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1.  Isoform-Specific Potentiation of Stem and Progenitor Cell Engraftment by AML1/RUNX1  
PLoS Medicine  2007;4(5):e172.
AML1/RUNX1 is the most frequently mutated gene in leukaemia and is central to the normal biology of hematopoietic stem and progenitor cells. However, the role of different AML1 isoforms within these primitive compartments is unclear. Here we investigate whether altering relative expression of AML1 isoforms impacts the balance between cell self-renewal and differentiation in vitro and in vivo.
Methods and Findings
The human AML1a isoform encodes a truncated molecule with DNA-binding but no transactivation capacity. We used a retrovirus-based approach to transduce AML1a into primitive haematopoietic cells isolated from the mouse. We observed that enforced AML1a expression increased the competitive engraftment potential of murine long-term reconstituting stem cells with the proportion of AML1a-expressing cells increasing over time in both primary and secondary recipients. Furthermore, AML1a expression dramatically increased primitive and committed progenitor activity in engrafted animals as assessed by long-term culture, cobblestone formation, and colony assays. In contrast, expression of the full-length isoform AML1b abrogated engraftment potential. In vitro, AML1b promoted differentiation while AML1a promoted proliferation of progenitors capable of short-term lymphomyeloid engraftment. Consistent with these findings, the relative abundance of AML1a was highest in the primitive stem/progenitor compartment of human cord blood, and forced expression of AML1a in these cells enhanced maintenance of primitive potential both in vitro and in vivo.
These data demonstrate that the “a” isoform of AML1 has the capacity to potentiate stem and progenitor cell engraftment, both of which are required for successful clinical transplantation. This activity is consistent with its expression pattern in both normal and leukaemic cells. Manipulating the balance of AML1 isoform expression may offer novel therapeutic strategies, exploitable in the contexts of leukaemia and also in cord blood transplantation in adults, in whom stem and progenitor cell numbers are often limiting.
The truncated "a" isoform of AML1 is shown to have the capacity to potentiate stem and progenitor cell engraftment, both of which are required for successful clinical transplantation.
Editors' Summary
Blood contains red blood cells (which carry oxygen round the body), platelets (which help the blood to clot), and white blood cells (which fight off infections). All these cells, which are regularly replaced, are derived from hematopoietic stem cells, blood-forming cells present in the bone marrow. Like all stem cells, hematopoietic stem cells self-renew (reproduce themselves) and produce committed progenitor cells, which develop into mature blood cells in a process called hematopoiesis. Many proteins control hematopoiesis, some of which are called transcription factors; these factors bind to DNA through their DNA-binding domain and then control the expression of genes (that is, how DNA is turned into proteins) through particular parts of the protein (their transcription regulatory domains). An important hematopoietic transcription factor is AML1—a protein first identified because of its involvement in acute myelogenous leukemia (AML, a form of blood cancer). Mutations (changes) in the AML1 gene are now known to be present in other types of leukemia, which are often characterized by overproliferation of immature blood cells.
Why Was This Study Done?
Because of AML1′s crucial role in hematopoiesis, knowing more about which genes it regulates and how its activity is regulated could provide clues to treating leukemia and to improving hematopoietic cell transplantation. Many cancer treatments destroy hematopoietic stem cells, leaving patients vulnerable to infection. Transplants of bone marrow or cord blood (the cord that links mother and baby during pregnancy contains peripheral blood stem cells) can replace the missing cells, but cord blood in particular often contains insufficient stem cells for successful transplantation. It would be useful, therefore, to expand the stem cell content of these tissues before transplantation. In this study, the researchers investigated the effect of AML1 on self-renewal and differentiation of hematopoietic stem and progenitor cells in the laboratory (in vitro) and in animals (in vivo). In particular, they have asked how two isoforms (closely related versions) of AML1 affect the ability of these cells to grow and differentiate (engraft) in mice after transplantation.
What Did the Researchers Do and Find?
The researchers artificially expressed AML1a and AML1b (both isoforms contain a DNA binding domain, but only AML1b has transcription regulatory domains) in mouse hematopoietic stem and progenitor cells and then tested the cells' ability to engraft in mice. AML1a-expressing cells engrafted better than unaltered cells and outgrew unaltered cells when transplanted as a mixture. AML1b-expressing cells, however, did not engraft. In vitro, AML1a-expressing cells grew more than AML1b-expressing cells, whereas differentiation was promoted in AML1b-expressing cells. To investigate whether the isoforms have the same effects in human cells, the researchers measured the amount of AML1a and AML1b mRNA (the template for protein production) made by progenitor cells in human cord blood. Although AML1b (together with AML1c, an isoform with similar characteristics) mRNA predominated in all the progenitor cell types, the relative abundance of AML1a was greatest in the stem and progenitor cells. Furthermore, forced expression of AML1a in these cells improved their ability to divide in vitro and to engraft in mice.
What Do These Findings Mean?
These findings indicate that AML1a expression increases the self-renewal capacity of hematopoietic stem and progenitor cells and consequently improves their ability to engraft in mice, whereas AML1b expression encourages the differentiation of these cell types. These activities are consistent with the expression patterns of the two isoforms in normal hematopoietic cells and in leukemic cells—the mutated AML made by many leukemic cells resembles AML1a. Because the AML1 isoforms were expressed at higher than normal levels in these experiments, the physiological relevance of these findings needs to be confirmed by showing that normal levels of AML1a and AML1b produce similar results. Nevertheless, these results suggest that manipulating the balance of AML1 isoforms made by hematopoietic cells might be useful clinically. In leukemia, a shift toward AML1b expression might slow the proliferation of leukemic cells and encourage their differentiation. Conversely, in cord blood transplantation, a shift toward AML1a expression might improve patient outcomes by expanding the stem and progenitor cell populations.
Additional Information.
Please access these Web sites via the online version of this summary at
Wikipedia has pages on hematopoiesis and hematopoietic stem cells (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The US National Cancer Institute has a fact sheet on bone marrow and peripheral blood stem cell transplantation (in English and Spanish) and information for patients and professionals on leukemia (in English)
The American Society of Hematology provides patient information about blood diseases, including information on bone marrow and stem cell transplantation
PMCID: PMC1868041  PMID: 17503961
2.  p27kip1 Maintains a Subset of Leukemia Stem Cells in the Quiescent State in Murine MLL-Leukemia 
Molecular oncology  2013;7(6):1069-1082.
