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Proteasomes are broadly expressed key components of the ubiquitin-dependent protein degradation pathway containing catalytically active subunits (β1, β2, and β5). LMP7 (β5i) is a subunit of the immunoproteasome, an inducible isoform that is predominantly expressed in hematopoietic cells. Clinically effective pan-proteasome inhibitors for the treatment of multiple myeloma (MM) nonselectively target LMP7 and other subunits of the constitutive proteasome and immunoproteasome with comparable potency, which can limit the therapeutic applicability of these drugs. Here, we describe the discovery and structure-based hit optimization of novel amido boronic acids, which selectively inhibit LMP7 while sparing all other subunits. The exploitation of structural differences between the proteasome subunits culminated in the identification of the highly potent, exquisitely selective, and orally available LMP7 inhibitor 50 (M3258). Based on the strong antitumor activity observed with M3258 in MM models and a favorable preclinical data package, a phase I clinical trial was initiated in relapsed/refractory MM patients.

Large multifunctional peptidase 7 (LMP7/β5i/PSMB8) is a proteolytic subunit of the immunoproteasome, which is predominantly expressed in normal and malignant hematolymphoid cells, including multiple myeloma, and contributes to the degradation of ubiquitinated proteins. Described herein for the first time is the preclinical profile of M3258; an orally bioavailable, potent, reversible and highly selective LMP7 inhibitor. M3258 demonstrated strong antitumor efficacy in multiple myeloma xenograft models, including a novel model of the human bone niche of multiple myeloma. M3258 treatment led to a significant and prolonged suppression of tumor LMP7 activity and ubiquitinated protein turnover and the induction of apoptosis in multiple myeloma cells both in vitro and in vivo. Furthermore, M3258 showed superior antitumor efficacy in selected multiple myeloma and mantle cell lymphoma xenograft models compared with the approved nonselective proteasome inhibitors bortezomib and ixazomib. The differentiated preclinical profile of M3258 supported the initiation of a phase I study in patients with multiple myeloma (NCT04075721).

Pharmacological inhibition of the canonical proteasome by inhibitors targeting its beta5 (PSMB5) subunit represent a cornerstone of the therapeutic management of plasma cell neoplasias. However, proteasome inhibitors have limited clinical applications in other hematologic malignancies. Notably, a broad spectrum of cell lines and patient-derived samples from lymphoma, leukemia and myeloma tend to express high levels of transcripts for and exhibit substantial activity of the immunoproteasome, an alternative proteasome particle which is enriched in hematopoietic/immune tissues. Established proteasome inhibitors used in clinical practice (e.g. bortezomib, carfilzomib, ixazomib) target both the canonical 20S proteasome and the immunoproteasome. We reasoned though that targeting of the immunoproteasome with more selective small molecule inhibitors that largely spare the canonical proteasome may provide insights into the specific functional role of the immunoproteasome in hematologic neoplasias, and importantly may also uncover underappreciated therapeutic opportunities related to targeting of hematologic neoplasias beyond myeloma, that might be more dependent on the immunoproteasome vs. the canonical proteasome; as well as potential benefits related to the therapeutic index of these agents. To address these questions, we characterized the activity of the previously disclosed selective immunoproteasome inhibitor M3258, which targets the LMP7 (beta5i subunit). The bortezomib-resistant cell line MM1VDR, which has a "gatekeeper" PSMB5 mutation preventing bortezomib binding to the canonical proteasome, remained sensitive to in vitro treatment with M3258, in further support of the functional specificity of this inhibitor. To obtain a comprehensive assessment of the activity of M3258 across hematologic malignancies, we studied a pool of 74 "DNA-barcoded" cell lines (PRISM system) from hematologic malignancies, including multiple myeloma (MM), leukemias and lymphomas, which