMc) through a loss of nucleolar localization signal accompanied by acquisition of new nuclear export signal. NPM mutations are heterozygous, so the other wild-type allele is consistently retained. NPMc binds to wild-type NPM through oligomerization domain and impairs its activity by delocalizing to the cytoplasm. It was reported that the NPM-null mice are early embryonic lethal and defective in primary hematopoiesis, suggesting important roles of NPM in early hematopoiesis. However, the molecular mechanism by which NPMc exerts its leukemogenic potential has never been established. Here we show that ectopic expression of NPMc, but not wild type (WT) NPM, in mouse bone marrow (BM) cells enhanced their colony formation activity in methylcellulose media. Increased expression of HoxA7, 9 and 10 genes were observed in cells expressing NPMc but not in those expressing WT NPM. It has been reported that the expression levels of HOXA genes are upregulated in various types of AML including NPMc+ AML. Since overexpression of HoxA9 immortalizes hematopoietic progenitor cells, our findings suggest that up-regulation of HoxA genes are involved in NPMc-mediated leukemogenesis. To clarify roles of NPMc in leukemogenesis, we purified the NPM protein complex and identified Y box-binding protein 1 (YB-1) as a binding partner for NPM. YB-1 belongs to the cold shock family and functions in gene transcription and RNA processing. YB-1 strongly bound to WT NPM but not to NPMc. In addition, interaction between YB-1 and NPM was impaired in the presence of NPMc. YB-1-deficient mice were embryonic lethal and their fetal liver were small. YB-1-deficient yolk sac cells showed decreased colony-forming activity, and decreased number of hematopoietic cells were observed when AGM region of YB-1-deficeint embryo were cultured on OP9 cells. Furthermore, expression of Hoxa9 was decreased in fetal liver cells derived from YB-1 knockout mice. To investigate the roles of YB-1 in NPMc-associated leukemogenesis, WT and YB-1-null E14.5 fetal liver cells were infected with retrovirus expressing NPMc. Analyses of colony-forming activity and mRNA expression showed that YB-1 was essential for NPMc-induced increases in colony formation activity as well as in expression of HoxA genes. However, YB-1 was not necessary for colony formation activity induced by other AML-associated fusion genes, such as AML1-MTG8 and MLL-AF10. These data indicate that YB-1 is specifically required for NPMc-induced leukemogenic transformation of hematopoietic cells. Disclosures: No relevant conflicts of interest to declare.

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A Novel Non-Genetic Photostable Approach to Labelling Acute Myeloid Leukemia and Bone Marrow Cells with Quantum Dots

Blood ◽

10.1182/blood.v120.21.4818.4818 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4818-4818

Author(s):

Yanwen Zheng ◽

Zhengwei Mao ◽

Bin Yin

Keyword(s):

Bone Marrow ◽

Quantum Dots ◽

Acute Myeloid Leukemia ◽

Blood Cells ◽

Myeloid Leukemia ◽

Hematopoietic Cells ◽

Leukemia Cell ◽

Leukemia Cells ◽

Dependent Manner ◽

Acute Myeloid

Abstract Abstract 4818 Acute myeloid leukemia (AML) is a detrimental disease with difficult diagnosis and treatment. Understanding the biology of AML at the molecular and cellular levels would be essential to successful management of the disease. However, the notoriously known difficulty in manipulation of leukemia cells has long hindered the dissection of AML pathogenesis. The advent of CdSe/ZnS quantum dots (QDs) represents an important advancement in the research field of nanotechnology, which have recently also been applied for imaging of live cells. Here, we have introduced a non-genetic approach of marking blood cells, by taking advantage of QD technology. We compared QDs complexed with different vehicles, including a peptide Tat (QDs-Tat), cationic polymer Turbofect (QDs-Tf) and liposome Lipofectamine 2000 (QDs-Lip), in their abilities to mark cells. QDs-Tat showed the highest efficiency in delivery into hematopoietic cells, among the three vehicles. We then examined QDs-Tat labelling of leukemia cell lines, and found that QDs-Tat could label 293T, bone marrow (BM) cells, THP-1, MEG-01 and HL-60 with a decreasing efficiency. The efficiency of QDs-Tat delivery was dependent on the concentration of QDs-Tat applied, but not the length of incubation time. In addition, more uniform intracellular distributions of QDs in 293T and leukemia cells were obtained with QDs-Tat, compared with the granule-like formation obtained with QDs-Lip. Clearly, QD fluorescence was sharp and tolerant to repetitive photo excitations, and could be detected in 293T for up to one week following labelling. In summary, our results suggest that QDs have provided a photostable, non-genetic and transient approach that labels normal and malignant hematopoietic cells in a cell type-, vehicle-, and QD concentration-dependent manner. We expect for potentially wide applications of QDs as an easy and fast tool assisting investigations of various types of blood cells in the near future. Disclosures: No relevant conflicts of interest to declare.

