ARTICLE https://doi.org/10.1038/s41467-019-11496-z OPEN The new-generation selective ROS1/NTRK inhibitor DS-6051b overcomes crizotinib resistant ROS1- G2032R mutation in preclinical models Ryohei Katayama 1, Bo Gong1,2, Noriko Togashi 3, Masaya Miyamoto3, Masaki Kiga3, Shiho Iwasaki3, Yasuki Kamai3, Yuichi Tominaga3, Yasuyuki Takeda3, Yoshiko Kagoshima3, Yuki Shimizu1,2, Yosuke Seto1, Tomoko Oh-hara1, Sumie Koike1, Naoki Nakao4, Hiroyuki Hanzawa4, Kengo Watanabe 3, Satoshi Yoda 5,6, Noriko Yanagitani7, Aaron N. Hata5,6, Alice T. Shaw5,6, Makoto Nishio7, Naoya Fujita1,2 & Takeshi Isoyama 3 1234567890():,; ROS1 gene rearrangement was observed in around 1–2 % of NSCLC patients and in several other cancers such as cholangiocarcinoma, glioblastoma, or colorectal cancer. Crizotinib, an ALK/ROS1/MET inhibitor, is highly effective against ROS1-rearranged lung cancer and is used in clinic. However, crizotinib resistance is an emerging issue, and several resistance mechanisms, such as secondary kinase-domain mutations (e.g., ROS1-G2032R) have been identified in crizotinib-refractory patients. Here we characterize a new selective ROS1/NTRK inhibitor, DS-6051b, in preclinical models of ROS1- or NTRK-rearranged cancers. DS-6051b induces dramatic growth inhibition of both wild type and G2032R mutant ROS1–rearranged cancers or NTRK-rearranged cancers in vitro and in vivo. Here we report that DS-6051b is effective in treating ROS1- or NTRK-rearranged cancer in preclinical models, including crizotinib-resistant ROS1 positive cancer with secondary kinase domain mutations especially G2032R mutation which is highly resistant to crizotinib as well as lorlatinib and entrectinib, next generation ROS1 inhibitors. 1 Division of Experimental Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo 135-8550, Japan. 2 Department of Medical Genome Science, Graduate School of Frontier Science, The University of Tokyo, Tokyo 108-8639, Japan. 3 Daiichi Sankyo Co., Ltd, Tokyo 140-8710, Japan. 4 Daiichi Sankyo RD Novare Co., Ltd, Tokyo 134-8630, Japan. 5 Massachusetts General Hospital Cancer Center, Boston, MA 02129, USA. 6 Department of Medicine, Harvard Medical School, Boston, MA 02115, USA. 7 Department of Thoracic Medical Oncology, the Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo 135-8550, Japan. Correspondence and requests for materials should be addressed to R.K. (email: ryohei. [email protected]) or to T.I. (email: [email protected]) NATURE COMMUNICATIONS | (2019) 10:3604 | https://doi.org/10.1038/s41467-019-11496-z | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-11496-z everal genetic alterations that aberrantly activate tyrosine Results Skinases have been identified as oncogenic drivers in non- DS-6051b Inhibits ROS1 Kinase in nanomolar concentrations. small cell lung cancer (NSCLC), and many tyrosine To obtain a potent and selective ROS1 inhibitor, high-throughput kinase inhibitors (TKIs) have been developed to target these inhibitor screening against ROS1 kinase was performed, and driver oncogene products1–5. Among the driver oncogenes, several hit compounds were identified. After robust chemical chromosomal rearrangements of anaplastic lymphoma kinase derivatization and characterization, we found a lead compound (ALK), ROS proto-oncogene 1 (ROS1),rearrangedduring that had inhibitory activities against ROS1 and NTRKs, and lead transfection (RET), and neurotrophic receptor tyrosine kinases optimization finally identified DS-6051b (Fig. 1a). DS-6051b (NTRKs) are each observed in 0.1–5% of NSCLC patients1,2,5,6. potently inhibited recombinant ROS1, NTRK1, and NTRK3 in ROS1 gene rearrangements are observed in ~1–2% of NSCLC sub-nanomolar concentration in an ATP-competitive manner patients and in cholangiocarcinoma, glioblastoma, ovarian, (Fig. 1b). Besides ROS1 and NTRKs, DS-6051b almost completely gastric, and colorectal cancers7.TheROS1oncogenicfusion inhibited ACK, ALK, DDR1, and LTK at 0.2 μM among 160 protein can be targeted by the ALK/ROS1/MET TKI crizoti- kinases in the presence of 1 mM ATP, but did not inhibit other nib8,9, which is approved for the treatment of metastatic 152 kinases strongly (Fig. 1c; Supplementary Fig 1a and Supple- ROS1-rearranged NSCLC. In a phase 1/2 clinical trial mentary Table 1a). In addition, DS-6051b inhibited NTRK2 with (PROFILE 1001), the overall response rate to crizotinib a few nanomolar IC50 values at approximately Km value for ATP, was 72%, and median progression-free survival reached and 40 nM of DS-6051b potently inhibited several other kinases, 19.2 months10. such as NuaK1, JAK2, and MUSK, at approximately Km value of In most cases of ROS1-rearranged NSCLC, crizotinib each kinase for ATP (Supplementary Table 1b). DS-6051b induces