were treated for 48hr with M3258. MM cell lines (9/16) were strongly represented among the sensitive lines which displayed 50% reduction in their cell viability in this analysis; and their responsiveness did not differ significantly from the M3258-responsive MM lines. Importantly, we observed no correlation between the responses of leukemia, lymphoma or MM lines to in vitro treatment with M3258 vs. clinically-relevant pulse of bortezomib. Furthermore, 7 of the 15 lymphoma/leukemia lines that were M3258-responsive had limited, if any, response to a clinically-relevant pulse of bortezomib. Taken together, these observations suggest that immunoproteasome inhibition with M3258 can be active against subsets of hematologic neoplasias with resistance/limited responsiveness to canonical proteasome inhibitors. To further examine the in vivo relevance of these observations, we studied the in vivo activity of M3258 against the MM.1S myeloma cell line, which has only moderate in vitro response to M3258 compared to other blood cancer cell lines tested in our "DNA-barcoded" pooled analysis. M3258 significantly increased the overall survival of mice with diffuse MM-bone lesions established after tail vein injection of MM.1S cells; and of mice harboring MM.1S lesions within a subcutaneously implanted ceramic scaffold-based system engineered to establish a bone marrow-like histological and functional unit with a "humanized" stromal compartment. The anti-tumor activity of M3258 in this scaffold model exhibited a trend for increased efficacy compared to twice weekly subcutaneous treatments with bortezomib. These results highlight that in vivo administration of M3258 exhibits anti-tumor activity against clinically-relevant models of MM lesions, even from cell lines that have modest in vitro responsiveness to this immunoproteasome inhibitor. Furthermore, our studies with a large panel of "DNA-barcoded" cell lines indicates that the anti-tumor effects of M3258 may extend beyond MM and into other classes of hematologic malignancies, including subsets of leukemias and lymphomas, identifying previously underappreciated therapeutic implications for the class of selective immunoproteasome inhibitors. Disclosures Walter: Merck Healthcare KGaA: Employment. Friese-Hamim:Merck Healthcare KGaA: Employment. Goodstal:EMD Serono Research and Development Institute: Employment. Sanderson:Merck Healthcare KGaA: Employment. Mitsiades:Takeda: Other: employment of a relative ; Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding.

Radiotherapy (RT) along with surgery is the mainstay of treatment in head and neck squamous cell carcinoma (HNSCC). Radioresistance represents a major source of treatment failure, underlining the urgent necessity to explore and implement effective radiosensitization strategies. The MET receptor widely participates in the acquisition and maintenance of an aggressive phenotype in HNSCC and modulates the DNA damage response following ionizing radiation (IR). Here, we assessed MET expression and mutation status in primary and metastatic lesions within a cohort of patients with advanced HNSCC. Moreover, we investigated the radiosensitization potential of the MET inhibitor tepotinib in a panel of cell lines, in vitro and in vivo, as well as in ex vivo patient-derived organotypic tissue cultures (OTC). MET was highly expressed in 62.4% of primary tumors and in 53.6% of lymph node metastases (LNM), and in 6 of 9 evaluated cell lines. MET expression in primaries and LNMs was significantly associated with decreased disease control in univariate survival analyses. Tepotinib abrogated MET phosphorylation and to distinct extent MET downstream signaling. Pretreatment with tepotinib resulted in variable radiosensitization, enhanced DNA damage, cell death, and G2–M-phase arrest. Combination of tepotinib with IR led to significant radiosensitization in one of two tested in vivo models. OTCs revealed differential patterns of response toward tepotinib, irradiation, and combination of both modalities. The molecular basis of tepotinib-mediated radiosensitization was studied by a CyTOF-based single-cell mass cytometry approach, which uncovered that MET inhibition modulated PI3K activity in cells radiosensitized by tepotinib but not in the resistant ones.