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Liposomal encapsulation of a synergistic molar ratio of cytarabine and daunorubicin enhances selective toxicity for acute myeloid leukemia progenitors as compared to analogous normal hematopoietic cells

Experimental Hematology ◽

10.1016/j.exphem.2011.04.001 ◽

2011 ◽

Vol 39 (7) ◽

pp. 741-750 ◽

Cited By ~ 45

Author(s):

Hyun Pyo Kim ◽

Brigitte Gerhard ◽

Troy O. Harasym ◽

Lawrence D. Mayer ◽

Donna E. Hogge

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Hematopoietic Cells ◽

Molar Ratio ◽

Selective Toxicity ◽

Acute Myeloid

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SETDB1 Represses Hox Gene Expression and Suppresses Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-117690 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1320-1320

Author(s):

James Ropa ◽

Nirmalya SAHA ◽

Andrew G. Muntean

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Colony Formation ◽

Myeloid Leukemia ◽

Target Genes ◽

Bone Marrow Cells ◽

Ex Vivo ◽

Hematopoietic Cells ◽

H3k9 Methylation ◽

Acute Myeloid

Abstract Epigenetic regulators play an important role in normal and malignant hematopoiesis. Epigenetic deregulation of the HOXA gene cluster drives transformation of about 50% of acute myeloid leukemia (AML), including those harboring MLL rearrangements and NPM mutations, as well as others. Expression of Hoxa9 and its co-factor Meis1 is sufficient to transform bone marrow into a lethal AML in mouse models. We previously demonstrated that the pro-leukemic genes Hoxa9 and Meis1 are critically regulated by the histone H3 Lysine 9 (H3K9) methyltransferase SETDB1. Recent studies show that SETDB1 is required for normal hematopoiesis and MLL-AF9 mediated leukemia (Koide, et al. Blood 2016). Our lab recently demonstrated that SETDB1 negatively regulates the expression of HoxA9 and Meis1 through deposition of promoter H3K9 methylation in MLL-AF9 AML cells (Ropa et al. Oncotarget 2018). Consistent with these data, HOXA9 and MEIS1 expression negatively correlates with SETDB1 expression in AML patient samples. Therefore, we investigated the biological impact of SETDB1 on AML. We first noted that expression of SETDB1 in AML patient samples is significantly lower compared to normal hematopoietic cells. Further, higher SETDB1 expression correlated with a significantly better overall survival (p=0.003) and lower expected hazard (HR=0.9/100RSEM; p=0.009) in AML patients compared with lower SETDB1 expression. These data are consistent with SETDB1 negatively regulating pro-leukemic genes and suggests that SETDB1 expression may be correlated with AML patient prognosis. We tested this directly by expressing high levels of SETDB1 in AML cells. Ex vivo assays show that retroviral overexpression of SETDB1 in MLL-AF9 AML cells leads to cell differentiation, decreased leukemia colony formation, and decreased cell proliferation. Consistent with the AML patient data, overexpression of SETDB1 significantly delays MLL-AF9 mediated leukemogenesis in vivo (p=0.01). Further, we observed a strong selective pressure against exogenous SETDB1 expression in moribund mice. Transcriptome analyses demonstrate that SETDB1 globally represses Hox and pluripotency gene programs. Strikingly, we found that SETDB1 represses many of the same genes that exhibit reduced promoter H3K9me3 in AML patient samples relative to CD34+ cells. These data point to a role for SETDB1 in negatively regulating pro-leukemic target genes and suppressing AML. We also explored how chemical and genetic inhibition of H3K9 methylation and Setdb1 affects AML initiation and maintenance. We first confirmed the previously reported requirement for Setdb1 in AML cell lines by genetically deleting both alleles of Setdb1 in MLL-AF9 cells, which resulted in a complete arrest of proliferation (Koide, et al. Blood 2016). Combined with our data presented above, these results suggest a narrow window of SETDB1 expression is maintained in AML cells. To achieve reduced (but not complete loss of) activity, we investigated how small molecule inhibition of H3K9 methylation (UNC0638) or shRNA mediated knock down of Setdb1 affects AML initiation. We observed increased ex vivo colony formation of normal ckit+ bone marrow cells upon shRNA mediated knockdown of Setdb1 or upon UNC0638 treatment. We hypothesized that this expansion of colony forming unit potential of hematopoietic cells may translate to increased transformation potential by leukemic oncogenes. Indeed, cells pretreated with UNC0638 followed by retroviral transduction with MLL-AF9 exhibit significantly higher capacity for leukemic colony formation than vehicle treated cells. These data are consistent with H3K9 methylation repressing genes required for AML transformation. Our data identified a narrow window of expression of SETDB1 in AML patient samples. SETDB1 expression is reduced in AML patients relative to normal cells and chemical inhibition of H3K9 methylation expands the pool of cells amenable to MLL-AF9 mediated transformation ex vivo. While inhibition of SETDB1 and other H3K9 methyltransferases has been suggested as a possible therapeutic strategy, our data suggests this may also prime bone marrow cells for transformation by inhibiting epigenetic processes that repress pro-leukemic target genes. Further investigation of the roles of SETDB1 and H3K9 methylation levels is necessary to determine the value of these epigenetic modifiers as therapeutic targets in AML and is currently ongoing. Disclosures No relevant conflicts of interest to declare.

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