Search

It is becoming increasingly clear that many solid tumors are not a homogeneous mass, but instead consist of highly heterogeneous microsystems comprised of multiple tumor subpopulations together with stromal cells. Such heterogeneity is often attributed to genetic and epigenetic alterations that promote functional specialization of different tumor subpopulations, thereby supporting the growth and differentiation of the primary tumor and subsequent invasion and metastases. This intratumoral complexity serves as a base for a range of intrinsic and acquired resistance mechanisms that are responsible for the failure of targeted therapies, especially in highly heterogeneous tumors such as lung cancer. JNJ-42756493 is a nanomolar inhibitor of fibroblast growth factor receptors (FGFRs) 1, 2, 3 and 4, that is currently being evaluated in Phase I clinical trials. As part of our ongoing preclinical program to elucidate potential mechanisms of resistance to JNJ-42756493, we have developed and characterized a range of cell-based models, representing different solid tumor histologies which show resistance to JNJ-42756493 and other FGFR inhibitors. Here, we describe the outgrowth of pre-existing subpopulations of cells that were intrinsically resistant to FGFR inhibition, from different FGFR1-amplified lung cancer subtypes: NCI-H1581, large cell (LCLC), and DMS114, small cell lung carcinoma (SCLC) cells. Following expansion of these resistant clones, a range of comparative ‘omics’ approaches (microarray analysis, RNA sequencing, shRNA knockdown screening, exome sequencing, and phospho RTK array analysis) were employed to explore the molecular mechanisms of resistance. Comprehensive profiling of these intrinsically resistant subpopulations has revealed that the LCLC and SCLC models have divergent mechanisms of resistance. Met pathway activation was found in LCLC model whilst IGF1R was found to be activated in the SCLC model resistant to FGFR inhibition. This is a novel observation of IGF1R pathway activation in FGFR1 amplified small cell lung cancer models that are resistant to FGFR inhibition. Blocking Met or IGF1R pathways with targeted therapies was observed to prevent the emergence of intrinsic resistance observed in these FGFR driven tumors. Follow-up experiments were performed in vitro and in vivo to confirm these intrinsic mechanisms of resistance and these data help build a rationale for combinations of targeted therapies in the clinic. Finally, our data indicates that pre-existing heterogeneity is likely to be a key driver for the appearance of resistance to targeted therapy in independent lung cancer models. Citation Format: Eleonora Jovcheva, Souichi Ogata, Kelly Van De Ven, Caroline Paulussen, Inez Van de Weyer, Hans De Wolf, Hugo Ceulemans, Steve McClue, Jorge Vialard, Timothy Perera. Identification of alternative mechanisms of resistance to FGFR inhibitor treatment in FGFR1-amplified large cell compared to FGFR1-amplified small cell lung cancer models. [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 LB-329. doi:10.1158/1538-7445.AM2014-LB-329

The fibroblast growth factor (FGF) signaling axis is increasingly implicated in tumorigenesis and chemoresistance. Alterations in FGFR family members including focal amplification of FGF receptor 1 (FGFR1), mutations in FGFR 2, 3 and 4, translocations involving FGFR 2 and FGFR3, as well as amplification or transcriptional upregulation of various ligand family members have been associated with tumor growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in subsets of various disease settings. A number of agents targeting the FGF signaling axis including small-molecule FGFR targeted agents, with diverse kinase inhibitory and pharmacological profiles, are currently in clinical development. JNJ-42756493 (first disclosure of the structure) has a pharmacological profile that is differentiated from other agents in this class currently under investigation. JNJ-42756493 displays single digit nanomolar FGFR (1, 2, 3 4) tyrosine kinase inhibitory activity. JNJ-42756493 inhibited recombinant FGFR kinase activity in vitro and suppressed FGFR signaling and growth in engineered cell lines and tumor cell lines dependent upon deregulated FGFR expression. JNJ-42756493 demonstrated highly specific tumor inhibitory effects in FGFR1-4 dependent cell lines, in vitro cell lines based xenografts and direct patient derived xenografts, with no discernible activity in models that were not dependent on FGFR signaling. JNJ-42756493 showed favorable drug like properties and displayed a high distribution to lung, liver and kidney tissue. JNJ-42756493 was well tolerated at efficacious doses and resulted in potent dose-dependent antitumor activity accompanied by pharmacodynamic modulation of tumor FGFR and downstream pathway components. Data presented here highlights JNJ-42756493 as a novel, highly potent and selective small-molecule inhibitor of all four known active FGFR kinase family members with potent antitumor activity against FGFR-dependent tumor models. These data, together with emerging observations from our ongoing Phase 1 clinical trial, position JNJ-42756493 as a differentiated FGFR 1, 2, 3 and 4 kinase inhibitor and support its continued clinical development in lung cancer and other malignancies associated with aberrant FGFR signaling. Citation Format: Timothy Perera, Eleanora Jovcheva, Jorge Vialard, Tinne Verhulst, Norbert Esser, Berthold Wroblowski, Ron Gilissen, Eddy Freyne, Peter King, Suso Platero, Olivier Querolle, Laurence Mevellec, Christopher Murray, Lynsey Fazal, Gordon Saxty, George Ward, Matthew Squires, Neil Thompson, David Newell, Patrick Angibaud. JNJ-42756493 is an inhibitor of FGFR-1, 2, 3 and 4 with nanomolar affinity for targeted therapy. [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 1738. doi:10.1158/1538-7445.AM2014-1738

