Inhibition Of Receptor Phosphorylation Research Articles

Fungal alkaloid malbrancheamide reorients the lipid binding domain of GRK5

G protein-coupled receptors (GPCRs) are the largest group of receptors involved in various types of signaling. GPCR signaling is regulated via receptor phosphorylation by G protein-coupled receptor kinases 5 (GRK5). Calmodulin (CaM), a universal Ca2+ sensor, inhibits receptor phosphorylation by binding to GRK5. However, the inhibitor malbrancheamide (MBC), which binds at CaM C-lobe, allows for receptor phosphorylation. To understand the phosphorylation mechanism by GRK5, we carried out a MD simulation of the CaM/GRK5 complex in the presence and absence of the MBC inhibitor. The lipid binding domain (LBD) of GRK5 adopted different positions in the presence and absence of inhibitor. Furthermore, the inhibitor MBC restricted the movement of the N-lobe tether (NLT) loop, probably blocking the autophosphorylation of GRK5. Communicated by Ramaswamy H. Sarma

Abstract B129: A bispecific antibody inhibits FGFR3 dimerization and is highly effective in FGFR3-driven tumor models

Abstract Background: The receptor tyrosine kinase (RTK) FGFR3 is frequently mutated in bladder cancer (~15-20% in muscle invasive bladder cancer, MIBC) and is a validated therapeutic target. Oncogenic alterations in FGFR3 include point mutations in the extracellular domain (S249C, R248C, G372C, Y375C) and gene fusions that place an oligomerization domain from a fusion partner at the FGFR3 C-terminus (e.g., FGFR3-TACC3). Although pan-FGFR tyrosine kinase inhibitors (TKI) have demonstrated clinical efficacy, toxicity and acquired resistance limit the benefit of these agents. While antibody-based therapy can offer superior selectivity compared to TKIs, conventional ligand-blocking antibodies are usually ineffective inhibitors of constitutively active RTKs. Further, the existence of multiple oncogenic variants of FGFR3 presents an additional challenge for antibody-mediated blockade. Results: We generated a tetravalent bispecific FGFR3xFGFR3 antibody that potently inhibits both FGFR3 point mutants and fusions with better efficacy than the conventional antibodies that we produced and a clinical-stage comparator antibody. Mechanistically, our bispecific antibody binds FGFR3 through two different epitopes located in immunoglobulin-like domains D1 and D2/3 respectively. It potently blocks the dimerization of FGFR3 S249C, the most common oncogenic variant, inhibiting receptor phosphorylation and downstream signaling. Additionally, our bispecific antibody inhibits the function of FGFR3 gatekeeper mutations (V557M/L) that are resistant to TKIs. In FGFR3-driven tumor models, our antibody is highly effective, providing efficacy similar to that of the FDA-approved TKI erdafitinib. Conclusion: We have characterized the properties of a tetravalent bispecific FGFR3xFGFR3 antibody that shows strong anti-tumor activity in models driven by FGFR3 S249C as well as by FGFR3-TACC3 fusion, outperforming a conventional antibody that was previously in clinical development. The activity of the bispecific antibody is superior to the mixture of the two parental antibodies and results from strong inhibition of FGFR3 dimerization. Our bispecific antibody provides a novel approach to achieve selective and potent inhibition of multiple oncogenic variants of FGFR3 in bladder cancer, including FGFR3 variants that are resistant to FGFR TKIs. Citation Format: Yan Yang, Naga Suhasini Avvaru, Zaoli Jiang, Nina Liu, Michael Rosconi, Bojie Zhang, Yinyin Li, Erica Ullman, John Chia-Yang Lin, Yang Shen, Christopher Daly. A bispecific antibody inhibits FGFR3 dimerization and is highly effective in FGFR3-driven tumor models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B129.

PLCγ1‑dependent invasion and migration of cells expressing NSCLC‑associated EGFR mutants.

The increased tyrosine kinase activity of non‑small cell lung cancer (NSCLC)‑associated epidermal growth factor receptor (EGFR) mutants results in deregulated pathways that contribute to malignant cell survival, tumor progression and metastasis. Previous studies investigating lung cancer‑associated EGFR have focused on the prognostic implications of receptor kinase mutations in patients with NSCLC; however, the role of EGFR mutations in tumor cell invasion and migration remains undetermined. The present study was designed to investigate the role of NSCLC‑associated mutant EGFR‑driven signaling pathways in cell proliferation and invasion. Non‑endogenous EGFR‑expressing 293cells stably expressing EGFR mutants that are sensitive or resistant to Food and Drug Administration(FDA)‑approved EGFR‑targeted tyrosine kinase inhibitors(TKIs) were used in the present study. The experiments demonstrated an increased phosphorylation of phospholipase (PLC)γ1, c‑Cbl, signal transducer and activator of transcription(Stat), extracellular regulated kinase (Erk)1/2, Akt, Shc and Gab1 proteins in cells expressing a mutant form, rather than the wild‑type receptor. As PLCγ1 is a known regulator of metastatic development, mutant receptor‑mediated PLCγ1 activation was further evaluated. To examine the effects of EGFR and PLCγ1 phosphorylation, the metastatic potential of cells expressing mutants was investigated using wound healing, Transwell cell migration and invasion assays. The inhibition of receptor phosphorylation with the 1st, 2nd and 3rdgeneration TKIs, gefitinib, afatinib, osimertinib, respectively, reduced PLCγ1 phosphorylation, and reduced the invasive and migratory potential of 293cells, confirming PLCγ1 as one of the probable downstream effectors of mutant EGFR signaling. However, the PLC inhibitor, U73122, inhibited cell migration and invasion without affecting EGFR signaling and PLCγ1 phosphorylation. Notably, U73122 reduced Akt and Erk1/2 phosphorylation within 25min of its application; however, 100%cell viability was recorded even after 48h. Upon further investigation, proliferative signaling pathways remained active at 48h, in accordance with cell viability. Therefore, the present study concludes that mutant receptor‑mediated PLCγ1 activation may play a significant role in the migration and invasion of NSCLC tumors; however, its regulatory role in tumor cell proliferation warrants further investigation and validation in lung tumor cell lines harboring EGFR mutations.