MLL (mixed-lineage leukemia)-fusion genes induce the development of leukemia through deregulation of normal MLL target genes, such as HOXA9 and MEIS1. Both HOXA9 and MEIS1 are required for MLL-fusion gene-induced leukemogenesis. Co-expression of HOXA9 and MEIS1 induces acute myeloid leukemia (AML) similar to that seen in mice in which MLL-fusion genes are over-expressed. p27kip1 (p27 hereafter), a negative regulator of the cell cycle, has also been defined as an MLL target, the expression of which is up-regulated in MLL leukemic cells (LCs). To investigate whether p27 plays a role in the pathogenesis of MLL-leukemia, we examined the effects of p27 deletion (p27-/-) on MLL-AF9 (MA9)-induced murine AML development. HOXA9/MEIS1 (H/M)-induced, p27 wild-type (p27+/+) and p27-/- AML were studied in parallel as controls. We found that LCs from both MA9-AML and H/M-AML can be separated into three fractions, a CD117-CD11bhi differentiated fraction as well as CD117+CD11bhi and CD117+CD11blo, two less differentiated fractions. The CD117+CD11blo fraction, comprising only 1-3% of total LCs, expresses higher levels of early hematopoietic progenitor markers but lower levels of mature myeloid cell markers compared to other populations of LCs. p27 is expressed and is required for maintaining the quiescent and drug-resistant states of the CD117+CD11blo fraction of MA9-LCs but not of H/M-LCs. p27 deletion significantly compromises the leukemogenic capacity of CD117+CD11blo MA9-LCs by reducing the frequency of leukemic stem cells (LSCs) but does not do so in H/M-LCs. In addition, we found that p27 is highly expressed and required for cell cycle arrest in the CD117-CD11bhi fraction in both types of LCs. Furthermore, we found that c-Myc expression is required for maintaining LCs in an undifferentiated state independently of proliferation. We concluded that p27 represses the proliferation of LCs, which is specifically required for maintaining the quiescent and drug-resistant states of a small subset of MA9-LSCs in collaboration with the differentiation blockage function of c-Myc.
PMCID: PMC3898829  PMID: 23988911
MLL-leukemia; p27kip1; leukemia stem cell; quiescence
3.  mTORC1 is essential for leukemia propagation but not stem cell self-renewal 
The Journal of Clinical Investigation  2012;122(6):2114-2129.
Although dysregulation of mTOR complex 1 (mTORC1) promotes leukemogenesis, how mTORC1 affects established leukemia is unclear. We investigated the role of mTORC1 in mouse hematopoiesis using a mouse model of conditional deletion of Raptor, an essential component of mTORC1. Raptor deficiency impaired granulocyte and B cell development but did not alter survival or proliferation of hematopoietic progenitor cells. In a mouse model of acute myeloid leukemia (AML), Raptor deficiency significantly suppressed leukemia progression by causing apoptosis of differentiated, but not undifferentiated, leukemia cells. mTORC1 did not control cell cycle or cell growth in undifferentiated AML cells in vivo. Transplantation of Raptor-deficient undifferentiated AML cells in a limiting dilution revealed that mTORC1 is essential for leukemia initiation. Strikingly, a subset of AML cells with undifferentiated phenotypes survived long-term in the absence of mTORC1 activity. We further demonstrated that the reactivation of mTORC1 in those cells restored their leukemia-initiating capacity. Thus, AML cells lacking mTORC1 activity can self-renew as AML stem cells. Our findings provide mechanistic insight into how residual tumor cells circumvent anticancer therapies and drive tumor recurrence.
PMCID: PMC3366413  PMID: 22622041
4.  Deacetylase inhibitors modulate proliferation and self-renewal properties of leukemic stem and progenitor cells 
Cell Cycle  2012;11(17):3219-3226.
Acute myeloid leukemia (AML) is a highly malignant disease that is not curable in the majority of patients. Numerous non-random genetic abnormalities are known, among which several translocations such as PLZF/RARα or AML1/ETO are known to aberrantly recruit histone deacetylases. Deacetylase inhibitors (DACi) are promising drugs leading to growth inhibition, cell cycle arrest, premature senescence and apoptosis in malignant cells. It is believed that DACi may have clinical efficacy by eradicating the most primitive population of leukemic stem and progenitor cells, possibly by interfering with self-renewal.
The aim of the study was to investigate the effects of DACi on leukemic stem and progenitor cells using murine transduction-transplantation models of hematopoietic cells harboring the leukemia-associated fusion proteins (LAFP) PLZF/RARα or a truncated AML1/ETO protein (AML1/ETO exon 9). We show that the self-renewal and short-term repopulation capacity of AML1/ETO- or PLZF/RARα-expressing Sca1+/lin- stem and progenitor cells are profoundly inhibited by clinically applicable concentrations of the DACi dacinostat and vorinostat. To further investigate the mechanisms underlying these effects, we examined the impact of DACi on the transcription factor c-MYC and the Polycomb group protein BMI1, which are induced by LAFP and involved in leukemic transformation. In AML1/ETO or PLZF/RARα-positive 32D cells, DACi-mediated antiproliferative effects were associated with downregulation of BMI1 and c-MYC protein levels. Similar effects were demonstrated in primary samples of cytogenetically defined high-risk AML patients. In conclusion, DACi may be effective as maintenance therapy by negatively interfering with signaling pathways that control survival and proliferation of leukemic stem and progenitor cells.
PMCID: PMC3466521  PMID: 22895185
acute myeloid leukemia; leukemic stem cells; deacetylase inhibitor; BMI1; self-renewal; short-term repopulation; dacinostat; vorinostat
5.  Protein Kinase Activity of Phosphoinositide 3-Kinase Regulates Cytokine-Dependent Cell Survival 
PLoS Biology  2013;11(3):e1001515.
The protein kinase activity of PI3K phosphorylates specific serine residues in growth factor receptors to promote cell survival; these events are constitutively activated in some leukemias.
The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathways in cancer.
