Novel FLT3 Mutation Shows Dominant Negative Effects on the Wild-Type FLT3 Receptor

Abstract Background The FMS-like tyrosine kinase-3 (FLT3) gene encodes for a receptor tyrosine kinase playing an important role in hematopoiesis. In acute leukemias it is one of the most frequently mutated genes. In this study we functionally characterized a novel frameshift deletion mutation of FLT3 found in a relapsed patient with acute myeloid leukemia (AML). The frameshift leads to a premature stop codon resulting in a truncated form of the receptor lacking most of the intracellular domains. Material and Methods FLT3 cDNA was expressed in the IL-3 dependent pro-B cell line Ba/F3 via a retroviral expression vector. The transduced cell lines were sorted by fluorescence-activated cell sorting (FACS). Stable expression of the receptor was confirmed on mRNA level by polymerase chain reaction (PCR) and on protein level by western blotting and cell surface expression of the receptor by flow cytometry. Cell proliferation assays were performed in presence or absence of IL-3 or FLT3-ligand. By western blotting receptor activation and its downstream signaling were analyzed. Ligand-binding of the receptor was shown via biotinylated and fluorescent FLT3-ligand-receptor-complexes by flow cytometry. Results and Discussion Stable expression of FLT3 Q569Vstop does not enable Ba/F3 cells to grow IL-3-independent. FLT3-ligand can still be bound by the mutated receptor but is not able to stimulate receptors´ signaling and growth of the cells. Furthermore coexpression of wild-type (WT) and mutant FLT3 receptor also abolishes the ability to stimulate the WT receptor with its ligand. This is confirmed by analyzing downstream signaling in the cells as MAPK is less phosphorylated in the FLT3-WT/Q569Vstop coexpressing cells than in FLT3-WT expressing cells alone. Conclusion Most of the FLT3 mutations are activating mutations leading to a constitutive activation of the receptor and ligand-independent growth. In our study we characterized a novel FLT3 mutation found in an AML patient which has not been described before. The resulting truncated receptor is still integrated in the plasma membrane and binds its ligand but its ability to be fully activated is completely lost. Furthermore coexpressed with a FLT3-WT receptor, it even prevents stimulation and activation of the WT receptor, thus acting in a dominant negative manner. How the truncated form of the receptor contributes to progression of acute leukemia is of great interest and will be further investigated. Disclosures No relevant conflicts of interest to declare.

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Selective disruption of E-cadherin function in early Xenopus embryos by a dominant negative mutant

Development ◽

10.1242/dev.120.4.901 ◽

1994 ◽

Vol 120 (4) ◽

pp. 901-909 ◽

Cited By ~ 10

Author(s):

E. Levine ◽

C.H. Lee ◽

C. Kintner ◽

B.M. Gumbiner

Keyword(s):

Early Embryo ◽

Dominant Negative ◽

Full Length ◽

Dominant Negative Mutant ◽

Wild Type ◽

Truncated Form ◽

Adhesive Properties ◽

Negative Mutant ◽

E Cadherin

E-cadherin function was disrupted in vivo in developing Xenopus laevis embryos through the expression of a mutant E-cadherin protein lacking its cytoplasmic tail. This truncated form of E-cadherin was designed to act as a dominant negative mutant by competing with the extracellular interactions of wild-type endogenous E-cadherin. Expression of truncated E-cadherin in the early embryo causes lesions to develop in the ectoderm during gastrulation. In contrast, expression of a similarly truncated N-cadherin protein failed to cause the lesions. The ectodermal defect caused by the truncated E-cadherin is rescued by overexpression of wild-type E-cadherin, by co-injection of full-length E-cadherin RNA along with the RNA for the truncated form. Overexpression of full-length C-cadherin, however, is unable to compensate for the disruption of E-cadherin function and can actually cause similar ectodermal lesions when injected alone, suggesting that there is a specific requirement for E-cadherin. Therefore, E-cadherin seems to be specifically required for maintaining the integrity of the ectoderm during epiboly in the gastrulating Xenopus embryo. Differential cadherin expression reflects, therefore, the requirement for distinct adhesive properties during different morphogenetic cell behaviors.