329 Background: Tepotinib, a potent and highly selective MET inhibitor, showed promising activity in advanced HCC with MET overexpression in two Phase Ib/II trials, which both met their primary endpoints. One trial enrolled Asian patients (pts) without prior systemic therapy (first line [1L]; NCT01988493) and one enrolled US/European pts with prior sorafenib (second line [2L]; NCT02115373). By investigator assessment, objective response rates and disease control rates with tepotinib in Phase II were 15.8% and 60.5% in 1L (n = 38), and 8.2% and 57.1% in 2L (n = 49). Outcomes appeared better in pts with METamp. Thus, we further investigated the preclinical and clinical activity of tepotinib in HCC, focusing on high-level METamp, which could be an oncogenic driver in this tumor type. Methods: Preclinical activity was assessed in 37 HCC pt-derived xenografts (PDXs) in nude mice treated with tepotinib (100 mg/kg, Days 1–5, every 7 days for five cycles; 6–12 replicates per PDX). Clinical activity was assessed by analyzing pts with METamp or high-level METamp (defined as mean MET gene copy number [GCN] ≥5 and ≥10, respectively; by fluorescence in situ hybridization), who received tepotinib 500 mg in Phase Ib or II of the 1L and 2L trials (n = 121). In the Phase Ib part of the 1L trial, pts could have received prior treatment. Results: Molecular profiling revealed high-level METamp in two of 37 HCC PDXs: LIM612 ( MET GCN 47.1) and LIPF210 ( MET GCN 44). Tepotinib induced significant tumor regression in both of these high-level METamp HCC PDX models (mean tumor volume reduction: 97% and 96%, respectively). Across the two trials, 15 pts treated with tepotinib 500 mg had METamp, of whom five had an objective response (one complete response [CR] and four partial responses [PRs]) and six had stable disease (SD) as best overall response. Four pts had high-level METamp (mean MET GCN 14.3, 18.1, 30.2, and 36.2), with best overall response of CR in one pt, PR in two pts and SD in one pt (Table). One pt with high-level METamp (mean MET GCN 14.3) treated with 1L tepotinib was still receiving treatment as of Sept 2020 (total tepotinib treatment duration: > 45 months). Conclusions: High-level METamp may be an oncogenic driver in HCC that sensitizes tumors to MET inhibition with tepotinib. Compared with MET overexpression, high-level METamp could be a better predictive biomarker for MET inhibitors in this setting. Clinical trial information: NCT01988493, NCT02115373. [Table: see text]

Background Patients with advanced non-small cell lung cancer (NSCLC) harboring oncogenic MET alterations, such as MET exon 14 skipping or MET amplification (MET amp), benefit from MET tyrosine kinase inhibitor (TKI) treatment. Brain metastases are common in patients with advanced NSCLC. In preclinical studies in rats, tepotinib had relatively high binding to brain tissue (fu br=0.4%, fu pl=4%) and free tepotinib concentrations in brain were 25% of the concentrations found in plasma (Kp u,u=0.25). In this study we investigated the efficacy of the MET TKIs tepotinib, capmatinib, savolitinib and crizotinib in mice implanted orthotopically with MET-dependent tumor explants derived from human NSCLC brain metastases. Methods The antitumor activity of tepotinib (30 mg/kg qd) was investigated in a screen of 21 subcutaneous lung cancer patient-derived xenograft (PDX) models of brain metastases grown in NOD-SCID mice (n=1/model). 20 of these tumors were retrospectively analyzed for cancer-specific mutations, gene copy number (GCN) and gene expression by Nanostring. 2 responding models, both found to harbor MET amp, were tested again subcutaneously in NOD-SCID mice treated with vehicle or tepotinib (125 mg/kg qd; n=5). Both models were orthotopically implanted into brains of NOD-SCID mice and tumor growth was monitored by MRI. Established tumors were treated with MET TKIs (n=10) tepotinib (125 mg/kg qd), capmatinib (30 mg/kg bid), savolitinib (60 mg/kg qd) or crizotinib (50 mg/kg qd). Results 2 of 21 subcutaneous PDX tumors (LU5406 and LU5349) regressed in response to tepotinib treatment. When implanted subcutaneously again into mice, both models regressed completely upon treatment with tepotinib. Molecular profiles revealed that these were the only tumors of the 20 models investigated by Nanostring that had MET amp (high-level increase in MET GCN of >10). Both models grew orthotopically when implanted into brains of mice. Contrast-weighted MRI indicated regions with intact and disrupted blood–brain barrier (BBB) in all implanted tumors. Tumor regression of orthotopic LU5406 tumors was observed with all MET TKIs (% median tumor volume changes [%TV]: tepotinib, –63%; capmatinib, –24%; savolitinib, –38%; crizotinib, –27%). In the orthotopic LU5349 tumors, treatment with crizotinib or savolitinib led to growth inhibition (%TV: +88% and –13%, respectively), whereas tepotinib and capmatinib induced tumor regression (%TV: –84% and –63%, respectively). Conclusions The heterogenous pattern of regions with intact and disrupted BBB in the orthotopic brain metastases models is thought to mirror the clinical situation. Tepotinib was efficacious in the two MET-driven orthotopic brain metastases PDX tumors characterized by pronounced tumor regression and may be relevant to patients with brain metastases. Relationships between efficacy and BBB leakiness will be discussed. Citation Format: Manja Friese-Hamim, Anderson Clark, Lindsey Crowley, Christof Reusch, Olga Bogatyrova, Claudia Wilm, Hong Zhang, Timothy Crandall, Jing Lin, Jianguo Ma, David Bachner, Jürgen Schmidt, Martin Schaefer, Christopher Stroh. Anti-tumor activity of tepotinib in orthotopic models of lung cancer patient-derived brain metastases with MET amplification [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3407.