Perturbation of the interactions of calmodulin with GRK5 using a natural product chemical probe

G protein-coupled receptor (GPCR) kinases (GRKs) are responsible for initiating desensitization of activated GPCRs. GRK5 is potently inhibited by the calcium-sensing protein calmodulin (CaM), which leads to nuclear translocation of GRK5 and promotion of cardiac hypertrophy. Herein, we report the architecture of the Ca2+·CaM-GRK5 complex determined by small-angle X-ray scattering and negative-stain electron microscopy. Ca2+·CaM binds primarily to the small lobe of the kinase domain of GRK5 near elements critical for receptor interaction and membrane association, thereby inhibiting receptor phosphorylation while activating the kinase for phosphorylation of soluble substrates. To define the role of each lobe of Ca2+·CaM, we utilized the natural product malbrancheamide as a chemical probe to show that the C-terminal lobe of Ca2+·CaM regulates membrane binding while the N-terminal lobe regulates receptor phosphorylation and kinase domain activation. In cells, malbrancheamide attenuated GRK5 nuclear translocation and effectively blocked the hypertrophic response, demonstrating the utility of this natural product and its derivatives in probing Ca2+·CaM-dependent hypertrophy.

Investigating Phosphorylation Differences in FFA4 Short and Long Isoforms

The G protein‐coupled receptor Free Fatty Acid Receptor 4 (FFA4) is an attractive drug target due to its profound influence on a variety of endocrine, inflammatory, and metabolic processes. Humans contain a unique long splice variant of FFA4, which contains an additional 16 amino acid insert within the 3rd intracellular loop, and has not been found to be expressed in any other mammals. Although the shorter FFA4 isoform (FFA4‐S) is ubiquitously expressed, human FFA4‐L has only been found in colorectal tissue. More interestingly, although both isoforms are agonized by the same ligands, upon agonism, FFA4‐L has been demonstrated to possess an intrinsic bias towards β‐arrestin signaling rather than coupling to G proteins. Since it is known that receptor phosphorylation is required for interaction with β‐arrestin, the purpose of the current study was to determine whether any differences exist between the long and short isoform of FFA4 in terms of basal, homologous, or heterologous phosphorylation.MethodsWhole cell phosphorylation assays using 32P‐labeled orthophosphate were performed in HEK293 cells transiently expressing FFA4 isoforms or mutants. Inquiries into G‐protein receptor kinase (GRK) activity were performed using small interfering RNA (siRNA) followed by whole cell phosphorylation assay. Inquiries into protein kinase A (PKA) or protein kinase C (PKC) activity utilized the adenylyl cyclase activator forskolin to induce PKA activity, PKA inhibitor H‐89, PKC activator phorbol‐myristol acetate (PMA), and PKC inhibitor BIMII. Arrestin recruitment assays were conducted using yellow fluorescence protein (YFP) tagged arrestin‐3 and visualized via confocal microscopy. Truncated mutants were generated by sequential deletions via overlap primers and PCR mutagenesis.ResultsSimilar to our previously reported results with FFA4‐S, homologous phosphorylation of FFA4‐L is mediated by GRK6, whereas heterologous phosphorylation is mediated by PKC. FFA4‐L has lower basal phosphorylation compared to FFA4‐S. Truncation of FFA4‐L at S356 fully inhibits receptor phosphorylation and abrogates arrestin recruitment, matching reported observations for FFA4‐S at corresponding S340.ConclusionThe same kinases are responsible for homologous and heterologous phosphorylation of FFA4‐S and FFA4‐L, while basal phosphorylation of FFA4‐L remains lower compared to FFA4‐S. The arrestin sensor for both isoforms is located on the C‐terminal tail of the receptor following S340 (FFA4‐S) or S356 (FFA4‐L).Support or Funding InformationNIH/NIDDK DK098730 to NHM and Diabetes Action Research and Education Foundation grants to NHMThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 4589: Perturbation of epidermal growth factor (EGF) receptor internalization by novel aspirin analogues in SW480 cells

