Expression Of Platelet-derived Growth Factor Receptor-alpha Research Articles

Suppression of Platelet-Derived Growth Factor Receptor-Alpha Overcomes Resistance to Trastuzumab through STAT3-Dependent IL-6 Reduction in HER2-Positive Breast Cancer Cells

Platelet-derived growth factor receptor (PDGFR) plays an essential role in the proliferation and invasion of malignant cancer cells. However, the functional role of PDGFR alpha (PDGFRA) in HER2-positive (HER2+) breast cancer has not been fully clarified yet. Thus, the objective of this study was to investigate the clinical significance of PDGFRA and the therapeutic potential of PDGFR inhibitors as part of an effort to overcome trastuzumab (TRZ) resistance. Aberrant PDGFRA expression is closely associated with decreased survival in HER2+ breast cancers. Therefore, we established BT474 trastuzumab-sensitive (TRZ_S) and trastuzumab-resistant (TRZ_R) cells to investigate the association between PDGFR signaling and TRZ resistance. We found that PDGFRA was significantly upregulated in the BT474 TRZ_R cells. In addition, IL-6 expression, which was also found to be upregulated in the TRZ_R cells, was induced by PDGFC, a ligand of PDGFR. Next, we investigated the effects of ponatinib and sunitinib, PDGFR inhibitors, on the BT474 TRZ_R and HCC1954 (TRZ-resistant cell line) cells. These inhibitors decreased cell viability and migration in a dose-dependent manner. Additionally, IL-6 expression was decreased by ponatinib in both the BT474 TRZ_R and HCC1954 cells. In contrast, IL-6 was not suppressed by TRZ, implying that the PDGFRA/STAT3/IL-6 axis is associated with resistance to TRZ. In addition, we found that STAT3 and ERK phosphorylation were increased in the BT474 TRZ_R cells. IL-6 expression was suppressed by a STAT3 inhibitor, indicating that IL-6 expression is modulated downstream of STAT3. Taken together, these results suggest that PDGFRA could serve as a therapeutic target to overcome TRZ resistance.

JUN and PDGFRA as Crucial Candidate Genes for Childhood Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder, characterized by marked genetic heterogeneity. In this study, two independent microarray datasets of cerebellum of ASD were integrative analyzed by NetworkAnalyst to screen candidate crucial genes. NetworkAnalyst identified two up-regulated genes, Jun proto-oncogene (JUN) and platelet derived growth factor receptor alpha (PDGFRA), as the most crucial genes in cerebellum of ASD patients. Based on KEGG pathway database, genes associated with JUN in the cerebellum highlight the pathways of Th17 cell differentiation and Th1 and Th2 cell differentiation. Genes associated with PDGFRA in the cerebellum were found enriched in pathways in EGFR tyrosine kinase inhibitor resistance and Rap1 signaling pathway. Analyzing all differentially expressed genes (DEGs) from the two datasets, Gene Set Enrichment Analysis (GSEA) brought out IL17 signaling pathway, which is related to the expression of JUN and PDGFRA. The ImmuCellAI found the elevated expression of JUN and PDGFRA correlating with increased Th17 and monocytes suggests JUN and PDGFRA may regulate Th17 cell activation and monocytes infiltrating. Mice model of maternal immune activation demonstrated that JUN and PDGFRA are up-regulated and related to the ASD-like behaviors that provide insights into the molecular mechanisms underlying the altered IL17 signaling pathway in ASD and may enable novel therapeutic strategies.

High PDGFRA Expression Does Not Serve As Effective Therapeutic Target in ERG-Deleted B-Cell Precursor Acute Lymphoblastic Leukemia

