© 2021. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2021) 14, dmm047779. doi:10.1242/dmm.047779 RESEARCH ARTICLE RET inhibition in novel patient-derived models of RET fusion- positive lung adenocarcinoma reveals a role for MYC upregulation Takuo Hayashi1,2,*,§§, Igor Odintsov1,2,§§, Roger S. Smith1,2,‡,§§, Kota Ishizawa2,§, Allan J. W. Liu3,4, Lukas Delasos3, Christopher Kurzatkowski1, Huichun Tai1,2, Eric Gladstone1,2, Morana Vojnic1,2, Shinji Kohsaka1,2,¶, Ken Suzawa1,**, Zebing Liu1,2,‡‡, Siddharth Kunte3, Marissa S. Mattar5, Inna Khodos6, Monika A. Davare6, Alexander Drilon3, Emily Cheng2, Elisa de Stanchina5, Marc Ladanyi1,2,¶¶,*** and Romel Somwar1,2,¶¶,*** ABSTRACT suppressed by treatment of cell lines with cabozantinib. MYC protein Multi-kinase RET inhibitors, such as cabozantinib and RXDX-105, are levels were rapidly depleted following cabozantinib treatment. Taken active in lung cancer patients with RET fusions; however, the overall together, our results demonstrate that cabozantinib is an effective response rates to these two drugs are unsatisfactory compared to other agent in preclinical models harboring RET rearrangements with three ′ targeted therapy paradigms. Moreover, these inhibitors may have different 5 fusion partners (CCDC6, KIF5B and TRIM33). Notably, we different efficacies against RET rearrangements depending on the identify MYC as a protein that is upregulated by RET expression and upstream fusion partner. A comprehensive preclinical analysis of downregulated by treatment with cabozantinib, opening up potentially the efficacy of RET inhibitors is lacking due to a paucity of disease new therapeutic avenues for the combinatorial targetin of RET fusion- models harboring RET rearrangements. Here, we generated two new driven lung cancers. The novel RET fusion-dependent preclinical patient-derived xenograft (PDX) models, one new patient-derived cell models described here represent valuable tools for further refinement line, one PDX-derived cell line, and several isogenic cell lines with RET of current therapies and the evaluation of novel therapeutic strategies. fusions. Using these models, we re-examined the efficacy and KEY WORDS: RET fusion PDX, MYC, RET inhibitor, Transcriptome mechanism of action of cabozantinib and found that this RET profiling, NSCLC inhibitor was effective at blocking growth of cell lines, activating caspase 3/7 and inhibiting activation of ERK and AKT. Cabozantinib INTRODUCTION treatment of mice bearing RET fusion-positive cell line xenografts and RET) two PDXs significantly reduced tumor proliferation without adverse The rearranged during transfection ( gene encodes a proto- toxicity. Moreover, cabozantinib was effective at reducing growth of a oncogene and was identified in 1985 (Takahashi et al., 1985). The lung cancer PDX that was not responsive to RXDX-105. gene maps to chromosome 10q11.2 and encodes a single-pass Transcriptomic analysis of lung tumors and cell lines with RET transmembrane receptor tyrosine kinase consisting of three alterations showed activation of a MYC signature and this was domains: an extracellular domain, a transmembrane domain and a tyrosine kinase domain. The primary RET ligands belong to the glial-derived neurotrophic factor (GDNF) family, which activates 1Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. 2Human Oncology and Pathogenesis Program, Memorial Sloan RET by first binding to their cognate receptor (GDNF receptor Kettering Cancer Center, New York, NY 10065, USA. 3Thoracic Oncology Service, alpha) and then the GDNF-GRFα complex dimerizes with RET, Division of Solid Tumor Oncology, Department of Medicine, Memorial Sloan leading to its autophosphorylation and activation. The phosphorylated Kettering Cancer Center, New York, NY 10065, USA. 4Faculty of Medicine, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China. 5Antitumor (phospho)-tyrosine residues on RET serve as docking sites for the Core Facility, Molecular Pharmacology Program, Memorial Sloan Kettering Cancer SH2 domains of several signaling molecules that activate downstream 6 Center, New York, NY 10065, USA. Department of Pediatrics, Oregon Health pathways (e.g. RAS/MAPK, PI3K/AKT) associated with cellular Sciences University, Portland, OR 97239, USA. *Present address: Department of Human Pathology, Graduate School of Medicine, proliferation, migration, and differentiation (Arighi et al., 2005; Phay Juntendo University, Bunkyo-ku, Tokyo, Japan. ‡Present address: Medical Scientist and Shah, 2010). Training Program, Northwestern University Feinberg School of Medicine, Chicago, IL, USA. §Present address: Department of Education and Support for Community Rearrangement of the RET protein through inter- or