| 1. |
- Emdal, Kristina B., et al.
(författare)
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Phosphoproteomics of primary AML patient samples reveals rationale for AKT combination therapy and p53 context to overcome selinexor resistance
- 2022
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 40:6
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Tidskriftsartikel (refereegranskat)abstract
- Acute myeloid leukemia (AML) is a heterogeneous disease with variable patient responses to therapy. Selinexor, an inhibitor of nuclear export, has shown promising clinical activity for AML. To identify the molecular context for monotherapy sensitivity as well as rational drug combinations, we profile selinexor signaling responses using phosphoproteomics in primary AML patient samples and cell lines. Functional phosphosite scoring reveals that p53 function is required for selinexor sensitivity consistent with enhanced efficacy of selinexor in combination with the MDM2 inhibitor nutlin-3a. Moreover, combining selinexor with the AKT inhibitor MK-2206 overcomes dysregulated AKT-FOXO3 signaling in resistant cells, resulting in synergistic anti-proliferative effects. Using high-throughput spatial proteomics to profile subcellular compartments, we measure global proteome and phospho-proteome dynamics, providing direct evidence of nuclear translocation of FOXO3 upon combination treatment. Our data demonstrate the potential of phosphoproteomics and functional phosphorylation site scoring to successfully pinpoint key targetable signaling hubs for rational drug combinations.
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| 2. |
- Arora, D, et al.
(författare)
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Protein tyrosine phosphatase DEP-1 controls receptor tyrosine kinase FLT3 signaling
- 2011
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 286:13, s. 10918-10929
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Tidskriftsartikel (refereegranskat)abstract
- Fms-like tyrosine kinase 3 (FLT3) plays an important role in hematopoietic differentiation and constitutively active FLT3 mutant proteins contribute to the development of acute myeloid leukemia (AML). Little is known about the protein tyrosine phosphatases (PTP) affecting the signaling activity of FLT3. To identify such PTP, myeloid cells expressing wild type FLT3 were infected with a panel of lentiviral pseudotypes carrying shRNA expression cassettes targeting different PTP. Out of 20 PTP tested, expressed in hematopoietic cells or presumed to be involved in oncogenesis or tumor suppression, DEP-1 (PTPRJ) was identified as a PTP negatively regulating FLT3 phosphorylation and signaling. Stable myeloid cell lines with strongly reduced DEP-1 levels showed site-selective hyperphosphorylation of FLT3. Similarly, acute depletion of DEP-1 in the human AML cell line THP-1 caused elevated FLT3 phosphorylation. Particularly, the sites pY589, pY591, and pY842 involved in the FLT3 ligand (FL) -mediated activation of FLT3 were hyperphosphorylated most. Enhanced FLT3 phosphorylation in DEP-1 depleted cells was accompanied by enhanced FLT3-dependent activation of ERK and cell proliferation. Overexpression of DEP-1 resulted in opposite effects on FL-mediated receptor phosphorylation and signaling activity. Furthermore, FL-mediated colony formation in methylcellulose of 32D cells expressing FLT3 was induced in response to shRNA-mediated DEP-1 knockdown. This transforming effect of DEP-1 knockdown was consistent with a moderately increased activation of STAT5 upon FL-stimulation, but did not translate into myeloproliferative disease formation in the 32D-C3H/HeJ mouse model. The data indicate that DEP-1 is negatively regulating FLT3 signaling activity and its loss may contribute to, but is not sufficient for leukemogenic cell transformation.
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| 3. |
- Breitenbuecher, Frank, et al.