Background and aimsSeveral oncogenic aberrations of receptor tyrosine kinases occur in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Here, we describe a subgroup of pediatric patients lacking common chromosomal aberrations but with high gene expression of platelet-derived growth factor receptor alpha (PDGFRA). Oncogenic fusions involving the tyrosine kinase domain of PDGFRA are recurrent in eosinophilic leukemia and are responsive to kinase inhibitors (Cools et al. N Engl J Med 2003). One case with a FIP1L1-PDGFRA fusion was reported in adult BCP-ALL (Roberts et al. J Clin Oncol 2016). Therefore, we tested for genomic aberrations underlying the overexpression of PDGFRA and evaluated the inhibition of PDGFRα as treatment option in pediatric BCP-ALL.MethodsExpression profiling was performed using Affymetrix U133 plus 2.0 arrays. BCR-ABL1 -like cases were identified using the 110 probe set expression signature (Den Boer et al. Lancet Oncol 2009). High PDGFRA was defined as log2 expression of probe set 203131_at >5.9. FISH was performed using Cytocell FIP1L1/CHIC2/PDGFRA probes. Targeted locus amplification (de Vree et al. Nat Biotechnol 2014) was performed using custom-designed primers. Cells were treated for 10 minutes with 10 ng/mL PDGF-BB with or without 10 µM imatinib or 1 µM CP673451 and analyzed by western blot. Cell viability after 4 day exposure to imatinib (0.2µM - 1.6µM - 12.5µM) or CP673451 (0.02µM - 0.16µM - 1.25µM) was assessed by flow cytometry.ResultsGene expression analysis of 654 pediatric ALL cases identified high expression of PDGFRA in 26 of 574 BCP-ALL cases (4.5%). High PDGFRA expression was exclusively found in cases belonging to the B-other (n=22) or BCR-ABL1 -like (n=4) subtypes, and not present in ETV6-RUNX1, high hyperdiploid, TCF3-PBX1, BCR-ABL1 and KMT2A -rearranged BCP-ALL subtypes or T-ALL. PDGFRA expression was confirmed by RT-qPCR, and also found in the BCP-ALL cell line Nalm6. Using RT-PCR we did not detect any of the six reported PDGFRA fusions, which involve FIP1L1, STRN, BCR, CDK5RAP2, ETV6, and KIF5B, nor did we detect novel structural aberrations affecting PDGFRA by FISH. We studied the PDGFRA locus at high resolution by targeted locus amplification. Following DNA cross-linking, digestion, and proximity-based re-ligation, targeted amplification was performed from viewpoints in intron 2 and exon 15 of PDGFRA. In the eosinophilic cell line EOL1 we confirmed an interstitial deletion on chromosome 4 which rearranges FIP1L1 intron 12 to PDGFRA exon 12. The results obtained from Nalm6 and primary BCP-ALL samples with high PDGFRA expression did not indicate genomic aberrations of PDGFRA.ERG deletions were present in 10 of 16 PDGFRA -high cases (63%), compared with 0% in PDGFRA -low BCP-ALL cases (n=71; p<0.001). Vice versa, all ERG-deleted cases expressed high levels of PDGFRA . Recently, a novel BCP-ALL subtype was described with rearrangements of the transcription factor DUX4 and deletion of ERG (Lilljebjörn et al. Nat Commun 2016; Yasuda et al. Nat Genet 2016; Zhang et al. Nat Genet 2016). Furthermore, the high PDGFRA-expressing cell line Nalm6 carries a DUX4 rearrangement (Yasuda et al. Nat Genet 2016) and PDGFRA is highly expressed in DUX4 BCP-ALL and its transcription start site directly bound by DUX4 (Zhang et al. Nat Genet 2016). Therefore, we regard it as highly likely that the PDGFRA-high cases represent this novel DUX4- rearranged subtype.Because transcription factors are difficult to target with small molecule inhibitors, PDGFRα inhibition could represent a treatment option. Exposure to the PDGFR inhibitors imatinib and CP673451 reduced phosphorylation of PDGFRα in Nalm6 and primary BCP-ALL cells. However, ex vivo drug exposure revealed no cytotoxic effect of the PDGFR inhibitors on PDGFRA-high BCP-ALL cells, either in the presence or absence of the ligand PDGF-BB. Interestingly, we found a marked ex vivo sensitivity towards prednisolone in PDGFRA-high cases, in line with the good prognosis reported for ERG -deleted/DUX4-rearranged BCP-ALL.ConclusionsWe found high PDGFRA expression, in the absence of genomic aberrations of the PDGFRA locus, in a subgroup of BCP-ALL patients with frequent ERG deletions. PDGFR inhibition caused abrogation of PDGFRα phosphorylation but was not cytotoxic in PDGFRA-high expressing BCP-ALL cells. Therefore, high PDGFRA expression does not serve as effective therapeutic target in ERG -deleted BCP-ALL. DisclosuresNo relevant conflicts of interest to declare.

Requirements for guinea pig cytomegalovirus tropism and antibody neutralization on placental amniotic sac cells

Congenital cytomegalovirus (cCMV) is a leading cause of birth defects. The guinea pig is the only small cCMV animal model. Guinea pig cytomegalovirus (GPCMV) encodes similar glycoprotein complexes to human CMV (HCMV) including gB and the gH-based pentamer complex (PC). In HCMV, both gB and PC are neutralizing antibody antigens. The relevance of GPCMV PC for virus tropism and vaccine target remains controversial. A novel guinea pig placental amniotic sac epithelial (GPASE) cell-line did not express viral cell receptor platelet derived growth factor receptor alpha (PDGFRA) and resulted in requirement for the PC for GPCMV infection unless PDGFRA was ectopically expressed. High titer anti-gB sera from a GPCMV gB vaccine study was evaluated for GPCMV neutralizing capability on GPASE cells in comparison to convalescent sera from GPCMV(PC+) or GPCMV(PC-) infected animals. Anti-gB sera neutralized fibroblast infection but was less effective compared to anti-GPCMV(PC-), which had antibodies to gH/gL. However, both anti-GPCMV(PC-) and anti-gB sera similarly had reduced neutralizing capability on GPASE and renal epithelial cells in comparison to anti-GPCMV(PC+) sera, which had additional antibodies to PC. Overall, results demonstrate the importance of the PC for GPCMV tropism to various cell types that lack PDGFRA expression and the limited ability of anti-gB sera to neutralize GPCMV on non-fibroblast cells despite the essential nature of gB glycoprotein.

A Functional Synonymous Variant in PDGFRA Is Associated with Better Survival in Acral Melanoma.

