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A) Structure of JNJ-42541707, a structurally related compound to JNJ-42756493. B) 72h growth inhibition (IC50) of JNJ-42541707 against 236 cancer cell lines from multiple origins color coded based on FGFR1,2,4 mRNA overexpression and FGFR WT.

In vitro kinase inhibition by DiscoverX KinomeScan assay. For details see (19)

Inhibitory activity of Brivanib and JNJ-42756943 in kinase (top) and BaF3 kinase dependent proliferation (bottom) assays and ratio of FGFRs/VEGFR2 activities.

JNJ-42756493 anti-proliferative activity against cancer cells lines from multiple origins.. Detailed data supporting Figure 2.

Erdafitinib (JNJ-42756493, BALVERSA) is a tyrosine kinase inhibitor indicated for the treatment of advanced urothelial carcinoma. In this work, a translational model-based approach to inform the choice of the doses in phase 1 trials is illustrated. A pharmacokinetic (PK) model was developed to describe the time course of erdafitinib plasma concentrations in mice and rats. Data from multiple xenograft studies in mice and rats were analyzed using the Simeoni tumor growth inhibition (TGI) model. The model parameters were used to derive a range of erdafitinib exposures that might inform the choice of the doses in oncology phase 1 trials. Conversion of exposures to doses was based on preliminary PK assessments from the first-in human (FIH) study. A one-compartment PK disposition model, with linear absorption and dose-dependent clearance, adequately described the PK data in both mice and rats via an allometric scaling approach. The TGI model was able to describe tumor growth dynamics, providing quantitative measurements of erdafitinib antitumor potency in mice and rats. Based on these estimates, ranges of efficacious unbound concentration were identified for erdafitinib in mice (0.642–5.364 μg/L) and rats (0.782–2.565 μg/L). Based on the FIH data, it was possible to transpose exposures into doses and doses of above 4 mg/day provided erdafitinib exposures associated with significant TGI in animals. The findings were in agreement with the results of the FIH trial, in which the first hints of clinical activities were observed at 6 mg. The successful modeling exercise of erdafitinib preclinical data showed how translational PK-PD modeling might be a tool to help to inform the choice of the doses in FIH studies.

The protein arginine methyltransferase 5 (PRMT5) methylates a variety of proteins involved in splicing, multiple signal transduction pathways, epigenetic control of gene expression, and mechanisms leading to protein expression required for cellular proliferation. Dysregulation of PRMT5 is associated with clinical features of several cancers, including lymphomas, lung cancer, and breast cancer. Here, we describe the characterization of JNJ-64619178, a novel, selective, and potent PRMT5 inhibitor, currently in clinical trials for patients with advanced solid tumors, non-Hodgkin's lymphoma, and lower-risk myelodysplastic syndrome. JNJ-64619178 demonstrated a prolonged inhibition of PRMT5 and potent antiproliferative activity in subsets of cancer cell lines derived from various histologies, including lung, breast, pancreatic, and hematological malignancies. In primary acute myelogenous leukemia samples, the presence of splicing factor mutations correlated with a higher ex vivo sensitivity to JNJ-64619178. Furthermore, the potent and unique mechanism of inhibition of JNJ-64619178, combined with highly optimized pharmacological properties, led to efficient tumor growth inhibition and regression in several xenograft models in vivo, with once-daily or intermittent oral-dosing schedules. An increase in splicing burden was observed upon JNJ-64619178 treatment. Overall, these observations support the continued clinical evaluation of JNJ-64619178 in patients with aberrant PRMT5 activity-driven tumors. ispartof: MOLECULAR CANCER THERAPEUTICS vol:20 issue:12 pages:2317-2328 ispartof: location:United States status: published

Fibroblast growth factor (FGF) signaling plays critical roles in key biological processes ranging from embryogenesis to wound healing and has strong links to several hallmarks of cancer. Genetic alterations in FGF receptor (FGFR) family members are associated with increased tumor growth, metastasis, angiogenesis, and decreased survival. JNJ-42756493, erdafitinib, is an orally active small molecule with potent tyrosine kinase inhibitory activity against all four FGFR family members and selectivity versus other highly related kinases. JNJ-42756493 shows rapid uptake into the lysosomal compartment of cells in culture, which is associated with prolonged inhibition of FGFR signaling, possibly due to sustained release of the inhibitor. In xenografts from human tumor cell lines or patient-derived tumor tissue with activating FGFR alterations, JNJ-42756493 administration results in potent and dose-dependent antitumor activity accompanied by pharmacodynamic modulation of phospho-FGFR and phospho-ERK in tumors. The results of the current study provide a strong rationale for the clinical investigation of JNJ-42756493 in patients with tumors harboring FGFR pathway alterations. Mol Cancer Ther; 16(6); 1010–20. ©2017 AACR.

