PDGFRα Activation Research Articles

PDGFRα Regulates Follicular Cell Differentiation Driving Treatment Resistance and Disease Recurrence in Papillary Thyroid Cancer

Dedifferentiation of follicular cells is a central event in resistance to radioactive iodine and patient mortality in papillary thyroid carcinoma (PTC). We reveal that platelet derived growth factor receptor alpha (PDGFRα) specifically drives dedifferentiation in PTC by disrupting the transcriptional activity of thyroid transcription factor-1 (TTF1). PDGFRα activation dephosphorylates TTF1 consequently shifting the localization of this transcription factor from the nucleus to the cytoplasm. TTF1 is required for follicular cell development and disrupting its function abrogates thyroglobulin production and sodium iodide transport. PDGFRα also promotes a more invasive and migratory cell phenotype with a dramatic increase in xenograft tumor formation. In patient tumors we confirm that nuclear TTF1 expression is inversely proportional to PDGFRα levels. Patients exhibiting PDGFRα at time of diagnosis are three times more likely to exhibit nodal metastases and are 18 times more likely to recur within 5years than those patients lacking PDGFRα expression. Moreover, high levels of PDGFRα and low levels of nuclear TTF1 predict resistance to radioactive iodine therapy. We demonstrate in SCID xenografts that focused PDGFRα blockade restores iodide transport and decreases tumor burden by >50%. Focused PDGFRα inhibitors, combined with radioactive iodine, represent an additional avenue for treating patients with aggressive variants of PTC.

Vascular Endothelial Cell Growth Factor A Acts via Platelet-Derived Growth Factor Receptor α To Promote Viability of Cells Enduring Hypoxia.

Vascular endothelial cell growth factor A (VEGF) is a biologically and therapeutically important growth factor because it promotes angiogenesis in response to hypoxia, which underlies a wide variety of both physiological and pathological settings. We report here that both VEGF receptor 2 (VEGFR2)-positive and -negative cells depended on VEGF to endure hypoxia. VEGF enhanced the viability of platelet-derived growth factor receptor α (PDGFRα)-positive and VEGFR2-negative cells by enabling indirect activation of PDGFRα, thereby reducing the level of p53. We conclude that the breadth of VEGF's influence extends beyond VEGFR-positive cells and propose a plausible mechanistic explanation of this phenomenon.

Structural and biochemical studies of the PDGFRA kinase domain

Platelet-derived growth factor receptor α (PDGFRA) is a Type III receptor tyrosine kinase, and this kinase is a target for treatment of gastrointestinal stromal tumors (GIST) as it is frequently mutated in these cancers. Most of the mutations that cause constitutive activation of PDGFRA occur in either the activation loop (A-loop) or in the juxtamembrane (JM) domain, such as the mutations D842V or V561D respectively. Treatment of PDGFRA-mutated GIST with imatinib is successful in some cases, but the D842V mutation is imatinib-resistant. To better understand the mechanism of PDGFRA drug-resistance, we have determined the crystal structure of the PDGFRA kinase domain in the auto-inhibited form, and studied the kinetics of the D842V mutation. Auto-inhibited PDGFRA is stabilized by the JM domain, which inserts into the active site of the kinase. The conserved residue Asp842 makes extensive contacts with several A-loop residues to maintain PDGFRA in the “DFG out” conformation, which stabilizes the kinase in the inactive state and facilitates the binding of imatinib. The D842V mutation would therefore be expected to activate the kinase and hinder the binding of drug through destabilizing the “DFG out” conformation. Furthermore, our kinetic data show that drug resistance in the D842V mutation may also in part result from its increased affinity for ATP. The PDGFRA kinase domain structure we report in this study has potential to facilitate development of new agents which can inhibit this kinase, including both its activating and drug-resistant mutations.

SHP-2-upregulated ZEB1 is important for PDGFRα-driven glioma epithelial-mesenchymal transition and invasion in mice and humans.

