Published OnlineFirst November 6, 2015; DOI: 10.1158/2159-8290.CD-15-0940 RESEARCH BRIEF Acquired Resistance to the TRK Inhibitor Entrectinib in Colorectal Cancer Mariangela Russo 1,2,3 , Sandra Misale 2 , G e W e i 4 , Giulia Siravegna 1,2 , Giovanni Crisafulli 2 , Luca Lazzari 1,2 , Giorgio Corti 2 , Giuseppe Rospo 2 , Luca Novara 2 , Benedetta Mussolin 2 , Alice Bartolini 2 , Nicholas Cam 4 , Roopal Patel 4 , Shunqi Yan 4 , Robert Shoemaker 4 , Robert Wild 4 , Federica Di Nicolantonio1,2 , Andrea Sartore - Bianchi 5 , Gang Li 4 , Salvatore Siena 5,6 , and Alberto Bardelli 1,2 ABSTRACT Entrectinib is a fi rst-in-class pan-TRK kinase inhibitor currently undergoing clini- cal testing in colorectal cancer and other tumor types. A patient with metastatic colorectal cancer harboring an LMNA–NTRK1 rearrangement displayed a remarkable response to treatment with entrectinib, which was followed by the emergence of resistance. To characterize the molecular bases of the patient’s relapse, circulating tumor DNA (ctDNA) was collected longitudinally during treatment, and a tissue biopsy, obtained before entrectinib treatment, was transplanted in mice (xenopatient), which then received the same entrectinib regimen until resistance developed. Genetic profi ling of ctDNA and xenopatient samples showed acquisition of two point mutations in the catalytic domain of NTRK1 , p.G595R and p.G667C. Biochemical and pharmacologic analysis in multiple preclini- cal models confi rmed that either mutation renders the TRKA kinase insensitive to entrectinib. These fi ndings can be immediately exploited to design next-generation TRKA inhibitors. SIGNIFICANCE: We provide proof of principle that analyses of xenopatients (avatar) and liquid biopsies allow the identifi cation of drug resistance mechanisms in parallel with clinical treatment of an individual patient. We describe for the fi rst time that p.G595R and p.G667C TRKA mutations drive acquired resistance to entrectinib in colorectal cancers carrying NTRK1 rearrangements. Cancer Discov; 6(1); 36–44. ©2015 AACR. See related commentary by Okimoto and Bivona, p. 14. INTRODUCTION ment is the most common mechanism of oncogenic acti- vation for this family of receptors, resulting in sustained TRK receptors are a family of tyrosine kinases that com- cancer cell proliferation through activation of MAPK and prises three members: TRKA, TRKB, and TRKC, encoded by AKT downstream pathways ( 1 ). Rearrangements of the the neurotrophic tyrosine kinase receptor, type 1 ( NTRK1 ), NTRK1 , NTRK2 , and NTRK3 genes occur across different NTRK2 , and NTRK3 genes, respectively. Genomic rearrange- tumors, including colorectal cancers ( 2 ). 1 Department of Oncology, University of Torino, Torino, Italy. 2 Candiolo Can- A. Bardelli and S. Siena share senior authorship of this article. 3 cer Institute, FPO, IRCCS, Torino, Italy. FIRC Institute of Molecular Oncol- Corresponding Author: Alberto Bardelli, Department of Oncology, Univer- 4 5 ogy (IFOM), Milano, Italy. Ignyta, Inc., San Diego, California. Department of sity of Torino, Candiolo Cancer Institute, FPO, IRCCS, strada provinciale Hematology and Oncology, Niguarda Cancer Center, Ospedale Niguarda Ca’ 142, km 3.95, Candiolo 10060, Torino, Italy. Phone: 39-011-9933235; 6 Granda, Milan, Italy. Università degli Studi di Milano, Milan, Italy. Fax: 39-011-9933225; E-mail: [email protected] Note: Supplementary data for this article are available at Cancer Discovery doi: 10.1158/2159-8290.CD-15-0940 Online (http://cancerdiscovery.aacrjournals.org/). © 2015 American Association for Cancer Research. M. Russo, S. Misale, and G. Wei contributed equally to this article. 36 | CANCER DISCOVERYJANUARY 2016 www.aacrjournals.org Downloaded from cancerdiscovery.aacrjournals.org on October 2, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst November 6, 2015; DOI: 10.1158/2159-8290.CD-15-0940 TRKA Mutations and Resistance to Entrectinib in Colorectal Cancer RESEARCH BRIEF Figure 1. Tracking resistance to TRKA inhibition in ctDNA of a patient with colorectal cancer. Top, CT scans of a patient with metastatic colorectal cancer harboring an LMNA–NTRK1 rearrangement were recorded at baseline (March 2014), at the time of partial response to the pan-TRK March 2014 April 2014 July 2014 inhibitor entrectinib (April 2014), and Entrectinib upon disease progression (July 2014). Bottom, longitudinal analysis of plasma ctDNA collected at different time Copies/mL LMNA–NTRK1 points throughout the treatment. Red 40,000 NTRK1 p.G595R 50 bars, absolute LMNA–NTRK1 copies in 1 mL of plasma; blue and black lines, 30,000 NTRK1 p.G667C NTRK1 p.G595R- and p.G667C-mutated 20,000 alleles (%), respectively. Average ± 40 SD of 3 independent experiments is 10,000 reported. Mutated