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HER2 G776S Mutation Promotes Oncogenic Potential in Colorectal Cancer Cells when Accompanied by Loss of APC Function

10.21203/rs.3.rs-594302/v1 ◽

2021 ◽

Author(s):

Yosuke Mitani ◽

Shinya Ohashi ◽

Osamu Kikuchi ◽

Yukie Nakai ◽

Tomomi Ida ◽

...

Keyword(s):

Colorectal Cancer ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitor ◽

Genome Sequencing ◽

Kinase Activity ◽

Kinase Inhibitor ◽

Wild Type ◽

Downstream Signaling ◽

Cancer Genome Sequencing ◽

Erk Phosphorylation

Abstract Background Clinical cancer genome sequencing detects oncogenic variants that are potential targets for cancer treatment, but it also detects variants of unknown significance that may interact and affect the pathophysiology of the tumor; however, these interactions are not fully understood. In this study, we examined the interactions of a minor HER2 mutation (G776S) and APC mutations, which were detected by cancer genome sequencing of samples from a patient with colorectal cancer. Methods We transfected HER2-G776S mutant- or HER2 wild type- expressing vectors into several cell lines, HeLa, FHC, CACO-2 and COLO-320, to evaluate their effects on HER2 phosphorylation and kinase activity, HER2 downstream signaling (phosphorylation of AKT and MAPK), and anchorage-independent growth ability. APC- knockout cells and APC overexpressing cells were established to investigate the effect of APC function on the HER2 signaling pathway. We also evaluated the efficacy of a HER2 tyrosine kinase inhibitor on xenograft tumors derived from HER2-G776S transfected cells. Results HER2 G776S mutation increased the kinase activity and phosphorylation of HER2 protein, but these effects were weaker than those of the other HER2 driver mutation. HER2 G776S did not activate HER2-downstream signal pathways, such as ERK and AKT phosphorylation, in cells with wild-type APC (HeLa and FHC cells). By contrast, HER2 G776S increased the activation of HER2 downstream signaling, especially ERK phosphorylation, and anchorage-independent cell growth in cells with an APC mutation (CACO-2 and COLO-320) and APC-knockout HeLa cells. Wild-type APC overexpression in HER2 G776S-transfected COLO-320 cells neutralized ERK phosphorylation. Loss of APC function increased Wnt pathway activity but also increased RAS–GTP, which increased ERK phosphorylation triggered by HER2 G776S transfection. Afatinib, a pan-HER tyrosine kinase inhibitor, inhibited tumor growth of HER2 G776S-transfected COLO-320 xenografts Conclusions HER2 G776S mutation acts as a weak oncogenic driver, but it also increases HER2–ERK signaling activity by increasing RAS–GTP production when APC function is simultaneously impaired. These results suggest that even weakly active mutations may be therapeutic targets, and the use of this strategy may contribute to the development of HER2-targeted therapy for colorectal cancer.

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304. DECIDUAL HtrA3 NEGATIVELY REGULATES TROPHOBLAST INVASION

Reproduction Fertility and Development ◽

10.1071/srb10abs304 ◽

2010 ◽

Vol 22 (9) ◽

pp. 104

Author(s):

H. Singh ◽

S. Makino ◽

Y. Endo ◽

G. Nie

Keyword(s):