PRMT5 is a type II methyltransferase that specifically adds methyl groups to arginine as a long-lasting post-translational modification. The PRMT5/MEP50 complex regulates a plethora of cellular processes, such as epigenetics and splicing, which are notable events involved in tumorigenesis. Although not frequently mutated or amplified in tumors, elevated PRMT5 protein levels in lung and hematologic cancers are correlated with poorer survival. The PRMT5 inhibitor JNJ-64619178 has been selected as a clinical candidate based on its high selectivity and potency (subnanomolar range) under different in vitro and cellular conditions, paired with favorable pharmacokinetics and safety properties. JNJ-64619178 binds into the SAM binding pocket and reaches the substrate binding pocket to inhibit PRMT5/MEP50 function in a time-dependent manner. Broad cell line panel profiling of JNJ-64619178 revealed a wide range of sensitivity, which is indicative of a genomic dependency instead of a general cytotoxic on-target consequence of PRMT5 inhibition. Further investigations indicate a synthetic lethal correlation between PRMT5 inhibition and key cancer driver pathways. JNJ-64619178, dosed orally (10 mg/kg, every day), showed selective and efficient blockage of the methylation of SMD1/3 proteins, which are crucial components of the spliceosome and substrates of PRMT5/MEP50. JNJ-64619178 also demonstrated tumor regression in a biomarker-driven human small cell lung cancer xenograft model (NCI-H1048) and prolonged tumor growth inhibition after dosing cessation. In rodent and nonrodent toxicology studies, a tolerated dose of JNJ-64619178 has been identified, with the observed toxicity consistent with on-target activity. In summary, JNJ-64619178 has a favorable preclinical package that supports clinical testing in patients diagnosed with lung cancer and hematologic malignancies. Citation Format: Dirk Brehmer, Tongfei Wu, Geert Mannens, Lijs Beke, Petra Vinken, Dana Gaffney, Weimei Sun, Vineet Pande, Jan-Willem Thuring, Hillary Millar, Italo Poggesi, Ivan Somers, An Boeckx, Marc Parade, Erika van Heerde, Thomas Nys, Carol Yanovich, Barbara Herkert, Tinne Verhulst, Marc Du Jardin, Lieven Meerpoel, Christopher Moy, Gaston Diels, Marcel Viellevoye, Wim Schepens, Alain Poncelet, Joannes T. Linders, Edward C. Lawson, James P. Edwards, Dushen Chetty, Sylvie Laquerre, Matthew V. Lorenzi. A novel PRMT5 inhibitor with potent in vitro and in vivo activity in preclinical lung cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr DDT02-04. doi:10.1158/1538-7445.AM2017-DDT02-04

Protein arginine methyltransferase 5 (PRMT5), a type II methyltransferase, is responsible for symmetric arginine di-methylation of multiple cellular proteins involved in the regulation of cellular transcription. PRMT5 is involved in cellular processes such as survival, proliferation, and apoptosis, and an elevated tumor PRMT5 protein level has recently been correlated with poor survival of cancer patients. JNJ-64619178, a selective PRMT5 inhibitor, showed inhibition of cellular growth in several cell lines representing multiple cancer histologies in vitro. From this, a broad selection of xenograft models was chosen to demonstrate potent anti-tumor efficacy. Xenograft models representing small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), acute myeloid leukemia (AML), and non-Hodgkin lymphoma were chosen to demonstrate anti-tumor efficacy. Biologically significant tumor growth inhibition up to 99% was observed in both solid and hematological xenograft models, including an aggressive disseminated model of AML, with oral doses of 1 to 10 mg/kg, once daily. Importantly, continued inhibition of tumor growth was observed for several weeks following dosing cessation. Dosing of JNJ-64619178 results in inhibition of Sym-Arg di-methylation of SMD1/3 proteins, core components of the spliceosome in the tumor, and general Sym-Arg di-methylation of serum proteins. These serve as pharmacodynamic markers of PRMT5 inhibition in xenograft models. Potent and prolonged inhibition of SMD1/3 di-methylation was observed in the SCLC model, during and after the dosing period. This has led to the exploration of alternative dosing regimens preclinically. PRMT5 inhibitor JNJ-64619178 is currently being investigated in a Phase I clinical trial, based on its high selectivity and potency, favorable pharmacokinetics and safety properties, and strong preclinical efficacy and pharmacodynamic data. Citation Format: Hillary J. Millar, Dirk Brehmer, Tinne Verhulst, Nahor Haddish-Berhane, Tony Greway, Dana Gaffney, An Boeckx, Erika Van Heerde, Thomas Nys, Joseph Portale, Ulrike Philippar, Tongfei Wu, Sylvie Laquerre, Kathryn Packman. In vivo efficacy and pharmacodynamic modulation of JNJ-64619178, a selective PRMT5 inhibitor, in human lung and hematologic preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 950.

