Identification of Existing Drugs That Effectively Target NTRK1 and ROS1 Rearrangements in Lung Cancer

Published OnlineFirst July 1, 2016; DOI: 10.1158/1078-0432.CCR-15-1601

Cancer Therapy: Preclinical Clinical Cancer Research Identiﬁcation of Existing Drugs That Effectively Target NTRK1 and ROS1 Rearrangements in Lung Cancer Curtis R. Chong1, Magda Bahcall1, Marzia Capelletti1, Takayuki Kosaka1, Dalia Ercan1, TaeboSim2,3, Lynette M. Sholl4, Mizuki Nishino5, Bruce E. Johnson1, Nathanael S.Gray6, and Pasi A. Janne€ 1,7

Abstract

Purpose: Efforts to discover drugs that overcome resistance to 200 nmol/L, respectively), entrectinib (IC50 ¼ 6/2,200/3,500 targeted therapies in patients with rare oncogenic alterations, such nmol/L), and PF-06463922 (IC50 ¼ 1/270/2 nmol/L). Cabozan- as NTRK1 and ROS1 rearrangements, are complicated by the cost tinib inhibited ROS1 autophosphorylation and downstream ERK and protracted timeline of drug discovery. activation in transformed Ba/F3 cells and in patient-derived Experimental Design: In an effort to identify inhibitors of tumor cell lines. The IGF-1R inhibitor BMS-536924 potently NTRK1 and ROS1, which are aberrantly activated in some patients inhibited CD74–NTRK1-transformed compared with parental with non–small cell lung cancer (NSCLC), we created and Ba/F3 cells (IC50 ¼ 19 nmol/L vs. > 470 nmol/L). A patient with screened a library of existing targeted drugs against Ba/F3 cells metastatic ROS1-rearranged NSCLC with progression on crizoti- transformed with these oncogenes. nib was treated with cabozantinib and experienced a partial Results: This screen identiﬁed the FDA-approved drug cabo- response. zantinib as a potent inhibitor of CD74–ROS1-transformed Ba/F3, Conclusions: While acquired resistance to targeted therapies is including the crizotinib-resistant mutants G2032R and L2026M challenging, this study highlights that existing agents may (IC50 ¼ 9, 26, and 11 nmol/L, respectively). Cabozantinib inhib- be repurposed to overcome drug resistance and identiﬁes cabo- ited CD74–ROS1-transformed Ba/F3 cells more potently zantinib as a promising treatment of ROS1-rearranged NSCLC than brigatinib (wild-type/G2032R/L2026M IC50 ¼ 30/170/ after progression on crizotinib. Clin Cancer Res; 1–10. 2016 AACR.

Introduction the clinical efficacy of these drugs (2). Beyond the more common NSCLC driver mutations in EGFR and KRAS, and ALK rearrange- Oncogenic driver mutations are found in approximately 50% ments, a significant, but albeit smaller, group of patients are of patients with non–small cell lung cancer (NSCLC; ref. 1). found to have alterations in ROS1, NTRK1, HER2, BRAF, RET, Patients with activating mutations in EGFR or ALK rearrange- and MET (1). ments respond to targeted therapies like erlotinib and crizotinib, The identification and regulatory approval of new therapies respectively, but the development of resistance ultimately limits directed against less common oncogenic drivers, and any resistance mechanisms that may emerge, is complicated by the high cost and protracted time required to develop a new drug. Devel- 1Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Boston, opment of a new cancer drug, including highly targeted agents, Massachusetts. 2Chemical Kinomics Research Center, Korea Institute of Science and Technology, Seoul, Republic of Korea. 3KU-KIST Graduate School of Con- may take up to 7 years and cost tens of millions of dollars (3). This verging Science and Technology, Seoul, Republic of Korea. 4Department of presents a challenge to the treatment of patients with rare onco- Pathology, Brigham and Women's Hospital, Boston, Massachusetts. 5Depart- genic drivers in lung cancer, who will eventually succumb to ment of Radiology, Brigham and Women's Hospital, Boston, Massachusetts, and disease progression due to different mechanisms of acquired 6 Dana-Farber Cancer Institute, Boston, Massachusetts. Department of Biological resistance. Chemistry and Molecular Pharmacology, Harvard Medical School, and Depart- One way to address this problem is to identify new uses for ment of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts. 7Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, Bos- existing drugs. Because the clinical and pharmacologic properties ton, Massachusetts. of existing drugs are defined, any new use may rapidly be brought to the clinic. Examples of off-target inhibition by kinase inhibitors Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). include crizotinib, which was initially developed to target MET, and later found to block ALK and ROS1 (4, 5), and imatinib, Corresponding Authors: Pasi A. Janne,€ Lowe Center for Thoracic Oncology, Dana- Farber Cancer Institute, 450 Brookline Avenue, LC4114, Boston, MA 02215. Phone: initially developed against ABL, and later found to inhibit PDGFR 617-632-6076; Fax: 617-582-7683; E-mail: [email protected]; and and KIT (6). Nathanael S. Gray, Department of Biological Chemistry, and Molecular Pharma- ROS1 was first reported as a potential driver oncogene in cology,360 LongwoodAvenue,LongwoodCenter,Room2209, Boston, MA02215. NSCLC in 2012. Patients with ROS1 rearrangements comprise Phone: 617-582-8590; Fax: 617-582-8615; E-mail: [email protected] approximately 1% to 3% of all NSCLC (5). Chromosomal rear- doi: 10.1158/1078-0432.CCR-15-1601 rangement with a variety of partners leads to constitutive activation of ROS1, and the hybrid kinase is able to drive proliferation

