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FLT3-JM-PMs induce IL-3-independent growth in Ba/F3 cells and hyperproliferation in response to FL. (A) Ba/F3 cells stably transduced with FLT3-WT, FLT3-ITD constructs (W51, NPOS, W78), FLT3-TKD constructs (D835Y, D835V) or one of the FLT3-JM-PM mutants (FLT3-V592A, FLT3-V579A, FLT3F594L, and FLT3-F590GY591D) were seeded at a density of 4 10 4 cells/mL in the absence or presence of IL-3. Viable cells were counted after 72 hours. The growth of cells in the presence of IL-3 was defined as 100% (control). All FLT3-JM-PM mutants showed a significantly higher proliferation rate compared with FLT3-WT (*P .05), although not as high as FLT3-ITD or FLT3-TKD. SD is indicated. (B) FLT3-WT and mutantexpressing Ba/F3 cells were seeded at a density of 4 10 4 cells/mL in the absence or presence of human recombinant FL (50 ng/mL). Viable cells were counted after 72 hours by trypan blue exclusion. The cell number of FLT3-WT cells after 72 hours was defined as 100%. SEM is indicated.
Source publication
In acute myeloid leukemia (AML), two clusters of activating mutations are known in the FMS-like tyrosine kinase-3 (FLT3) gene: FLT3-internal tandem duplications (FLT3-ITDs) in the juxtamembrane (JM) domain in 20% to 25% of patients, and FLT3 point mutations in the tyrosine-kinase domain (FLT3-TKD) in 7% to 10% of patients, respectively. Here, we ha...
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Fms-like tyrosine kinase 3 (FLT3) receptor mutations as internal tandem duplication (ITD) or within the kinase domain are detected in up to 35% of patients with acute myeloid leukemia (AML). N-benzoyl staurosporine (PKC412), a highly effective inhibitor of mutated FLT3 receptors, has significant antileukemic efficacy in patients with FLT3-mutated A...
Citations
... Primers, including divergent primers for rt-circRNAs, are listed in Supplementary Table 1. [27]. Expression of FLT3 was confirmed by RT-qPCR and Western blot as described elsewhere [28]. ...
... In addition, missense mutations were identified either close or within the JXM domain, including Y579A, V592A, V592D and K663R. Alterations at these positions were demonstrated to be activating mutations possibly reducing the stability of the inhibitory conformation of the JXM domain, making it more accessible for autophosphorylation [27,38,41]. Moreover, 2 unstudied mutations were also identified, Y589D and G846D, which were selected for further in vitro studies due to their recurrence in childhood ALL (Supplementary Table 3) and their location at key positions along FLT3 receptor. ...
Background
Alterations of FLT3 are among the most common driver events in acute leukaemia with important clinical implications, since it allows patient classification into prognostic groups and the possibility of personalising therapy thanks to the availability of FLT3 inhibitors. Most of the knowledge on FLT3 implications comes from the study of acute myeloid leukaemia and so far, few studies have been performed in other leukaemias.
Methods
A comprehensive genomic (DNA-seq in 267 patients) and transcriptomic (RNA-seq in 160 patients) analysis of FLT3 in 342 childhood acute lymphoblastic leukaemia (ALL) patients was performed. Mutations were functionally characterised by in vitro experiments.
Results
Point mutations (PM) and internal tandem duplications (ITD) were detected in 4.3% and 2.7% of the patients, respectively. A new activating mutation of the TKD, G846D, conferred oncogenic properties and sorafenib resistance. Moreover, a novel alteration involving the circularisation of read-through transcripts (rt-circRNAs) was observed in 10% of the cases. Patients presenting FLT3 alterations exhibited higher levels of the receptor. In addition, patients with ZNF384 - and MLL/KMT2A -rearranged ALL, as well as hyperdiploid subtype, overexpressed FLT3.
Discussion
Our results suggest that specific ALL subgroups may also benefit from a deeper understanding of the biology of FLT3 alterations and their clinical implications.