Author Summary
The ability of cells to survive in the absence of proliferation (cell division), differentiation (cell maturation) or activation allows tissues to maintain cell populations that are poised for rapid responses to damage, infections, or other physiological demands. While this “survival-only” response is fundamental to all physiological processes, the underlying mechanisms are not understood. Many growth factors are potent regulators of cell survival through their ability to bind specific cell surface receptors, which in turn activate specialized enzymes called kinases. Phosphoinositide 3-kinase (PI3K) is a dual specificity kinase that is known to be involved in cell survival and malignant transformation, and it is able to phosphorylate both lipid and protein substrates. While the PI3K lipid kinase activity has been extensively studied, the functional significance of its protein kinase activity remains unclear. Here we show that PI3K protein kinase activity can directly phosphorylate growth factor receptors on human hematopoietic (blood) cells to promote a “survival-only” response. We further show that the protein kinase activity of PI3K can be hijacked to result in uncontrolled growth factor receptor phosphorylation and the deregulated survival of leukemic cells. Our studies provide the first evidence that the protein kinase activity of PI3K can control cell survival and that this activity may be deregulated in cancer.
PMCID: PMC3601961  PMID: 23526884
6.  Role of Misfolded N-CoR Mediated Transcriptional Deregulation of Flt3 in Acute Monocytic Leukemia (AML)-M5 Subtype 
PLoS ONE  2012;7(4):e34501.
The nuclear receptor co-repressor (N-CoR) is a key component of the generic multi-protein complex involved in transcriptional control. Flt3, a key regulator of hematopoietic cell growth, is frequently deregulated in AML (acute myeloid leukemia). Here, we report that loss of N-CoR-mediated transcriptional control of Flt3 due to misfolding, contributes to malignant growth in AML of the M5 subtype (AML-M5). An analysis of hematopoietic genes in AML cells led to the identification of Flt3 as a transcriptional target of N-CoR. Flt3 level was inversely related to N-CoR status in various leukemia cells. N-CoR was associated with the Flt3 promoter in-vivo, and a reporter driven by the Flt3 promoter was effectively repressed by N-CoR. Blocking N-CoR loss with Genistein; an inhibitor of N-CoR misfolding, significantly down-regulated Flt3 levels regardless of the Flt3 receptor mutational status and promoted the differentiation of AML-M5 cells. While stimulation of the Flt3 receptor with the Flt3 ligand triggered N-CoR loss, Flt3 antibody mediated blockade of Flt3 ligand-receptor binding led to N-CoR stabilization. Genetic ablation of N-CoR potentiated Flt3 ligand induced proliferation of BA/F3 cells. These findings suggest that N-CoR-induced repression of Flt3 might be crucial for limiting the contribution of the Flt3 signaling pathway on the growth potential of leukemic cells and its deregulation due to N-CoR loss in AML-M5, could contribute to malignant growth by conferring a proliferative advantage to the leukemic blasts. Therapeutic restoration of N-CoR function could thus be a useful approach in restricting the contribution of the Flt3 signaling pathway in AML-M5 pathogenesis.
PMCID: PMC3326026  PMID: 22514634
7.  The role, mechanism and potentially therapeutic application of microRNA-29 family in acute myeloid leukemia 
Cell Death and Differentiation  2013;21(1):100-112.
Abnormal proliferation, apoptosis repression and differentiation blockage of hematopoietic stem/progenitor cells have been characterized to be the main reasons leading to acute myeloid leukemia (AML). Previous studies showed that miR-29a and miR-29b could function as tumor suppressors in leukemogenesis. However, a comprehensive investigation of the function and mechanism of miR-29 family in AML development and their potentiality in AML therapy still need to be elucidated. Herein, we reported that the family members, miR-29a, -29b and -29c, were commonly downregulated in peripheral blood mononuclear cells and bone marrow (BM) CD34+ cells derived from AML patients as compared with the healthy donors. Overexpression of each miR-29 member in THP1 and NB4 cells markedly inhibited cell proliferation and promoted cell apoptosis. AKT2 and CCND2 mRNAs were demonstrated to be targets of the miR-29 members, and the role of miR-29 family was attributed to the decrease of Akt2 and CCND2, two key signaling molecules. Significantly increased Akt2, CCND2 and c-Myc levels in the AML cases were detected, which were correlated with the decreased miR-29 expression in AML blasts. Furthermore, a feed-back loop comprising of c-Myc, miR-29 family and Akt2 were found in myeloid leukemogenesis. Reintroduction of each miR-29 member partially corrected abnormal cell proliferation and apoptosis repression and myeloid differentiation arrest in AML BM blasts. An intravenous injection of miR-29a, -29b and -29c in the AML model mice relieved leukemic symptoms significantly. Taken together, our finding revealed a pivotal role of miR-29 family in AML development and rescue of miR-29 family expression in AML patients could provide a new therapeutic strategy.
PMCID: PMC3857615  PMID: 24076586
acute myeloid leukemia; cell proliferation and apoptosis; miR-29 family; diagnostic markers; therapeutic targets
8.  Dendritic Cell-Based Immunotherapy for Myeloid Leukemias 
Acute and chronic myeloid leukemia (AML, CML) are hematologic malignancies arising from oncogene-transformed hematopoietic stem/progenitor cells known as leukemia stem cells (LSCs). LSCs are selectively resistant to various forms of therapy including irradiation or cytotoxic drugs. The introduction of tyrosine kinase inhibitors has dramatically improved disease outcome in patients with CML. For AML, however, prognosis is still quite dismal. Standard treatments have been established more than 20 years ago with only limited advances ever since. Durable remission is achieved in less than 30% of patients. Minimal residual disease (MRD), reflected by the persistence of LSCs below the detection limit by conventional methods, causes a high rate of disease relapses. Therefore, the ultimate goal in the treatment of myeloid leukemia must be the eradication of LSCs. Active immunotherapy, aiming at the generation of leukemia-specific cytotoxic T cells (CTLs), may represent a powerful approach to target LSCs in the MRD situation. To fully activate CTLs, leukemia antigens have to be successfully captured, processed, and presented by mature dendritic cells (DCs). Myeloid progenitors are a prominent source of DCs under homeostatic conditions, and it is now well established that LSCs and leukemic blasts can give rise to “malignant” DCs. These leukemia-derived DCs can express leukemia antigens and may either induce anti-leukemic T cell responses or favor tolerance to the leukemia, depending on co-stimulatory or -inhibitory molecules and cytokines. This review will concentrate on the role of DCs in myeloid leukemia immunotherapy with a special focus on their generation, application, and function and how they could be improved in order to generate highly effective and specific anti-leukemic CTL responses. In addition, we discuss how DC-based immunotherapy may be successfully integrated into current treatment strategies to promote remission and potentially cure myeloid leukemias.