Abstract BACKGROUND Colorectal cancer (CRC) is a global phenomenon with over 135 million new cases and over 600,000 deaths worldwide annually. The EGFR regulates normal growth and differentiation of cells, however its dysregulation is the cause of a wide range of cancers, including CRC. Activation of the EGFR requires phosphorylation at specific sites, which then leads to the recruitment of proteins and ultimately its internalization. The use of low-dose aspirin as a preventative agent is a controversial topic, particularly as the mechanism of action is hotly disputed. We have previously determined that novel aspirin analogues dramatically reduce EGFR phosphorylation in SW480 CRC cells; these cells express neither COX-1 nor COX-2, the perceived target for aspirin-like compounds. We have seen these aspirin analogues to also perturb the internalization of this receptor. METHODS SW 480 CRC cells were cultured in Leibovitz's L-15 medium on glass slides and starved for 48 hours. The cells were then treated with 0.5 mM aspirin analogues (ortho-aspirin, meta-aspirin, para-aspirin, ortho-thioaspirin, meta-thioaspirin and para-thioaspirin) for 30 minutes and then treated with EGF-tagged with AlexaFluor 555 for an hour. The reaction was the quenched with cold PBS, fixed and mounted on microscope slides with VectaShield + DAPI. Confocal microscopy was used to study the effect of these aspirin analogues on EGFR internalization. RESULTS EGF increased phosphorylation at all sites tested. We have peviously shown that these effects were prevented by preincubation with thioaspirins more potently than with aspirin and its positional isomers. These novel thioaspirins are also more potent in inhibiting proliferation of SW480 CRC cells. We now show these analogues prevent the internalization of EGFR. Instead they permit the formation of clusters. Aspirin and its analogues can perturb EGFR internalization and signalling in SW 480 cells. CONCLUSION The EGFR signalling pathway is a target for aspirin and aspirin analogues. The inhibition of receptor phosphorylation is accompanied by the loss of receptor internalization. Citation Format: Asma’U I.J. Bashir, Sarah Jones, Christopher J. Perry, Stephen T. Safrany, Iain D. Nicholl. Perturbation of epidermal growth factor (EGF) receptor internalization by novel aspirin analogues in SW480 cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4589.

Gβ promotes pheromone receptor polarization and yeast chemotropism by inhibiting receptor phosphorylation.

Gradient-directed cell migration (chemotaxis) and growth (chemotropism) are processes that are essential to the development and life cycles of all species. Cells use surface receptors to sense the shallow chemical gradients that elicit chemotaxis and chemotropism. Slight asymmetries in receptor activation are amplified by downstream signaling systems, which ultimately induce dynamic reorganization of the cytoskeleton. During the mating response of budding yeast, a model chemotropic system, the pheromone receptors on the plasma membrane polarize to the side of the cell closest to the stimulus. Although receptor polarization occurs before and independently of actin cable-dependent delivery of vesicles to the plasma membrane (directed secretion), it requires receptor internalization. Phosphorylation of pheromone receptors by yeast casein kinase 1 or 2 (Yck1/2) stimulates their internalization. We showed that the pheromone-responsive Gβγ dimer promotes the polarization of the pheromone receptor by interacting with Yck1/2 and locally inhibiting receptor phosphorylation. We also found that receptor phosphorylation is essential for chemotropism, independently of its role in inducing receptor internalization. A mathematical model supports the idea that the interaction between Gβγ and Yck1/2 results in differential phosphorylation and internalization of the pheromone receptor and accounts for its polarization before the initiation of directed secretion.

Abstract 1696: Blocking activity of a novel anti-MET humanized monoclonal antibody, KTN0216, is enhanced by IgG2 isotype in HGF-dependent and Met-amplified tumors

Abstract Aberrant activation of MET kinase can be mediated by ligand-dependent mechanisms through paracrine or autocrine stimulation by its ligand, HGF, or by ligand-independent mechanisms through gene amplification, over-expression or mutation. Both mechanisms of activating MET have been associated with promoting tumor growth and progression. The discovery of antagonistic MET antibodies has been challenging as many anti-MET antibodies promote kinase activation that leads to cell proliferation and migration. We have systematically screened MET antibodies using cell based functional assays and identified a number of antibodies that potently block MET-dependent cell proliferation, cell scattering as well as tumor growth in vivo. One such antibody, KTN-MET-IgG1, binds to the Sema/PSI domain and prevents HGF-MET interaction; additionally it induces receptor ubiqutination and degradation. To evaluate if the IgG isotype influences activity on MET activation and tumor growth we grafted the binding domains of this antibody onto 2 other IgG backbones: KTN-MET-IgG2 and KTN-MET-IgG4. Surprisingly, grafting the antibody variable regions onto the IgG2 framework dramatically enhanced the anti-MET activity in vitro and in vivo. Comparing to KTN-MET-IgG1 and KTN-MET-IgG4 antibodies with KTN-MET-IgG2 antibody, it demonstrated significantly better inhibition of receptor phosphorylation and cell proliferation in MET amplified tumor cell lines (EBC1, SNU5) and comparable activity in HGF-dependent tumor cell lines (A549, U87MG). KTN-MET-IgG2 treatment of tumor bearing mice achieved potent and prolonged suppression of tumor growth using the U87MG glioblastoma xenograft model and tumor growth regression with tumors remaining below detection for over 90 days post last dosing using the SNU5 gastric cancer xenograft model. In summary, we have found a novel IgG2 anti-MET antibody that inhibits tumor growth driven by HGF-dependent MET activation and MET amplification. A model to help explain the contribution of IgG2 isotype to enhanced blocking activity will be presented. The humanized version of KTN-MET-IgG2, KTN0216, is currently in preclinical development to evaluate tumor types best suited for KTN0216 treatment. Citation Format: Sreekala Mandiyan, Brett S. Robinson, Lida Kimmel, Gerald McMahon, Yaron Hadari, Yan Yang. Blocking activity of a novel anti-MET humanized monoclonal antibody, KTN0216, is enhanced by IgG2 isotype in HGF-dependent and Met-amplified tumors. [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 1696. doi:10.1158/1538-7445.AM2015-1696