Gliomas are highly malignant brain tumors that are highly invasive and resistant to conventional therapy. Receptor tyrosine kinases (RTKs) such as PDGFRα (platelet-derived growth factor receptor-α), which show frequent aberrant activation in gliomas, are associated with a process of epithelial–mesenchymal transition (EMT), a cellular alteration that confers a more invasive and drug-resistant phenotype. Although this phenomenon is well documented in human cancers, the processes by which RTKs including PDGFRα mediate EMT are largely unknown. Here, we report that SHP-2 (encoded by PTPN11) upregulates an EMT inducer, ZEB1, to mediate PDGFRα-driven glioma EMT, invasion and growth in glioma cell lines and patient-derived glioma stem cells (GSCs) using cell culture and orthotopic xenograft models. ZEB1 and activated PDGFRα were coexpressed in invasive regions of mouse glioma xenografts and clinical glioma specimens. Glioma patients with high levels of both phospho-PDGFRα (p-PDGFRα) and ZEB1 had significantly shorter overall survival compared with those with low expression of p-PDGFRα and ZEB1. Knockdown of ZEB1 inhibited PDGFA/PDGFRα-stimulated glioma EMT, tumor growth and invasion in glioma cell lines and patient-derived GSCs. PDGFRα mutant deficient of SHP2 binding (PDGFRα-F720) or phosphoinositide 3-kinase (PI3K) binding (PDGFRα-F731/42), knockdown of SHP2 or treatments of pharmacological inhibitor for PDGFRα-signaling effectors attenuated PDGFA/PDGFRα-stimulated ZEB1 expression, cell migration and GSC proliferation. Importantly, SHP-2 acts together with PI3K/AKT to regulate a ZEB1-miR-200 feedback loop in PDGFRα-driven gliomas. Taken together, our findings uncover a new pathway in which ZEB1 functions as a key regulator for PDGFRα-driven glioma EMT, invasiveness and growth, suggesting that ZEB1 is a promising therapeutic target for treating gliomas with high PDGFRα activation.

Minicircle DNA-mediated endothelial nitric oxide synthase gene transfer enhances angiogenic responses of bone marrow-derived mesenchymal stem cells.

BackgroundNon-viral-based gene modification of adult stem cells with endothelial nitric oxide synthase (eNOS) may enhance production of nitric oxide and promote angiogenesis. Nitric oxide (NO) derived from endothelial cells is a pleiotropic diffusible gas with positive effects on maintaining vascular tone and promoting wound healing and angiogenesis. Adult stem cells may enhance angiogenesis through expression of bioactive molecules, and their genetic modification to express eNOS may promote NO production and subsequent cellular responses.MethodsRat bone marrow-derived mesenchymal stem cells (rBMSCs) were transfected with a minicircle DNA vector expressing either green fluorescent protein (GFP) or eNOS. Transfected cells were analysed for eNOS expression and NO production and for their ability to form in vitro capillary tubules and cell migration. Transcriptional activity of angiogenesis-associated genes, CD31, VEGF-A, PDGFRα, FGF2, and FGFR2, were analysed by quantitative polymerase chain reaction.ResultsMinicircle vectors expressing GFP (MC-GFP) were used to transfect HEK293T cells and rBMSCs, and were compared to a larger parental vector (P-GFP). MC-GFP showed significantly higher transfection in HEK293T cells (55.51 ± 3.3 %) and in rBMSC (18.65 ± 1.05 %) compared to P-GFP in HEK293T cells (43.4 ± 4.9 %) and rBMSC (15.21 ± 0.22 %). MC-eNOS vectors showed higher transfection efficiency (21 ± 3 %) compared to P-eNOS (9 ± 1 %) and also generated higher NO levels. In vitro capillary tubule formation assays showed both MC-eNOS and P-eNOS gene-modified rBMSCs formed longer (14.66 ± 0.55 mm and 13.58 ± 0.68 mm, respectively) and a greater number of tubules (56.33 ± 3.51 and 51 ± 4, respectively) compared to controls, which was reduced with the NOS inhibitor L-NAME. In an in vitro wound healing assay, MC-eNOS transfected cells showed greater migration which was also reversed by L-NAME treatment. Finally, gene expression analysis in MC-eNOS transfected cells showed significant upregulation of the endothelial-specific marker CD31 and enhanced expression of VEGFA and FGF-2 and their corresponding receptors PDGFRα and FGFR2, respectively.ConclusionsA novel eNOS-expressing minicircle vector can efficiently transfect rBMSCs and produce sufficient NO to enhance in vitro models of capillary formation and cell migration with an accompanying upregulation of CD31, angiogenic growth factor, and receptor gene expression.

Frequency and clinicopathologic profile of PIK3CA mutant GISTs: molecular genetic study of 529 cases

Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors usually driven by the mutational activation of receptor tyrosine kinases, KIT, or PDGFRA. Oncogenic activation of phosphatidylinositide-3-kinase (PI3K), a downstream effector in the KIT signaling pathway, has been identified in different types of cancer, with the PI3K 110α subunit encoded by PIK3CA being a common mutational target. In this study, the mutational hotspot in the PIK3CA kinase domain encoded by exon 20 was evaluated in 529 imatinib-naive GISTs using PCR amplification and Sanger sequencing. Eight mutations (two co-existing in one tumor) were identified. Subsequently, The cobas PIK3CA Mutation Test was employed to evaluate mutational hotspots in exons 1, 4, 7, and 9 in 119 PIK3CA exon 20-wild type tumors. In two cases, mutations in exons 1 and 9 were identified. In one GIST, previously undetected by Sanger sequencing, the exon 20 mutation was discovered. Altogether, eight primary and two metastatic GISTs carried PIK3CA mutations. The size of primary PIK3CA-mutant GISTs was ≥14 cm (mean size 17 cm), and mitotic activity varied from 0 to 72 per 50HPF (mean 5/50HPF). Follow-up data showed short survival in 6 of 7 studied cases. Detection of PIK3CA mutations in large or metastatic KIT-mutant GISTs may suggest that PIK3CA-mutant clones have a proliferative advantage during disease progression. Tyrosine kinase inhibitors have been successfully used in GIST treatment. However, resistance frequently develops due to secondary KIT mutations or activation of downstream to KIT signaling pathways, such as the PI3K/AKT/mTOR pathway. PIK3CA mutations similar to the ones detected in GISTs have been shown to cause such activation. Therefore, genotyping of PIK3CA in GISTs might help to pinpoint primary and metastatic tumors with the potential to develop resistance to tyrosine kinase inhibitors and guide therapy with PI3K inhibitors.

C-Kit non-mutated metastatic melanoma showing positive response to Nilotinib

Melanoma is an aggressive tumor with advanced disease characterized by widespread metastatic lesions and the tumor has traditionally been resistant to most forms of treatment. Indeed, metastatic melanoma has a very poor prognosis with a median survival time of 8-9 months and an estimated 3-year survival rate of less than 15%. Recent advances in our understanding of the genetic profile of melanoma cells and the molecular factors that drive malignant transformation have resulted in the identification of numerous new therapeutic targets. KIT is an established therapeutic target in cancers with activating mutations of KIT, such as gastrointestinal stromal tumors (GIST), and considerable efficacy has been achieved with various small molecule inhibitors of KIT including imatinib mesylate. Nilotinib is an inhibitor of ligand-induced PDGFRα and PDFGRβ kinase activity and autophosphorylation of constitutively activated KIT harboring exon 13 or exon 11 mutations (IC50 values of 0.2 and 0.027 μmol/L, respectively), with efficacy comparable to that of imatinib. We report a case of non-kit mutated metastatic vaginal melanoma showing impressive response to nilotinib.

TMOD-14CHARACTERIZING SIGNALING PATHWAYS IN A MOUSE MODEL OF GLIOBLASTOMA

TMOD-14. CHARACTERIZING SIGNALING PATHWAYS IN A MOUSE MODEL OF GLIOBLASTOMA Matthaeus Ware, Kaitlin Thomas, Jessica DePetro, Carmen Binding, Michael Blough, and Gregory Cairncross; Southern Alberta Cancer Research Institute, University of Calgary, Calgary, AB, Canada INTRODUCTION: Glioblastoma (GBM) is a fatal brain cancer, characteristically ‘driven’ by mutations in multiple tyrosine kinase and tumour suppressor pathways. We have developed a new murine model of GBM in which the sequence of molecular events and signaling pathways that underlie human GBM can be explored. In our model, cells from the subventricular zone (SVZ) of p53-/mice become growth factor independent when cultured in serum-free media supplemented with platelet-derived growth factor-AA (PDGF-AA). When these cells are implanted into the brains of immunecompetent p53 wild-type mice they form tumours that closely resemble human GBM. Here, we explored the mechanism of sustained cell proliferation in the transformed state. METHODS: Phospho-receptor tyrosine kinase (RTK) arrays were used to evaluate RTK phosphorylation, non-RTK arrays to examine pathway activation, and PDGFR-a inhibitors to assess the role of PDGFR-a in sustaining cell viability in pre-transformed and transformed cells. RESULTS: PDGFR-a was phosphorylated in pre-transformed SVZ cells and remained phosphorylated in transformed cells proliferating in the absence of exogenous PDGF-AA. An inhibitor of PDGFR-a that blocked PDGF-AA binding decreased the viability of pre-transformed SVZ cells but had no effect on transformed cells. In contrast, Imatinib, an inhibitor of PDGFR-a phosphorylation, blocked the proliferation of both. Downstream proteins, ERK1/2 and WNK1, and the transcription factor, CREB, were activated in transformed cells. CONCLUSION: In a PDGF-AA initiated mouse model of GBM, exogenous growth factor independent proliferation of p53-null SVZ cells and tumorigenicity are associated with persistent phosphorylation (i.e., activation) of PDGFR-a. The finding that transformed cells are insensitive to inhibition by a PDGFR-a blocking antibody, but respond to Imatinib, is consistent with receptor autophosphorylation. These observations may be applicable to human GBM where over-expression of PDGF-AA appears to be an early event in the pathogenesis of proneural and other subtypes of GBM. Neuro-Oncology 17:v226–v229, 2015. doi:10.1093/neuonc/nov237.14 Published by Oxford University Press on behalf of the Society for Neuro-Oncology 2015.