(författare)
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A novel molecular mechanism of primary resistance to FLT3-kinase inhibitors in AML
- 2009
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Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 113:17, s. 4063-4073
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Tidskriftsartikel (refereegranskat)abstract
- Currently, FLT3 tyrosine kinase inhibitors (TKIs) are emerging as the most promising drug therapy to overcome the dismal prognosis of acute myelogenous leukemia (AML) patients harboring internal tandem duplications (ITDs) of FLT3. However, up-front drug resistance occurs in approximately 30% of patients, and molecular mechanisms of resistance are poorly understood. Here, we have uncovered a novel mechanism of primary resistance to FLT3 TKIs in AML: an FLT3 receptor harboring a nonjuxtamembrane ITD atypically integrating into the beta-2 sheet of the first kinase domain (FLT3_ITD627E) induces dramatic up-regulation of the anti-apoptotic myeloid cell leukemia 1 protein (MCL-1). Using RNA interference technology, deregulated MCL-1 protein expression was shown to play a major role in conferring the resistance phenotype of 32D_ITD627E cells. Enhanced and sustained binding of the adaptor protein GRB-2 to the FLT3_ITD627E receptor is involved in MCL-1 up-regulation and is independent from TKI (PKC412)-induced inhibition of the receptor kinase. Thus, we describe a new mechanism of primary resistance to TKIs, which operates by reprogramming local and distant signal transduction events of the FLT3 tyrosine kinase. The data presented suggest that particular ITDs of FLT3 may be associated with rewired signaling and differential responsiveness to TKIs. (Blood. 2009; 113: 4063-4073)
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| 4. |
- Heiss, Elke, et al.
(författare)
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Identification of Y589 and Y599 in the juxamembrane domain of Flt3 as ligand-induced autophosphorylation sites involved in binding of Src family kinases and the protein tyrosine phosphatase SHP2
- 2006
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Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 108:5, s. 1542-1550
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Tidskriftsartikel (refereegranskat)abstract
- Early signal relay steps upon ligand binding to the receptor tyrosine kinase Flt3, i.e. sites of Flt3 autophosphorylation and subsequent docking partners, are mainly unresolved. By immunoprecipitation of specific tryptic peptides contained in the juxtamembrane region of human Flt3 and subsequent radiosequencing we identified the tyrosine residues 572, 589, 591 and 599 as in vivo autophosphorylation sites. Focusing on Y589 and Y599, we examined Flt3-ligand-mediated responses in WT-Flt3-, Y589F-Flt3- and Y599F-Flt3-expressing 32D cells. Compared to WT-Flt3-32D cells upon ligand-stimulation, 32DY589F- Flt3 showed enhanced Erk activation and proliferation/survival whereas 32DY599F-Flt3 cells hereby displayed substantially diminished responses. Both pY589 and pY599 were identified as association sites for signal relay molecules including Src family kinases and SHP2. Consistently, 32D-Y589F-Flt3 and 32D-Y599F-Flt3 showed decreased FL-triggered activation of Src family kinases. Interference with the Srcdependent negative regulation of Flt3 signaling may account for the enhanced mitogenic response of Y589F-Flt3. Y599 was additionally found to interact with the protein tyrosine phosphatase SHP2 in a phosphorylation dependent manner. As Y599F-Flt3-32D was unable to associate with and to phosporylate SHP2 and since silencing of SHP2 in WT-Flt3-expressing cells mimicked the Y599F-Flt3-phenotype we hypothesize that recruitment of SHP2 to pY599 contributes to FL-mediated Erk activation and proliferation.
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| 5. |
- Kharazi, Shabnam, et al.
(författare)
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Impact of gene dosage, loss of wild-type allele, and FLT3 ligand on Flt3-ITD-induced myeloproliferation
- 2011
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Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 118:13, s. 3613-3621
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Tidskriftsartikel (refereegranskat)abstract
- Acquisition of homozygous activating growth factor receptor mutations might accelerate cancer progression through a simple gene-dosage effect. Internal tandem duplications (ITDs) of FLT3 occur in approximately 25% cases of acute myeloid leukemia and induce ligand-independent constitutive signaling. Homozygous FLT3-ITDs confer an adverse prognosis and are frequently detected at relapse. Using a mouse knockin model of Flt3-internal tandem duplication (Flt3-ITD)-induced myeloproliferation, we herein demonstrate that the enhanced myeloid phenotype and expansion of granulocyte-monocyte and primitive Lin(-)Sca1(+)c-Kit(+) progenitors in Flt3-ITD homozygous mice can in part be mediated through the loss of the second wild-type allele. Further, whereas autocrine FLT3 ligand production has been implicated in FLT3-ITD myeloid malignancies and resistance to FLT3 inhibitors, we demonstrate here that the mouse Flt3(ITD/ITD) myeloid phenotype is FLT3 ligand-independent. (Blood. 2011; 118(13):3613-3621)
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| 6. |
- Masson, Kristina, et al.
