A combinatorial strategy for treating KRAS-mutant lung cancer

Abstract

Therapeutic targeting of KRAS-mutant lung adenocarcinoma represents a major goal of clinical oncology. KRAS itself has proved difficult to inhibit, and the effectiveness of agents that target key KRAS effectors has been thwarted by activation of compensatory or parallel pathways that limit their efficacy as single agents. Here we take a systematic approach towards identifying combination targets for trametinib, a MEK inhibitor approved by the US Food and Drug Administration, which acts downstream of KRAS to suppress signalling through the mitogen-activated protein kinase (MAPK) cascade. Informed by a short-hairpin RNA screen, we show that trametinib provokes a compensatory response involving the fibroblast growth factor receptor 1 (FGFR1) that leads to signalling rebound and adaptive drug resistance. As a consequence, genetic or pharmacological inhibition of FGFR1 in combination with trametinib enhances tumour cell death in vitro and in vivo. This compensatory response shows distinct specificities: it is dominated by FGFR1 in KRAS-mutant lung and pancreatic cancer cells, but is not activated or involves other mechanisms in KRAS wild-type lung and KRAS-mutant colon cancer cells. Importantly, KRAS-mutant lung cancer cells and patients’ tumours treated with trametinib show an increase in FRS2 phosphorylation, a biomarker of FGFR activation; this increase is abolished by FGFR1 inhibition and correlates with sensitivity to trametinib and FGFR inhibitor combinations. These results demonstrate that FGFR1 can mediate adaptive resistance to trametinib and validate a combinatorial approach for treating KRAS-mutant lung cancer.

Figures

Extended Data Figure 1. A synthetic lethal RNAi screen identifies different MAPK signaling effectors and FGFR1 as sensitizers to MEK inhibition in KRAS-mutant lung cancer cells

a, Library features and schematic of the TRMPV-Neo vector. b, Schematic outline of the synthetic lethal RNAi screen for identifying sensitizers to trametinib in KRAS-mutant lung cancer cells. c, Clonogenic assay of KRAS-mutant lung cancer cell lines (H23, H460, and H2030) cultured in the presence of increasing concentrations of trametinib. d, Proliferation assay of H23 and H2030 cells in the presence of increasing concentrations of trametinib for 4 passages. Data presented as mean of two independent replicates. e, Immunoblot analysis of KRAS-mutant lung cancer cell lines treated with 25 nM of trametinib for 48 hr. f, g, Scatter plots illustrating the correlation of normalized reads per shRNA between replicates at the beginning of the experiment (d) and replicates at different time points in the absence (left panel) or presence (right panel) of trametinib (25 nM) (e). h, Scatter plot illustrating the fold change in the relative abundance of each shRNA in the library after ten population doublings on doxycycline in the absence or presence of trametinib (25 nM) in H23 cells. Two shRNAs for FGFR1, CRAF, BRAF, and ERK2 were identified as selectively depleted in trametinib-treated cells. For gel source data, see supplementary Fig. 1.

Extended Data Figure 2. Suppression of FGFR1 and different MAPK signaling effectors reduces the proliferation and viability of KRAS-mutant lung cancer cells treated with trametinib

a, Quantification of fluorescent cells in competitive proliferation assays in H2030 (upper) and A549 (lower) cells transduced with non-targeting control (Ren) or the indicated shRNAs. Data presented as mean (n = 2). Unpaired two-tailed t-test. ns: not significant, *P<0.05, **P<0.01. b, Immunoblot of H23 and H2030 cells pretreated with 25 nM trametinib for various times and subsequently treated with 200 nM trametinib for 2 hours. c, Immunoblot of H23 cells transduced with doxycycline-inducible shRNAs targeting CRAF and BRAF and treated with trametinib (25 nM) and doxycycline for the times shown. H23 cells were pretreated with trametinib for 4 days, followed by treatment with doxycycline and trametinib for 4 days. d, Clonogenic assay of H23 cells transduced with BRAF, CRAF, ERK2, and non-targeting control shRNAs, and cultured with DMSO or trametinib (25 nM) for 10 days. Relative growth of DMSO- (grey bars) and trametinib-treated cells (blue and red bars) is shown (right). Data presented as mean ± s.d. (n = 3). For gel source data, see supplementary Fig. 1.