PMCID: PMC3876024  PMID: 24427158
dendritic cells; immunotherapy; active; myeloid leukemia; minimal residual disease; leukemia stem cells
9.  Identification of Chromatin Remodeling Genes Arid4a and Arid4b as Leukemia Suppressor Genes 
Leukemia evolves through a multistep process from premalignancy to malignancy. Epigenetic alterations, including histone modifications, have been proposed to play an important role in tumorigenesis. The involvement of two chromatin remodeling genes, retinoblastoma-binding protein 1 (Rbbp1/Arid4a) and Rbbp1-like 1 (Rbbp1l1/Arid4b), in leukemogenesis was not characterized.
The leukemic phenotype of mice deficient for Arid4a with or without haploinsufficiency for Arid4b was investigated by serially monitoring complete blood counts together with microscopic histologic analysis and flow cytometric analysis of bone marrow and spleen from the Arid4a−/− mice or Arid4a−/−Arid4b+/− mice. Regulation in bone marrow cells of downstream genes important for normal hematopoiesis was analyzed by reverse transcription–polymerase chain reaction. Genotypic effects on histone modifications were examined by western blotting and immunofluorescence analysis. All statistical tests were two-sided.
Young (2–5 months old) Arid4a-deficient mice had ineffective blood cell production in all hematopoietic lineages. Beyond 5 months of age, the Arid4a−/− mice manifested monocytosis, accompanied by severe anemia and thrombocytopenia. These sick Arid4a−/− mice showed bone marrow failure with myelofibrosis associated with splenomegaly and hepatomegaly. Five of 42 Arid4a−/− mice and 10 of 12 Arid4a−/−Arid4b+/− mice progressed to acute myeloid leukemia (AML) and had rapid further increases of leukocyte counts. Expression of Hox genes (Hoxb3, Hoxb5, Hoxb6, and Hoxb8) was decreased in Arid4a-deficient bone marrow cells with or without Arid4b haploinsufficiency, and FoxP3 expression was reduced in Arid4a−/−Arid4b+/− bone marrow. Increases of histone trimethylation of H3K4, H3K9, and H4K20 (fold increases in trimethylation = 32, 95% confidence interval [CI] = 27 to 32; 45, 95% CI = 41 to 49; and 2.2, 95% CI = 1.7 to 2.7, respectively) were observed in the bone marrow of Arid4a-deficient mice.
Arid4a-deficient mice initially display ineffective hematopoiesis, followed by transition to chronic myelomonocytic leukemia (CMML)–like myelodysplastic/myeloproliferative disorder, and then transformation to AML. The disease processes in the Arid4a-deficient mice are very similar to the course of events in humans with CMML and AML. This mouse model has the potential to furnish additional insights into the role of epigenetic alterations in leukemogenesis, and it may be useful in developing novel pharmacological approaches to treatment of preleukemic and leukemic states.
PMCID: PMC2528019  PMID: 18728284
10.  RepSox Slows Decay of CD34+ Acute Myeloid Leukemia Cells and Decreases T Cell Immunoglobulin Mucin-3 Expression 
To facilitate development of therapies that target leukemic stem/progenitor cells (LPCs), in vitro ways to enhance the survival and immunogenicity of a patient's CD34+ acute myeloid leukemia (AML) cells were explored. RepSox was identified as a candidate cell-engineering tool because it slows in vitro decay of CD34+ AML cells (which often contain LPCs) and accelerates loss of the immune checkpoint receptor T cell immunoglobulin mucin-3 (Tim-3).
Despite initial response to therapy, most acute myeloid leukemia (AML) patients relapse. To eliminate relapse-causing leukemic stem/progenitor cells (LPCs), patient-specific immune therapies may be required. In vitro cellular engineering may require increasing the “stemness” or immunogenicity of tumor cells and activating or restoring cancer-impaired immune-effector and antigen-presenting cells. Leukapheresis samples provide the cells needed to engineer therapies: LPCs to be targeted, normal hematopoietic stem cells to be spared, and cancer-impaired immune cells to be repaired and activated. This study sought to advance development of LPC-targeted therapies by exploring nongenetic ways to slow the decay and to increase the immunogenicity of primary CD34+ AML cells. CD34+ AML cells generally displayed more colony-forming and aldehyde dehydrogenase activity than CD34− AML cells. Along with exposure to bone marrow stromal cells and low (1%–5%) oxygen, culture with RepSox (a reprogramming tool and inhibitor of transforming growth factor-β receptor 1) consistently slowed decline of CD34+ AML and myelodysplastic syndrome (MDS) cells. RepSox-treated AML cells displayed higher CD34, CXCL12, and MYC mRNA levels than dimethyl sulfoxide-treated controls. RepSox also accelerated loss of T cell immunoglobulin mucin-3 (Tim-3), an immune checkpoint receptor that impairs antitumor immunity, from the surface of AML and MDS cells. Our results suggest RepSox may reduce Tim-3 expression by inhibiting transforming growth factor-β signaling and slow decay of CD34+ AML cells by increasing CXCL12 and MYC, two factors that inhibit AML cell differentiation. By prolonging survival of CD34+ AML cells and reducing Tim-3, RepSox may promote in vitro immune cell activation and advance development of LPC-targeted therapies.
PMCID: PMC4073822  PMID: 24855276
Acute myeloid leukemia; Cancer stem cells; Immunotherapy; Immunogenicity; Tim-3; CD34+
11.  A Histone Acetyltransferase p300 Inhibitor C646 Induces Cell Cycle Arrest and Apoptosis Selectively in AML1-ETO-Positive AML Cells 
PLoS ONE  2013;8(2):e55481.
AML1-ETO fusion protein (AE) is generated by t(8;21)(q22;q22) chromosomal translocation, which is one of the most frequently observed structural abnormalities in acute myeloid leukemia (AML) and displays a pivotal role in leukemogenesis. The histone acetyltransferase p300 promotes self-renewal of leukemia cells by acetylating AE and facilitating its downstream gene expression as a transcriptional coactivator, suggesting that p300 may be a potential therapeutic target for AE-positive AML. However, the effects of p300 inhibitors on leukemia cells and the underlying mechanisms have not been extensively investigated. In the current study, we analyzed the anti-leukemia effects of C646, a selective and competitive p300 inhibitor, on AML cells. Results showed that C646 inhibited cellular proliferation, reduced colony formation, evoked partial cell cycle arrest in G1 phase, and induced apoptosis in AE-positive AML cell lines and primary blasts isolated from leukemic mice and AML patients. Nevertheless, no significant inhibitory effects were observed in granulocyte colony-stimulating factor-mobilized normal peripheral blood stem cells. Notably, AE-positive AML cells were more sensitive to lower C646 doses than AE-negative ones. And C646-induced growth inhibition on AE-positive AML cells was associated with reduced global histone H3 acetylation and declined c-kit and bcl-2 levels. Therefore, C646 may be a potential candidate for treating AE-positive AML.