Protein arginine methyltransferase 5 (PRMT5) is an epigenetic enzyme with oncogenic properties. JNJ-64619178 (JNJ178) is a potent, selective, structurally unique PRMT5 inhibitor with good preclinical efficacy in inhibiting the growth of hematologic and solid tumor cell lines. Toxicology studies showed that JNJ178 decreased reticulocytes and neutrophils in rats and dogs. The objectives of translational modeling and simulation were to understand the exposure-response relationship of both safety and efficacy and provide guidance to the first-in-human clinical development of JNJ178. Experimental data for the PK/PD (Pharmacokinetics/Pharmacodynamics) modeling included: plasma concentration after single dose of JNJ178 in non-tumor bearing mice, plasma concentration and PD markers of dimethylation of arginine (%SDMA in plasma and %SMD1/3-Me2 in tumor, respectively) after multiple doses (1 to 10 mg/kg) QD (once daily) of JNJ178 in H1048 (human small cell lung carcinoma) xenografts, and tumor volume in four xenograft mouse models (A427, human lung carcinoma; H441, human lung adenocarcinoma; H520, human squamous cell lung carcinoma; and H1048). Plasma PK were first described by a standard two-compartment model and used as a driver of PD and tumor volume (efficacy). Plasma and tumor PD were modeled using an indirect response model. A hybrid tumor growth coupled with transit compartment mediated tumor killing model was used to fit the tumor volume data. To predict the safety profile of JNJ178, lifespan based indirect response model for erythropoiesis and Friberg myelosuppression model were used to simulate hemoglobin and neutrophil kinetics in human. The PK/PD model described the data well and validated the hypothesis that PD is driven by trough concentration. Based on the exposure-response relationship from the four xenograft models, the trough concentration needed to achieve tumor stasis for mouse was determined. In addition, the level of inhibition in tumor and plasma PD marker that was associated with tumor stasis was identified. Together with human PK parameters scaled using allometry, the dose range needed to achieve target therapeutic exposure for a typical human subject was predicted. Simulation results from erythropoiesis and Friberg myelosuppression models informed the optimal doing schedules for certain dose levels that would allow hematological toxicity to be manageable with 1.0 x 109/L neutrophil counts at all times. Overall, a translational modeling and simulation approach that considers safety and efficacy has been instrumental in the design of the first-in-human clinical development of PRMT5 inhibitor JNJ178 regarding selection of dose and schedule. Citation Format: Yue Guo, Nahor Haddish-Berhane, Hillary J. Millar, Tinne Verhulst, Tony Greway, Junguo Zhou, Loeckie DeZwart, Dana Gaffney, Joseph Portale, Dirk Brehmer, An Boeckx, Erika Van Heerde, Daniele Ouellet. Translational efficacy and safety modeling and simulation to support the clinical development of JNJ-64619178, a PRMT5 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3905.

