TRK fusions are found in a variety of cancer types, lead to oncogenic addiction, and strongly predict tumor-agnostic efficacy of TRK inhibition1,2,3,4,5,6,7,8. With the recent approval of the first selective TRK inhibitor, larotrectinib, for patients with any TRK-fusion-positive adult or pediatric solid tumor, to identify mechanisms of treatment failure after initial response has become of immediate therapeutic relevance. So far, the only known resistance mechanism is the acquisition of on-target TRK kinase domain mutations, which interfere with drug binding and can potentially be addressable through second-generation TRK inhibitors9,10,11. Here, we report off-target resistance in patients treated with TRK inhibitors and in patient-derived models, mediated by genomic alterations that converge to activate the mitogen-activated protein kinase (MAPK) pathway. MAPK pathway-directed targeted therapy, administered alone or in combination with TRK inhibition, re-established disease control. Experimental modeling further suggests that upfront dual inhibition of TRK and MEK may delay time to progression in cancer types prone to the genomic acquisition of MAPK pathway-activating alterations. Collectively, these data suggest that a subset of patients will develop off-target mechanisms of resistance to TRK inhibition with potential implications for clinical management and future clinical trial design.
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All genomic results and associated clinical data for all patients in this study are publically available in the cBioPortal for Cancer Genomics at http://cbioportal.org/msk-impact. All relevant cell-free DNA sequencing data are included in the paper and/or supplementary files.
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This study was funded by the National Cancer Institute (NCI) under the MSKCC Support Grant/Core Grant (P30 CA008748) and the R01CA226864 (to M.S. and A.D.). This work was also partially funded by the Cycle for Survival (to A.D.) and LOXO Oncology. The study was also supported by NIH T32 CA009207 (to A.M.S). A.M.S. is a recipient of the ASCO Young Investigator Award. E.C. is a recipient of a MSK society scholar prize.
M.S. is on the Advisory Board of the Bioscience Institute and Menarini Ricerche, has received research funds from Puma Biotechnology, Daiichi-Sankio, Targimmune, Immunomedics, and Menarini Ricerche, is a co-founder of Medendi Medical Travel, and in the past 2 years has received honoraria from Menarini Ricerche and ADC Pharma. A.D. has honoraria from Medscape, OncLive, PeerVoice, Physician Education Resources, Tyra Biosciences, Targeted Oncology, MORE Health, Research to Practice, Foundation Medicine, PeerView, AstraZeneca, Genentech/Roche, Bayer, and has consulting roles at Ignyta, Loxo Oncology, TP Therapeutics, AstraZeneca, Pfizer, Blueprint Medicines, Genentech/Roche, Takeda, Helsinn Therapeutics, BeiGene, Hengrui Therapeutics, Exelixis, and Bayer. D.M.H. reports personal fees from Atara Biotherapeutics, Chugai Pharma, CytomX Therapeutics, Boehringer Ingelheim, and AstraZeneca and research funding from Puma Biotechnology, AstraZeneca, and Loxo Oncology. R.Y. has received research support from GlaxoSmithKline, Novartis and Array and consulting fees from GlaxoSmithKline. J.F.H. has received honoraria from Medscape, the European Society of Medical Oncology, and Axiom Biotechnologies, as well as research funding from Bayer. R.S. has received research funding from Helsinn Therapeutics. M.F.B. has received honoraria for advisory board participation from Roche and research support from Illumina. M.L. has received honoraria for ad hoc advisory board participation from AstraZeneca, Bristol-Myers Squibb, Takeda, and Bayer, and research support from LOXO Oncology (for expanded Archer targeted RNAseq testing) and Helsinn Therapeutics. P.R. has received consulting fees from Novartis.
Peer review information: Joao Monteiro was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Hotspots mutations in KRAS and BRAF confer resistance to TRK inhibitors in patients and preclinical models.
a, Representative scans of Patient 1 at baseline, 4 weeks on larotrectinib treatment (responding) and at progression. Targeted sequencing of the tumor at progression identified a BRAF V600E mutation (red square). b, cfDNA analysis confirmed the emergence of BRAF V600E and identified a subclonal KRAS G12D mutation. c, Emergence of a BRAF V600E mutation in the larotrectinib-resistant PDXs presented in Fig. 1b demonstrated by Sanger sequencing and IHC staining using a BRAF V600E specific antibody to detect the mutant protein. d, Representative scans of Patient 2 at baseline, 4 weeks on LOXO-195 treatment (responding) and at progression. Targeted sequencing of the tumor at progression identified a KRAS G12A mutation (white square). e, cfDNA analysis confirmed the emergence of KRAS G12A. f, Sanger sequencing demonstrating the emergence of a KRAS G12D mutation in a LMNA-NTRK1, NTRK1 G595R positive primary CRC cell line treated with increasing concentrations of LOXO-195 for 4 months until the development of resistance. g, Cell proliferation on the LMNA-NTRK1, NTRK1 G595R and the LMNA-NTRK1, NTRK1 G595R, KRAS G12D primary cell lines treated for 72 hours with increasing concentrations (ranging from 0 to 1,000 nM) of LOXO-195. Data are presented as mean±SD of two biological replicates.
