Akt-Induced Phosphorylation of N-CoR at Serine 1450 Contributes to Its Misfolded Conformational Dependent Loss (MCDL) in Acute Myeloid Leukemia of the M5 Subtype

The nuclear receptor co-repressor (N-CoR) is a key component of the generic co-repressor complex that plays an important role in the control of cellular growth and differentiation. As shown by us recently, the growth suppressive function of N-CoR largely relies on its capacity to repress Flt3, a key regulator of cellular gorwth during normal and malignant hematopoesis. We further demonstrated how de-repression of Flt3 due to the misfolded conformation dependent loss (MCDL) of N-CoR contributed to malignant growth in acute myeloid leukemia (AML). However, the molecular mechanism underlying the MCDL of N-CoR and its implication in AML pathogenesis is not fully understood. Here, we report that Akt-induced phosphorylation of N-CoR at the consensus Akt motif is crucial for its misfolding and subsequent loss in AML (AML-M5). N-CoR displayed significantly higher level of serine specific phosphorylation in almost all AML-M5 derived cells and was subjected to processing by AML-M5 specific aberrant protease activity. To identify the kinase linked to N-CoR phosphorylation, a library of activated kinases was screened with the extracts of AML cells; leading to the identification of Akt as the putative kinase linked to N-CoR phosphorylation. Consistent with this finding, a constitutively active Akt consistently phosphorylated N-CoR leading to its misfolding; while the therapeutic and genetic ablation of Akt largely abrogated the MCDL of N-CoR in AML-M5 cells. Site directed mutagenic analysis of N-CoR identified serine 1450 as the crucial residue whose phosphorylation by Akt was essential for the misfolding and loss of N-CoR protein. Moreover, Akt-induced phosphorylation of N-CoR contributed to the de-repression of Flt3, suggesting a cross talk between Akt signaling and N-CoR misfolding pathway in the pathogenesis of AML-M5. The N-CoR misfolding pathway could be the common downstream thread of pleiotropic Akt signaling activated by various oncogenic insults in some subtypes of leukemia and solid tumors.     

Ectopic expression of Flt3 kinase inhibits proliferation and promotes cell death in different human cancer cell lines

Stable ectopic expression of Flt3 receptor tyrosine kinase is usually performed in interleukin 3 (IL-3)-dependent murine cell lines like Ba/F3, resulting in loss of IL-3 dependence. Such high-level Flt3 expression has to date not been reported in human acute myeloid leukemia (AML) cell lines, despite the fact that oncogenic Flt3 aberrancies are frequent in AML patients. We show here that ectopic Flt3 expression in different human cancer cell lines might reduce proliferation and induce apoptotic cell death, involving Bax/Bcl2 modulation. Selective depletion of Flt3-expressing cells occurred in human AML cell lines transduced with retroviral Flt3 constructs, shown here using the HL-60 leukemic cell line. Flt3 expression was investigated in two cellular model systems, the SAOS-2 osteosarcoma cell line and the human embryonic kidney HEK293 cell line, and proliferation was reduced in both systems. HEK293 cells underwent apoptosis upon ectopic Flt3 expression and cell death could be rescued by overexpression of Bcl-2. Furthermore, we observed that the Flt3-induced inhibition of proliferation in HL-60 cells appeared to be Bax-dependent. Our results thus suggest that excessive Flt3 expression has growth-suppressive properties in several human cancer cell lines.     