Abstract 4170: The role of VEGF-C for cell viability, tumor growth and bevacizumab resistance in glioblastoma multiforme

Abstract Although treatment with the VEGF-A targeting antibody Bevacizumab has resulted in substantial response rates in patients with the brain tumor Glioblastoma Multiforme, patients invariable progress. The VEGF-A receptor VEGFR2, has in addition to be expressed on endothelial cells, recently been shown also to be expressed by GBM tumor cells. Autocrine VEGF-A/VEGFR2 signaling has been identified in GBM cells, but contradicting results exist for the effects of inhibiting VEGF-A and VEGFR2 respectively; indicating that VEGFR2 in GBM at least partly is activated by other factors than VEGF-A. Study aim was to search for other factors responsible for VEGFR2 activation in GBM cells and to examine their role for GBM growth and response to Bevacizumab. To study VEGFR2 regulation we examined a VEGFR2-positive and a VEGFR2-negative GBM cell culture, which both expressed VEGF-A. In line with others, we found that VEGFR2 phosphorylation could be stimulated by recombinant VEGF-A, and found that inhibition of receptor phosphorylation by SU1498 resulted in significantly reduced proliferation of the VEGFR2-positive cells, while inhibition of the VEGF-A expressed by the cells using Bevacizumab only had minimal effect on proliferation. Examinations revealed that the VEGFR2-positive cells also were positive for the VEGF variant VEGF-C. Addition of recombinant VEGF-C protein to the VEGFR2-positive cells could stimulate VEGFR2 phosphorylation, while inhibition of VEGF-C using siRNA constructs resulted in reduced in vitro growth of VEGFR2-positive cells. Further, when injected into the brains of mice, VEGF-C-siRNA transfected cells resulted in reduced tumor growth and increased survival compared to control cells. Moreover, measurement of the VEGF-C mRNA level in 19 GBM patient tumors showed that all tumors were positive for VEGF-C expression, although the level was varying. To examine if VEGF-C expression represents a possible mechanism behind insensitivity towards Bevacizumab therapy, we are currently investigating the expression of VEGF-C in paired samples from GBM patients taken before and after Bevacizumab therapy and in xenograft tumors from mice receiving this treatment. In conclusion, our current results show that VEGF-C is of importance for GBM cell viability and tumor growth presumable due to its ability to stimulate autocrine activation of VEGFR2. VEGF-C expression therefore could respresent a possible mechanism behind Bevacizumab resistance. An update on this will be presented. Citation Format: Signe R. Michaelsen, Mette K. Nedergaard, Thomas Urup, Mette Villingshoej, Andreas Kjaer, Lara Perryman, Janine T. Erler, Ulrik Lassen, Hans S. Poulsen. The role of VEGF-C for cell viability, tumor growth and bevacizumab resistance in glioblastoma multiforme. [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 4170. doi:10.1158/1538-7445.AM2015-4170

Salicin from Willow Bark can Modulate Neurite Outgrowth in Human Neuroblastoma SH-SY5Y Cells.

Salicin from willow bark has been used throughout centuries in China and Europe for the treatment of pain, headache, and inflammatory conditions. Recently, it could be demonstrated that salicin binds and activates the bitter taste receptor TAS2R16. Studies on rodent tissues showed the general expression of bitter taste receptors (TAS2Rs) in rodent brain. Here, we demonstrate the expression of hTAS2R16 in human neuronal tissues and the neuroblastoma cell line SH-SY5Y. The functionality was analyzed in the neuroblastoma cell line SH-SY5Y after stimulation with salicin, a known TAS2R16 agonist. In this setting salicin induced in SH-SY5Y cells phosphorylation of ERK and CREB, the key transcription factor of neuronal differentiation. PD98059, an inhibitor of the ERK pathway, as well as probenecid, a TAS2R16 antagonist, inhibited receptor phosphorylation as well as neurite outgrowth. These data show that salicin might modulate neurite outgrowth by bitter taste receptor activation.

