Platelet-derived Growth Factor Receptor Family Research Articles

Computational evidence for multi-layer crosstalk between the cadherin-11 and PDGFR pathways

Various cell surface receptors play an important role in the differentiation and self-renewal of human mesenchymal stem cells (hMSCs). One example of such receptors are the cadherins, which maintain cell–cell adhesion and mechanically couple cells together. Recently, cadherin-11, which is a member of the type II classical cadherin family, has been shown to be involved in the fate commitment of hMSCs. Interestingly, cadherin-11 has no known intrinsic signaling activity and is thought to affect cell behavior via interactions with other cell surface receptors. Members of the platelet-derived growth factor receptor (PDGFR) family are hypothesized to be one of the interaction partners of cadherin-11. Experiments confirmed that PDGFR-α binding to extracellular cadherin-11 regions increases the PDGFR-α activity, whereas the interaction between PDGFR-β and cadherin-11 suppresses the activity of the growth factor receptor. Cadherin-11 knockdown experiments also decreased cell proliferation. These interactions between cadherin-11 and PDGFRs indicate a crosstalk between these receptors and their downstream signaling activities but the nature of this crosstalk is not entirely known. In this study, we used a computational model to represent the experimentally proven interactions between cadherin-11 and the two PDGFRs and we inspected whether the crosstalk also exists downstream of the signaling initiated by the two receptor families. The computational framework allowed us to monitor the relative activity levels of each protein in the network. We performed model simulations to mimic the conditions of previous cadherin-11 knockdown experiments and to predict the effect of crosstalk on cell proliferation. Overall, our predictions suggest the existence of another layer of crosstalk, namely between β-catenin (downstream to cadherin-11) and an ERK inhibitor protein (e.g. DUSP1), different than the crosstalk at the receptor level between cadherin-11 and PDGFR-α and -β. By investigating the multi-level crosstalk between cadherin and PDGFRs computationally, this study contributes to an improved understanding of the effect of cell surface receptors on hMSCs proliferation.

Synthesis and Development of Highly Selective Pyrrolo[2,3-d]pyrimidine CSF1R Inhibitors Targeting the Autoinhibited Form.

Colony-stimulating factor-1 receptor (CSF1R) is a receptor tyrosine kinase that controls the differentiation and maintenance of most tissue-resident macrophages, and the inhibition of CSF1R has been suggested as a possible therapy for a range of human disorders. Herein, we present the synthesis, development, and structure-activity relationship of a series of highly selective pyrrolo[2,3-d]pyrimidines, showing subnanomolar enzymatic inhibition of this receptor and with excellent selectivity toward other kinases in the platelet-derived growth factor receptor (PDGFR) family. The crystal structure of the protein and 23 revealed that the binding conformation of the protein is DFG-out-like. The most promising compounds in this series were profiled for cellular potency and subjected to pharmacokinetic profiling and in vivo stability, indicating that this compound class could be relevant in a potential disease setting. Additionally, these compounds inhibited primarily the autoinhibited form of the receptor, contrasting the behavior of pexidartinib, which could explain the exquisite selectivity of these structures.

Dasatinib inhibits proliferation of liver cancer cells, but activation of Akt/mTOR compromises dasatinib as a cancer drug.

Dasatinib is a multi-target protein tyrosine kinase inhibitor. Due to its potent inhibition of Src, Abl, the platelet-derived growth factor receptor (PDGFR) family kinases, and other oncogenic kinases, it has been investigated as a targeted therapy for a broad spectrum of cancer types. However, its efficacy has not been significantly extended beyond leukemia. The mechanism of resistance to dasatinib in a wide array of cancers is not clear. In the present study, we investigated the effect of dasatinib on hepatocellular carcinoma cell growth and explored the underlying mechanisms. Our results showed that dasatinib potently inhibited the proliferation of SNU-449 cells, but not that of other cell lines, such as SK-Hep-1, even though it inhibited the phosphorylation of Src on both negative and positive regulation sites in all these cells. Dasatinib activated the phosphoinositide-dependent protein kinase1 (PDK1)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway in SK-Hep-1 cells, but not in SNU-449 cells. Blocking the Akt/mTOR signaling pathway strongly promoted the efficacy of dasatinib in SK-Hep-1 cells. In SNU-449 cells, dasatinib promoted apoptosis and the cleavage of caspase-3 and caspase-7, induced cell cycle arrest in the G1 phase, and inhibited the expression of Cyclin-dependent kinase (CDK4)/6/CyclinD1 complex. These findings demonstrate that dasatinib exerts its anti-proliferative effect on hepatocellular cell proliferation by blocking the Src family kinases; however, it causes Akt activation, which compromises dasatinib as an anti-cancer drug.

