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Published OnlineFirst November 19, 2019; DOI: 10.1158/1535-7163.MCT-18-1274

MOLECULAR CANCER THERAPEUTICS | CANCER BIOLOGY AND TRANSLATIONAL STUDIES

Targeting the MET Receptor Tyrosine Kinase as a Strategy for Radiosensitization in Locoregionally Advanced Head and Neck Squamous Cell Carcinoma Lluís Nisa1,2,3, Paola Francica1,2, Roland Giger3, Matus Medo1,2, Olgun Elicin2, Manja Friese-Hamim4, Claudia Wilm4, Christopher Stroh4, Beat Bojaxhiu2, Aurelie Quintin1,2, Marco D. Caversaccio3, Matthias S. Dettmer5,Melanie Buchwalder3,5, Tess M. Brodie1,2,6, Daniel M. Aebersold1,2, Yitzhak Zimmer1,2, Thomas E. Carey7,8, and Michaela Medova1,2

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

Introduction in which activation of oncogenes, accelerated tumor cell repopulation, hypoxia, proﬁcient DNA damage repair, and tumor–stroma interac- Head and neck squamous cell carcinoma (HNSCC) is a leading tions among others play crucial roles (3–5). cause of cancer with a yearly incidence of more than 650,000 new cases In this context, oncogenic signaling through MET, the RTK for and over 350,000 cancer-related deaths per year (1). Patients with scatter-factor/hepatocyte growth factor (SF/HGF), contributes to advanced disease are treated with radiotherapy (RT), alone or in increased cell growth and proliferation, angiogenesis, invasiveness, combination with systemic agents, exclusively or following sur- migration, and metastatic potential (6). Moreover, accumulating gery (1, 2). Consequently, resistance to RT represents a major cause evidence situates MET as a relevant actor in the cellular DNA damage of treatment failure in patients with persistent and relapsing HNSCC. response (DDR) in various preclinical cancer models (7–10). Cur- Radioresistance in turn is a complex and multifactorial phenomenon, rently, nearly 160 clinical trials study a battery of MET inhibitors alone or in combination with other therapeutic modalities in various cancers (11). In HNSCC, both MET and SF/HGF overexpression and 1 Department for BioMedical Research, Inselspital, Bern University Hospital, and paracrine loops enhance invasive features and potentially confer 2 University of Bern, Bern, Switzerland. Department of Radiation Oncology, therapeutic resistance to cytotoxic drugs (12, 13). Moreover, Saintigny Inselspital, Bern University Hospital, and University of Bern, Bern, Switzerland. 3Department of Otorhinolaryngology – Head and Neck Surgery, Inselspital, Bern and colleagues suggested that MET activation represents an early University Hospital, and University of Bern, Bern, Switzerland. 4Translational driver and a target for chemoprevention of oral cancers (14). To date, Innovation Platform Oncology, Merck HealthCare KGaA, Darmstadt, Germany. however, the effects of MET targeting along with ionizing radiation 5Institute of Pathology, University of Bern, Bern, Switzerland. 6Mass Cytometry (IR), the most common treatment modality for HNSCC, have not been 7 Facility, University of Zurich, Zurich, Switzerland. Department of Otolaryngol- comprehensively explored (13, 15, 16). – ogy Head and Neck Surgery, University of Michigan School of Medicine, Ann Here, we investigate the prevalence and impact of MET expres- Arbor, Michigan. 8Comprehensive Cancer Center, University of Michigan School of Medicine, Ann Arbor, Michigan. sion and mutations in primary tumors (PT) and lymph node metastases (LNM) in a cohort of advanced-stage HNSCC patients Note: Supplementary data for this article are available at Molecular Cancer treated with RT. Moreover, we evaluate the effects of MET targeting Therapeutics Online (http://mct.aacrjournals.org/). in combination with IR in a panel of representative HNSCC cell Corresponding Author: Michaela Medova, Inselspital, Bern University Hospital, lines both in vitro and in vivo,aswellasinex vivo organotypic tissue and University of Bern, Murtenstrasse 35, 3008 Bern, Switzerland. Phone: 41- cultures (OTC) derived from patients with HNSCC. We show that 3163-23565; Fax: 41-3163-23297; E-mail: [email protected] MET expression in LNMs is associated with decreased disease-free – Mol Cancer Ther 2020;19:614 26 survival and that MET targeting along with IR leads to heteroge- doi: 10.1158/1535-7163.MCT-18-1274 neous responses, potentially resulting in radiosensitization in high 2019 American Association for Cancer Research. MET-expressing models.

