Ligand-induced Flt3-downregulation modulates cell death associated proteins and enhances chemosensitivity to idarubicin in THP-1 acute myeloid leukemia cells (original) (raw)
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Survey of Activated FLT3 Signaling in Leukemia
PLoS ONE, 2011
Activating mutations of FMS-like tyrosine kinase-3 (FLT3) are found in approximately 30% of patients with acute myeloid leukemia (AML). FLT3 is therefore an attractive drug target. However, the molecular mechanisms by which FLT3 mutations lead to cell transformation in AML remain unclear. To develop a better understanding of FLT3 signaling as well as its downstream effectors, we performed detailed phosphoproteomic analysis of FLT3 signaling in human leukemia cells. We identified over 1000 tyrosine phosphorylation sites from about 750 proteins in both AML (wild type and mutant FLT3) and B cell acute lymphoblastic leukemia (normal and amplification of FLT3) cell lines. Furthermore, using stable isotope labeling by amino acids in cell culture (SILAC), we were able to quantified over 400 phosphorylation sites (pTyr, pSer, and pThr) that were responsive to FLT3 inhibition in FLT3 driven human leukemia cell lines. We also extended this phosphoproteomic analysis on bone marrow from primary AML patient samples, and identify over 200 tyrosine and 800 serine/threonine phosphorylation sites in vivo. This study showed that oncogenic FLT3 regulates proteins involving diverse cellular processes and affects multiple signaling pathways in human leukemia that we previously appreciated, such as Fc epsilon RI-mediated signaling, BCR, and CD40 signaling pathways. It provides a valuable resource for investigation of oncogenic FLT3 signaling in human leukemia.
FLT3 ligand causes autocrine signaling in acute myeloid leukemia cells
Blood, 2004
The FLT3 receptor tyrosine kinase is highly expressed in most acute leukemias and frequently mutated in acute myeloid leukemia (AML). The mutated form of the receptor is constitutively activated and known to play an important role in AML, but the activation state of the overexpressed wild-type (wt) receptor is, at present, unknown. In this study, we examined the activation state of the wild-type receptor in AML. We found that the wild-type receptor was constitutively phosphorylated/activated in 8 of 12 primary AML samples and 4 of 13 leukemia cell lines. To explain why wtFLT3 is often activated, we investigated the expression of its ligand, FL, by these same cells. Coexpression of FL with FLT3 was a universal finding in both primary AML samples and leukemic-derived cell lines. To further prove that autocrine signaling was accounting for the activation, we showed that conditioned media but not fresh media was able to activate FLT3. In addition, an antibody that blocks binding of liga...
Blood, 2002
Activating mutations of the protein tyrosine kinase (PTK) FLT3 can be found in approximately 30% of patients with acute myeloid leukemia (AML), thereby representing the most frequent single genetic alteration in AML. These mutations occur in the juxtamembrane (FLT3 length mutations; FLT3-LMs) and the second tyrosine kinase domain of FLT3-TKD and confer interleukin 3 (IL-3)–independent growth to Ba/F3 cells. In the mouse bone marrow transplantation model, FLT3-LMs induce a myeloproliferative syndrome stressing their transforming activity in vivo. In this study, we analyzed the pro-proliferative and antiapoptotic potential of FLT3 in FLT3-LM/TKD-mutation–transformed Ba/F3 cells and AML-derived cell lines. The PTK inhibitor SU5614 has inhibitory activity for FLT3 and selectively induces growth arrest, apoptosis, and cell cycle arrest in Ba/F3 and AML cell lines expressing a constitutively activated FLT3. In addition, the compound reverts the antiapoptotic and pro-proliferative activity...