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AZD7451, and ARRY-470, are among at least nine compounds Translational Relevance undergoing clinical development. We identiﬁed cabozantinib as a potent inhibitor of wild- In an effort to further accelerate the development of drugs that type ROS1 kinase and the L2026M and G2032R mutants that target ROS1 and NTRK1 in lung cancer, including resistant var- confer acquired clinical resistance to crizotinib by screening a iants of ROS1, a library of existing targeted therapies was screened library of existing targeted therapies. A patient with ROS1- for inhibition of these two kinases. The collection of existing rearranged non–small cell lung cancer who progressed on targeted therapies includes 290 compounds that target 104 dif- crizotinib was treated with cabozantinib and experienced a ferent signaling pathways (Supplementary Fig. S1; Supplementary partial response. Cabozantinib is a more potent inhibitor of File 1), and are in different stages of clinical development, from G2032R and L2026M mutant, crizotinib-resistant ROS1 com- preclinical to FDA approved. To discover ROS1 and NTRK1 kinase pared with brigatinib, entrectinib, and PF-06463922, and inhibitors, this collection, the Dana-Farber Targeted Therapy should be evaluated in ROS1-rearranged crizotinib-resistant Library, was used to assess the antiproliferative activity on the patients. The identiﬁcation of new uses for existing targeted oncogene-dependent cell line Ba/F3 transformed with either therapies in such patients is a way to rapidly deliver new CD74–ROS1 or CD74–NTRK1. We selected a repurposing strategy treatments to the clinic, while avoiding the high cost of de because, in contrast to ALK-rearranged and EGFR-mutant novo drug development. This strategy may also be deployed to NSCLCs, for which second- and third-generation inhibitors that overcome resistance to targeted therapies across a variety of overcome resistance have entered the clinic (16, 17), no such oncogene-addicted cancers. targeted second-line therapies exist for patients with ROS1 rearrangements after progression on crizotinib.

Materials and Methods

of oncogene-dependent cells (5). In addition to NSCLCs, ROS1 Proliferation assays rearrangements are reported in glioblastoma, cholangiocarci- Ba/F3 cells (immortalized murine bone-marrow-derived pro-B CD74–ROS1 CD74–NTRK1 noma, gastric adenocarcinoma, ovarian serous tumors, inflam- cells) transfected with either or were matory myofibroblastic tumors, and chronic myelomonocytic used for screening and were prepared as previously described (14). leukemia (7). An expanded version of the KIN001 library, which is composed of After preclinical studies demonstrated that crizotinib inhi- commercially available and in-house kinase inhibitors, was used bits ROS1 kinase (5, 8), a phase II study of NSCLC patients for screening (18). High-throughput screening was performed in fi with ROS1 rearrangements treated with crizotinib was initi- duplicate in 384-well plates with 1,000 cells/well and a nal < ated. This study demonstrated an impressive 72% response DMSO concentration of 0.5%. Cells and drugs were incubated rate to crizotinib treatment with a 19.2-month progression- for 72 hours, and viability was measured using CellTiter Glo. IC50 free survival (9). Despite the success of crizotinib in ROS1- experiments were performed in triplicate, were calculated using a – rearranged NSCLC, patients inevitably develop acquired resis- sigmoidal, non-linear dose response model in GraphPad Prism, tance through either the L2026M "gatekeeper" mutation, and are presented as the mean of 3 separate experiments. The alteration of another active site residue (G2032R), or unde- CUTO-3 cell line was the generous gift of Dr. Robert C. Doebele fined changes (10). (University of Colorado Division of Medical Oncology). The A number of methods have been used to design more potent UMG-118 and HCC-78 cell lines were obtained from ATCC inhibitors or to overcome crizotinib resistance in ROS1-rear- and passaged in our laboratory for less than 6 months; ATCC fi ranged NSCLC, including de novo drug discovery (11) and uses short tandem repeat pro ling for cell line authentication. screens of existing kinase inhibitors (12, 13). The c-MET and VEGFR-2 inhibitor foretinib potently inhibits wild-type Western blot analysis and G2032R-mutant ROS1 (12). Foretinib reached phase II Cells were incubated with inhibitor for 6 hours. Cell lysate clinical trials in breast and lung cancers. However, because of (prepared by 30 minute incubation with 0.1% Triton X-100 þ its lack of clinical activity in unselected populations, its clinical protease inhibitor) was loaded into each well of a 4% to 12% Bis- development was discontinued. There are two ROS1-specific Tris gel. Gels were transferred to PVDF membranes, blocked with inhibitors undergoing clinical development: the dual ALK1/ 5% milk, and incubated with a 1:1,000 dilution of primary ROS1 inhibitor PF-06463922 (NCT01970865) and entrectinib antibody and a 1:5,000 secondary antibody solution. (RXDX-101/NMS E628), a pan-TRK, ALK, and ROS1 inhibitor (STARTRK-1, NCT02097810). Structural modeling NTRK1 (TRKA, neurotrophic tyrosine kinase receptor 1) Molecular docking studies of cabozantinib on the wild-type fusions with different partners, including CD74 and MPRIP, were ROS1 and point-mutated (L2026M and G2032R) kinase domain identified in lung cancer in 2013. NTRK1 rearrangements are were performed. In order to construct a homology model, struc- thought to occur in 1% to 3% of patients with NSCLC (14). tures of the wild-type ROS1 and point-mutated (L2026M and Under normal circumstances, the TRK family of kinases plays a G2032R) ROS1 kinase domain, the 3D structure of the template role in the development of the central and peripheral nervous kinase c-Met in the DFG-out conformations (PDB accession code: systems and in pain sensation (15). NTRK1 fusions have also been 3LQ8) was retrieved from the Protein Data Bank. Sequence identified in thyroid, colon, and ovarian cancer, melanoma, alignments of ROS1 and the template protein were generated cholangiocarcinoma, glioblastoma, and acute myeloid leukemia using the Discovery Studio 4.1 package. Three-dimensional (3D) (15). The activity of the fusion kinase is inhibited by crizotinib model structures of wild-type and point-mutated (L2026M and and lestaurtinib, and TRK-specific inhibitors, such as entrectinib, G2032R) ROS1 kinase domain were built-up using the Modeler in

