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Results for “internal tandem duplication (ITD)”

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At least 19 recordsLinked to original sources
PubMed2007

A neoepitope generated by an FLT3 internal tandem duplication (FLT3-ITD) is recognized by leukemia-reactive autologous CD8+ T cells.

The FLT3 receptor tyrosine kinase is expressed in more than 90% of acute myelogeneous leukemias (AMLs), up to 30% of which carry an internal tandem duplication (ITD) within the FLT3 gene. Although varying duplication sites exist, most FLT3-ITDs affect a single protein domain. We analyzed the FLT3-ITD of an AML patient for encoding HLA class I-restricted immunogenic peptides. One of the tested peptides (YVDFREYEYY) induced in vitro autologous T-cell responses restricted by HLA-A*0101 that were also detectable ex vivo. These peptide-reactive T cells recognized targets transfected with the patient's FLT3-ITD, but not wild-type FLT3, and recognized the patient's AML cells. Our results demonstrate that AML leukemic blasts can in principle process and present immunogenic FLT3-ITD neoepitopes. Therefore, FLT3-ITD represents a potential candidate target antigen for the immunotherapy of AML.

Amino Acid Sequence↗
PubMed2001

FLT3, RAS, and TP53 mutations in elderly patients with acute myeloid leukemia.

The prevalence and significance of genetic abnormalities in older patients with acute myeloid leukemia (AML) are unknown. Polymerase chain reactions and single-stranded conformational polymorphism analyses were used to examine 140 elderly AML patients enrolled in the Southwest Oncology Group study 9031 for FLT3, RAS, and TP53 mutations, which were found in 34%, 19%, and 9% of patients, respectively. All but one of the FLT3 (46 of 47) mutations were internal tandem duplications (ITDs) within exons 11 and 12. In the remaining case, a novel internal tandem triplication was found in exon 11. FLT3 ITDs were associated with higher white blood cell counts, higher peripheral blast percentages, normal cytogenetics, and less disease resistance. All RAS mutations (28 of 28) were missense point mutations in codons 12, 13, or 61. RAS mutations were associated with lower peripheral blast and bone marrow blast percentages. Only 2 of 47 patients with FLT3 ITDs also had a RAS mutation, indicating a significant negative association between FLT3 and RAS mutations (P =.0013). Most TP53 mutations (11 of 12) were missense point mutations in exons 5 to 8 and were associated with abnormal cytogenetics, especially abnormalities in both chromosomes 5 and 7. FLT3 and RAS mutations were not associated with inferior clinical outcomes, but TP53 mutations were associated with a worse overall survival (median 1 versus 8 months, P =.0007). These results indicate that mutations in FLT3, RAS, or TP53 are common in older patients with AML and are associated with specific AML phenotypes as defined by laboratory values, cytogenetics, and clinical outcomes. (Blood. 2001;97:3589-3595)

Aged↗
PubMed2026

NOTCH3 Internal Tandem Duplication Defines a Novel Oncogenic Activation Mechanism of NOTCH Signaling.

NOTCH signaling is activated in tumors through multiple mechanisms, including mutations, gene rearrangements, and gene amplification. We report a novel activation mechanism, an internal tandem duplication (ITD) near the NOTCH3 negative regulatory region (NRR), found in a myogenic mesenchymal neoplasm. This 17-amino acid residue duplication disrupts the tightly autoinhibited structure surrounding the S2 cleavage site, resulting in ligand-independent S2 cleavage and constitutive pathway activation, as demonstrated by increased expression of the NOTCH3 target gene HES1. Cells expressing NOTCH3-ITD showed increased nuclear localization of the receptor and exhibited malignant phenotypes, including enhanced proliferation and migration. Together, these findings support the oncogenic role of NOTCH3-ITD.

Receptor, Notch3↗
PubMed2004

Evolution of FLT3-ITD and D835 activating point mutations in relapsing acute myeloid leukemia and response to salvage therapy.