Western Blotting ◽

Protease Activity ◽

Specific Inhibitor ◽

First Trimester ◽

Dominant Negative ◽

Trophoblast Invasion ◽

Wild Type ◽

Placental Development ◽

Endometrial Stromal Cells ◽

Decidual Cells

Controlled trophoblast invasion cell into the maternal decidua (interstitial invasion) is important for placental development. Abnormalities in the invasion process may lead to pregnancy complications. Decidua secrets many factors to control trophoblast invasion. Serine protease HtrA3 is highly expressed in the decidual cells in the late secretory phase of the menstrual cycle and throughout pregnancy. It is highly expressed in first trimester in most trophoblast cell types, but not in the invading interstitial trophoblast. HtrA3 and its family members are down-regulated in a number of cancers and are proposed as tumor suppressors. We hypothesized that HtrA3 is an inhibitor of trophoblast invasion. The current study aimed to investigate whether HtrA3 secreted by decidual cells regulates trophoblast invasion. Human endometrial stromal cells (HESC) were decidualised with estradial, medroxyprogesterone acetate and cyclic AMP. Real-time RT-PCR, western blotting and immunocytochemistry demonstrated that decidualisation increased HtrA3 mRNA and protein expression. HtrA3 was also detected by western blotting in the conditioned media (CM) of decidualised HESC (96h), confirming its secretory nature. For functional studies, wild type and protease inactive mutant HtrA3 were produced using wheat germ cell-free technology. The mutant has negligible protease activity and significantly inhibited the wild type protease activity, supporting its dominant-negative inhibition and utility as a specific inhibitor of the wild type protein. CM of decidualised HESC suppressed invasion of trophoblast HTR-8 cells, whereas inhibition of HtrA3 in the decidual HESC CM by exogeneous addition of HtrA3 mutant increased trophoblast HTR-8 cell invasion. These results strongly support our hypothesis that decidual HtrA3 negatively regulates trophobalst invasion.

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TheXenopusreceptor tyrosine kinase Xror2 modulates morphogenetic movements of the axial mesoderm and neuroectoderm via Wnt signaling

Development ◽

10.1242/dev.129.22.5227 ◽

2002 ◽

Vol 129 (22) ◽

pp. 5227-5239 ◽

Cited By ~ 4

Author(s):

Hiroki Hikasa ◽

Mikihito Shibata ◽

Ichiro Hiratani ◽

Masanori Taira

Keyword(s):

Tyrosine Kinase ◽

Wnt Signaling ◽

Cell Polarity ◽

Planar Cell Polarity ◽

Dominant Negative ◽

Treated Animal ◽

Convergent Extension ◽

Wild Type ◽

Morphogenetic Movements ◽

Planar Cell Polarity Pathway

The Spemann organizer plays a central role in neural induction, patterning of the neuroectoderm and mesoderm, and morphogenetic movements during early embryogenesis. By seeking genes whose expression is activated by the organizer-specific LIM homeobox gene Xlim-1 in Xenopusanimal caps, we isolated the receptor tyrosine kinase Xror2. Xror2 is expressed initially in the dorsal marginal zone, then in the notochord and the neuroectoderm posterior to the midbrain-hindbrain boundary. mRNA injection experiments revealed that overexpression of Xror2 inhibits convergent extension of the dorsal mesoderm and neuroectoderm in whole embryos, as well as the elongation of animal caps treated with activin, whereas it does not appear to affect cell differentiation of neural tissue and notochord. Interestingly, mutant constructs in which the kinase domain was point-mutated or deleted (named Xror2-TM) also inhibited convergent extension, and did not counteract the wild-type, suggesting that the ectodomain of Xror2 per se has activities that may be modulated by the intracellular domain. In relation to Wnt signaling for planar cell polarity, we observed: (1) the Frizzled-like domain in the ectodomain is required for the activity of wild-type Xror2 and Xror2-TM; (2) co-expression of Xror2 with Xwnt11, Xfz7, or both,synergistically inhibits convergent extension in embryos; (3) inhibition of elongation by Xror2 in activin-treated animal caps is reversed by co-expression of a dominant negative form of Cdc42 that has been suggested to mediate the planar cell polarity pathway of Wnt; and (4) the ectodomain of Xror2 interacts with Xwnts in co-immunoprecipitation experiments. These results suggest that Xror2 cooperates with Wnts to regulate convergent extension of the axial mesoderm and neuroectoderm by modulating the planar cell polarity pathway of Wnt.