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Discovery Studio 4.1 package, and were further refined by using JP1540 vector using Cre recombination (New England Biolabs) the CHARMM force field. The 3D structure of cabozantinib was and transformed into E. coli. Plasmid was purified and then built using a Maestro build panel and energy-minimized using the transfected using the FuGene reagent (Promega) into 293 cells. Impact module of Maestro in the Schrodinger€ suite program. Protein expression and kinase activity were assessed as described Ligand docking onto the active site of wild-type and point- above. mutated (L2026M and G2032R) ROS1 kinase domain was carried out using the Schrodinger€ docking program, Glide. The best- Results docked poses were selected as the lowest Glide score. Identification of targeted agents that inhibit ROS1 and NRTK1 Genetic profiling kinase activity OncoPANEL is a hybrid capture-targeted next-generation A library of 290 agents that target 104 different cellular path- sequencing assay to detect somatic mutations, copy number ways was created (Supplementary Fig. S1A and S1B; Supplemen- variations, and structural variants in tumor DNA extracted from tary File S1A). Compounds active against more than one pathway fresh, frozen, or formalin-fixed paraffin-embedded samples (19). are represented for each unique target; for example, crizotinib is DNA was isolated from tissue containing at least 20% tumor included as both a MET and ALK inhibitor. This collection, the nuclei and analyzed by massively parallel sequencing using a Dana-Farber Targeted Therapy Library (DFTTL), contains 148 solution-phase Agilent SureSelect hybrid capture kit and an Illu- preclinical compounds (51%), 31 drugs that entered phase I mina HiSeq 2500 sequencer. The studies were performed in a clinical trials (11%), 50 drugs in phase II clinical trials (18%), Clinical Laboratory Improvement Amendments (CLIA)-certified 30 drugs tested in phase III clinical trials (10%), and 31 drugs laboratory, and the resulting sequences were interpreted by a approved by the FDA (10%), including 25 of the 32 targeted board-certified anatomic pathologist. The patient provided con- agents approved by the FDA in cancer (78%). Others have sent to clinical and research use of the genomic findings, which screened libraries of targeted agents on ROS1 (12, 20), and the were approved by the Dana-Farber Harvard Cancer Center Insti- overlap between libraries is summarized in Supplementary tutional Review Board. Fig. S2. The DFTTL was screened at 1 mmol/L concentration on Ba/F3 Creation of NTRK1 gatekeeper mutation cells transformed with CD74–ROS1 or CD74–NTRK1 fusions; the The pDNR-dual plasmid containing wild-type CD74–NTRK1 parental Ba/F3 cell line grown in the presence of IL3 was screened kinase was used for site-directed mutagenesis of Phe589 to Val. in parallel. A cutoff of 50% inhibition, represented as the percent The mutated CD74–NTRK1 fusion was then transferred to the viability parental Ba/F3 percent viability transformed Ba/F3,

Figure 1. A, Top inhibitors of Ba/F3 transformed with CD74–ROS1, color coded by clinical development status: FDA-approved (green), phase III (yellow), phase II (blue), phase I (purple), and preclinical (gray). B, Top inhibitors of Ba/F3 transformed with CD74–NTRK1, color coded by clinical development status: FDA-approved (green), phase III (yellow), phase II (blue), phase I (purple), and preclinical (gray).

