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Translational Cancer Mechanisms and Therapy

Amplification of Wild-type KRAS Imparts Resistance to Crizotinib in MET Exon 14 Mutant Non–Small Cell Lung Cancer

Magda Bahcall, Mark M. Awad, Lynette M. Sholl, Frederick H. Wilson, Man Xu, Stephen Wang, Sangeetha Palakurthi, Jihyun Choi, Elena V. Ivanova, Giulia C. Leonardi, Bryan C. Ulrich, Cloud P. Paweletz, Paul T. Kirschmeier, Masayuki Watanabe, Hideo Baba, Mizuki Nishino, Rebecca J. Nagy, Richard B. Lanman, Marzia Capelletti, Emily S. Chambers, Amanda J. Redig, Paul A. VanderLaan, Daniel B. Costa, Yu Imamura and Pasi A. Jänne
DOI: 10.1158/1078-0432.CCR-18-0876 Published December 2018

Abstract

Purpose: MET inhibitors can be effective therapies in patients with MET exon 14 (METex14) mutant non–small cell lung cancer (NSCLC). However, long-term efficacy is limited by the development of drug resistance. In this study, we characterize acquired amplification of wild-type (WT) KRAS as a molecular mechanism behind crizotinib resistance in three cases of METex14-mutant NSCLC and propose a combination therapy to target it.

Experimental Design: The patient-derived cell line and xenograft (PDX) DFCI358 were established from a crizotinib-resistant METex14-mutant patient tumor with massive focal amplification of WT KRAS. To characterize the mechanism of KRAS-mediated resistance, molecular signaling was analyzed in the parental cell line and its KRAS siRNA-transfected derivative. Sensitivity of the cell line to ligand stimulation was assessed and KRAS-dependent expression of EGFR ligands was quantified. Drug combinations were screened for efficacy in vivo and in vitro using viability and apoptotic assays.

Results: KRAS amplification is a recurrent genetic event in crizotinib-resistant METex14-mutant NSCLC. The key characteristics of this genetic signature include uncoupling MET from downstream effectors, relative insensitivity to dual MET/MEK inhibition due to compensatory induction of PI3K signaling, KRAS-induced expression of EGFR ligands and hypersensitivity to ligand-dependent and independent activation, and reliance on PI3K signaling upon MET inhibition.

Conclusions: Using patient-derived cell line and xenografts, we characterize the mechanism of crizotinib resistance mediated by KRAS amplification in METex14-mutant NSCLC and demonstrate the superior efficacy of the dual MET/PI3K inhibition as a therapeutic strategy addressing this resistance mechanism.

This article is featured in Highlights of This Issue, p. 5785

Translational Relevance

MET exon 14 mutations (METex14) have recently been recognized as a targetable oncogenic driver in non–small cell lung cancer (NSCLC), and these cancers inevitably acquire resistance to MET inhibitors. Although secondary mutations in the MET kinase domain that confer resistance to MET inhibitors have been reported, characterization of mechanisms involving parallel signaling in METex14-mutant NSCLC have not yet been described. Our study describes one such mechanism and proposes a therapeutic strategy to target it.

Introduction

Genotype-directed therapy is the standard of care for patients with advanced non–small cell lung cancer (NSCLC; refs. 1–3). However, the vast majority of patients ultimately develop acquired drug resistance to targeted therapies (4–7). The mechanistic understanding of drug resistance has been instrumental in the development of therapeutic strategies to overcome or prevent acquired resistance (8–11).

MET encodes the tyrosine kinase proto-oncogene c-MET, the receptor for HGF. MET exon 14 mutations (METex14) have only recently been appreciated as relatively frequent alterations in NSCLC (3%–6% of all NSCLC) with sensitivity to tyrosine kinase inhibitors (TKI; refs. 12–15). Exon 14 carries the tyrosine 1003 (Y1003) residue necessary for the binding of the E3 ubiquitin ligase CBL. Amino acid substitution at Y1003 or mutations or deletions in METex14 or its flanking introns that cause in-frame skipping of METex14 eliminates the CBL-binding site and leads to increased MET protein stability and effector signaling (13, 16). Despite dramatic responses seen in MET-amplified or METex14-mutant NSCLC patients treated with MET-directed TKIs, acquired resistance inevitably develops. To date, only a handful of studies have characterized the mechanisms of resistance to MET inhibitors, all of which are attributed to different secondary mutations in the MET tyrosine kinase domain, believed to interfere with adequate binding of the drug to the kinase (4, 17–19).

In this study, we identify acquired wild-type (WT) KRAS amplification in three cases of crizotinib-resistant METex14-mutant NSCLC. Using a cell line (CL) and xenograft derived from one of these patients at the time of crizotinib resistance, we demonstrate the unique mechanisms by which KRAS amplification leads to drug resistance, and develop a therapeutic approach to treat these resistant cancers.

Materials and Methods

Patients

All patient studies were conducted in accordance with the Declaration of Helsinki and the Dana-Farber/Harvard Cancer Center Institutional Review Board (IRB)–approved clinical research correlative protocols, which allow for the collection and reporting of clinical, pathologic, and genomic characteristics from participants. All patients included in the study provided a written informed consent.

Next-generation sequencing, plasma genotyping, and identification of MET exon 14 skipping mutation

Methods for targeted next-generation sequencing (NGS) were previously reported (15). Cell-free circulating tumor DNA was isolated from blood and analyzed using the Guardant360 assay (Guardant Health, Inc.) as previously reported (20). The skipping of exon 14 in MET was initially investigated by RT-PCR, carried out using the QuanTitec Reverse Transcription Kit (Qiagen; see Supplementary Materials and Methods).

FISH analysis

FISH analysis was performed on formalin-fixed, paraffin-embedded (FFPE) tissue using chromosome 7 centromere (CEN7), chromosome 8 centromere (CEN8), and chromosome 12 centromere (CEN12), KRAS, EGFR, BRAF, and MYC bacterial artificial chromosomes labeled as FISH probes (Chromosomescience laboratory, Sappro, Japan; see Supplementary Materials and Methods).

Antibodies, compounds, oligonucleotides, and other reagents

For more details, refer to Supplementary Table S1.

IHC studies

FFPE tissue was subjected to IHC for KRAS (1:200, ab55391; Abcam) or phospho-ERK (1:400, 4370S; Cell Signaling Technology; see Supplementary Materials and Methods).

KRAS copy number and mRNA quantification

RNA and DNA from the frozen cell pellet of processed DFCI358 pleural fluid and DFCI358 CL were extracted using Trizol according to manufacturer's instructions. qRT-PCR was performed using inventoried or Made to Order Assays-on-Demand provided by Applied Biosystems (see Supplementary Materials and Methods).

CL generation and maintenance

DFCI358 CL was derived from a pleural effusion (PE) harvested from a crizotinib-resistant patient (Case #1; see Supplementary Materials and Methods) and maintained in complete RPMI1640. Patient-derived xenograft (PDX) DFCI358 CL (DFCI358 PDX) was established from a freshly resected tumor expanded in a mouse according to the described PDX generation protocol (see Supplementary Materials and Methods).

