scholarly journals The Receptor Tyrosine Kinase RON and Its Isoforms as Therapeutic Targets in Ewing Sarcoma

Cancers ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 904
Author(s):  
Philipp Berning ◽  
Carolin Hennemann ◽  
Claudia Tulotta ◽  
Christiane Schaefer ◽  
Birgit Lechtape ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Ewing Sarcoma ◽  
Dna Methyltransferase ◽  
Short Form ◽  
Metastatic Progression ◽  
Zebrafish Model ◽  
Resistance To Insulin

The receptor tyrosine kinase (RTK) RON is linked to an aggressive metastatic phenotype of carcinomas. While gaining interest as a therapeutic target, RON remains unstudied in sarcomas. In Ewing sarcoma, we identified RON among RTKs conferring resistance to insulin-like growth factor-1 receptor (IGF1R) targeting. Therefore, we explored RON in pediatric sarcoma cell lines and an embryonic Tg(kdrl:mCherry) zebrafish model, using an shRNA-based approach. To examine RON–IGF1R crosstalk, we employed the clinical-grade monoclonal antibody IMC-RON8, alone and together with the IGF1R-antibody IMC-A12. RON silencing demonstrated functions in vitro and in vivo, particularly within micrometastatic cellular capacities. Signaling studies revealed a unidirectional IGF1-mediated cross-activation of RON. Yet, IMC-A12 failed to sensitize cells to IMC-RON8, suggesting additional mechanisms of RON activation. Here, RT-PCR revealed that childhood sarcomas express short-form RON, an isoform resistant to antibody-mediated targeting. Interestingly, in contrast to carcinomas, treatment with DNA methyltransferase inhibitor did not diminish but increased short-form RON expression. Thus, this first report supports a role for RON in the metastatic progression of Ewing sarcoma. While principal molecular functions appear transferrable between carcinomas, Ewing sarcoma and possibly more common sarcoma subtypes, RON highlights that specific regulations of cellular networks and isoforms require better understanding to successfully transfer targeting strategies.

The Multi-Targeted Receptor Tyrosine Kinase Inhibitor, ABT-869, Induces Apoptosis of AML Cells Both In Vitro and In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 616-616 ◽  
Author(s):  
Deepa B. Shankar ◽  
Jenny C. Chang ◽  
Bertrand Parcells ◽  
Salemiz Sandoval ◽  
Junling Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Receptor Tyrosine Kinase ◽  
Scid Mice ◽  
Parp Cleavage