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was used to identify hits for further characterization. Ba/F3 cells ment, such as ROS1 (12, 20, 21). Compounds showing > 50% have been extensively used in kinase drug discovery, including inhibition for ROS1 (n ¼ 44) and NTRK1 (n ¼ 19) are shown by screens for compounds targeting kinases activated by rearrange- stage of clinical development in Fig. 1, with IC50 values for hits

Figure 2. A, Inhibition of Ba/F3 transformed with CD74–NTRK1 proliferation by BMS- * 536924 (red, ,IC50 ¼ 28 nmol/L), * crizotinib (green, ,IC50 ¼ 75 nmol/L), * lestaurtinib (CEP-701, blue, ,IC50 ¼ 15 * nmol/L), and dovitinib (black, ,IC50 ¼ 69 nmol/L) and of Ba/F3 parental cells

by BMS-536924 (red, &,IC50 ¼ 2.75 mmol/L), crizotinib (green, &,IC50 ¼ 15.4 mmol/L), lestaurtinib (CEP-701,

blue &,IC50 ¼ 3.4 mmol/L), and dovitinib (black, &,IC50 ¼ 4.2 mmol/L). B, Inhibition of NTRK1 autophosphorylation and downstream signaling in CD74–NTRK1-transformed NIH-3T3 cells. C, Inhibition of NTRK1 autophosphorylation in CUTO-3 tumor cells derived from a patient harboring an MPRIP–NTRK1 rearrangement. D, Inhibition of CUTO-3 cells by crizotinib * (green, ,IC50 ¼ 462 nmol/L), * lestaurtinib (CEP-701, blue, ,IC50 ¼ 3.5 nmol/L), BMS-536924 (red, *, * IC50 ¼ 34 nmol/L), dovitinib (black, , IC50 ¼ 170 nmol/L). E, Phe589 is postulated to be the "gatekeeper" residue in the NTRK1 kinase active site and is modeled using space-ﬁlling representation. F, Mutation of the putative "gatekeeper" residue Phe589 to Val does not abrogate inhibition of TRK autophosphorylation at residues 490 and 674/675 for lestaurtinib or BMS-536924.