Interplay of protein misfolding pathway and unfolded-protein response in acute promyelocytic leukemia

Protein misfolding has traditionally been linked to the pathogenesis of various neurodegenerative diseases. However, emerging evidence from various laboratories, including ours, suggests that protein misfolding may also play a fundamental role in some malignancies, particularly those caused by fusion oncoprotein generated from chromosomal translocation. Promyelocytic leukemia (PML) fused to the retinoic acid receptor (RAR) is a fusion oncoprotein linked to the transformation of acute promyelocytic leukemia (APL), and is not only a misfolded protein itself, but also promotes misfolding of nuclear receptor corepressor (N-CoR) protein, a corepressor essential for the growth-suppressive function of several tumor-suppressor proteins. PML–RAR promotes misfolding of N-CoR by inducing aberrant post-translational modification, which destabilizes its core and promotes instability. Misfolded N-CoR, thus, contributes to differentiation arrest and survival of APL cells through loss-of-function and aberrant gain-of-function properties. Therapeutic restoration of N-CoR conformation and function with conformation-modifying agents not only releases this differentiation arrest but also sensitizes APL cells to programmed cell death. These findings illustrate the potential of the misfolded N-CoR protein as a conformation-based drugable molecular target for APL, and highlights the promise of various conformation-modifying agents as novel therapeutics for APL. Protein conformational rearrangement, resulting from an inherited or acquired genetic alteration, could be a common pathological phenomenon contributing to transformation in different types of leukemias and solid tumors and, therefore, could serve as a common ground for designing a unifying diagnostic as well as therapeutic approach for a widely diverse disease such as cancer. To that end, APL could serve as a model for the development of a novel conformation-based therapeutic approach for other malignant diseases.     

Src-Like Adaptor Protein (SLAP) Binds to the Receptor Tyrosine Kinase Flt3 and Modulates Receptor Stability and Downstream Signaling

Fms-like tyrosine kinase 3 (Flt3) is an important growth factor receptor in hematopoiesis. Gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia (AML). Src-like adaptor protein (SLAP) is an interaction partner of the E3 ubiquitin ligase Cbl that can regulate receptor tyrosine kinases-mediated signal transduction. In this study, we analyzed the role of SLAP in signal transduction downstream of the type III receptor tyrosine kinase Flt3. The results show that upon ligand stimulation SLAP stably associates with Flt3 through multiple phosphotyrosine residues in Flt3. SLAP constitutively interacts with oncogenic Flt3-ITD and co-localizes with Flt3 near the cell membrane. This association initiates Cbl-dependent receptor ubiquitination and degradation. Depletion of SLAP expression by shRNA in Flt3-transfected Ba/F3 cells resulted in a weaker activation of FL-induced PI3K-Akt and MAPK signaling. Meta-analysis of microarray data from patient samples suggests that SLAP mRNA is differentially expressed in different cancers and its expression was significantly increased in patients carrying the Flt3-ITD mutation. Thus, our data suggest a novel role of SLAP in different cancers and in modulation of receptor tyrosine kinase signaling apart from its conventional role in regulation of receptor stability.     

Hematopoietic growth factors in patients receiving intensive chemotherapy for malignant disorders: studies of granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and Flt-3 ligand (Flt3L)

The levels of hematopoietic growth factors in patients receiving intensive chemotherapy for malignant disorders were investigated using a variety of approaches. Firstly, serum levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF and Flt3-ligand (Flt3L) were examined in acute leukemia patients with treatment-induced cytopenia and complicating bacterial infections. Increased serum levels of both G-CSF and Flt3-ligand (Flt3L) were detected when these patients developed therapy-induced leukopenia, whereas GM-CSF levels were low or undetectable. Development of complicating bacterial infections then increased the serum levels of both G- and GM-CSF, and the Flt3L levels remained high during the infections. Secondly, release of growth factors was characterized for clonogenic T cells that remained in the circulation of acute leukemia patients with chemotherapy-induced cytopenia. CD4(+) and CD8(+) T cells from these patients released high levels of GM-CSF, relatively low levels of IL-3 secretion having been detected, and only a minority of the clones released detectable amounts of Flt3L. Thus, circulating T cells may contribute to the high systemic growth factor levels in cytopenic patients. Thirdly, plasma levels of GM-CSF and interleukin-3 (IL-3) were examined in patients with malignant disorders who received chemotherapy plus G-CSF for stem cell mobilization. Increased levels of GM-CSF and Flt3L were detected both in the patients' plasma and in the stem cell grafts. Despite the increased growth factor levels in neutropenic patients with complicating infections, the occurrence of febrile neutropenia did not have a major impact on normal hematopoietic reconstitution (i.e. duration of treatment-induced neutropenia) after intensive chemotherapy for acute myelogenous leukemia.     