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Materials and Methods Fungizone 0.25 mg/mL; Gibco, Invitrogen Corp.), and nonessential Sample collection, inclusion criteria, TMA construction, and IHC aminoacids (1% vol/vol; Sigma). SCC-61, SQ-20B, and HN5 cells were provided by Prof. M. Pruschy (University of Zurich, Switzer- Patients' tissue and data were collected according to a predefined land) and no further authentication was performed. University of protocol approved by the regional ethical committee (Kantonale Michigan Squamous Cell Carcinoma (UM-SCC) cell lines were Ethikkomission Bern; Protocol Nr.: 050/14). Written informed obtained from Prof. T. Carey's laboratory (University of Michigan), consent has been obtained from the patients and the studies were cultured and authenticated as previously reported (20). More conducted in accordance with recognized ethical guidelines (Dec- detailed information about these cell lines is provided in Supple- laration of Helsinki). Formalin-fixed paraffin-embedded (FFPE) mentary Methods. All cells are MET wild-type. For all experiments, archival blocks from HNSCC patients treated between 2004 and the cells were maintained in culture upon thawing for up to 8 2012 at Inselspital Bern were selected according to the following passages (4 weeks). inclusion criteria: (i) histologically proven squamous cell carcinoma The MET tyrosine kinase inhibitor tepotinib (or MSC2156119J; of the oral cavity, oropharynx, larynx and hypopharynx; (ii) pres- ref. 21) was kindly provided by Merck HealthCare KGaA (22). ence of lymph node metastases involvement; (iii) primary neck PHA665752 (23) was purchased from Pfizer. Both drugs were dissection; (iv) primary or postoperative RT to the PT and the neck dissolved in DMSO, and DMSO concentrations were normalized ( concomitant chemotherapy or cetuximab); (v) complete follow- for all treatment conditions. Recombinant human SF/HGF was up data for at least 2 years after the end of therapy for event-free purchased from R&D Systems and prepared following the manu- patients; (vi) >18 years of age. facturer's instructions. Treatments were carried out as described in A tissue microarray (TMA) including PTs and/or LNMs, as well as Results. normal tissue (from histologically normal salivary glands harvested All in vitro experiments were performed at least 3 times unless during neck dissection), was constructed using a new-generation tissue otherwise indicated. arrayer as previously reported (17). At least two punches per sample were included to minimize sampling bias. For IHC and H&E staining, 5-mm sections were cut from TMAs. Irradiation In vitro ex vivo 137 Sections were deparaffinized and rehydrated. IHC was performed and IR was delivered using a Cs research irradiator with the BOND RX automated system (Leica Biosystems). Endog- (MDS Nordion) with a dose rate of 0.75 Gy/min. Irradiation was carried out at the indicated time points following tepotinib treatment enous peroxidase activity was blocked with H2O2 3% for 4 minutes. Sections were incubated for 30 minutes with specific primary of cells or OTCs. antibodies at room temperature: MET (Invitrogen, clone 3D4) and Ki67 (Cell Signaling Technology, clone D2H10). Antibody detec- Immunoblotting and antibodies tion was performed with the Bond Polymer Refine Detection kit Immunoblotting was performed as previously described (18). Over- (Leica Biosystems, DS9800) according to the manufacturer's night incubation with the following primary antibodies took place: instructions. Expression of p-MET could not be detected with two p-Y1234/Y1235 MET (p-MET), MET, p-Ser473 AKT, and p-Thr202/ different commercially available antibodies in FFPE tissues in the Tyr204 ERK (all from Cell Signaling Technology). Anti-b-actin was presence of positive controls (from fresh-frozen tissues). Semiquan- obtained from Millipore Corporation and pSer139 H2AX from titative assessment of IHC stainings was performed as previously Upstate Biotechnology Inc. Membranes were incubated with appro- described (18). priate secondary antibodies, and signals were detected with the ECL kit (Amersham Pharmacia Biotech) or with infrared fluorescence with an MET mutational screening Odyssey imager (Li-Cor Biosciences). Densitometry values were The presence of 34 previously reported MET gene mutations was determined using ImageJ and b-actin was used for value normalization determined using the iPLEX Assay (Agena GmbH), with genomic (imagej.nih.gov/ij/). DNA isolated from 400 FFPE blocks as described in Supplementary Methods. Clonogenic assays Colony-forming assays were performed using standard methodol- HPV genotyping ogy (24). The surviving fraction was normalized to the plating A total of 100 ng of genomic DNA were used to perform efficiency (PE ¼ colonies formed/cells plated). Radiosensitization was polymerase chain reactions (PCR) with PGMY primers (19). HPV evaluated using the radiation enhancement ratio (RER). Briefly, the genotype was determined using the Linear Array HPV Genotyping RER is the ratio between area under the curve (AUC) of two treatment Test (Roche Molecular Diagnostics). This test allows detection of 16 conditions. A RER significantly superior to 1 according to one-sample t high-risk (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73, 82) test is deemed to indicate radiosensitization (25). and 21 low-risk HPV types (6, 11, 26, 40, 42, 53, 54, 55, 61, 62, 64,

67, 69, 70, 71, 72, 81, 83, 84, IS39, and CP6108). For HPV-negative Cell proliferation and EC50 value determination ﬁ cases, beta-globin of 400 bp was ampli ed by PCR to test the Maximal effective concentration (EC50) values were determined as integrity of extracted DNA. previously described (24).

Cell lines and reagents Cell viability, cell death, senescence, and cell-cycle analysis FaDu cells were purchased from the ATCC and Detroit-562 cells Caspase-3 enzymatic activity was determined by using a ﬂuorogenic from Cell Line Services GmbH and authenticated by the suppliers; assay based on the caspase-3–speciﬁc substrate Ac-DEVD-AMC both lines were grown in MEM (Sigma) supplemented with fetal calf (Calbiochem). Cell death/viability was assessed with the Live/Dead serum (FCS 10% vol/vol; Sigma), antibiotic–antimycotic (penicillin Assay Kit (Molecular Probes) following the manufacturer's instruc- 100 U/mL, streptomycin sulfate 100 U/mL, amphotericin B as tions (18). Staining for senescence-associated b-galactosidase was