Internal tandem duplications (ITDs) of the juxtamembrane region of the FLT3 tyrosine kinase receptor are the most frequent genetic alterations in acute myeloid leukemia (AML). The presence of this mutation has been recognized as an independent poor prognostic factor. In this study, we compared the FLT3 mutational status between diagnosis and subsequent relapses in 31 patients with AML. At diagnosis, seven patients were identified to contain FLT3-ITD mutations and one patient to harbor the D835 mutation. Five patients remained FLT3-ITD positive throughout the disease course (+/+). Three patients lost the FLT3 gene mutation at first (one FLT3-ITD, one D835 mutation), or second relapse (one FLT3-ITD) (+/-). One additional patient lost a small FLT3-ITD positive clone at relapse and at the same time gained an apparently unrelated FLT3-ITD positive clone. One patient without FLT3 mutation at diagnosis relapsed with an FLT3-ITD mutation (-/+). A shorter median duration of first remission (6 months versus 11.5 months) and a higher relapse rate after salvage therapy (e.g. allogeneic peripheral blood stem cell transplantation) resulted in a lower leukemia-free survival in the FLT3 mutated group (11% versus 31%). The loss of a clone with a mutation in the FLT3 gene at relapse did not improve the prognosis.

Acute Disease↗
PubMed2003

An innovative phase I clinical study demonstrates inhibition of FLT3 phosphorylation by SU11248 in acute myeloid leukemia patients.

PURPOSE: Obtaining direct and rapid proof of molecular activity in early clinical trials is critical for optimal clinical development of novel targeted therapies. SU11248 is an oral multitargeted kinase inhibitor with selectivity for fms-related tyrosine kinase 3/Flk2 (FLT3), platelet-derived growth factor receptor alpha/beta, vascular endothelial growth factor receptor 1/2, and KIT receptor tyrosine kinases. FLT3 is a promising candidate for targeted therapy in acute myeloid leukemia (AML), because activating mutations occur in up to 30% of patients. We conducted an innovative single-dose clinical study with a primary objective to demonstrate inhibition of FLT3 phosphorylation by SU11248 in AML. EXPERIMENTAL DESIGN: Twenty-nine AML patients each received a single dose of SU11248, escalated from 50 to 350 mg, in increments of 50 mg and cohorts of three to six patients. FLT3 phosphorylation and plasma pharmacokinetics were evaluated at seven time points over 48 h after SU11248 administration, and FLT3 genotype was determined. Study drug-related adverse events occurred in 31% of patients, mainly grade 1 or 2 diarrhea and nausea, at higher dose levels. RESULTS: Inhibition of FLT3 phosphorylation was apparent in 50% of FLT3-wild-type (WT) patients and in 100% of FLT3-mutant patients. FLT3 internal tandem duplication (ITD) mutants showed increased sensitivity relative to FLT3-WT, consistent with preclinical predictions. The primary end point, strong inhibition of FLT3 phosphorylation in >50% patients, was reached in 200 mg and higher dose cohorts. Downstream signaling pathways were also inhibited; signal transducer and activator of transcription 5 (STAT5) was reduced primarily in internal tandem duplication patients and at late time points in FLT3-WT patients, whereas extracellular signal-regulated kinase (ERK) activity was reduced in the majority of patients, independent of FLT3 inhibition. CONCLUSIONS: This novel translational study bridges preclinical models to the patient setting and provides the first evidence of anti-FLT3 activity in patients. Proof of target inhibition accomplishes a crucial milestone in the development of novel oncology therapeutics.

Administration, Oral↗
PubMed2006

ATRA can enhance apoptosis that is induced by Flt3 tyrosine kinase inhibition in Flt3-ITD positive cells.