alleles (%) 4,000 30 3,000 LMNA–NTRK1 2,000 20 1,000 Copies/mL 400 300 10 200 100 0 0 19 MAR 9 APR 23 APR 7 MAY 21 MAY 18 JUN 14 JUL 2014 2014 2014 2014 2014 2014 2014 Entrectinib (RXDX-101, previously known as NMS-E628) entrectinib on an intermittent dosing schedule of 4 days on/3 is a potent pan-TRK, ALK, and ROS1 inhibitor, currently days off for 3 weeks followed by a week break in every 28-day undergoing phase I clinical trials ( 3 ). During treatment with cycle ( 4 ). Treatment was remarkably effective and well tolerated, entrectinib, a patient with metastatic colorectal cancer har- leading to a partial response (PR) with 30% tumor shrinkage boring an LMNA–NTRK1 rearrangement showed a remarka- of multiple liver metastases that was demonstrated by an early ble response. We reasoned that, as it has been shown for most CT scan assessment performed after 30 days of treatment. The targeted agents, response to entrectinib might be limited in clinical response lasted 4 months, followed by the emergence of time due to the emergence of acquired resistance. Nothing drug resistance as evaluated by Response Evaluation Criteria in is presently known about the mechanisms of resistance to Solid Tumor (RECIST) progression ( Fig. 1 , top). entrectinib and consequently further lines of treatment are not available. We postulated that it might be possible to Emergence of NTRK1 Mutations in ctDNA during identify the resistance mechanism(s) while the patient was Entrectinib Treatment being treated by analyzing circulating tumor DNA (ctDNA) To unveil the molecular basis of acquired resistance to and developing a xenopatient (avatar). TRKA inhibition, we analyzed ctDNA, a form of liquid biopsy ( 8 ) we previously optimized to detect and monitor drug RESULTS resistance in patients treated with targeted agents ( 9, 10 ). ctDNA extracted from plasma samples collected before treat- Acquired Resistance to TRKA Inhibition in a ment initiation and at clinical relapse was subjected to molecu- Patient with Colorectal Cancer lar profi ling using the IRCC-TARGET panel, a next-generation A molecular screen identifi ed a genetic rearrangement sequencing (NGS) platform based on 226 cancer-related genes involving exon 10 of NTRK1 and exon 11 of the LMNA genes which we optimized to detect with high sensitivity mutations ( 4 ) in a patient with metastatic colorectal cancer whose dis- in ctDNA ( 10 ). Profi ling of ctDNA at entrectinib resistance ease was intrinsically resistant to fi rst-line FOLFOX, second- revealed two novel NTRK1 genetic alterations in the kinase line FOLFIRI/cetuximab, and third-line irinotecan. We and oth- domain of the protein, p.G595R and p.G667C, which were ers have previously reported that colorectal cancer cell models not detected in ctDNA obtained before initiation of therapy harboring NTRK1 translocations are sensitive to NTRK1 silenc- (Supplementary Tables S1 and S2). To monitor the NTRK1 - ing and to TRKA (protein encoded by the NTRK1 gene) kinase mutated alleles in the patient’s plasma collected throughout inhibition ( 5–7 ). Based on this, the patient was enrolled in the the treatment, droplet digital PCR (ddPCR; refs. 11, 12 ) assays phase I ALKA clinical trial (EudraCT Number 2012-000148-88) were designed for both mutations. As a means of tracking the of the pan-TRK kinase inhibitor entrectinib, a fi rst-in-class drug overall disease, a ddPCR assay was also optimized to detect the currently undergoing clinical testing ( 3 ). The patient received LMNA–NTRK1 rearrangement in ctDNA. JANUARY 2016CANCER DISCOVERY | 37 Downloaded from cancerdiscovery.aacrjournals.org on October 2, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst November 6, 2015; DOI: 10.1158/2159-8290.CD-15-0940 RESEARCH BRIEF Russo et al. A 4,000 Figure 2. Resistance to entrectinib in xenopatient and colorectal cancer cell models carrying NTRK1 Entrectinib translocations. A, bioptic specimen obtained from a thin needle biopsy of a patient with metastatic colorec- Vehicle 60 mg/kg tal cancer harboring an LMNA–NTRK1 rearrangement 3,000 #1 #1 was fi rst implanted subcutaneously in an immunocom- ) 3 #2 #4 promised mouse and then expanded in multiple mice upon successful engraftment. Mice were treated with #3 #5 dosage levels and schedules (60 mg/kg, 4 days/week) #6 2,000 that yielded clinically relevant exposure achievable Treatment #7 in patients. After 3 weeks of treatment, a mouse (#4) in the treated arm relapsed. Blue and red lines, start vehicle- and entrectinib-treated mice, respectively. B, proliferation assay of KM12 (carrying a TPM3– Tumor volume (mm volume Tumor 1,000 NTRK1 rearrangement) R1 cells made resistant to a low dose of entrectinib (300 nmol/L). Cell viability was assessed by measuring ATP content after 5 days