小分子 Fms 样酪氨酸激酶 3 抑制剂的研究进展

Fms 样酪氨酸激酶 3（FLT3）是一种重要的Ⅲ型受体酪氨酸激酶,对造血细胞和淋巴细胞的增殖起关键作用,其突变以及过度表 达是造成多种恶性肿瘤的关键因素。通过外源性抑制剂阻断细胞增殖信号的传导来促使肿瘤细胞凋亡是当前治疗肿瘤的重要手段。FLT3 小 分子抑制剂作为一类重要的外源性受体酪氨酸激酶抑制剂已应用于多种恶性肿瘤的治疗并引起广泛关注。综述近 5 年来 FLT3 小分子抑制剂 的研究进展。     

The juxtamembrane domain in ETV6/FLT3 is critical for PIM-1 up-regulation and cell proliferation

We recently reported that the ETV6/FLT3 fusion protein conferred interleukin-3-independent growth on Ba/F3 cells. The present study has been conducted to assess role of the juxtamembrane domain of FLT3 for signal transduction and cell transformation. The wild-type ETV6/FLT3 fusion protein in transfected cells was a constitutively activated tyrosine kinase that led to up-regulation of PIM-1 and activations of STAT5, AKT, and MAPK. Deletion of the juxtamembrane domain abrogated interleukin-3-independent growth of the transfected cells and PIM-1 up-regulation, whereas it retained compatible levels of phosphorylations of STAT5, AKT, and MAPK. Further deletion of N-terminal region of the tyrosine kinase I domain of FLT3 completely abolished these phosphorylations. Our data indicate that the juxtamembrane domain of FLT3 in ETV6/FLT3 fusion protein is critical for cell proliferation and PIM-1 up-regulation that might be independent of a requirement for signaling through STAT5, MAPK, and AKT pathways.     

MiR-424 and miR-155 deregulated expression in cytogenetically normal acute myeloid leukaemia: correlation with NPM1 and FLT3 mutation status

Background

Gain-of-function mutations of tyrosine kinase FLT3 are frequently found in acute myeloid leukemia (AML). This has made FLT3 an important marker for disease diagnosis and a highly attractive target for therapeutic drug development. This study is intended to generate a sensitive substrate for assays of the FLT3 enzymatic activity.

Methods

We expressed in Escherichia coli cells a glutathione S-transferase (GST) fusion protein designated GST-FLT3S, which contains a peptide sequence derived from an autophosphorylation site of FLT3. The protein was used to analyze tyrosine kinase activity of baculovirus-expressed FLT3 and crude cell extracts of bone marrow cells from AML patients. It was also employed to perform FLT3 kinase assays for FLT3 inhibitor screening.

Results

GST-FLT3S in solution or on beads was strongly phosphorylated by recombinant proteins carrying the catalytic domain of wild type FLT3 and FLT3D835 mutants, with the latter exhibiting much higher activity and efficiency. GST-FLT3S was also able to detect elevated tyrosine kinase activity in bone marrow cell extracts from AML patients. A small-scale inhibitor screening led to identification of several potent inhibitors of wild type and mutant forms of FLT3.

Conclusions

GST-FLT3S is a sensitive protein substrate for FLT3 assays. It may find applications in diagnosis of diseases related to abnormal FLT3 activity and in inhibitor screening for drug development.     

Crystal Structure of the FLT3 Kinase Domain Bound to the Inhibitor Quizartinib (AC220)

More than 30% of acute myeloid leukemia (AML) patients possess activating mutations in the receptor tyrosine kinase FMS-like tyrosine kinase 3 or FLT3. A small-molecule inhibitor of FLT3 (known as quizartinib or AC220) that is currently in clinical trials appears promising for the treatment of AML. Here, we report the co-crystal structure of the kinase domain of FLT3 in complex with quizartinib. FLT3 with quizartinib bound adopts an “Abl-like” inactive conformation with the activation loop stabilized in the “DFG-out” orientation and folded back onto the kinase domain. This conformation is similar to that observed for the uncomplexed intracellular domain of FLT3 as well as for related receptor tyrosine kinases, except for a localized induced fit in the activation loop. The co-crystal structure reveals the interactions between quizartinib and the active site of FLT3 that are key for achieving its high potency against both wild-type FLT3 as well as a FLT3 variant observed in many AML patients. This co-complex further provides a structural rationale for quizartinib-resistance mutations.     