A Noncanonical Flt3ITD/NF-κB Signaling Pathway Represses DAPK1 in Acute Myeloid Leukemia
Clinical Cancer Research, 2011
Purpose: Death-associated protein kinase 1 (DAPK1), a tumor suppressor, is a rate-limiting effector in an endoplasmic reticulum (ER) stress-dependent apoptotic pathway. Its expression is epigenetically suppressed in several tumors. A mechanistic basis for epigenetic/transcriptional repression of DAPK1 was investigated in certain forms of acute myeloid leukemia (AML) with poor prognosis, which lacked ER stress-induced apoptosis. Experimental Design: Heterogeneous primary AMLs were screened to identify a subgroup with Flt3ITD in which repression of DAPK1, among NF-κB–and c-Jun–responsive genes, was studied. RNA interference knockdown studies were carried out in an Flt3ITD+ cell line, MV-4-11, to establish genetic epistasis in the pathway Flt3ITD–TAK1–DAPK1 repression, and chromatin immunoprecipitations were carried out to identify proximate effector proteins, including TAK1-activated p52NF-κB, at the DAPK1 locus. Results: AMLs characterized by normal karyotype with Flt3ITD were found ...
Oncotarget, 2016
Constitutive activation of the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3), via co-expression of its ligand or by genetic mutation, is common in acute myeloid leukemia (AML). In this study we show that FLT3 activation inhibits the activity of the tumor suppressor, protein phosphatase 2A (PP2A). Using BaF3 cells transduced with wildtype or mutant FLT3, we show that FLT3-induced PP2A inhibition sensitizes cells to the pharmacological PP2A activators, FTY720 and AAL(S). FTY720 and AAL(S) induced cell death and inhibited colony formation of FLT3 activated cells. Furthermore, PP2A activators reduced the phosphorylation of ERK and AKT, downstream targets shared by both FLT3 and PP2A, in FLT3/ITD+ BaF3 and MV4-11 cell lines. PP2A activity was lower in primary human bone marrow derived AML blasts compared to normal bone marrow, with blasts from FLT3-ITD patients displaying lower PP2A activity than WT-FLT3 blasts. Reduced PP2A activity was associated with hyperphosphorylation ...
A Noncanonical Flt3ITD/NF-κB Signaling Pathway Represses DAPK1 in Acute Myeloid Leukemia
Clinical Cancer Research, 2012
Purpose: Death-associated protein kinase 1 (DAPK1), a tumor suppressor, is a rate-limiting effector in an endoplasmic reticulum (ER) stress-dependent apoptotic pathway. Its expression is epigenetically suppressed in several tumors. A mechanistic basis for epigenetic/transcriptional repression of DAPK1 was investigated in certain forms of acute myeloid leukemia (AML) with poor prognosis, which lacked ER stress-induced apoptosis. Experimental Design: Heterogeneous primary AMLs were screened to identify a subgroup with Flt3ITD in which repression of DAPK1, among NF-kB-and c-Jun-responsive genes, was studied. RNA interference knockdown studies were carried out in an Flt3ITD þ cell line, MV-4-11, to establish genetic epistasis in the pathway Flt3ITD-TAK1-DAPK1 repression, and chromatin immunoprecipitations were carried out to identify proximate effector proteins, including TAK1-activated p52NF-kB, at the DAPK1 locus. Results: AMLs characterized by normal karyotype with Flt3ITD were found to have 10-to 100-fold lower DAPK1 transcripts normalized to the expression of c-Jun, a transcriptional activator of DAPK1, as compared with a heterogeneous cytogenetic category. In addition, Meis1, a c-Jun-responsive adverse AML prognostic gene signature was measured as control. These Flt3ITD þ AMLs overexpress relB, a transcriptional repressor, which forms active heterodimers with p52NF-kB. Chromatin immunoprecipitation assays identified p52NF-kB binding to the DAPK1 promoter together with histone deacetylase 2 (HDAC2) and HDAC6 in the Flt3ITD þ human AML cell line MV-4-11. Knockdown of p52NF-kB or its upstream regulator, NF-kBinducing kinase (NIK), de-repressed DAPK1. DAPK1-repressed primary Flt3ITD þ AMLs had selective nuclear activation of p52NF-kB. Conclusions: Flt3ITD promotes a noncanonical pathway via TAK1 and p52NF-kB to suppress DAPK1 in association with HDACs, which explains DAPK1 repression in Flt3ITD þ AML. Clin Cancer Res; 18(2); 360-9. Ó2011 AACR.