Extended Data Figure 3. ERK inhibitor SCH772984 enhances the antiproliferative effects of trametinib in KRAS-mutant lung cancer cells

a, Clonogenic assay of H2030 (upper) and H460 (lower) cells treated with increasing concentrations of trametinib, ERK inhibitor SCH772984, or their combination as indicated. Percent inhibition at each concentration of the drugs in H23, H2030, and H460 cells is presented (right). Data presented as mean of three independent experiments (n = 3). b, Immunoblot analysis of H2030 cells treated with trametinib (25 nM), SCH772984 (500 nM), or their combination for the times shown. H2030 cells were pretreated with trametinib for 4 days, followed by treatment with SCH772984 and trametinib for 2 days. c, Cell viability of H23, H2030, and H460 cells treated with increasing doses of trametinib, ERK inhibitor SCH772984, or their combination for 10 days. Data presented as mean ± s.d. (n = 3). The concentration of trametinib that inhibited cell proliferation by 50% (GI50) was calculated in the absence or the presence of increasing concentrations of SCH772984 (bottom). For gel source data, see supplementary Fig. 1. Source data for Extended Data Figure 3.

Extended Data Figure 4. Feedback activation of FGFR1 signaling leads to adaptive resistance to trametinib in KRAS-mutant lung cancer cells

a, Immunoblot analysis of KRAS-mutant lung cancer cell lines H23 and H2030 treated with 25 nM trametinib for various times. b, c, d, qRT-PCR for FGFR1 and FGF2 in A549 (b), H2030 (c) and H460 (d) cells treated with trametinib for the indicated times. Data presented as mean normalized for FGFR1 and FGF2 expression ± s.d. (n = 3). e, Immunoblot analysis of A549, H2030, and H358 cells treated with trametinib (25 nM) for various times. f, Quantification of fluorescent cells in competitive proliferation assays in A549, H358, and H460 cells transduced with doxycycline-inducible non-targeting control (Ren) or FGFR1 shRNAs. Data presented as mean ± s.d. (n = 3). g, qRT-PCR for FGFR1 in H23 cells transduced with non-targeting control and FGFR1 shRNAs. Data presented as mean normalized for FGFR1 expression ± s.d. (n = 3). h, Quantification of fluorescent cells in competitive proliferation assays in A549 cells transduced with non-targeting control (Ren) or the indicated shRNAs. Data presented as mean ± s.d. (n = 3). i, qRT-PCR for FGFR2, FGFR3, and FRS2 in A549 cells transduced with non-targeting control, FGFR2, FGFR3 and FRS2 shRNAs. Data presented as mean normalized for FGFR2, FGFR3, and FRS2 expression ± s.d. (n = 3). b–d, paired two-tailed t-test. f–i, unpaired two-tailed t-test. ns: not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For gel source data, see supplementary Fig. 1.

Extended Data Figure 5. Trametinib-induced phosphorylation of FRS2 predicts sensitivity to MEK and FGFR1 combined inhibition

a, Competitive proliferation assays in the indicated KRAS-mutant cancer cell lines transduced with doxycycline-inducible non-targeting control (Ren) or FGFR1 shRNAs. Data presented as mean ± s.d. (n = 3). b, A panel of lung (H1975, H1650, Ludlu-1, H1703, and H1299), pancreas (MIAPACA, PANC1), and colorectal (SW620, SW480, and DLD1) cancer cell lines were treated with 25 nM trametinib for various times. Lysates were subject to immunoblot analysis with the indicated antibodies. c, Scatter plot illustrating the correlation between depletion of FGFR1 shRNAs-expressing cells and fold change in FRS2 phosphorylation following trametinib treatment in human cancer cells lines (n = 15). d, Representation of the fold change in FRS2 phosphorylation following treatment with trametinib for 12 days in human cancer cell lines (n = 15). a, d, unpaired two-tailed t-test. c, two-tailed pearson’s correlation. **P<0.01, ***P<0.001, ****P<0.0001. For gel source data, see supplementary Fig. 1.

Extended Data Figure 6. Trametinib in combination with ponatinib synergizes at inhibiting cell proliferation of KRAS-mutant lung cancer cells

a, Serum starved H23 (left panel) and 3T3 (right panel) cells were pre-treated with increasing concentration of ponatinib for 24 hr (1, 30, 100, and 300 nM), followed by stimulation with FGF2 (50 ng/ml) for 10 min. Immunoblot analysis for the indicated antibodies is shown. b, Immunoblot analysis of H2030 cells treated with trametinib (25 nM), ponatinib (750 nM), or their combination for the times shown. Cells were pretreated with trametinib for 4 days, followed by co-treatment with ponatinib and trametinib for 2 days. c, Clonogenic assay of H2030, A549, H2009, and H460 cells treated with increasing concentrations of trametinib, ponatinib, or their combination as indicated. d, Percentage of cell growth inhibition at each concentration of trametinib, ponatinib, or their combination in A549, H2009, and H460 cells after is shown. Data presented as mean of three independent experiments (n = 3). e, Combination Index (CI) scores for H23, H2030, A549, H2009, and H460 cells treated with trametinib in combination with ponatinib at the indicated concentrations. Each CI score represents data from at least three independent experiments. For gel source data, see supplementary Fig. 1. Source data for Extended Data Figure 6.