Activation of the MET/HGF pathway has been linked to tumor initiation, metastasis, angiogenesis and resistance to therapeutic agents. Here we present pharmacological characterization of OMO-1 (formerly JNJ-38877618), a potent, highly selective, orally bioavailable MET kinase inhibitor with nM binding affinity (Kd=1.4 nM) and enzyme inhibitory activity against wt and M1268T mutant MET (2 and 3 nM IC50). MET inhibitory effects were assessed in proliferation, colony formation and motility assays. OMO-1 displayed nM potency against MET Ampl/mutant and therapy resistant models. In vivo, OMO-1 induced complete inhibition of tumor growth in 3 models: the SNU5 MET amp gastric, U87-MG HGF autocrine glioblastoma and Hs746T MET exon 14 skipping mutant gastric cancer. OMO-1 induced regression of large MET amplified EBC-1 SqNSCLC where OMO-1 led to dose- and time-dependent inhibition of MET kinase activation, with the duration of target shut down considerably exceeding plasma exposure times. Combination treatments were well tolerated and improved EGFR targeted therapy. Although single agent OMO-1 had no effect on NSCLC HCC827 EGFR, combination with Erlotinib led to delayed onset of tumor recurrence. The acquired EGFR inhibitor resistant model HCC827-ER1 was determined to be MET amplified. OMO-1 and erlotinib both inhibited tumor growth of this model whilst combination induced tumor regression. In an EGFR inhibitor resistant PDX having MET amplification, single agent OMO-1 caused tumour stasis whereas MetMab/erlotinib only led to tumor growth delay. The potent preclinical activity we have observed, supports ongoing clinical development of OMO-1 in patients with MET pathway-driven tumors. Citation Format: Marion Libouban, Eleonora Jovcehva, Desiree De Lange, Boudewijn Janssens, Tinne Verhulst, Souichi Ogata, Berthold Wroblowski, Laurence Mevellec, Tianbao Lu, Glen Clack, Timothy Perera. OMO-1, a potent, highly selective, orally bioavailable, MET kinase inhibitor with a favorable preclinical toxicity profile, shows both monotherapy activity, against MET pathway-driven tumors, and EGFR TKI combination activity in acquired resistance models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4791.

Tineke Casneuf,1 Amy E Axel,2 Peter King,2 John D Alvarez,2 Jillian L Werbeck,3 Tinne Verhulst,1 Karin Verstraeten,1 Brett M Hall,2 A Kate Sasser2 1Janssen Research and Development, Beerse, Belgium; 2Janssen Research and Development, Spring House, PA, 3LabConnect LLC, Seattle, WA, USAIntroduction: Interleukin-6 (IL-6) is an important growth factor for estrogen receptor-α (ERα)-positive breast cancer, and elevated serum IL-6 is associated with poor prognosis.Methods: The role of the phosphorylated signal transducer and activator of transcription 3 pathway was investigated in ERα-positive breast cancer. A panel of cell lines was treated with exogenous IL-6. An IL-6 specific gene signature was generated by profiling ten ERα-positive breast cancer cell lines alone or following treatment with 10 ng/mL recombinant IL-6 or human marrow stromal cell-conditioned media, with or without siltuximab (a neutralizing anti-IL-6 antibody) and grown in three-dimensional tumor microenvironment-aligned cultures for 4 days, 5 days, or 6 days. The established IL-6 signature was validated against 36 human ERα-positive breast tumor samples with matched serum. A comparative MCF-7 xenograft murine model was utilized to determine the role of IL-6 in estrogen-supplemented ERa-positive breast cancer to assess the efficacy of anti-IL-6 therapy in vivo.Results: In eight of nine ERα-positive breast cancer cell lines, recombinant IL-6 increased phosphorylation of tyrosine 705 of STAT3. Differential gene expression analysis identified 17 genes that could be used to determine IL-6 pathway activation by combining their expression intensity into a pathway activation score. The gene signature included a variety of genes involved in immune cell function and migration, cell growth and apoptosis, and the tumor microenvironment. Validation of the IL-6 gene signature in 36 matched human serum and ERα-positive breast tumor samples showed that patients with a high IL-6 pathway activation score were also enriched for elevated serum IL-6 (≥10 pg/mL). When human IL-6 was provided in vivo, MCF-7 cells engrafted without the need for estrogen supplementation, and addition of estrogen to IL-6 did not further enhance engraftment. Subsequently, we prophylactically treated mice at MCF-7 engraftment with siltuximab, fulvestrant, or combination therapy. Siltuximab alone was able to blunt MCF-7 engraftment. Similarly, siltuximab alone induced regressions in 90% (9/10) of tumors, which were established in the presence which were established in the presence of hMSC expressing human IL-6 and estrogen.Conclusion: Given the established role for IL-6 in ERα-positive breast cancer, these data demonstrate the potential for anti-IL-6 therapeutics in breast cancer.Keywords: breast cancer, estrogen receptor, gene signature, paracrine IL-6, siltuximab