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The Src Related Tyrosine Kinase Lyn Mediates Proliferative Signals from Leukemia-Related Flt3 Mutants.

Blood ◽

10.1182/blood.v104.11.2562.2562 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2562-2562

Author(s):

Lisa J. Robinson ◽

Jia Xue ◽

Seth J. Corey

Keyword(s):

Growth Factor ◽

Tyrosine Kinase ◽

Kinase Inhibitors ◽

Src Kinase ◽

Leukemic Cell ◽

Flt3 Ligand ◽

Myeloproliferative Disease ◽

Wild Type ◽

Acute Leukemias ◽

Molecular Abnormalities

Abstract Fms-like tyrosine kinase-3 (Flt3) is a hematopoietic growth factor receptor that regulates the survival and proliferation of myeloid and B-cell precursors. Mutations of Flt3 resulting in constitutive activation are the most common known molecular abnormalities in acute myeloid leukemia. Cells expressing activated Flt3 mutants show enhanced survival and proliferation, and leukemia-associated Flt3 mutations produced a fatal myeloproliferative disorder, in a mouse model (Kelly et al. Blood 2002). Nevertheless, the signaling pathways mediating Flt3 effects on proliferation are incompletely understood. Src kinases, such as Lyn, have also been linked to myeloproliferative disease, but the participation of Src-related kinases in Flt3 signaling has not been established. We therefore determined whether Src-family kinases were activated by Flt3 in human leukemic cell lines that express either wild-type or mutant Flt3. Flt3-ligand stimulation of THP1 cells, which express wild-type Flt3, significantly increased the phosphorylation of Lyn, the principal Src-family kinase found in these cells. In MV4;11 cells, which express a constitutively-active tandem duplication mutant of Flt3, Lyn phosphorylation is constitutively elevated. In both cases, a Lyn phosphorylation state specific antibody confirmed phosphorylation at tyrosine 397 of human Lyn; phosphorylation at this site is indicative of Lyn kinase activation. To determine whether the increase in Lyn phosphorylation seen in MV4;11 cells could be attributed specifically to effects of mutant Flt3, we examined Lyn phosphorylation in the murine leukemic Baf3 cells transduced with either wild-type or mutant human Flt3. Lyn phosphorylation and activation in Baf3 cells expressing the Flt3 mutant was significantly increased, compared to basal Lyn phosphorylation in Baf3 cells expressing wild-type Flt3 or vector alone. This result suggested that the constitutive Lyn phosphorylation seen in MV4;11 cells could be caused by mutant Flt3. In Baf3 cells expressing wild-type Flt3, Lyn phosphorylation was stimulated by Flt3-ligand treatment as expected. The Baf3 cell line is growth factor dependent, requiring IL-3 for survival and proliferation. However, as previously reported, expression of activated Flt3 mutants render the cells growth factor independent. We found that the Src-family specific inhibitors PP1 and PP2, but not the inactive analog PP3, inhibited the growth factor-independent proliferation of Baf3 cells expressing mutant Flt3. Moreover, anti-sense oligonucleotides that specifically reduced Lyn expression also inhibited proliferation of cells expressing Flt3 mutants. Previous studies identified Stat5 and Erk as mediators of the proliferation signal from mutant Flt3. We therefore examined the effects of Src kinase inhibitors on the activation of these signaling proteins by mutant Flt3. No change in Stat5 phosphorylation was detected, but inhibition of Lyn did inhibit Erk phosphorylation. These results suggest that Lyn is upstream from Erk in a Flt3 signaling pathway important for proliferation. Flt3 activation of Lyn also raises the possibility that Src kinase inhibitors could have therapeutic efficacy in Flt3-related acute leukemias.

