Timothy Pietro Suren Perera

Amplification of the MET Receptor Drives Resistance to Anti-EGFR Therapies in Colorectal Cancer

EGF receptor (EGFR)-targeted monoclonal antibodies are effective in a subset of metastatic colorectal cancers. Inevitably, all patients develop resistance, which occurs through emergence of KRAS mutations in approximately 50% of the cases. We show that amplification of the MET proto-oncogene is associated with acquired resistance in tumors that do not develop KRAS mutations during anti-EGFR therapy. Amplification of the MET locus was present in circulating tumor DNA before relapse was clinically evident. Functional studies show that MET activation confers resistance to anti-EGFR therapy both in vitro and in vivo. Notably, in patient-derived colorectal cancer xenografts, MET amplification correlated with resistance to EGFR blockade, which could be overcome by MET kinase inhibitors. These results highlight the role of MET in mediating primary and secondary resistance to anti-EGFR therapies in colorectal cancer and encourage the use of MET inhibitors in patients displaying resistance as a result of MET amplification.

CD74-NRG1 fusions in lung adenocarcinoma

UNLABELLED We discovered a novel somatic gene fusion, CD74-NRG1, by transcriptome sequencing of 25 lung adenocarcinomas of never smokers. By screening 102 lung adenocarcinomas negative for known oncogenic alterations, we found four additional fusion-positive tumors, all of which were of the invasive mucinous subtype. Mechanistically, CD74-NRG1 leads to extracellular expression of the EGF-like domain of NRG1 III-β3, thereby providing the ligand for ERBB2-ERBB3 receptor complexes. Accordingly, ERBB2 and ERBB3 expression was high in the index case, and expression of phospho-ERBB3 was specifically found in tumors bearing the fusion (P < 0.0001). Ectopic expression of CD74-NRG1 in lung cancer cell lines expressing ERBB2 and ERBB3 activated ERBB3 and the PI3K-AKT pathway, and led to increased colony formation in soft agar. Thus, CD74-NRG1 gene fusions are activating genomic alterations in invasive mucinous adenocarcinomas and may offer a therapeutic opportunity for a lung tumor subtype with, so far, no effective treatment. SIGNIFICANCE CD74–NRG1 fusions may represent a therapeutic opportunity for invasive mucinous lung adenocarcinomas, a tumor with no effective treatment that frequently presents with multifocal unresectable disease.

The phosphatase and tensin homolog regulates epidermal growth factor receptor (EGFR) inhibitor response by targeting EGFR for degradation

The phosphatase and tensin homolog (PTEN) is a tumor suppressor that is inactivated in many human cancers. PTEN loss has been associated with resistance to inhibitors of the epidermal growth factor receptor (EGFR), but the molecular basis of this resistance is unclear. It is believed that unopposed phosphatidylinositol-3-kinase (PI3K) activation through multiple receptor tyrosine kinases (RTKs) can relieve PTEN-deficient cancers from their “dependence” on EGFR or any other single RTK for survival. Here we report a distinct resistance mechanism whereby PTEN inactivation specifically raises EGFR activity by impairing the ligand-induced ubiquitylation and degradation of the activated receptor through destabilization of newly formed ubiquitin ligase Cbl complexes. PTEN-associated resistance to EGFR kinase inhibitors is phenocopied by expression of dominant negative Cbl and can be overcome by more complete EGFR kinase inhibition. PTEN inactivation does not confer resistance to inhibitors of the MET or PDGFRA kinase. Our study identifies a critical role for PTEN in EGFR signal termination and suggests that more potent EGFR inhibition should overcome resistance caused by PI3K pathway activation.

Met signaling regulates growth, repopulating potential and basal cell-fate commitment of mammary luminal progenitors: implications for basal-like breast cancer

Basal-like breast cancer is an aggressive subtype of mammary carcinoma. Despite expressing basal markers, typical of mammary stem cells, this tumor has been proposed to originate from luminal progenitors, which are downstream of stem cells along the mammary epithelial hierarchy. This suggests that committed luminal progenitors may reacquire basal, stem-like characteristics, but the mechanisms that regulate this transition remain unclear. Using mouse models, we found that luminal progenitors express high levels of the Met receptor for hepatocyte growth factor (HGF), as compared with the other mammary epithelial sub-populations. Constitutive activation of Met led luminal progenitors to attain stem cell properties, including enhanced clonogenic activity in vitro and de novo ability to reconstitute mammary glands in repopulation assays in vivo. Moreover, in response to Met signaling, luminal progenitors gave rise to hyperplastic ductal morphogenesis and preferentially underwent basal lineage commitment at the expense of luminal cell-fate specification. Opposite and symmetric results were produced by systemic pharmacological inhibition of Met. Hence, Met signaling targets luminal progenitors for expansion, impairs their differentiation toward the mature luminal phenotype and enables their commitment toward the basal lineage. These results emphasize a critical role for Met in promoting deregulated proliferation and basal plasticity of normal luminal progenitors in the mammary gland, a complex of events that may be required for sustaining the functional and phenotypic properties of basal-like breast tumors.