... These non-ITD mutations, with clinical and biological features similar to FLT3-ITD variants, were identified in ~2% of all AML cases but their role still remains controversial. 2 However, the precise characterization of these mutations is crucial to expand the potential use of TKI in these cases. Herein, we studied a case harbouring a FLT3 deletion/ insertion to functionally characterize its impact on FLT3 signalling and explore TKI vulnerabilities. ...
... The off-target mutation mechanisms are represented by the stimulation of FLT3 downstream signaling pathways, such as the JAK/STAT5 or PI3K/ AKT/mTOR pathway in mutated FLT3 AML cells, and like these cascades resulted in lessening the cytotoxic efficacy of FLT3 inhibitors (such as Midostaurin, Sorafenib, and Quizartinib) on AML cell lines [22,23]. Also, off-target mutations include specific mutations such as TET2, RAS, and IDH1/2, activation of SYK and AXL (like Midostaurin and Quizartinib), upregulation of Pim and FLT3 ligand (FL) (such as Lestaurtinib, Midostaurin, Sorafenib, and Quizartinib), and preservation of FLT3-ITD AML cells in bone marrow microenvironment (like Quizartinib, Sorafenib, and Gilteritinib) [24][25][26][27][28]. About 30% of recently identified AML cases were recently accompanied by FLT3 mutations [10]. ...
... Mutations in both FLT3-ITD and tyrosine kinase regions could facilitate the activation of FLT3 kinase in the absence of FL and consequently promote cell propagation and survival, resulting in AML. The phenomenon of point mutations' incidence in the FLT3 juxtamembrane region (FLT3-JM-PMs) is another mode of FLT3 mutations, which affords lower levels of the receptor autophosphorylation and its down proceeding mediator STAT5 in comparison with FLT3-ITD and FLT3 tyrosine region mutations [28]. ...
Fms-like tyrosine kinase 3 (FLT3) mutation mechanisms are among the most common genetic abnormalities detected in about 30% of acute myeloid leukemia (AML) patients. These mutations are accompanied by poor clinical response, although all these progressions in identifying and interpreting biological AML bio-targets. Several small structured FLT3 inhibitors have been ameliorated to struggle against AML. Despite all these developments regarding these inhibitors, the Overall survival rate is about five years or more in less than one-third of diagnosed AML patients. Midostaurin was the first FDA-approved FLT3 inhibitor in 2017 in the United States and Europe for AML remedy. Next, Gilteritinib was an FDA-approved FLT3 inhibitor in 2018 and in the next year, Quizartinib was approved an as FLT3 inhibitor in Japan. Interestingly, indole-based motifs had risen as advantaged scaffolds with unusual multiple kinase inhibitory activity. This review summarises indole-based FLT3 inhibitors and related scaffolds, including FDA-approved drugs, clinical candidates, and other bioactive compounds. Furthermore, their chemotypes, mechanism of action, and interaction mode over both wild and mutated FLT3 target proteins had been judgmentally discussed. Therefore, this review could offer inspiring future perspectives into the finding of new FLT3-related AML therapies.
... 24 Subsequently, additional activating mutations at positions N676, N841 and Y842 and some point mutations in the JMD were also identified because of expanded detection scopes or more accurate PCR product detection. 18,19,25,26 Frohling et al. identified 12 non-canonical variants of FLT3 in 222 AML patients who were present at not only JMD and TKD1/2 but also extra-cellular and transmembrane domains using high-throughput DNA sequence analysis of the entire coding region. 17 Furthermore, the introduction of NGS allowing more sensitive detection in blood disease research and diagnostic laboratories in recent years identified an increased amount of FLT3 non-canonical mutations provided insights into the FLT3 mutation landscape. ...