The ability of drugs to penetrate into cells and tissues is a fundamentally important property that is essential for the efficacy of drugs targeting intracellular processes. Many weakly basic small molecule drugs are known to accumulate extensively in acidic sub-cellular compartments such as lysosomes, through a process called ion trapping that is dependent on the physico-chemical properties of a compound. ‘Lysosomal uptake’ has been shown to significantly influence the activity of many agents, including anticancer drugs, both positively and negatively. Here we describe the unique physico-chemical properties of JNJ-42756493, a potent inhibitor of fibroblast growth factor receptors (FGFRs) 1, 2, 3 and 4, currently undergoing clinical testing for the treatment of malignancies with FGFR pathway activating alterations. JNJ-42756493 has weakly basic and lipophilic characteristics, is highly permeable, and is fluorescent when exposed to UV light. The fluorescent property of JNJ-42756493 enabled rapid and convenient visualization of the drug (spatial and temporal localization) within the lysosomes of cultured cells exposed to JNJ-42756493. In order to evaluate the influence of this lysosomotropic property on the activity of JNJ-42756493, FGFR phosphorylation was monitored in a series of wash-out experiments performed in various cellular models with FGFR1 or FGFR2 amplifications. This comparative washout analysis demonstrated prolonged FGFR kinase inhibition by JNJ-42756493 in comparison to other FGFR inhibitors lacking lysosomotropic properties. This qualitative fluorescence-based data was complemented by accurate quantification of the amount of compound taken up by the cells through the use of radiolabelled JNJ-42756493. Our results unequivocally demonstrate that JNJ-42756493 accumulates at high concentrations within the lysosomal compartment of cells without undergoing metabolism. The continued, long lasting effects on the target FGF receptor kinase inhibition following drug washout, suggest a novel intrinsic slow release of the unmodified drug from these intracellular stores. The lysosomotropic property of JNJ-42756493 is proposed to be a major contributor to the efficient and persistent inhibition of FGFR phosphorylation leading to potent cellular and in vivo activity. Citation Format: Eleonora Jovcheva, Caroline Paulussen, Patrick Van Bergen, Yolanda Chong, Jorge Vialard, Laurence Mevellec, Patrick Angibaud, Timothy Perera. Unique physicochemical properties of the potent FGFR 1, 2, 3 and 4 inhibitor JNJ-42756493 contribute to prolonged target shutdown. [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 LB-233. doi:10.1158/1538-7445.AM2014-LB-233

