Tyrosine 842 in the activation loop is required for full transformation by the oncogenic mutant FLT3-ITD
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Julhash Kazi
PROVIDER: E-MTAB-5258 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:FLT3 is the most frequently mutated gene in AML â up to 40% of AML harbor an activating mutation within FLT3 gene. Though AML is a relatively rare disease, such a high mutability rate, as observed with FLT3 gene, is striking. To elucidate the molecular background of this phenomenon, we have established nine unique FLT3/ITD - carrying 32D cell lines and a set of controls, and subjected them to whole genome expression analysis and 2DE LC/MS proteomics. Data obtained on this so far largest set of ITD mutants indicates that in comparison to the wild type FLT3 expressing 32D cells and transduction controls, FLT3/ITD positive cells exhibit less mature expression profiles resembling ST-HSC and MkEP/CMP/LMPP progenitors. We hypothesize that FLT3/ITD might contribute not only to the proliferative advantage of FLT3/ITD positive cells, but also to their reprogramming towards less differentiated stages, thus strengthening their malignant properties. This finding might explain the pronounced mutation rate of the aberrantly expressed FLT3 gene in AML, and, also, the inferior prognosis of FLT3/ITD positive AML patients. Moreover, the microarray data has revealed biological differences among individual ITD variants â a finding supporting the recent clinical data on the prognostic impact of the size of individual ITDs. Keywords: genetic modification Nine stable 32D cell lines harboring unique human FLT3/ITD mutants, two parental 32 cell lines, two 32D stable cell lines harboring cloning vector only, and two 32D cell lines stably expressing human wild type FLT3 were subjected to the microarray analysis.
2010-04-08 | E-GEOD-12766 | biostudies-arrayexpress
Project description:Transcriptional profiling of murine bone marrow c-kit+, Sca-1+ lineage neative (KSL) cells from p21CDKN1a-/- and p21+/+ overexpressing Flt3/ITD. The goal was to determine the effect on global gene expression by loss of p21 in Flt3/ITD transformed KSL cells Internal tandem duplication (ITD) mutations in the Flt3 gene (Flt3-ITD) are associated with poor prognosis in patients with acute myeloid leukemia (AML). Few inhibitors of Flt3-ITD are effective against Flt3-ITD+ AML due to the development of drug-resistance. In this study, we demonstrate that Flt3-ITD activates a novel pathway involving p21Cdkn1a (p21) and pre-B cell leukemia transcription factor 1 (Pbx1) that attenuates Flt3-ITD cell proliferation and is involved in the development drug resistance. Flt3-ITD up-regulated p21 expression in mouse bone marrow c-kit+-Sca-1+-Lin- (KSL) cells and in Ba/F3 cells. Loss of p21 expression enhanced growth factor-independent proliferation and sensitivity to cytarabine as a consequence of enriching the S+G2/M phase population concomitant with a significant increase in the expression of Pbx1, but not Evi-1, in Flt3-ITD+ cells. This enhancement of cell proliferation by loss of p21 was partially abrogated when Pbx1 expression was silenced in Flt3-ITD+ primary bone marrow colony-forming cells (CFCs) and Ba/F3 cells. Antagonizing Flt3-ITD using AC220, a selective inhibitor of Flt3-ITD, decreased the expression of p21, coincident with the up-regulation of Pbx1 mRNA and a rapid decline in the number of viable Flt3-ITD+ Ba/F3 cells, however the cells eventually became refractory to AC220. Overexpressing p21 in Flt3-ITD+ Ba/F3 cells delayed the emergence of cells refractory to AC220, whereas silencing p21 accelerated their development. These data demonstrate that Flt3-ITD is capable of inhibiting the proliferation of Flt3-ITD+ cells through the p21/Pbx1 axis and that antagonizing Flt3-ITD contributes to the subsequent development of cells refractory to Flt3-ITD inhibitor by disrupting p21 expression. biological replicates: 3 KSL cell replicates overexpressing ITD-Flt3 from p21+/+ and p21-/- cells, 1 KSL cell replicate from p21+/+ and p21-/- cells
2015-11-20 | E-GEOD-75200 | biostudies-arrayexpress
Project description:The functional relevance of many microRNAs in the context of tumor biology remains unclear. Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to test the impact of individual microRNAs on the growth of FLT3-ITD positive leukemia cells. This approach identified both evolutionarily conserved and non-conserved human microRNAs that function to suppress or promote tumor cell growth, revealing that microRNAs are extensively integrated into the molecular networks that control tumor cell physiology. Our study describes a powerful genetic approach by which the function of individual microRNAs can be assessed on a global level, and its use will rapidly advance our understanding of how microRNAs contribute to human disease. Loss-of-function CRISPR-Cas9 screen identifies genes whose loss leads to increased or decreased FLT3-ITD+ cell growth over 23 day time-course
2016-07-01 | E-GEOD-71539 | biostudies-arrayexpress
Project description:Internal tandem duplications in the tyrosine kinase receptor FLT3 (FLT3-ITD) are among the most common lesions in acute myeloid leukemia (AML) and there exists a need for new forms of treatment. Using ex vivo drug sensitivity screening, we found that FLT3-ITD+ patient cells are particularly sensitive to HSP90 inhibitors. While it is well known that HSP90 is important for FLT3-ITD stability, we found that HSP90 family members play a much more complex role in FLT3-ITD signaling than previously appreciated. First, we found that FLT3-ITD activates the unfolded protein response (UPR), leading to increased expression of GRP94/HSP90B1. GRP94 rewires FLT3-ITD signaling by binding and retaining FLT3-ITD in the ER, where it aberrantly activates downstream signaling pathways. Second, HSP90 family proteins protect FLT3-ITD+ AML cells against apoptosis by alleviating proteotoxic stress, and treatment with HSP90 inhibitors results in proteotoxic overload that triggers UPR-induced apoptosis. Importantly, leukemic stem cells are strongly dependent upon HSP90 for their survival, and the HSP90 inhibitor ganetespib causes leukemic stem cell exhaustion in mouse PDX models. Taken together, our study reveals a molecular basis for HSP90 addiction of FLT3-ITD+ AML cells and provides a rationale for including HSP90 inhibitors in the treatment regime for FLT3-ITD+ AML.