Pdgfra and Pdgfrb Genetically Interact in the Murine Neural Crest Cell Lineage to Regulate Migration and Proliferation.

Cranial neural crest cells (cNCCs) are migratory, multipotent cells that originate from the forebrain to the hindbrain and eventually give rise to the cartilage and bone of the frontonasal skeleton, among other derivatives. Signaling through the two members of the platelet-derived growth factor receptor (PDGFR) family of receptor tyrosine kinases, alpha and beta, plays critical roles in the cNCC lineage to regulate craniofacial development during murine embryogenesis. Further, the PDGFRs have been shown to genetically interact during murine craniofacial development at mid-to-late gestation. Here, we examined the effect of ablating both Pdgfra and Pdgfrb in the murine NCC lineage on earlier craniofacial development and determined the cellular mechanisms by which the observed phenotypes arose. Our results confirm a genetic interaction between the two receptors in this lineage, as phenotypes observed in an allelic series of mutant embryos often worsened with the addition of conditional alleles. The defects observed here appear to stem from aberrant cNCC migration, as well as decreased proliferation of the facial mesenchyme upon combined decreases in PDGFRα and PDGFRβ signaling. Importantly, we found that PDGFRα plays a predominant role in cNCC migration whereas PDGFRβ primarily contributes to proliferation of the facial mesenchyme past mid-gestation. Our findings provide insight into the distinct mechanisms by which PDGFRα and PDGFRβ signaling regulate cNCC activity and subsequent craniofacial development in the mouse embryo.

The emerging complexity of PDGFRs: activation, internalization and signal attenuation.

The platelet-derived growth factor receptor (PDGFR) family of receptor tyrosine kinases allows cells to communicate with the environment to regulate diverse cellular activities. Here, we highlight recent data investigating the structural makeup of individual PDGFRs upon activation, revealing the importance of the whole receptor in the propagation of extracellular ligand binding and dimerization. Furthermore, we review ongoing research demonstrating the significance of receptor internalization and signal attenuation in the regulation of PDGFR activity. Interactions with internalization machinery, signaling from endosomes, receptor degradation and receptor recycling are physiological means by which cells fine-tune PDGFR responses to growth factor stimulation. In this review, we discuss the biophysical, structural, in silico and biochemical data that have provided evidence for these mechanisms. We further highlight the commonalities and differences between PDGFRα and PDGFRβ signaling, revealing critical gaps in knowledge. In total, this review provides a conclusive summary on the state of the PDGFR field and underscores the need for novel techniques to fully elucidate the mechanisms of PDGFR activation, internalization and signal attenuation.

Concentration-dependent effects of CSF1R inhibitors on oligodendrocyte progenitor cells ex vivo and in vivo

Microglia are the principal resident immune cells in the central nervous system (CNS) and play important roles in CNS development, maintenance and repair. The survival and development of microglia depends on colony-stimulating factor 1 receptor (CSF1R), a member of the platelet-derived growth factor receptor (PDGFR) family of tyrosine kinases. Recently pharmacological CSF1R inhibition has been used to investigate the effects of microglial depletion in numerous animal models of CNS disease. However, the effects of CSF1R inhibitors on other cell types in the CNS remains incompletely characterized. In this report, we compared the effect of two commonly used CSF1R inhibitors, PLX5622 and PLX3397, on microglia and oligodendrocyte progenitor cell (OPC) numbers. In ex vivo cerebellar slices and adult mouse brain, both PLX compounds caused robust microglia loss; the kinetics of microglial depletion was more rapid with PLX5622. While high-doses of PLX5622 and PLX3397 reduced OPC number in primary cultures in vitro and ex vivo, low-doses of PLX5622 did not affect the number of OPCs or mature oligodendroglia in culture or in vivo. In adult mice, treatment with PLX5622 had no effect on OPC numbers for 7 days; however, a mild reduction was observed after 21 days in some CNS regions. In contrast, PLX3397 caused significant OPC loss after 7 days of treatment, despite only modest microglia depletion. Neither PLX compound had a remarkable effect on mature oligodendrocytes or myelin protein expression following long-term oral administration. Our results show that CSF1R inhibition with PLX5622 can selectively deplete microglia ex vivo and in vivo without affecting OPC number, demonstrating that microglia are not essential for OPC viability in ex vivo slice cultures or adult CNS tissues.