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done after 7 days of treatment as previously described (18). Images Single-cell mass cytometry analysis were acquired with a Leica DC 300F inverted microscope at 40 Multitarget analysis of relevant proteins and phosphoproteins magnification. involved in intracellular signaling pathways was performed as previ- For cell-cycle analysis, cells were plated in 10-cm dishes and treated ously described by means of single-cell mass cytometry (CyTOF; as indicated for 2 days. At the endpoint, cells were fixed and stained Supplementary Methods; ref. 27). with propidium iodide and acquired on LSRII flow cytometer (Beckton Dickinson). Cell cycle was evaluated by Dean-Jett-Fox using the Statistical analysis FlowJo software (FlowJo LLC; ref. 18). Summary statistics were calculated and presented as relative averages standard deviation (SD). For intergroup comparisons, if – t Cell migration not otherwise stated, Mann Whitney test or one-way ANOVA – Cell migration was evaluated with the Oris Cell Migration Assembly followed by Tukey Kramer multiple comparison tests were performed in vivo Kit (AMS Biotechnology) after 3 days of treatment. Pictures were using GraphPad Prism 7 or SPSS. To compare growth curves, captured at baseline and at experimental endpoint (i.e., confluent two-way ANOVA followed by Bonferroni multiple comparisons was – controls) using a Leica DC 300F microscope. The invaded area in each used. Univariate survival analysis was plotted according to the Kaplan treatment condition was determined with the ImageJ software (imagej. Meier method and compared with the log-rank test. Multivariable nih.gov/ij/). survival analysis was performed using Cox proportional hazards survival regression. All P values were two-sided. An alpha level <0.05 denoted statistical significance. In vivo tumor growth delay xenograft models and phospho- histone H3 IHC The antitumor efficacy of tepotinib (MSC2156119J) in combi- Results nation with irradiation was investigated in mouse xenograft models Prognostic impact of MET expression and mutational status in fi using immunode cient mice. All mice were housed in a pathogen- patients with locoregionally advanced HNSCC treated with free barrier room in the animal care facility at the Merck Health- definitive radiotherapy Care KGaA and handled using aseptic procedures. All procedures High MET expression in HNSCC has been reported at rates wereapprovedbythelocalauthority.CD-1femalenudemice ranging between 55% and 85% of cases, with inconsistent corre- (Charles River Laboratories) or HSD-athymic female nude mice lation between MET expression and both several clinicopathologic (Envigo) were injected subcutaneously at the lower back with the characteristics and patients' outcome (15). Patterns of differential human head and neck cancer cell lines FaDu and Detroit-562 MET expression in PTs and LNMs have not been previously (5 106 cells in 100 mL PBS, 1:1 matrigel). As soon as the tumors explored in HNSCC. Here we assessed MET expression in 798 reached the linear growth phase (FaDu tumors: 90–225 mm3, tissues (250 PTs, 317 LNMs, and 231 normal tissues). MET Detroit 562 tumors: 100–200 mm3), tumor-bearing mice was highly expressed in 62.4% of PTs, 53.63% of LNMs, and only (10 mice/group) were irradiated with an X-RAD 320 biological 3.39% normal tissue samples (Supplementary Fig. S2A and S2B). irradiator (2 Gy, 5 days on/2 days off) followed by daily admin- Expression of MET was significantly higher in PTs than LNMs istration of tepotinib (125 mg/kg, per os) or vehicle (20% (P ¼ 0.040), and in both PTs and LNMs significantly higher than in kolliphor/80% 100 mmol/L sodium-acetate buffer, pH 5.5). Body normal tissues (P < 0.001). We next evaluated MET expression weight and tumor size (length [L] and width [W]) were measured with clinicopathologic features and outcome in a cohort of 89 twice weekly. The tumor volume was calculated using the formula patients with HNSCC for whom matched sets of PTs–LNMs– L W2/2. At the end of the treatment period, animals were normal tissues and complete clinical data were available (Supple- euthanized, and samples of the tumor tissue were collected and mentary Table S1). Median follow-up after therapy was formalin-fixed. Histone H3 phosphorylation was analyzed in FFPE 31.03 months (range, 3.23–165.30), and patients' median age was tumor tissue as described in Supplementary Methods. 60.02 years (range, 20.07–88.08). High MET expression was detected only in PTs or only in LNMs in 25.84% of the cases, Preparation and culture of OTCs and manual TMA construction low in both in 23.60% and high in both in 50.56% (Fig. 1A and B). We generated 300-mm OTCs with a Vibratome VT1200 (Leica MET expression levels were not significantly different across the Microsystems) from freshly resected fragments of HNSCCs as various HNSCC anatomic subsites (Supplementary Fig. S2C) and previously described (Supplementary Methods; ref. 26). A total of did not correlate with any specific clinical or pathologic character- 34 fresh patient samples were initially collected but 8 had to be istics (Supplementary Table S1). Regarding outcome, high MET excluded due to lack of tumor tissue, significant morphologic expression in LNMs and both primaries and LNMs was signifi- alterations upon culture, or loss of Ki67-positive nuclei (>10%) in cantly associated with decreased regional recurrence-free survival vehicle-treated OTCs. Culture contamination occurred in 2 con- (P ¼ 0.026 and P ¼ 0.024, respectively; Fig. 1C and D). No secutive cases within the first 48 hours of treatment. Altogether, independent risk factors were identified on the various multivar- OTCs from 24 patients were included in the final TMAs (Supple- iable analyses performed. mentary Fig. S1A), comprising 2 to 6 punches (median 4) per Previous studies in HNSCC and other tumor entities showed that uncultured tumor and 2 to 5 punches (median 2) for OTCs MET mutations tend to cluster in the juxtamembrane and extracellular (Supplementary Fig. S1B). Methodological validation was per- domains (13, 28). In our cohort of 223 patients with locoregionally formed using 10 tissues to evaluate morphologic changes and advanced HNSCC, we identified 5 different mutations in 10 patients variations of MET and Ki67 expression during culture (Supple- (prevalence 4.48%), located in the extracellular domain of MET mentary Fig. S1C–S1F). TMAs were manually constructed as (E168D, n ¼ 1 patient; N375S, n ¼ 4), the juxtamembrane domain described in Supplementary Methods. (R998C, n ¼ 1; T1010I, n ¼ 3), and the tyrosine kinase domain