HER-3 peptide vaccines/mimics: Combined therapy with IGF-1R, HER-2, and HER-1 peptides induces synergistic antitumor effects against breast and pancreatic cancer cells

The human epidermal growth factor receptor 3 (HER-3/ErbB3) is a unique member of the human epidermal growth factor family of receptors, because it lacks intrinsic kinase activity and ability to heterodimerize with other members. HER-3 is frequently upregulated in cancers with epidermal growth factor receptor (EGFR/HER-1/ErbB1) or human epidermal growth factor receptor 2 (HER-2/ErBB2) overexpression, and targeting HER-3 may provide a route for overcoming resistance to agents that target EGFR or HER-2. We have previously developed vaccines and peptide mimics for HER-1, HER-2 and vascular endothelial growth factor (VEGF). In this study, we extend our studies by identifying and evaluating novel HER-3 peptide epitopes encompassing residues 99–122, 140–162, 237–269 and 461–479 of the HER-3 extracellular domain as putative B-cell epitopes for active immunotherapy against HER-3 positive cancers. We show that the HER-3 vaccine antibodies and HER-3 peptide mimics induced antitumor responses: inhibition of cancer cell proliferation, inhibition of receptor phosphorylation, induction of apoptosis and antibody dependent cellular cytotoxicity (ADCC). Two of the HER-3 epitopes 237–269 (domain II) and 461–479 (domain III) significantly inhibited growth of xenografts originating from both pancreatic (BxPC3) and breast (JIMT-1) cancers. Combined therapy of HER-3 (461–471) epitope with HER-2 (266–296), HER-2 (597–626), HER-1 (418–435) and insulin-like growth factor receptor type I (IGF-1R) (56–81) vaccine antibodies and peptide mimics show enhanced antitumor effects in breast and pancreatic cancer cells. This study establishes the hypothesis that combination immunotherapy targeting different signal transduction pathways can provide effective antitumor immunity and long-term control of HER-1 and HER-2 overexpressing cancers.

Abstract 1753: GLPG1790: The first Ephrin (EPH) receptor tyrosine kinase inhibitor for the treatment of triple negative breast cancer

Abstract Receptor tyrosine kinases define a clinically relevant class of targets in the field of cancer. Here we report the discovery of a pre-clinical drug candidate directed against the EPH receptor family. Members of this family of receptor tyrosine kinases are over-expressed in diverse cancer types. GLPG1790 is a small molecule, nanomolar inhibitor of various EPH receptors kinases. The compound displayed a remarkable efficacy by once-daily oral administration in a mouse EPHA2 expressing xenograft model (MDA-MB-231). A rapid dose-dependent reduction of tumor growth was achieved, with full inhibition at the oral dose of 30 mg/kg/d. GLPG1790 efficacy at this dose was similar to that of Paclitaxel administered at its maximum tolerated dose. A series of experiments was initiated to confirm the mechanism of action of this compound. GLPG1790 inhibits human EPHA2 kinase activity with an IC50 of 11 nM in a biochemical assay. In the human MDA-MB-231 breast cancer cell line, that expresses a high level of EPHA2 protein, GLPG1790 inhibits receptor phosphorylation with an IC50 of 260 nM, and anchorage-independent growth with similar potency. In addition, in vivo target engagement was demonstrated in the mouse MDA-MB-231 xenograft model. GLPG1790 inhibited EPHA2 receptor phosphorylation after single oral administration of 30 and 100 mg/kg doses. The extent of the effects observed on EPHA2 phosphorylation correlated with intra-tumoral GLPG1790 concentration. Moreover, the MAPK pathway, known to be a major driver of proliferation of this cell line, was inhibited both in in vitro cellular assays and in xenograft target engagement studies. Flow cytometric analyses revealed a cell cycle arrest at the G0/G1 phase for MDA-MB-231 cells treated with GLPG1790. EPHA2 knock-down-based experiments further support the EPH-driven mode of action of GLPG1790. All together these data stimulate the development of GLPG1790 in triple negative breast cancer. This novel mechanism of action is under investigation in other cancer types overexpressing EPH's (melanoma, pancreatic, ovarian, prostatic and colorectal cancers). Citation Format: Philippe Pujuguet, Filip Beirinckx, Carole Delachaume, Jacques Huck, Ellen Van der Aar, Reginald Brys, Luc Van Rompaey, Piet Wigerinck, Laurent Saniere. GLPG1790: The first Ephrin (EPH) receptor tyrosine kinase inhibitor for the treatment of triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1753. doi:10.1158/1538-7445.AM2014-1753