Hs746T and H596 were a generous gift from the Belfer Institute for Advanced Science (2016). Calu-6 cells were purchased from ATCC (2015). Hs746T were maintained in DMEM supplemented with 10% FBS, streptomycin, and penicillin. Remaining CLs were maintained in RPMI1640 supplemented with 10% FBS, streptomycin, and penicillin. Hs746T were authenticated before banking in 2012 and used for this study immediately after thawing in 2016; Calu-6 were authenticated within 6 months of being used for this study, using the Promega GenePrint 10 System at the RTSF Genomics Core at Michigan State University in 2016. All CLs used in the study were routinely tested for mycoplasma, using the Mycoplasma Plus PCR Primer Set (Agilent).

PC9 (EGFR del19), A549 (KRAS G12S), HCC827 (EGFR del19), and H1975 (EGFR L858R) were maintained in complete RPMI1640. The PC9 cells were obtained from Dr. Nishio (Kinki University, Osaka, Japan; 2005), the others from ATCC. PC9, HCC827, and H1975 were authenticated in 2014; A549 in 2016, and used within 6 months of being authenticated.

MTS growth and viability assay

Growth and inhibition of growth was assessed by MTS assay according to previously established methods (21). All experimental points were set up in 6 to 12 wells and all presented data are representative of several replicates (see Supplementary Methods).

Growth and apoptosis assay

Cells undergoing drug treatments were continuously monitored for growth and apoptosis using the CellEvent Caspase-3/7 Green Detection Reagent (Life Technologies) and Incucyte ZOOM (Essen BioScience; see Supplementary Materials and Methods).

Drug treatment and Western blotting

Cells were treated with drugs at concentrations and for durations specific to each experiment as indicated in figure legends. Western blotting and immunoblotting were done as described previously (10). Blots were developed on Amersham Imager 600 (GE Healthcare Life Sciences; see Supplementary Materials and Methods for details).

siRNA knockdown studies

The ON-TARGETplus Human KRAS SMARTpool siRNA, ON-TARGETplus Non-targeting Pool control and ON-TARGETplus Human MET SMARTpool siRNA (Dharmacon) were used for all siRNA experiments. Drug treatments or IncuCyte assays were conducted 72 hours after transfection or 96 hours after transfection where overnight serum starvation was required (see Supplementary Materials and Methods for details).

KRAS overexpression

For stable KRAS overexpression, Hs746T cells were transduced with a KRAS or empty vector lentiviral construct according to standard procedures (see Supplementary Materials and Methods for details; ref. 22).

Coimmunoprecipitation and active KRAS studies

Cells were lysed in the MB lysis buffer (50 mmol/L HEPES pH 7.5; 50 mmol/L NaCl; 1% glycerol, 0.3% NP-40; 1.5 nmol/L MgCl), containing phosphatase and protease inhibitors. Lysates for the detection of GTP-bound RAS/KRAS were further processed using 300 μg lysate and the Active Ras Detection Kit (Cell Signaling Technology) adhering to the manufacturer's protocol. Mouse anti-p110 (Santa Cruz Biotechnology) was used for coimmunoprecipitation (co-IP). Mouse anti-p110 (Santa Cruz Biotechnology), rabbit anti-KRAS (Santa Cruz Biotechnology), rabbit anti-cRaf (Cell Signaling Technology); mouse anti-RAS (Cell Signaling Technology) were used for immunoblotting (see Supplementary Materials and Methods for details).

Human phospho-RTK array

For experiments in Fig. 2J and Supplementary Fig. S3B, cells were seeded at 4 × 106 cells/100 mm2 dish, let attach overnight, and treated with 0.1 μmol/L of DMSO, each drug alone, or in combination for 6 hours. Using the Human Phospho-RTK Array Kit (R&D Systems) and following the manufacturer's protocol, cells were lysed and 1 mg of protein used to hybridize to antibody membranes (see Supplementary Materials and Methods for details).

Ligand stimulation studies

Seventy-two hours posttransfection, cells were starved for 24 hours in serum-free RPMI1640. Cells in Fig. 4A were treated for 30 minutes at 37°C with FBS, recombinant EGF, insulin, or IGF-1 prepared in serum-free RPMI1640 at the indicated concentrations. Cells in Fig. 4B and C were treated with EGF or FBS at the indicated concentrations for 10 minutes at 37°C. After the 10-minute treatment, EGF/FBS was washed off with PBS and cells were returned to 37°C for another 20 minutes, for a total of 30 minutes. Cells in Fig. 4H were starved and treated with supernatants (normalized to cell number) collected from DFCI358 NS Control or KRAS KD for 30 minutes. Following ligand treatment, cells were washed in ice-cold PBS, lysed in the MB buffer, and processed as outlined elsewhere.

EGFR ligand quantification

For experiments in Fig. 4D and G, 0.75 or 1.25 × 106 cells/dish were plated in 60 mm2 dishes for transfection with control or KRAS siRNA, respectively, and transfected as described previously. Seventy-two hours posttransfection, media were replaced with 3.5 mL fresh complete RPMI1640. Supernatants were collected, filtered, and snap-frozen 24 hours later. Each experiment was done in biological triplicates. For experiments in Fig. 4F, G, and J, cells were treated with 1 μmol/L drug in 3.5 mL complete RPMI1640 for 24 hours. Ligand concentrations were measured in cell culture media using an immunoassay based on the acoustic membrane microparticles (AMMP) technology (23). Cells were trypsinized and counted for the purposes of normalization (see Supplementary Materials and Methods for details).

DFCI358 PDX in vivo efficacy and pharmacodynamics studies

All in vivo mouse studies were performed at the Dana-Farber Cancer Institute (DFCI; Boston, MA), in accordance with the guidelines approved by the Institutional Animal Care and Use Committee of DFCI. All mice were housed in a pathogen-free environment at DFCI animal facility and handled in strict accordance with good animal practice as defined by the office of laboratory animal welfare. Ten-week-old female NOD.Cg-Prkdc< scid>IL2rg< tm1Wjl>/Szj (NSG) mice were purchased from The Jackson Laboratory. DFCI358 PDX was built from patient-derived core biopsy specimen obtained with patient consent under an IRB approved protocol.

Expanded DFCI358 PDX tumors were implanted in the right flank of each mouse. When tumors reached 70 to 150 mm3 or 100 to 200 mm3 for the copanlisib or alpelisib study, respectively, mice were randomized to groups of n = 6 mice/arm (copanlisib study) or n = 5 mice/arm (alpelisib study) and treated for 2 to 3 weeks. Tumor growth was monitored until the average tumor volume (TV) reached 2,000 mm3. Tumor size and body weight were monitored twice weekly. TV was calculated as follows: TV (mm3) = length × width × width × 0.5. Statistical analysis was performed using the Kruskal–Wallis/Dunn post hoc test. P ≤ 0.05 was considered statistically significant. Pharmacodynamics study was performed following a 3-day treatment (see Supplementary Materials and Methods and Supplementary Table S2 for details on drug dosing).