Over-expression of FoxM1 is associated with adverse prognosis and FLT3-ITD in acute myeloid leukemia

Forkhead box M1 (FoxM1) drives cell cycle progression and the prevention of growth arrest and is over-expressed in many human malignancies. However, the characteristics of FoxM1 in acute myeloid leukemia (AML) are not clearly understood. We investigated the expression level of FoxM1 and analyzed the correlation of FoxM1 expression with AML patient characteristics and prognoses. Changes in FoxM1 expression were detected after MV4–11 cells, which have an internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 gene (FLT3-ITD), and control THP1 cells (encoding wild-type FLT3) were treated with the FLT3 receptor tyrosine kinase inhibitor AC220 (quizartinib) or FLT3 ligand (FL). Finally, we determined the apoptosis rates after the addition of the FoxM1 inhibitor thiostrepton (TST) to AML cells with or without FLT3-ITD. The expression of FoxM1 in AML patients was correlated with the presence of FLT3-ITD, genetic groups, and possibly overall survival. Inhibition of FLT3-ITD by AC220 down-regulated FoxM1 expression in MV4–11 cells, and stimulation of FLT3 by FL up-regulated FoxM1 expression in MV4–11 and THP1 cells. TST induced the apoptosis of MV4–11 and THP1 cells in a dose-dependent manner. Thus, FoxM1 is a potential prognostic marker and a promising therapeutic target in AML.     

Identification of 2-acylaminothiophene-3-carboxamides as potent inhibitors of FLT3

A series of 2-acylaminothiophene-3-carboxamides has been identified which exhibit potent inhibitory activity against the FLT3 tyrosine kinase. Compound 44 inhibits the isolated enzyme (IC50 = 0.027 microM) and blocks the proliferation of MV4-11 cells (IC50 = 0.41 microM). Structure-activity relationship studies within this series are described in the context of a proposed binding model within the ATP binding site of the enzyme.     

The combination of FLT3 and SYK kinase inhibitors is toxic to leukaemia cells with CBL mutations

Mutations in the E3 ubiquitin ligase CBL, found in several myeloid neoplasms, lead to decreased ubiquitin ligase activity. In murine systems, these mutations are associated with cytokine‐independent proliferation, thought to result from the activation of hematopoietic growth receptors, including FLT3 and KIT. Using cell lines and primary patient cells, we compared the activity of a panel of FLT3 inhibitors currently being used or tested in AML patients and also evaluated the effects of inhibition of the non‐receptor tyrosine kinase, SYK. We show that FLT3 inhibitors ranging from promiscuous to highly targeted are potent inhibitors of growth of leukaemia cells expressing mutant CBL in vitro, and we demonstrate in vivo efficacy of midostaurin using mouse models of mutant CBL. Potentiation of effects of targeted FLT3 inhibition by SYK inhibition has been demonstrated in models of mutant FLT3‐positive AML and AML characterized by hyperactivated SYK. Here, we show that targeted SYK inhibition similarly enhances the effects of midostaurin and other FLT3 inhibitors against mutant CBL‐positive leukaemia. Taken together, our results support the notion that mutant CBL‐expressing myeloid leukaemias are highly sensitive to available FLT3 inhibitors and that this effect can be significantly augmented by optimum inhibition of SYK kinase.     