Fibroblast growth factors (FGFs) and their receptors (FGFR1 through 4) regulate a variety of key cellular processes, including proliferation, migration, survival, and differentiationa. Aberrant activation of FGF/FGFR is strongly implicated in oncogenic signalling in many tumor types. This has stimulated the development of a number of FGFR inhibitors, with diverse kinase inhibition and pharmacological profiles that are currently being evaluated in clinical studies. We conducted a fragment screening campaign and this resulted in identification of a 6-aminoquinoxalinyl fragment with a binding affinity in the micromolar range. Structure-guided medicinal chemistry led to the identification of a novel quinoxaline-based chemical series with nanomolar affinity for FGFR1, 2, 3, and 4, activity in cells, and selectivity with respect to VEGFR-2. Further optimisation resulted in the generation of JNJ-42756493, a compound with favourable drug-like properties that demonstrated strong anti-tumoral activity in a FGFR2-dependent SNU-16 human gastric carcinoma xenograft model. This report represents the first disclosure of the structure-activity relationships as well as the chemical synthesis pathway of the JNJ-42756493 series and illustrates how a fragment-based drug discovery approach has been efficiently used to discover FGFR1-4 inhibitors with nanomolar affinity. aTurner, N. and Grose, R. Nat. Rev. Cancer, 2010, 10, 116-129. Citation Format: Patrick R. Angibaud, Laurence Mevellec, Gordon Saxty, Christophe Adelinet, Rhalid Akkari, Valerio Berdini, Pascal Bonnet, Marine Bourgeois, Xavier Bourdrez, Anne Cleasby, Helene Colombel, Imre Csoka, Werner Embrechts, Eddy Freyne, Ronaldus Gilissen, Eleonora Jovcheva, Peter King, Jean Lacrampe, Delphine Lardeau, Yannick Ligny, Steve Mcclue, Lieven Meerpoel, David R. Newell, Martin Page, Alexandra Papanikos, Elisabeth Pasquier, Isabelle Pilatte, Virginie Poncelet, Olivier Querolle, David C. Rees, Sharna Rich, Bruno Roux, Elodie Sement, Yvan Simonnet, Matthew Squires, Virginie Tronel, Tinne Verhulst, Jorge Vialard, Marc Willems, Steven J. Woodhead, Berthold Wroblowski, Christopher W. Murray, Timothy Perera. Discovery of JNJ-42756493, a potent fibroblast growth factor receptor (FGFR) inhibitor using a fragment based approach. [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 4748. doi:10.1158/1538-7445.AM2014-4748

2501 Background: JNJ-42756493 is an orally bioavailable FGFR 1, 2, 3 and 4 inhibitor with nanomolar antitumor activity in cell lines and in vivo models with FGFR pathway aberration. Methods: This f...

Recent data in a number of tumour types has implicated Fibroblast Growth Factor (FGF) and Fibroblast Growth Factor receptor (FGFR) signalling as being key to the molecular pathology of cancer. A fragment screening campaign was conducted against the tyrosine kinase domain of FGFR1 to detect low molecular weight compounds that bound to the hinge region of the kinase. The screening produced several fragment inhibitors (molecular weight The poster will focus on the description of previously undescribed compounds bearing an imidazo[1,2-a]pyridine core scaffold where selectivity versus other protein kinases, for example FLT3, is obtained using the X-ray crystal structure and structure-based design. In summary we will illustrate how X-ray crystallography and fragment-based drug design (FBDD) can be used to discover compounds with activity in an FGFR driven xenograft model when dosed by the oral route. 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 1361. doi:10.1158/1538-7445.AM2011-1361

Recent data in a number of tumour types has implicated Fibroblast Growth Factor (FGF) and Fibroblast Growth Factor receptor (FGFR) signalling as being key to the molecular pathology of cancer. FGFR is a receptor tyrosine kinase which activates the extracellular signal-regulated kinase / mitogen-activated protein kinase and the protein kinase B / Akt pathways which promote cell growth and survival. Amplification, over-expression or activating mutations of fibroblast growth factor receptors have been associated with bladder tumours, multiple myeloma, hormone-refractory prostate cancer and breast cancer. Multiple lead series of FGFR inhibitors were developed using Astex's fragment based medicinal chemistry approach, Pyramid™, linked to high throughput X-ray Crystallography. We describe here the characterisation of some examples of these lead molecules. In particular we detail the pharmacological profile of a compound from one of these lead series that demonstrated activity against FGFR 1-4 with an IC50 10 fold lower in cell lines lacking FGFR expression. We demonstrate inhibition of FGFR 2 and 3 phosphorylation in gastric and multiple myeloma cell lines respectively with associated inhibition of downstream signalling pathways. This lead molecule has an excellent pharmacokinetic profile and high oral bioavailibility in mice and rats. In xenograft models in mice where aberrant FGF signalling underlies tumour pathology, tumour growth inhibition is observed at doses of 100mg/kg /day orally for 21 days. This xenograft efficacy was observed in several models, with significantly lower activity in models where aberrant FGF signalling is not involved in tumour pathology. This suggests that the mechanism of action is consistent with FGFR inhibition. The pharmacological profile in these models is also distinct from other broader spectrum receptor tyrosine kinase inhibitors. The pre-clinical data shown here suggests that such compounds warrant further investigation pre-clinically and may benefit patients whose disease is driven by FGFR activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3626.