(författare)
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A role of Gab2 association in Flt3 ITD mediated Stat5 phosphorylation and cell survival.
- 2009
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Ingår i: British Journal of Haematology. - : Wiley. - 0007-1048 .- 1365-2141. ; 146, s. 193-202
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Tidskriftsartikel (refereegranskat)abstract
- Summary The haematopoietic growth factor receptor Flt3 has been implicated as major cause of transformation in acute myeloid leukaemia. Intracellular signals mediated by wild-type Flt3 are involved in cell differentiation and survival whereas signalling via the mutant Flt3 ITD (internal tandem duplication) promotes enhanced cell growth. In this study, we identified tyrosines 768, 955 and 969 of Flt3 as phosphorylation sites and mediators of growth factor receptor binding protein 2 (Grb2) interaction, leading to the association of Grb2 associated binder 2 (Gab2) and contributing to proliferation and survival. Ba/F3 cells were transfected with either the wild-type Flt3 or the ITD, with or without a triple mutation of the Grb2 binding sites, and characterised in terms of proliferation and viability. Interestingly, the Flt3 ITD promoted increased survival but after introducing the triple mutation, this phenotype was lost. When looking into different downstream pathways, this effect was mainly caused by decreased phosphoinositide 3-kinase and Stat5 signalling, and the Flt3 ITD carrying the Grb2 binding mutations showed less Akt and Stat5 activation compared to the regular Flt3 ITD receptor. These findings not only reveal novel phosphorylation sites in Flt3 but contribute to the understanding of the molecular mechanism by which Flt3 ITD functions in pathological conditions.
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| 7. |
- Masson, Kristina, et al.
(författare)
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Direct binding of Cbl to Tyr(568) and Tyr(936) of the stem cell factor receptor/c-Kit is required for ligand-induced ubiquitination, internalization and degradation
- 2006
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Ingår i: Biochemical Journal. - 0264-6021. ; 399, s. 59-67
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Tidskriftsartikel (refereegranskat)abstract
- The ubiquitin E3 ligase Cbl has been shown to negatively regulate tyrosine kinase receptors, including the stem cell factor receptor/c-Kit. Impaired recruitment of Cbl to c-Kit results in a deregulated positive signalling that eventually can contribute to carcinogenesis. Here, we present results showing that Cbl is activated by the SFKs (Src family kinases) and recruited to c-Kit in order to trigger receptor ubiquitination. We demonstrate that phosphorylated Tyr(568) and Tyr(936) in c-Kit are involved in direct binding and activation of Cbl and that binding of the TKB domain (tyrosine kinase binding domain) of Cbl to c-Kit is specified by the presence of an isoleucine or leucine residue in position +3 to the phosphorylated tyrosine residue on c-Kit. Apart from the direct association between Cbl and c-Kit, we show that phosphorylation of Cbl by SFK members is required for activation of Cbl to occur. Moreover, we demonstrate that Cbl mediates mono-ubiquitination of c-Kit and that the receptor is subsequently targeted for lysosomal degradation. Taken together, our findings reveal novel insights into the mechanisms by which Cbl negatively regulates c-Kit-mediated signalling.
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| 8. |
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| 9. |
- Masson, Kristina, et al.
(författare)
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Oncogenic signaling from the hematopoietic growth factor receptors c-Kit and Flt3.