Abstract Children with acute myeloid leukemia (AML) have less than 60% overall survival despite aggressive chemotherapy and bone marrow transplantation. Only one third of the adult patients diagnosed with AML will be cured. AML blast cells from up to 30% of patients express a constitutively active receptor tyrosine kinase, FLT3-ITD, which contains an internal tandem duplication in the juxtamembrane domain. Patients with FLT3-ITD have a worse prognosis. ABT-869 is a novel multi-targeted small molecule inhibitor of receptor tyrosine kinases and is a potent inhibitor of FLT3, c-Kit, and all members of the VEGF and PDGF receptor families. To determine the effects of ABT-896 on AML cells, we treated AML cell lines, primary cells, and tumors in xenograft models with varying concentrations of the drug. In vitro viability assays showed that ABT-869 inhibited the growth of two different cell lines, MV-4-11 (human AML cell line that expresses FLT3-ITD) and BAF3-ITD (murine B-cell line stably transfected with the FLT3-ITD) at an IC50 of 10nM. ABT-869 was also effective against another mutation of FLT3, D835V, but at higher concentrations (IC50 of 100nM). Phosphorylation of FLT3 and activation of downstream signaling molecules, STAT5 and ERK, were inhibited by ABT-869 in a concentration-dependent manner. Cells were also stained with Annexin V-FITC and Propidium Iodide, and analyzed using FACS. ABT-869 induced apoptosis, caspase-3 activation, and PARP cleavage after 48 hours. To examine the in vitro effects of ABT-869 on normal hematopoietic progenitor cells, we performed methylcellulose-based colony assays with human bone marrow. No significant difference was observed in the number and type of colonies formed using BM cells treated with ABT-869 or control, up to a concentration of 1 micromolar. These results suggest that ABT-869 is not toxic to normal bone marrow progenitor cells at concentrations that are effective against AML cells. To examine the effects of ABT-869 in vivo, we treated SCID mice injected with MV-4-11, Baf3-ITD, Baf3-D835V, or Baf3-WT cells, with oral preparations of ABT-869. Complete regression of MV-4-11 tumors was observed in mice treated with ABT-869 at 20 and 40 mg/kg/day. No adverse effects were detected in the peripheral blood counts, bone marrow, spleen or liver. Histology of the tumors from the control-treated group showed a high degree of proliferation by Ki-67 staining, increased mitotic figures, and a well-defined tumor mass. In contrast, the tumors from mice treated with ABT-869 showed a number of apoptotic bodies by TUNEL staining and the presence of reactive, inflammatory cells. Interestingly, we also observed that mice that received ABT-869 the day after injection of AML cells remained tumor-free for over 2 months in contrast to the mice receiving the vehicle alone. Inhibition of FLT3 phosphorylation was demonstrated in the tumors from mice treated with ABT-869. We are evaluating the activity of ABT-869 treatment of SCID mice injected with Baf3-ITD, Baf3-D835V, or Baf3-WT cells. NOD-SCID mouse models are currently being used to analyze the effects of ABT-869 on primary AML cells in vivo. Our preclinical studies demonstrate that ABT-869 is effective and nontoxic, and provide rationale for the treatment and prevention of relapse in AML patients.

Preclinical Testing of a Novel Axl-Kinase Inhibitor in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1799-1799
Author(s):  
Maria Göbel ◽  
Michael Möllmann ◽  
Andre Görgens ◽  
Ulrich Dührsen ◽  
Andreas Hüttmann ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Cell Polarization ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Blue Staining ◽  
Nsg Mice

Abstract Abstract 1799 The receptor tyrosine kinase Axl belongs to the TAM (Tyro-3, Axl and Mer) family and is involved in the progression of several human malignancies including chronic lymphocytic leukemia (CLL), where it is has been found to be overexpressed in comparison to normal B-cells. An increasing body of evidence suggests that Axl acts as an oncogene which increases the survival, proliferation, metastatic potential and chemotherapy resistance of tumor cells. Hence, it has been recently identified as a potential therapeutic target in a wide range of tumor entities with deregulated Axl expression including prostate cancer, glioma, lung cancer and CLL. Here, we investigated two different Axl inhibitors for their potential to inhibit the migratory capacity and survival of leukemic cells in preclinical CLL models. In vitro studies: Freshly isolated PBMC (>90% CD5+CD19+) from CLL patients were incubated in serum free medium for 48h containing concentrations series of 2 different Axl inhibitors: BMS777607, a previously published inhibitor of the MET kinase family, and LDC2636, a novel inhibitor of the TAM receptor tyrosine kinase (RTK) family with high affinity to Axl. Viability of CLL cells was assessed by trypan blue staining and flow cytometry employing annexin V staining. Since a polarized phenotype is required for migration, cell polarization was analyzed by time-lapse video-microscopy. We detected cytotoxic effects in a patient dependent manner that were more prevalent in LDC2636 as compared to BMS777607 treated cells (LD50= 1.4 μM vs. 5.2 μM, p<0.004, n=5). Cell polarization of the remaining viable cells was significantly reduced in a dose dependent fashion in comparison to vehicle only controls (LDC2636 IC50 = 7.2 μM, p<0.00001; BMS777607: IC50=6.2μM; p=0.0004). Of note, both Axl inhibitors exhibited significantly weaker effects on both, the viability and cell polarization of normal PBMC over the whole concentration range tested (p<0.05, n=5). In vivo studies: To verify our hypothesis that reduced cell polarization results in decreased homing of leukemic cells in vivo we employed a recently developed adoptive transfer model of CLL. In this model NOD/SCID/gcnull(NSG) mice were pre-treated with a single intraperitoneal bolus of LDC2636 or BMS777607 (20 mg/kg) and subsequently transplanted with primary CLL cells. Both Axl inhibitors significantly reduced the homing capacity of CLL cells to the bone marrow of NSG mice by 43% and 59%, respectively, compared to vehicle treated controls (LDC2636: p=0.046, BMS777607 p=0.0077; n=3). These data demonstrate that Axl inhibitors exert potent in vitro and in vivo activity against human CLL cells, which is caused at least in part by the suppression of CLL homing to their supportive stromal niches. Disclosures: No relevant conflicts of interest to declare.