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Figure 1. Patterns of MET expression and impact on survival in patients with locoregionally advanced HNSCC treated with deﬁnitive radiotherapy. A, Patterns of MET receptor expression in coupled HNSCC primaries and LNMs. B, Frequency of differential expression patterns. Low MET expression in primaries and matched LN metastasis was seen in 23.6% of the cases, high expression only in primaries or LN metastasis in 12.36% and 13.48%, respectively, and in both primaries and LN metastasis in 50.56%. Kaplan–Meier estimates illustrating regional recurrence-free survival according to MET expression in LNs (C) and primaries and LNs (D).

(M1178I; n ¼ 1; Supplementary Fig. S2D). When present, mutations of 5 cell lines, whereas the remaining 2 cell lines had detectable but low were detected in both primaries and LNMs, without significant p-MET levels (Fig. 2A). MET phosphorylation could be effectively differences in terms of mutated allele's abundance or clinicopathologic abrogated by using MET inhibitors (Fig. 2A) and, even though effects features (Supplementary Fig. S2E–S2G). on both ERK and AKT phosphorylation were usually modest, tepotinib (50 nmol/L) tended to be more effective than PHA665752 Expression of MET and effects of MET inhibition in HNSCC cell (300 nmol/L; Fig. 2A). Consequently, for subsequent assays we used lines the more novel MET inhibitor tepotinib, currently under clinical The biological effects of MET targeting were evaluated with two development in phase I and II trials (22). Although EC50 values for distinct specific MET inhibitors (PHA665752 and tepotinib) in an tepotinib were generally on the low micromolar range (Supplementary initial panel of 5 HNSCC cell lines. Total MET as well as p-MET could Fig. S3A–S3E), tepotinib at a concentration of 100 nmol/L was be detected in all cell lines (Fig. 2A). High levels of the activated MET sufficient to significantly impair plating efficiency in all 3 HNSCC receptor as inferred from its phosphorylation status were observed in 3 cell lines with higher p-MET levels (Supplementary Fig. S3F).

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Figure 2. Expression of MET and effect of MET inhibition in HNSCC cell lines. A, The effect of MET inhibitors PHA665752 (300 nmol/L) and tepotinib (50 nmol/L) was tested by immunoblotting total MET, active p-MET, and phosphorylation of downstream signaling molecules ERK and AKT after 16 hours of treatment. B, Effect of MET inhibitor tepotinib (50 nmol/L) in patient-paired cell lines UM-SCC-10A, UM-SCC- 10B, UM-SCC-81A, and UM-SCC-81B. Densitometric values of p-MAPK and p-AKT were normalized using b-actin.

In addition, expression of MET and p-MET as well as effects of whether treatment with tepotinib prior to or following IR had an tepotinib on MET downstream signaling was assessed in two pairs impact on colony formation in UM-SCC-81A and UM-SCC-81B cells. of patient-matched UM-SCC cell lines: UM-SCC-10A (primary The two treatment schedules had no significant impact on results laryngeal tumor) and UM-SCC-10B (lymph node metastasis), and (Supplementary Fig. S4A and S4B), and further assays were conducted UM-SCC-81A (primary laryngeal) and UM-SCC-81B (second oro- by pretreating the samples. pharyngeal PTs after radiotherapy; Fig. 2B). All 4 cell lines Pretreatment with tepotinib (50 nmol/L) significantly sensitized 8 of expressed considerable p-MET levels that were decreased upon the 9 cell lines evaluated in this study to IR when assessing their MET inhibition by tepotinib, however with moderate impact on survival, however with very variable degrees of radiosensitization p-AKT and p-ERK activation (Fig. 2B). (significant RERs 1.08–1.23). Indeed, Detroit-562, UM-SCC-81A, and MET's ligand SF/HGF is a well-established motogenic molecule that UM-SCC-81B cell lines displayed RERs that were significant but very has been shown to protect neoplastic cells from cytotoxic agents while close to 1, denoting poor radiosensitization with 50 nmol/L of tepo- promoting invasive features such as cell migration (7, 29). As men- tinib. In contrast, combination of IR with tepotinib 100 nmol/L tioned, SF/HGF-MET paracrine loops in a background of receptor/ resulted in higher RERs, except in FaDu and UM-SCC-10B (the latter ligand overexpression are a common pathogenic feature in consistently did not reach a significant RER when comparing tepotinib HNSCC (12). We investigated the effect of MET inhibition on 50 vs. 100 nmol/L). An increase in tepotinib concentration from 50 to migration in cell lines with high basal p-MET levels (FaDu and 100 nmol/L further enhanced the radiosensitizing effect of MET Detroit-562) and in cells with low basal p-MET levels stimulated with inhibition only in 4 of 9 cell lines in a dose-dependent manner, this SF/HGF (SCC-61 and HN5). Tepotinib impaired migration in a dose- effect not being marked in UM-SCC-10A with an RER of 1.037 dependent manner in FaDu and Detroit-562 (Supplementary Fig. S3G (Fig. 3A; Supplementary Fig. S4C). and S3H). Moreover, as expected, SF/HGF significantly stimulated cell Based on the potential radiosensitization in vitro, we evaluated migration both without and with IR, an effect that could be fully tepotinib along with a clinically relevant fractionated IR dose of 2 Gy in abrogated by MET inhibition (Supplementary Fig. S3I). subcutaneous xenografts. FaDu and Detroit-562 were used for in vivo experiments. Animals were irradiated daily 5 days a week with a 2-day Tepotinib results in differential degrees of radiosensitization in hiatus. Tepotinib was administered daily. Tepotinib alone did HNSCC in vitro, in vivo, and in patient-derived OTCs not display a significant effect on FaDu cell lines but it did in MET signaling potentially confers resistance to IR as well as other Detroit-562 (P < 0.05, Fig. 3B and C). Concerning IR, both xenograft cytotoxic therapeutic modalities in several cancer models (7–10). Here, models showed significant responses to this modality alone (P < 0.001). we sought to evaluate the ability of MET inhibition to radiosensitize Nevertheless, although IR resulted in tumor regression in Detroit-562 HNSCC cells by conducting clonogenic assays. As a first step, we tested xenografts, it led to baseline tumor sizes in the FaDu model. Given the