- 2009
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Ingår i: Cellular Signalling. - : Elsevier BV. - 1873-3913 .- 0898-6568. ; 21, s. 1717-1726
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Forskningsöversikt (refereegranskat)abstract
- Signal transduction in response to growth factors is a strictly controlled process with networks of feedback systems, highly selective interactions and finely tuned on-and-off switches. In the context of cancer, detailed signaling studies have resulted in the development of some of the most frequently used means of therapy, with several well established examples such as the small molecule inhibitors imatinib and dasatinib in the treatment of chronic myeloid leukemia. Impaired function of receptor tyrosine kinases is implicated in various types of tumors, and much effort is put into mapping the many interactions and downstream pathways. Here we discuss the hematopoietic growth factor receptors c-Kit and Flt3 and their downstream signaling in normal as well as malignant cells. Both receptors are members of the same family of tyrosine kinases and crucial mediators of stem-and progenitor-cell proliferation and survival in response to ligand stimuli from the surrounding microenvironment. Gain-of-function mutations/alterations render the receptors constitutively and ligand-independently activated, resulting in aberrant signaling which is a crucial driving force in tumorigenesis. Frequently found mutations in c-Kit and Flt3 are point mutations of aspartic acid 816 and 835 respectively, in the activation loop of the kinase domains. Several other point mutations have been identified, but in the case of Flt3, the most common alterations are internal tandem duplications (ITDs) in the juxtamembrane region, reported in approximately 30% of patients with acute myeloid leukemia (AML). During the last couple of years, the increasing understanding of c-Kit and Flt3 signaling has also revealed the complexity of these receptor systems. The impact of gain-of-function mutations of c-Kit and Flt3 in different malignancies is well established and shown to be of clinical relevance in both prognosis and therapy. Many inhibitors of both c-Kit or Flt3 or of their downstream substrates are in clinical trials with encouraging results, and targeted therapy using a combination of such inhibitors is considered a promising approach for future treatments.
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| 10. |
- Masson, Kristina
(författare)
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Studies on the normal and oncogenic signaling networks from the hematopoietic growth factor receptors c-Kit and Flt3
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The closely related receptor tyrosine kinases c-Kit and Flt3 are mainly expressed in hematopoietic cells and are involved in driving cell proliferation and promoting survival in response to growth factor stimulation. Several gain-of-function mutations of both c-Kit and Flt3 have been found in malignancies like acute myeloid leukemia, gastrointestinal stroma tumors and small cell lung carcinomas, and the constitutively activated receptors are very potent oncogenes. We have studied the detailed signal transduction pathways downstream of c-Kit and Flt3, in order to understand the exact mechanism by which processes such as cell growth and differentiation are regulated. We looked into the negative regulation of RTKs by focusing on the E3 ubiquitin ligase Cbl and its interaction with c-Kit, and found that this interaction was dependent on Src family kinases to target the receptor for lysosomal degradation. Src is found to be involved in the development of a variety of tumors and this mechanism is crucial for a balanced signaling. In Flt3, we identified several novel phosphorylation sites involved in Src binding and activation as well as the binding of the adaptor proteins Grb2/Gab2. Upon Y-to-F point mutations of these residues, we investigated the downstream signaling pathways affected and the biological outcomes of this. Moreover, we compared phosphorylation and signaling patterns between the wild-type and the mutated versions of Flt3. The most common mutation in Flt3, an internal tandem duplication (ITD) in the juxtamembrane region of the receptor, is found in about 30% of patients with AML and is correlated to a worse prognosis. The ITD receptor activates the downstream signaling protein Stat5, an important mediator of survival, whereas the wild-type Flt3 does not. We found that this activation of Stat5 to some extent is induced by the function of Gab2 and its recruitment to the Flt3 receptor, and that this as an important pathway for cell viability and proliferation. By using phospho-specific Flt3 antibodies produced in-house, we compared the site-specific phosphorylation and kinetics of wild-type Flt3 with the two most common mutants in AML; D853Y and the ITD. We found both quantitative and qualitative differences, indicating that the oncogenic receptors act through specific signaling pathways, which provides potential therapeutic targets.
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