Results

Recurrent amplification of WT KRAS in 3 patients with METex14-mutant NSCLC who developed acquired resistance to crizotinib

We identified 3 METex14 patients treated with crizotinib, all of whom developed amplification of WT KRAS in their drug-resistant tumor (Supplementary Figs. S1 and S2). Patient #1 (MET c.2903_3028+67del193insA) had an initial marked response to crizotinib (Fig. 1A) followed by disease progression within 4 months (Fig. 1A) of crizotinib treatment. Targeted NGS on thoracentesis and a repeat biopsy of an enlarging left cervical lymph node revealed no secondary mutations in MET but revealed marked focal WT KRAS amplification (estimated 55 copies by NGS), which was not present in either of the pre-crizotinib lung or adrenal samples (Fig. 1B). Acquired KRAS amplification was further confirmed by FISH, and a marked acquired increase in KRAS and phospho-ERK expression was demonstrated by IHC (Fig. 1B). Plasma-based cell-free DNA NGS at the time of crizotinib resistance also demonstrated high-level focal KRAS amplification with no other significant copy number gains or genomic mutations in a targeted 73-gene panel (Fig. 1C). MET expression was detected in both the pre-crizotinib and post-crizotinib samples, and none of the samples displayed MET amplification by NGS or FISH (Supplementary Fig. S1).

Figure 1.
Figure 1.

Acquired WT KRAS amplification in 3 patients with MET exon 14 mutant NSCLC at the time of crizotinib resistance. A, Axial CT images of the upper abdomen for Case #1 showing a liver metastasis at baseline (left), which significantly decreased in response to crizotinib at 2 months of therapy (middle). Fused coronal image of the PET-CT scan obtained at 4 months of therapy (right) demonstrated FDG-avid, enlarging mediastinal adenopathy (arrow) and a new loculated left PE (asterisk) from which the DFCI358 CL was derived. B, NGS (left) copy number plots of the KRAS locus on chromosome 12 from two crizotinib-naïve tumors from Case #1 and the crizotinib-resistant supraclavicular lymph node (bottom left) with estimated 55 to 75 copies of KRAS. FISH for KRAS (red) and centromere 12 (CEN12, green) is shown at low and high power for the crizotinib naïve and resistant samples. IHC for KRAS and phospho-ERK (pERK) are also shown. C, Plasma gene copy number for several genes are displayed for Case #1 from blood taken at the time of crizotinib resistance. D, Coronal CT images for Case #2 showing a consolidated mass in the left upper lobe of the lung at baseline (left), which responded to crizotinib at 2 months of therapy (middle); however, progression with regrowth of left lung tumor was noted at 14 months indicative of the development of acquired resistance (right). E, NGS copy number plots of the KRAS locus on chromosome 12 and the EGFR locus on chromosome 7 are shown for the pre- and post-crizotinib samples for Case #2. Post-crizotinib sample shows estimated 25 copies of KRAS in the upper lobe. KRAS IHC before and after crizotinib treatment are also shown. F, KRAS (red) and CEN12 (green) FISH (top left) and EGFR (red) and centromere 7 (CEN7, green) FISH (top right) are shown in the crizotinib-naïve tumor. Dual KRAS/EGFR (red/green, respectively) FISH in the crizotinib-resistant tumor is shown (bottom). G, Axial CT images of the chest for Case #3 showing bilateral lung masses and a nodule at baseline (left), which partially responded to crizotinib (middle), followed by growth of one of the nodules in the right lower lobe (right, arrow), which was biopsied at the time of acquired resistance. H, NGS copy number plots of the KRAS and EGFR loci are shown for the pre- and post-crizotinib samples for Case #3. Post-crizotinib sample shows estimated 21 copies of KRAS. KRAS IHC before and after crizotinib treatment are also shown. FISH for KRAS/CEN12 (red/green, respectively), EGFR/CEN7 (yellow/green, respectively) and dual KRAS/EGFR (red/green) are shown in the crizotinib naïve and crizotinib-resistant tumor samples.

Patient #2 (MET c.2888-20_2888-10del) experienced a dramatic initial response to crizotinib but developed disease progression after 14 months and died shortly thereafter (Fig. 1D). At autopsy, three different crizotinib-resistant lesions were analyzed by NGS (Fig. 1E; Supplementary Fig. S2), none of which contained a MET secondary mutation (data not shown). All three demonstrated evidence of WT KRAS amplification not present in the pre-crizotinib treated tumor, and increased KRAS expression by IHC (Fig. 1E; Supplementary Fig. S2). In addition, they contained variable degree of WT EGFR amplification (Supplementary Fig. S2). Dual color FISH detecting the KRAS and EGFR amplifications further revealed that they were located in different cells within the drug-resistant tumor (Fig. 1F).

Patient #3 (MET c.3028 +1G>T) showed a partial response after 2 months of treatment with crizotinib but progressed 4 months later with a growth of a lung lesion (Fig. 1G). Targeted NGS of a growing lesion revealed no secondary mutations in MET, but compared with the pre-crizotinib tumor specimen, there was evidence of amplification of WT KRAS and EGFR (Fig. 1H). Genomic amplification of KRAS and EGFR was confirmed by FISH and detected in nonoverlapping cells (Fig. 1H).

KRAS amplification uncouples MET from downstream effectors in crizotinib-resistant cells

A PE collected from patient #1 following disease progression was used to generate the DFCI358 CL and at the same time lymph node biopsy was used to also establish the DFCI358 PDX model. The CL retained the high level of KRAS amplification and expression observed in the PE (Fig. 2A). We analyzed the pMET status and downstream signaling in the DFCI358 CL upon crizotinib treatment and observed high levels of activated MET at baseline and complete MET inhibition with as little as 10 nmol/L of crizotinib, implying a MET-independent mechanism of resistance (Fig. 2B). In contrast, MAPK/ERK and AKT pathway activation was only minimally inhibited at well above the clinically achievable concentration of 57 nmol/L free drug (Fig. 2B; ref. 24), demonstrating that the activation of these pathways was partially uncoupled from that of MET. This was consistent with the failure of crizotinib to inhibit cell proliferation even at micromolar concentrations (Fig. 2C), similar to the crizotinib-resistant METex14/PI3K-mutant H596 (homozygous MET c.3251spl+1 G>T; ref. 25; PIK3CA E545K mutation) and in contrast to the gastric crizotinib sensitive METex14-mutant Hs746T (MET c.3082+1G>T) CLs (Fig. 2C). MET siRNA further inhibited proliferation of Hs746T, but not of DFCI358 (Fig. 2D). To investigate the functional involvement of KRAS in crizotinib resistance, we downregulated KRAS using a siRNA (knockdown; KD) and compared these cells to those expressing nonsilencing siRNA control (NS Control). KRAS siRNA treatment was effective in downregulating KRAS expression and activation in the DFCI358 cells to levels observed in the crizotinib-sensitive METex14-mutant Hs746T cells (Fig. 2E; refs. 26, 27). We demonstrated that parental DFCI358, NS control DFCI358, and the DFCI358 PDX CL exhibited high baseline KRAS activation, as evidenced by the levels of GTP-bound KRAS precipitating with the fusion protein GST-Raf1-RBD and that the levels were comparable with those seen in the KRAS G12C-mutant CL Calu-6 (Fig. 2E). In addition, the amplified KRAS DFCI358 cells exhibited upregulation of negative regulators of the RAS and RTK signaling, DUSP6 and SPRY4, respectively, a feature shared with mutant KRAS CLs, including Calu-6. In contrast, the levels of both negative regulators were low in the Hs746T cells and in the KRAS KD DFCI358 derivative. The high GTP-bound state of KRAS at baseline is further indicative of functional involvement of amplified KRAS in the signaling of DFCI358.