1820PD - Synergistic anti-cancer activity of tyrosine kinase inhibitors and paclitaxel with radiation on anaplastic thyroid cancer in vitro and in vivo

Background: Anaplastic thyroid carcinoma (ATC) although rare is the most deadly form of thyroid cancer. The goal of this study was to investigate the anti-tumor activities of paclitaxel with radiation and in combination with tyrosine kinase inhibitors (TKI) in anaplastic thyroid cancer cells in vitro and in vivo. Methods: Three ATC cell lines were exposed to TKI in the presence or absence of paclitaxel with radiation and cell viability was determined by MTT assay. Effects of combined treatment on cell cycle and intracellular signaling pathways were assessed by flow cytometry and western blot analysis. The ATC cell lines xenograft model was used to examine the anti-tumor activity in vivo. Results: Our data showed that paclitaxel with radiation and TKIs synergistically decreased cell viability in ATC cells, and also significantly increased apoptotic cell death in these cells, as proved by the cleavage of caspase-3 and DNA fragmentation. Paclitaxel and TKI with radiation combination reduced anti-apoptotic factor in ATC. Thus, TKI that targeted the vascular endothelial growth factor receptor family (VEGFR-2 and -3) and platelet-derived growth factor receptor family (PDGFR-beta and Kit), which play key roles in tumor progression and angiogenesis. Combination therapy with paclitaxel and TKI with radiation significantly decreased vessel density, and most significantly reduced tumor volume and increased survival in ATC xenografts. Conclusions: These results propose that paclitaxel and TKI with radiation has significant anti-cancer activity in preclinical models, potentially suggesting a new clinical approach for patients of advanced thyroid cancer type. Legal entity responsible for the study: Yong Sang Lee. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.

Preliminary in vitro and in vivo investigation of a potent platelet derived growth factor receptor (PDGFR) family kinase inhibitor

Aberrant expression of wild-type and mutant forms of the platelet-derived growth factor receptor (PDGFR) family of receptor tyrosine kinases has been implicated in various oncologic indications such as leukemias, gliomas, and soft tissue sarcomas. Clinically used kinase inhibitors imatinib and sunitinib are potent inhibitors of wild-type PDGFR family members, but show reduced binding to mutant forms. Here we describe compound 5 which binds to both wild-type and oncogenic mutant forms of PDGFR family members, and demonstrates both cellular and in vivo activity.

Platelet-Derived Growth Factor Receptor Activation Promotes the Prodestructive Invadosome-Forming Phenotype of Synoviocytes from Patients with Rheumatoid Arthritis.