intra- Medicine, Tohoku Medical Megabank Organization, Tohoku University, Sendai, chromosomal translocation, in which the kinase domain of RET is Japan. ¶Present address: Division of Cellular Signaling, National Cancer Center fused with another gene, results in a fusion oncogene Research Institute, Tokyo, Japan. **Present address: Department of Thoracic, RET Breast and Endocrinological Surgery, Okayama University Graduate School of (Stransky et al., 2014). Fusion of the kinase domain of to the Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan. ‡‡Present 5′ untranslated region (5′-UTR) of CCDC6 was initially discovered address: Department of Molecular Pathology, Renji Hospital, Medical School of in papillary thyroid carcinoma (Fusco et al., 1987). Subsequently, Shanghai Jiao Tong University, Shanghai, China. §§These authors contributed equally to this work fusion of RET with various other upstream partners were discovered ¶¶Co-senior authors in lung (Li et al., 2017; Lipson et al., 2012; Suehara et al., 2012; ***Authors for correspondence ([email protected]; [email protected]) Takeuchi et al., 2012) and other cancers (Le Rolle et al., 2015; Paratala et al., 2018). In non-small cell lung carcinoma (NSCLC), M.L., 0000-0001-9055-7213 RET fusion with 5′-UTRs have been described for at least twelve KIF5B CCDC6 NCOA4 MYO5C EPFA5 This is an Open Access article distributed under the terms of the Creative Commons Attribution partner genes, i.e. , , , , , License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, TRIM33, CLIP1, ERC1, PICALM, FRMD4A, RUFY2 and TRIM24 distribution and reproduction in any medium provided that the original work is properly attributed. (Ferrara et al., 2018). The N-terminal partner of RET in all fusion Handling Editor: Elaine R. Mardis variants described to date contributes to a protein-protein interaction Received 13 October 2020; Accepted 8 December 2020 (i.e. coiled-coil) domain that aids dimerization of the fusion oncoprotein, Disease Models & Mechanisms 1 RESEARCH ARTICLE Disease Models & Mechanisms (2021) 14, dmm047779. doi:10.1242/dmm.047779 allowing for constitutive RET activation by autophosphorylation RESULTS (Ju et al., 2012). The tumorigenic potential of RET fusion proteins Generation and characterization of novel RET-rearranged has been demonstrated in vitro and in vivo (Li et al., 2017; Takeuchi cell lines et al., 2012). In addition to gene rearrangements, RET can also be To perform preclinical investigation of RET inhibitors, we compiled activated by mutations within the kinase domain in medullary a collection of RET-driven cell lines. This collection included the thyroid carcinoma (Jhiang, 2000). previously reported LC-2/ad cell line that harbors a CCDC6-RET Oncogenic RET is an actionable therapeutic target that is found in fusion (Li et al., 2017) (xxxxx) as well as two novel patient-derived 1-2% of NSCLCs and 9% of thyroid cancers (Elisei et al., 2013; cell lines generated by us, i.e. ECLC5B harboring TRIM33-RET and Zehir et al., 2017). In NSCLCs, several small-molecule RET PDX-derived lung adenocarcinoma cells harboring KIF5B-RET inhibitors, such as the multi-kinase inhibitors (MKIs) cabozantinib (LUAD-0002AS1). The patient demographic details and genomic (Drilon et al., 2016), sunitinib (Gautschi et al., 2017), lenvatinib profiling of the novel models by the MSK-IMPACT platform is (Gautschi et al., 2017), vandetanib (Yoh et al., 2017), RXDX-105 given in Table S1 and Table S2, respectively. Additionally, we have (Drilon et al., 2019a), or selective inhibitors, such as selpercatinib established isogenic cell lines by overexpressing the CCDC6-RET (LOXO-292) (Drilon et al., 2019b, 2020) and pralsetinib (BLU- fusion gene in 3T3 murine fibroblasts (NIH 3T3) and immortalized 667) (Gainor et al., 2019), have been tested in individual patients or human broncho-epithelial cells (HBECp) yielding 3T3-RET and clinical trials. HBECp-RET1, respectively. We first examined expression of the MKIs, including cabozantinib, RXDX-105, vandetanib and respective RET fusion mRNA by PCR using primers that bind to 5′ sunitinib, in patients with RET-rearranged NSCLC [overall partners and exon 12 of RET, respectively (Fig. 1A). Protein response rate (ORR): 18%-37%; median progression-free survival expression and phosphorylation were confirmed by western blotting (PFS): 2.3-5.5 months)] (Drilon et al., 2016; Gautschi et al., 2017)], (Fig. 1B). We used antibodies against total RET and against RET is clearly inferior compared with responses and survival outcomes fusion protein phosphorylated at tyrosine residue Y905 (p-RET seen with selective tyrosine kinase inhibitors (TKIs) and other Y905), and found multiple bands in lysates extracted from some cell