PMCID: PMC3563640  PMID: 23390536
12.  Independent oncogenic and therapeutic significance of phosphatase PRL-3 in FLT3-ITD–negative acute myeloid leukemia 
Cancer  2014;120(14):2130-2141.
Internal tandem duplication of FMS-like tyrosine kinase (FLT3-ITD) is well known to be involved in acute myeloid leukemia (AML) progression, but FLT3-ITD–negative AML cases account for 70% to 80% of AML, and the mechanisms underlying their pathology remain unclear. This study identifies protein tyrosine phophatase PRL-3 as a key mediator of FLT3-ITD–negative AML.
A total of 112 FLT3-ITD–negative AML patients were sampled between 2010 and 2013, and the occurrence of PRL-3 hyperexpression in FLT3-ITD–negative AML was evaluated by multivariate probit regression analysis. Overexpression or depletion of endogenous PRL-3 expression with the specific small interfering RNAs was performed to investigate the role of PRL-3 in AML progression. Xenograft models were also used to confirm the oncogenic role of PRL-3.
Compared to healthy donors, PRL-3 is upregulated more than 3-fold in 40.2% of FLT3-ITD–negative AML patients. PRL-3 expression level is adversely correlated to the overall survival of the AML patients, and the AML relapses accompany with re-upregulation of PRL-3. Mechanistically, aberrant PRL-3 expression promoted cell cycle progression and enhanced the antiapoptotic machinery of AML cells to drug cytotoxicity through downregulation of p21 and upregulation of Cyclin D1 and CDK2 and activation of STAT5 and AKT. Depletion of endogenous PRL-3 sensitizes AML cells to therapeutic drugs, concomitant with apoptosis by upregulation of cleaved PARP (poly ADP ribose polymerase) and apoptosis-related caspases. Xenograft assays further confirmed PRL-3’s oncogenic role in leukemogenesis.
Our results demonstrated that PRL-3 is a novel independent crucial player in both FLT3-ITD–positive and FLT3-ITD–negative AML and could be a potential therapeutic target. Cancer 2014;120:2130–2141. © 2014 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society.
FLT3-ITD–negative acute myeloid leukemia (AML) accounts for up to approximately 70% to 80% of all cases. This study demonstrates that PRL-3, an independent driver in FLT3-ITD–negative AML, is adversely correlated to patient survival. Mechanistically, PRL-3 can promote AML cell cycle progression and render antiapoptosis features to AML cells, suggesting it could be an independent factor for AML diagnosis and therapy.
PMCID: PMC4231236  PMID: 24737397
acute myeloid leukemia; PRL-3; FLT3-ITD–negative; apoptosis; drug resistance; cell proliferation
13.  Accelerated Telomere Shortening Precedes Development of Therapy-Related Myelodysplasia or Acute Myelogenous Leukemia After Autologous Transplantation for Lymphoma 
Journal of Clinical Oncology  2009;27(5):791-798.
Therapy-related myelodysplasia or acute myelogenous leukemia (t-MDS/AML) is a lethal complication of autologous hematopoietic stem-cell transplantation (aHCT) for Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL). Here, we investigated the hypothesis that accelerated telomere shortening after aHCT could contribute to the development of t-MDS/AML.
Patients and Methods
A prospective longitudinal cohort was constructed to investigate the sequence of cellular and molecular abnormalities leading to development of t-MDS/AML after aHCT for HL/NHL. This cohort formed the sampling frame for a nested case-control study to compare changes in telomere length in serial blood samples from patients who developed t-MDS/AML with matched controls who did not develop t-MDS/AML.
An initial increase in telomere length at day 100 after aHCT was followed by an accelerated telomere shortening in t-MDS/AML patients when compared with controls. These telomere alterations preceded the onset of t-MDS and were independent of other known risk factors associated with development of t-MDS/AML on multivariate analysis. Additionally, we observed reduced generation of committed progenitors in patients who developed t-MDS/AML, indicating that these telomere alterations were associated with reduced regenerative capacity of hematopoietic stem cells.
The development of t-MDS/AML after aHCT is associated with and preceded by markedly altered telomere dynamics in hematopoietic cells. Accelerated telomere loss in patients developing t-MDS/AML may reflect increased clonal proliferation and/or altered telomere regulation in premalignant cells. Genetic instability associated with shortened telomeres may contribute to leukemic transformation in t-MDS/AML.
PMCID: PMC2645091  PMID: 19124806
14.  Persistent Transactivation by Meis1 Replaces Hox Function in Myeloid Leukemogenesis Models: Evidence for Co-Occupancy of Meis1-Pbx and Hox-Pbx Complexes on Promoters of Leukemia-Associated Genes 
Molecular and Cellular Biology  2006;26(10):3902-3916.
Homeobox transcription factors Meis1 and Hoxa9 promote hematopoietic progenitor self-renewal and cooperate to cause acute myeloid leukemia (AML). While Hoxa9 alone blocks the differentiation of nonleukemogenic myeloid cell-committed progenitors, coexpression with Meis1 is required for the production of AML-initiating progenitors, which also transcribe a group of hematopoietic stem cell genes, including Cd34 and Flt3 (defined as Meis1-related leukemic signature genes). Here, we use dominant trans-activating (Vp16 fusion) or trans-repressing (engrailed fusion) forms of Meis1 to define its biochemical functions that contribute to leukemogenesis. Surprisingly, Vp16-Meis1 (but not engrailed-Meis1) functioned as an autonomous oncoprotein that mimicked combined activities of Meis1 plus Hoxa9, immortalizing early progenitors, inducing low-level expression of Meis1-related signature genes, and causing leukemia without coexpression of exogenous or endogenous Hox genes. Vp16-Meis1-mediated transformation required the Meis1 function of binding to Pbx and DNA but not its C-terminal domain (CTD). The absence of endogenous Hox gene expression in Vp16-Meis1-immortalized progenitors allowed us to investigate how Hox alters gene expression and cell biology in early hematopoietic progenitors. Strikingly, expression of Hoxa9 or Hoxa7 stimulated both leukemic aggressiveness and transcription of Meis1-related signature genes in Vp16-Meis1 progenitors. Interestingly, while the Hoxa9 N-terminal domain (NTD) is essential for cooperative transformation with wild-type Meis1, it was dispensable in Vp16-Meis1 progenitors. The fact that a dominant transactivation domain fused to Meis1 replaces the essential functions of both the Meis1 CTD and Hoxa9 NTD suggests that Meis-Pbx and Hox-Pbx (or Hox-Pbx-Meis) complexes co-occupy cellular promoters that drive leukemogenesis and that Meis1 CTD and Hox NTD cooperate in gene activation. Chromatin immunoprecipitation confirmed co-occupancy of Hoxa9 and Meis1 on the Flt3 promoter.