Purpose: Polymorphisms of genes in the platelet-derived growth factor (PDGF) signaling pathway have been found to predict cutaneous melanoma (CM) survival, but their clinical effects in acral melanoma (AM) patients have not been explored. The aim of this study was to characterize the functional effect of the tag single-nucleotide polymorphism (SNP) rs2228230:C>T and assess its association with clinical outcomes in AM patients.Methods: The effect of rs2228230:C>T on mRNA structures and codon usage values were evaluated using in silico analyses. PDGF receptor alpha (PDGFRA) expression vectors with the rs2228230:C or rs2228230:T allele were constructed to evaluate the expression and signaling activity of PDGFRA. The expression of PDGFRA in AM samples was measured using in situ RNAscope hybridization and immunohistochemical staining. The association of the rs2228230 genotype with survival was analyzed in two independent AM cohorts.Results: In silico analyses indicated that the rs2228230:T allele increases the minimum free energy and reduces synonymous codon usage. The rs2228230:T allele decreased the expression of PDGFRA by reducing the stability of its mRNA and protein as well as the signaling activity of the MAPK and PI3K/AKT pathways. PDGFRA mRNA and protein expression was significantly reduced in AM tissues with the rs2228230:T allele. The progression-free survival and overall survival of AM patients with the rs2228230:T allele were significantly longer than those of patients with the CC genotype.Conclusion: Our study indicated that rs2228230:T can reduce the expression of PDGFRA and downstream signaling activity and is associated with better survival in AM patients.

Imatinib +/- Brentuximab Vedotin Induces Sustained Complete Remission in Chemotherapy-Resistant Anaplastic Large Cell Lymphoma Expressing PDGFR

Background. Anaplastic lymphoma kinase positive systemic anaplastic large cell lymphoma (ALK+ ALCL) represents an aggressive T-cell malignancy that primarily affects children and younger adults. Upon treatment with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or CHOP-like regimens its prognosis is relatively favourable compared to other aggressive T-cell lymphomas, however, the rare event of disease reoccurrence remains particularly challenging. ALCL is CD30 positive, and mono treatment with brentuximab vedotin (BrV) can achieve long term remissions in only few relapsed patients not receiving consolidating stem cell transplantation (Pro B. et al. Blood 2017). A subset of ALCL patients express platelet derived growth factor receptor (PDGFR) alpha or beta in their tumour cells which is induced by ALK via AP-1 transcription factor activation. We previously demonstrated that PDGFR blockade by the Abl/c-Kit/PDGFR kinase inhibitor imatinib is an effective treatment concept for ALK+ ALCL in experimental mouse models (Laimer D. et al. Nature Med. 2012). Here we aimed to assess the prognostic value of PDGFR expression and to probe the clinical value of its targeting by imatinib in ALK+ ALCL patients. Methods. The impact of PDGFR expression to predict clinical outcome was analyzed in samples of 98 ALK+ ALCL patients included in the studies NHL-BFM 90, 95 and ALCL99 between 1992 and 2006. Six patients (age 18-45) with chemo-refractory ALK+ ALCL were prospectively treated with imatinib plus (4) or minus (2) BrV. 33% (2/6) had previously received salvage treatment with stem cell transplantation (SCT). Three of the patients had been enrolled in a prospective single-arm study combining imatinib and BrV, a trial that was terminated due to poor patient recruitment (AGMT ALCL1 trial, EudraCT No.: 2013-003505-26, supported by Takeda®). PDGFR alpha and beta expression analyses were performed on tumour samples of treated patients. Results. ALK+ ALCL patients with high PDGFR expression on tumor cells (n=11) had a significantly lower event-free survival rate (EFS) at 5 years compared to patients with no or low expression (n=87) (27±13% versus 70±5%, respectively, p&lt;0.001, figure 1a). Four of the six patients with relapsed/ refractory ALK+ ALCL responded to imatinib +/- BrV with a rapid metabolic complete remission (CR). At a median clinical follow-up of 52 months (range 20 to108) all responding patients demonstrated an ongoing CR without SCT even after imatinib was discontinued with an average time on imatinib of 49 weeks (range 32 to 130; figure 1b). Half (2/4) of responding patients received no or only 1 infusion of BrV; both non-responding patients had received BrV. Importantly, despite the few patients that could be prospectively treated, PDGFR expression status had a significant impact on clinical course. All patients (4/4) who expressed either PDGFR alpha or beta in tumour cells and in the lymph node microenvironment responded with an ongoing metabolic CR, whereas the two patients whose lymphoma cells had negative PDGFR expression failed to respond (EFS: 4 versus 210 weeks, p = 0.018; figure 1c). Conclusions. 1) PDGFR expression is a poor risk factor in ALK+ ALCL. 2) Imatinib alone or in combination with BrV overcomes chemo-resistance conferred by PDGFR expression. 3) Imatinib treatment can be discontinued with ongoing CR. 4) These observations suggest that PDGFR expressing ALK+ ALCL patients might benefit from early treatment with imatinib. Disclosures Staber: Takeda-Millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; MSD: Honoraria, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Klapper:Roche, Takeda, Amgen, Regeneron: Honoraria, Research Funding. Mayerhoefer:Siemens: Research Funding, Speakers Bureau; BMS: Speakers Bureau. Greil:Gilead: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Ratiopharm: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Eisai: Honoraria; Genentech: Honoraria, Research Funding; Janssen-Cilag: Honoraria; GSK: Research Funding; Sandoz: Honoraria; Roche: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Consultancy, Honoraria; MSD: Consultancy, Honoraria, Research Funding; Merck: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Sanofi Aventis: Honoraria; Boehringer Ingelheim: Honoraria; Mundipharma: Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Jaeger:Novartis, Roche, Sandoz: Consultancy; AbbVie, Celgene, Gilead, Novartis, Roche, Takeda Millennium: Research Funding; Amgen, AbbVie, Celgene, Eisai, Gilead, Janssen, Novartis, Roche, Takeda Millennium, MSD, BMS, Sanofi: Honoraria; Celgene, Roche, Janssen, Gilead, Novartis, MSD, AbbVie, Sanofi: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Imatinib - bcr-abl Inhibitor