Fibroblast-like synoviocytes (FLS) play a major role in invasive joint destruction in rheumatoid arthritis (RA). This prodestructive phenotype has been shown to involve autocrine TGF-β that triggers formation of matrix-degrading invadosomes through molecular mechanisms that are not fully elucidated. The platelet-derived growth factor (PDGF) receptor (PDGFR) family of receptor tyrosine kinases (RTK) has been shown to cooperate with TGF-β in various pathological conditions. We therefore sought to determine whether RTK activity played a role in invadosome biogenesis. We demonstrated that, among the common RTKs, PDGFR-αβ was specifically phosphorylated in FLS from RA patients. Phosphorylation of PDGFR-αβ was also elevated in RA synovial tissues. Interference with PDGFR activation or PDGF neutralization inhibited invadosome formation in RA synoviocytes, indicating the presence of an autocrine PDGFR activation loop that involved endogenous PDGF. Among the PDGF-A-D isoforms, only PDGF-B was found both significantly elevated in FLS lines from RA patients, and related to high-invadosome forming cells. Addition of TGF-β upregulated invadosome formation, PDGF-B mRNA expression, and phosphorylation of PDGFR. All of these functions were efficiently suppressed by TGF-β neutralization or interference with the Smad/TβR1or PI3K/Akt pathway. Among the class 1 PI3K family proteins known to be expressed in RA synoviocytes, PI3Kα was selectively involved in PDGF-B expression, whereas both PI3Kα and PI3Kδ participated in invadosome formation. Our findings demonstrate that PDGFR is a critical RTK required for the prodestructive phenotype of RA synovial cells. They also provide evidence for an association between autocrine TGF-β and PDGFR-mediated invadosome formation in RA synoviocytes that involves the production of PDGF-B induced by TGF-β.

In silico exploration of c-KIT inhibitors by pharmaco-informatics methodology: pharmacophore modeling, 3D QSAR, docking studies, and virtual screening.

c-KIT is a component of the platelet-derived growth factor receptor family, classified as type-III receptor tyrosine kinase. c-KIT has been reported to be involved in, small cell lung cancer, other malignant human cancers, and inflammatory and autoimmune diseases associated with mast cells. Available c-KIT inhibitors suffer from tribulations of growing resistance or cardiac toxicity. A combined in silico pharmacophore and structure-based virtual screening was performed to identify novel potential c-KIT inhibitors. In the present study, five molecules from the ZINC database were retrieved as new potential c-KIT inhibitors, using Schrödinger's Maestro 9.0 molecular modeling suite. An atom-featured 3D QSAR model was built using previously reported c-KIT inhibitors containing the indolin-2-one scaffold. The developed 3D QSAR model ADHRR.24 was found to be significant (R2 = 0.9378, Q2 = 0.7832) and instituted to be sufficiently robust with good predictive accuracy, as confirmed through external validation approaches, Y-randomization and GH approach [GH score 0.84 and Enrichment factor (E) 4.964]. The present QSAR model was further validated for the OECD principle 3, in that the applicability domain was calculated using a "standardization approach." Molecular docking of the QSAR dataset molecules and final ZINC hits were performed on the c-KIT receptor (PDB ID: 3G0E). Docking interactions were in agreement with the developed 3D QSAR model. Model ADHRR.24 was explored for ligand-based virtual screening followed by in silico ADME prediction studies. Five molecules from the ZINC database were obtained as potential c-KIT inhibitors with high in -silico predicted activity and strong key binding interactions with the c-KIT receptor.

Nintedanib: from discovery to the clinic.

Nintedanib (BIBF1120) is a potent, oral, small-molecule tyrosine kinase inhibitor, also known as a triple angiokinase inhibitor, inhibiting three major signaling pathways involved in angiogenesis. Nintedanib targets proangiogenic and pro-fibrotic pathways mediated by the VEGFR family, the fibroblast growth factor receptor (FGFR) family, the platelet-derived growth factor receptor (PDGFR) family, as well as Src and Flt-3 kinases. The compound was identified during a lead optimization program for small-molecule inhibitors of angiogenesis and has since undergone extensive clinical investigation for the treatment of various solid tumors, and in patients with the debilitating lung disease idiopathic pulmonary fibrosis (IPF). Recent clinical evidence from phase III studies has shown that nintedanib has significant efficacy in the treatment of NSCLC, ovarian cancer, and IPF. This review article provides a comprehensive summary of the preclinical and clinical research and development of nintedanib from the initial drug discovery process to the latest available clinical trial data.

The fibroblast growth factor receptor genetic status as a potential predictor of the sensitivity to CH5183284/Debio 1347, a novel selective FGFR inhibitor.