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Targeting BCR-ABL and Its Downstream Signaling Cascade as Therapy for Chronic Myelogenous Leukemia.

Blood ◽

10.1182/blood.v104.11.2964.2964 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2964-2964 ◽

Cited By ~ 1

Author(s):

Nicholas J. Donato ◽

Ji Wu ◽

Ling Y. Kong ◽

Feng Meng ◽

Waldemar Priebe ◽

...

Keyword(s):

Gene Expression ◽

Cell Growth ◽

Tyrosine Kinase ◽

Point Mutations ◽

Myelogenous Leukemia ◽

Therapeutic Activity ◽

Wild Type ◽

Growth And Survival ◽

Downstream Signaling ◽

Kinase Inhibitory Activity

Abstract BCR-ABL is an unregulated tyrosine kinase expressed as a consequence of chromosomal translocation in chronic myelogenous leukemia (CML). The tyrosine kinase activity of BCR-ABL activates signaling cascades that induce cytokine independence and transformation of myeloid progenitor cells. Targeted inhibition of this kinase with specific inhibitors (imatinib or BMS-354825) is a very effective therapy for some CML patients but resistance to these agents (through point mutations and other mechanisms) leads to advanced disease with very few therapeutic options. An alternate therapeutic strategy is to reduce BCR-ABL expression or its critical downstream signaling elements important for transformation. We examined BCR-ABL signaling elements and gene expression changes that occur in CML cells following kinase inhibition by imatinib in newly established imatinib sensitive and resistant cells to identify critical signaling elements involved in CML cell death. Imatinib rapidly and progressively suppressed c-myc expression in imatinib sensitive but not resistant cells prior to the onset of apoptosis. These results suggested that c-myc expression was regulated by BCR-ABL signaling and may play a role in CML tumorigenicity. To confirm a role for c-myc in CML cell growth and/or survival, c-myc expression was specifically down-regulated by siRNA using a novel electroporation instrument (AMAXA) that permits high level gene transfer with limited toxicity in CML cell lines. Jak2 siRNA was used as a control. c-myc, but not Jak2 siRNA, suppressed c-myc expression and cell growth and survival in both imatinib sensitive and resistant CML cells, suggesting that targeted suppression of c-myc may have therapeutic activity against both kinase inhibitor sensitive and resistant CML cells. Since the tyrphostin AG490 was previously shown to inhibit c-myc expression in CML cells through its inhibitory effects on Jak2, we screened a series of > 200 AG490 derivatives for their ability to rapidly reduce c-myc expression in hematological malignancies. After several rounds of testing we synthesized an agent (WP-1066) capable of rapid c-myc downregulation (beginning 1–5 min after treatment with 1–2 microM) but poor Jak2 kinase inhibitory activity (IC50 > 100 microM). These results suggested a more direct effect of WP-1066 on c-myc protein expression than AG490 and mechanistic studies suggest that WP-1066 reduces c-myc protein stability but does not affect c-myc gene expression. In BCR-ABL expressing cells WP-1066 rapidly reduced c-myc protein levels in CML cells and inhibited the growth and survival of cell lines or patient specimens expressing wild-type or mutant forms of BCR-ABL that effect tyrosine kinase inhibitory activity (T315I in BV-173R cells). Equal concentrations of imatinib or WP-1066 reduced BCR-ABL activation and downstream signaling (Stat5 phosphorylation) in CML cells. However, WP-1066 differed from imatinib in its ability to downregulate BCR-ABL protein expression without affects on c-abl or Stat5 expression. Similar results were obtained in clinical specimens taken from patients with BCR-ABL point mutations that mediate imatinib (or BMS-354825) resistance. Nude mouse studies demonstrated that WP-1066 reduced the growth of K562 tumors to an extent similar to that of imatinib. Together these results suggest that WP-1066 downregulates BCR-ABL and c-myc expression, induces apoptosis in CML cells expressing wild-type or mutant BCR-ABL and may have therapeutic activity in imatinib (or BMS-354825) resistant CML tumors.