Figure 2.
Figure 2.

KRAS-amplified crizotinib-resistant cells exhibit enhanced KRAS/MAPK/ERK signaling. A, Real-time qPCR quantification of WT KRAS copy number and transcript in DFCI358 tumor tissue, patient-derived DFCI358 CL, and nonamplified lung cancer CLs for comparison. B, Western blot analysis of MET inhibition and sustained downstream signaling in DFCI358 CL following 6-hour crizotinib treatment. C, Dose–response curves of crizotinib-resistant DFCI358, H596, and crizotinib sensitive Hs746T METex14-mutant CLs treated with crizotinib for 3 days. D, IncuCyte analysis of proliferation as measured by confluence per well of DFCI358 or Hs746T cells, each transduced with either non-silencing siRNA (NS Control) or MET siRNA pool (MET KD), each at 50 nmol/L siRNA. Error bars represent SEM of nine technical and three biological replicates in a 12-well format. E, Western blot analysis of GTP-bound KRAS or pan RAS following pull-down with active RAS-reactive GST-Raf1-RBD fusion protein in parental, nonsilencing control siRNA (NS Control), or KRAS siRNA transduced DFCI358 (KRAS KD), DFCI358 PDX CL, Hs746T, and Calu-6 CLs. GTP loaded controls illustrate the maximum achievable GTP-bound KRAS levels. Baseline shows GTP-RAS levels without GTP incubation. αTubulin was used as a loading control. F, Western blot analysis of DFCI358 cells treated with dose escalated crizotinib (criz), trametinib (tram), or crizotinib combined with 0.1 μmol/L trametinib, for 6 hours. HSP90 was used as a loading control. G, Dose–response growth and viability curve of DFCI358 cells treated with dose-escalated crizotinib alone or in combination with trametinib for 3 days, assessed by MTS assay. H, Dose–response curve of DFCI358 transduced with NS Control or KRAS siRNA (KRAS KD), treated with dose-escalated crizotinib for 3 days and assessed by MTS assay. I, IncuCyte analysis of proliferation as measured by confluence per well of vehicle or crizotinib treated DFCI358 NS Control or KRAS KD cells. Error bars represent SEM of nine technical and three biological replicates. J, Quantification of phospho-RTK array signals (shown in Supplementary Fig. S3B) following a 6-hour treatment with DMSO, crizotinib (criz), trametinib (tram), or the combination. The mean signal (n = 2) was calculated and normalized to the mean reference signal (n = 6). Error bars represent SD of the mean.

MEK inhibition or KRAS KD fails to restore sensitivity to crizotinib due to activation of PI3K/AKT signaling

Given that the MAPK/ERK pathway is one of the major KRAS effectors, we assessed whether inhibiting MEK alone, using trametinib, could restore sensitivity to crizotinib. Although trametinib completely inhibited pERK1/2 at a concentration of 10 nmol/L (Fig. 2F), treatment with trametinib alone and in combination with crizotinib prominently induced PI3K/AKT, and to a lesser extent JAK/STAT3 pathway signaling (Fig. 2F), but did not increase pS6 levels, the target of the mTOR pathway (Supplementary Fig. S3A). A growth viability assay demonstrated that the sensitivity of the DFCI358 cells to crizotinib in the presence of trametinib was limited (Fig. 2G). Next, we examined whether the KD of KRAS, used as a surrogate for KRAS inhibition, could overcome crizotinib resistance. The viability assay showed a comparable IC50 between the control and the KD cells with crizotinib treatment (Fig. 2H), however, the KRAS KD cells exhibited a substantially reduced growth rate at baseline (Fig. 2I), suggesting strong dependence on amplified KRAS. To identify potential mechanism(s) leading to PI3K/AKT activation following drug treatment in the parental or KRAS KD CLs, we performed a phospho-receptor tyrosine kinase (RTK) array following crizotinib and/or trametinib treatment, and identified the EGFR, HER2, IGF1R, and insulin receptor (IR) as RTKs that remained activated following drug treatment (Fig. 2J; Supplementary Fig. S3B).

To further differentiate between the relative contributions of amplified KRAS and the above identified RTKs to PI3K/AKT pathway induction upon pharmaceutical or siRNA-mediated MAPK/ERK inhibition, and by so doing implicate amplified KRAS in resistance to crizotinib ± trametinib, we compared the signaling responses of DFCI358 NS Control and KRAS KD following pharmaceutical inhibition of MET, MEK, EGFR/HER2, IGF1R/IR, and the combinations thereof using crizotinib, trametinib, lapatinib, and linsitinib, respectively (Supplementary Fig. S3C, 3A–G). Comparing the levels of RTK induction between crizotinib-treated NS Control or KRAS KD as determined by quantification of the immunoblot in Fig. 3A, we observe that KRAS KD rescues from MET inhibition via PI3K/AKT by inducing stimuli upstream of PI3K, namely IRS1 and HER2 (Fig. 3B–D). Despite the relatively higher contribution of these stimuli in prosurvival signaling in KRAS KD cells than in the NS Control, the NS Control cells still exhibit a substantially higher induction of AKT upon the inhibition of MET and MAPK/ERK (Fig. 3E), implicating amplified KRAS in the molecular rewiring and maintenance of prosurvival signaling via PI3K/AKT in these cells. Consistent with this conclusion is the finding that pShc, an adaptor upstream of MAPK/ERK, is induced to higher levels in the NS Control than in the KRAS KD cells (Fig. 3F). Finally, both DFCI358 derivatives induce JAK/STAT signaling in response to MET/MAPK/ERK inhibition (Fig. 3G); however, the cause and effect of this induction remains unclear.

Figure 3.
Figure 3.