SU11652 Inhibits tyrosine kinase activity of FLT3 and growth of MV-4-11 cells

FLT3-ITD and FLT3-TKD mutations are frequently found in acute myeloid leukemia (AML). This makes tyrosine kinase FLT3 a highly attractive target for therapeutic drug development. However, effective drugs have not yet emerged. This study is intended to identify and to characterize new FLT3 inhibitors. By using the protein substrate GST-FLT3S to analyze kinase activity of recombinant proteins carrying the catalytic domain of wild type and mutant forms of FLT3, we screened a chemical library containing 80 known protein kinase inhibitors. We identified SU11652 as a potent FLT3 inhibitor and further employed FLT3-ITD-positive MV- 4–11 cells to study its effects on cell growth, apoptosis, cell cycles, and cell signaling. SU11652 strongly inhibited the activity of wild type, D835Y, and D835H mutant forms of FLT3 with IC50 values of 1.5, 16, and 32 nM, respectively. It effectively blocked the growth of FLT3-ITD -positive MV-4-11 cells at nanomolar concentrations but exhibited much less effects on several other cells which do not carry mutations of FLT3. SU11652 inhibited growth of MV-4-11 cells by inducing apoptosis, causing cell cycle arrest, and blocking activation of the ERK, Akt, and STAT signaling pathways. SU11652 is a potent FLT3 inhibitor which selectively targets FLT3-ITD-positive cells. It should serve as a good candidate for development of therapeutic drugs to treat AML.     

Novel Oncogenic Mutations of CBL in Human Acute Myeloid Leukemia That Activate Growth and Survival Pathways Depend on Increased Metabolism

Acute myeloid leukemia (AML) is characterized by multiple mutagenic events that affect proliferation, survival, as well as differentiation. Recently, gain-of-function mutations in the α helical structure within the linker sequence of the E3 ubiquitin ligase CBL have been associated with AML. We identified four novel CBL mutations, including a point mutation (Y371H) and a putative splice site mutation in AML specimens. Characterization of these two CBL mutants revealed that coexpression with the receptor tyrosine kinases FLT3 (Fms-like tyrosine kinase 3) or KIT-induced ligand independent growth or ligand hyperresponsiveness, respectively. Growth of cells expressing mutant CBL required expression and kinase activity of FLT3. In addition to the CBL-dependent phosphorylation of FLT3 and CBL itself, transformation was associated with activation of Akt and STAT5 and required functional expression of the small GTPases Rho, Rac, and Cdc42. Furthermore, the mutations led to constitutively elevated intracellular reactive oxygen species levels, which is commonly linked to increased glucose metabolism in cancer cells. Inhibition of hexokinase with 2-deoxyglucose blocked the transforming activity of CBL mutants and reduced activation of signaling mechanisms. Overall, our data demonstrate that mutations of CBL alter cellular biology at multiple levels and require not only the activation of receptor proximal signaling events but also an increase in cellular glucose metabolism. Pathways that are activated by CBL gain-of-function mutations can be efficiently targeted by small molecule drugs.     

Selective cytotoxic mechanism of GTP-14564, a novel tyrosine kinase inhibitor in leukemia cells expressing a constitutively active Fms-like tyrosine kinase 3 (FLT3)

The receptor tyrosine kinase FLT3 is constitutively activated by an internal tandem duplication (ITD) mutation within the juxtamembrane domain in 20-30% of patients with acute myeloid leukemia. In this study, we identified GTP-14564 as a specific kinase inhibitor for ITD-FLT3 and investigated the molecular basis of its specificity. GTP-14564 inhibited the growth of interleukin-3-independent Ba/F3 expressing ITD-FLT3 at 1 microM, whereas a 30-fold higher concentration of GTP-14564 was required to inhibit FLT3 ligand-dependent growth of Ba/F3 expressing wild type FLT3 (wt-FLT3). However, this inhibitor suppressed the kinase activities of wt-FLT3 and ITD-FLT3 equally, suggesting that the signaling pathways for proliferation differ between wt-FLT3 and ITD-FLT3. Analysis of downstream targets of FLT3 using GTP-14564 revealed STAT5 activation to be essential for growth signaling of ITD-FLT3. In contrast, wt-FLT3 appeared to mainly use the MAPK pathway rather than the STAT5 pathway to transmit a proliferative signal. Further analysis demonstrated that the first two tyrosines in an ITD were critical for STAT5 activation and growth induction but that all of the tyrosines in the juxtamembrane region were dispensable in terms of the proliferation signals of wt-FLT3. These results indicate that an ITD mutation in FLT3 elicits an aberrant STAT5 activation that results in increased sensitivity to GTP-14564. Thus, FLT3-targeted inhibition is an attractive approach, with the potential for selective cytotoxicity, to the treatment of ITD-FLT3-positive acute myeloid leukemia.     