Oligomerization of the extracellular domain of Boss enhances its binding to the Sevenless receptor and its antagonistic effect on R7 induction

1998 ◽  
Vol 111 (6) ◽  
pp. 737-747 ◽  
Author(s):  
E.A. Sevrioukov ◽  
J.H. Walenta ◽  
A. Sunio ◽  
M. Phistry ◽  
H. Kramer
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Compound Eye ◽  
Transmembrane Domain ◽  
Extracellular Domain ◽  
Antagonistic Effect ◽  
Lac Repressor ◽  
Receptor Complexes

In the developing compound eye of Drosophila, neuronal differentiation of the R7 photoreceptor cell is induced by the interaction of the receptor tyrosine kinase Sevenless with its ligand Bride of sevenless (Boss), which is expressed on the neighboring R8 cell. Boss is an unusual ligand of a receptor tyrosine kinase: it is composed of a large extracellular domain, a transmembrane domain with seven membrane-spanning segments and a cytoplasmic tail. Expression of a monomeric, secreted form of the extracellular domain of Boss is not sufficient for Sevenless activation, and instead acts as a weak antagonist. Because oligomerization appears to be a critical step in the activation of receptor tyrosine kinases, we used oligomerized forms of the Boss extracellular domain to test their ability to bind to Sevenless in vivo and restore R7 induction in vivo. Oligomerization was achieved by fusion to the leucine zipper of the yeast transcription factor GCN4 or to the tetramerization helix of Lac repressor. Binding of these multivalent proteins to Sevenless could be detected in vitro by immunoprecipitation of cross-linked ligand/receptor complexes and in vivo by receptor-dependent ligand localization. However, neither R8-specific or ubiquitous expression of multivalent Exboss ligands rescued the boss phenotype. Instead, these ligands acted as competitive inhibitors for wild-type Boss protein and thereby suppressed R7 induction. Therefore the role of the transmembrane or cytoplasmic domains of Boss in the activation of the Sev receptor cannot be replaced by oligomerization.

Inhibiting the IGF-1 Receptor Tyrosine Kinase with Picropodophillin: An In Vitro and In Vivo Study in the 5T33MM Mouse Model.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 640-640
Author(s):  
Karin Vanderkerken ◽  
Eline Menu ◽  
Thomas Stromberg ◽  
Hendrik De Raeve ◽  
Kewal Asosingh ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Mouse Model ◽  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Microvessel Density ◽  
Kinase Inhibitor ◽  
High Dose