Extended Data Figure 7. Distinct FGFR1 inhibitors sensitize murine and human KRAS-mutant cancer cells to trametinib

a, Clonogenic assay of a murine lung cancer cell line harboring KrasG12D and Trp53R270H mutations (KP cell line), and human KRAS-mutant pancreatic cancer cell lines (MIAPACA and PANC1). Tumor cells were cultured with increasing concentrations of trametinib, ponatinib, or their combination as indicated. b, Percentage of cell growth inhibition at each concentration of trametinib, ponatinib, or their combination in KP, MIAPACA, and PANC1 cells is shown. Data presented as mean of three independent replicates (n = 3). c, Combination Index (CI) scores for KP, MIAPACA, and PANC1 cells treated with trametinib in combination with ponatinib at the indicated concentrations. Each CI score represents data from at least three independent experiments (n = 3). d, Clonogenic assay of H23, H2030, and H460 cells cultured with increasing concentrations of trametinib alone or in combination with FGFR1 inhibitors BGJ398 (1.5 µM) or AZD4547 (2 µM). e, Percentage of cell growth inhibition at each concentration of trametinib alone or in combination with BGJ398 (1.5 µM) or AZD4547 (2 µM) in H23, H2030, and H460 cells is shown. Data presented as mean of three independent replicates (n = 3). Source data for Extended Data Figure 7.

Extended Data Figure 8. The magnitude of trametinib-induced FRS2 phosphorylation correlates with the sensitivity to trametinib and FGFR1 combined inhibition in human cancer cells

a, Dot plot illustrating the sensitivity increase to trametinib after the treatment with AZD4547 (2.5 µM) in a panel of KRAS mutant (n=15) and KRAS wild type (n=15) cancer cell lines. Data presented as mean of two independent replicates (n = 2). b, Scatter plot illustrating the correlation between fold increase in sensitivity to trametinib after treatment with AZD4547 (2.5 µM) or ponatinib (100 nM) and fold change in FRS2 phosphorylation following trametinib treatment in a panel of human cancer cells lines. c, Immunoblot analysis of a panel of human cancer cells treated with trametinib (25 nM) for 6 days. a, unpaired two-tailed t-test. b, two-tailed pearson’s correlation. ns: not significant, **P<0.01, ***P<0.001, ****P<0.0001. For gel source data, see supplementary Fig. 1.

Extended Data Figure 9. Ponatinib prevents trametinib-induced reactivation of MAPK and PI3K signaling. Upregulation of distinct RTKs in KRAS-mutant lung cancer cells after trametinib treatment

a, Immunoblot analysis of H2030 transduced with PTEN and non-targeting control shRNAs, and treated with trametinib (25 nM) for the times shown. b, Clonogenic assay of H2030 (left) and H460 (middle) cells transduced with PTEN and non-targeting control shRNAs. Cells were treated with ponatinib alone (300 nM) or in combination with trametinib at the indicated concentrations. Quantification of the relative cell growth of H460 cells is shown (right). Data presented as mean of two independent experiments. c, Immunoblot analysis of H2030 transduced with PTEN and non-targeting control shRNAs, and treated with trametinib (25 nM) alone or in combination with ponatinib (750 nM) for the times shown. PTEN suppression did not affect ERK signaling or its inhibition following trametinib treatment but instead activated AKT and, more importantly, attenuated the ability of ponatinib to suppress trametinib-induced increase in pAKT. d, AnnexinV/PI double staining assay of H23 cells treated with vehicle, trametinib (25 nM) alone or in combination with ponatinib (300 nM) or SCH772984 (1 µM) for the times shown (n = 3). e, f, qRT-PCR for EGFR, MET, and ERBB2 in H23 (e) and H2030 (f) cells treated with trametinib for 0, 2, and 4 days. Data presented as mean normalized for EGFR, MET, and ERBB2 expression ± s.d. (n = 3). g, Immunoblot analysis of H23 cells treated with 25 nM of trametinib for various times. h, Immunoblot analysis of serum starved H2030 cells pre-treated with 500 nM or 1 µM of gefitinib, crizotinib, CP-724714, or afatinib for 12 hr, followed by stimulation with EGF, HGF, NRG1, or their combination (50 ng/ml) for 10 min. b, e, f, unpaired two-tailed t-test. ns: not significant, *P<0.05, **P<0.01, ***P<0.001. For gel source data, see supplementary Fig. 1.