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available in Supplementary Files 1B and 1C. Screen results by incubated with increasing concentrations of cabozantinib or pathway are presented Supplementary Fig. S3. foretinib, a decrease is seen in the phosphorylation of ROS1 and ERK (Fig. 3B). In contrast, crizotinib inhibits the phosphorylation of ROS1 and ERK in the wild-type CD74–ROS1 Ba/F3 cells only. Identification of NTRK1 inhibitors Brigatinib inhibits the phosphorylation of ROS1 and ERK in Dovitinib and BMS-536924 selectively inhibited the prolifer- CD74–ROS1 wild-type and L2026M mutant–transformed Ba/F3 ation of CD74–NTRK1-transformed Ba/F3 cells but not the paren- cells, with less activity seen in the CD74–ROS1 G2032R-trans- tal, interleukin-dependent Ba/F3 cells (Fig. 2A). Lestaurtinib formed Ba/F3 cells. PF-06463922 inhibited wild-type and (CEP-701) is an FLT3, JAK2, TrkA/B/C inhibitor structurally L2026M-mutant ROS1 autophosphorylation and ERK activation, similar to staurosporine that is undergoing clinical trials in acute with little effect on signaling in the G2032R mutant, and entrec- myelogenous leukemia (22), and was used as a positive control tinib was effective only at blocking activity of wild-type ROS1 for NTRK1 inhibition. Dovitinib is an FGFR3 inhibitor in phase III signaling (Supplementary Fig. S5). clinical trials for renal cell carcinoma that reaches a peak plasma level with oral dosing of 568 nmol/L observed in clinical trials (23), and BMS-536924 is an ATP-competitive IGF1-1R inhibitor. Inhibition of ROS1 signaling in human cancer cells bearing NTRK1 undergoes autophosphorylation at tyrosine residues ROS1 rearrangements 490, 674, and 675, and this leads to activation of ERK via The effect of cabozantinib, crizotinib, and foretinib were next phosphorylation (15). In NIH-3T3 cells transformed with studied in the NSCLC HCC78 cell line, which expresses the FLAG-tagged CD74–NTRK1, both BMS-536924 and lestaurtinib SLC34A2–ROS1 fusion, and in the UMG-118 glioblastoma cell inhibit NTRK1 autophosphorylation (Fig. 2B). Inhibition of line, which expresses the FIG–ROS1 fusion. As seen in the CD74– NTRK1 autophosphorylation by dovitinib was weaker than ROS1-transformed Ba/F3 cells, brigatinib, cabozantinib, crizoti- would be suggested by its IC50 value. The CUTO-3 cell line was nib, and foretinib showed similar inhibition of HCC78 prolifer- derived from a patient with lung adenocarcinoma and contains an ation (Fig. 3C), ROS1 autophosphorylation, and downstream MPRIP–NTRK1 rearrangement (14). BMS-536924, dovitinib, and ERK activation (Fig. 3D). The IC50 for inhibition of HCC78 lestaurtinib inhibited proliferation of CUTO-3 cells (IC50 ¼ 34 observed for these drugs is similar to that reported by others nmol/L, 170 nmol/L, and 3 nmol/L, respectively; Fig. 2C). As seen (12), and is up to 150-fold less potent than in CD74–ROS1- in 3T3 cells transformed with CD74–NTRK1, BMS-536924 and transformed Ba/F3 cells, likely due to activation of other signaling lestaurtinib inhibited NTRK1 autophosphorylation in CUTO-3 pathways. Inhibition of ROS1 autophosphorylation and ERK cells (Fig. 2D). Dovitinib again showed partial inhibition of activation is also seen in the UMG-118 cell line (Supplementary NTRK1 autophosphorylation at 1,000 nmol/L in CUTO-3 cells, Fig. S6). suggesting that blockade of other targets may account for the inhibition observed. Modeling of cabozantinib in the ROS1 active site reveals the Mutations in the active site "gatekeeper" residue decrease or molecular basis for inhibition of wild-type and mutant kinase abolish the efficacy of inhibitors that bind to the active site of may To define the molecular basis for inhibition, cabozantinib was kinases. The residue Phe589 was identified as the putative gate- modeled into the active site of ROS1 (Fig. 3E). In this structure, the keeper residue in the NTRK1 kinase active site based on the quinoline nitrogen of cabozantinib makes a hinge contact with available crystal structure (Fig. 2E). This residue was mutated to Met2029, an amide group attached to the bridge phenyl ring valine, and the effect of inhibitors identified in our screen was forms a pair of hydrogen bonds with the backbone carbonyl assessed on NTRK1 autophosphorylation in 293 cells transiently group of Gly2101 and the e-amino group of Lys1980. Further transfected with both wild-type and F589V CD74–NTRK1. As seen contacts are made with a hydrogen bond between the amide in Fig. 2F, the F589V mutation did not affect inhibition of NTRK1 group attached to the tail phenyl ring and the carboxyl group of autophosphorylation by lestaurtinib or BMS-536924. Asp2102, and a p–p stacking interaction between the phenyl ring and Phe2103. In G2032R- and L2026M-mutant ROS1, while Lead compounds inhibit ROS1 signaling there is no hydrogen bond between the amide group attached The most potent agents identified were cabozantinib, foretinib, to the tail phenyl ring and the carboxyl group of Asp2102, the and brigatinib (AP26113) for ROS1, with little inhibition seen of other interactions are preserved. By preserving most of the inter- the parental Ba/F3 cells by these compounds (Fig. 3; Table 1; actions seen in the wild-type kinase, cabozantinib is able to Supplementary Fig. S4). Cabozantinib and foretinib exhibited achieve potent inhibition of both the G2032R and L2026M potent activity against both wild-type ROS1 and the two reported mutants. kinase mutants that confer acquired resistance to crizotinib, the G2032R and L2026M mutations, as previously reported (20). In Cabozantinib demonstrates antitumor activity in a patient with contrast, PF-06463922, which is in phase II clinical trials of ALK- ROS1-rearranged NSCLC who progressed on crizotinib and and ROS1-rearranged NSCLC, potently inhibited wild-type and multiple lines of cytotoxic chemotherapy L2026M-mutant ROS1, but demonstrated > 250-fold less potent Given the efficacy of cabozantinib in preclinical models of inhibition of the G2032R mutant. Entrectinib inhibited wild-type ROS1-rearranged NSCLC, a 53-year-old woman with ROS1-rear- ROS1 with an IC50 of 6 nmol/L, with little inhibition (IC50 > 1 ranged metastatic lung adenocarcinoma who developed acquired mmol/L) seen for the G2032R or L2026M mutants. resistance to crizotinib was identified and eventually treated with ROS1 undergoes autophosphorylation at tyrosine 2274, and cabozantinib. The patient was a never smoker who presented with this stimulates cell proliferation via activation of multiple down- cough and fatigue in November 2011 and was found to have stream pathways, including ERK, PI3K, and AKT (7). When CD74– diffuse tumor involvement of the left lung with extensive medi- ROS1 wild-type, G2032R- or L2026M-transformed Ba/F3 cells are astinal adenopathy, enlarged intra-abdominal lymph nodes, and

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Table 1. Inhibition of Ba/F3 cells transformed with CD74–ROS1 kinase