Abstract Multiple myeloma (MM) represents a B-cell malignancy, characterized by monoclonal proliferation of plasma cells in the bone marrow (BM) and is associated with osteolysis and angiogenesis. Insulin-like growth factor-1 (IGF-1), produced by the BM stromal cells, has been described as an important factor in the survival, proliferation and migration of MM cells. The latter process is involved in the homing of the MM cells to the BM. IGF-1 also induces VEGF secretion by the MM cells, thus stimulating angiogenesis in the BM. As IGF-1 is a pleiotropic factor in MM, therapeutic strategies targeting the IGF-1R may be effective as anti-tumor treatments. In this work we investigated the effect of an IGF-1 receptor tyrosine kinase inhibitor (picropodophyllin or PPP1) in the murine, syngeneic 5T33MM model of multiple myeloma. This mouse model is representative for the human disease and can combine in vitro and in vivo studies. We first investigated the effects of PPP on the MM cells in vitro. We and others have previously demonstrated that IGF-1 induced ERK activation, involved in VEGF secretion and proliferation. When the 5T33MM cells were preincubated with 1microM PPP, Western blot analysis demonstrated the blocking of this activation. Furthermore, when the 5T33MM cells were preincubated with PPP for 30 min, IGF-1 induced VEGF secretion and proliferation of the 5T33MM cells were completely blocked. Next, we used the tyrosine kinase inhibitor PPP in vivo. 5T33MM cells were injected intravenously in C57BLKaLwRij mice and the development of the disease was monitored by measuring the serum paraprotein concentration. Mice were either treated with a low (17mM, IP, twice a day) or a high dose of PPP (50mM, IP, twice a day) or with the vehicle (DMSO/oil 9/1) from the day of injection with 5T33MM onward. At week 3, vehicle controls showed signs of morbidity and were sacrificed. The presence of tumor was measured by assessing serum paraprotein concentrations and determining the proportion of idiotype positive cells in the BM by flow cytometry. Angiogenesis was assessed by measuring the microvessel density on CD31 stained paraffin sections. The tumor burden in the bone marrow in the PPP treated mice was 77% lower than in vehicle treated animals (p< 0,0001) and the serum paraprotein concentration was 90% lower (p< 0,0001). The microvessel density in the BM of the PPP treated group was reduced by 60% (p< 0,02). In a separate survival experiment the mice were either treated with the vehicle or with the high dose (50mM) of PPP, from the time of tumor injection. Kaplan-Meier analysis demonstrated a significant increase in survival after treatment with PPP when compared with vehicle (28 vs. 18 days, p<0,001). These data demonstrate that the IGF-1RTK inhibitor PPP possesses strong anti-tumor activity, as demonstrated both in vitro and in vivo in a syngeneic model of multiple myeloma, and may therefore be an effective therapeutic candidate for MM treatment.

Multi-Targeted Receptor Tyrosine Kinase Inhibitor, ABT-869, Induces Apoptosis and Suppresses Proliferation of Ba/F3 FLT-3 ITD Mutant Cells in Vitro and in Vivo through Inhibition of FLT3 and AKT.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1938-1938
Author(s):  
Jenny E Hernandez ◽  
Joan Zape ◽  
Keith Glaser ◽  
Elliot Landaw ◽  
Cecilia Fu ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Lines ◽  
Receptor Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Mutant Cell ◽  
Vehicle Control ◽  
Ic50 Value ◽  
Mutant Cells