Extended Data Fig. 2 Radiologic response to combined RAF/MEK inhibition in Patient 1 correlates with decreased allele frequency of the TRK fusion in cfDNA.
Graph depicting the allele frequencies of truncal NTRK fusion in the cfDNA of the CTRC-NTRK1 positive pancreatic adenocarcinoma) patient (Patient 1) while treated with LOXO-195 and the combination of dabrafenib and trametinib. The time on treatment, best clinical response (SD: stable disease based on RECIST v1.1 criteria) and the time of progression (POD) for each of the indicated therapeutic regimens are displayed.
Extended Data Fig. 3 TRK inhibition enhances the anti-tumor effect of the combination of RAF and MEK blockade in TRK fusion-positive preclinical models harboring a BRAF V600E mutation.
a, Activity of dual RAF/MEK inhibition (dabrafenib ranging from 50 to 500 nM and trametinib 1 and 5 nM) in the absence (left panel) or presence (right panel) of the TRK inhibitor [larotrectinib or LOXO-195 (25 nM)] on the proliferation of LMNA-NTRK1 and LMNA-NTRK1, NTRK1 G595R CRC cell lines transduced with the BRAF V600E mutation. Two biological replicates were performed. b, Western blot analysis on the same cell lines treated for 4 hours as indicated (larotrectinib/LOXO-195 = 25 nM, trametinib = 5 nM, dabrafenib = 100 nM, the combination of dabrafenib = 100 nM and trametinib = 5 nM or the triple therapy at two different concentrations of larotrectinib/LOXO-195= 10 and 25 nM, respectively). The triple therapy is more potent than the combination of anti RAF/MEK alone in inhibiting MEK, ERK and AKT. Two biological replicates were performed. c, Efficacy of the triple therapy (larotrectinib + debrafenib + trametinib) against the Patient 1-derived PDX that harbors a V600E mutation. The triple therapy is significantly more efficacious than the combination of dabrafenib and trametinib alone in inhibiting tumor growth (P = 0.000001). A minimum of six animals per group [vehicle (n = 7), larotrectinib (n = 6), dabrafenib + trametinib (n = 7) and larotrectinib + dabrafenib + trametinib (n = 6)] were used. Two-tailed unpaired t-test was used to evaluate significant differences in the tumor volumes. Data are presented as mean±SEM.
Extended Data Fig. 4 Radiologic response to combined TRK/MET inhibition in Patient 3 correlates with decreased allele frequency of the targeted alterations in cfDNA.
a, Graph depicting the allele frequencies of the truncal NTRK fusion in the cfDNA of the PLEKHA6-NTRK1 positive cholangiocarcinoma patient (Patient 3) while treated with LOXO-195 and the combination of LOXO-195 and crizotinib. The time on treatment, best clinical response (SD: stable disease based on RECIST v1.1 criteria) and the time of progression (POD) for each of the indicated therapeutic regimens are displayed. b, Copy number plots from this patient demonstrating disappearance of the MET amplification on treatment and reemergence at the time of disease progression.
Extended Data Fig. 5 Dual TRK and MEK blockade inhibits growth of the LOXO-195 resistant LMNA-NTRK1, NTRK1 G595R, KRAS G12D cancer cell line.
a, Western blot from the two colorectal cancer cell lines LMNA-NTRK1, NTRK1 G595R and LMNA-NTRK1, NTRK1 G595R, KRAS G12D, treated as indicated. LOXO-195 (50 nM), MEK-162 (50 nM) or the combination of both drugs (195 + 162) were administered at the indicated time and protein lysates were probed with the indicated antibodies. While LOXO-195 was sufficient to inhibit both phospo-TRK and phospho-ERK in the KRAS wild type cell line, the combination of LOXO-195 and MEK-162 was required for this dual inhibition in the KRAS G12D mutated cell line. Three biological replicates were performed. b, Proliferation assays on the same cell lines (labeled NTRK1 G595R and KRAS G12D, respectively) treated for 72 hours with LOXO-195 (125 nM), MEK-162 (25 nM) or their combination. Data are presented as mean±SD of four biological replicates. Two-tailed unpaired t-test was used to evaluate significant differences in % of viable cells. P values < 0.05 were considered statistically significant.
Extended Data Fig. 6 Radiologic and cfDNA correlates in a LOXO-195 resistant CRC patient treated with the combination of LOXO-195 and trametinib.
Graph depicting the dynamics of select mutations detected in the cfDNA of the LMNA-NTRK1, G595R mutated colorectal cancer patient while treated on targeted therapy (LOXO + tram: LOXO-195 + trametinib). The time on treatment, best clinical response (PR: partial response based on RECIST v1.1 criteria) and the time of progression (POD) for each of the indicated therapeutic regimens are displayed. Representative scans of Patient 2 are presented at baseline and at progression (4 weeks) with the combination of LOXO-195 and trametinib.
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Cocco, E., Schram, A.M., Kulick, A. et al. Resistance to TRK inhibition mediated by convergent MAPK pathway activation. Nat Med 25, 1422–1427 (2019). https://doi.org/10.1038/s41591-019-0542-z
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