Ba/F3 cells expressing ETV6-PDGFRb and FIP1L1-PDGFRa treated or not with Glivec
ABSTRACT: Gene expression profiles in Ba/F3 cells expressing ETV6-PDGFRB, FIP1L1-PDGFRA or a control vector, treated or not with imatinib (Glivec) Ba/F3 cells expressing FIP1L1-PDGFRA or ETV6-PDGFRB were cultured in the presence or absence of imatinib for 4 hours before RNA extraction followed by hybridization on Affymetrix microarrays. In a control condition Ba/F3 cells were cultured in the presence of IL3 in the absence or in the presence of imatinib for 4 hours before RNA extraction. 4 hours treatment with imatinib in Ba/F3 cells expressing ETV6-PDGFRB, FIP1L1-PDGFRA or a control vector
Project description:Gene expression profiles in Ba/F3 cells expressing ETV6-PDGFRB, FIP1L1-PDGFRA or a control vector, treated or not with imatinib (Glivec) Overall design: Ba/F3 cells expressing FIP1L1-PDGFRA or ETV6-PDGFRB were cultured in the presence or absence of imatinib for 4 hours before RNA extraction followed by hybridization on Affymetrix microarrays. In a control condition Ba/F3 cells were cultured in the presence of IL3 in the absence or in the presence of imatinib for 4 hours before RNA extraction. 4 hours treatment with imatinib in Ba/F3 cells expressing ETV6-PDGFRB, FIP1L1-PDGFRA or a control vector
Project description:Leukemia is a heterogeneous disease commonly associated with recurrent chromosomal translocations that involve tyrosine kinases including BCR-ABL, TEL-PDGFRB and TEL-JAK2. Most studies on the activated tyrosine kinases have focused on proximal signaling events, but little is known about gene transcription regulated by these fusions.Oligonucleotide microarray was performed to compare mRNA changes attributable to BCR-ABL, TEL-PDGFRB and TEL-JAK2 after 1 week of activation of each fusion in Ba/F3 cell lines. Imatinib was used to control the activation of BCR-ABL and TEL-PDGFRB, and TEL-JAK2-mediated gene expression was examined 1 week after Ba/F3-TEL-JAK2 cells were switched to factor-independent conditions.Microarray analysis revealed between 800 to 2000 genes induced or suppressed by two-fold or greater by each tyrosine kinase, with a subset of these genes commonly induced or suppressed among the three fusions. Validation by Quantitative PCR confirmed that eight genes (Dok2, Mrvi1, Isg20, Id1, gp49b, Cxcl10, Scinderin, and collagen V?1(Col5a1)) displayed an overlapping regulation among the three tested fusion proteins. Stat1 and Gbp1 were induced uniquely by TEL-PDGFRB.Our results suggest that BCR-ABL, TEL-PDGFRB and TEL-JAK2 regulate distinct and overlapping gene transcription profiles. Many of the genes identified are known to be involved in processes associated with leukemogenesis, including cell migration, proliferation and differentiation. This study offers the basis for further work that could lead to an understanding of the specificity of diseases caused by these three chromosomal translocations.
Project description:BACKGROUND: Rapid identification of diverse fusion genes with involvement of PDGFRA or PDGFRB in eosinophilia-associated myeloproliferative neoplasms is essential for adequate clinical management but is complicated by the multitude and heterogeneity of partner genes and breakpoints. DESIGN AND METHODS: We established a generic quantitative reverse transcriptase polymerase chain reaction to detect overexpression of the 3'-regions of PDGFRA or PDGFRB as a possible indicator of an underlying fusion. RESULTS: At diagnosis, all patients with known fusion genes involving PDGFRA (n=5; 51 patients) or PDGFRB (n=5; 7 patients) showed significantly increased normalized expression levels compared to 191 patients with fusion gene-negative eosinophilia or healthy individuals (PDGFRA/ABL: 0.73 versus 0.0066 versus 0.0064, P<0.0001; PDGFRB/ABL: 196 versus 3.8 versus 5.85, P<0.0001). The sensitivity and specificity of the activation screening test were, respectively, 100% and 88.4% for PDGFRA and 100% and 94% for PDGFRB. Furthermore, significant overexpression of PDGFRB was found in a patient with an eosinophilia-associated myeloproliferative neoplasm with uninformative cytogenetics and an excellent response to imatinib. Subsequently, a new SART3-PDGFRB fusion gene was identified by 5'-rapid amplification of cDNA ends polymerase chain reaction (5'-RACE-PCR). CONCLUSIONS: Quantitative reverse transcriptase polymerase chain reaction analysis is a simple and useful adjunct to standard diagnostic assays to detect clinically significant overexpression of PDGFRA and PDGFRB in eosinophilia-associated myeloproliferative neoplasms or related disorders.