2020-09-02 | PXD009423 | Pride
Project description:Cytogenetically normal acute myeloid leukemia (CN-AML) represents nearly 50% of human acute myeloid leukemia (AML) cases with a 5-year overall survival of approximately 30%. In CN-AML with poorer prognosis, mutations in the de novo DNA methyltransferase (DNMT3A) and the FMS-like tyrosine kinase 3 (Flt3) commonly co-occur (1-3). We demonstrate that mice with Flt3-internal-tandem duplication (Flt3ITD) and inducible deletion of Dnmt3a spontaneously develop a rapidly-lethal, completely-penetrant, and transplantable AML of normal karyotype. These murine AML retain a single Dnmt3a floxed allele, revealing the oncogenic potential of Dnmt3a haploinsufficiency. FLT3-ITD/DNMT3A-mutant primary human and murine AML demonstrate a similar pattern of global DNA methylation. In the murine model, rescuing DNMT3A expression was accompanied by DNA re-methylation and loss of clonogenic potential, suggesting that Dnmt3a-mutant oncogenic effects are reversible. Differentially methylated genomic regions were associated with changes in the expression of nearby genes. Moreover, dissection of the cellular architecture of the AML model using single-cell RNA-Seq, flow cytometry and colony assays identified clonogenic subpopulations that differentially express genes that are sensitive to the methylation of nearby genomic loci and varied in response to Dnmt3a levels. Thus, Dnmt3a haploinsufficiency transforms Flt3ITD myeloproliferative disease by modulating methylation-sensitive gene expression within a clonogenic AML subpopulation. To identify the gene expression changes associated with Dnmt3a loss of function in human and murine Flt3-ITD and Dnmt3a-mutant AML (Bulk RNA-Seq).
2016-03-27 | E-GEOD-77846 | biostudies-arrayexpress
Project description:Acute Myeloid Leukaemia (AML) carries a 5 year survival rate of just 24%. Toxic chemotherapy regimens remain the backbone of standard of care for AML. The KIT tyrosine kinase is a recognised AML oncogene, associated with poor outcome. We recently identified DNA-PK as a novel therapeutic target in FLT3 mutant AML. The similarity between KIT and FLT3 regulated signalling pathways led us to investigate DNA-PK in KIT-mutant AML.
2023-01-26 | PXD030005 | Pride
Project description:Cytogenetically normal acute myeloid leukemia (CN-AML) represents nearly 50% of human acute myeloid leukemia (AML) cases with a 5-year overall survival of approximately 30%. In CN-AML with poorer prognosis, mutations in the de novo DNA methyltransferase (DNMT3A) and the FMS-like tyrosine kinase 3 (Flt3) commonly co-occur (1-3). We demonstrate that mice with Flt3-internal-tandem duplication (Flt3ITD) and inducible deletion of Dnmt3a spontaneously develop a rapidly-lethal, completely-penetrant, and transplantable AML of normal karyotype. These murine AML retain a single Dnmt3a floxed allele, revealing the oncogenic potential of Dnmt3a haploinsufficiency. FLT3-ITD/DNMT3A-mutant primary human and murine AML demonstrate a similar pattern of global DNA methylation. In the murine model, rescuing DNMT3A expression was accompanied by DNA re-methylation and loss of clonogenic potential, suggesting that Dnmt3a-mutant oncogenic effects are reversible. Differentially methylated genomic regions were associated with changes in the expression of nearby genes. Moreover, dissection of the cellular architecture of the AML model using single-cell RNA-Seq, flow cytometry and colony assays identified clonogenic subpopulations that differentially express genes that are sensitive to the methylation of nearby genomic loci and varied in response to Dnmt3a levels. Thus, Dnmt3a haploinsufficiency transforms Flt3ITD myeloproliferative disease by modulating methylation-sensitive gene expression within a clonogenic AML subpopulation. To identify the gene expression changes associated with Dnmt3a loss of function in human and murine Flt3-ITD and Dnmt3a-mutant AML (Single Cell RNA-Seq).
2016-03-27 | E-GEOD-77847 | biostudies-arrayexpress
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2020-04-27 | PXD013402 | Pride
Project description:Abnormalities in the FLT3 signaling pathway play an integral role in AML disease relapse and drug resistance. Developing new and specific FLT3 tyrosine kinase inhibitors for use in combination to induction therapy is an important step to reduce disease relapse and achieve clinical remission. To develop potent FLT3 TKI requires sensitive in vitro assay that depended on efficient FLT3 artificial substrates, which there are none reported for FLT3 WT and kinase variants. The kinase assay linked with phosphoproteomics was applied as a high throughput technique to increase the known FLT3 kinase substrates (WT, ITD and D835Y) that were used to identify the FLT3 kinase variant’s preferred kinase sequence using the KINATEST-ID substrate predictive pipeline. The identified substrate sequence was used to synthesize and validate pan-FLT3 artificial substrates to monitor in vitro kinase activity in the presence of clinically relevant FLT3 TKI.
2019-01-07 | PXD011478 | Pride