IC50 (mmol/L) Wild-type ROS G2032R L2026M Parental Ba/F3 [Plasma] (mmol/L) Alectinib >10 >10 4.2 >10 1.4 (600 mg) Brigatinib 0.03 0.17 0.2 1.54 N/A (phase I/II) Cabozantinib 0.009 0.026 0.011 >10 1.08 Ceritinib 0.23 2.2 0.09 >10 1.4 Crizotinib 0.044 2.7 0.127 >10 0.61 Entrectinib 0.006 2.2 3.5 >10 N/A Foretinib 0.008 0.09 0.008 2.1 0.34–0.9 PF-06463922 0.001 0.27 0.002 >10 N/A (phase I)

a single metastasis in the liver (T4N3M1b). She underwent 6 12 (Supplementary Fig. S7) and EZR intron 10, and other geno- cycles of cisplatin þ pemetrexed followed by 6 cycles of mainte- mic alterations of unclear significance. In this patient, the ROS1 nance pemetrexed with a partial response. After progression in translocation may involve a ROS1 (exon 1–33)–RNPC (exon 12– October 2012 she received 6 cycles of carboplatin þ gemcitabine 15) fusion and an EZR–ROS1 (exon 34–43) fusion. The EZR– with erlotinib maintenance. Crizotinib treatment was initiated in ROS1 fusion is commonly observed in patients with ROS1-rear- September 2013 after biopsy of a left lower lobe nodule done ranged NSCLC and is predicted to be oncogenic as the ROS1 following progression on erlotinib revealed a ROS1 rearrange- kinase domain is located after exon 36 (7). ment in 18 of 50 nuclei. Restaging scans showed a partial response, which lasted until October 2014 when she developed Discussion worsening dyspnea and cough. A computed tomography scan showed multiple new pulmonary nodules and opacities as well as To accelerate discovery of new therapeutics for ROS1- and a dominant mass in the left upper lobe (Fig. 4A and B), and NTRK1-rearranged lung cancer, a library of existing targeted thoracentesis yielded 2.5 L of malignant pleural fluid. agents was screened. Compounds were selected for further study In December 2014, she was initiated on cabozantinib at 140 mg based on potency, selectivity, and clinical availability. daily, based on the preclinical work by us and others (20) Cabozantinib was the most promising ROS1 inhibitor identi- demonstrating inhibition of the wild-type, G2032R, and fied because it inhibits both the wild-type ROS1 and the G2032R L2026M ROS1 kinase. The patient was informed of the off-label and L2026M mutants that confer resistance to crizotinib. use of cabozantinib. Approximately 2 weeks later, the dose was Unlike brigatinib, entrectinib, and PF-06463922, three drugs in reduced to 120 mg daily, then 80 mg daily, and then cabozantinib clinical development as ROS1 inhibitors, cabozantinib is equally was held after approximately 3 weeks on treatment due to grade III potent against wild-type and crizotinib-resistant (G2032R and palmar-plantar erythrodysesthesia and grade II fatigue. A CT scan L2026M) ROS1. Brigatinib and PF-06463922 were not present in performed because of dyspnea 4 weeks after initiation of cabo- our initial library, and were used to compare the efficacy of zantinib revealed bilateral segmental pulmonary emboli and inhibition of wild-type ROS1 and the G2032R and L2026M resolution of the dominant mass in the left upper lobe, which mutations with cabozantinib. Cabozantinib is able to achieve had previously measured 4.3 cm, and reduction in the right lower potent inhibition of wild-type and crizotinib-resistant ROS1 by lobe lesion from 1.7 cm to 1.4 cm (Fig. 4C and D). The pulmonary maintaining most of the interactions in the enzyme active site. embolism was medically managed, and the palmar-plantar ery- Cabozantinib is currently FDA approved for the treatment of throdysesthesia improved with the use of gabapentin, urea cream, medullary thyroid carcinoma. Kinase inhibition occurs at con- and clobetasol. Cabozantinib was restarted at 20 mg daily. A centrations 50-fold less than the peak plasma level obtained repeat chest CT scan performed 2 weeks later and approximately 8 through FDA-approved oral dosing (Table 1). As an FDA- weeks after initiation of cabozantinib treatment demonstrated approved drug, cabozantinib has been extensively studied in the maintenance of the disease response with slightly increased clinic. In medullary thyroid cancer, cabozantinib increases the bilateral pleural effusions. The cabozantinib dose was subse- progression-free survival (11.2 months vs. 4 months) compared quently increased to 40 mg daily. After uptitration of the dose with placebo (24). Cabozantinib has also been studied in prostate to 60 mg daily, the grade III palmar-plantar erythrodysesthesia cancer (25), RET-rearranged NSCLC, and renal cell carcinoma returned, and the patient elected to discontinue cabozantinib. She (26). Of 3 patients with RET-rearranged NSCLC treated with subsequently showed symptomatic and radiographic disease cabozantinib, 2 patients experienced a partial response and 1 progression, approximately 10 weeks after initiating cabozanti- patient had stable disease (27). In the phase III thyroid cancer nib, and 3 weeks after its discontinuation. study, adverse events including diarrhea, palmar-plantar erythro- Sequencing of tumor cells from the effusion that developed dysesthesia, weight loss, anorexia, and fatigue led to dose reduc- after crizotinib resistance revealed a wild-type ROS1 kinase tions or discontinuation in 79% and 16% of patients, respectively domain, rearrangement of ROS1 intron 33 with RNPC3 intron (24).