Histone H3F3A and HIST1H3B K27M mutations define two subgroups of diffuse intrinsic pontine gliomas with different prognosis and phenotypes.

Diffuse intrinsic pontine glioma (DIPG) is the most severe paediatric solid tumour, with no significant therapeutic progress made in the past 50 years. Recent studies suggest that diffuse midline glioma, H3-K27M mutant, may comprise more than one biological entity. The aim of the study was to determine the clinical and biological variables that most impact their prognosis. Ninety-one patients with classically defined DIPG underwent a systematic stereotactic biopsy and were included in this observational retrospective study. Histone H3 genes mutations were assessed by immunochemistry and direct sequencing, whilst global gene expression profiling and chromosomal imbalances were determined by microarrays. A full description of the MRI findings at diagnosis and at relapse was integrated with the molecular profiling data and clinical outcome. All DIPG but one were found to harbour either a somatic H3-K27M mutation and/or loss of H3K27 trimethylation. We also discovered a novel K27M mutation in HIST2H3C, and a lysine-to-isoleucine substitution (K27I) in H3F3A, also creating a loss of trimethylation. Patients with tumours harbouring a K27M mutation in H3.3 (H3F3A) did not respond clinically to radiotherapy as well, relapsed significantly earlier and exhibited more metastatic recurrences than those in H3.1 (HIST1H3B/C). H3.3-K27M-mutated DIPG have a proneural/oligodendroglial phenotype and a pro-metastatic gene expression signature with PDGFRA activation, while H3.1-K27M-mutated tumours exhibit a mesenchymal/astrocytic phenotype and a pro-angiogenic/hypoxic signature supported by expression profiling and radiological findings. H3K27 alterations appear as the founding event in DIPG and the mutations in the two main histone H3 variants drive two distinct oncogenic programmes with potential specific therapeutic targets.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-015-1478-0) contains supplementary material, which is available to authorized users.

Abstract 985: Stromal PDGFR-α activation stalls mammary ductal development and increases tumorigenic potential of mammary epithelia

Abstract One of the major confounding factors in the treatment of breast cancer is the cellular heterogeneity of the breast. An important cellular component of the normal breast as well as tumor stroma is the fibroblast. Fibroblasts are cells of mesenchymal origin that express platelet derived growth factor receptor alpha (PDGFR-α), a pharmaceutically targetable receptor tyrosine kinase. While activation of PDGFR-α was shown to be a poor prognostic factor in glioblastoma and gastro-intestinal stromal tumors, the role of stromal PDGFR-α signaling in mammary development and carcinomas is yet to be explored. To elucidate the role of stromal PDGFR-α signaling in this context, we utilized mice harboring the activating Pdgfra-D842V mutation knocked into the endogenous Pdgfra locus under a Lox-STOP-Lox cassette. To obtain selective activation of PDGFR-α in the fibroblastic stroma, these mice were crossed with fibroblast specific Fsp-cre mice. Whole mount carmine alum staining revealed that the D842V mutants have severely impaired mammary ductal development. Further analyses at the histological level revealed that the D842V glands have increased extra-cellular matrix (ECM) deposition (Masson's trichrome) likely due to the observable increase in fibroblasts (vimentin positivity). Immuno-fluorescent dual staining for keratin 8 and Ki67 demonstrated that stromal PDGFR-α activation increases the proliferation of neighboring mammary ductal cells, potentially making them more tumorigenic. To gain insight into the molecular mechanisms underlying this stromal-epithelial cross-talk, primary mammary epithelial cells and fibroblasts were isolated for downstream assays. Western blot analyses showed that mutant mammary fibroblasts have increased levels of activated PLC-γ, AKT and JNK which are known oncogenic pathways in the stroma. Gene expression arrays followed by global pathway analyses showed that pro-tumorigenic processes such as proliferation and metastasis are enriched in the epithelium from the mutant mice, while pro-tumor pathways such as KRAS and JNK were found to be enriched in the mutant fibroblasts. Interestingly, Pdgfra-D842V mutant mice have significantly increased stiffness as measured by atomic force microscopy of naïve tissue. This result implies that the observed increase in fibroblast number and resulting ECM deposition is sufficient to alter the entire mammary tissue. Since breast density correlates with higher breast cancer risk and tumor invasiveness, this phenotype reveals a novel function for stromal PDGFR-α signaling in breast cancer susceptibility. Studies are underway to evaluate whether these mice are predisposed to mammary tumor formation. Given our present data, we conclude that activation of stromal PDGFR-α signaling not only abrogates mammary development, but also makes the mammary ductal epithelium more tumorigenic. Citation Format: Anisha Mathur, Gina M. Sizemore, Subhasree Balakrishnan, Vasudha C. Shukla, Maria Cuitino, Anthony J. Trimboli, Samir Ghadiali, Gustavo W. Leone, Michael C. Ostrowski. Stromal PDGFR-α activation stalls mammary ductal development and increases tumorigenic potential of mammary epithelia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 985. doi:10.1158/1538-7445.AM2015-985