The FGF receptors (FGFR) are tyrosine kinases that are constitutively activated in a subset of tumors by genetic alterations such as gene amplifications, point mutations, or chromosomal translocations/rearrangements. Recently, small-molecule inhibitors that can inhibit the FGFR family as well as the VEGF receptor (VEGFR) or platelet-derived growth factor receptor (PDGFR) family displayed clinical benefits in cohorts of patients with FGFR genetic alterations. However, to achieve more potent and prolonged activity in such populations, a selective FGFR inhibitor is still needed. Here, we report the identification of CH5183284/Debio 1347, a selective and orally available FGFR1, FGFR2, and FGFR3 inhibitor that has a unique chemical scaffold. By interacting with unique residues in the ATP-binding site of FGFR1, FGFR2, or FGFR3, CH5183284/Debio 1347 selectively inhibits FGFR1, FGFR2, and FGFR3 but does not inhibit kinase insert domain receptor (KDR) or other kinases. Consistent with its high selectivity for FGFR enzymes, CH5183284/Debio 1347 displayed preferential antitumor activity against cancer cells with various FGFR genetic alterations in a panel of 327 cancer cell lines and in xenograft models. Because of its unique binding mode, CH5183284/Debio 1347 can inhibit FGFR2 harboring one type of the gatekeeper mutation that causes resistance to other FGFR inhibitors and block FGFR2 V564F-driven tumor growth. CH5183284/Debio 1347 is under clinical investigation for the treatment of patients harboring FGFR genetic alterations.

Randomized Phase II Open-Label Study of mFOLFOX6 in Combination With Linifanib or Bevacizumab for Metastatic Colorectal Cancer

BackgroundAlthough CRC is the third most commonly diagnosed cancer in the United States, second-line CRC treatment is limited. In this trial we examined the efficacy and safety of linifanib, an oral, potent, selective tyrosine kinase inhibitor of vascular endothelial growth factor and platelet-derived growth factor receptor families, with mFOLFOX6, compared with bevacizumab and mFOLFOX6, in previously treated metastatic CRC. Patients and MethodsOne hundred forty-eight patients with advanced CRC previously treated with fluoropyrimidine or irinotecan received bevacizumab (10 mg/kg, intravenous), low-dose linifanib (7.5 mg), or high-dose linifanib (12.5 mg), with mFOLFOX6. The primary end point was progression-free survival (PFS). Secondary objectives included overall survival (OS), objective response rate (ORR), and safety. ResultsNo statistically significant differences in PFS occurred between bevacizumab and linifanib doses (low, hazard ratio [HR], 1.453 [95% confidence interval [CI], 0.830-2.539]; high, HR, 1.257 [95% CI, 0.672-2.351]). Median OS values were similar for bevacizumab and high-dose linifanib (bevacizumab, 16.5 months [95% CI, 13.0-not available]; high-dose linifanib, 16.4 months [95% CI, 11.9-21.7]; low-dose linifanib, 12.0 months [95% CI, 10.1-13.0]). ORRs were similar (bevacizumab, 34.7% [95% CI, 21.7-49.6]; low-dose linifanib, 24.0% [95% CI, 13.1-38.2]; high-dose linifanib, 22.4% [95% CI, 11.8-36.6]). Median cycles of 5-fluorouracil were reduced in the linifanib arms, versus the bevacizumab arm. Grade 3/4 adverse event occurrences were more frequent with linifanib. Palmar-plantar erythrodysesthesia, hypothyroidism, and thrombocytopenia were more common with high-dose linifanib than bevacizumab. ConclusionCombining linifanib with mFOLFOX6 as a second-line treatment for metastatic CRC did not improve PFS, radiographic findings, or duration of response versus bevacizumab and mFOLFOX6.

Genetic screening and functional characterization of PDGFRB mutations associated with basal ganglia calcification of unknown etiology.