PMCID: PMC1488994  PMID: 16648484
15.  Acute myeloid leukemia arising from a donor derived premalignant hematopoietic clone: A possible mechanism for the origin of leukemia in donor cells 
Leukemia Research Reports  2014;3(2):38-41.
During recent years, it has become increasingly evident that donor leukemia following allogeneic transplant may be more common then realized in the past. We identified five cases of potential donor leukemia cases during past five years. The precise mechanism of the origin of such leukemias, however, remains poorly defined. In this short communication, we report a well documented case of donor-derived de novo acute myeloid leukemia (AML) that developed fourteen years after allogeneic stem cell transplantation for treatment induced AML for his primary malignancy Immunoblastic lymphoma. This case allows us to postulate a possible mechanism of the origin of donor leukemia. The de novo AML clone contained a distinct cytogenetic abnormality, trisomy 11, which was simultaneously detected in preserved peripheral blood obtained at the time of transplantation as well as in the current bone marrow from an otherwise clinically and phenotypically normal donor. The findings from this unique case, provides insight into the process of leukemogenesis, and suggests that the sequence of events leading to leukemogenesis in this patient involved the senescence/apoptosis of normal donor hematopoietic cells due to telomere shortening resulting in the selective proliferation and transformation of this clone with MLL (mixed-lineage leukemia) gene amplification.
•Donor leukemia following allogeneic bone marrow transplantation may not be as rare.•Acute leukemia arises from a quiescent preexisting donor derived hematopoietic clone.•Telomere erosion and replicative stress in normal donor cells results in senescence.•Selective proliferation of clone with MLL amplification results in donor leukemia.•The results provide mechanistic insight into leukemogenesis in donor derived cells.
PMCID: PMC4050285  PMID: 24918066
Donor leukemia; Acute secondary leukemia; Telomere length in translantation; Allogeneic bone marrow transplantation; Origin of donor leukemia mechanism
16.  Sulindac Sulfide Reverses Aberrant Self-Renewal of Progenitor Cells Induced by the AML-Associated Fusion Proteins PML/RARα and PLZF/RARα 
PLoS ONE  2011;6(7):e22540.
Chromosomal translocations can lead to the formation of chimeric genes encoding fusion proteins such as PML/RARα, PLZF/RARα, and AML-1/ETO, which are able to induce and maintain acute myeloid leukemia (AML). One key mechanism in leukemogenesis is increased self renewal of leukemic stem cells via aberrant activation of the Wnt signaling pathway. Either X-RAR, PML/RARα and PLZF/RARα or AML-1/ETO activate Wnt signaling by upregulating γ-catenin and β-catenin. In a prospective study, a lower risk of leukemia was observed with aspirin use, which is consistent with numerous studies reporting an inverse association of aspirin with other cancers. Furthermore, a reduction in leukemia risk was associated with use of non-steroidal anti-inflammatory drug (NSAID), where the effects on AML risk was FAB subtype-specific. To better investigate whether NSAID treatment is effective, we used Sulindac Sulfide in X-RARα-positive progenitor cell models. Sulindac Sulfide (SSi) is a derivative of Sulindac, a NSAID known to inactivate Wnt signaling. We found that SSi downregulated both β-catenin and γ-catenin in X-RARα-expressing cells and reversed the leukemic phenotype by reducing stem cell capacity and increasing differentiation potential in X-RARα-positive HSCs. The data presented herein show that SSi inhibits the leukemic cell growth as well as hematopoietic progenitors cells (HPCs) expressing PML/RARα, and it indicates that Sulindac is a valid molecular therapeutic approach that should be further validated using in vivo leukemia models and in clinical settings.
PMCID: PMC3139642  PMID: 21811629
17.  TGF-β-Neutralizing Antibody 1D11 Enhances Cytarabine-Induced Apoptosis in AML Cells in the Bone Marrow Microenvironment 
PLoS ONE  2013;8(6):e62785.
Hypoxia and interactions with bone marrow (BM) stromal cells have emerged as essential components of the leukemic BM microenvironment in promoting leukemia cell survival and chemoresistance. High levels of transforming growth factor beta 1 (TGFβ1) produced by BM stromal cells in the BM niche regulate cell proliferation, survival, and apoptosis, depending on the cellular context. Exogenous TGFβ1 induced accumulation of acute myeloid leukemia (AML) cells in a quiescent G0 state, which was further facilitated by the co-culture with BM-derived mesenchymal stem cells (MSCs). In turn, TGFβ-neutralizing antibody 1D11 abrogated rhTGFβ1 induced cell cycle arrest. Blocking TGFβ with 1D11 further enhanced cytarabine (Ara-C)–induced apoptosis of AML cells in hypoxic and in normoxic conditions. Additional constituents of BM niche, the stroma-secreted chemokine CXCL12 and its receptor CXCR4 play crucial roles in cell migration and stroma/leukemia cell interactions. Treatment with 1D11 combined with CXCR4 antagonist plerixafor and Ara-C decreased leukemia burden and prolonged survival in an in vivo leukemia model. These results indicate that blockade of TGFβ by 1D11 and abrogation of CXCL12/CXCR4 signaling may enhance the efficacy of chemotherapy against AML cells in the hypoxic BM microenvironment.