Among activating Flt3 mutations that have been shown in 25-30% of acute myeloid leukaemia (AML) Flt3-internal tandem duplication (ITD) mutations are predominant. We investigated the influence of all-trans-retinoic acid (ATRA) and granulocyte colony stimulating factor (G-CSF) for their effects on differentiation and apoptosis in human cell lines with different Flt3 variants (THP-1 versus MV4-11 and MOLM13) dependent on the inhibition of Flt3 tyrosine kinase by the bis(lH-2-indolyl)methanone D-65476. While myeloid differentiation was not observed in both Flt3-ITD cell lines (MV4-11 and MOLM13), we demonstrate an enhanced proapoptotic effect of D-65476 in the presence of ATRA that was restricted to the Flt3-ITD expressing cells. The combined treatment with ATRA and D-65476 also led to a pronounced down-regulation of surviv in on mRNA and protein level in Flt3-ITD but not in Flt3 wildtype expressing cells (THP-1). Surprisingly, there was no differential expression of important proteins like Bcl-X(L), Bcl-2 or Bax that might explain enhanced apoptosis. Furthermore, Akt phosphorylation after stimulation with Flt3 ligand dependent on D-65476 was not affected by pretreatment with ATRA. We suggest that regulation of inhibitors of apoptosis might play a crucial role how ATRA can increase the proapoptotic effect of Flt3 inhibitors in myeloid leukemia cells expressing Flt3-ITD. This effect can potentially be exploited for the treatment of Flt3-ITD positive acute myeloid leukemia.

Apoptosis↗
PubMed2005

Minimal residual disease based on patient specific Flt3-ITD and -ITT mutations in acute myeloid leukemia.

We present our first experiences with determination of minimal residual disease (MRD) based on patient specific Flt3-ITD (internal tandem duplication) mutations. We analysed MRD status of 11 AML patients in a retrospective investigation and its potential impact on the follow up of these patients. In five out of six patients with a positive Flt3-ITD based MRD status a relapse of AML was observed in the follow up while one patient lacks a clinical relapse so far. In contrast, four out of five patients with a negative MRD status remain free of disease. One of these patients relapsed with a switch of FAB subtype including loss of Flt3-ITD mutation. Furthermore, in one patient we could identify a Flt3-ITT (internal tandem triplication mutation).

Adult↗
PubMed2005

AML-associated Flt3 kinase domain mutations show signal transduction differences compared with Flt3 ITD mutations.

Activating mutations of Flt3 are found in approximately one third of patients with acute myeloid leukemia (AML) and are an attractive drug target. Two classes of Flt3 mutations occur: internal tandem duplications (ITDs) in the juxtamembrane and point mutations in the tyrosine kinase domain (TKD). We and others have shown that Flt3-ITD induced aberrant signaling including strong activation of signal transducer and activator of transcription 5 (STAT5) and repression of CCAAT/estradiol-binding protein alpha (c/EBPalpha) and Pu.1. Here, we compared the signaling properties of Flt3-ITD versus Flt3-TKD in myeloid progenitor cells. We demonstrate that Flt3-TKD mutations induced autonomous growth of 32D cells in suspension cultures. However, in contrast to Flt3-ITD and similar to wild-type Flt3 (Flt3-WT), Flt3-TKD cannot support colony formation in semisolid media. Also, in contrast to Flt3-ITD, neither Flt3-WT nor Flt3-TKD induced activation or induction of STAT5 target genes. Flt3-TKD also failed to repress c/EBPalpha and Pu.1. No significant differences were observed in receptor autophosphorylation and the phosphorylation of Erk-1 and -2, Akt, and Shc. Importantly, TKD but not ITD mutations were a log power more sensitive toward the tyrosine kinase inhibitor protein kinase C 412 (PKC412) than Flt3-WT. In conclusion, Flt3-ITD and Flt3-TKD mutations display differences in their signaling properties that could have important implications for their transforming capacity and for the design of mutation-specific therapeutic approaches.

Acute Disease↗
PubMed2006

Newly identified c-KIT receptor tyrosine kinase ITD in childhood AML induces ligand-independent growth and is responsive to a synergistic effect of imatinib and rapamycin.