M3258 is an orally bioavailable, potent, selective, reversible inhibitor of the large multifunctional peptidase 7 (LMP7, β5i, PSMB8) proteolytic subunit of the immunoproteasome; a crucial component of the cellular protein degradation machinery, which is highly expressed in malignant hematopoietic cells including multiple myeloma. M3258 was previously shown to deliver strong in vivo preclinical efficacy in multiple myeloma xenograft models, as well as a more benign non-clinical safety profile compared to approved pan-proteasome inhibitors, exemplified by a lack of effects on the central and peripheral nervous systems and cardiac and respiratory organs. Here we describe preclinical PK/PD and PK/efficacy modelling which led to a prediction of the PK profile, and the efficacious and safe dose ranges of M3258 in human which were used to guide the design of the phase I dose-escalation trial of M3258 in >3 line relapsed/refractory multiple myeloma (RRMM) patients. Mouse, rat, dog and monkey PK, plasma protein binding and intrinsic clearance data were used to estimate a half-life of approximately 6 hours for M3258 in human. The human total clearance and volume of distribution for M3258 were predicted to be 0.033 L/h/kg and 0.28 L/kg, respectively, whilst oral bioavailability was estimated to be above 80%. LMP7 proteolytic activity was assessed as a PD readout in human multiple myeloma tumor cells xenografted to mice as well as in dog peripheral blood mononuclear cells (PBMCs). A strong PK/PD relationship was observed for M3258 across both species. LMP7 inhibition by M3258 also correlated strongly with anti-tumor efficacy in multiple myeloma xenografts, with maximal efficacy observed at M3258 exposure delivering sustained inhibition of tumor LMP7 activity. Quantitative PK/PD/efficacy modeling predicted the biologically efficacious dose (BED) of M3258 upon oral application to be between 10 - 90 mg daily in human. By incorporating data from nonclinical safety studies, these data suggest an attractive human PK profile of M3258, enabling oral application, as well as an improved human therapeutic index compared to approved pan-proteasome inhibitors. M3258 is being investigated in a phase I, first-in-man, 2-part, open label clinical study designed to determine the safety, tolerability, PK, PD and early signs of efficacy of M3258 as a single agent (dose-escalation) and co-administered with dexamethasone (dose-expansion) in participants with RRMM whose disease has progressed following > 3 prior lines of therapy and for whom no effective standard therapy exists. Integration of these data will guide the selection of the BED for potential further clinical development of M3258. Disclosures Lignet: Merck Healthcare KGaA: Employment. Esdar:Merck Healthcare KGaA: Employment. Friese-Hamim:Merck Healthcare KGaA: Employment. Becker:Merck Healthcare KGaA: Employment, Other: Holding shares with a value below 1000-USD. Drouin:EMD Serono Research and Development Institute: Employment. El Bawab:Merck Healthcare KGaA: Employment. Goodstal:EMD Serono Research and Development Institute: Employment. Gimmi:Merck Healthcare KGaA: Employment. Haselmayer:Merck Healthcare KGaA: Employment. Jährling:Merck Healthcare KGaA: Employment. Sanderson:Merck Healthcare KGaA: Employment. Sloot:Merck Healthcare KGaA: Employment. Stinchi:Merck Healthcare KGaA: Employment. Victor:Merck Healthcare KGaA: Employment. Walter:Merck Healthcare KGaA: Employment. Rohdich:Merck Healthcare KGaA: Employment.