Protective function of pirfenidone and everolimus on the development of chronic allograft rejection after experimental lung transplantation.

Long-term survival of lung allografts is limited by chronic rejection (CR). Oxidative stress (OxS) plays a central role in the development of CR. We investigated the influence of pirfenidone (alone or in combination with everolimus) on OxS and CR. A rat model of left lung allo-transplantation (F344-to-WKY) was used to evaluate the effects of pirfenidone alone [0,85% in chow from postoperative day (POD) -3 to 20/60] and in combination with everolimus [2,5 mg/kg bw daily from POD 7 to 20/60]. Allografts of non-treated animals, everolimus treated animals and right, non-transplanted lungs were used as references. Immunohistology of myeloperoxidase (MPO), haemoxygenase-1 (HO-1), iron and platelet-derived-growth-factor-receptor-alpha (PDGFR-a) were performed. On POD 20, all groups showed severe acute rejection (ISHLT A3-4/B1R-B2R). Groups treated with pirfenidone showed a lower interstitial inflammatory infiltration and a lower participation of highly fibrotic degenerated vessels (ISHLT-D2R). In the long term follow up (POD 60), pirfenidone alone significantly reduced chronic airway rejection (ISHLT-C; p≤0.05), interstitial fibrosis (IF; p≤0.05), content of collagen (p≤0.05), expression of PDGFR-a (p≤0.05) and the deposition of iron (p≤0.05). All groups treated with pirfenidone showed a high expression of the cytoprotective enzyme HO-1 (p≤0.05). The additional application of everolimus resulted in a significant decrease of chronic airway rejection (ISHLT-C; p≤0.05), vasculopathy (ISHLT; p≤0.05) and IF (p≤0.05). In conclusion, early application of pirfenidone inhibited the progression of CR by its anti-fibrotic and anti-oxidative properties. The additional application of an m-TOR-inhibitor increased the anti-fibrotic effects of pirfenidone which resulted in a reduction of CR after experimental LTx.

Differential Expression of miR-34c and Its Predicted Target Genes in Testicular Tissue at Different Development Stages of Swine.

To verified the target genes of miR-34c, bioinformatics software was used to predict the targets of miR-34c. Three possible target genes of miR-34c related to spermatogenesis and male reproductive development: zinc finger protein 148 (ZNF148), kruppel-like factor 4 (KLF4), and platelet-derived growth factor receptor alpha (PDGFRA) were predicted. Then, the expression of miR-34c and its target genes were detected in swine testicular tissue at different developmental stages by quantitative polymerase chain reaction. The results suggested that the expression of PDGFRA has the highest negative correlation with miR-34c. Then immunohistochemical staining was done to observe the morphology of swine testicular tissue at 2-days and 3, 4, 5-months of age, which indicated that PDGFRA was mainly expressed in the support cells near the basement membrane during the early development stages of testicular tissue, but that the expression of PDGFRA was gradually reduced in later stages. Therefore, western blot analyzed that the highest expression of PDGFRA was generated in 2-days old testicular tissues and the expression levels reduced at 3 and 4-months old, which correlated with the results of immunohistochemical staining. In conclusion, PDGFRA is a target gene of miR-34c.

PDGF-D expression is down-regulated by TGFβ in fibroblasts.

Transforming growth factor-β (TGFβ) is a key mediator of fibrogenesis. TGFβ is overexpressed and activated in fibrotic diseases, regulates fibroblast differentiation into myofibroblasts and induces extracellular matrix deposition. Platelet-derived growth factor (PDGF) is also a regulator of fibrogenesis. Some studies showed a link between TGFβ and PDGF in certain fibrotic diseases. TGFβ induces PDGF receptor alpha expression in scleroderma fibroblasts. PDGF-C and -D are the most recently discovered ligands and also play a role in fibrosis. In this study, we report the first link between TGFβ and PDGF-D and -C ligands. In normal fibroblasts, TGFβ down-regulated PDGF-D expression and up-regulated PDGF-C expression at the mRNA and protein levels. This phenomenon is not limited to TGFβ since other growth factors implicated in fibrosis, such as FGF, EGF and PDGF-B, also regulated PDGF-D and PDGF-C expression. Among different kinase inhibitors, only TGFβ receptor inhibitors and the IκB kinase (IKK) inhibitor BMS-345541 blocked the effect of TGFβ. However, activation of the classical NF-κB pathway was not involved. Interestingly, in a model of lung fibrosis induced by either bleomycin or silica, PDGF-D was down-regulated, which correlates with the production of TGFβ and other fibrotic growth factors. In conclusion, the down-regulation of PDGF-D by TGFβ and other growth factors may serve as a negative feedback in the network of cytokines that control fibrosis.