Fms‐like tyrosine kinase 3 (FLT3) is frequently mutated in haematological malignancies. Although canonical FLT3 mutations including internal tandem duplications (ITDs) and tyrosine kinase domains (TKDs) have been extensively studied, little is known about the clinical significance of non‐canonical FLT3 mutations. Here, we first profiled the spectrum of FLT3 mutations in 869 consecutively newly diagnosed acute myeloid leukaemia (AML), myelodysplastic syndrome and acute lymphoblastic leukaemia patients. Our results showed four types of non‐canonical FLT3 mutations depending on the affected protein structure: namely non‐canonical point mutations (NCPMs) (19.2%), deletion (0.7%), frameshift (0.8%) and ITD outside the juxtamembrane domain (JMD) and TKD1 regions (0.5%). Furthermore, we found that the survival of patients with high‐frequency (>1%) FLT3‐NCPM in AML was comparable to those with canonical TKD. In vitro studies using seven representative FLT3‐deletion or frameshift mutant constructs showed that the deletion mutants of TKD1 and the FLT3‐ITD mutant of TKD2 had significantly higher kinase activity than wild‐type FLT3, whereas the deletion mutants of JMD had phosphorylation levels comparable with wild‐type FLT3. All tested deletion mutations and ITD were sensitive to AC220 and sorafenib. Collectively, these data enrich our understanding of FLT3 non‐canonical mutations in haematological malignancies. Our results may also facilitate prognostic stratification and targeted therapy of AML with FLT3 non‐canonical mutations.
... 22,23 Rarer point mutations and smaller insertions/deletions have also been identified within the TKD and other domains (extracellular and JM domains). 21,[24][25][26] All these point mutations result in loss of auto-inhibition and constitutive activation of downstream proliferative signaling cascades ( Figure 1). ...
FMS-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in acute myeloid leukemia (AML). Approximately 30% of the adult cases harbor an internal tandem duplication (FLT3-ITD) and 5–10% a tyrosine kinase domain (TKD) amino acid substitution (FLT3-TKD). The treatment paradigm of AML patients harboring FLT3 mutations (30%) has been modified by the discovery of tyrosine kinase inhibitors. First- and second-generation inhibitors classify FLT3 inhibitors according to FLT3 specificity: first-generation FLT3 inhibitors include sorafenib and midostaurin and second-generation inhibitors are represented by quizartinib, gilteritinib and crenolanib, among others. Activity of these inhibitors depends on their mechanism of receptor binding (active vs inactive conformation) and efficacy against the FLT3-ITD and -TKD mutations (type 1 inhibitors are active both on FLT3-ITD and TKD, whereas type 2 inhibitors are active only on FLT3-ITD). The FLT3 inhibitors sorafenib, midostaurin, quizartinib and gilteritinib have been tested in monotherapy in several settings including refractory or relapsed AML (R/R AML), post-transplant maintenance as well as in combination with intensive chemotherapy (ICT) or non-intensity regimens. The results of published randomized studies support the use of sorafenib in a post-transplant setting (SORMAIN trial), midostaurin in combination with ICT based (RATIFY trial) and gilteritinib for R/R AML (ADMIRAL trial). Gilteritinib in combination with hypomethylating agent as well as quizartinib are not supported by solid randomized trial results for their use in FLT3-mutated AML patients.
... In addition to typical ITD mutations, previous reports indicated the presence of rare non-ITD mutations (small deletions and point mutations) in the FLT3 JMD of AML patients (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). However, due to the low frequency of these alterations, there are so far only casuistic reports available, with limited information on molecular and clinical associations. ...
... Compared to AML, higher rates (~4-fold) of JMD non-ITD mutations were previously detected in patients with acute lymphoblastic leukemia (ALL) (8). Among non-ITD mutations in our cohort, the point mutations Y572C, V592G (9), L576Q (10), G583S (8), Y591H (11), and V592A (12,13) as well as the deletion EY598_599del (14) were previously recognized as gain-of-function mutations that result in constitutive kinase activation and stimulate AML growth through aberrant STAT5 signaling (9,12,14). In addition, in one patient we identified a novel and likely damaging (PolyPhen score = 1) point mutation at Y599N (30% VAF), not previously reported in the literature. ...
... Compared to AML, higher rates (~4-fold) of JMD non-ITD mutations were previously detected in patients with acute lymphoblastic leukemia (ALL) (8). Among non-ITD mutations in our cohort, the point mutations Y572C, V592G (9), L576Q (10), G583S (8), Y591H (11), and V592A (12,13) as well as the deletion EY598_599del (14) were previously recognized as gain-of-function mutations that result in constitutive kinase activation and stimulate AML growth through aberrant STAT5 signaling (9,12,14). In addition, in one patient we identified a novel and likely damaging (PolyPhen score = 1) point mutation at Y599N (30% VAF), not previously reported in the literature. ...