A Noncanonical Flt3ITD/NF- B Signaling Pathway Represses DAPK1 in Acute Myeloid Leukemia
Clinical Cancer Research, 2012
Purpose: Death-associated protein kinase 1 (DAPK1), a tumor suppressor, is a rate-limiting effector in an endoplasmic reticulum (ER) stress-dependent apoptotic pathway. Its expression is epigenetically suppressed in several tumors. A mechanistic basis for epigenetic/transcriptional repression of DAPK1 was investigated in certain forms of acute myeloid leukemia (AML) with poor prognosis, which lacked ER stress-induced apoptosis. Experimental Design: Heterogeneous primary AMLs were screened to identify a subgroup with Flt3ITD in which repression of DAPK1, among NF-kB-and c-Jun-responsive genes, was studied. RNA interference knockdown studies were carried out in an Flt3ITD þ cell line, MV-4-11, to establish genetic epistasis in the pathway Flt3ITD-TAK1-DAPK1 repression, and chromatin immunoprecipitations were carried out to identify proximate effector proteins, including TAK1-activated p52NF-kB, at the DAPK1 locus. Results: AMLs characterized by normal karyotype with Flt3ITD were found to have 10-to 100-fold lower DAPK1 transcripts normalized to the expression of c-Jun, a transcriptional activator of DAPK1, as compared with a heterogeneous cytogenetic category. In addition, Meis1, a c-Jun-responsive adverse AML prognostic gene signature was measured as control. These Flt3ITD þ AMLs overexpress relB, a transcriptional repressor, which forms active heterodimers with p52NF-kB. Chromatin immunoprecipitation assays identified p52NF-kB binding to the DAPK1 promoter together with histone deacetylase 2 (HDAC2) and HDAC6 in the Flt3ITD þ human AML cell line MV-4-11. Knockdown of p52NF-kB or its upstream regulator, NF-kBinducing kinase (NIK), de-repressed DAPK1. DAPK1-repressed primary Flt3ITD þ AMLs had selective nuclear activation of p52NF-kB. Conclusions: Flt3ITD promotes a noncanonical pathway via TAK1 and p52NF-kB to suppress DAPK1 in association with HDACs, which explains DAPK1 repression in Flt3ITD þ AML. Clin Cancer Res; 18(2); 360-9. Ó2011 AACR.
2008
hematopoietic progenitors carrying oncogenic FLT3 inhibitor-induced apoptosis of human leukemic cells and transduced BH3-only protein Bim more critical than Puma in tyrosine kinase http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub\_requests Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml Information about subscriptions and ASH membership may be found online at: digital object identifier (DOIs) and date of initial publication. the indexed by PubMed from initial publication. Citations to Advance online articles must include final publication). Advance online articles are citable and establish publication priority; they are appeared in the paper journal (edited, typeset versions may be posted when available prior to Advance online articles have been peer reviewed and accepted for publication but have not yet Abstract Constitutively activating internal tandem duplications (ITD) of FLT3 are the most common mutations in acute myeloid leukemia (AML) and correlate with poor prognosis. Receptor tyrosine kinase inhibitors targeting FLT3 have developed as attractive treatment options. Because relapses occur after initial responses, identification of FLT3-ITD-mediated signaling events are important to facilitate novel therapeutic interventions. Here, we have determined the growth-inhibitory and proapototic mechanisms of two small molecule inhibitors of FLT3, AG1295 or PKC412, in hematopoietic progenitor cells, human leukemic cell lines, and primary AML cells expressing FLT3-ITD. Inactivation of the PI3-kinase pathway, but not of Ras-MAP kinase signaling, was essential to elicit cytotoxic responses. Both compounds induced upregulation of proapoptotic BH3-only proteins Bim and Puma, and subsequent cell death. However, only silencing of Bim, or its direct transcriptional activator FOXO3a, abrogated apoptosis efficiently. Similar findings were made in bone marrow cells from genetargeted mice lacking Bim and/or Puma infected with FLT3-ITD and treated with inhibitor,