Large multifunctional peptidase 7 (LMP7, β5i, PSMB8) is a chymotrypsin-like proteolytic subunit of the immunoproteasome, which degrades ubiquitinated proteins and generates peptides for presentation on MHC class I. In contrast to the constitutive proteasome, which is broadly expressed, the immunoproteasome is specifically present in normal and malignant hematopoietic cells and can be induced in non-hematopoietic cells by inflammatory stimuli such as IFNγ. Pan-proteasome inhibitors like Bortezomib, which is approved in multiple myeloma and mantle cell lymphoma, indiscriminately inhibit the proteolytic activities of multiple subunits of the constitutive proteasome and immunoproteasome, including LMP7. Widespread proteasome inhibition is believed to underpin the adverse safety profiles of these agents and limit their therapeutic potential. Selective LMP7 targeting could achieve improved antitumor activity as a result of enhanced target inhibition, meanwhile circumventing the dose-limiting severe toxicities associated with pan-proteasome inhibitors. Based on the aforementioned hypothesis, a drug discovery program was initiated to identify selective inhibitors of LMP7. This led to the discovery of M3258, a covalent-reversible, potent, selective and orally-bioavailable inhibitor of LMP7. M3258 demonstrated strong in vivo antitumor activity, up to complete regression, in multiple myeloma xenograft models at daily oral doses as low as 1 mg/kg. This was associated with significant and prolonged suppression of tumor LMP7 activity. Furthermore, M3258 was efficacious in several multiple myeloma models that were refractory to Bortezomib. Subacute GLP toxicology studies with M3258, applied orally on a once-daily schedule, identified the lymphatic and hematopoietic systems in rat and dog and intestinal system in dog alone as main target organs. Importantly, M3258 was without effect on the peripheral and central nervous systems and cardiac and respiratory organs. Overall, M3258 demonstrated a superior preclinical therapeutic window and more restricted spectrum of toxicities compared to pan-proteasome inhibitors. Supported by robust preclinical data, clinical phase I assessment of M3258 in multiple myeloma patients is planned to begin in 2019. Citation Format: Michael Sanderson, Michael Busch, Christina Esdar, Manja Friese-Hamim, Mireille Krier, Jianguo Ma, Djordje Musil, Felix Rohdich, Willem Sloot, Gina Walter, Ugo Zanelli, Oliver Schadt, Markus Klein. First-time disclosure of M3258: A selective inhibitor of the immunoproteasome subunit LMP7 with potential for improved therapeutic utility in multiple myeloma compared to pan-proteasome inhibitors [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 DDT02-01.

Purpose: To explore in a panel of patient-derived xenograft models of human non–small cell lung cancer (NSCLC) whether high EGFR expression, was associated with cetuximab activity. Experimental Design: NSCLC patient-derived xenograft models (n = 45) were implanted subcutaneously into panels of nude mice and randomization cohorts were treated with either cetuximab, cisplatin, cisplatin plus cetuximab, vehicle control, or else were left untreated. Responses according to treatment were assessed at week 3 by analyzing the relative change in tumor volume and an experimental analogue of the Response Evaluation Criteria in Solid Tumors (RECIST) guidelines. An EGFR IHC score was calculated for each patient-derived xenograft model and response was assessed according to EGFR expression level. Results: When tumors were stratified into high and low EGFR expression groups (IHC score threshold 200; scale 0–300), a stronger antitumor activity was seen in the high EGFR expression group compared with the low EGFR expression group in both the cetuximab monotherapy and cisplatin plus cetuximab combination therapy settings. For tumors treated with cisplatin plus cetuximab, the objective response rate was significantly higher in the high EGFR expression group compared with the low EGFR expression group (68% vs. 29%). Objective response rates were similar in high and low expression groups for tumors treated with cisplatin alone (27% vs. 24%, respectively). Conclusion: Cetuximab activity in NSCLC patient-derived xenograft models was demonstrated clearly only in tumors that expressed high levels of EGFR, as defined by an IHC score of ≥200. Clin Cancer Res; 20(17); 4478–87. ©2014 AACR.