FLT3-ITD mutations are common druggable alterations in patients with acute myeloid leukemia (AML) and associated with poor prognosis. Beside typical ITD mutations, point mutations and deletions in the juxtamembrane domain (JMD) have been observed. However, due to the low frequency of these alterations, there is only limited information on molecular and clinical associations. To evaluate the prognostic impact of non-ITD mutations in the FLT3 JMD region, we analyzed a large cohort of 1,539 adult AML patients treated in different protocols of the Study Alliance Leukemia, using next-generation sequencing. Non-ITD point mutations and deletions within the FLT3 JMD were identified with a prevalence of ~1.23% (n = 19). Both FLT3-ITD and non-ITD mutations were associated with a higher rate of NPM1 (42%–61%; p < 0.001) and DNMT3A mutations (37%–43%; p < 0.001), as well as an increased percentage of peripheral blood (54%–65%) and bone marrow blast cells (74%; p < 0.001), compared to FLT3-wild-type patients. Most significantly, AML patients with FLT3 non-ITD mutations had a higher rate of concomitant KMT2A-PTD mutations (37.5%; p < 0.001) as compared to FLT3-ITD (7%) or FLT3-wild-type cases (4.5%). In a multivariable analysis, FLT3 non-ITD mutations were not an independent prognostic factor. However, patients with dual FLT3 non-ITD and KMT2A-PTD mutations showed a trend for inferior outcome, which points at a functional interaction in this subset of AML.
... The most common codon affected by point mutation is D835, located near the activation loop, which can undergo a variety of mutations, including D835V, H, E, and N, all of which lead to FLT3 activation. The other less common activating point mutations in FLT3 include S451F, Y572C, V579A, F590G, Y591D, V592A, V592G, F594L, K663Q, N676K, R834Q, N841I, N841K and Y842C ( Figure 3) [40,[66][67][68][69][70][71][72]. The type of mutation influences the downstream signalling pathways that are activated. ...
Acute myeloid leukemia (AML) is a highly heterogeneous malignancy characterized by the clonal expansion of myeloid stem and progenitor cells in the bone marrow, peripheral blood, and other tissues. AML results from the acquisition of gene mutations or chromosomal abnormalities that induce proliferation or block differentiation of hematopoietic progenitors. A combination of cytogenetic profiling and gene mutation analyses are essential for the proper diagnosis, classification, prognosis, and treatment of AML. In the present review, we provide a summary of genomic abnormalities in AML that have emerged as both markers of disease and therapeutic targets. We discuss the abnormalities of RARA, FLT3, BCL2, IDH1, and IDH2, their significance as therapeutic targets in AML, and how various mechanisms cause resistance to the currently FDA-approved inhibitors. We also discuss the limitations of current genomic approaches for producing a comprehensive picture of the activated signaling pathways at diagnosis or at relapse in AML patients, and how innovative technologies combining genomic and functional methods will improve the discovery of novel therapeutic targets in AML. The ultimate goal is to optimize a personalized medicine approach for AML patients and possibly those with other types of cancers.
... All classes of mutations (missense, deletion, insertions, and nonsense) have been identified (Table 1); however, there are limited functional data for the JMD mutations, especially the deletions. 12,[14][15][16][17][18][19][20][21][22] Existing functional data suggest that any changes in the wild-type JMD sequence are activating, similar to effects of the JMD-lengthening FLT-3/ITD mutations. Pharmacologic studies suggest that targeting of the other classes of JMDaltering mutations with small-molecule inhibitors may be possible, but the pattern of sensitivity to inhibition varies. ...