Abstract 791: BLU-285, the first selective inhibitor of PDGFRα D842V and KIT Exon 17 mutants

Abstract The advent of imatinib therapy in gastrointestinal stromal tumor (GIST) has been transformative, altering the treatment of advanced disease in the majority of patients from supportive care and poor prognosis to a median 24-month progression free survival in the first line setting. However, for 20% of patients with gastric GIST, imatinib shows no therapeutic benefit. These patients are characterized by the amino acid substitution D842V in the PDGFRα protein; this mutation results in ligand-independent activation of PDGFRα and is insensitive to all small molecule inhibitors approved for GIST. When metastatic, the prognosis for PDGFRα D842V GIST is dire, with median overall survival of 12 months. Starting with a novel chemical library optimized for kinase selectivity and potency, we have identified BLU-285, a small molecule kinase inhibitor that potently inhibits PDGFRα D842V mutant activity in vitro (IC50 = 0.5 nM) and PDGFRα D842V autophosphorylation in the cellular setting (IC50 = 30 nM). We have observed that in addition to PDGFRα D842V, BLU-285 is also a potent inhibitor of the analogous KIT mutation, D816V in KIT Exon 17 (IC50 = 0.5nM). KIT D816 mutations have been identified in several human diseases such as systemic mastocytosis (SM), acute myeloid leukemia (AML), and as resistance mutations in second and third line GIST, after imatinib and sunitinib failure. Cellular assays measuring inhibition of KIT mutant autophosphorylation confirm the activity of BLU-285 against the KIT D816 mutants D816V (HMC1.2 cells, IC50 = 3 nM) and D816Y (P815 cells, IC50 = 22 nM) as well as other KIT Exon 17 mutants such as N822K (Kasumi cells, IC50 = 40 nM) found in treatment-refractory GIST. In vivo, BLU-285 is a well-tolerated, orally bioavailable agent that achieves dose dependent tumor growth inhibition in a D816Y-driven xenograft model. A PK-PD-efficacy relationship with BLU-285 has been established demonstrating that tumor regression results from >90% target suppression and is observed with 30 mg/kg once daily dosing. Furthermore, in non-clinical toxicology studies, projected human efficacious exposures were well tolerated. With potent activity against PDGFRα D842V and KIT Exon 17 mutants, BLU-285 targets previously unaddressed genomic drivers of disease and provides promise for the treatment of PDGFRα D842V-driven GIST or SM, where more than 90% of patients carry the KIT D816V mutation. Besides single agent activity, the highly selective BLU-285 offers an opportunity for combination with other agents in GIST to cover the entirety of KIT primary and resistance mutants. This approach of pairing potent, selective kinase inhibitors with defined disease-driving kinase mutants is designed to fulfill the promise of personalized medicine and, if clinically proven, may provide maximum benefit to patients. BLU-285 is scheduled to enter first-in-human phase 1 clinical trials for both the GIST and SM patient populations in the first half of 2015. Citation Format: Erica K. Evans, Brian L. Hodous, Alexandra K. Gardino, Alison Davis, Julia Zhu, Adam Shutes, Joseph L. Kim, Kevin J. Wilson, Doug Wilson, Yulian Zhang, Tat Chu, Nancy E. Kohl, Vivek Kadambi, Timothy Guzi, Christoph Lengauer. BLU-285, the first selective inhibitor of PDGFRα D842V and KIT Exon 17 mutants. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 791. doi:10.1158/1538-7445.AM2015-791

Platelet Derived Growth Factor Has a Role in Pressure Induced Bladder Smooth Muscle Cell Hyperplasia and Acts in a Paracrine Way