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A Novel Mutation 774del4 in IFNGR1, Which Produces Truncated Form of Interferon γ Receptor 1, Causes a Dominant-Negative Effect on Interferon γ Signal Transduction.

Blood ◽

10.1182/blood.v108.11.1246.1246 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1246-1246

Author(s):

Satoshi Okada ◽

Nobutsune Ishikawa ◽

Kenichirou Shirao ◽

Hiroshi Kawaguchi ◽

Miyuki Tsumura ◽

...

Keyword(s):

Cell Surface ◽

Interferon Γ ◽

Dominant Negative ◽

Hek293 Cells ◽

Dominant Negative Effect ◽

Heterozygous Mutation ◽

Wild Type ◽

Truncated Form ◽

Mycobacterium Infections ◽

Ifn Γ

Abstract Patients with interferon γ receptor 1 (IFNγR1) deficiency are characterized by disseminated Bacilli Calmette-Guérin (BCG) infections and severe non-tuberculosis mycobacterium infections, and show remarkable genetic heterogeneity. It is known as one of inherited immunodeficiency disorders with recessive or dominant form. So far known dominant forms of IFNγR1 deficiency are associated with heterozygous mutation in the intracellular domain of IFNγR1, among of which 818del4 is known as a major mutation in IFNGR1, leading to frameshift and premature stop codon. We describe here a novel heterozygous IFNGR1 mutation in a patient with recurrent mycobacterium infections, resulting in impairment of receptor degradation. The proband, a 12-year old Japanese girl, developed multiple osteomyelitis due to M. avium infection. She had suffered from BCG lymphadenitis, which occurred 2 months after BCG vaccination, for 3 years. DNA sequence analysis of IFNGR1 demonstrated that the patient had a heterozygous mutation 774del4, producing a truncated protein lacking intracellular component of IFNγR1. IFNγR1 was overexpressed on the surface of CD14-positive cells in the peripheral blood of the patient, and STAT1 phosphorylation was partially defected in response to high dose IFN-γ stimulation. Impaired TNF-α production in response to IFN-γ stimulation was also observed. T cells from the patient presented decreased IFN-γ production in response to IL-12 stimulation. In order to define the characterization of this truncated form of IFNγR1, we cloned 774del4 and 818del4 mutations as well as the wild-type into mammalian expression vector. These constructs were transfected into HEK293 cells. Cycloheximide (CHX) treatment enabled detection of IFNγR1 expression by flow cytometry and Western blot analysis. In contrast to the wild-type IFNγR1 protein, which rapidly disappeared after CHX treatment, 774del4 mutant protein was stably retained on the cell surface as was observed in 818del4 mutant. These observations suggest that 774del4 mutant causes overexpression of dominant-negative form of IFNγR1 on the cell surface through impairment of receptor degradation.

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Mer Receptor Tyrosine Kinase Is Over-Expressed In and Contributes to Oncogenesis In Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v118.21.1390.1390 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1390-1390

Author(s):

Alisa B. Lee-Sherick ◽

Kristen M. Eisenman ◽

Susan Sather ◽

Deborah DeRyckere ◽

Jennifer Schlegel ◽

...