Curcumin sensitizes acute promyelocytic leukemia cells to unfolded protein response-induced apoptosis by blocking the loss of misfolded N-CoR protein

Acute promyelocytic leukemia (APL) is characterized by accumulation of apoptosis-resistant immature promyelocytic cells in the bone marrow and peripheral blood. We have shown that endoplasmic reticulum (ER)-associated degradation (ERAD) and protease-mediated degradation of misfolded nuclear receptor corepressor (N-CoR) confer resistance to unfolded protein response (UPR)-induced apoptosis in APL. These findings suggest that therapeutic inhibition of N-CoR misfolding or degradation may promote growth arrest in APL cells by sensitizing them to UPR-induced apoptosis. On the basis of this hypothesis, we tested the effects of several known protein conformation-modifying agents on the growth and survival of APL cells and identified curcumin, a natural component of turmeric, as a potent growth inhibitor of APL cells. Curcumin selectively inhibited the growth and promoted apoptosis in both primary and secondary leukemic cells derived from APL. The curcumin-induced apoptosis of APL cells was triggered by an amplification of ER stress, possibly from the accumulation of misfolded N-CoR protein in the ER. Curcumin promoted this net accumulation of aberrantly phosphorylated misfolded N-CoR protein by blocking its ERAD and protease-mediated degradation, which then led to the activation of UPR-induced apoptosis in APL cells. The activation of UPR by curcumin was manifested by phosphorylation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) and eukaryotic translation initiation factor 2 alpha (eIF2α), and upregulation of C/EBP homologous protein (CHOP) and GADD34, the principal mediators of proapoptotic UPR. These findings identify the therapeutic potential of curcumin in APL and further establish the rationale of misfolded N-CoR protein as an attractive molecular target in APL.     

Efficient Production of Bioactive Recombinant Human Flt3 Ligand in <Emphasis Type="Italic">E. coli</Emphasis>

Flt3 ligand (FL) is an early-acting hematopoietic cytokine that stimulates the proliferation and differentiation of hematopoietic progenitor cells by activating its cognate receptor, Flt3. Recently, FL was shown to potently contribute to the development and expansion of antigen-presenting dendritic cells and CD34⁺ natural killer cell progenitors in vivo. Here, we report a comprehensive method for the production of bioactive recombinant human FL (rhFL) in E. coli, suitable for structural, biophysical and physiological studies. A soluble form of human FL capable of binding to the Ftl3 receptor could be overexpressed in the E. coli strain Rosetta-gami(DE3) as inclusion bodies. We have established protocols for the efficient in vitro refolding and ensuing purification of rhFL to homogeneity (>95%), with yields approaching 5 mg of pure rhFL per liter of culture. The ability of rhFL to adopt a bioactive conformation was confirmed via a cell-proliferation assay and the activation of the Flt3 receptor in the human leukemic cell line, OCI-AML3.     