Crizotinib/trametinib-treated NS Control DFCI358 and crizotinib-treated KRAS KD DFCI358 induce PI3K/AKT via distinct mechanisms. A, Western blot analysis of DFCI358 transduced with NS Control or KRAS siRNA (KRAS KD) treated with each drug at 0.5 μmol/L, for 6 hours. HSP90 was used as a loading control. B, Quantification of Western blot analysis signals shown in A for indicated markers in crizotinib-treated KRAS KD DFCI358 cells. Ratios of crizotinib-treated KRAS KD to crizotinib-treated NS Control values were calculated to determine fold-change (induction of each marker) upon KRAS KD, which was plotted as Log2 for each marker. C–G, Quantification of Western blot signals shown in A for pHER2 (C), pIRS1 (D), pAKT (E), pShc (F), and pSTAT3 (G). The induction of each marker in response to MET/MAPK/ERK inhibition by pharmaceutical (NS Control) and/or KRAS KD methods relative to MET inhibition only (NS Control) was assessed by calculating the ratios of crizotinib-treated KRAS KD or crizotinib/trametinib-treated NS Control relative to crizotinib-treated NS Control values for each marker. Additional inhibition of the ERBB family (lapatinib) and/or insulin family (linsitinib) receptors was assessed for the relative involvement of these pathways in resistance to MET/MAPK/ERK inhibition. The fold change was expressed as Log2 for each marker and treatment condition. H, Co-IP study using an anti-pan-p110 antibody or IgG control in DFCI358 treated with DMSO, crizotinib (criz), trametinib (tram), crizotinib + trametinib (criz+tram), or copanlisib (copan), each at 0.5 mM for 6 hours. Interactions of p110 with KRAS or c-Raf were assessed by immunoblotting the immunoprecipitate against KRAS or c-Raf antibodies, respectively. Immunoblot against p110 was used to assess total levels of immunoprecipitated p110. Whole cell lysates (WCL) were blotted against targets of interest. I and J, Quantification of IP Western blot signals shown in H, expressed as Log2 of fold change, where fold change is the ratio of each value relative to DMSO-treated control, normalized to total immunoprecipitated p110 levels and corrected for IgG background.

The catalytic subunit of PI3K, p110, can be directly engaged and activated by KRAS (28). To further study the relationship of amplified KRAS and PI3K/AKT signaling, we evaluated the levels of KRAS-bound p110 (pan) using co-IP studies following drug treatments (Fig. 3H). An immunoblot quantification revealed that upon crizotinib treatment, the interaction between KRAS and p110 decreased, consistent with our previous data implicating the MAPK/ERK pathway in crizotinib resistance. In contrast, MEK ± MET inhibition led to enhanced interaction between KRAS and p110, suggesting that the relatively low efficacy of MEK inhibition can be attributed to a compensatory induction of p110 by KRAS. Inhibiting PI3K with copanlisib slightly diminished the p110/KRAS complex levels, possibly accounting for the compensatory increase in MAPK/ERK signaling (Fig. 3I). In addition, p110 was found in complex, directly or indirectly, with c-Raf regardless of drug treatment (Fig. 3J). Our findings suggest that MEK inhibition or KRAS KD lead to compensatory activation of PI3K/AKT signaling, suggesting that this pathway may need to be inhibited to restore crizotinib sensitivity.

KRAS-amplified cells are hypersensitive to growth factor stimulation

To understand how amplified KRAS may govern downstream signaling in the context of MET inhibition, we exposed serum-starved NS Control and KRAS KD DFCI358 to EGF, insulin, or IGF1, the stimulatory ligands for RTKs whose activation was maintained following crizotinib treatment in Fig. 2J (Fig. 4A). NS Control cells exhibited marked activation of pMEK, pERK1/2, and pAKT in response to both low and high concentrations of EGF and FBS, whereas KRAS KD cells demonstrated a substantially attenuated response under the same conditions. Interestingly, EGF at 100 ng/mL not only activated EGFR but also led to massive induction of insulin receptor substrate 1 (IRS1), independently of IGF1R activation. EGF further increased the levels of phosphorylated MET precursor. Finally, although we observed no RTK induction in response to insulin, KRAS KD DFCI358 exhibited enhanced reliance on the PI3K/AKT pathway compared with the NS Control, as evidenced by increased pAKT following IGF1 treatment. In light of these results, we sought to determine the lowest concentration of EGF or FBS sufficient for activation of signaling downstream of KRAS (Fig. 4B and C). Starved NS Control cells exhibited prominent activation of MEK and ERK1/2 with EGF concentrations as low as 0.002 ng/mL, whereas the KRAS KD equivalent required a minimum of 2 ng/mL EGF, a 1,000-fold difference, to fully activate its downstream effectors (Fig. 4B). Similar trends were observed with pMEK following treatment with serially diluted FBS, even though the dynamics of ERK1/2 activation was similar between the NS and KD cells, after accounting for the difference in the absolute pERK1/2 levels (Fig. 4C). These studies suggest that amplified KRAS imparts resistance, in part, by rendering cells hyper-sensitive to ligand stimulation. In light of previously published reports demonstrating that ERK1/2 transcriptionally induces the expression of EGFR ligands, including TGFα, amphiregulin (AREG), and heparin-binding EGF-like growth factor (HB-EGF; refs. 29, 30), we also sought to determine whether amplified KRAS may enhance autocrine signaling in DFCI358. Supernatants from cultured NS Control and KRAS KD DFCI358 cells were subjected to quantification of secreted EGFR ligands AREG, TGFα, epiregulin (EREG), EGF; the MET specific ligand HGF; the HER3/4 ligand neuregulin 1 (NRG1; Fig. 4D). NS Control cells expressed high levels of all of these ligands, and KD of KRAS resulted in a significant decrease in AREG (P < 0.011), TGFα (P < 0.0006), and EREG (P < 0.0001), respectively, which are EGFR ligands shown to promote EGFR recycling, unlike EGF known to induce degradation (31, 32). In contrast, KRAS KD DFCI358 exhibited an increase in secreted EGF (P < 0.005) and HGF (P < 0.0007), respectively, although the absolute EGF levels remained low. Although the mechanistic basis and biological implications of this increase remain unclear, it may represent a compensatory response to the attenuated MAPK/ERK signaling in KRAS KD cells. NRG1 expression remained unchanged in KRAS KD cells.

Figure 4.
Figure 4.