Keyword(s):

Flow Cytometry ◽

Targeted Therapy ◽

Tyrosine Kinase ◽

Western Blot ◽

Cell Survival ◽

Cell Lines ◽

Wild Type ◽

Knock Down ◽

Acute Myelogenous

Abstract Abstract 1390 The abnormal activation of tyrosine kinases in pediatric leukemias has been associated with a poor prognosis, and provides a potential focus for targeted therapy. Pediatric acute myelogenous leukemia (AML) is known to be particularly difficult to treat successfully. The development of therapy for AML targeted against a specific cancer-promoting signaling pathway would potentially allow for a more efficacious clinical response with less therapy-associated toxicity. The Mer Tyrosine Kinase (TK), a transmembrane receptor in the TAM family, is known to regulate intracellular pathways promoting cell survival and proliferation in a number of malignancies, but has not previously been explored in AML. We assessed the prevalence of Mer TK expression in AML. Western blot and flow cytometric analysis demonstrated aberrant expression of Mer TK in 80% (13 of 15) of AML cell lines. Similarly, greater than 85% (24 of 28) of samples from newly diagnosed pediatric AML patients expressed Mer TK on leukemic blasts. In addition, 5 of 6 pediatric patients with relapsed or refractory AML had increased or equivalent Mer expression by flow cytometry relative to diagnostic samples. To assess whether Mer plays a role in proliferation in AML, we investigated downstream signaling pathways in the Nomo-1 and Kasumi-1 AML cell lines. Phosphoarray and western blot analysis demonstrated increased phospho-Erk 1/2, phospho-Akt, phospho-mTOR and phospho-MSK1 following treatment with Gas6, the Mer ligand. These data demonstrate activation of pathways which are known to aid in malignant cell survival. To assess the effect of Mer TK inhibition on myeloblast phenotype, we used two different shRNA constructs to decrease expression of Mer by >50% in the Nomo-1 and Kasumi-1 cell lines. The ability of these cell lines to evade apoptosis was determined by flow cytometry following staining with propidium iodide and Yo-Pro-1-iodide. Compared to wild-type Nomo-1 and Kasumi-1, the cell lines expressing decreased levels of Mer demonstrated two to four times more apoptosis in response to serum starvation (p<0.5). Additionally, myeloblast proliferative capacity was assessed using methylcellulose colony forming assays. Compared to wild-type, the AML cell lines expressing reduced levels of Mer demonstrated a 40–70% decrease in total colony forming units (p<0.5). To explore how knockdown of Mer affects myeloblast survival in vivo, we used a mouse xenograft model. Sub-lethally irradiated NSG mice were injected intravenously with wild-type Nomo-1 or Mer knock-down Nomo-1 lines and tumor-free survival was determined. Kaplan-Meier curves were generated and demonstrated a statistically significant difference in survival between mice injected with wild-type Nomo-1 cells and those injected with a Nomo-1 Mer knock-down cell line (20 versus 43 days, p<0.1). These data demonstrate a role for Mer in acute myelogenous leukemogenesis in vivo and suggest that inhibition of Mer TK may have a clinically significant effect in patients as a targeted therapy in the treatment of human AML. Disclosures: No relevant conflicts of interest to declare.

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β-Chemokine Receptor CCR5 Signals Via the Novel Tyrosine Kinase RAFTK

Blood ◽

10.1182/blood.v91.3.791.791_791_797 ◽

1998 ◽

Vol 91 (3) ◽

pp. 791-797 ◽

Cited By ~ 2

Author(s):

Ramesh K. Ganju ◽

Parmesh Dutt ◽

Lijun Wu ◽

Walter Newman ◽

Hava Avraham ◽

...

Keyword(s):

Tyrosine Kinase ◽

Chemokine Receptors ◽

Growth Regulation ◽

Dominant Negative ◽

Downstream Signaling ◽

Inflammatory Protein ◽

Ccr5 Receptor ◽

Mitogen Activated Protein ◽

Chemokine Receptor Ccr5 ◽

Receptor Ccr5

Chemokine receptors are coupled to G-proteins and their activation results in prominent changes in cell migration and growth. The downstream signaling pathways that mediate these effects of chemokines are largely uncharacterized. Macrophage inflammatory protein 1β (MIP 1β) binding to its cognate receptor CCR5 resulted in activation of the related adhesion focal tyrosine kinase (RAFTK), with subsequent activation of the cytoskeletal protein paxillin and the downstream transcriptional activators, c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) and p38 mitogen-activated protein (MAP) kinase. Inhibition of RAFTK by a dominant-negative kinase mutant markedly attenuated JNK/SAPK activity. Thus, RAFTK appears to provide a functional “bridge” for the transmission of CCR5 receptor signaling to the cytoskeleton and nucleus, primary sites of chemotaxis and growth regulation.