P01.18 * AUTOCRINE VEGFC-VEGFR2 SIGNALING IS OF IMPORTANCE FOR THE GROWTH OF GLIOBLASTOMA MULTIFORME

BACKGROUND: Recent research shows that the angiogenic mediator Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) is expressed in Glioblastoma Multiforme (GBM) cells and that inhibition of VEGFR2 results in reduced growth of GBM cells. Although autocrine VEGFA-VEGFR2 signaling has been identified in these GBM cells, VEGFA inhibition has very limited effect on growth in the cells, indicating that activation of VEGFR2 in GBM cells not solely is dependent on VEGFA. METHODS: Material consisted of 5 GBM cell cultures established under stem cell conditions and tumors from 18 GBM patients. Angiogenesis-related genes were deregulated using SU1498, recombinant VEGFA-165 and VEGFC protein and VEGFC specific siRNA and a corresponding control. Cell viability was measured using MTT assay, mRNA expression by Q-PCR, protein expression by western blotting and tumor growth by bioluminescence imaging. RESULTS: Examination of a panel of GBM cell cultures identified one culture positive for VEGFR2, while the receptor was undetectable in the remaining cultures. As shown by others, we found that inhibition of receptor phosphorylation by SU1498 resulted in significantly reduced proliferation of the VEGFR2-positive cells. Although VEGFR2 phoshorylation could be stimulated by recombinant VEGFA, inhibition of the VEGFA expressed by the cells using Bevacizumab only had minimal effect on proliferation. Examination of the expression of a range of genes revealed that the VEGFR2 positive cells also were positive for the VEGF variant VEGFC. Addition of recombinant VEGFC protein to the VEGFR2 positive cells resulted in VEGFR2 phosphorylation, while inhibition of VEGFC using specific siRNA constructs resulted in significantly reduced in vitro growth of VEGFR2 positive cells. Moreover, when transplanted as orthotopic brain tumors significantly reduced tumor growth was seen for VEGFC siRNA transfected cells as compared to control cells. In parallel, the median survival was increased from 34 days in mice injected with control cells to 55 days in mice injected with VEGFC siRNA transfected cells. Further, to establish the relevance of VEGFC in GBM patient tumors in general, we measured the VEGFC mRNA level in 18 GBM tumors. Although the level was varying, all examined tumors were positive for VEGFC expression. CONCLUSION: GBM cells can in addition to VEGFA also be dependent on VEGFC for VEGFR2 activation, cell viability and tumor growth.

Role of FK506 binding protein 12 in morphine-induced μ-opioid receptor internalization and desensitization

Agonist-activated μ-opioid receptor (OPRM1) undergoes robust receptor phosphorylation by G protein-coupled receptor kinases and subsequent β-arrestin recruitment, triggering receptor internalization and desensitization. Morphine, a widely prescribed opioid, induces receptor phosphorylation inefficiently. Previously we reported that FK506 binding protein 12 (FKBP12) specifically interacts with OPRM1 and such interaction attenuates receptor phosphorylation and facilitates morphine-induced recruitment and activation of protein kinase C. In the current study, we demonstrated that the association of FKBP12 with OPRM1 also affects morphine-induced receptor internalization and G protein-dependent adenylyl cyclase desensitization. Morphine induced faster receptor internalization and adenylyl cyclase desensitization in cells expressing OPRM1 with Pro353 mutated to Ala (OPRM1P353A), which does not interact with FKBP12, or in the presence of FK506 which dissociates the receptor–FKBP12 interaction. Furthermore, knockdown of cellular FKBP12 level by siRNA accelerated morphine-induced receptor internalization and adenylyl cyclase desensitization. Our study further demonstrated that peptidyl prolyl cis–trans isomerase activity of FKBP12 probably plays a role in inhibition of receptor phosphorylation. In the view that internalized receptor recycles and thus counteracts the development of analgesic tolerance, receptor's association with FKBP12 could also contribute to the development of morphine tolerance through modulation of receptor trafficking.

Recombinant Human Kallistatin Inhibits Angiogenesis by Blocking VEGF Signaling Pathway

Kallistatin has been recognized as an endogenous angiogenic inhibitor. However, the underlying molecular mechanism remains poorly understood. Taking it into account that vascular endothelial growth factor (VEGF) has been implicated in all aspects of normal and pathological vasculogenesis and angiogenesis. In this study, we investigated whether VEGF signaling pathway was impacted by the anti-angiogenic effect of recombinant human kallistatin (rhKal). We found that the rhKal inhibited proliferation as well as induced apoptosis of cultured human umbilical vein endothelial cells (HUVECs) in both concentration- and time-dependent manners. The rhKal also suppressed the VEGF-induced migration and tube formation of HUVECs. Furthermore, our data revealed that the rhKal suppressed the VEGF165-stimulated tyrosine phosphorylation of VEGFR-2 as well as its downstream signal molecular activation. The inhibition of receptor phosphorylation was correlated with a decrease in VEGF-triggered phosphorylation of angiogenesis signal molecules AKT and ERK, but not stress-related JNK. Taken together, these findings added the knowledge for us to understand the anti-angiogenic mechanism of kallistatin, which suggested that the rhKal could be worth as a candidate compound for further development for the purpose of anti-angiogenic therapies.