KRAS-amplified cells are hypersensitive to growth factor stimulation. A, Western blot analysis of serum-starved growth factor induced DFCI358 transduced with either NS Control or KRAS siRNA (KRAS KD). αTubulin was used as a loading control. B, Western blot analysis of serum-starved DFCI358 transduced with either NS Control or KRAS siRNA (KRAS KD) and induced with EGF at increasing concentrations. HSP90 was used as a loading control. p-MEK (top) and pERK1/2 (bottom) Western blot signals were quantified and plotted relative to untreated NS Control values. C, Western blot analysis of serum-starved DFCI358 transduced with either NS Control or KRAS siRNA (KRAS KD) and induced with FBS at increasing concentrations. HSP90 was used as a loading control. p-MEK (lower left) and pERK1/2 (lower right) Western blot analysis signals were quantified in Image Studio Lite and plotted relative to the value of untreated NS Control. D, AMMP based quantification of ligands in cell culture media conditioned by NS Control or KRAS KD DFCI358 cells, expressed as mean concentration of three biological and three technical replicates each. Error bars represent SEM. Asterisks denote statistically significant difference. E, Western blot analysis of DFCI358 cells treated with crizotinib (criz), gefitinib (gef), or crizotinib/gefitinib combination, each at 1 μmol/L, for 6 hours. GTP-KRAS levels were determined from a GST-Raf1-RBD pull-down, using the same lysates. HSP90 was used as a loading control. F, AMMP-based quantification of ligands in cell culture media conditioned by DFCI358 cells treated with each drug at 1 μmol/L, for 6 hours, expressed as mean concentration of three technical replicates. Error bars represent SEM. Only ligands and conditions with values above level of detection (LOD) are shown. G, AMMP-based quantification of ligands in cell culture media conditioned by DFCI358 cells treated with each drug at 0.5 μmol/L, for 24 hours, expressed as mean concentration of two to three technical replicates. Error bars represent SEM. Only ligands and conditions with values above LOD are shown. H, Starved DFCI358 cells were treated with PBS (uninduced) or supernatants collected from either DFCI358 NS Control or DFCI358 KRAS KD. Western blot analysis shows induction of pEGFR and pERK1/2 in DFCI358 treated with supernatant from NS Control, whereas pAKT is induced by KRAS KD supernatant. I, Dose–response growth and viability curve of empty vector–transduced (JP1698) and KRAS-FLAG overexpressing (KRAS FL) Hs746T derivatives treated with dose-escalated crizotinib for 3 days, assessed by MTS assay. J, AMMP-based quantification of ligands in cell culture media conditioned by empty vector-transduced (JP1698) or KRAS-FLAG overexpressing (KRAS FL) Hs746T derivatives treated with crizotinib at 0.5 μmol/L, for 24 hours, expressed as mean concentration of two to three technical replicates. Error bars represent standard error of the mean. Only ligands and conditions with values above LOD are shown. Asterisks denote statistically significant difference.

To further examine the role of EGFR activation in crizotinib resistance, we subjected DFCI358 to the combination of the MET inhibitor crizotinib and/or the EGFR inhibitor gefitinib. The combination acutely reduced the levels of all, active MET, EGFR, HER2 (an EGFR dimerization partner), and IRS1, suggesting crosstalk and interdependence between the receptors (Fig. 4E). More importantly, even though all tested RTKs were strongly inhibited by the combination, the levels of GTP-bound KRAS, pMEK, and pERK1/2 remained high, further supporting our observations that KRAS amplification renders cells hypersensitive to upstream stimuli and/or leads to a partial uncoupling of the MAPK/ERK pathway from activated RTKs, akin to mutant KRAS signaling (Fig. 4E). In addition, the quantification of EGFR ligands expressed by DFCI358 following crizotinib/gefitinib treatment demonstrated that both, EGFR and to a lesser extent MET, cooperate with amplified KRAS to promote autocrine signaling by sustaining EGFR ligand production, as evidenced by a decrease in EGFR ligand levels upon inhibition of EGFR and/or MET (Fig. 4F) and upon KRAS KD and/or MET/MEK inhibition (Fig. 4G). Supernatant collected from NS Control cells induced pEGFR/pERK1/2, whereas that from KRAS KD cells induced pAKT signaling in starved DFCI358 as expected (Fig. 4H). Together these data suggest that KRAS amplification partially uncouples RTKs from MAPK/ERK signaling and brings about heightened expression of three EGFR agonists that promote receptor recycling. Moreover, we speculate that following downregulation of KRAS expression, these cells increase HGF and EGF production via an unknown mechanism to activate MET and EGFR/IRS1, as a compensation for the loss of KRAS expression. Finally, a GST–Raf1–RBD fusion protein pull-down showed that in the context of estimated approximately 186-copy KRAS amplification and long-term drug treatment (Supplementary Fig. S4A), especially when compared with an acute drug treatment (Supplementary Fig. S4B), none of the tested drug combinations appreciably decreased the levels of GTP-bound KRAS, despite a modest short-term decrease in pERK1/2 achieved by the gefitinib combinations (Supplementary Fig. S4B). A kinetics analysis of the GTP-KRAS levels in the DFCI358 CL undergoing a combined crizotinib/gefitinib treatment over time further illustrates that the inhibition of KRAS is modest and transient, limited to approximately 1 to 6 hours of drug exposure, and followed by compensatory KRAS reactivation at longer time points (Supplementary Fig. S4C). These data suggest a rapid and dynamic rebound in GTP-KRAS in response to drug pressure, to sustain signaling via MEK or PI3K.

To further study the role of KRAS amplification in crizotinib resistance in METex14-mutant models, we engineered the crizotinib-sensitive Hs746T CL to overexpress WT KRAS (Supplementary Fig. S4D) and evaluated its crizotinib sensitivity. As assessed by the growth and viability assay, KRAS overexpression imparted a degree of resistance to crizotinib in this CL (Fig. 4I). In addition, quantification of EGFR ligands revealed that upon crizotinib treatment, Hs746TKRAS cells sustained higher expression of AREG as compared with the vector-transduced Hs746T (Fig. 4J). The effect of KRAS overexpression in Hs746T is likely less pronounced due to the cellular context and lower relative KRAS expression as compared with KRAS-amplified DFCI358.

Inhibition of PI3K/AKT signaling restores sensitivity to crizotinib in METex14 KRAS-amplified models in vitro and in vivo

A model of crizotinib and crizotinib/trametinib resistance in parental, NS Control, and KRAS KD DFCI358 derivatives proposes possible mechanisms and hints at alternative approaches to target them (Fig. 5). In search of a therapeutic combination to overcome KRAS amplification–mediated crizotinib resistance, we evaluated proliferation and apoptosis of DFCI358 cells during treatment with MET, ERBB family, MEK, PI3K, and IGF1R/IR inhibitors, either alone or in combination. Among the tested treatments, combining crizotinib with the pan PI3K inhibitor copanlisib even at low concentrations (100 nmol/L) potently induced cellular apoptosis (Fig. 6A). Strikingly, even copanlisib alone showed modest pro-apoptotic activity. In comparison, no other crizotinib combination was effective at inducing apoptosis in vitro (Fig. 6A), including with trametinib, which showed only an antiproliferative effect (Fig. 6B). Of note is that alpelisib, a p110α selective inhibitor, was ineffective at this concentration in vitro (Fig. 6A), although some efficacy was observed at higher concentrations when combined with crizotinib in vitro (data not shown). Both DFCI358 and Hs746TKRAS cells responded modestly to single-agent copanlisib and were strongly inhibited by the crizotinib/copanlisib combination (Fig. 6C). The inhibitory effects of the combination were somewhat less pronounced in the Hs746TKRAS, presumably because of our limited ability to overexpress KRAS. We further show that p110 inhibition alone leads to transient suppression of the MAPK/ERK pathway, as evidenced by the temporary decreases in active c-Raf, MEK1/2, and ERK1/2 (Fig. 6D and E).

Figure 5.
Figure 5.

Model of resistance to MET and MET/MAPK/ERK inhibition in DFCI358. A, At baseline, mutant MET in parental DFCI358 cells activates KRAS, which in turn amplifies downstream MAPK/ERK signaling resulting in enhanced transcription and release of EGFR ligands. B, Upon MET inhibition in parental DFCI358 cells, amplified KRAS sustains MAPK/ERK signaling via EGFR-ligand stimulated EGFR and other activated RTKs. C, Upon dual MET/MEK inhibition in KRAS-amplified NS Control DFCI358 cells, cell viability is sustained by the PI3K/AKT pathway activated directly by amplified KRAS. D, With KRAS downregulation and MET inhibition in KRAS KD DFCI358 cells, resulting in diminished MAPK/ERK signaling, cell viability is sustained by the PI3K/AKT pathway activated by RTKs, such as IR/IGF1R, HER2, and EGFR.