marked tumor shrinkage. However, the majority of inhibited the growth of the CD74-ROS1 overexpressed Ba/F3 patients will develop resistance to crizotinib within a few years cells and the ROS1 fusion-positive HCC78 cancer cell line with a of treatment. Several secondary mutations in the ROS1 tyr- lower IC50 than crizotinib (Fig. 1d, e). Consistent with the cell osine kinase domain have been identified from the molecular viability assay data, DS-6051b potently inhibited autopho- analysis of crizotinib-refractory ROS1-rearranged NSCLC sphorylation of ROS1 in CD74-ROS1–expressed Ba/F3 cells and cancer patient samples11,12. The ROS1 G2032R mutation is the SLC34A2-ROS1 harboring HCC78 cells at approximately analogous to the ALK G1202R mutation identified in crizoti- single-digit to double-digit nanomolar concentration (Fig. 1f, g). nib-, ceritinib-, and alectinib-resistant ALK-rearranged lung cancers. ROS1 G2032R is also analogous to the G595R muta- Antitumor activity of DS-6051b in ROS1-rearranged PDC tion in NTRK1 identified in entrectinib-resistant NTRK1- models. For further evaluation of DS-6051b efficacy, we estab- rearranged cancer. lished several ROS1-rearranged cancer cell lines from ROS1 We previously reported that the ALK G1202R mutation con- fusion-positive lung cancer patients and tested the efficacy. Since fers high-level resistance to first- and second-generation ALK no CD74-ROS1 rearrangement-harboring cancer cell line was inhibitors examined13, but is sensitive to the third-generation publicly available, two CD74-ROS1 and one EZR-ROS1- ALK/ROS1 inhibitor lorlatinib (PF-06463922)14. Lorlatinib has rearranged cancer cells were established from the pleural effu- been reported to inhibit ROS1 G2032R, but its half-maximal sions of three TKI-naive patients (JFCR-165, JFCR-168, and inhibitory concentration (IC ) is much higher with ROS1 50 MGH-193-1). These patient-derived cells were sensitive to DS- G2032R compared with ALK G1202R15. We previously found 6051b, crizotinib, lorlatinib, cabozantinib, ceritinib, brigatinib, or that cabozantinib, a clinically approved multi-kinase inhibitor for entrectinib. Lorlatinib has the lowest, and DS-6051b the second thyroid and kidney cancers, is active against ROS1 G2032R lowest IC among the inhibitors tested (Fig. 2a–c). Consistent in vitro, but yet to be evaluated in vivo or in clinical setting16.As 50 with the cell viability assay data, DS-6051b, crizotinib, lorlatinib, ROS1 G2032R is the most common mechanism of resistance to ceritinib, cabozantinib, or foretinib potently inhibited autopho- crizotinib in ROS1-rearranged NSCLC11, the identification of sphorylation of ROS1 in JFCR-165, JFCR-168, and MGH193-1B therapeutic strategies to overcome ROS1 G2032R is critically cells (Fig. 2d–f). Next, we used a glioblastoma U-118 MG cell line, important. harboring the FIG-ROS1 fusion gene, and DS-6051b treatment The NTRK gene family consists of NTRK1, NTRK2, and dose dependently inhibited autophosphorylation of ROS1 in U- NTRK3. It encodes a receptor tyrosine kinase, and plays an 118-MG cells in vitro (Fig. 2g). For in vivo experiments, DS- important role in the nervous system in a ligand-dependent 6051b treatment effectively inhibited tumor growth at ≥25 mg/kg manner. However, in cancer, NTRK is often observed as a fusion DS-6051b without significant body weight loss (Fig. 2h; Supple- oncogene, such as TPM3-NTRK1, which induces constitutive mentary Fig. 5a). In addition, we evaluated the efficacy of DS- activation of NTRK1 tyrosine kinase resulting in cancer pro- 6051b using lung cancer PDX model harboring CD74-ROS1 gression. Since the growth of NTRK fusion-positive cancers fusion gene in vivo, and DS-6051b treatment markedly inhibited depends on TRK tyrosine kinase activity, TRK tyrosine kinase tumor growth at ≥10 mg/kg DS-6051b without significant body inhibitors induce a marked tumor growth inhibition17. Indeed, weight loss (Fig. 2i; Supplementary Fig. 5b). several TRK inhibitors, such as entrectinib or larotrectinib, have demonstrated remarkable responses in NTRK-rearranged cancers across various cancer types, including lung, colorectal, thyroid, DS-6051b potently inhibits NTRK-rearranged cancer in vivo. and pediatric cancers18,19. KM12 colorectal cancer cells are known to harbor the TPM3- Here, we develop and characterize a novel ROS1/NTRK inhi- NTRK1 fusion gene, and are addicted to TPM3-NTRK1-mediated bitor, DS-6051b, which is a potent and selective ROS1 and TRK growth signaling. In our previous study, we developed Ba/F3 cells family inhibitor capable of inhibiting ROS1 G2032R and other expressing TPM3-NTRK1 and identified multiple NTRK1 crizotinib-resistant ROS1 mutants. We investigate the preclinical inhibitor-resistance