Bladder outlet obstruction is a finding in many urological disorders, leading to bladder wall hyperplasia. We investigated platelet derived growth factor and its receptor in human bladder smooth muscle cells and urothelial cells exposed to hydrostatic pressure or PDGF invitro. Bladder smooth muscle cells and urothelial cells were exposed to elevated hydrostatic pressure for 1 hour. The expression of PDGF and PDGFR was evaluated using reverse transcriptase-polymerase chain reaction and Western blot analysis. Pressure or PDGF induced proliferation of bladder smooth muscle cells with or without pretreatment with lovastatin or imatinib was measured by enzyme-linked immunosorbent assay. PDGFRα was knocked down with siRNA. After hydrostatic pressure bladder smooth muscle cells showed increased PDGFRα and β expression. PDGF was not expressed in bladder smooth muscle cells. Urothelial cells showed no expression of PDGFR but PDGF expression was noted. Western blot analysis of bladder smooth muscle cells revealed a pressure induced increase in PDGFR in the membrane fraction. Phosphorylation of PDGFR occurred with pressure induction. Bladder smooth muscle cell proliferation was increased in pressure and PDGF mediated fashion. Pretreatment with lovastatin or imatinib prevented proliferation. There was no cell proliferation after PDGFRα knockdown. Increased expression and phosphorylation of PDGFR in bladder smooth muscle cells after hydrostatic pressure suggests a pivotal role of the PDGF pathway in pressure induced hyperplasia of bladder smooth muscle cells. PDGF expressed in urothelial cells may act in a paracrine way. Cholesterol depletion, inhibition of receptor tyrosine kinase activity and knockdown of PDGFRα in bladder smooth muscle cells prevent pressure and PDGF mediated cell proliferation. Targeting PDGFR seems a promising way to influence pressure induced bladder wall hyperplasia.

RasGAP Promotes Autophagy and Thereby Suppresses Platelet-Derived Growth Factor Receptor-Mediated Signaling Events, Cellular Responses, and Pathology.

Platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) make profound contributions to both physiology and pathology. While it is widely believed that direct (PDGF-mediated) activation is the primary mode of activating PDGFRs, the discovery that they can also be activated indirectly begs the question of the relevance of the indirect mode of activating PDGFRs. In the context of a blinding eye disease, indirect activation of PDGFRα results in persistent signaling, which suppresses the level of p53 and thereby promotes viability of cells that drive pathogenesis. Under the same conditions, PDGFRβ fails to undergo indirect activation. In this paper, we report that RasGAP (GTPase-activating protein of Ras) prevented indirect activation of PDGFRβ. RasGAP, which associates with PDGFRβ but not PDGFRα, reduced the level of mitochondrion-derived reactive oxygen species, which are required for enduring activation of PDGFRs. Furthermore, preventing PDGFRβ from associating with RasGAP allowed it to signal enduringly and drive pathogenesis of a blinding eye disease. These results indicate a previously unappreciated role of RasGAP in antagonizing indirect activation of PDGFRβ, define the underlying mechanism, and raise the possibility that PDGFRβ-mediated diseases involve indirect activation of PDGFRβ.

Genetic events that limit the efficacy of MEK and RTK inhibitor therapies in a mouse model of KRAS-driven pancreatic cancer.

Mutated KRAS (KRAS*) is a fundamental driver in the majority of pancreatic ductal adenocarcinomas (PDAC). Using an inducible mouse model of KRAS*-driven PDAC, we compared KRAS* genetic extinction with pharmacologic inhibition of MEK1 in tumor spheres and in vivo. KRAS* ablation blocked proliferation and induced apoptosis, whereas MEK1 inhibition exerted cytostatic effects. Proteomic analysis evidenced that MEK1 inhibition was accompanied by a sustained activation of the PI3K-AKT-MTOR pathway and by the activation of AXL, PDGFRa, and HER1-2 receptor tyrosine kinases (RTK) expressed in a large proportion of human PDAC samples analyzed. Although single inhibition of each RTK alone or plus MEK1 inhibitors was ineffective, a combination of inhibitors targeting all three coactivated RTKs and MEK1 was needed to inhibit proliferation and induce apoptosis in both mouse and human low-passage PDAC cultures. Importantly, constitutive AKT activation, which may mimic the fraction of AKT2-amplified PDAC, was able to bypass the induction of apoptosis caused by KRAS* ablation, highlighting a potential inherent resistance mechanism that may inform the clinical application of MEK inhibitor therapy. This study suggests that combinatorial-targeted therapies for pancreatic cancer must be informed by the activation state of each putative driver in a given treatment context. In addition, our work may offer explanative and predictive power in understanding why inhibitors of EGFR signaling fail in PDAC treatment and how drug resistance mechanisms may arise in strategies to directly target KRAS.