Activating mutations of c-KIT lead to ligand-independent growth. Internal tandem duplications (ITDs) of exon 11, which encodes the juxtamembrane domain (JMD), are constitutively activating mutations found in 7% of gastrointestinal stromal tumors (GISTs) but have not been described in childhood acute myeloid leukemia (AML). DNA and cDNA from 60 children with AML were screened by polymerase chain reaction (PCR) for mutations of the JMD. A complex ITD (kit cITD) involving exon 11 and exon 12 was identified with a relative frequency of 7% (4/60). The human kit cITDs were inserted into the murine c-Kit backbone and expressed in Ba/F3 cells. KIT cITD induced factorindependent growth and apoptosis resistance, and exhibited constitutive autophosphorylation. KIT cITD constitutively activated the PI3K/AKT pathway and phosphorylated STAT1, STAT3, STAT5, and SHP-2. Imatinib (IM) or rapamycin (Rap) led to complete inhibition of growth, with IC50 values at nanomolar levels. IM and Rap synergistically inhibited growth and surmounted KIT cITD-induced apoptosis resistance. IM but not LY294002 inhibited phosphorylation of STAT3 and STAT5, suggesting aberrant cross talk between PI3K- and STAT-activating pathways. The findings presented may have immediate therapeutic impact for a subgroup of childhood AML-expressing c-KIT mutations.

Acute Disease↗
PubMed2006

Roles of tyrosine 589 and 591 in STAT5 activation and transformation mediated by FLT3-ITD.

Acquired mutations in the FLT3 receptor tyrosine kinase are common in acute myeloid leukemia and result in constitutive activation. The most frequent mechanism of activation is disruption of the juxtamembrane autoregulatory domain by internal tandem duplications (ITDs). FLT3-ITDs confer factor-independent growth to hematopoietic cells and induce a myeloproliferative syndrome in murine bone marrow transplant models. We and others have observed that FLT3-ITD activates STAT5 and its downstream effectors, whereas ligand-stimulated wild-type FLT3 (FLT3WT) does not. In vitro mapping of tyrosine phosphorylation sites in FLT3-ITD identified 2 candidate STAT5 docking sites within the juxtamembrane domain that are disrupted by the ITD. Tyrosine to phenylalanine substitution of residues 589 and 591 in the context of the FLT3-ITD did not affect tyrosine kinase activity, but abrogated STAT5 activation. Furthermore, FLT3-ITD-Y589/591F was incapable of inducing a myeloproliferative phenotype when transduced into primary murine bone marrow cells, whereas FLT3-ITD induced myeloproliferative disease with a median latency of 50 days. Thus, the conformational change in the FLT3 juxtamembrane domain induced by the ITD activates the kinase through dysregulation of autoinhibition and results in qualitative differences in signal transduction through STAT5 that are essential for the transforming potential of FLT3-ITD in vivo.

Animals↗
PubMed2002

Suppression of myeloid transcription factors and induction of STAT response genes by AML-specific Flt3 mutations.

The receptor tyrosine kinase Flt3 is expressed and functionally important in early myeloid progenitor cells and in the majority of acute myeloid leukemia (AML) blasts. Internal tandem duplications (ITDs) in the juxtamembrane domain of the receptor occur in 25% of AML cases. Previously, we have shown that these mutations activate the receptor and induce leukemic transformation. In this study, we performed genome-wide parallel expression analyses of 32Dcl3 cells stably transfected with either wild-type or 3 different ITD isoforms of Flt3. Comparison of microarray expression analyses revealed that 767 of 6586 genes differed in expression between FLT3-WT- and FLT3-ITD-expressing cell lines. The target genes of mutationally activated Flt3 resembled more closely those of the interleukin 3 (IL-3) receptor than those of ligand-activated Flt3. The serine-threonine kinase Pim-2 was up-regulated on the mRNA and the protein level in Flt3-ITD-expressing cells. Further experiments indicated that Pim-2 function was important for clonal growth of 32D cells. Several genes repressed by the mutations were found to be involved in myeloid gene regulation. Pu.1 and C/EBPalpha, both induced by ligand-activation of wild-type Flt3, were suppressed in their expression and function by the Flt3 mutations. In conclusion, internal tandem duplication mutations of Flt3 activate transcriptional programs that partially mimic IL-3 activity. Interestingly, other parts of the transcriptional program involve novel, IL-3-independent pathways that antagonize differentiation-inducing effects of wild-type Flt3. The identification of the transcriptional program induced by ITD mutations should ease the development of specific therapies.