Protein-tyrosine phosphatase DEP-1 controls receptor tyrosine kinase FLT3 signaling

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. 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 32D myeloid cell lines with strongly reduced DEP-1 levels showed site-selective hyperphosphorylation of FLT3. In particular, the sites pTyr-589, pTyr-591, and pTyr-842 involved in the FLT3 ligand (FL)-mediated activation of FLT3 were hyperphosphorylated the most. Similarly, acute depletion of DEP-1 in the human AML cell line THP-1 caused elevated FLT3 phosphorylation. Direct interaction of DEP-1 and FLT3 was demonstrated by "substrate trapping" experiments showing association of DEP-1 D1205A or C1239S mutant proteins with FLT3 by co-immunoprecipitation. Moreover, activated FLT3 could be dephosphorylated by recombinant DEP-1 in vitro. Enhanced FLT3 phosphorylation in DEP-1-depleted cells was accompanied by enhanced FLT3-dependent activation of ERK and cell proliferation. Stable overexpression of DEP-1 in 32D cells and transient overexpression with FLT3 in HEK293 cells resulted in reduction of FL-mediated FLT3 signaling activity. Furthermore, FL-stimulated colony formation of 32D cells expressing FLT3 in methylcellulose 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 that its loss may contribute to but is not sufficient for leukemogenic cell transformation.     

MicroRNA-16 Is Down-Regulated in Mutated FLT3 Expressing Murine Myeloid FDC-P1 Cells and Interacts with Pim-1

Activating mutations in the receptor tyrosine kinase FLT3 are one of the most frequent somatic mutations in acute myeloid leukemia (AML). Internal tandem duplications of the juxtamembrane region of FLT3 (FLT3/ITD) constitutively activate survival and proliferation pathways, and are associated with a poor prognosis in AML. We suspected that alteration of small non-coding microRNA (miRNA) expression in these leukemia cells is involved in the transformation process and used miRNA microarrays to determine the miRNA signature from total RNA harvested from FLT3/ITD expressing FDC-P1 cells (FD-FLT3/ITD). This revealed that a limited set of miRNAs appeared to be affected by expression of FLT3/ITD compared to the control group consisting of FDC-P1 parental cells transfected with an empty vector (FD-EV). Among differentially expressed miRNAs, we selected miR-16, miR-21 and miR-223 to validate the microarray data by quantitative real-time RT-PCR showing a high degree of correlation. We further analyzed miR-16 expression with FLT3 inhibitors in FLT3/ITD expressing cells. MiR-16 was found to be one of most significantly down-regulated miRNAs in FLT3/ITD expressing cells and was up-regulated upon FLT3 inhibition. The data suggests that miR-16 is acting as a tumour suppressor gene in FLT3/ITD-mediated leukemic transformation. Whilst miR-16 has been reported to target multiple mRNAs, computer models from public bioinformatic resources predicted a potential regulatory mechanism between miR-16 and Pim-1 mRNA. In support of this interaction, miR-16 was shown to suppress Pim-1 reporter gene expression. Further, our data demonstrated that over-expression of miR-16 mimics suppressed Pim-1 expression in FD-FLT3/ITD cells suggesting that increased miR-16 expression contributes to depletion of Pim-1 after FLT3 inhibition and that miR-16 repression may be associated with up-regulated Pim-1 in FLT3/ITD expressing cells.     

Tricyclic quinoxalines as potent kinase inhibitors of PDGFR kinase,Flt3 and Kit

Here we report on novel quinoxalines as highly potent and selective inhibitors of the type III receptor tyrosine kinases PDGFR, FLT3, and KIT. These compounds, tricyclic quinoxalines, were generated in order to improve bioavailability over the highly hydrophobic bicyclic quinoxalines. Four of the highly active compounds were characterized in detail and are shown to inhibit PDGFR kinase activity of the isolated receptor as well as in intact cells in the sub-micromolar concentration range. We show that the most active inhibitor (compound 13, AGL 2043) is approximately 15-20 times more potent than its isomer (compound 14, AGL 2044). We therefore compared the three dimensional structures of the two compounds by X-ray crystallography. These compounds are also highly effective in blocking the kinase activity of FLT3, KIT, and the oncogenic protein Tel-PDGFR in intact cells. These compounds are potent inhibitors of the proliferation of pig heart smooth muscle cells. They fully arrest the growth of these cells and the effect is fully reversible. The chemical, biochemical and cellular properties of these compounds as well as the solubility properties make them suitable for development as anti-restenosis and anti-cancer agents.     