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Ki23819 (KRN383•HCl) Inhibits Kinase Activity of Wild Type and Mutant FLT3 Receptor Tyrosine Kinase In Vitro.

Blood ◽

10.1182/blood.v104.11.1168.1168 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1168-1168

Author(s):

Yukiko Komeno ◽

Mineo Kurokawa ◽

Yoichi Imai ◽

Masataka Takeshita ◽

Tomoko Matsumura ◽

...

Keyword(s):

Tyrosine Kinase ◽

Western Blotting ◽

Receptor Tyrosine Kinase ◽

Kinase Activity ◽

Kinase Assay ◽

Wild Type ◽

Direct Inhibition ◽

Flt3 Inhibitor ◽

In Vitro Kinase Assay

Abstract [Background] FLT3 is a class III receptor tyrosine kinase which is widely expressed on hematopoietic stem/progenitor cells. Two types of constitutively active FLT3 mutations have been reported to be expressed on a subset of leukemic cells; internal tandem duplications (ITD) and kinase domain mutations. The former are associated with poor prognosis in acute myeloid leukemia (AML) patients. Although several inhibitors targeting FLT3-ITD are tested in clinical trials, their cytotoxic effects are still unsatisfactory. Innate and acquired resistance is also a problem to be solved. [Purpose] To screen a novel potent FLT3 inhibitor and characterize its in vitro activity. [Materials and Methods] MOLM13 and MV4-11 cells, human leukemia cell lines expressing FLT3-ITD, were exposed to candidate compounds for 48 hours, and cytotoxic effect was assessed by colorimetric assay. Inhibitory effect on autophosphorylation was evaluated by immunoprecipitation and Western blotting. These effects were also tested in 32D cells engineered to express wild type FLT3 (FLT3-WT) or FLT3-ITD. FLT3-WT was activated with 50 ng/ml FLT3 ligand for 15 min. Proapoptotic effect was confirmed by flow cytometry with Annexin V staining. In vitro kinase assay was performed to demonstrate direct inhibition of tyrosine kinase activity of FLT3-ITD. Inhibitory effects on downstream signaling molecules, ERK and STAT5, were assessed by Western blotting. [Results] Among candidates for VEGFR inhibitors from a library, a quinoline-urea derivative Ki23819 (KRN383•HCl) was identified to specifically inhibit proliferation and induce apoptosis to MOLM13 and MV4-11 cells. Ki23819 inhibited proliferation of MV4-11 cells more effectively than SU11248, a precedent FLT3 inhibitor (IC50 <1 nM vs 3~10 nM). Similar results were obtained when MOLM13 cells were used. Ki23819 inhibited autophosphorylation of both ligand-activated FLT3-WT and FLT3-ITD (IC50 30 nM and 3 nM, respectively), and abrogated IL-3-independent proliferation of 32D cells expressing FLT3-ITD (IC50 3~10 nM). In vitro kinase assay demonstrated direct inhibition of kinase activity of FLT3-ITD (IC50 7.8 nM). This compound also inhibited ERK and STAT5 constitutively activated by FLT3-ITD. The IC50 for inhibition of phosphorylation in 32D FLT3-ITD cells was 3 nM for both proteins, which is equivalent to that for inhibition of FLT3-ITD autophosphorylation. [Conclusion] Ki23819 is a novel and potent candidate for antileukemic agents against FLT3-ITD positive AML. In vivo activity of KRN383, the free base of Ki23819, is also to be reported in this ASH meeting (Nishiyama et al.).

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