Project description:In chronic eosinophilic leukemia, the transforming oncoprotein FIP1L1-PDGFRA is a major target of therapy. In most patients, the tyrosine kinase inhibitor (TKI) imatinib induces complete remission. For patients who are intolerant or resistant, novel TKIs have been proposed. We examined the in vitro effects of 14 kinase blockers on growth and function of EOL-1 cells, a FIP1L1-PDGFRA(+) eosinophil cell line. Major growth-inhibitory effects were seen with all PDGFR-blocking agents, with IC50 values in the low nanomolar range: ponatinib, 0.1-0.2 nmol/L; sorafenib, 0.1-0.2 nmol/L; masitinib, 0.2-0.5 nmol/L; nilotinib, 0.2-1.0 nmol/L; dasatinib, 0.5-2.0 nmol/L; sunitinib, 1-2 nmol/L; midostaurin, 5-10 nmol/L. These drugs were also found to block activation of PDGFR-downstream signaling molecules, including Akt, S6, and STAT5 in EOL-1 cells. All effective TKIs produced apoptosis in EOL-1 cells as determined by microscopy, Annexin-V/PI, and caspase-3 staining. In addition, PDGFR-targeting TKIs were found to inhibit cytokine-induced migration of EOL-1 cells. In all bioassays used, ponatinib was found to be the most potent compound in EOL-1 cells. In addition, ponatinib was found to downregulate expression of the activation-linked surface antigen CD63 on EOL-1 cells and to suppress the growth of primary neoplastic eosinophils. We also examined drug effects on Ba/F3 cells expressing two clinically relevant, imatinib-resistant, mutant forms of FIP1L1-PDGFRA, namely T674I and D842V. Strong inhibitory effects on both mutants were seen only with ponatinib. In summary, novel PDGFR-targeting TKIs may be alternative agents for the treatment of patients with imatinib-resistant chronic eosinophilic leukemia. Although several different PDGFR-targeting agents are effective, the most potent drug appears to be ponatinib.