ALK gene aberrations and the JUN/JUNB/PDGFR axis in metastatic NSCLC.

Anaplastic lymphoma kinase (ALK) gene aberrations are found in several tumor types including anaplastic large cell lymphoma (ALCL) and non-small cell lung cancer (NSCLC). Crizotinib, an inhibitor of ALK-fusion proteins, has shown clinical activity, but resistance mechanisms limit long-lasting disease control. It has been shown that ALK-NPM fusion upregulates platelet-derived growth factor receptor beta (PDGFRB) via JUN and transcription factor Jun B (JUNB) in ALCL. PDGFRB inhibition has been identified as therapy option for ALK-positive ALCL. Here, we investigated the ALK/JUN/JUNB/transcription factor Jun C (JUNC)/PDGFR axis in metastatic NSCLC with regard to ALK aberrations. We performed immunohistochemical analysis of platelet-derived growth factor receptor alpha (PDGFRA), PDGFRB, JUNB and JUNC expression in formalin-fixed, paraffin-embedded specimens of 15 NSCLC brain metastases (5 ALK translocations, 3 of them involving EML4, 5 ALK amplifications, 5 without ALK aberrations). We did not find a statistically significant difference in expression of PDGFRA, PDGFRB, JUNB or JUNC in tumor samples with normal ALK status, ALK amplification or ALK translocations (Kruskal-Wallis test p>0.05). Interestingly, PDGFRB was not expressed in tumor cells (but in vascular and stromal cells) in any of our cases. Our data argue against PDGFRB activation in association with ALK gene aberrations in metastatic NSCLC and thus seem to imply differential pathobiological roles of ALK alterations in lung cancer and lymphoma, possibly depending on different fusion partner genes. These results may be relevant for targeted therapies, as they indicate that inhibition of PDGFRB in ALK translocated NSCLC seems to be no therapeutic opportunity.

Platelet-derived growth factor alpha mediates the proliferation of peripheral T-cell lymphoma cells via an autocrine regulatory pathway

Peripheral T-cell lymphomas not otherwise specified (PTCL/NOS) are very aggressive tumors characterized by consistent aberrant expression of platelet-derived growth factor receptor alpha (PDGFRA). In this study, we aimed to identify the determinants of PDGFRA activity in PTCL/NOS and to elucidate the biological consequences of its activation. We observed overexpression of the PDGFRA gene by gene expression profiling in most of the tested PTCLs and confirmed the expression of PDGFRA and phospho-PDGFRA using immunohistochemistry. The integrity of the PDFGRA locus was demonstrated using several different approaches, including massive parallel sequencing and Sanger sequencing. PDGF-AA was found to be expressed and secreted by PTCL/NOS cells and to be necessary and sufficient for PDGFRA phosphorylation ex vivo by sustaining an autocrine stimulation. We documented consistently high PDGF-A expression in primary biopsies and patients' plasma and tracked PDGFRA signaling in primary tumors, achieving evidence of its activation. Indeed, we found that STAT1 and STAT5 are implicated in PDGFRA signaling transduction. Finally, we demonstrated that PDGFRA activation supported tumor cell proliferation and provided the first evidence of the anti-lymphoma activity of PDGRA inhibition in a PTCL/NOS patient. Altogether, our data demonstrated that PDGFRA activity fosters PTCL/NOS proliferation via an autocrine loop.

Better Prognosis of Patients with Glioma Expressing FGF2-Dependent PDGFRA Irrespective of Morphological Diagnosis

Signaling of platelet derived growth factor receptor alpha (PDGFRA) is critically involved in the development of gliomas. However, the clinical relevance of PDGFRA expression in glioma subtypes and the mechanisms of PDGFRA expression in gliomas have been controversial. Under the supervision of morphological diagnosis, analysis of the GSE16011 and the Repository of Molecular Brain Neoplasia Data (Rembrandt) set revealed enriched PDGFRA expression in low-grade gliomas. However, gliomas with the top 25% of PDGFRA expression levels contained nearly all morphological subtypes, which was associated with frequent IDH1 mutation, 1p LOH, 19q LOH, less EGFR amplification, younger age at disease onset and better survival compared to those gliomas with lower levels of PDGFRA expression. SNP analysis in Rembrandt data set and FISH analysis in eleven low passage glioma cell lines showed infrequent amplification of PDGFRA. Using in vitro culture of these low passage glioma cells, we tested the hypothesis of gliogenic factor dependent expression of PDGFRA in glioma cells. Fibroblast growth factor 2 (FGF2) was able to maintain PDGFRA expression in glioma cells. FGF2 also induced PDGFRA expression in glioma cells with low or non-detectable PDGFRA expression. FGF2-dependent maintenance of PDGFRA expression was concordant with the maintenance of a subset of gliogenic genes and higher rates of cell proliferation. Further, concordant expression patterns of FGF2 and PDGFRA were detected in glioma samples by immunohistochemical staining. Our findings suggest a role of FGF2 in regulating PDGFRA expression in the subset of gliomas with younger age at disease onset and longer patient survival regardless of their morphological diagnosis.