Analysis of Activated Platelet-Derived Growth FactorβReceptor and Ras-MAP Kinase Pathway in Equine Sarcoid Fibroblasts

Equine sarcoids are skin tumours of fibroblastic origin affecting equids worldwide. Bovine papillomavirus type-1 (BPV-1) and, less commonly, type-2 are recognized as etiological factors of sarcoids. The transforming activity of BPV is related to the functions of its major oncoprotein E5 which binds to the platelet-derived growth factor β receptor (PDGFβR) causing its phosphorylation and activation. In this study, we demonstrate, by coimmunoprecipitation and immunoblotting, that in equine sarcoid derived cell lines PDGFβR is phosphorylated and binds downstream molecules related to Ras-mitogen-activated protein kinase-ERK pathway thus resulting in Ras activation. Imatinib mesylate is a tyrosine kinase receptors inhibitor which selectively inhibits the activation of PDGFβR in the treatment of several human and animal cancers. Here we show that imatinib inhibits receptor phosphorylation, and cell viability assays demonstrate that this drug decreases sarcoid fibroblasts viability in a dose-dependent manner. This study contributes to a better understanding of the molecular mechanisms involved in the pathology of sarcoids and paves the way to a new therapeutic approach for the treatment of this common equine skin neoplasm.

Molecular characterization of novel trispecific ErbB-cMet-IGF1R antibodies and their antigen-binding properties

Therapeutic antibodies are well established drugs in diverse medical indications. Their success invigorates research on multi-specific antibodies in order to enhance drug efficacy by co-targeting of receptors and addressing key questions of emerging resistance mechanisms. Despite challenges in production, multi-specific antibodies are potentially more potent biologics for cancer therapy. However, so far only bispecific antibody formats have entered clinical phase testing. For future design of antibodies allowing even more targeting specificities, an understanding of the antigen-binding properties of such molecules is crucial. To this end, we have generated different IgG-like TriMAbs (trispecific, trivalent and tetravalent antibodies) directed against prominent cell surface antigens often deregulated in tumor biology. A combination of surface plasmon resonance and isothermal titration calorimetry techniques enables quantitative assessment of the antigen-binding properties of TriMAbs. We demonstrate that the kinetic profiles for the individual antigens are similar to the parental antibodies and all antigens can be bound simultaneously even in the presence of FcγRIIIa. Furthermore, cooperative binding of TriMAbs to their antigens was demonstrated. All antibodies are fully functional and inhibit receptor phosphorylation and cellular growth. TriMAbs are therefore ideal candidates for future applications in various therapeutic areas.

Abstract A27: PI3K/Akt pathway-dependent and -independent mechanisms of resistance to EGFR tyrosine kinase inhibitors in glioblastoma

Abstract EGFR-targeted therapies such as the tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib have had limited success clinically for the treatment of glioblastoma (GBM). Interestingly, some of the most common mechanisms of resistance to these agents in other solid tumors are rarely present in GBM. In an effort to identify and characterize molecular mechanisms of acquired resistance to EGFR TKIs in GBM, we utilized TKI-sensitive, transformed ink4a/arf −/− astrocytes overexpressing the constitutively active mutant ΔEGFR (or EGFRvIII). To model TKI-resistance in vitro, cells were seeded in soft agar in the presence of gefitinib or erlotinib for 2 weeks; during this time, colony formation was inhibited by TKIs. The dose was escalated, and after 3 weeks, TKI-resistant colonies began to emerge and were isolated to generate clonal cell lines. TKI resistance was verified in vitro as well as in vivo using an orthotopic intracranial model. In three clonally derived TKI-resistant lines, ΔEGFR expression was not altered, and gefitinib inhibited receptor phosphorylation. Two of these three cell lines demonstrated decreased PTEN expression and increased, sustained Akt phosphorylation, indicating a failure of TKIs to suppress signaling through the oncogenic PI3K/Akt pathway. Importantly, this phenotype mimics a known mechanism, as PTEN loss has been implicated in resistance to EGFR TKIs in GBM. The third TKI-resistant cell line had no change in PTEN expression, and Akt phosphorylation was completely inhibited by gefitinib. Interestingly, TKI-resistance was irreversible in those cells with decreased PTEN, and reversible in cells having no change in PTEN expression. In fact, resensitized cells demonstrated ΔEGFR and ERK phosphorylation that was several-fold greater than that of parental cells, as well as increased sensitivity to gefitinib and erlotinib. Interestingly, autophagy was dramatically elevated in all of these TKI-resistant lines, and the cell line that was resensitized to TKIs displayed a significant reduction in autophagy compared to its resistant counterpart. Overall, we have generated and characterized a model system for the identification of mechanisms of resistance to EGFR TKIs in GBM. We have identified at least two distinct resistance scenarios in cells with wild-type PTEN: an irreversible mechanism involving decreased PTEN expression, and another mechanism that is reversible and does not appear to directly involve the PI3K pathway. These findings suggest that reversibility of TKI-resistance following drug removal may depend on mechanism of resistance, which has potential clinical implications. Moreover, the fact that increased autophagy is a common feature suggests a potential therapeutic opportunity for targeting the autophagic process that may apply despite apparent differences in molecular mechanism. This work forms the basis for our effort to uncover novel targets or approaches to therapy that may increase the utility of these drugs for treatment of GBM.