Project description:In gastrointestinal muscles, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) is predominantly expressed by interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor-? (PDGFRA) polypeptide is expressed by so-called fibroblast-like cells. KIT and PDGFRA have been reported to be coexpressed in ICC precursors and gastrointestinal stromal tumors (GISTs), which originate from the ICC lineage. PDGFRA signaling has been proposed to stimulate growth of GISTs that express mutant KIT, but the effects and mechanisms of selective blockade of PDGFRA are unclear. We investigated whether inhibiting PDGFRA could reduce proliferation of GIST cells with mutant KIT via effects on the KIT-dependent transcription factor ETV1.We studied 53 gastric, small intestinal, rectal, or abdominal GISTs collected immediately after surgery or archived as fixed blocks at the Mayo Clinic and University of California, San Diego. In human GIST cells carrying imatinib-sensitive and imatinib-resistant mutations in KIT, PDGFRA was reduced by RNA interference (knockdown) or inhibited with crenolanib besylate (a selective inhibitor of PDGFRA and PDGFRB). Mouse ICC precursors were retrovirally transduced to overexpress wild-type Kit. Cell proliferation was analyzed by methyltetrazolium, 5-ethynyl-2'-deoxyuridine incorporation, and Ki-67 immunofluorescence assays; we also analyzed growth of xenograft tumors in mice. Gastric ICC and ICC precursors, and their PDGFRA(+) subsets, were analyzed by flow cytometry and immunohistochemistry in wild-type, Kit(+/copGFP), Pdgfra(+/eGFP), and NOD/ShiLtJ mice. Immunoblots were used to quantify protein expression and phosphorylation.KIT and PDGFRA were coexpressed in 3%-5% of mouse ICC, 35%-44% of ICC precursors, and most human GIST samples and cell lines. PDGFRA knockdown or inhibition with crenolanib efficiently reduced proliferation of imatinib-sensitive and imatinib-resistant KIT(+)ETV1(+)PDGFRA(+) GIST cells (50% maximal inhibitory concentration = 5-32 nM), but not of cells lacking KIT, ETV1, or PDGFRA (50% maximal inhibitory concentration >230 nM). Crenolanib inhibited phosphorylation of PDGFRA and PDGFRB, but not KIT. However, Kit overexpression sensitized mouse ICC precursors to crenolanib. ETV1 knockdown reduced KIT expression and GIST proliferation. Crenolanib down-regulated ETV1 by inhibiting extracellular-signal-regulated kinase (ERK)-dependent stabilization of ETV1 protein and also reduced expression of KIT and PDGFRA.In KIT-mutant GIST, inhibition of PDGFRA disrupts a KIT-ERK-ETV1-KIT signaling loop by inhibiting ERK activation. The PDGFRA inhibitor crenolanib might be used to treat patients with imatinib-resistant, KIT-mutant GIST.
Project description:BACKGROUND: Activating mutations in Kit receptor tyrosine kinase or the related platelet-derived growth factor receptor (PDGFR) play an important role in the pathogenesis of gastrointestinal stromal tumors (GIST). METHODS: This study investigated the activity of motesanib, an inhibitor of vascular endothelial growth factor receptors (VEGFR) 1, 2, and 3; PDGFR; and Kit, against primary activating Kit mutants and mutants associated with secondary resistance to imatinib. Single- and double-mutant isoforms of Kit were evaluated for their sensitivity to motesanib or imatinib in autophosphorylation assays and in Ba/F3 cell proliferation assays. RESULTS: Motesanib inhibited Kit autophosphorylation in CHO cell lines expressing primary activating mutations in exon 9 (AYins503-504, IC50 = 18 nM) and exon 11 (V560 D, IC50 = 5 nM; Delta552-559, IC50 = 1 nM). Motesanib also demonstrated activity against kinase domain mutations conferring imatinib resistance (V560D/V654A, IC50 = 77 nM; V560D/T670I, IC50 = 277 nM; Y823 D, IC50 = 64 nM) but failed to inhibit the imatinib-resistant D816V mutant (IC50 > 3000 nM). Motesanib suppressed the proliferation of Ba/F3 cells expressing Kit mutants with IC50 values in good agreement with those observed in the autophosphorylation assays. CONCLUSIONS: In conclusion, our data suggest that motesanib possesses inhibitory activity against primary Kit mutations and some imatinib-resistant secondary mutations.
Project description:Stimulatory antiplatelet derived growth factor receptor α (PDGFRA) antibodies have been associated with extensive chronic graft-versus-host disease (cGVHD). We performed a phase 1 dose escalation trial of imatinib in corticosteroid-dependent/refractory cGVHD to assess the safety of imatinib and test the hypothesis that abrogation of PDGFRA signaling can ameliorate the manifestations of cGVHD. Fifteen patients were enrolled. Mean follow-up time was 56.6 weeks (range, 18-82.4 weeks). Imatinib 400 mg daily was associated with more frequent moderate to life-threatening adverse events than 200 mg daily. The main adverse events were nausea, edema, confusion, diarrhea, liver function test elevation, fatigue, and myalgia. The overall response rate was 40% (6 of 15). The treatment failure rate was 40% (6 of 15). Twenty percent (3 of 15) of subjects had stable disease. Of 4 subjects with phospho-PDGFRA and phospho-PDGFRB immunohistochemistry studies before and after treatment, inhibition of phosphorylation was observed in 3 but correlated with response in one. Anti-PDGFRA antibodies were observed in 7 of 11 evaluable subjects but correlated with clinical activity in 4. We conclude that cGVHD responds to imatinib through multiple pathways that may include PDGFRA signal transduction. This study is registered at www.clinicaltrials.gov as #NCT00760981.