Abstract FLT3 is a receptor tyrosine kinase of the subclass III family that plays a vital role in the regulation of differentiation, proliferation and survival of normal hematopoietic cells. FLT3 mutations are often found in patients with acute myelogenous leukemia (AML) and confer a poor prognosis. Of these mutations, 15–35% are FLT3 ITD (internal tandem duplication) mutations and 5–7% are point mutations in the FLT3 kinase activation loop, e.g. D835V. We are studying the signaling pathways associated with a small molecule multi-targeted receptor tyrosine kinase inhibitor (RTKI), ABT-869. To determine the effects of ABT-869 in vitro and in vivo, a Ba/F3 mouse pro-B lymphocytic cell line harboring the FLT-3 ITD or FLT-3 D835V mutation was used as an isolated FLT-3 mutant model system. In vitro, ABT-869 is effective in inhibiting the proliferation of Ba/F3 Flt-3 ITD mutant cells (IC50 value of 1 nM) when compared to Ba/F3 Flt-3 D835V mutant (IC50 value between 1 and 10 μM) and Ba/F3 Flt-3 wildtype (WT) cells (IC50 value of 10 μM). Annexin V and propidium iodide staining of cells revealed that an increase in apoptosis occurred in Ba/F3 Flt-3 ITD mutant cells treated with 1μM ABT-869 for 24 hours (42.8%) when compared to untreated (4.7%) or vehicle control (4.0%) cells. Ba/F3 Flt-3 D835V mutant cell lines demonstrated a 12.5% rate of apoptosis at 1μM, compared to untreated (1.99%) and vehicle control (2.1%) cell lines. Propidium iodide staining of treated Ba/F3 Flt-3 WT cell lines revealed no difference in apoptosis when compared to untreated Ba/F3 Flt-3 WT cells or DMSO controls. PARP cleavage was observed in Ba/F3 FLT-3 ITD mutant cells, following 6 hours of treatment with 1 to 100 nM ABT-869, whereas no cleavage was observed in Ba/F3 WT cells treated with ABT-869. To study the effects of ABT-869 in vivo, we treated SCID mice injected with Ba/F3 Flt-3 ITD, Ba/F3 Flt-3 D835V, or Ba/F3 Flt-3 WT cells and monitored disease progression using bioluminescence imaging. The mice injected with the Ba/F3 FLT-3 ITD mutant cells and treated with vehicle control developed metastases and had a median survival time of 2 weeks. In contrast, the ABT-869 treated group had slower disease progression with median survival of 6.2 weeks (P&lt;0.008). Both control and treated mice injected with Ba/F3 FLT-3 D835V mutant cell lines developed metastases and had similar survival (median 1.7 and 1.9 weeks, respectively). Survival times of control and treated mice injected with Ba/F3 FLT-3 WT cells were also similar (median 8.4 and 8.1 weeks, respectively). Previous work identified that ABT-869 induced apoptosis of acute myeloid leukemia cells through inhibition of FLT-3 reception phosphorylation, which is observed as early as 3 hours after treatment. In Ba/F3 cells expressing FLT-3 ITD, ABT-869 also inhibited phosphorylation of AKT, which is upstream of the pro-apoptotic protein Bad. Therefore, our preclinical data suggest that ABT-869 induces apoptosis of FLT-3 ITD mutant cells both in vitro and in vivo. These studies provide rationale for the treatment of acute leukemia patients harboring the FLT3-ITD mutation with ABT-869 and the potential benefit of combining small molecule inhibitors that target both RTKs and AKT.

EphA2: expression in the renal medulla and regulation by hypertonicity and urea stress in vitro and in vivo

2005 ◽  
Vol 288 (4) ◽  
pp. F855-F866 ◽  
Author(s):  
Hongshi Xu ◽  
Wei Tian ◽  
Jessie N. Lindsley ◽  
Terry T. Oyama ◽  
Juan M. Capasso ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Transcriptional Activation ◽  
Egf Receptor ◽  
Collecting Duct ◽  
Renal Medulla ◽  
Epithelial Tissue ◽  
Promoter Fragment

EphA2, a member of the large family of Eph receptor tyrosine kinases, is highly expressed in epithelial tissue and has been implicated in cell-cell and cell-matrix interactions, as well as cell growth and survival. Expression of EphA2 mRNA and protein was markedly upregulated by both hypertonic stress and by elevated urea concentrations in cells derived from the murine inner medullary collecting duct. This upregulation likely required transactivation of the epidermal growth factor (EGF) receptor tyrosine kinase and metalloproteinase-dependent ectodomain cleavage of an EGF receptor ligand, based on pharmacological inhibitor studies. A human EphA2 promoter fragment spanning nucleotides −4030 to +21 relative to the putative EphA2 transcriptional start site was responsive to tonicity but insensitive to urea. A promoter fragment spanning −1890 to +128 recapitulated both tonicity- and urea-dependent upregulation of expression, consistent with transcriptional activation. Neither the bona fide p53 response element at approximately −1.5 kb nor a pair of putative TonE elements at approximately −3 kb conferred the tonicity responsiveness. EphA2 mRNA and protein were expressed at low levels in rat renal cortex but at high levels in the collecting ducts of the renal medulla and papilla. Water deprivation in rats increased EphA2 expression in renal papilla, whereas dietary supplementation with 20% urea increased EphA2 expression in outer medulla. These data indicate that transcription and expression of the EphA2 receptor tyrosine kinase are regulated by tonicity and urea in vitro and suggest that this phenomenon is also operative in vivo. Renal medullary EphA2 expression may represent an adaptive response to medullary hypertonicity or urea exposure.

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