Recent data obtained in several tumor types have identified Fibroblast Growth Factor signaling as a key factor in the molecular pathology of a number of cancers1. This has stimulated the development of a number of agents that block this pathway, including FGFR kinase inhibitors with diverse inhibition and pharmacological profiles that are currently being evaluated in clinical studies. We recently reported that a quinoxaline moiety can efficiently bind the hinge region of FGFR kinase catalytic sites. In continuation of our efforts to discover additional FGFR1-4 inhibitors, we have identified a quinazolinone scaffold as a novel FGFR kinase hinge binder. Initial hits were optimized into compounds displaying nanomolar affinity for FGFR1-4, potent activity in FGFR driven cells and efficacy in a Ba/F3-FGFR3 xenograft model. This report represents the first disclosure of the structure-activity relationships as well as the chemical synthesis pathways of these novel quinazolinone-based FGFR1-4 inhibitors. Turner N. and Grose R. Nature Reviews-Cancer 2010, 10, 116-129 Citation Format: Olivier Querolle, Patrick Angibaud, Hélène Colombel, Virginie Caron, Isabelle Pilatte, Virginie Poncelet, Norbert Esser, Ron Gilissen, Peter King, Lieven Meerpoel, Tinne Verhulst, Berthold Wroblowski, Jorge Vialard, Chris W. Murray, David C. Rees, Anita Reningovolo, Gordon Saxty. Discovery of quinazolinones as fibroblast growth factor receptor (FGFR1-4) kinase inhibitors. [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 3640. doi:10.1158/1538-7445.AM2015-3640

The fibroblast growth factor receptor (FGFR) tyrosine kinase family members, FGFR1, 2, 3 and 4, have roles in a variety of key cellular processes, including proliferation, migration, survival, and differentiation1. Aberrant activation of FGFRs through mutation, amplification, chromosomal translocation, and ligand up-regulation being strongly implicated in oncogenic signalling in many tumour types, has triggered efforts to identify selective FGFR inhibitors. As a result, several potent FGFR kinase inhibitors are currently being evaluated in clinical studies across many tumor types, including non-small cell lung, breast and bladder cancers. We have designed novel 1,5 and 1,7-naphthyridine derivatives that are potent kinase inhibitors of all FGFR family members in enzymatic and cellular systems. Initial hits were further optimized to increase potency and ADME properties leading to identification of a novel 1,5-naphthyridine-based chemical series with nanomolar affinity for FGFR1, 2, 3, and 4, activity in cells, and selectivity with respect to VEGFR-2. In vivo screening using an FGFR3-driven xenograft model revealed efficacious compounds that could be explored further as antitumoral agents. This report represents the first disclosure of the structure-activity relationship and synthesis pathway of novel naphthyridine chemical series displaying nanomolar affinity for FGFRs1, 2, 3 and 4. 1 Dieci M. V., Ardenos M., Andre F., Soria J.C. Cancer Discovery. 2013, 3(3) 264-279. Citation Format: Patrick R. Angibaud, Michel Obringer, Julien Marin, Matthieu Jeanty, Norbert Esser, Ron Gilissen, Peter King, Lieven Meerpoel, Olivier Querolle, David C. Rees, Bruno Roux, Gordon Saxty, Tinne Verhulst, Berthold Wroblowski, Christopher C. Murray, Jorge Vialard. Identification of naphthyridines as potent inhibitors of fibroblast growth factor receptor kinase family. [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 3641. doi:10.1158/1538-7445.AM2015-3641

The naturally occurring cyclic tetrapeptide chlamydocin is a very potent inhibitor of cell proliferation. Here we show that chlamydocin is a highly potent histone deacetylase (HDAC) inhibitor, inhibiting HDAC activity in vitro with an IC50 of 1.3 nM. Like other HDAC inhibitors, chlamydocin induces the accumulation of hyperacetylated histones H3 and H4 in A2780 ovarian cancer cells, increases the expression of p21(cip1/waf1), and causes an accumulation of cells in G(2)/M phase of the cell cycle. In addition, chlamydocin induces apoptosis by activating caspase-3, which in turn leads to the cleavage of p21(cip1/waf1) into a 15-kDa breakdown product and drives cells from growth arrest into apoptosis. Concomitant with the activation of caspase-3 and cleavage of p21(cip1/waf1), chlamydocin decreases the protein level of survivin, a member of the inhibitor of apoptosis protein family that is selectively expressed in tumors. Although our data indicate a potential link between degradation of survivin and activation of the apoptotic pathway induced by HDAC inhibitors, stable overexpression of survivin does not suppress the activation of caspase-3 or cleavage of p21(cip1/waf1) induced by chlamydocin treatment. The decrease of survivin protein level is mediated by degradation via proteasomes since it can be inhibited by specific proteasome inhibitors. Taken together, our results show that induction of apoptosis by chlamydocin involves caspase- dependent cleavage of p21(cip1/waf1), which is strikingly associated with proteasome-mediated degradation of survivin.