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Figure 3. Radiosensitization potential of tepotinib in vitro and in vivo. A, Radiation survival curves. HNSCC cell lines were treated with increasing concentrations of tepotinib (50 nmol/L and 100 nmol/L) for 16 hours prior to IR and surviving fraction normalized to plating efﬁciency and corrected to drug toxicity has been determined. B, Tumor growth delay curves of in vivo models generated with FaDu and Detroit-562 cell lines. C, Tumor sizes in each treatment condition at the endpoint. D, IHC staining of phospho-histone H3 as a surrogate of proliferation. P values: , <0.05; , <0.001.

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strong response of Detroit-562 to IR alone, combination with Supplementary Methods. The basal expression levels of all tested tepotinib did not have an additional effect in these xenografts proteins differed in both cell lines,especiallyEGFR,EphA2,p-S6, (Fig. 3B and C). In contrast, combination of IR and tepotinib in and p-p53. Importantly, treatments had basically no effect on FaDu xenografts led to significantly higher levels of tumor control HN5, but were able to modulate several proteins in UM-SCC- (P < 0.001, Fig. 3B and C). Phospho-histone H3 was used as 10B. Most relevant perhaps was the downregulation of EphA2 and surrogate for proliferation, equally confirming a more marked p-S6 upon MET inhibition (whether alone or in combination with resistance to IR in FaDu models, an effect that could be countered IR; Supplementary Fig. S6B and S6C). Next, we combined the by combination with tepotinib (Fig. 3D). results of the live/dead assays presented in Fig. 5B (combination Next, we evaluated the radiosensitization potential of MET vs. IR alone) with the CyTOF data and used logistic regression to targeting in translationally relevant patient-derived OTCs, a model model the cell death probability. We finally computed the death that allows preserving tumoral histoarchitecture ex vivo (26). For odds ratio (OR) between the 10th and 90th percentiles of the this purpose, after an incubation timeof6to12hoursinculture transformed protein expression values. Figure 5C shows the pro- medium, OTCs were treated with tepotinib 1 mmol/L (drug con- teins from the CyTOF panel with the largest OR values in the centration was increased as compared with in vitro experiments in tepotinib-radiosensitized cell line UM-SCC-10B (a full list of ORs order to ensure appropriate tissue penetration) or DMSO for for each protein is provided in Supplementary Table S2). In this 16 hours and subsequently irradiated (6 Gy). OTCs were cultured model, an OR above 1 indicates that increased expression of the for a total time of 72 hours since the beginning of treatment. protein is associated (although not necessarily in a causal way) with Evaluation of proliferation patterns upon different therapeutic cell death, whereas an OR below 1 implies the opposite. Our model modalities revealed substantial heterogeneity in responses (Supple- points out that expression of the RTK EphA2 and the PI3K mentary Fig. S5A). Ki67 expression was significantly reduced upon downstream effector p-S6 seem to be associated with cell survival IR and combination of MET inhibition and IR when compared with upon IR. As expected, expression of activated p-p53 and Ki67 was vehicle (DMSO) or tepotinib alone, but combination of the two associated with cell death. The analysis results for the 19 proteins perturbations was not globally superior to IR as single therapy. included in the CyTOF panel are shown in Supplementary Fig. S6D. Importantly, however, when considering individual OTCs and The relevance of EGFR expression being associated with cell death clustering those with similar responses to IR/combination, four is difficult to interpret from a biological perspective. However, basic patterns emerged (Fig. 4A and B): (i) OTCs sensitive to IR not because EGFR expression can be a mechanism of compensation further sensitized by concomitant MET inhibition (6/24); (ii) OTCs upon MET inhibition (31), it could be hypothesized that upon MET sensitive to IR further sensitized by MET inhibition (4/24); (iii) inhibition and/or IR, EGFR expression occurs in a cell about to OTCs not significantly responsive to either IR or combination (8/ engage in cell death. In such a scenario, our model would interpret 24); and (iv) OTCs initially resistant to IR sensitized by concomitant EGFR expression not as a prosurvival mechanism, but as an event MET inhibition (6/24). High MET expression was observed in associated with cell death. tissues both responsive and resistant to MET inhibition, but pattern To further explore the effects of tepotinib in HNSCC cell lines, cell- 4 contained only MET-high tissues (Fig. 4C). These results suggest cycle analysis was carried out in cells