In clinical practice, acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC) causes EGFR TKI treatment failure. Several resistance mechanisms have been identified; EGFR T790M mutation and aberrant activation of c-Met through c-Met gene amplification and/or HGF upregulation are the two most common mechanisms. We have previously shown that combining the highly selective c-Met inhibitor tepotinib with 1st or 2nd-generation EGFR TKIs overcomes gefitinib resistance caused only by c-MET amplification. Third-generation EGFR TKIs such as rociletinib have been developed specifically to overcome T790M-mediated resistance to EGFR TKIs. We hypothesized that combining a 3rd-generation EGFR TKI with tepotinib could overcome resistance to a 1st or 2nd-generation EGFR TKI (e.g. gefitinib) regardless of the mechanism of resistance. We assessed the efficacy of tepotinib and a Merck-synthesized version of rociletinib (MSR) as monotherapy or in combination in a NSCLC cell line xenograft model (HCC827-GR-T790M) with an EGFR del 19 mutation, c-Met amplification, and exogenous T790M expression, and in a patient-derived xenograft model (DFCI081) with an EGFR del 19 mutation and c-Met amplification, but not a T790M mutation. Single-agent tepotinib, erlotinib, and afatinib did not show antitumor activity in HCC827-GR-T790M xenografts and tumors progressed on treatment (median tumor volume [TV] changes >73%). The combinations of tepotinib + erlotinib and tepotinib + afatinib delayed tumor regrowth more than single-agent therapy, but were no more effective than single-agent MSR. In contrast, the combination of tepotinib + MSR had strong antitumor activity in this model with double resistance due to c-Met amplification and exogenous T790M expression, resulting in complete tumor regression (median TV change -97%). In the patient-derived xenograft model DFCI081, tumors progressed under single-agent therapy with the EGFR TKIs erlotinib, afatinib, and MSR, as expected given that this cell line expresses c-Met. In contrast, tepotinib induced complete tumor regression as monotherapy and when combined with each of the EGFR TKIs (median TV change -100%). Complete regression was maintained until the end of the 60-day observation period after treatment termination. These findings are compatible with the hypothesis that combining tepotinib with a 3rd-generation EGFR TKI to overcome double resistance to 1st and 2nd-generation TKIs mediated by T790M and c-Met in NSCLC is more effective than using either agent alone. Furthermore, whereas EGFR TKIs were not effective in tumor models showing c-Met amplification and EGFR del 19 mutation but not T790M mutation, ie resistance is c-Met mediated, tepotinib monotherapy induced complete tumor regression. Citation Format: Manja Friese-Hamim, Giuseppe Locatelli, Friedhelm Bladt. Combination of c-Met inhibitor tepotinib (MSC2156119J) and a third-generation EGFR inhibitor can overcome double resistance mediated by EGFR T790M mutation and c-Met amplification in non-small cell lung cancer models. [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 4663.

The therapeutic success of EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC) is hampered in a subset of patients by the development of drug resistance through the EGFR T790M ‘gate keeper’ mutation. Recent investigations suggest that aberrant activation of the c-Met receptor through c-Met amplification and/or HGF upregulation may be an additional mechanism for acquired resistance. Combination of an EGFR TKI with a c-Met inhibitor could, therefore, delay and/or prevent the emergence of c-Met-driven resistance. The aim of our study was to evaluate the efficacy of MSC2156119J, a highly selective small molecule c-Met inhibitor, as monotherapy or in combination with EGFR TKIs (erlotinib, gefitinib and afatinib) in human xenograft models transplanted with two different primary NSCLC explants, LU342 and LU0858. While both tumors express the activated EGFR mutant form, LU342 expresses low levels of c-Met, while LU0858 expresses high levels of c-Met and carries an amplified c-Met locus. Single-agent MSC2156119J was inactive against the low c-Met-expressing LU342 explant and tumors progressed on treatment (median tumor volume [TV] change >73%). Erlotinib treatment resulted in tumor regression, with median TV change of -93% and tumor growth delay (TGD) of 56 days. The combination of MSC2156119J and erlotinib was more active, delaying tumor regrowth by 73 days compared to erlotinib monotherapy. Administration of afatinib at 5mg/kg was highly active in this model, inducing complete tumor regression (median TV change -100%), similar to the combination of afatinib and MSC2156119J. In contrast, the LU0858 explant, which carries an amplified c-Met locus, was sensitive to MSC2156119J at a dose of 100mg/kg 5 days on and 2 days off (5 on/2 off), inducing tumor stasis with a median TV change