Recent data in a number of tumour types has implicated Fibroblast Growth Factor (FGF) and Fibroblast Growth Factor receptor (FGFr) signalling as being key to the molecular pathology of cancer. This poster will describe fragment based drug discovery using biophysical screening to identify initial fragments. Subsequently, in the fragments-to-leads stage a detailed structural understanding of the binding interactions between the fragment and its target protein utilised X-ray crystallography and NMR. Starting with different fragments allows several lead series to be identified, often by synthesizing only small numbers of compounds. A fragment screening campaign was conducted against the FGFr-1 to detect very low molecular weight compounds that bound to the hinge region of the kinase. The screening produced several fragment molecules (Molecular Weight This poster represents first disclosure of the structure of the lead series and illustrates how a fragment-based drug discovery approach can be efficiently used to discover compounds advanced nanomolar compounds with oral bioavailability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5778.

Constitutively active receptor tyrosine kinases (RTKs) are known oncogenic drivers and provide valuable therapeutic targets in many cancer types. However, clinical efficacy of RTK inhibitors is limited by intrinsic and acquired resistance. To identify genes conferring resistance to inhibition of the MET RTK, we conducted a forward genetics screen in the GTL-16 gastric cancer cell line, carrying MET amplification and exquisitely sensitive to MET inhibition. Cells were transduced with three different retroviral cDNA expression libraries and selected for growth in the presence of the MET inhibitor PHA-665752. Selected cells displayed robust and reproducible enrichment of library-derived cDNAs encoding truncated forms of RAF1 and BRAF proteins, whose silencing reversed the resistant phenotype. Transduction of naive GTL-16 cells with truncated, but not full length, RAF1 and BRAF conferred in vitro and in vivo resistance to MET inhibitors, which could be reversed by MEK inhibition. Induction of resistance by truncated RAFs was confirmed in other MET-addicted cell lines, and further extended to EGFR-addicted cells. These data show that truncated RAF1 and BRAF proteins, recently described as products of genomic rearrangements in gastric cancer and other malignancies, have the ability to render neoplastic cells resistant to RTK-targeted therapy.

Glioblastoma (GBM) contains stem‐like cells (GSCs) known to be resistant to ionizing radiation and thus responsible for therapeutic failure and rapidly lethal tumor recurrence. It is known that GSC radioresistance relies on efficient activation of the DNA damage response, but the mechanisms linking this response with the stem status are still unclear. Here, we show that the MET receptor kinase, a functional marker of GSCs, is specifically expressed in a subset of radioresistant GSCs and overexpressed in human GBM recurring after radiotherapy. We elucidate that MET promotes GSC radioresistance through a novel mechanism, relying on AKT activity and leading to (i) sustained activation of Aurora kinase A, ATM kinase, and the downstream effectors of DNA repair, and (ii) phosphorylation and cytoplasmic retention of p21, which is associated with anti‐apoptotic functions. We show that MET pharmacological inhibition causes DNA damage accumulation in irradiated GSCs and their depletion in vitro and in GBMs generated by GSC xenotransplantation. Preclinical evidence is thus provided that MET inhibitors can radiosensitize tumors and convert GSC‐positive selection, induced by radiotherapy, into GSC eradication. ![][1] GBM radioresistance relies on the inherent properties of its stem‐like cell population (GSCs). MET, which encodes for the HGF receptor, is shown to be a functional marker of GSC radioresistance and a therapeutic target for GSC radiosensitization. [1]: /embed/graphic-1.gif