treated with either tepotinib (100 – that increased MET expression, while necessary in order to achieve nmol/L), IR (6 Gy), or their combination for 48 hours. IR led to G2 M radiosensitization, is not sufficient for this effect. It is important to accumulation in both cell lines, whereas combination of tepotinib and – note that p-MET expression could not be detected (discussed IR further increased the G2 M fraction only in UM-SCC-10B cells below). Finally, tepotinib alone at a concentration of 1 mmol/L did (Fig. 6A). As IR-induced prolonged G2-arrest has been shown to fi – – notresultinsigni cant decrease of Ki67 expression in any of the potentially induce G2 M checkpoint regulated senescence (32) and patterns (Supplementary Fig. S5B). we previously demonstrated that MET targeting enhances IR-induced senescence in gastric models featuring MET oncogene addiction (18), Tepotinib enhances IR-induced cell death, DNA damage, and we retrospectively reviewed the morphology of irradiated but living senescence in HNSCC cell lines UM-SCC-10B cells in live–dead assays, noticing a flattened and Radionsensitization following EGFR targeting in HNSCC swollen shape suggestive of senescence. Based on these observations, has previously been shown to be the consequence of enhanced we performed senescence-associated b-galactosidase (SA-b-gal) stain- apoptosis and DNA damage (30). To assess whether MET inhibi- ings in UM-SCC-10B and could show that in addition to induction of tion enhanced IR-induced cell death in HNSCC models, cells were cell death, IR significantly increased the percentage of SA-b-gal– exposed to tepotinib (100 nmol/L) or DMSO for 6 hours prior to IR positive cells, which was further increased upon addition of MET (6 Gy), and caspase-3 enzymatic activity was determined 3 days inhibition (Fig. 6B). after treatment. As expected, IR significantly induced caspase-3 Finally, we assessed whether tepotinib enhanced IR-induced DNA activation in all 9 evaluated cell lines (Fig. 5A). Combination of damage, thus leading to the observed radiosensitization and increase in tepotinib and IR was significantly superior to IR alone in inducing cell death and cellular senescence. Phosphorylated levels of the histone caspase-3 activity in 6 of 9 cell lines (all of which have high basal variant H2AX at Ser139 (gH2AX) were used as surrogate marker of levels of p-MET). The findings from caspase-3 assays were further DNA double-strand breaks (33). UM-SCC-10B and HN5 cells were confirmed with live/dead assays (Fig. 5B; Supplementary Fig. S6A). lysed at 2 and 8 hours after IR. Pretreatment with tepotinib resulted in To elucidate potential molecules and pathways involved in radio- increased levels of gH2AX 2 hours after IR in both cell lines, but sensitization, we selected UM-SCC-10B as a model of MET inhi- whereas gH2AX levels in HN5 cells considerably decreased 6 hours bition–radiosensitized cell line and HN5 (nonsensitized by MET later, tepotinib (100 nmol/L) pretreatment in UM-SCC-10B cells led to inhibition). Cells were treated with either vehicle or tepotinib (100 slightly more sustained H2AX phosphorylation after 8 hours, indi- nmol/L) 16 hours prior to IR. To capture early (though not cating slower resolution of DNA damage (Fig. 6C). Similar findings immediate) events, cells were lysed 6 hours following IR and were observed in UM-SCC-81A and UM-SCC-81B (Supplementary subjected to CyTOF as described in Materials and Methods and Fig. S6E).

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Figure 4. Patterns of response to MET inhibition and IR in OTCs. A, Representative images of tumors of each pattern, displaying MET expression in baseline tumors. B, Quantiﬁcation of variations in percentage of Ki67-positive tumors in each pattern. P values: , <0.05; , <0.01; , <0.001 (one-sample t tests were used to determine whether values were signiﬁcantly different to 0). C, Frequency of MET-low/MET-high tumors in each pattern of response (P value according to Fisher exact test).

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Figure 5. Tepotinib enhances IR-induced cell death in MET-overexpressing cell lines. A, Cells were treated with tepotinib (100 nmol/L) for 16 hours prior to IR (6 Gy). Caspase-3 enzymatic activity was determined 72 hours after irradiation. B, Representative images (10 magniﬁcation) of live–dead assays. Viable (calcium AM-positive) cells appear in green, whereas dead (ethidium homodimer-1 (EthD-1)–positive) appear in red. The percentage of dead cells was determined in each treatment condition (P values: , <0.05; , <0.01; , <0.001). C, Results of logistic regression analysis of CyTOF data displaying the 4 proteins with the largest cell-death OR values (an OR value higher than 1 indicates that the protein's increased level is associated with a higher probability of cell death).