of 19% and TGD of 34 days. While gefitinib and afatinib alone at doses of 150mg/kg 5 on/2 off and 5mg/kg 5 on/2 off, respectively, were inactive (median TV changes >73%), in combination with MSC2156119J tumor growth inhibition was enhanced, resulting in tumor regression (median TV changes of -64% and -85%, respectively). In comparison, tumors treated with platinum-based doublet chemotherapy (cisplatin 5mg/kg QW/pemetrexed 200mg/kg QW) progressed, with TGD of 16 days. When MSC2156119J was combined with cisplatin/pemetrexed, antitumor activity was not significantly enhanced compared to single-agent MSC2156119J (median TV change -32%, TGD 30 days). Together, these findings are compatible with the hypothesis that inhibition of the c-Met pathway prevents and/or delays the emergence of c-Met-driven resistance in EGFR-mutant NSCLC. Furthermore, the enhancement of antitumor activity in the low c-Met-expressing LU342 model when MSC2156119J was combined with erlotinib suggests that the combination of an EGFR TKI + MSC2156119J may also be active in tumors with low levels of c-Met expression. Citation Format: Friedhelm Bladt, Manja Friese-Hamim, Andree Blaukat. Activity of MSC2156119J in non-small cell lung cancer models with activating EGFR mutation. [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 2591. doi:10.1158/1538-7445.AM2015-2591

The mesenchymal-epithelial transition factor (c-Met) receptor, also known as hepatocyte growth factor receptor (HGFR), controls morphogenesis, a process physiologically required for embryonic development and tissue repair. Aberrant c-Met activation is associated with human malignancies, including hepatocellular carcinomas (HCC). The aim of this study was to evaluate the effect of a novel, highly selective c-Met inhibitor, MSC2156119J (EMD 1214063), in hepatocellular cancer models. MHCC97H is a human HCC cell line co-expressing c-Met and HGF, and exhibiting high metastatic potential. The effect of MSC2156119J on MHCC97H was evaluated in a Balb/c nu/nu murine xenograft model in a subcutaneous and orthotopic setting. Tumor-bearing mice were orally treated with 100 mg/kg MSC2156119J (5 days on/2 days off). In the subcutaneous setting, MSC2156119J resulted in tumor regression in 10/10 mice, achieving tumor eradication in 9/10 mice. In the orthotopic model, MHCC97H tumor fragments proliferate in the liver and invariably metastasize to the lung (100%). MSC2156119J treatment started 7 days after tumor fragment implantation. After 5 weeks of treatment, mice were necropsied and a series of parameters were assessed. Primary tumor size and weight, and circulating Alpha-Feto-Protein levels were significantly lower in mice treated with MSC2156119J compared to controls (p To better predict the clinical efficacy of MSC2156119J, 9 patient-derived primary HCC explants models were used. The explants were subdivided into 3 groups, based on their c-Met levels and HGF expression. Each group comprised 3 tumors expressing high, intermediate, and low levels of c-Met and HGF, respectively. Mice were treated with MSC2156119J, sorafenib, or a combination of both. MSC2156119J (100 mg/kg/5 out of 7 days) strongly inhibited tumor growth in 4/9 models (TGD range of 370% to 41%). Analysis of intratumoral c-Met, phospho c-Met and HGF levels indicated that explants with high levels of c-Met and HGF were more sensitive to MSC2156119J than low-expressing models. In 3/4 responsive models, MSC2156119J exhibited a better anti-tumor activity than sorafenib, while sorafenib was more efficacious in a model characterized by intermediate c-Met/HGF expression. MSC2156119J was not efficacious in models exhibiting low or no signs of c-Met signalling. When used in combination, MSC2156119J did not enhance the activity of sorafenib. MSC2156119J monotherapy was well tolerated while sorafenib alone and in combination with MSC2156119J induced significant body weight loss. Overall, these data indicate that MSC2156119J may be a valuable therapeutic option for liver cancers with high levels of c-Met expression/activation. Citation Format: Friedhelm Bladt, Andree Blaukat, Dieter Dorsch, Manja Friese-Hamim, Michael Meyring, Oliver Schadt. The c-Met inhibitor MSC2156119J effectively inhibits growth of liver cancer models. [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 925. doi:10.1158/1538-7445.AM2013-925