Figure 3. A, Inhibition of Ba/F3 transformed with CD74–ROS1 wild-type (red, *), G2032R (green, *), L2026M (blue, *), and Ba/F3 parental cells (black, *). B, Effect of drugs on ROS1 autophosphorylation (pY2274) and downstream activation of ERK signaling. C, Inhibition of the HCC78 NSCLC cell line by brigatinib (blue, * * * * ,IC50 ¼ 2.2 mmol/L), cabozantinib (red, ,IC50 ¼ 1.36 mmol/L), crizotinib (green, ,IC50 ¼ 1.6 mmol/L), and foretinib (black, ,IC50 ¼ 0.46 mmol/L); data for alectinib (IC50 ¼ 0.75 mmol/L), brigatinib (IC50 ¼ 2.2 mmol/L), ceritinib (IC50 ¼ 0.53 mmol/L), entrectinib (IC50 ¼ 0.45 mmol/L), and PF-06463922 (IC50 10 mmol/L) not shown. D, Inhibition of ROS signaling in HCC78 cells. E, Molecular modeling of cabozantinib into the active site of ROS1 kinase domain, with dashed lines indicating hydrogen bonds.

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Figure 4. A and B, Computed tomography (CT) of the chest prior to cabozantinib therapy demonstrated a dominant mass in the left upper lobe (B, black arrow), measuring 4.3 cm in the longest diameter with patchy opacities (A, arrowheads) and a 1.7-cm discrete nodule in the right lower lobe (B, white arrow). Note bilateral small pleural effusions. C and D, On a chest CT scan performed due to dyspnea 4 weeks after the initiation of cabozantinib therapy, the dominant lesion and surrounding tumor burden in the left upper lobe has mostly resolved with minimal residual opacities (C), indicating marked response to therapy. The right lower lobe lesion has also decreased, measuring 1.4 cm (arrow, D). Bilateral segmental pulmonary emboli are also noted (not shown), accounting for the clinical symptoms.

Based on the preclinical work presented here and in the sion on an EGFR TKI, cabozantinib 40 mg daily with erlotinib available literature (20), off-label cabozantinib treatment was resulted in an 11% response rate (29), and this was attributed used in a ROS1-rearranged NSCLC patient who had progressed to targeting of resistance pathways, including MET. It is possible after 13 months of crizotinib treatment and 3 other forms of that cabozantinib was active against a secondary pathway that systemic therapy. Cabozantinib was chosen over cytotoxic che- caused resistance, in addition to more potently inhibiting wild- motherapy because the patient's lung cancer had previously type ROS1 kinase. progressed on cisplatin þ pemetrexed, carboplatin þ gemcita- Our patient had two ROS1 rearrangements involving intron 33 bine, erlotinib, and crizotinib. The FDA-approved dose of 140 mg with EZR, which has previously been reported (9), and with daily was chosen given the patient's symptomatic progression on RNPC3 (RNA-binding region containing 3) on chromosome crizotinib. Her symptoms of cough and dyspnea rapidly 1p21, which, to our knowledge, has not been previously reported. improved on the 140-mg cabozantinib dose, and radiographic CD74 is the most common ROS1 fusion partner among the 11 improvement was apparent 4 weeks later. This is consistent with different partners that have been reported (7, 9). The RNPC3 gene the prompt responses seen to erlotinib/gefitinib and crizotinib in encodes a 517 amino acid protein, RNA binding protein 40 (also EGFR-mutant and ALK-rearranged NSCLC. known as U11/U12 snRNP 65k), that is a component of the U12- Unfortunately, it appeared that the therapeutic window of dependent (minor) spliceosome (30). This protein complex cabozantinib overlapped with toxic levels in our patient. In the removes U12-type introns, which are thought to comprise < clinical trial of cabozantinib in medullary thyroid cancer, 79% of 0.1% of all human introns (30). The relative abundance of tumor patients had dose reductions, and 65% of patients had doses held cells with the EZR–ROS1 and RNPC3–ROS1 fusion is unclear, as is (24). The clinical trial of cabozantinib in prostate cancer whether both are expressed in tumor cells versus a heterogeneous attempted to address this issue by using either 40-mg or 100-mg population of tumor cells expressing either the EZR or RNPC doses (25). The rate of dose reduction was 84% in the 100-mg fusion. group and 31% in the 40-mg group, with discontinuation rates of Based on our preclinical and clinical observations, a potential 25% and 18%, respectively. future clinical trial should be initiated to specifically to examine Ceritinib, an ALK inhibitor, is effective in crizotinib resistant the ef ficacy of cabozantinib in patients with progression on cancers that do or do not contain an ALK secondary mutation crizotinib. If ROS1 rearrangements comprise 1% to 3% of all (17). The activity in the latter subgroup may be due to either the NSCLC, this population could include nearly 7,000 patients increased potency of ceritinib on ALK and/or the inhibition of annually in the United States, and a substantially larger number other kinases, such as IGFR1, inhibited by ceritinib but not around the world. crizotinib (28). Analogously, the increased potency of cabozan- Our screen also identified BMS-536924 as a potential NTRK1 tinib on ROS1 may be the reason for the clinical benefit in our inhibitor. Mutation of the putative gatekeeper Phe589 (F589V) patient. failed to abrogate inhibition of NTRK1 autophosphorylation for Analysis of the patient's tumor DNA using targeted next- both BMS-536924 and lestaurtinib in 293 cells transformed with generation sequencing did not reveal any variants that could mutant CD74–NTRK1, suggesting that both agents may still retain explain resistance to crizotinib. Specifically, we did not identify the ability to inhibit NTRK1 and downstream signaling, including in our patient the G2032R or L2026M mutations that confer ERK even in the presence of the gatekeeper mutation. resistance to crizotinib, while maintaining sensitivity to cabo- A second potential explanation for these observations is that zantinib. In patients with EGFR-mutant NSCLC and progres- inhibition of CD74–NTRK1 F589V Ba/F3 cells by BMS-536924