PDGFRA is essential for the development of a novel cardiac cell type with stem-like properties

Background We recently described a novel adult stem-like cell. Cardiac mesenchymal stromal cells (cMSCs) are phenotypically similar to MSCs from other organs, showing long-term growth and broad differentiation potential in vitro. In vivo transplantation assays demonstrate cardiomyocyte, endothelial and smooth muscle differentiation. Platelet-derived growth factor receptor alpha (PDGFRA) marks cMSCs, and demonstrates their epicardial location during embryogenesis. Over 100 clinical trials are currently using MSCs, however their natural role/s remain largely unknown, as are genes required for their development and maintenance. Aims To determine the role of PDGFRA in cMSC development. Methods Mouse embryos were genetically modified to express either no, or low levels of PDGFRA in the epicardium, or to express a long-lived fluorescent protein (GFP) from Pdgfra in order to trace progeny of PDGFRA-expressing cells. Embryos were analysed by confocal immunofluorescence microscopy and flow cytometry. cMSCs were quantified by colony forming assays. Results Three classes of epicardial-derived cells were identified with varying Pdgfra-GFP, PDGFRA and PDGFRB expression. Mice mutant for Pdgfra were deficient in two of these populations, and in cMSCs. Pdgfra-GFP was traced into coronary smooth muscle cells. Conclusions PDGFRA is essential for cMSC development. The data supports cMSC as progenitors for coronary smooth muscle. We recently described a novel adult stem-like cell. Cardiac mesenchymal stromal cells (cMSCs) are phenotypically similar to MSCs from other organs, showing long-term growth and broad differentiation potential in vitro. In vivo transplantation assays demonstrate cardiomyocyte, endothelial and smooth muscle differentiation. Platelet-derived growth factor receptor alpha (PDGFRA) marks cMSCs, and demonstrates their epicardial location during embryogenesis. Over 100 clinical trials are currently using MSCs, however their natural role/s remain largely unknown, as are genes required for their development and maintenance. To determine the role of PDGFRA in cMSC development. Mouse embryos were genetically modified to express either no, or low levels of PDGFRA in the epicardium, or to express a long-lived fluorescent protein (GFP) from Pdgfra in order to trace progeny of PDGFRA-expressing cells. Embryos were analysed by confocal immunofluorescence microscopy and flow cytometry. cMSCs were quantified by colony forming assays. Three classes of epicardial-derived cells were identified with varying Pdgfra-GFP, PDGFRA and PDGFRB expression. Mice mutant for Pdgfra were deficient in two of these populations, and in cMSCs. Pdgfra-GFP was traced into coronary smooth muscle cells. PDGFRA is essential for cMSC development. The data supports cMSC as progenitors for coronary smooth muscle.

PDGFRA is essential for the development of a novel cardiac cell type with stem-like properties

Background We recently described a novel adult stem-like cell. Cardiac mesenchymal stromal cells (cMSCs) are phenotypically similar to MSCs from other organs, showing long-term growth and broad differentiation potential in vitro. In vivo transplantation assays demonstrate cardiomyocyte, endothelial and smooth muscle differentiation. Platelet-derived growth factor receptor alpha (PDGFRA) marks cMSCs, and demonstrates their epicardial location during embryogenesis. Over 100 clinical trials are currently using MSCs, however their natural role/s remain largely unknown, as are genes required for their development and maintenance. Aims To determine the role of PDGFRA in cMSC development. Methods Mouse embryos were genetically modified to express either no, or low levels of PDGFRA in the epicardium, or to express a long-lived fluorescent protein (GFP) from Pdgfra in order to trace progeny of PDGFRA-expressing cells. Embryos were analysed by confocal immunofluorescence microscopy and flow cytometry. cMSCs were quantified by colony forming assays. Results Three classes of epicardial-derived cells were identified with varying Pdgfra-GFP, PDGFRA and PDGFRB expression. Mice mutant for Pdgfra were deficient in two of these populations, and in cMSCs. Pdgfra-GFP was traced into coronary smooth muscle cells. Conclusions PDGFRA is essential for cMSC development. The data supports cMSC as progenitors for coronary smooth muscle.

The effect of crenolanib (CP-868596) on phosphorylation of the imatinib-resistant D842V PDGFRA activating mutation associated with advanced gastrointestinal stromal tumors.