Acute Disease↗
PubMed2004

Suppression of leukemia expressing wild-type or ITD-mutant FLT3 receptor by a fully human anti-FLT3 neutralizing antibody.

FMS-like tyrosine kinase 3 (FLT3), a class III receptor tyrosine kinase, is expressed at high levels in the blasts of approximately 90% of patients with acute myelogenous leukemia (AML). Internal tandem duplications (ITDs) in the juxtamembrane domain and point mutations in the kinase domain of FLT3 are found in approximately 37% of AML patients and are associated with a poor prognosis. We report here the development and characterization of a fully human anti-FLT3 neutralizing antibody (IMC-EB10) isolated from a human Fab phage display library. IMCEB10 (immunoglobulin G1 [IgG1], kappa) binds with high affinity (KD=158 pM) to soluble FLT3 in enzyme-linked immunosorbent assay (ELISA) and to FLT3 receptor expressed on the surfaces of human leukemia cell lines. IMC-EB10 blocks the binding of FLT3 ligand (FL) to soluble FLT3 in ELISA and competes with FL for binding to cell-surface FLT3 receptor. IMC-EB10 treatment inhibits FL-induced phosphorylation of FLT3 in EOL-1 and EM3 leukemia cells and FL-independent constitutive activation of ITD-mutant FLT3 in BaF3-ITD and MV4;11 cells. Activation of the downstream signaling proteins mitogen-activated protein kinase (MAPK) and AKT is also inhibited in these cell lines by antibody treatment. The antibody inhibits FL-stimulated proliferation of EOL-1 cells and ligand-independent proliferation of BaF3-ITD cells. In both EOL-1 xenograft and BaF3-ITD leukemia models, treatment with IMC-EB10 significantly prolongs the survival of leukemia-bearing mice. No overt toxicity is observed with IMC-EB10 treatment. Taken together, these data demonstrate that IMC-EB10 is a specific and potent inhibitor of wild-type and ITD-mutant FLT3 and that it deserves further study for targeted therapy of human AML.

Animals↗
PubMed2005

FLT3-ITD and tyrosine kinase domain mutants induce 2 distinct phenotypes in a murine bone marrow transplantation model.

Activating mutations of the Fms-like tyrosine kinase 3 (FLT3) receptor are the most common genetic alteration in acute myeloid leukemia (AML). Two distinct groups of FLT3 mutations are found: internal tandem duplications (ITDs) of the juxtamembrane region and point mutations within the tyrosine kinase domain (TKD). Recently, point mutations within the activation loop of FLT3 have also been described in childhood acute lymphoblastic leukemia (ALL). FLT3-ITD has been shown to induce a myeloproliferative syndrome in a murine bone marrow transplantation model. The phenotype of FLT3-TKD in mice has not yet been investigated. We transduced murine bone marrow with retrovirus-expressing FLT3-TKD mutants or FLT3-ITD and transplanted these cells into lethally irradiated mice. Mice that received a transplant of FLT3-ITD developed an oligoclonal myeloproliferative disease as previously described. In contrast, FLT3-TKD mutants induced an oligoclonal lymphoid disorder with longer latency and distinct hematologic manifestations: importantly, induction of the lymphoid phenotype was not due to a low number of transplanted cells. The lymphoid manifestation and longer latency of FLT3-TKD compared with FLT3-ITD mutants together with the lack of influence of FLT3-TKD mutations on the clinical outcome of patients with AML suggest differences in cell signaling between FLT3-TKD mutants and FLT3-ITDs. Indeed strong signal transducers and activators of transcription 5 (STAT5) activation could only be demonstrated for FLT3-ITDs.

Animals↗
PubMed2005

FLT3-ITD-, but not BCR/ABL-transformed cells require concurrent Akt/mTor blockage to undergo apoptosis after histone deacetylase inhibitor treatment.