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NTRK1 and ROS1 Inhibitors in NSCLC

and lestaurtinib is due to an off-target effect of these inhibitors. In intellectual property on EGFR mutations licensed to Lab Corp. No potential order to differentiate between these two possibilities, additional conflicts of interest were disclosed by the other authors. biochemical studies, to determine binding of BMS-536924 and lestaurtinib to NTRK1 and NTRK1 F589V, and mutagenesis stud- Authors' Contributions ies, to introduce additional gatekeeper residues and solvent front Conception and design: C.R. Chong, M. Capelletti, M. Nishino, N.S. Gray, mutations, will need to be performed. Given the ATP binding site P.A. J€anne homology observed among NTRK family members (31), the Development of methodology: C.R. Chong inhibitors reported here could be tested for inhibition of NTRK2 Acquisition of data (provided animals, acquired and managed patients, and NTRK3 in future studies. provided facilities, etc.): C.R. Chong, M. Bahcall, M. Capelletti, T. Kosaka, Genetic sequencing efforts are identifying ever more rare sub- M. Nishino, B.E. Johnson, N.S. Gray fi Analysis and interpretation of data (e.g., statistical analysis, biostatistics, sets of patients with actionable driver oncogenes. The identi ca- computational analysis): C.R. Chong, D. Ercan, T. Sim, L.M. Sholl, B.E. tion of new uses for existing targeted therapies in such patients is a Johnson, P.A. J€anne way to rapidly deliver new treatments to the clinic, while avoiding Writing, review, and/or revision of the manuscript: C.R. Chong, M. Bahcall, the high cost of de novo drug development. This strategy may also M. Capelletti, T. Sim, M. Nishino, B.E. Johnson, N.S. Gray, P.A. J€anne be deployed to overcome resistance to targeted therapies across a Administrative, technical, or material support (i.e., reporting or organizing variety of oncogene-addicted cancers. As new uses are discovered data, constructing databases): C.R. Chong for existing targeted therapies, patients should be encouraged to participate in clinical trials of these agents, rather than be used as a Grant Support rationale for off-protocol, non-trial use of such drugs. This study is supported by grants from the NIH RO1CA136851 (P.A. J€anne and N.S. Gray), R01CA135257 (P.A. J€anne), 1K23CA157631 (M. Nishino) and Disclosure of Potential Conflicts of Interest 1 U54 HL127365-01 (N.S. Gray), the Cammarata Family Foundation Research € M. Nishino is a consultant/advisory board member for Bristol-Myers Squibb. Fund (M. Capelletti and P.A. Janne), and the Nirenberg Fellowship at Dana- € B.E. Johnson has ownership interest (including patents) in KEW Group, is a Farber Cancer Institute (M. Capelletti and P.A. Janne). C.R. Chong is the consultant/advisory board member for AstraZeneca, Chugai, Clovis, Eli Lilly, recipient of funding from Uniting Against Lung Cancer, a Conquer Cancer KEW Group, Merck, Novartis, and Transgenomics, has provided expert testi- Foundation/ASCO Young Investigator Award, and a post-doctoral fellowship mony for Genentech and reports receiving post-marketing royalties for EGFR from the American Cancer Society (PF-14-020-01-CDD). mutation testing from Dana-Farber Cancer Institute. P.A. Janne reports receiving The costs of publication of this article were defrayed in part by the payment of advertisement commercial research grants from Astellas and AstraZeneca, has ownership page charges. This article must therefore be hereby marked in interest (including patents) in Gatekeeper Pharmaceuticals, and is a consul- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tant/advisory board member for AstraZeneca, Boehringer Ingelheim, Chugai, Clovis, Genentech, Merrimack Pharmaceuticals, Pfizer, and Sanofi, and reports Received July 7, 2015; revised June 1, 2016; accepted June 12, 2016; receiving post-marketing royalties from Dana-Farber Cancer Institute owned published OnlineFirst July 1, 2016.

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Identification of Existing Drugs That Effectively Target NTRK1 and ROS1 Rearrangements in Lung Cancer

Curtis R. Chong, Magda Bahcall, Marzia Capelletti, et al.

Clin Cancer Res Published OnlineFirst July 1, 2016.

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