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

Chromosomal rearrangements resulting in oncogenic fusion proteins containing the ROS1 receptor tyrosine kinase have been described in subsets of a variety of human malignancies including non-small-cell lung cancer (NSCLC), cholangiocarcinoma, and glioblastoma multiforme. Promising clinical responses have been observed in patients bearing tumors with ROS1 fusions treated with Xalkori, an ALK/MET kinase inhibitor that also inhibits Ros1. However, resistance has been observed and a ROS1 kinase domain mutation, G2032R, was identified in a ROS1 fusion positive NSCLC patient who developed resistance to Xalkori treatment. Clinical experience with other receptor tyrosine kinase inhibitors suggests that additional resistance mutations are likely to arise, highlighting the need for therapeutic agents that can overcome this type of resistance. We identified a novel chemical series of potent and selective Ros1 inhibitors with a unique DFG-out binding mode. The structure of human Ros1 in a complex with a ligand from this chemical class was confirmed by X-ray crystallography. Here we describe the structure-activity relationships and synthesis route for this chemical series. A representative compound from this series inhibited isolated recombinant Ros1 kinase activity with an IC50 of approximately 30 nM. This compound inhibited less than 6% of kinases in a panel of 400 at 1 μM concentration. Growth of Ba/F3 cells engineered to express Ros1 autophosphorylation in HCC78 NSCLC cells that harbor a SLC34A2-ROS1 fusion were inhibited at similar concentrations as the isolated protein. This activity translated into potent Ba/F3-Ros1 tumor growth inhibition in mice. This compound was also active on Ba/F3 cells containing Ros1 with a mutation in the gatekeeper residue, L2026M, in cell growth assays in vitro and tumor growth in vivo. Furthermore, this unique binding mode provides scope for activity on additional Ros1 mutations that confer resistance to Xalkori, such as the clinically relevant G2032R mutation. The results shown here describe a novel chemical series with a unique binding mode that has potential for activity in Ros1 driven tumors with mutations that confer resistance to Xalkori. Citation Format: Laurence Mevellec, Berthold Wroblowski, Ron Gilissen, Sophie Descamps, Elisabeth Pasquier, Christophe Adelinet, Marine Bourgeois, Guillaume Mercey, Matthieu Jeanty, Thierry Jousseaume, Aurélie Luguern, Javier Astray Gandara, Said Akzinnay, Etienne Daras, Inge Boeckx, Nele Van Slycken, Mariette Bekkers, Jeroen Van De Ven, Tinne Verhulst, Lieven Meerpoel, Jorge Vialard. Discovery of potent and selective Ros1 inhibitors with a unique DFG-out binding mode. [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 4745. doi:10.1158/1538-7445.AM2014-4745

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

Plasma cell malignancies, such as multiple myeloma, are characterized by an extensively developed endoplasmic reticulum (ER) to accommodate the high secretion rate of immunoglobulins. An adaptive stress response mechanism, termed the unfolded protein response (UPR), is markedly elevated in multiple myeloma cells to ensure a homeostatic balance between ER burden and ER capacity. One branch of the UPR consists of the activation of PERK under chronic ER stress: PERK phosphorylates and impairs the function of the translation initiation factor, eIF2α, thereby downregulating global protein synthesis, and reducing the secretory burden on the ER. To test the therapeutic potential of PERK inhibitors in multiple myeloma, we identified two chemically diverse PERK inhibitors: both are sub-nM inhibitors of PERK, and have a >100-fold window against other kinases (including other eIF2α kinases). These compounds inhibit phosphorylation of eIF2α at 10-20nM (IC50) in HEK293 cells, incubated with the ER stressor tunicamycin. Both PERK inhibitors were selectively anti-proliferative in an ER-stressed epithelial cancer model (A549 cells with tunicamycin) at nM concentrations, but not in the absence of ER stress. Furthermore, in the absence of an exogenous ER stressor, both PERK inhibitors induced ER stress (eg, as evidenced by induction of the pro-apoptotic CHOP gene) selectively in multiple myeloma cell lines (and certain B-cell lymphoma lines) at low nM concentrations but not in normal or malignant epithelial cells (induction of CHOP at 10 μM). The magnitude of this induction was comparable to well-established ER stressors, such as bortezomib or tunicamycin, and correlated closely with reduced proliferation in malignant B-cell lines. The potent induction of CHOP occurred both in vitro and upon oral dosing of mice, with a xenografted multiple myeloma tumour (JIM-1); however, in both instances, the induction of ER stress was maximal at a dose corresponding to approximately 50-75% inhibition of PERK, whereas, remarkably, at dose levels corresponding to more complete PERK inhibition (as evidenced by inhibition of P-eIF2α), CHOP induction (and ER-stress induction in general as determined by a genome-wide expression profile) was reduced, eventually down to baseline levels. Consistently, both PERK inhibitors resulted in a biphasic concentration-dependent reduction of proliferation of certain myeloma and B-cell lymphoma lines. Hence, PERK inhibition triggers a biphasic induction of ER stress and inhibition of cell proliferation, selectively in B-cell malignancies. The possible underlying mechanism as well as the therapeutic implications of these findings will be discussed. Citation Format: Matthias Versele, Tamara Geerts, Jeroen Van De Ven, Ilse Van den Wyngaert, Peter Vermeulen, Inge Beerden, Danielle Peeters, Johnny Liebregts, Kurt Van Baelen, Cedric Simillion, Boudewijn Janssen, Tinne Verhulst, Norbert Esser, Christophe Meyer, Ian Stansfield, James Bischoff. Selective PERK kinase inhibition triggers a biphasic concentration-dependent induction of ER stress in multiple myeloma and B-cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2926. doi:10.1158/1538-7445.AM2013-2926