Three causal genes for idiopathic basal ganglia calcification (IBGC) have been identified. Most recently, mutations in PDGFRB, encoding a member of the platelet-derived growth factor receptor family type β, and PDGFB, encoding PDGF-B, the specific ligand of PDGFRβ, were found implicating the PDGF-B/PDGFRβ pathway in abnormal brain calcification. In this study, we aimed to identify and study mutations in PDGFRB and PDGFB in a series of 26 patients from the Mayo Clinic Florida Brain Bank with moderate to severe basal ganglia calcification (BCG) of unknown etiology. No mutations in PDGFB were found. However, we identified one mutation in PDGFRB, p.R695C located in the tyrosine kinase domain, in one BGC patient. We further studied the function of p.R695C mutant PDGFRβ and two previously reported mutants, p.L658P and p.R987W PDGFRβ in cell culture. We show that, in response to PDGF-BB stimulation, the p.L658P mutation completely suppresses PDGFRβ autophosphorylation, whereas the p.R695C mutation results in partial loss of autophosphorylation. For the p.R987W mutation, our data suggest a different mechanism involving reduced protein levels. These genetic and functional studies provide the first insight into the pathogenic mechanisms associated with PDGFRB mutations and provide further support for a pathogenic role of PDGFRB mutations in BGC.

Pooled Population Pharmacokinetic Analysis of Phase I, II and III Studies of Linifanib in Cancer Patients

Linifanib is a multi-targeted receptor tyrosine kinase inhibitor, which can inhibit members of the vascular endothelial growth factor and platelet-derived growth factor receptor families. The objective of this analysis was to characterize the population pharmacokinetics of linifanib in cancer patients. We pooled 7,351 linifanib plasma concentrations from 1,010 cancer patients enrolled in 13 clinical studies. Population pharmacokinetic modelling was performed using NONMEM version 7.2. The covariates that were screened included the cancer type, co-medications, creatinine clearance, formulation, fed status, liver function markers (bilirubin, blood urea nitrogen [BUN], aspartate aminotransferase [AST], alanine aminotransferase [ALT]), albumin, age, sex, race, body weight, surface area and body mass index. A two-compartment model with first-order absorption and disposition best described linifanib pharmacokinetics. An increase in body weight was associated with less than proportional increases in volumes of distribution. Subjects with hepatocellular carcinoma and renal cell carcinoma were estimated to have 63 and 86% larger volumes of distribution, respectively, than subjects with the other cancer types. Females had 25% slower oral clearance (CL/F) than males, while subjects with colorectal cancer had 41% faster CL/F than other subjects. For linifanib bioavailability, subjects with refractory acute myeloid leukaemia or myelodysplastic syndrome had 43% lower bioavailability, evening doses were associated with 27% lower bioavailability than morning doses, and administration of linifanib under fed conditions decreased the bioavailability by 14%. Finally, the oral solution formulation showed two-fold faster absorption than the tablet formulations. The use of mixed-effects modelling allowed robust assessment of the impact of the concomitant effects of body size, different cancer types, formulation, diurnal variation, sex and food on linifanib pharmacokinetics. The developed population pharmacokinetic model describes linifanib concentrations adequately and can be used to conduct simulations or to evaluate the linifanib exposure-response relationship.

Therapeutic effects of sunitinib, one of the anti-angiogenetic drugs, in a murine arthritis

Objectives. The purpose of this study was to confirm the inhibitory effects of sunitinib, an angiogenesis inhibitor that targets tyrosine kinases of vascular endothelial growth factor receptor (VEGFR) family and platelet-derived growth factor receptor (PDGFR) family, on arthritis in mice with type II collagen-induced arthritis (CIA).Methods. Sunitinib at a concentration of 30 or 60 mg/kg/day was intraperitoneally administered to mice with CIA. We compared the changes in arthritis score over time, pathological score, bone density, and microvascular density in synovial membrane between the vehicle and treatment groups.Results. In the sunitinib-treated groups, the arthritis score decreased in a dose-dependent manner in comparison with that in the vehicle group. Furthermore, improvement in the pathological score, inhibitory tendency of loss in the bone density, and a decrease in the synovial microvascular density were also observed in the sunitinib-treated groups.Conclusions. Sunitinib remarkably inhibited arthritis, particularly synovial angiogenesis in a murine CIA model. This compound may be useful for treating arthritis.