The FMS-like tyrosine kinase 3 (FLT-3) is the most frequently mutated gene in acute myeloid leukemia (AML), a high-risk feature, and now the target of tyrosine kinase inhibitors (TKIs), which are approved and in development. The most common mutation is the internal tandem duplication (ITD). We present a novel mutation, FLT-3/Q575Δ, identified in a patient with AML through next-generation sequencing (NGS). This mutation is activating, drives downstream signaling comparable to FLT-3/ITD, and can be targeted using available FLT-3 TKIs. We present the results of a systematic analysis that identified Y572Δ, E573Δ, and S574Δ as similarly activating and targetable deletions located in the FLT-3 juxtamembrane domain (JMD). These mutations target key residues in the JMD involved in the interactions within FLT-3 that regulate its activation. Our results suggest a new class of FLT-3 mutations that may have an impact on patient care and highlight the increasing importance of a systematic understanding of FLT-3 mutations other than ITD. It is likely that, as NGS becomes more commonly used in the diagnosis of patients with AML, these and other activating mutations will be discovered with increasing frequency.
... Both these genetic alterations are gain of function mutations, leading to constitutive FLT3 activation and, consequently, aberrant cell proliferation. ITD mutations represent the most common type of FLT3 mutations with a percentage of about 25% of all AML cases [11], while point mutations are identified in 2% to 10% of all AML patients and are localized in the JMD or in the TKDs domains [18][19][20]. Among the different point mutations, the Asp 835 residue in the activation loop is the predominant FLT3 genetic alteration. ...
... In Table 2, we have summarized the target specificity of FLT3 inhibitors which are currently under evaluation in clinical trials. [19,27]). Yellow dots indicate point mutations, whereas amino acid residues in red indicate the residues wherein ITD mutations are frequently located. ...
FLT3 mutations are the most frequently identified genetic alterations in acute myeloid leukemia (AML) and are associated with poor clinical outcome, relapse and chemotherapeutic resistance. Elucidating the molecular mechanisms underlying FLT3-dependent pathogenesis and drug resistance is a crucial goal of biomedical research. Given the complexity and intricacy of protein signaling networks, deciphering the molecular basis of FLT3-driven drug resistance requires a systems approach. Here we discuss how the recent advances in mass spectrometry (MS)-based (phospho) proteomics and multiparametric analysis accompanied by emerging computational approaches offer a platform to obtain and systematically analyze cell-specific signaling networks and to identify new potential therapeutic targets.
... Known potential mechanisms of TKI resistance include up-regulation of FLT3L or FLT3 expression, activation of parallel pro-survival pathways, modulation of antiapoptotic pathways, and additional FLT3 point mutations 43 . Different juxtamembrane (JM) or activation loop mutations in the FLT3 gene result in varying levels of constitutive FLT3 kinase activity, thus driving diverse levels of persistent signaling by the STAT5, PI3K, and MAPK pathways 38,44,45 . To deter resistance mutations, second-generation TKIs effective against both JM and activation loop mutations such as crenolanib, quizartinib, and gilteritinib are currently being tested in clinical trials, and gilteritinib has recently received FDA approval for treatment of relapsed FLT3 mutant AML 11,46,47 . ...
Internal tandem duplication (-ITD) mutations of Fms-like tyrosine kinase 3 (FLT3) provide growth and pro-survival signals in the context of established driver mutations in FLT3 mutant acute myeloid leukemia (AML). Maternal embryonic leucine zipper kinase (MELK) is an aberrantly expressed gene identified as a target in AML. The MELK inhibitor OTS167 induces cell death in AML including cells with FLT3 mutations, yet the role of MELK and mechanisms of OTS167 function are not understood. OTS167 alone or in combination with tyrosine kinase inhibitors (TKIs) were used to investigate the effect of OTS167 on FLT3 signaling and expression in human FLT3 mutant AML cell lines and primary cells. We describe a mechanism whereby OTS167 blocks FLT3 expression by blocking FLT3 translation and inhibiting phosphorylation of eukaryotic initiation factor 4E–binding protein 1 (4E-BP1) and eukaryotic translation initiation factor 4B (eIF4B). OTS167 in combination with TKIs results in synergistic induction of FLT3 mutant cell death in FLT3 mutant cell lines and prolonged survival in a FLT3 mutant AML xenograft mouse model. Our findings suggest signaling through MELK is necessary for the translation and expression of FLT3-ITD, and blocking MELK with OTS167 represents a viable therapeutic strategy for patients with FLT3 mutant AML.