Figure 6.
Figure 6.

Inhibition of PI3K/AKT signaling restores sensitivity to crizotinib in METdel14 KRAS-amplified cells. A, IncuCyte analysis of rate of apoptosis normalized to confluence, expressed as the mean apoptosis reached per condition and time point in DFCI358 cells. Error bars, SEM. B, IncuCyte analysis of proliferation as measured by confluence per well of TKI-treated DFCI358 cells. Error bars, SEM (n = 6 or 12). Abbreviations: crizotinib, criz; gefitinib, gef; trametinib, tram; linsitinib, lins; alpelisib, alp; lapatinib, lap; copanlisib, copan. C, Dose–response growth and viability curve of DFCI358 NS Control or KRAS KD and Hs746TJP1698 or Hs746TKRAS FL derivatives treated with dose-escalated crizotinib ± 0.05 or 1 μmol/L copanlisib or dose-escalated copanlisib alone for 3 days, assessed by MTS assay. D, Western blot analysis showing a time course of copanlisib treatment in DFCI358 (250 nmol/L) with respect to the inhibition of downstream effectors. E, Quantification of Western blot analysis signals shown in D for indicated markers. Ratios of phospho signals at each timepoint relative to those at time = 0 were calculated to determine fold-change (suppression of each marker) upon copanlisib treatment, which was plotted as log2 for each marker. F, DFCI358 PDX study comparing the efficacy of each indicated single agent and drug combination. Asterisks denote statistically significant difference. G, Pharmacodynamics of the DFCI358 PDX study evaluating target inhibition following a 3-day treatment. Graph shows quantification of average Western blot analysis signals for indicated markers. Inhibition relative to vehicle average value is expressed as log2 for each marker. HSP90 or KRAS was used as a loading control. H, DFCI358 PDX study comparing the efficacy of each indicated single agent and drug combination. Asterisks denote statistically significant difference.

Informed by our in vitro data, we performed an in vivo study using the DFCI358 PDX and evaluated both efficacy and pharmacodynamics of the crizotinib/copanlisib combination cotargeting MET and PI3K. The crizotinib/copanlisib combination was well tolerated. Although neither of the single agents achieved tumor regression within the course of the study (14 days), the combination of crizotinib and copanlisib was more efficacious than crizotinib alone at inhibiting tumor growth (%TGI of 90.2 vs. 69.4, respectively; Fig. 6F). In vivo efficacy of the combination was lower than expected, we believe, due to suboptimal availability of copanlisib when administered as a mannitol suspension intraperitoneally, a conclusion further supported by the incomplete pAKT inhibition achieved within the pharmacodynamic study (Fig. 6G). In a separate study, the combination of crizotinib and the selective PI3Kα inhibitor alpelisib maintained tumor stasis (%TGI of 93.68 for the combination vs. 31.62 for crizotinib; P ≤ 0.0249) for the duration of the study (21 days; Fig. 6H), further strengthening our conclusions, despite this combination being less efficacious in vitro at low concentrations than crizotinib/copanlisib (Fig. 6A and B and data not shown). We confirmed that PDX tumors retained KRAS amplification and overexpression (Supplementary Fig. S5).

Discussion

METex14-expressing NSCLCs are clinically sensitive to MET TKIs (12–15). Unfortunately, as with many other genotype-directed TKI therapies, the benefit of a single-agent approach is almost always limited by the acquisition of secondary kinase domain resistance mutations and/or activation of compensatory pathways. Here we describe three cases of crizotinib-resistant METex14-mutant NSCLC where resistant tumors presented with a high copy number gain of the WT KRAS allele. We developed and characterized, both in vitro and in vivo, a unique patient-derived model from one of those patients, and implicated amplified KRAS in crizotinib resistance in this patient. We define three hallmarks of KRAS-mediated resistance in this context: (i) relative insensitivity to dual MET/MEK inhibition due to strong compensatory induction of PI3K, (ii) KRAS-induced expression of EGFR ligands and hypersensitivity to ligand-dependent and independent activation, and (iii) reliance on PI3K signaling upon MET inhibition.

Both de novo and acquired KRAS mutations have been shown to impart resistance to single-agent EGFR-targeted therapies in colorectal cancers (23–35) by constitutively activating downstream signaling despite complete inhibition of the driver RTK. Amplified WT KRAS can impart resistance to cetuximab in colorectal cancer (35) and dual KRAS/MET amplification can confer resistance in MET-amplified CLs chronically exposed to MET TKIs (36). WT KRAS amplification has been implicated in the resistance of the ALK-rearranged H3122 CL chronically treated with ALK TKIs in vitro, leading to MAPK pathway reactivation (37).

Both the DFCI358 PE and primary CL contain a massive amplification in KRAS. Intriguingly, the levels of GTP-bound KRAS in DFCI358 were not linearly proportional to the gene copy number and were like those observed in a KRAS-mutant model. Given that overexpression/overactivation of KRAS may be toxic, the RTK/RAS/ERK signaling must be tightly regulated by feedback and feedforward loops (38). Indeed, we observed heightened expression of negative ERK1/2 and RTK regulators, DUSP6 and SPRY4, respectively, in KRAS-amplified DFCI358, DFCI358 PDX, and KRAS-mutant Calu-6, but not in KRAS KD DFCI358 or crizotinib sensitive Hs746T. This finding, in part, explains the nonlinear relationship between the KRAS copy number and the levels of activated KRAS, MEK, and ERK1/2 in DFCI358 seen throughout the study. It is likely that the MAPK/ERK baseline activation in DFCI358 is regulated to a tolerable level that allows cells to bypass RTK inhibition with the minimum amount of upstream stimulus necessary. This is evident in the relatively lower levels of activated RTKs in DFCI358 NS Control, compared with the KRAS KD cells, following serum starvation, as well as upregulation of SPRY4 in KRAS-amplified cells, dampening RTK activation and/or GTP-KRAS formation. Nevertheless, given the large reservoir of inactive KRAS available for compensatory activation in response to RTK inhibition, kinase-based strategies to address KRAS-mediated resistance are likely to pose a challenge. As illustrated by the sustained GTP-KRAS levels following long-term exposure to an array of inhibitor combinations, the compensatory activation of copious KRAS reserves interferes with the effectiveness of most tested drug combinations. Our conclusion is further strengthened by the ability of the crizotinib/gefitinib combination to better inhibit GTP-RAS and ERK1/2 when administered acutely (3 hours), before KRAS rebound thwarts its effectiveness (Supplementary Fig. S4).