Blockade of the PDGFR family together with SRC leads to diminished proliferation of colorectal cancer cells

Among the family of receptor tyrosine kinases (RTKs), platelet-derived growth factor receptor (PDGFR) has attracted increasing attention as a potential target of anti-tumor therapy in colorectal cancer (CRC). To study the function of PDGFRβ in CRC cell lines, SW480, DLD-1 and Caco-2 cells showing high PDGFRβ expression were used for receptor down-regulation by small interfering RNA (siRNA) and using the pharmacological inhibitor of PDGFRβ Ki11502. Blockade of PDGFRβ using both approaches led to moderate inhibition of proliferation and diminished activation of the downstream PI3K-signaling pathway in all three cell lines. Surprisingly, incubation with Ki11502 resulted in an arrest of SW480 cells in the G2 phase of the cell cycle, whereas the siRNA approach did not result in this effect. To address this difference, we analyzed the involvement of the PDGFRβ family member c-KIT in Ki11502 effectiveness, but siRNA and proliferation studies in SW480 and DLD-1 cells could not prove the involvement of c-KIT inactivation during Ki11502 treatment. Hence, an RTK activation antibody array on SW480 cells led us to the identification of the non-receptor tyrosine kinase SRC, which is inactivated after Ki11502 treatment but not after the siRNA approach. Further studies using the SRC-specific inhibitor PP2 showed that SRC inhibition upon treatment with the inhibitor Ki11502 is responsible for the observed effects of Ki11502 in SW480 and DLD-1 CRC cells. In summary, our results demonstrate that the inhibition of PDGFRβ alone using siRNA has only moderate cellular effects in CRC cell lines; however, the multi-target inhibition of PDGFRβ, c-KIT and SRC, e.g., using Ki11502, represents a promising therapeutic intervention for the treatment of CRC.

Preclinical Characterization of ABT-348, a Kinase Inhibitor Targeting the Aurora, Vascular Endothelial Growth Factor Receptor/Platelet-Derived Growth Factor Receptor, and Src Kinase Families

ABT-348 [1-(4-(4-amino-7-(1-(2-hydroxyethyl)-1H-pyrazol-4-yl)thieno[3,2-c]pyridin-3-yl)phenyl)-3-(3-fluorophenyl)urea] is a novel ATP-competitive multitargeted kinase inhibitor with nanomolar potency (IC(50)) for inhibiting binding and cellular autophosphorylation of Aurora B (7 and 13 nM), C (1 and 13 nM), and A (120 and 189 nM). Cellular activity against Aurora B is reflected by inhibition of phosphorylation of histone H3, induction of polyploidy, and inhibition of proliferation of a variety of leukemia, lymphoma, and solid tumor cell lines (IC(50) = 0.3-21 nM). In vivo inhibition of Aurora B was confirmed in an engrafted leukemia model by observing a decrease in phosphorylation of histone H3 that persisted in a dose-dependent manner for 8 h and correlated with plasma concentration of ABT-348. Evaluation of ABT-348 across a panel of 128 kinases revealed additional potent binding activity (K(i) < 30 nM) against vascular endothelial growth factor receptor (VEGFR)/platelet-derived growth factor receptor (PDGFR) families and the Src family of cytoplasmic tyrosine kinases. VEGFR/PDGFR binding activity correlated with inhibition of autophosphorylation in cells and inhibition of vascular endothelial growth factor (VEGF)-stimulated endothelial cell proliferation (IC(50) ≤ 0.3 nM). Evidence of on-target activity in vivo was provided by the potency for blocking VEGF-mediated vascular permeability and inducing plasma placental growth factor. Activity against the Src kinase family was evident in antiproliferative activity against BCR-ABL chronic myeloid leukemia cells and cells expressing the gleevec-resistant BCR-ABL T315I mutation. On the basis of its unique spectrum of activity, ABT-348 was evaluated and found effective in representative solid tumor [HT1080 and pancreatic carcinoma (MiaPaCa), tumor stasis] and hematological malignancy (RS4;11, regression) xenografts. These results provide the rationale for clinical assessment of ABT-348 as a therapeutic agent in the treatment of cancer.