We have shown that the oral pan-HDAC inhibitor, JNJ-26481585, is a potent inhibitor of in vitro growth in a broad panel of solid and hematological tumor cell lines and western blot analysis confirmed modulation of HDAC1, 3 and 6 substrates (Arts et al, 2009). Inhibition of HDAC1 and 3 is associated with transcriptional suppression of target genes and regulation of tumor growth; modulation of HDAC6, impacting tubulin and Hsp90, results in depletion of oncogenic Hsp90 client proteins and potentiates tubulin acetylation with paclitaxel (Marcus et al, 2005; Valenzuela-Fernandez et al, 2008; Ai et al, 2009). We hypothesized that combinations between taxanes and JNJ-26481585 could result in enhanced tumor cell killing and describe our observations from such combinations in PC3-M prostate xenografts as a model of castrate resistant disease. We have previously shown that JNJ-26481585 has significant activity in xenograft models of prostate cancer, including PC3-M. In comparative studies in mice, JNJ-26481585 has favorable tissue distribution, achieving higher levels of exposure than panobinostat and vorinostat in tissues including e.g. prostate (2-fold and 3-fold, respectively); a sustained PD response is also observed in lung and skin (suppression of Ki67 for 24h; elevation of AcH3). We further demonstrate that the levels of JNJ-26481585 observed in orthotopically implanted PC3-M tumors are superior to those of panobinostat or vorinostat (5-fold and 13-fold, respectively). Thus we selected this orthotopic model in order to define further how the distribution of JNJ-26481585 to target tissue contributes to efficacy. We found that the activity of taxotere alone was surpassed by that of JNJ-26481585 alone (tumor growth delay (TGD) of 5d for taxotere, and 15d for JNJ-26481585; vs vehicle) and significant TGD was observed from the combination of the agents (with 1mpk JNJ-26481585 = 20d; with 3mpk JNJ-26481585 = beyond study end). The role of HDAC inhibition in the stability and regulation of the androgen receptor has recently been described as dependent on both HDAC1, 3 and 6 activities (Welsbie et al, 2009; Ai et al, 2009), which gives further support to our rationale for combining agents with JNJ-26481585. We hypothesized that JNJ-26481585 would be an efficacious agent when combined with abiraterone, an inhibitor of androgen synthesis. In LnCAP and VCaP prostate cancer cells, we showed by western blot analysis that exposure to JNJ-26481585 resulted in a dose dependent reduction in AR levels and profiling on Affymetrix microarrays also showed modulation of the expression of AR responsive genes e.g. KLKs and TMPRSS2. Consistent with a sustained PD response in tissues, we see continued reduction in AR levels and expression of AR dependent genes up to 24h after removal of compound. We continue to investigate the combination of JNJ-26481585 and abiraterone in vivo as our data provide strong support for the combination in prostate cancer therapy. 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 2606. doi:10.1158/1538-7445.AM2011-2606