Chromosome 5q deletion and epigenetic suppression of the gene encoding alpha-catenin (CTNNA1) in myeloid cell transformation

Interstitial loss of all or part of the long arm of chromosome 5, or del(5q), is a frequent clonal chromosomal abnormality in human myelodysplastic syndrome (MDS, a preleukemic disorder) and acute myeloid leukemia (AML), and is thought to contribute to the pathogenesis of these diseases by deleting one or more tumor-suppressor genes. Although a major commonly deleted region (CDR) has been delineated on chromosome band 5q31.1 (refs. 3-7), attempts to identify tumor suppressors within this band have been unsuccessful. We focused our analysis of gene expression on RNA from primitive leukemia-initiating cells, which harbor 5q deletions, and analyzed 12 genes within the CDR that are expressed by normal hematopoietic stem cells. Here we show that the gene encoding alpha-catenin (CTNNA1) is expressed at a much lower level in leukemia-initiating stem cells from individuals with AML or MDS with a 5q deletion than in individuals with MDS or AML lacking a 5q deletion or in normal hematopoietic stem cells. Analysis of HL-60 cells, a myeloid leukemia line with deletion of the 5q31 region, showed that the CTNNA1 promoter of the retained allele is suppressed by both methylation and histone deacetylation. Restoration of CTNNA1 expression in HL-60 cells resulted in reduced proliferation and apoptotic cell death. Thus, loss of expression of the alpha-catenin tumor suppressor in hematopoietic stem cells may provide a growth advantage that contributes to human MDS or AML with del(5q).     

Expression patterns of leptin receptor (OB-R) isoforms and direct in vitro effects of recombinant leptin on OB-R, leptin expression and cytokine secretion by human hematopoietic malignant cells

Several studies have implicated leptin in the pathophysiology of neoplasias. We investigated the direct effect of leptin on malignant hematopoietic tissue that included: primary acute myeloid leukemia (AML) cells, leukemic cell lines and bone marrow biopsies from multiple myeloma (MM) patients. PBMC, T-cells, B-cells and monocytes from healthy subjects served as controls. We defined the patterns of OB-R isoform expression in AML cells and leukemic cell lines in comparison to control cells by RT-PCR. rLeptin upregulated the expression of OB-R and endogenous leptin in AML blasts and certain cell lines but not in control cells. Cytometric Bead Array analysis of pro- and anti-inflammatory cytokines showed that rleptin upregulates IL-6 secretion by AML cells, various cytokines by the leukemic cell lines tested and IL-10 secretion by control PBMC, contributed by monocytes. Western immunoblotting revealed that the effect of rleptin was independent of JAK-2/phospho-JAK-2 protein levels. Finally, MM biopsies stained positive for leptin and, to a lesser extend, OB-R. Immunoreactivity was confined mostly to the nucleus of the myeloma cells. Normal myelocytes, promyelocytes and megakaryocytes stained weakly positive, and erythroid cells were constantly negative. We propose that the leptin/OB-R system is strongly and directly involved in supporting the growth of hematopoietic malignancies.     

Expression patterns of leptin receptor (OB-R) isoforms and direct in vitro effects of recombinant leptin on OB-R,leptin expression and cytokine secretion by human hematopoietic malignant cells

Several studies have implicated leptin in the pathophysiology of neoplasias. We investigated the direct effect of leptin on malignant hematopoietic tissue that included: primary acute myeloid leukemia (AML) cells, leukemic cell lines and bone marrow biopsies from multiple myeloma (MM) patients. PBMC, T-cells, B-cells and monocytes from healthy subjects served as controls. We defined the patterns of OB-R isoform expression in AML cells and leukemic cell lines in comparison to control cells by RT-PCR. rLeptin upregulated the expression of OB-R and endogenous leptin in AML blasts and certain cell lines but not in control cells. Cytometric Bead Array analysis of pro- and anti-inflammatory cytokines showed that rleptin upregulates IL-6 secretion by AML cells, various cytokines by the leukemic cell lines tested and IL-10 secretion by control PBMC, contributed by monocytes. Western immunoblotting revealed that the effect of rleptin was independent of JAK-2/phospho-JAK-2 protein levels. Finally, MM biopsies stained positive for leptin and, to a lesser extend, OB-R. Immunoreactivity was confined mostly to the nucleus of the myeloma cells. Normal myelocytes, promyelocytes and megakaryocytes stained weakly positive, and erythroid cells were constantly negative. We propose that the leptin/OB-R system is strongly and directly involved in supporting the growth of hematopoietic malignancies.     