10012 Background: 8-10% of gastrointestinal stromal tumors (GIST) have activating mutations of the platelet-derived growth factor receptor alpha (PDGFRA) gene. The most common PDGFRA mutation is the D842V mutation encoded by exon 18. This gain-of-function mutation results in constitutive tyrosine kinase activity. Currently approved tyrosine kinase inhibitors (TKIs), such as imatinib (IM) or sunitinib, have little to no in vitro activity against this mutation. Clinically, these drugs are ineffective for the treatment of patients whose GIST has a D842V mutation. In addition, the D842V mutation can develop as a secondary IM resistance mutation during treatment of patients with primary IM-sensitive GIST or hypereosinophillic syndrome. Crenolanib (formerly CP-868596) is an orally bioavailable, highly potent and selective PDGFR TKI. Phase I trials of crenolanib (CR) have shown a favorable toxicity profile, and achievable serum concentrations as high as 2,000 nM. Methods: Mutant PDGFRα isoforms were expressed by transient transfection of Chinese hamster ovary cells. The transfected cells were treated with various concentrations of CR or IM. Protein lysates were immunoprecipitated with an anti-PDGFRA antibody, followed by sequential immunoblotting for activated- and total-PDGFRA. IC50 (50% inhibitory concentration) was measured by densitometry of the phosphoPDGFRA bands and normalization using total PDGFRA expression. Results: CR was effective in blocking the activity of single or compound PDGFRA D842V mutant kinases. In contrast, IM had no significant activity against these same mutant kinases. Conclusions: GISTs with PDGFRA D842V mutations are clinically resistant to currently approved TKIs. CR blocks the kinase activity of PDGFRA D842V mutants (single and compound) at clinically achievable concentrations, providing a potential new therapeutic modality for GIST patients. A phase II clinical study of CR for treatment of GIST patients with primary or secondary PDGFRA D842V mutation is currently being initiated. Mutation CR IC50(nM) IM IC50(nM) PDGFRA WT 13 30 PDGFRA D842V 20 >1,000 PDGFRA V561D + D842V 35 >1,000 PDGFRA T674I + D842V 26 >1,000

Abstract 3586: CP-868,596, a highly potent PDGFR inhibitor, inhibits phosphorylation of the imatinib-resistant PDGFRA D842V activating mutation associated with advanced GIST

Abstract Background: 8-10% of gastrointestinal stromal tumors (GIST) have activating mutations of the platelet-derived growth factor receptor alpha (PDGFRA) kinase. The most common PDGFRA mutation is the D842V substitution that is encoded by exon 18. This gain-of-function mutation results in auto-phosphorylation and constitutive activation of PDGFRA kinase activity. Type II receptor tyrosine kinase (RTK) inhibitors, such as imatinib and sunitinib, which only bind to the inactive conformation of the RTK, have little or no in vitro activity against this mutation. Clinically, these drugs are not effective for the treatment of GIST patients with D842V mutation. In addition, the D842V mutation can develop as a secondary imatinib resistant mutation during treatment of GISTs with primary imatinib-sensitive PDGFRA mutations (e.g. primary exon 12 mutations), or treatment of patients with hypereosinophillic syndrome with translocations involving PDGFRA. CP-868,596 is an orally bioavailable, highly potent and selective PDGFR TKI. CP-868,596 is a benzimidazole compound that has IC50s of 0.9 nM and 1.8 nM for PDGFRA and PDGFRB, respectively. Phase I trials of CP-868,596 have shown good oral bioavailability, a favorable toxicity profile, and achievable serum concentrations as high as 2000 nM. Methods: Mutant PDGFRA isoforms were expressed by transient transfection of Chinese Hamster ovary cells. The transfected cells were treated with various concentrations of CP-868,596 before preparation of protein lystates. PDGFRA protein was assayed for activation status (phosphorylation) by immunoprecipitation using an anti-PDGFRA antibody, followed by sequential immunoblotting for phospho PDGFRA (using antiphosphotyrosine antibody) or total PDGFRA (anti-PDGFRA monoclonal antibody). IC50 was measured by densitometry of the phospho PDGFRA bands and normalization using total PDGFRA expression (to correct for variations in loading of PDGFRA protein in the various lanes). Results: CP-868,596 inhibited the phosphorylation of wild type PDGFRA at an IC50 of 10 nM and PDGFRA D842V with an IC50 between 10 to 30 nM. Imatinib was ineffective in blocking PDGFRA D842V phosphorylation in these experiments (IC50 &amp;gt; 1000 nM). Profiling of CP-868,596 against other GIST-relevant primary and secondary PDGFRA mutations is ongoing and will be reported. Discussion: GISTs due to D842V activating mutations of PDGFRA gene are clinically resistant to imatinib, sunitinib, and other commercially available tyrosine kinase inhibitors. CP-868,596 blocks phosphorylation of the PDGFRA D842V mutant at clinically achievable concentrations, providing a potentially new therapeutic modality for GIST patients. A clinical trial in GIST patients with primary or secondary PDGFRA D842V mutations is being initiated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3586. doi:10.1158/1538-7445.AM2011-3586

Post-Transcriptional Regulation of Osteoblastic Platelet-Derived Growth Factor Receptor-Alpha Expression by Co-Cultured Primary Endothelial Cells

Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in osteoblast function. Inhibition of PDGFR activity leads to a suppression of osteoblast proliferation, whereas mineralized matrix production is enhanced. In previous experiments, we showed that co-cultivation of human primary endothelial cells and human primary osteoblasts (hOBs) leads to a cell contact-dependent downregulation of PDGFR-α expression in the osteoblasts. In this study, we investigated this effect in more detail, revealing that human umbilical vein endothelial cell (HUVEC)-mediated PDGFR-α downregulation is dependent on time and cell number. This effect was specific to endothelial cells and was not observed when hOBs were co-cultured with human primary chondrocytes or fibroblasts. Likewise, HUVEC-mediated suppression of PDGFR-α expression was only seen in hOBs and mesenchymal stem cells but not in immortalized osteoblastic cell lines. Functional inhibition of gap junctional communication between HUVECs and hOBs by 18α-glycyrrhetinic acid had no effect on HUVEC-mediated PDGFR-α downregulation, whereas inhibition of p38 mitogen-activated protein kinase (MAPK) prevented the HUVEC-mediated reduction in osteoblastic PDGFR-α expression. To delineate the molecular mechanism underlying the PDGFR-α downregulation, we examined the effect of HUVEC co-cultivation on osteoblastic PDGFR-α promoter activity as well as mRNA stability. Co-cultivation of HUVECs with hOBs significantly shortened the half-life of osteoblastic PDGFR-α mRNA, but did not decrease its promoter activity. In summary, our data show that PDGFR-α is downregulated in hOBs by co-cultivation with human primary endothelial cells through a p38 MAPK-dependent post-transcriptional mechanism.