Abstract 853: Ponatinib, a potent pan-BCR-ABL inhibitor, retains activity against gatekeeper mutants of FLT3, RET, KIT, PDGFRα/β and FGFR1

Abstract Introduction: Ponatinib was designed as a highly potent inhibitor of native BCR-ABL as well as the T315I gatekeeper mutant that is resistant to all approved therapies, and has demonstrated substantial activity in patients with native or mutant BCR-ABL in a phase 1 trial. Activity against T315I is due to a key design feature, the triple bond linker, which enforces ligand linearity in this sterically demanding region of the binding site. Ponatinib also inhibits the in vitro kinase activity of a number of other clinically relevant kinases (FLT3, RET, KIT, PDGFRα, PDGFRα, and FGFR1; IC50s of 0.2 to 13 nM), many of which have developed resistance to existing therapies via mutation of the gatekeeper residue. Here we assessed whether ponatinib might represent a more general solution to gatekeeper mutant inhibition of these and other clinically important kinases by virtue of its design features. Results: The cellular activity of ponatinib was assessed using Ba/F3 cells transformed to IL-3 independence by expression of an activated kinase (via fusion to a TEL dimerization domain). Ponatinib potently inhibited viability of cells expressing activated FLT3, RET, KIT, PDGFRα, PDGFRα and FGFR1 with IC50s of 15, 8, 7, 1, 2 and 21 nM, respectively. In contrast, ponatinib did not affect viability of parental Ba/F3 cells grown in the presence of IL-3 (IC50 >1000 nM). Kinase variants in which the gatekeeper residue was mutated to isoleucine, leucine or methionine (all by a single nucleotide change from the native sequence) were then transformed into Ba/F3 cells. All of these variants have been shown previously, or in our studies, to confer substantial resistance (i.e. >50-fold) to other targeted agents. Ponatinib maintained substantial activity against all variants tested. The smallest potency reduction (2- to 9-fold) was seen in variants mutated to isoleucine, with FLT3F691I, KITT670I, PDGFRαT674I, and PDGFRαT681I being inhibited with IC50s of 62, 26, 2 and 17 nM, respectively. A slightly greater potency reduction (10- to 22-fold) was seen in sterically more demanding leucine or methionine mutations, with RETV804M, RETV804L and FGFR1V561M being inhibited with IC50s of 84, 178 and 202 nM, respectively. In all cell lines, cell viability effects were accompanied by inhibition of receptor phosphorylation. Conclusion: Ponatinib is a potent inhibitor of activated variants of FLT3, RET, KIT, PDGFRα, PDGFRα, and FGFR1, in addition to BCR-ABL. Ponatinib also maintains substantial activity against gatekeeper variants shown to mediate resistance to other targeted agents, inhibiting their activity with IC50s that are within range of known clinically achievable plasma concentrations. These results suggest that ponatinib may offer a more general solution to gatekeeper mutant inhibition and support clinical testing in a variety of cancers driven by activated kinases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 853. doi:1538-7445.AM2012-853

Abstract B73: Molecular mechanisms of glioma resistance to EGFR tyrosine kinase inhibitors.

Abstract EGFR-targeted therapies such as the tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib have had limited success clinically in glioblastoma (GBM). However, some of the most common mechanisms of resistance to these agents in other solid tumors are rarely present in GBM. In an effort to identify molecular mechanisms of acquired resistance to EGFR TKIs in GBM, we utilized ink4a/arf −/− astrocytes over-expressing EGFR and having wild-type or deleted PTEN. To model TKI-resistance in vitro, cells were seeded in soft agar with 1 μM gefitinib or erlotinib for 2 weeks; during this time the growth of colonies was inhibited. The dose was escalated to 2 μM and after 3 weeks, TKI-resistant colonies began to form and were isolated to generate clonal cell lines. In three PTEN wild-type TKI-resistant lines, EGFR expression was not altered and gefitinib inhibited receptor phosphorylation in a manner identical to that observed in parental cells, thereby ruling out the possibility that the cells are resistant because the drug cannot reach its site of action and inhibit its target. Interestingly, two of these three cell lines demonstrated decreased PTEN expression and increased AKT phosphorylation. The remaining cell line had no change in total PTEN levels, and appeared identical to parental cells with regard to AKT phosphorylation. Removal of resistant cells from the maintenance dose of TKI resulted in restoration of EGFR and ERK phosphorylation to a level several-fold greater than that in parental cells. Notably, the TKI-resistant phenotype was reversible, and these cells became significantly more sensitive to gefitinib and erlotinib than parental cells. Overall, we have generated and characterized a model system for the identification of mechanisms of resistance to EGFR TKIs in GBM. We have identified at least two different resistance scenarios in cells with wild-type PTEN: decrease in PTEN expression, and another as-of-yet unknown mechanism that does not appear to involve changes in the PI3K pathway. The reversibility of resistance and subsequent increased sensitivity to TKIs have potential clinical implications. This work forms the basis for our effort to uncover novel targets or approaches to therapy that may increase the utility of these drugs for treatment of GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B73.