A preclinical study demonstrating the efficacy of nilotinib in inhibiting the growth of pediatric high-grade glioma

Solid tumors arising from malignant transformation of glial cells are one of the leading causes of central nervous system tumor-related death in children. Recurrence in spite of rigorous surgical and chemoradiation therapies remains a major hurdle in management of these tumors. Here, we investigate the efficacy of the second-generation receptor tyrosine kinase inhibitor nilotinib as a therapeutic option for the management of pediatric gliomas. We have utilized two independent pediatric high-grade glioma cell lines with either high platelet-derived growth factor receptor alpha (PDGFRα) or high PDGFRβ expression in in vitro assays to investigate the specific downstream effects of nilotinib treatment. Using in vitro cell-based assays we show that nilotinib inhibits PDGF-BB-dependent activation of PDGFRα. We further show that nilotinib is able to decrease cell proliferation and anchorage-independent growth via suppression of AKT and ERK1/2 signaling pathways. Our results suggest that nilotinib may be effective for management of a PDGFRα-dependent group of pediatric gliomas.Electronic supplementary materialThe online version of this article (doi:10.1007/s11060-015-1744-y) contains supplementary material, which is available to authorized users.

Double minute amplification of mutant PDGF receptor α in a mouse glioma model.

In primary brain tumors, oncogenes are frequently amplified and maintained on extrachromosomal DNA as double minutes (DM), but the underlying mechanisms remain poorly understood. We have generated a mouse model of malignant glioma based on knock-in of a mutant PDGF receptor α (PDGFRα) that is expressed in oligodendrocyte precursor cells (OPCs) after activation by a Cre recombinase. In the tumor suppressor INK4/Arf−/− background, mutant animals frequently developed brain tumors resembling anaplastic human gliomas (WHO grade III). Besides brain tumors, most animals also developed aggressive fibrosarcomas, likely triggered by Cre activation of mutant PDGFRα in fibroblastic cell lineages. Importantly, in the brain tumors and cell lines derived from brain tumor tissues, we identified a high prevalence of DM Pdgfra gene amplification, suggesting its occurrence as an early mutational event contributing to the malignant transformation of OPCs. Amplicons extended beyond the Pdgfra locus and included in some cases neighboring genes Kit and Kdr. Our genetically defined mouse brain tumor model therefore supports OPC as a cell of origin for malignant glioma and offers an example of a defined temporal sequence of mutational events, thus providing an entry point for a mechanistic understanding of DM gene amplification and its functionality in gliomagenesis.

PDGFRβ and oncogenic mutant PDGFRα D842V promote disassembly of primary cilia through a PLCγ- and AURKA-dependent mechanism.

Primary cilia are microtubule-based sensory organelles projecting from most quiescent mammalian cells, which disassemble in cells cultured in serum-deprived conditions upon re-addition of serum or growth factors. Platelet-derived growth factors (PDGF) are implicated in deciliation, but the specific receptor isoforms and mechanisms involved are unclear. We report that PDGFRβ promotes deciliation in cultured cells and provide evidence implicating PLCγ and intracellular Ca(2+) release in this process. Activation of wild-type PDGFRα alone did not elicit deciliation. However, expression of constitutively active PDGFRα D842V mutant receptor, which potently activates PLCγ (also known as PLCG1), caused significant deciliation, and this phenotype was rescued by inhibiting PDGFRα D842V kinase activity or AURKA. We propose that PDGFRβ and PDGFRα D842V promote deciliation through PLCγ-mediated Ca(2+) release from intracellular stores, causing activation of calmodulin and AURKA-triggered deciliation.

Use of human embryonic stem cells to model pediatric gliomas with H3.3K27M histone mutation.

Over 70% of diffuse intrinsic pediatric gliomas, an aggressive brainstem tumor, harbor heterozygous mutations that create a K27M amino acid substitution (methionine replaces lysine 27) in the tail of histone H3.3. The role of the H3.3K27M mutation in tumorigenesis is not fully understood. Here, we use a human embryonic stem cell system to model this tumor. We show that H3.3K27M expression synergizes with p53 loss and PDGFRA activation in neural progenitor cells derived from human embryonic stem cells, resulting in neoplastic transformation. Genome-wide analyses indicate a resetting of the transformed precursors to a developmentally more primitive stem cell state, with evidence of major modifications of histone marks at several master regulator genes. Drug screening assays identified a compound targeting the protein menin as an inhibitor of tumor cell growth in vitro and in mice.