Leukemias are differentially sensitive to histone deacytelase inhibitor (HDI)-induced apoptosis, but molecular reasons for this remain unclear. We here show that BCR/ABL-, but not FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD)-transformed 32D cells or primary acute myeloid leukemia (AML) blasts undergo apoptosis after treatment with the HDI valproic acid (VPA) plus all-trans retinoic acid (VPA/ATRA). A particular VPA/ATRA responsiveness of Philadelphia chromosome-positive (Ph+) acute lymphatic leukemia (ALL) was confirmed in a therapy-refractory patient in vivo. HDI-stimulated apoptosis in Ph+ cells was caspase dependent, but independent from Akt pathway inhibition. Conversely, separate blockage of the Akt/mTor-signaling pathway was a prerequisite for overcoming apoptosis resistance to VPA/ATRA in FLT3-ITD cells, and primary AML blasts (n = 9). In conclusion, constitutive Akt activation causes apoptosis resistance to VPA/ATRA in AML, but not in Ph+ leukemia. This warrants the application of HDI-based therapies in poor-risk Ph+ ALL, and the use of Akt/mTor inhibitors to overcome HDI resistance in AML.

Antineoplastic Agents↗
PubMedDate not supplied

FLT3 overexpression in acute promyelocytic leukemia patients without detectable FLT3-ITD or codon 835-836 mutations: a pilot study.

BACKGROUND: Activating mutations of the FLT3 receptor tyrosine kinase are common in acute promyelocytic leukemia (APL) but have uncertain prognostic significance. Information regarding FLT3 expression levels in APL without FLT3 mutations is lacking. MATERIALS AND METHODS: Using RT-PCR, mutation analysis of the FLT3 gene, regarding internal tandem duplications (ITDs) and codon 835-836 point mutations, was performed and real-time PCR was carried out to determine the level of FLT3 expression in 11 APL patients at diagnosis and 5 in haematological remission with molecularly detectable disease. RESULTS: High levels of FLT3 transcript, at least a 10-fold increase compared to the normal controls, were found at diagnosis in all 3 mutated cases and in 2 patients without detectable FLT3 mutations. CONCLUSION: FLT3 overexpression can be documented in patients without FLT3 mutations. These patients might benefit from treatment using specific FLT3 tyrosine kinase inhibitors. Larger studies are needed to evaluate the clinical and biological significance of FLT3 overexpression in the absence of FLT3 mutations.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2006

Targeting PIM kinases impairs survival of hematopoietic cells transformed by kinase inhibitor-sensitive and kinase inhibitor-resistant forms of Fms-like tyrosine kinase 3 and BCR/ABL.

Previous studies have shown that activation of the signal transducer and activator of transcription 5 (STAT5) plays an essential role in leukemogenesis mediated through constitutive activated protein tyrosine kinases (PTK). Because PIM-1 is a STAT5 target gene, we analyzed the role of the family of PIM serine/threonine kinases (PIM-1 to PIM-3) in PTK-mediated transformation of hematopoietic cells. Ba/F3 cells transformed to growth factor independence by various oncogenic PTKs (TEL/JAK2, TEL/TRKC, TEL/ABL, BCR/ABL, FLT3-ITD, and H4/PDGFbetaR) show abundant expression of PIM-1 and PIM-2. Suppression of PIM-1 activity had a negligible effect on transformation. In contrast, expression of kinase-dead PIM-2 mutant (PIM-2KD) led to a rapid decline of survival in Ba/F3 cells transformed by FLT3-ITD but not by other oncogenic PTKs tested. Coexpression of PIM-1KD and PIM-2KD abrogated growth factor-independent growth of Ba/F3 transformed by several PTKs, including BCR/ABL. Targeted down-regulation of PIM-2 by RNA interference (RNAi) selectively abrogated survival of Ba/F3 cells transformed by various Fms-like tyrosine kinase 3 (FLT3)-activating mutants [internal tandem duplication (ITD) and kinase domain] and attenuated growth of human cell lines containing FLT3 mutations. Interestingly, cells transformed by FLT3 and BCR/ABL mutations that confer resistance to small-molecule tyrosine kinase inhibitors were still sensitive to knockdown of PIM-2, or PIM-1 and PIM-2 by RNAi. Our observations indicate that combined inactivation of PIM-1 and PIM-2 interferes with oncogenic PTKs and suggest that PIMs are alternative therapeutic targets in PTK-mediated leukemia. Targeting the PIM kinase family could provide a new avenue to overcome resistance against small-molecule tyrosine kinase inhibitors.