PMCID: PMC3695026  PMID: 23826077
18.  Rearrangement and expression of the immunoglobulin μ-chain gene in human myeloid cells 
Immunoglobulin (Ig), a characteristic marker of B cells, has been reported to be expressed in epithelial cells, with a suggested role in their growth and survival. We have previously reported that IgG heavy chain is expressed in acute myeloid leukemia (AML), but not in the monocytes or neutrophils from patients with non-hematopoietic neoplasms or healthy controls. In the present study, we assessed IgM heavy chain expression and repertoire in human myeloid cells. We detected VHμDJHμ rearrangement and expression in 7/7 AML cell lines, 7/14 primary myeloblasts from AML patients, and interestingly, 8/20 monocytes and 3/20 neutrophils from patients with non-hematopoietic neoplasms and healthy individuals. We also found evidence of somatic hypermutation of the variable (V) gene segments in AML-derived IgM gene rearrangements but not in IgM from monocytes or neutrophils from patients with non-hematopoietic neoplasms and healthy individuals. Furthermore, IgM VHμDJHμ gene rearrangements in AML cell lines, primary myeloblasts, and monocytes and neutrophils from patients with non-hematopoietic neoplasms showed a restricted V usage and repertoire, whereas the VHμDJHμ gene rearrangements in monocytes and neutrophils from healthy individuals displayed more diversity. Anti-human IgM inhibited cell proliferation, but did not induce apoptosis in AML cell lines. Our findings suggest that AML-derived IgM might be a novel AML-related molecule that is involved in leukemogenesis and AML progression and might serve as a useful molecular marker for designing targeted therapy and monitoring minimal residual disease.
PMCID: PMC4002143  PMID: 24141767
acute myeloid leukemia; IgM; VHμDJHμ gene rearrangements
19.  Overexpression of the local bone marrow renin-angiotensin system in acute myeloid leukemia. 
OBJECTIVES: Local bone marrow renin-angiotensin system (RAS) is an autocrine-paracrine system affecting hematopoiesis. Angiotensin II stimulates the proliferation of bone marrow and umbilical cord blood hematopoietic progenitors. Angiotensin-converting enzyme (ACE) hyperfunction may lead to the acceleration of negative hematopoietic regulator peptide, AcSDKP, metabolism, which in turn lowers its level in the bone marrow microenvironment, finally removing the antiproliferative effect of AcSDKP on the hematopoietic cells and blasts. The aim of this study is therefore to search those major RAS components simultaneously in the leukemic blast cells taken from the bone marrow of patients with acute myeloid leukemia (AML). METHODS: Bone marrow aspiration materials were obtained from 10 patients with AML (8 males, 2 females; median age 48.5 years) and 8 patients with nonmalignant hematological disorders (6 males, 2 females; median age 45 years). EDTA-treated bone marrow samples were stored at -70 degrees C until analysis. Total RNA was extracted from 200-microl bone marrow samples by High Pure RNA Isolation Kit. RESULTS: The medians of expression ratios of AML patient samples have been found 0.736 (IQR 1.359), 0.540 (IQR 0.725), and 0.075 (IQR 0.002) for ACE, ANG and REN genes, respectively. All three gene expressions were found to be significantly higher in the bone marrow samples of AML patients. CONCLUSION: In this study, the expression of the mRNAs of the major RAS components-namely ACE, renin and angiotensinogen-in human bone marrow samples were quantified by reverse transcription-polymerase chain reaction (RT-PCR) to confirm the presence of the local bone marrow RAS. Elucidation of the pathological activity of the local RAS-mediated regulation of the leukemogenesis is both pathobiologically and clinically important, since the angiotensin peptides represent a molecular target in the disease management.
PMCID: PMC2569610  PMID: 17304969
20.  In Vitro Transformation of Primary Human CD34+ Cells by AML Fusion Oncogenes: Early Gene Expression Profiling Reveals Possible Drug Target in AML 
PLoS ONE  2010;5(8):e12464.
Different fusion oncogenes in acute myeloid leukemia (AML) have distinct clinical and laboratory features suggesting different modes of malignant transformation. Here we compare the in vitro effects of representatives of 4 major groups of AML fusion oncogenes on primary human CD34+ cells. As expected from their clinical similarities, MLL-AF9 and NUP98-HOXA9 had very similar effects in vitro. They both caused erythroid hyperplasia and a clear block in erythroid and myeloid maturation. On the other hand, AML1-ETO and PML-RARA had only modest effects on myeloid and erythroid differentiation. All oncogenes except PML-RARA caused a dramatic increase in long-term proliferation and self-renewal. Gene expression profiling revealed two distinct temporal patterns of gene deregulation. Gene deregulation by MLL-AF9 and NUP98-HOXA9 peaked 3 days after transduction. In contrast, the vast majority of gene deregulation by AML1-ETO and PML-RARA occurred within 6 hours, followed by a dramatic drop in the numbers of deregulated genes. Interestingly, the p53 inhibitor MDM2 was upregulated by AML1-ETO at 6 hours. Nutlin-3, an inhibitor of the interaction between MDM2 and p53, specifically inhibited the proliferation and self-renewal of primary human CD34+ cells transduced with AML1-ETO, suggesting that MDM2 upregulation plays a role in cell transformation by AML1-ETO. These data show that differences among AML fusion oncogenes can be recapitulated in vitro using primary human CD34+ cells and that early gene expression profiling in these cells can reveal potential drug targets in AML.
PMCID: PMC2929205  PMID: 20805992
21.  (Lymph)angiogenic influences on hematopoietic cells in acute myeloid leukemia 
The purpose of this review is to provide an overview of the effect of (lymph)angiogenic cytokines on hematopoietic cells involved in acute myeloid leukemia (AML). Like angiogenesis, lymphangiogenesis occurs in pathophysiological conditions but not in healthy adults. AML is closely associated with the vasculature system, and the interplay between lymphangiogenic cytokines maintains leukemic blast survival in the bone marrow (BM). Once AML is induced, proangiogenic cytokines function as angiogenic or lymphangiogenic factors and affect hematopoietic cells, including BM-derived immune cells. Simultaneously, the representative cytokines, VEGFs and their receptors are expressed on AML blasts in vascular and osteoblast niches in both the BM and the peripheral circulation. After exposure to (lymph)angiogenic cytokines in leukemogenesis and infiltration, immune cell phenotypes and functions are affected. These dynamic behaviors in the BM reflect the clinical features of AML. In this review, we note the importance of lymphangiogenic factors and their receptors in hematopoietic cells in AML. Understanding the functional characterization of (lymph)angiogenic factors in the BM niche in AML will also be helpful in interrupting the engraftment of leukemic stem cells and for enhancing immune cell function by modulating the tumor microenvironment.