Assessing the Activity of Cediranib, a VEGFR-2/3 Tyrosine Kinase Inhibitor, against VEGFR-1 and Members of the Structurally Related PDGFR Family

Cediranib is a potent inhibitor of the VEGF receptor (VEGFR)-2 and VEGFR-3 tyrosine kinases. This study assessed the activity of cediranib against the VEGFR-1 tyrosine kinase and the platelet-derived growth factor receptor (PDGFR)-associated kinases c-Kit, PDGFR-α, and PDGFR-β. Cediranib inhibited VEGF-A-stimulated VEGFR-1 activation in AG1-G1-Flt1 cells (IC(50) = 1.2 nmol/L). VEGF-A induced greatest phosphorylation of VEGFR-1 at tyrosine residues Y1048 and Y1053; this was reversed by cediranib. Potency against VEGFR-1 was comparable with that previously observed versus VEGFR-2 and VEGFR-3. Cediranib also showed significant activity against wild-type c-Kit in cellular phosphorylation assays (IC(50) = 1-3 nmol/L) and in a stem cell factor-induced proliferation assay (IC(50) = 13 nmol/L). Furthermore, phosphorylation of wild-type c-Kit in NCI-H526 tumor xenografts was reduced markedly following oral administration of cediranib (≥1.5 mg/kg/d) to tumor-bearing nude mice. The activity of cediranib against PDGFR-β and PDGFR-α was studied in tumor cell lines, vascular smooth muscle cells (VSMC), and a fibroblast line using PDGF-AA and PDGF-BB ligands. Both receptor phosphorylation (IC(50) = 12-32 nmol/L) and PDGF-BB-stimulated cellular proliferation (IC(50) = 32 nmol/L in human VSMCs; 64 nmol/L in osteosarcoma cells) were inhibited. In vivo, ligand-induced PDGFR-β phosphorylation in murine lung tissue was inhibited by 55% following treatment with cediranib at 6 mg/kg but not at 3 mg/kg or less. In contrast, in C6 rat glial tumor xenografts in mice, ligand-induced phosphorylation of both PDGFR-α and PDGFR-β was reduced by 46% to 61% with 0.75 mg/kg cediranib. Additional selectivity was showed versus Flt-3, CSF-1R, EGFR, FGFR1, and FGFR4. Collectively, these data indicate that cediranib is a potent pan-VEGFR kinase inhibitor with similar activity against c-Kit but is significantly less potent than PDGFR-α and PDGFR-β.

Abstract 1133: LRIG2 promotes PDGF induced experimental glioma

Abstract Leucine-rich and immunoglobulin-like domains protein 2 (LRIG2) is a transmembrane protein, whose expression is associated with poor survival of oligodendroglioma patients. This is in contrast with findings for the homologous protein LRIG1, a negative regulator of growth factor signaling, whose expression is associated with good survival in several different malignancies. Here, we address the role of LRIG2 in an experimental glioma model and its possible regulation of growth factor receptors belonging to the epidermal growth factor receptor (EGFR) and platelet-derived growth factor (PDGF) receptor (PDGFR) families. Gliomas were induced in Ntv-a mice by intracranial injection of PDGFB-expressing RCAS viruses. All injected Lrig2-wild-type mice developed oligodendroglioma-like brain tumors of grade II/III (82%) or glioblastoma-like tumors of grade IV (18%), whereas Lrig2-deficient mice only developed grade II/III tumors (77%) or no detectable tumors (23%). A role for Lrig2 in PDGFR signaling was shown in mouse embryonic fibroblast (MEF) cell lines. MEF cells from Lrig2-deficient mice were less sensitive to PDGFB-induced cell proliferation than MEF cells from wild-type mice. In transfected HEK-293T cells, LRIG2 and LRIG3, in contrast to LRIG1, did not negatively regulate the protein levels of EGFR, ErbB2, PDGFRα, or PDGFRβ. Intriguingly, LRIG2 and LRIG3 instead up-regulated the protein levels of PDGFRα. In summary, we show that LRIG2 promoted the genesis and/or progression of PDGF-induced oligodendroglioma and positively regulated PDGFRα protein levels. This further supports the notion that LRIG proteins play important roles in cancer biology and suggests that LRIG1 and LRIG2/LRIG3 may have opposing functions. 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 1133. doi:10.1158/1538-7445.AM2011-1133