PM-23 * EXPLORATION OF BASIC AND THERAPEUTIC MECHANISMS IN NOVEL MODELS OF RTK/RAS-NETWORK-DRIVEN GLIOBLASTOMA

Glioblastoma (GBM) remains fatal despite intensive treatments including standard-of-care surgery, radiation, and chemotherapy. Modern targeted therapies have also failed to produce effective results in clinical trials, possibly due to a lack of relevant preclinical models for drug discovery/assessment. Approximately 90% GBMs harbor aberrations in RTK/RAS-signaling, including EGFR amplification/gain-of-function mutations (57%), PDGFRa amplification (10%), and NF1 deletion/loss-of-function mutations (10%). KRAS mutations also occur, but at low frequency (1%). Frequent alteration of RTK/RAS-signaling emphasizes a great need for several distinct RTK/RAS-network-driven preclinical GBM models that mirror the range of human GBM lesions, since therapeutic responses may differ depending on the major network driver in a given tumor. We have recently generated a genetically-engineered mouse model of GBM via adult-inducible astrocyte-specific perturbation of RB, KRAS and PTEN. The triple mutant mice develop GBMs that faithfully recapitulate the histopathology and molecular characteristics of human primary GBMs. Interestingly, without RAS activation, only low-grade disease is induced. We established astrocytes from the triple mutant mouse model [RB-inactivated/KRASG12D/+/PTEN+/-; RKP astrocytes)] that elicit GBM upon intracranial transplantation, as well as the RB/PTEN double mutant mice [RB-inactivated/PTEN+/-; RP astrocytes], which do not produce GBM. Thus, the RP cells provide an ideal platform for evaluation of multiple RAS-activating events in causation of GBM. We are currently introducing the most frequent events found in human GBMs, including EGFR, ERFRvIII and PDGFRa activation and NF1 deficiency, into RP cells in vitro. The ability of each event to convert the RP cells into GBM-forming astrocytes by orthotopic transplantation is being assessed. This novel in vitro-to-allograft approach provides direct comparisons of etiologies driven by various RAS-activating events in the same genetic background and will potentially lead to basic discovery of distinct mechanisms in GBM tumorigenesis. Furthermore, this approach will generate relevant panels of RTK/RAS-network-driven GBM models for robust preclinical therapeutic discovery and biomarker development.

ABCG2 AND MDR PROMOTE BRAINSTEM GLIOMAGENESIS AND LIMIT THE EFFICACY OF DASATINIB IN A DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) MOUSE MODEL

BACKGROUND: DIPG is an incurable tumor that arises in the brainstem of children. Our laboratory generates murine DIPGs by expressing PDGF-B and deleting p53 in nestin progenitors of the neonatal brainstem using the RCAS/tv-a system. Dasatinib, an inhibitor of PDGFR-A and SRC activation, has been evaluated in clinical trials for children with DIPG but with disappointing results. As dasatinib is a substrate of the ABC transporters ABCG2 and MDR, we hypothesized that ABCG2 and MDR may limit the efficacy of dasatinib in DIPG. METHODS: We generated murine DIPGs driven by PDGF-B and p53 loss in ABC WT (ABCG2+/+; MDRa+/+; MDRb+/+) and ABC KO (ABCG2-/-; MDRa-/-; MDRb-/-) mice. Next, we investigated whether dasatinib is efficacious in ABC KO and ABC WT DIPG-bearing mice. We treated ABC WT DIPG-bearing mice with dasatinib alone, elacridar (a dual ABCG2/MDR inhibitor) alone, and dasatinib in combination with elacridar. We also measured dasatinib levels in the tumor and plasma of all of the different tumor cohorts. Lastly, we evaluated the blood-brain-barrier (BBB) of our DIPG mouse model with Dextran Texas Red. RESULTS: Surprisingly, we observed a significant difference in tumor latency between untreated ABC KO and untreated ABC WT mice in this PDGF-B; p53 deficient DIPG model. Median survival of DIPG-bearing ABC KO was longer than DIPG-bearing ABC WT: 43 days vs. 34 days respectively (p = 0.02). We observed that dasatinib almost doubled the survival of DIPG-bearing mice in the ABC KO model: 44 days vs. 80 days (p = 0.0004). Dasatinib modestly prolonged survival in ABC WT mice: 33 days vs. 42 days (p < 0.0001). Co-treatment with elacridar significantly increased the efficacy of dasatinib in the ABC WT model 42 days vs. 59 days (p < 0.0001). However, elacridar alone did not significantly prolong survival in the ABC WT mice 33 days vs. 34 days (p = 0.38). Pharmacokinetic analysis demonstrated that genetic ablation of the ABC transporters or inhibition of ABC transporters with elacridar significantly increased dasatinib delivery into normal brain but not into tumor. Dextran Texas Red studies demonstrated a disrupted BBB in DIPG models independent of ABC transporters. CONCLUSIONS: 1. ABC transporters promote brainstem gliomagenesis 2. ABC transporters regulate dasatinib delivery into normal brain but not into the tumor as BBB is disrupted in our DIPG mouse model 3. ABC transporter activity limits the efficacy of dasatinib in vivo. Our results suggest that ABCG2 and MDR inhibitors may have a therapeutic role in the treatment of DIPG. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.