Resistance to TRK inhibition mediated by convergent MAPK pathway activation

Abstract

TRK fusions are found in a variety of cancer types, lead to oncogenic addiction, and strongly predict tumor-agnostic efficacy of TRK inhibition^1-8. 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 inhibitors^9-11. 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.

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 1,000nM) 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= 25nM, trametinib= 5nM, 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 (50nM), MEK-162 (50nM) 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 (125nM), MEK-162 (25nM) 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.

Fig. 1:. Alterations in the MAPK pathway or an upstream receptor tyrosine kinase confer resistance to TRK inhibitors in patients and preclinical models.

a, Schematic showing acquired BRAF V600E and KRAS G12D mutations in a CTRC-NTRK1-positive pancreatic adenocarcinoma patient with acquired resistance to the first-generation TRK inhibitor, larotrectinib. b, Tumor growth of larotrectinib-sensitive patient-derived xenografts established from this patient’s tumor treated with larotrectinib (200mg/Kg, 5 days/week). Genotyping at the time of acquired resistance identified outgrowth of a BRAF V600E-positive clone. c, Western blot and cell viability assay of a TPR-NTRK1, NTRK1 G595R pancreatic cancer cell line with ectopic expression of BRAF V600E and treated with 50nM of LOXO-195 for 24 (WB) or 72 (cell viability) hours. Total and phosphorylated proteins detected are indicated. Two biological replicates were performed for each experiment. d, Schematic showing presence of KRAS G12A and G12D mutations in a LMNA-NTRK1 fusion-positive colorectal cancer patient with acquired resistance to LOXO-195. Note that KRAS G12D emerged in cfDNA upon further disease progression (17 months on LOXO-195 therapy). e, f, Western blot for MAPK effectors and cell proliferation curves of a LMNA-NTRK1 (e) and a LMNA-NTRK1, NTRK1 G595R (f) colorectal cancer cell lines with ectopic expression of KRAS G12A and G12D, treated as indicated. Data are presented as mean ± SD. Two-tailed unpaired t-test was used to evaluate significant differences in % of viable cells. * indicates differences that were considered statistically significant (P<0.05). Exact P values are P=0.000000000000001 for the LMNA-NTRK1 cell line and P=0.000000001115265 for the LMNA-NTRK1, NTRK1 G595R cell line. Two biological replicates were performed for each experiment. g, Schematic showing acquired MET amplification in a PLEKHA6-NTRK1 fusion-positive cholangiocarcinoma patient with acquired resistance to entrectinib. h, Representative fluorescence in situ hybridization (FISH) and i, Immunohistochemistry (IHC) images of the pre- and post-entrectinib tumor biopsies from this patient. Lower panels show confirmed acquisition of MET amplification in the post-entrectinib sample (h) and increased MET and pERK staining by IHC (i). FISH and IHC were performed two independent times and similar results were obtained.

Fig. 2:. Tailored combinatorial therapies are effective against tumors that developed bypass resistance to TRK inhibitors.

a,b, upper panel, Graph depicting the dynamics of select mutations detected in the cfDNA of the CTRC-NTRK1 positive pancreatic adenocarcinoma (Patient 1, a) and PLEKHA6-NTRK1 positive cholangiocarcinoma (Patient 3, b) patients while treated with a series of targeted therapies. middle panel, The time on treatment, best clinical response achieved (PR: partial 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. Lower panel, Representative scans from the patients at baseline and on treatment with the combination of dabrafenib + trametinib (a) and LOXO-195 + crizotinib (b), respectively.

Fig. 3:. Dual TRK and MEK blockade is required to inhibit tumor growth in TRK fusion-positive models that acquired MAPK alterations.

a, Western blots from the two colorectal cancer cell lines LMNA-NTRK1, NTRK1 G595R and LMNA-NTRK1, NTRK1 G595R, KRAS G12D, treated as indicated. LOXO-195 (50nM), trametinib (10nM) or the combination of both drugs (195 + tram) 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 trametinib 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 (125nM), trametinib (2nM) 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. c, In vivo efficacy of LOXO-195 (100mg/Kg BID 5 days a week), trametinib (1mg/kg 4 days a week) or their combination on xenofgrafts established from the LMNA-NTRK1, NTRK1 G595R, KRAS G12D cell line (vehicle n=5, LOXO-195 n=6, trametinib n=5, LOXO-195 + trametinib n=6). d, In vivo efficacy of LOXO-195 (100mg/Kg BID 5 days a week), trametinib (3mg/kg 4 days a week) or their combination on PDXs established from the KRAS G12A positive liver biopsy collected at the time of LOXO-195 progression from patient 2 (Figure 1d; vehicle n=4, LOXO-195 n=5, trametinib n=5, LOXO-195 + trametinib n=5). e, In vivo efficacy of larotrectinib (200mg/kg daily 5 days a week), trametinib (1mg/kg daily 4 days per week) and the combination of both drugs in larotrectinib-resistant PDXs established from Patient 1 (Patient 1-derived PDX, BRAF V600E, Figure 1a; vehicle n=8, larotrectinib n=8, trametinib n=8, larotrectinib + trametinib n=7). f, In vivo efficacy of larotrectinib (200mg/kg daily 5 days a week), trametinib (1mg/kg 4 days a week) and the combination of both drugs in larotrectinib-sensitive PDXs established from Patient 1 (Patient 1-derived PDX, Figure 1a; note that trametinib was also tested in combination with larotrectinib at half of the dose: 0.5mg/kg 4 days a week, orange line; vehicle n=6, larotrectinib n=8, trametinib n=7, larotrectinib + trametinib (1mg/kg) n=7, larotrectinib + trametinib (0.5mg/kg) n=7). Combination therapy prevents the development of primary or acquired resistance (ongoing at 3 months). Two-tailed unpaired t-test was used to evaluate significant differences in the tumor volumes. Data are presented as mean±SEM. P values <0.05 were considered statistically significant.