Project description:GZD824 is a novel third-generation BCR-ABL inhibitor. It entered Phase II clinical trials in China and Phase Ib clinical trials in USA in 2019 for treatment of patients with resistant chronic myeloid leukemia (CML). We found that at concentrations below 10 nM, GZD824 significantly suppresses FLT3, FGFR1 and PDGFR? kinase activities and inhibits their signal pathways in MV4-11Flt3-ITD, KG-1FGFR1OP2-FGFR1 and EOL-1FIP1L1-PDGFRa leukemia cells. It selectively inhibits the growth of MV4-11Flt3-ITD, KG-1FGFR1OP2-FGFR1 and EOL-1FIP1L1-PDGFRa cells, and also effectively suppresses the growth of Ba/F3-FLT3-ITD cells harboring F691I and other mutations with IC50 values <10 nM. GZD824 induces G0/G1 phase arrest and apoptosis in MV4-11, KG-1 and EOL-1 cells and activates cleavage of caspase-3 and PARP. In MV4-11, Ba/F3-ITD-F691I and KG-1 mouse xenograft models, GZD824 at 10 or 20 mg/kg, q2d, p.o. almost completely eradicates tumors. It also inhibits the viability of primary leukemic blasts from a FLT3-ITD positive AML patient but not those expressing native FLT3. Thus GZD824 suppresses leukemia cells of FLT3-ITD-driven AML and other hematologic malignancies driven by FGFR1 or PDGFRa, and it may be considered to be a novel agent for the treatment of leukemia.
Project description:ENDOGLIN/CD105 (ENG) is a transmembrane glycoprotein and an auxiliary unit of the transforming growth factor-β (TGF-β); receptor, expressed predominantly in vascular endothelium. Noteworthy, Eng mRNA expression has been reported also in Kit(+) interstitial cells of Cajal (ICC) in the mouse intestine. Gastrointestinal stromal tumours (GIST) are thought to derive from ICC. Here we have investigated Eng expression in the Kit(K641E) mouse GIST model, in human GIST and in the Ba/F3 cell model. In wild type (WT) mouse antrum, Eng immunoreactivity (-ir) was detected in CD34(+) /CD31(+) endothelium and in Kit(+) ICC. In Kit(K641E) mice, hyperplasia of Kit(+) cells made Eng-ir even more evident. Quantitative PCR confirmed the increased expression of Eng transcript in Kit(K641E) mice. On human GIST TMA, 26/49 cases stained positive for ENG. Strong ENG staining was associated with malignant and high-risk tumours. ENG negative cases were predominantly of the epithelioid type or harboured PDGFRA mutation. In vitro, Eng mRNA was up-regulated in Ba/F3 cell lines stably expressing various oncogenic Kit mutations (K641E, del559, del814). This effect appeared to be independent of Kit activation, as neither the stimulation of WT Kit by its ligand SCF, nor the inhibition of Kit autophosphorylation by imatinib mesylate in oncogenic mutants, altered Eng expression. Elevated Eng expression in Kit oncogenic mutants appeared rather to be indirectly mediated by DNA hypomethylation, because treatment with the demethylating agent 5-Aza/dC increased Eng mRNA expression in Kit(WT) cells. ENG expression in ICC and in GIST deserves further consideration as ENG is emerging as a potential target for cancer therapy.