Development of an adenoviral vector system with adenovirus serotype 35 tropism; efficient transient gene transfer into primary malignant hematopoietic cells

BACKGROUND: A paucity of coxsackie adenovirus receptor (CAR) hampers the adenovirus serotype 5 (Ad5)-based vector-mediated gene transfer into malignant hematopoietic cells. Fiber-retargeted adenoviral vectors with species B tropism can potentially bypass the CAR requirement and facilitate efficient gene transfer into malignant hematopoietic cells. METHODS: For feasible generation of fiber-retargeted adenoviral vectors, we have modified the versatile AdEasy system with a chimeric fiber gene encoding the Ad5 fiber tail domain and Ad35 fiber shaft and knob domains. An Ad5-based vector encoding the green fluorescent protein (GFP) gene under the control of the PGK promoter with Ad35 fiber receptor specificity was generated (Ad5F35-GFP). The Ad5F35-GFP vector-mediated gene transfer efficiency was compared with a fiber non-modified Ad5-GFP vector, which also encodes the GFP gene under the control of the PGK promoter. RESULTS: We demonstrated that a variety of Ad5-refractory malignant myeloid and B lymphoid cell lines were highly permissive to the Ad5F35-GFP vector infection. Importantly, primary chronic myeloid leukemic (CML) cells and chronic lymphocytic leukemia (CLL) B cells were superiorly transduced by the Ad5F35-GFP vector at a multiplicity of infection (MOI) of 100 compared with the Ad5-GFP vector. CONCLUSIONS: Our study will facilitate the generation of fiber-retargeted adenoviral vectors and enable transient genetic manipulation of primary malignant hematopoietic cells.     

TIM-3 as a therapeutic target for malignant stem cells in acute myelogenous leukemia

Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. Recent studies have shown that many AML LSC-specific surface antigens could be such candidates. T cell immunoglobulin mucin-3 (TIM-3) is expressed on LSCs in most types of AML, except for acute promyelocytic leukemia, but not on normal hematopoietic stem cells (HSCs). In mouse models reconstituted with human AML LSCs or human hematopoietic stem cells, a human TIM-3 mouse IgG2a antibody with complement-dependent and antibody-dependent cellular cytotoxic activities eradicates AML LSCs in vivo but does not affect normal human hematopoiesis. Thus, TIM-3 is one of the promising targets to eradicate AML LSCs.     

Mechanism of activation of human c-KIT kinase by internal tandem duplications of the juxtamembrane domain and point mutations at aspartic acid 816

The proto-oncogene c-KIT receptor has been implicated as an essential component in the activation of leukemic cells. The internal tandem duplication (ITD) of c-KIT has also been identified as a predominant cause of acute myeloid leukemia (AML), although its role in the activation process is still unclear. To investigate the biological mechanisms of c-KIT activation, we generated a c-KIT receptor bearing two different immunological tags, HA and Flag tags. In this study, we demonstrated that the mutant (Mt)-ITD and Asp816 (D816Y) c-KIT receptors spontaneously formed dimers and that these Mt-ITD forms of c-KIT displayed high levels of phosphorylation and increased cellular tyrosine phosphorylation. The amount of wild-type homodimers increased following the addition of the c-KIT ligand, while the level of mutant homodimers was less affected by the addition of the c-KIT ligand. Furthermore, we demonstrated that Mt-ITD and activating point mutations of D816Y induced constitutive activation of c-KIT kinase in the absence of ligand in COS-1 cells. These data suggest a novel mechanism for the regulation of cell growth autonomy. Overall, our study suggests that c-KIT activation might have significant effects on hematopoietic cells and might help to improve our understanding of the pathogenesis of systemic mast cell disease, gastrointestinal stromal tumors and AML and potentially lead to the development of novel therapeutic approaches.