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MET-Mediated Radiosensitization in Head and Neck Cancer

Figure 6. Mechanisms of radiosensitization upon MET targeting in vitro. A, Representative flow cytometry profiles of UM-SCC-10B and HN5 cells treated with tepotinib (100 nmol/L) or DMSO 16 hours prior to IR (6 Gy). UM-SCC-10B but not HN5 showed increased G2–M arrest upon combination of tepotinib and IR. B, Representative images (10 magnification) of SA-b-gal stainings in UM-SCC-10B cells, as relative average SD of 3 independent experiments (P values: , <0.05; , <0.01; , <0.001; calculated with one-way ANOVA). C, Immunoblot evaluation of gH2AX as a surrogate marker of DNA double-strand breaks. UM-SCC-10B and HN5 cells were exposed to tepotinib (100 nmol/L) or DMSO for 16 hours and then irradiated (6 Gy). Cells were lysed after 2 and 8 hours.

Discussion that presence of LNMs is per se one of the most significant In this study, we evaluate the clinical impact of MET expression in prognostic factors in HNSCC and that all patients in our cohort PTs and LNMs as well as the potential radiosensitizing effect of MET had LNMs, it is likely that the multivariable survival analysis did not inhibition in preclinical HNSCC models. reflect classic findings in cohorts of patients encompassing both The MET RTK and its ligand SF/HGF are commonly over- early- and late-stage HNSCC (38). Consequently, our results are to expressed in HNSCC, and their high expression is in turn correlated be interpreted within the frame of aggressive locoregionally with poor prognosis (13, 34–37). In terms of potential use of advanced disease, the presentation most likely to require multi- MET expression as a biomarker in HNSCC, our findings demon- modal therapeutic approaches. strate that MET expression in PTs and LNMs is associated with We also report the findings of a MET mutational screening decreased rates of regional disease control in univariable survival approach in a large cohort of primary HNSCCs and LNMs analyses. Classic prognostic factors were not significant indepen- (patient-matched in 79.37% of the cases). In line with previous studies, dent risk factors in our multivariable disease control analysis. These MET mutations were found in almost 5% of patients, clustering results are most likely due to the specific composition of our study predominantly in the extracellular and juxtamembrane receptor cohort, in which only patients with locoregionally advanced domains. The presence of mutations did not correlate with specific HNSCC treated with neck dissection and RT were included. Given clinicopathologic features or survival (13, 39–41). Moreover,