Our study provides a line of evidence implicating amplified WT KRAS in crizotinib resistance, in part, by rendering cells hypersensitive to ligand stimulation (Fig. 4A–C and H). This quality of amplified KRAS signaling could, in and of itself, confer resistance to crizotinib by transducing ligand-dependent activation of expressed RTKs even in the absence of appreciable amounts of stimulating ligands. Indeed, the upregulation of EGFR ligands by the MAPK/ERK pathway has long been recognized (30, 39, 40). In colorectal cancer, both TGFα and AREG were overexpressed and shown to promote crosstalk between EGFR and MET, conferring EGFR blockade resistance (41, 42). Conversely, EGF and TGFα were implicated in EGFR-mediated phosphorylation of MET (43, 44). In DFCI358, augmented EGFR ligand production is of particular significance, given the intricate crosstalk involving RTKs of the ERBB family and MET (38, 45, 46). Intriguingly, 2 of the 3 patients (Figs. 1E, F, H; Supplementary Fig. S2) showed evidence of WT EGFR amplification, in nonoverlapping cells with KRAS amplification, suggesting this may be a complementary mechanism of activating EGFR signaling leading to drug resistance, where KRAS-amplified cells activate EGFR-amplified cells via paracrine signaling. Morgillo and colleagues reported that in NSCLC, erlotinib increased the levels of EGFR/IGF1R heterodimer, activating IGF1R and its downstream effectors, thereby compromising the antitumor activity of erlotinib (47). This or a similar mechanism may be partially responsible for the relative long-term ineffectiveness of the crizotinib/gefitinib combination seen in vitro (Supplementary Fig. S4A and S4C).

Our studies implicate KRAS/MAPK/ERK signaling as the main pathway mediating crizotinib resistance in the presence of KRAS amplification. However, because MEK inhibition strongly induces prosurvival PI3K/AKT signaling, in contrast to PI3K inhibition which triggers apoptosis presumably via temporary suppression of MAPK/ERK (Fig. 6D and E; ref. 48), and because all of the ligand-activated RTKs signal through PI3K, concomitant inhibition of MET and PI3K emerged as the most effective treatment strategy both in vitro and in vivo (Fig. 6). Although the number of resistant METex14-mutant cases studied to date is still small requiring a larger study of drug-resistant patients, our findings suggest that acquired WT KRAS amplification may emerge as one of the more common mechanisms of resistance, in addition to MET secondary mutations. Whether the recurrent nature of KRAS amplification observed in METex14 patients is due to an inherent predisposition of these cancers to developing such amplification remains to be determined. Continued systematic genomic studies of tumors from patients with METex14-mutant NSCLC will be necessary to establish the frequency of different acquired resistance mechanisms and to determine what proportion of patients may benefit from an alternative MET inhibitor (in case of a MET secondary mutation) or require a combination treatment approach.

Disclosure of Potential Conflicts of Interest

M.M. Awad is a consultant/advisory board member for Pfizer, Novartis, and Mirati. F.H. Wilson reports receiving commercial research grants from Agios and is a consultant/advisory board member for Loxo Oncology. C.P. Paweletz is a consultant/advisory board member for Dropworks. M. Nishino reports receiving commercial research grants to their institution from Toshiba Medical Systems. R.J. Nagy holds ownership interest (including patents) in Guardant Health. R.B. Lanman holds ownership interest (including patents) in and is an employee of Guardant Health. A.J. Redig reports receiving speakers bureau honoraria from Medtronic and AstraZeneca, and is a consultant/advisory board member for Ariad, Roche, and Boehringer-Ingelheim. P.A. Janne reports receiving commercial research grants from AstraZeneca, Daiichi Sankyo, Astellas Pharmaceuticals, Eli Lilly, and PUMA; holds ownership interest (including patents) in Gatekeeper Pharmaceuticals; and is a consultant/advisory board member for AstraZeneca, Boehringer-Ingelheim, Pfizer, Roche/Genentech, Merrimack Pharmaceuticals, Chugai, Ariad, Ignyta, LOXO Oncology, and Mirati Therapeutics. No potential conflicts of interest were disclosed by the other authors.

Authors' Contributions

Conception and design: M. Bahcall, M.M. Awad, M. Nishino, M. Capelletti, P.A. Jänne

Development of methodology: M. Bahcall, M.M. Awad, M. Xu, S. Palakurthi, J. Choi, E.V. Ivanova, R.B. Lanman, P.A. Jänne

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Bahcall, M.M. Awad, L.M. Sholl, F.H. Wilson, M. Xu, S. Palakurthi, E.V. Ivanova, B.C. Ulrich, C.P. Paweletz, P.T. Kirschmeier, M. Watanabe, H. Baba, M. Nishino, R.J. Nagy, R.B. Lanman, M. Capelletti, A.J. Redig, P.A. VanderLaan, D.B. Costa, Y. Imamura

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Bahcall, M.M. Awad, L.M. Sholl, F.H. Wilson, M. Xu, S. Palakurthi, E.V. Ivanova, R.B. Lanman, M. Capelletti, A.J. Redig, D.B. Costa, P.A. Jänne

Writing, review, and/or revision of the manuscript: M. Bahcall, M.M. Awad, L.M. Sholl, M. Xu, G.C. Leonardi, C.P. Paweletz, M. Nishino, R.J. Nagy, R.B. Lanman, M. Capelletti, A.J. Redig, P.A. VanderLaan, D.B. Costa, P.A. Jänne

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Bahcall, M.M. Awad, S. Wang, G.C. Leonardi, E.S. Chambers

Study supervision: M. Bahcall, M.M. Awad, P.T. Kirschmeier, P.A. Jänne

Other (cell line establishment): J. Choi

Acknowledgments

This work was supported by The American Cancer Society (CRP-17-111-01-CDD to P.A. Jänne), the National Cancer Institute (R01CA135257; R01CA222823 to P.A. Jänne), the Conquer Cancer Foundation of the American Society of Clinical Oncology (to M.M. Awad), the Mugar Family Fund (to P.A. Jänne), and the Goldstein Family Fund (to P.A. Jänne).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

  • Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

  • M. Bahcall and M.M. Awad are co-first authors of this article.

  • Received March 16, 2018.
  • Revision received June 19, 2018.
  • Accepted July 23, 2018.
  • Published first August 2, 2018.

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Amplification of Wild-type KRAS Imparts Resistance to Crizotinib in MET Exon 14 Mutant Non–Small Cell Lung Cancer
Magda Bahcall, Mark M. Awad, Lynette M. Sholl, Frederick H. Wilson, Man Xu, Stephen Wang, Sangeetha Palakurthi, Jihyun Choi, Elena V. Ivanova, Giulia C. Leonardi, Bryan C. Ulrich, Cloud P. Paweletz, Paul T. Kirschmeier, Masayuki Watanabe, Hideo Baba, Mizuki Nishino, Rebecca J. Nagy, Richard B. Lanman, Marzia Capelletti, Emily S. Chambers, Amanda J. Redig, Paul A. VanderLaan, Daniel B. Costa, Yu Imamura and Pasi A. Jänne
Clin Cancer Res December 1 2018 (24) (23) 5963-5976; DOI: 10.1158/1078-0432.CCR-18-0876
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