A preclinical algorithm of soluble surrogate biomarkers that correlate with therapeutic inhibition of the MET oncogene in gastric tumors

The MET oncogene is amplified in a fraction of human gastric carcinoma cell lines, with consequent overexpression and constitutive activation of the corresponding protein product, the Met tyrosine kinase receptor. This genetically driven hyperactivation of Met is necessary for cancer cell growth and survival, so that Met pharmacological blockade results in cell‐cycle arrest or apoptosis (oncogene addiction). MET gene amplification also occurs in vivo in a number of human gastric carcinomas, and clinical trials are now ongoing to assess the therapeutic efficacy of Met inhibitors in this type of malignancy. The aim of our study was to identify a preclinical algorithm of soluble surrogate biomarkers indicative of response to Met inhibition in gastric tumors, as a potential tool to integrate imaging criteria during patient follow‐up. We started from a survey of candidate molecules based on antibody proteomics and gene expression profiling; after ELISA validation and analytical quantification, four biomarkers were identified that appeared to be strongly and consistently modulated by Met inhibition in a panel of Met‐addicted gastric carcinoma cell lines, but not in Met‐independent cell lines. Pharmacologic blockade of Met using specific small‐molecule inhibitors led to reduced secretion of IL‐8, GROα and the soluble form of uPAR and to increased production of IL‐6 both in vitro (in culture supernatants) and in vivo (in the plasma of xenografted mice). If confirmed in patients, this information might prove useful to monitor clinical response to Met‐targeted therapies in MET‐amplified gastric carcinomas.

Heat-shock protein 27 (HSP27, HSPB1) is up-regulated by MET kinase inhibitors and confers resistance to MET-targeted therapy

The tyrosine kinase encoded by the MET oncogene is activated by gene mutation or amplification in tumors, which in most instances maintain addiction, i.e., dependency, to MET activation. This makes MET an attractive candidate for targeted therapies. Here we show that, in 3/3 MET‐addicted human gastric cancer cell lines, MET kinase inhibition resulted in a 3‐ to 4‐fold increased expression of the antiapoptotic small heat‐shock protein of 27 kDa (HSP27, HSPB1). HSP27 increase depended on the inhibition of the MEK/ERK pathway and on heat‐shock factor 1 (HSF1) and hypoxia‐inducible factor‐1α (HIF‐1α) regulation. Importantly, HSP27‐silenced MET‐addicted cells underwent 2‐ and 3‐fold more apoptosis following MET inhibition in vitro and in vivo, respectively. Likewise, in human cancer cells susceptible to epidermal growth factor receptor (EGFR) inhibition, EGFR inhibitors induced HSP27 expression and were strengthened by HSP27 suppression. In control cell lines that were not affected by drugs targeting MET or EGFR, these drugs did not induce HSP27 increase. Therefore, in cancer therapies targeting the MET pathway, the induction of HSP27 might limit the efficacy of anti‐MET agents. As HSP27 increase also impairs the effectiveness of EGFR inhibitors and is known to protect cells from chemotherapeutics, the induction of HSP27 by targeted agents might strongly affect the success of combination treatments.—Musiani, D., Konda, J. D., Pavan, S., Torchiaro, E., Sassi, F., Noghero, A., Erriquez, J., Perera, T., Olivero, M., Di Renzo, M. F. Heat‐shock protein 27 (HSP27, HSPB1) is up‐regulated by MET kinase inhibitors and confers resistance to MET‐targeted therapy. FASEB J. 28, 4055‐4067 (2014). www.fasebj.org

Discovery and pharmacological characterization of JNJ-42756493 (erdafitinib), a functionally selective small molecule FGFR family inhibitor

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.

C-met inhibition blocks bone metastasis development induced by renal cancer stem cells.

Cancer stem cells (CSCs) are key players in bone metastasis. In some renal tumors CSCs overexpress the HGF receptor c-MET, speculating that c-MET targeting could lead to bone metastasis inhibition. To address this hypothesis we isolated renal CD105+/CD24−CSCs, expressing c-MET receptor from a primary renal carcinoma. Then, to study their ability to metastasize to bone, we injected renal CSCs in NOD/SCID mice implanted with a human bone and we tested the effect of a c-MET inhibitor (JNJ-38877605) on bone metastasis development. JNJ-38877605 inhibited the formation of metastases at bone implant site. We showed that JNJ-38877605 inhibited the activation of osteoclasts induced by RCC stem cells and it stimulated osteoblast activity, finally resulting in a reduction of bone turnover consistent with the inhibition of bone metastases. We measured the circulating levels of osteotropic factors induced by RCC stem cells in the sera of mice treated with c-Met inhibitor, showing that IL-11 and CCL20 were reduced in mice treated with JNJ-38877605, strongly supporting the involvement of c-MET in the regulation of this process. To address the clinical relevance of c-MET upregulation during tumor progression, we analysed c-MET in renal cancer patients detecting an increased expression in the bone metastatic lesions by IHC. Then, we dosed CCL20 serum levels resulting significantly increased in patients with bone metastases compared to non-metastatic ones. Collectively, our data highlight the importance of the c-MET pathway in the pathogenesis of bone metastases induced by RCC stem cells in mice and humans.

Abstract 1738: JNJ-42756493 is an inhibitor of FGFR-1, 2, 3 and 4 with nanomolar affinity for targeted therapy

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

Abstract 4748: Discovery of JNJ-42756493, a potent fibroblast growth factor receptor (FGFR) inhibitor using a fragment based approach

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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