Mutation-Free Expression of c-Kit and PDGFRA in Phyllodes Tumors of the Breast

Purpose: Phyllodes tumors (PTs) of the breast have been classified as benign, borderline, or malignant based on their histopathologic features. However, predicting clinical behavior based on these features has proven to be difficult given that local recurrence occurs in both benign and malignant PTs. Recurrence has been shown to mirror the histologic pattern of the primary tumor or to show dedifferentiation. The aim of this study was to assess the value of the histopathologic parameters, expression or mutation of c-Kit and platelet derived growth factor receptor alpha (PDGFRA) in predicting tumor recurrence. Methods: Representative areas from 39 benign, 16 borderline, and 12 malignant PTs were selected for construction of tissue microarrays. Immunohistochemical analyses for p53, Ki-67, c-Kit, and PDGFRA were performed and SSCP-PCR analysis was carried out to identify mutations in exons 9, 11, 13, and 17 of the c-Kit gene and exons 12 and 18 of the PDGFRA gene. Clinicopathologic features, including tumor recurrence and margin status, were also evaluated. Results: Of the 67 PTs, 11 cases (16.4%) recurred from 3 to 92 months following initial diagnosis (4 benign, 2 borderline, and 5 malignant). One benign PT case recurred as a borderline tumor and two borderline PT cases recurred as malignancies. Three patients died of malignant PT. No mutations of the c-Kit or PDGFRA genes were found and there was no statistically significant association of either p53 or p16 immunostaining with recurrent disease (p>0.05). However, histologic grade (p=0.033), margin status (p< 0.001), Ki-67 (p=0.012), c-Kit (p=0.002), and PDGFRA (p= 0.007) stromal immunopositivity were significantly correlated with recurrence. Conclusion: Even though positive or close margins were significantly associated with tumor recurrence, stromal c-Kit, PDGFRA positivity, and the Ki-67 index were useful for predicting recurrent PTs. Despite this, no c-Kit or PDGFRA mutations were found.

Amplification and overexpression of KIT, PDGFRA, and VEGFR2 in medulloblastomas and primitive neuroectodermal tumors

Medulloblastomas (MB) and primitive neuroectodermal tumors (PNET) are the most common malignant brain tumors in children. These two tumor types are histologically similar, but have different genetic backgrounds and clinical outcomes. Other brain tumors, such as gliomas, frequently have coamplification and overexpression of receptor tyrosine kinases KIT, platelet-derived growth factor receptor alpha (PDGFRA), and vascular endothelial growth factor receptor 2 (VEGFR2). We investigated protein expression and gene copy numbers of KIT, PDGFRA, and VEGFR2 in 41 MB and 11 PNET samples by immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH). KIT and PDGFRA expression was detected in both MBs and PNETs, whereas VEGFR2 expression was weak in these tumors. KIT, PDGFRA, and VEGFR2 amplifications were all present in 4% of MBs/PNETs, and KIT amplification was associated with concurrent PDGFRA and VEGFR2 amplifications (P <or= 0.001). Most strikingly, increased gene copy number of PDGFRA was associated with poor overall survival (P = 0.027). We suggest that coamplification of PDGFRA or VEGFR2 with KIT may be clinically useful novel molecular markers in MBs and PNETs.

Autopsy case of primary small cell carcinoma of the urinary bladder: KIT and PDGFRA expression and mutations

The author reports a rare autopsy case of primary small cell carcinoma of the urinary bladder with an emphasis on KIT and platelet-derived growth factor receptor-alpha (PDGFRA) genes. An 81-year-old man was admitted to Shizuoka City Shimizu Hospital because of dysuria. Cytology of the urine showed small carcinoma cells. Transurethral bladder tumorectomy indicated small cell carcinoma, and the patient was treated by radiation and chemotherapy. The patient died, however, of systemic metastasis 2 months after the first presentation. An autopsy showed a bladder small cell carcinoma and it systemic metastasis. Immunohistochemistry of the tumorectomy and autopsy specimens was positive for cytokeratins, neuron-specific enolase, synaptophysin, CD56, KIT and PDGFRA, and negative for chromogranin and thyroid transcriptional factor-1. Polymerase chain reaction-direct sequencing showed no mutations of KIT (exons 9, 11, 13 and 17) or PDGFRA (exons 12 and 18) genes in the tumorectomy specimens and was non-contributory due to DNA damage in autopsy specimens. The present case is the first case of primary small cell carcinoma of the urinary bladder involving examination of KIT and PDGFRA expression and KIT and PDGFRA gene mutations.