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abundance of mutated alleles in PTs did not significantly differ from signaling to sustain growth, proliferation, and other oncogenic fea- that in LNMs. tures (46). Some of these alterations confer extreme sensitivity to MET In this study, we provide the first evaluation of the radiosensitization inhibitors. In models featuring such exquisite sensitivity to potential of tepotinib, a novel and highly selective MET inhibitor, in MET inhibitors (referred to as oncogene addiction), targeting MET HNSCC (22). So far, the role of MET targeting as a strategy for results in effective radiosensitization (8–10, 18). Consequently, the radiosensitization in HNSCC has not been comprehensively investi- presence of such lesions could be used as a pretherapeutic strati- gated. To the best of our knowledge, only a study by Baschnagel and fication marker. In HNSCC, however, preclinical findings by us and colleagues (16) using the multikinase inhibitor crizotinib, which others do not support the notion of MET oncogene addic- reported negative findings in vivo, has addressed this issue. Discor- tion (13, 24). In order to identify pathways potentially contributing dance between such negative results and our present findings is most to MET inhibition–mediated radiosensitization, we explored the likely due to differences in experimental design, models used (and activation status of several signaling pathways in cell lines by means specifically evaluation of OTCs in our study), treatments and potential of CyTOF. This approach demonstrated that MET inhibition could use of crizotinib instead of a selective MET inhibitor. To the best of our modulate PI3K activity in the tepotinib-radiosensitized cell line knowledge, we provide the first evidence of MET inhibition-based UM-SCC-10B, but not in the resistant HN5 cells. The seeming radiosensitization in an in vivo HNSCC. relevance of PI3K signaling in mediating responses to MET target- We found that tepotinib effectively blocked MET phosphorylation ing in HNSCC stands in contrast with previous findings in gastric and reduced colony-forming ability in HNSCC cell lines. In combi- cancer models, suggesting the MAPK pathway as the link between nation with IR, tepotinib led to differential degrees of radiosensitiza- MET inhibition and radiosensitization (18). This observation indi- – tion by enhancing IR-induced cell death, G2 M cell-cycle arrest, cates that different tumor entities and even different tumor types senescence, and impairing repair of DNA double-strand in sensitive within an anatomic location may selectively use and rely upon cell lines. An interesting observation was that, although tepotinib alone different signaling nodes to regulate the cellular stress response did not have any significant impact on cell proliferation at submicro- following IR. Consequently, determining activation status or molar concentrations, concentrations of 50 to 100 nmol/L were expression abundance of selected markers in each tumor entity sufficient to result in radiosensitization. It can be argued that exper- could be beneficial for pretherapeutic stratification. imental settings and evaluated endpoints partly account for such Finally, our results both in vivo and in OTCs indicate that MET observation, as metabolism-based cell proliferation assays evaluate expression seems to be necessary but not sufficient to predict radio- short-term effects, whereas clonogenic assays focus on long-term sensitization. A limitation that needs to be acknowledged was the fact outcome at the cellular level. Nevertheless, our results underline that that p-MET expression could not at all be detected in archival FFPEs or MET signaling plays an important role in the stress-and-response OTCs, most likely due to a suboptimal tissue fixation protocol in spite process following IR (42). Indeed, in baseline conditions, HNSCCs do of using a robust and thoroughly standardized staining used for not seem to strongly rely on MET signaling. This is probably the result routine histopathologic diagnosis (47). Consequently, we are not able of the well-known RTK-mediated cooperation and redundancy phe- to elaborate on any associations between OTCs' responses and MET nomenon in HNSCC (15, 31, 43). In the presence of IR-induced activation status. genotoxic stress, however, prosurvival cell signaling circuitry under- goes significant rewiring. In such conditions, impairment of MET Disclosure of Potential Conflicts of Interest signaling may represent a major disadvantage at least in a subset of O. Elicin is a consultant at the advisory board meeting of AstraZeneca and Serono. HNSCCs, ultimately resulting in radiosensitization (31, 42–44). Sig- M. Friese-Hamim is an Associate Director In Vivo Pharmacology Oncology for nificantly, our CyTOF analysis pointed out the EphA2 RTK as a new Merck Healthcare KGaA. C. Wilm is principal scientist at Merck Healthcare KGaA. C. Stroh is Director Translational and Biomarker Research at Merck KGaA. No potential potential compensatory mechanism upon MET inhibition. conflicts of interest were disclosed by the other authors. The heterogeneity in responses to targeted therapy is most relevantinclinicaltermsasitisestimatedthatthevastmajority Authors’ Contributions of new anticancer drugs fail in clinical trials, an observation partly Conception and design: L. Nisa, D.M. Aebersold, Y. Zimmer, M. Medova attributed to the preclinical models used (45). Indeed, cell lines and Development of methodology: L. Nisa, R. Giger, O. Elicin, C. Wilm, M. Buchwalder, their derived murine xenografts, whereas of immense discovery and D.M. Aebersold, Y. Zimmer mechanistic value, fail to recapitulate the complexity of human Acquisition of data (provided animals, acquired and managed patients, provided neoplasms and their heterogeneity among other features (26). We facilities, etc.): L. Nisa, P. Francica, R. Giger, O. Elicin, M. Friese-Hamim, C. Wilm, opted to further explore the findings in cell lines and xenografts C. Stroh, B. Bojaxhiu, M.D. Caversaccio, M.S. Dettmer, M. Buchwalder, T.M. Brodie using OTCs from patients with HNSCC. OTCs allow preservation Analysis and interpretation of data (e.g., statistical analysis, biostatistics, in situ ex vivo computational analysis): L. Nisa, P. Francica, M. Medo, M. Friese-Hamim, of tumor complexity in conditions. Even though the C. Stroh, Y. Zimmer, M. Medova ability of OTCs to predict actual clinical responses remains to be Writing, review, and/or revision of the manuscript: L. Nisa, R. Giger, O. Elicin, demonstrated, and in spite of the fact that OTCs allow only short- M. Friese-Hamim, C. Stroh, M.D. Caversaccio, M.S. Dettmer, M. Buchwalder, term readouts, our current findings suggest the potential benefitof D.M. Aebersold, Y. Zimmer, T.E. Carey, M. Medova combined MET inhibition with IR seen in cell lines in vitro and Administrative, technical, or material support (i.e., reporting or organizing data, in vivo and in particular subsets of HNSCC patients. Moreover, the constructing databases): L. Nisa, R. Giger, B. Bojaxhiu, A. Quintin, M.D. Caversaccio, M.S. Dettmer, M. Medova ensemble of our preclinical observations strongly emphasizes the Study supervision: L. Nisa, Y. Zimmer, M. Medova importance of pretherapeutic stratification. Other (provided cell line models and read and approved manuscript): T.E. Carey In this respect, the genomic background has been shown to determine to a great extent responses to RTK targeting in several tumor Acknowledgments fi fi entities. Indeed, gene ampli cation, activating mutations, and speci c We cordially thank Dr. Vinko Tosevski for assistance with establishment of mass lesions such as MET exon 14 skipping, as well as RNA overexpression, cytometry measurements and Prof. Manfred Claassen for helpful advises on single- have all been shown to render malignant cells dependent on MET cell data analysis. We are gratefully indebted to Mr. Bruno Streit and Dr. Wieslawa

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MET-Mediated Radiosensitization in Head and Neck Cancer

Blank-Liss for their outstanding technical help. The assistance of Mrs. Irina Ciorba- The costs of publication of this article were defrayed in part by the payment of page Nisa, MSc, in production of this manuscript, is most gratefully acknowledged. This charges. This article must therefore be hereby marked advertisement in accordance work was supported by the Research Grant of the Inselspital for Young Clinicians (to with 18 U.S.C. Section 1734 solely to indicate this fact. L. Nisa), by a Bernese Cancer League grant and by the Stiftung zur Krebsbek€ampfung (both to M. Medova). These funding sources had no role in study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the Received November 13, 2018; revised August 19, 2019; accepted November 14, decision to submit the article for publication. 2019; published ﬁrst November 19, 2019.

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Targeting the MET Receptor Tyrosine Kinase as a Strategy for Radiosensitization in Locoregionally Advanced Head and Neck Squamous Cell Carcinoma

Lluís Nisa, Paola Francica, Roland Giger, et al.

Mol Cancer Ther 2020;19:614-626. Published OnlineFirst November 19, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-18-1274

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