PMCID: PMC4262793  PMID: 25412683
22.  MYCN Transgenic Zebrafish Model with the Characterization of Acute Myeloid Leukemia and Altered Hematopoiesis 
PLoS ONE  2013;8(3):e59070.
Amplification of MYCN (N-Myc) oncogene has been reported as a frequent event and a poor prognostic marker in human acute myeloid leukemia (AML). The molecular mechanisms and transcriptional networks by which MYCN exerts its influence in AML are largely unknown.
Methodology/Principal Findings
We introduced murine MYCN gene into embryonic zebrafish through a heat-shock promoter and established the stable germline Tg(MYCN:HSE:EGFP) zebrafish. N-Myc downstream regulated gene 1 (NDRG1), negatively controlled by MYCN in human and functionally involved in neutrophil maturation, was significantly under-expressed in this model. Using peripheral blood smear detection, histological section and flow cytometric analysis of single cell suspension from kidney and spleen, we found that MYCN overexpression promoted cell proliferation, enhanced the repopulating activity of myeloid cells and the accumulation of immature hematopoietic blast cells. MYCN enhanced primitive hematopoiesis by upregulating scl and lmo2 expression and promoted myelopoiesis by inhibiting gata1 expression and inducing pu.1, mpo expression. Microarray analysis identified that cell cycle, glycolysis/gluconeogenesis, MAPK/Ras, and p53-mediated apoptosis pathways were upregulated. In addition, mismatch repair, transforming and growth factor β (TGFβ) were downregulated in MYCN-overexpressing blood cells (p<0.01). All of these signaling pathways are critical in the proliferation and malignant transformation of blood cells.
The above results induced by overexpression of MYCN closely resemble the main aspects of human AML, suggesting that MYCN plays a role in the etiology of AML. MYCN reprograms hematopoietic cell fate by regulating NDRG1 and several lineage-specific hematopoietic transcription factors. Therefore, this MYCN transgenic zebrafish model facilitates dissection of MYCN-mediated signaling in vivo, and enables high-throughput scale screens to identify the potential therapeutic targets.
PMCID: PMC3598662  PMID: 23554972
23.  MicroRNA-146a and AMD3100, two ways to control CXCR4 expression in acute myeloid leukemias 
Blood Cancer Journal  2011;1(6):e26-.
CXCR4 is a negative prognostic marker in acute myeloid leukemias (AMLs). Therefore, it is necessary to develop novel ways to inhibit CXCR4 expression in leukemia. AMD3100 is an inhibitor of CXCR4 currently used to mobilize cancer cells. CXCR4 is a target of microRNA (miR)-146a that may represent a new tool to inhibit CXCR4 expression. We then investigated CXCR4 regulation by miR-146a in primary AMLs and found an inverse correlation between miR-146a and CXCR4 protein expression levels in all AML subtypes. As the lowest miR-146a expression levels were observed in M5 AML, we analyzed the control of CXCR4 expression by miR-146a in normal and leukemic monocytic cells and showed that the regulatory miR-146a/CXCR4 pathway operates during monocytopoiesis, but is deregulated in AMLs. AMD3100 treatment and miR-146a overexpression were used to inhibit CXCR4 in leukemic cells. AMD3100 treatment induces the decrease of CXCR4 protein expression, associated with miR-146a increase, and increases sensitivity of leukemic blast cells to cytotoxic drugs, this effect being further enhanced by miR-146a overexpression. Altogether our data indicate that miR-146a and AMD3100, acting through different mechanism, downmodulate CXCR4 protein levels, impair leukemic cell proliferation and then may be used in combination with anti-leukemia drugs, for development of new therapeutic strategies.
PMCID: PMC3255264  PMID: 22829170
CXCR4; miR-146a; AMD3100; acute myeloid leukemia
24.  CDX2-driven leukemogenesis involves KLF4 repression and deregulated PPARγ signaling 
Aberrant expression of the homeodomain transcription factor CDX2 occurs in most cases of acute myeloid leukemia (AML) and promotes leukemogenesis, making CDX2, in principle, an attractive therapeutic target. Conversely, CDX2 acts as a tumor suppressor in colonic epithelium. The effectors mediating the leukemogenic activity of CDX2 and the mechanism underlying its context-dependent properties are poorly characterized, and strategies for interfering with CDX2 function in AML remain elusive. We report data implicating repression of the transcription factor KLF4 as important for the oncogenic activity of CDX2, and demonstrate that CDX2 differentially regulates KLF4 in AML versus colon cancer cells through a mechanism that involves tissue-specific patterns of promoter binding and epigenetic modifications. Furthermore, we identified deregulation of the PPARγ signaling pathway as a feature of CDX2-associated AML and observed that PPARγ agonists derepressed KLF4 and were preferentially toxic to CDX2+ leukemic cells. These data delineate transcriptional programs associated with CDX2 expression in hematopoietic cells, provide insight into the antagonistic duality of CDX2 function in AML versus colon cancer, and suggest reactivation of KLF4 expression, through modulation of PPARγ signaling, as a therapeutic modality in a large proportion of AML patients.
PMCID: PMC3533294  PMID: 23202735
25.  The AML1-ETO fusion gene and the FLT3 length mutation collaborate in inducing acute leukemia in mice 
Journal of Clinical Investigation  2005;115(8):2159-2168.
The molecular characterization of leukemia has demonstrated that genetic alterations in the leukemic clone frequently fall into 2 classes, those affecting transcription factors (e.g., AML1-ETO) and mutations affecting genes involved in signal transduction (e.g., activating mutations of FLT3 and KIT). This finding has favored a model of leukemogenesis in which the collaboration of these 2 classes of genetic alterations is necessary for the malignant transformation of hematopoietic progenitor cells. The model is supported by experimental data indicating that AML1-ETO and FLT3 length mutation (FLT3-LM), 2 of the most frequent genetic alterations in AML, are both insufficient on their own to cause leukemia in animal models. Here we report that AML1-ETO collaborates with FLT3-LM in inducing acute leukemia in a murine BM transplantation model. Moreover, in a series of 135 patients with AML1-ETO–positive AML, the most frequently identified class of additional mutations affected genes involved in signal transduction pathways including FLT3-LM or mutations of KIT and NRAS. These data support the concept of oncogenic cooperation between AML1-ETO and a class of activating mutations, recurrently found in patients with t(8;21), and provide a rationale for therapies targeting signal transduction pathways in AML1-ETO–positive leukemias.
PMCID: PMC1174917  PMID: 16025155

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