The tyrosine kinase CSK associates with FLT3 and c-Kit receptors and regulates downstream signaling

Type III receptor tyrosine kinases (RTKs), FLT3 and c-Kit play important roles in a variety of cellular processes. A number of SH2-domain containing proteins interact with FLT3 and c-Kit and regulate downstream signaling. The SH2-domain containing non-receptor protein tyrosine kinase CSK is mainly studied in the context of regulating Src family kinases. Here we present an additional role of this kinase in RTK signaling. We show that CSK interacts with FLT3 and c-Kit in a phosphorylation dependent manner. This interaction is facilitated through the SH2-domain of CSK. Under basal conditions CSK is mainly localized throughout the cytosolic compartment but upon ligand stimulation it is recruited to the inner side of cell membrane. CSK association did not alter receptor ubiquitination or phosphorylation but disrupted downstream signaling. Selective depletion of CSK using siRNA, or inhibition with CSK inhibitor, led to increased phosphorylation of Akt and Erk, but not p38, upon FLT3 ligand (FL) stimulation. Stem cell factor (SCF)-mediated Akt and Erk activation was also elevated by CSK inhibition. However, siRNA mediated CSK knockdown increased SCF stimulated Akt phosphorylation but decreased Erk phosphorylation. CSK depletion also significantly increased both FL- and SCF-induced SHC, Gab2 and SHP2 phosphorylation. Furthermore, CSK depletion contributed to oncogenic FLT3- and c-Kit-mediated cell proliferation, but not to cell survival. Thus, the results indicate that CSK association with type III RTKs, FLT3 and c-Kit can have differential impact on receptor downstream signaling.     

Expression cloning of oligomerization-activated genes with cell-proliferating potency by pseudotype retrovirus vector

We developed a method of clone proliferation promoting fusion genes whose proteins were activated by protein oligomerization through the helix-loop-helix region (PNT domain) of TEL. We inserted a cDNA library downstream of the PNT domain with a retrovirus vector. The resulting retrovirus infected cytokine-dependent 32D cells and cells with cytokine-independent growth were analyzed for the inserted cDNA. We cloned 25 independent fusion genes including seven kinds of partner genes. Six of the seven were a fusion of TEL with protein tyrosine kinase, LYN, HCK, FGR, SYK, FLT3, and TYK2. A serine/threonine kinase, ARAF1, was also found to fuse with TEL. These kinase fusion proteins included kinase domains with proper reading frames. These fusions may be a useful model for clarifying the downstream signal transduction of constitutive active kinase and this expression cloning method may provide a new tool with which to study cell proliferation signalling.     

Lestaurtinib potentiates TRAIL‐induced apoptosis in glioma via CHOP‐dependent DR5 induction

Lestaurtinib, also called CEP‐701, is an inhibitor of tyrosine kinase, causes haematological remission in patients with AML possessing FLT3‐ITD (FLT3 gene) internal tandem duplication and strongly inhibits tyrosine kinase FLT3. Treatment with lestaurtinib modulates various signalling pathways and leads to cell growth arrest and programmed cell death in several tumour types. However, the effect of lestaurtinib on glioma remains unclear. In this study, we examined lestaurtinib and TRAIL interactions in glioma cells and observed their synergistic activity on glioma cell apoptosis. While U87 and U251 cells showed resistance to TRAIL single treatment, they were sensitized to apoptosis induced by TRAIL in the presence of lestaurtinib because of increased death receptor 5 (DR5) levels through CHOP‐dependent manner. We also demonstrated using a xenograft model of mouse that the tumour growth was absolutely suppressed because of the combined treatment compared to TRAIL or lestaurtinib treatment carried out singly. Our findings reveal a potential new strategy to improve antitumour activity induced by TRAIL in glioma cells using lestaurtinib through a mechanism dependent on CHOP.