Animals↗
PubMed2003

Mutations in the tyrosine kinase domain of FLT3 define a new molecular mechanism of acquired drug resistance to PTK inhibitors in FLT3-ITD-transformed hematopoietic cells.

Activating mutations in the juxtamembrane domain (FLT3-length mutations, FLT3-LM) and in the protein tyrosine kinase domain (TKD) of FLT3 (FLT3-TKD) represent the most frequent genetic alterations in acute myeloid leukemia (AML) and define a molecular target for therapeutic interventions by protein tyrosine kinase (PTK) inhibitors. We could show that distinct activating FLT3-TKD mutations at position D835 mediate primary resistance to FLT3 PTK inhibitors in FLT3-transformed cell lines. In the presence of increasing concentrations of the FLT3 PTK inhibitor SU5614, we generated inhibitor resistant Ba/F3 FLT3-internal tandem duplication (ITD) cell lines (Ba/F3 FLT3-ITD-R1-R4) that were characterized by a 7- to 26-fold higher IC50 (concentration that inhibits 50%) to SU5614 compared with the parental ITD cells. The molecular characterization of ITD-R1-4 cells demonstrated that specific TKD mutations (D835N and Y842H) on the ITD background were acquired during selection with SU5614. Introduction of these dual ITD-TKD, but not single D835N or Y842H FLT3 mutants, in Ba/F3 cells restored the FLT3 inhibitor resistant phenotype. Our data show that preexisting or acquired mutations in the PTK domain of FLT3 can induce drug resistance to FLT3 PTK inhibitors in vitro. These findings provide a molecular basis for the evaluation of clinical resistance to FLT3 PTK inhibitors in patients with AML.

Acute Disease↗
PubMed2002

PML/RARalpha and FLT3-ITD induce an APL-like disease in a mouse model.

Acute promyelocytic leukemia (APL) cells invariably express aberrant fusion proteins involving the retinoic acid receptor alpha (RARalpha). The most common fusion partner is promyelocytic leukemia protein (PML), which is fused to RARalpha in the balanced reciprocal chromosomal translocation, t(15;17)(q22:q11). Expression of PML/RARalpha from the cathepsin G promoter in transgenic mice causes a nonfatal myeloproliferative syndrome in all mice; about 15% go on to develop APL after a long latent period, suggesting that additional mutations are required for the development of APL. A candidate target gene for a second mutation is FLT3, because it is mutated in approximately 40% of human APL cases. Activating mutations in FLT3, including internal tandem duplication (ITD) in the juxtamembrane domain, transform hematopoietic cell lines to factor independent growth. FLT3-ITDs also induce a myeloproliferative disease in a murine bone marrow transplant model, but are not sufficient to cause AML. Here, we test the hypothesis that PML/RARalpha can cooperate with FLT3-ITD to induce an APL-like disease in the mouse. Retroviral transduction of FLT3-ITD into bone marrow cells obtained from PML/RARalpha transgenic mice results in a short latency APL-like disease with complete penetrance. This disease resembles the APL-like disease that occurs with long latency in the PML/RARalpha transgenics, suggesting that activating mutations in FLT3 can functionally substitute for the additional mutations that occur during mouse APL progression. The leukemia is transplantable to secondary recipients and is ATRA responsive. These observations document cooperation between PML/RARalpha and FLT3-ITD in development of the murine APL phenotype.

Animals↗