We have recently reported the identification and characterization of JNJ-26481585, as a “second generation” oral pan-HDAC inhibitor. The compound has shown potent in vitro activity against solid and hematological tumor cell lines and significant induction of apoptosis was observed, consistent with the potency of JNJ-26481585 for Class I HDACs. In dose response to JNJ-26481585, western blot analysis of A2780 cell extracts confirmed modulation of HDAC1-3 substrates and also HDAC6 substrates. Inhibition of HDAC6, the deacetylase for tubulin and Hsp90, results in depletion of oncogenic Hsp90 client proteins, decreased cell motility, and potentiation of therapeutics such as taxanes. We hypothesized that rational combinations with taxanes and JNJ-26481585 could result in enhanced tumor cell killing and describe our observations from the combination of JNJ-26481585 in PC3-M prostate tumors. Combinations of JNJ-26481585 and taxanes were performed by co-administration or 24h separation of agents in A549, NCI-H460, NCI-H1373, NCI-H1703, NCI-H322 and NCI H-1650 lung cell lines; breast cell lines, MDA-MB-231, T47D, BT474 and MCF-7 cells; and prostate cell lines PC3M, DU-145, 22Rv1 and LnCap. We will present data on the outcome of these combinations, including additive and synergistic effects. The in vitro data encouraged us to explore the effects of the compound in vivo and in prostate models where we already had excellent single agent activity for JNJ-26481585 in DU-145, PC-3 and the PC-3M orthotopic model (significant T/C values in each model and at doses substantially below MTD). In PC3-M xenograft models, the combination of 1.25, 5 or 10mpk JNJ-26481585 (Q2D) and taxotere (Q7D), was more effective than either agent alone and resulted in dose dependent tumor regression and substantial extension of time to relapse. We then selected the more challenging PC-3M orthotopic mouse model to further define the contribution to efficacy from the excellent tissue distribution of our agent. Comparing 5mpk taxotere (Q7D), with 1 or 3mpk of JNJ-26481585 (QD) or the combination of agents, the activity of taxotere alone was surpassed by either dose of HDAC inhibitor alone and substantial tumor growth delay was observed from the combination of 3mpk JNJ-26481585 and taxotere; activity greater than predicted from the in vitro experiments. Finally, although HDAC6 inhibition could directly explain potentiation of taxotere by JNJ-26481585 in vitro, we believe the substantial effects in vivo may be due to the anti-angiogenic effect observed for our agent. We present also our data showing anti-angiogenic effects of JNJ-26481585 in PC3-M tumors, consistent with this hypothesis. Our data support the further use of more complex models, reflective of human disease in preclinical assessment and also support further exploration of this agent in rational combinations with SOC agents. JNJ-26481585 is currently in Phase I clinical trials. 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 5443.

JNJ-38877605 is a potent and selective Met receptor tyrosine kinase inhibitor that has substantial pre-clinical antitumor activity. We recently described JNJ-38877605 as an ATP-competitive, inhibitor of the catalytic activity of Met kinase (IC50 4 nM) with a unique binding mode that leads to selectivity, exhibiting a 600-fold selectivity (vs cFMS IC50 2.6μM; the next potently inhibited kinase) for Met compared with a panel of 250 diverse tyrosine and serine-threonine kinases. Previously, JNJ-38877605 was shown to inhibit Met phosphorylation in tumor xenografts up to 16 h following a single oral dose, and that inhibition of receptor phosphorylation was associated with dose-dependent tumor growth inhibition. Herein we describe the activity of JNJ-38877605, on a broader range of preclinical solid tumor xenograft models, at doses below MTD and with different schedules of administration. In addition, we now also show data with primary patient-tumor derived xenografts where we have dosed JNJ-39977605 at the MTD using gastric, colorectal and liver metastatic (derived from primary CRC) samples. JNJ-38877605 was observed to induce significant tumor regression in large well established MET gene amplified gastric cancer models and in Met pathway activated (autocrine or paracrine) models. Significant growth inhibition was achieved when dosing JNJ-38877605 at MTD (and lower doses) with T/C < 42% in tumors of MKN-45, GTL-16, SNU-5 and Kato II met gene amplified models. Significant inhibition was also observed in K-ras mt (dependent) NCI-H441 NSCLC xenografts. Regression of U87 MG PTEN negative glioblastoma tumors (autocrine loop), grown either as subcutaneous or orthotopic models was also observed. Using primary patient derived xenograft models, we have shown that differential expression of the c-Met/HGF axis and epithelial-mesenchymal transition (EMT)-related gene markers between primary colorectal carcinomas and liver metastases in primary tissue, play a role in the activity of this compound in vivo. The strong preclinical properties of the JNJ-38877605 compound has resulted in the selection of this potent and uniquely selective Met inhibitor for clinical evaluation. JNJ-38877605 is currently in Phase I clinical trials. 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 3628.