c-Met is a receptor tyrosine kinase that has hepatocyte growth factor as its ligand. Evidence from biochemical and human genetic studies indicate that c-Met is one of the most frequently activated tyrosine kinases in human cancer. Dysregulated c-Met signaling can result in the development of tumors highly dependent on c-Met. Furthermore, enhanced c-Met signaling is likely involved in conferring resistance to epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) inhibitors. The orally available EMD 1214063 and EMD 1204831 inhibit c-Met activity in a potent and highly selective fashion, and are currently being evaluated in cancer patients. Here we report the activity of these compounds in combination with inhibitors of EGFR and VEGF in various tumor xenograft models. EMD 1214063 was tested alone and in combination with EGFR inhibitors (erlotinib and cetuximab) in xenografts of erlotinib-resistant tumor cells, namely the lung cancer H1975 and the NCI-H441 non-small-cell lung cancer (NSCLC) cell lines. The H1975 cell line expresses moderate amounts of c-Met, and EMD 1214063 was inactive as a single agent (treatment group/control group [T/C] 78%; tumor growth delay [TGD] 2 days). Cetuximab was active with a T/C of 13% inducing a TGD of 21 days. The combination of EMD 1214063 and cetuximab was active with a T/C of α1% and a TGD of 42 days. The NCI-H441 cell line is characterized by constitutive c-Met overexpression and is sensitive to c-Met inhibitors in vivo. EMD 1214063 (100 mg/kg) was active inducing a T/C of 20%, while erlotinib alone (30 mg/kg) was not efficacious. However, the combination of EMD 1214063 with erlotinib enhanced tumor growth inhibition, induced partial tumor regression in 2/9 mice, and delayed tumor re-growth. These data are compatible with the hypothesis that cMet pathway activation confers resistance to EGFR inhibitors. We additionally investigated the combination of either EMD 1214063 or EMD 1204831 with VEGF inhibitors in NSCLC models. In NCI-H441 xenografts, EMD 1204831 produced a modest T/C (66%) at the suboptimal dose of 25 mg/kg/bid, similarly to aflibercept at 40 mg/kg (T/C 55%). In contrast, the combination of both compounds yielded a T/C of 1% with partial responses in 2/10 mice. In the EBC-1 model, treatment with EMD 1214063 (10 mg/kg) or with the antiVEGF antibody B20-4.1 (20 mg/kg) as single agents resulted in a T/C of β21% (with partial regression in 1/10 mice) and 19%, respectively. In contrast, the combination of EMD 1214063 and B20-4.1 exhibited enhanced anti-tumor activity with a T/C of −72% and partial regressions in 10/10 mice. These data support the hypothesis that EMD 1214063 or EMD 1204831 may be effective in NSCLC patients in combination with VEGF-targeting agents. In conclusion, our findings provide a rationale to evaluate the efficacy of the combination of EMD 1214063 or EMD 1204831 with EGFR and VEGF inhibitors in NSCLC patients. 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 1787. doi:1538-7445.AM2012-1787

c-Met, the receptor for hepatocyte growth factor (HGF), is a well characterized receptor tyrosine kinase, which is crucial for cell functions, such as survival, proliferation, motility and migration. Activating point mutations of c-Met have been identified in human malignancies, including renal carcinoma and lung cancer and constitutive activation of the HGF/c-Met pathway leads to tumor development and progression in cancer animal models. In the light of these findings, c-Met has become a key target for oncology therapeutics. In order to develop c-Met specific inhibitors, we have conducted a biochemical HTS run with the recombinant kinase domain of c-Met. This approach has enabled us to identify a new class of c-Met inhibitors. Our report will focus on pharmacological and pre-clinical data, characterising EMD1214063, a promising therapeutic candidate currently under clinical testing. Inhibition of the c-Met kinase by EMD1214063 was potent and highly selective. In in vitro kinase reactions and binding assays, EMD1214063 at a dose of 1μM, inhibited only 5 additional kinases in a panel of more than 250 different potential off-targets more than 50%. In vitro, EMD1214063 interfered with survival, anchorage-independent growth and HGF-induced migration of tumor cells, by blocking HGF-dependent as well as constitutive phosphorylation of c-Met. In vivo, oral administration of EMD1214063 resulted in a strong inhibition - ranging from delayed growth to complete regression - of HGF-dependent and -independent c-Met driven tumor xenografts. Such anti-tumor activity was observed at a broad dose range, suggesting that EMD1214063 has a wide therapeutic index. The inhibitor was well tolerated, as indicated by the lack of significant weight loss in treated mice. The mechanism of action of EMD1214063 was investigated in vivo in a series of PK/PD studies. Administration of a single dose of EMD1214063 induced a complete and long-lasting inhibition of c-Met phosphorylation in the tumor xenografts. Furthermore, it strongly reduced the plasma levels of tumor-derived IL-8. c-Met phosphorylation and plasma IL-8 levels decreased as a function of the drug concentration in the tumor and plasma. Taken together, the high c-Met selectivity, potency and in vivo efficacy of EMD1214063 render this compound a very promising candidate for the treatment of patients bearing c-Met-driven tumors. 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 3622.