Extended Data Figure 10. Unresponsiveness of KRAS-mutant lung cancer cells to MEK inhibitor trametinib is predominantly mediated by feedback activation of FGFR1 signaling

a, Clonogenic assay of H23 and H2030 cells treated with increasing concentration of trametinib alone or in combination with 500 nM crizotinib, gefitinib, CP-724714, and afatinib, or 300 nM ponatinib. Percent inhibition at each concentration of the drugs in H23, H460, and H2030 cells is presented (right). Data presented as mean of at least two independent experiments (n = 2). b, Combination Index (CI) scores for H23, H460, and H23 cells treated with trametinib in combination with crizotinib, gefitinib, CP-724714, afatinib, and ponatinib at the indicated concentrations. Each CI score represents data from at least two independent experiments (n = 2). c, Immunoblot of H23 and H2030 treated with trametinib (25 nM), crizotinib (1 µM), gefitinib (1 µM), CP-724714 (1 µM), and ponatinib (750 nM) for 48 hours. d, Immunoblot analysis of H2030 treated with trametinib (25 nM), crizotinib (1 µM), gefitinib (1 µM), CP-724714 (1 µM), ponatinib (750 nM), or their combination for the times shown. Cells were pretreated with trametinib for 4 days, followed by co-treatment with RTK inhibitors and trametinib for 2 days. For gel source data, see supplementary Fig. 1. Source data for Extended Data Figure 10.

Extended Data Figure 11. Suppression of FGFR1 cooperates with trametinib to inhibit growth of KRAS-mutant lung tumors

a, b, Mice bearing H23 (a) or H2030 (b) xenografts transduced with FGFR1 or non-targeting control shRNAs were treated with either vehicle or trametinib (3 mg/kg). For H23 xenografts, a waterfall representation of the best response for each tumor is shown (n = 8 per group) (a). For H2030 xenografts, the tumor volumes are shown as a function of time after treatment. Error bars represent mean ± s.e.m. (n ≥ 4 per group) (b). c, Mice bearing A549 and H2122 xenografts, and JHU-LX55a patient-derived xenograft tumors were treated with vehicle, trametinib (3 mg/kg), ponatinib (30 mg/kg), or both drugs in combination. A waterfall representation of the best response for each tumor is shown. (n ≥ 6 per group). d, Body weight of mice bearing A549 xenografts and treated with vehicle, trametinib (3 mg/kg), ponatinib (30 mg/kg), or both drugs in combination for the indicated times (n ≥ 6 per group). e, KrasG12D; Trp53−/− genetically engineered mice harboring lung adenocarcinomas were treated with vehicle, trametinib (3 mg/kg), ponatinib (30 mg/kg), or both drugs in combination for 7 weeks. A waterfall representation of the response for each tumor after 7 weeks of treatment is shown (n ≥ 5). f, Representative hematoxylin and eosin stains of pancreatic tumor tissue resulting from orthotopic transplantation of GEMM-KPCflox/+ PDAC organoids. Mice were treated with vehicle, trametinib (3 mg/kg), ponatinib (30 mg/kg), or both drugs in combination. A black asterisk indicates necrosis. g, Immunoblot analysis of tumor tissue from mice bearing JHU-LX55a patient-derived xenografts treated with vehicle, trametinib (3 mg/kg), ponatinib (30 mg/kg), or both drugs in combination for 18 days. a–c, e, Unpaired two-tailed t-test. ns: not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For gel source data, see supplementary Fig. 1. Source data for Extended Data Figure 11.

Figure 1. Suppression of MAPK signaling effectors and FGFR1 sensitizes KRAS-mutant lung cells to trametinib

a, Relative abundance of each shRNA in the library in vehicle- or trametinib-treated H23 cells after ten population doublings on doxycycline. The mean of three (vehicle) and two (trametinib) replicates is plotted. Positive and negative controls included shRNAs targeting RPA1 and CDK11A (Red circles), and renilla (REN) luciferase (Green circles). b, Quantification of fluorescent cells in competitive proliferation assays in H23 cells transduced with non-targeting control (Ren) or the indicated shRNAs. Data presented as mean (n = 2). ns: not significant, *P<0.05, **P<0.01 (unpaired two-tailed t-test). c, Immunoblot of KRAS-mutant lung cells treated with 25 nM trametinib for various times. d, Immunoblot of H23 cells transduced with a doxycycline-inducible shRNA targeting ERK2 and treated with trametinib (25 nM) and doxycycline for the times shown. e, Immunoblot of H23 cells treated with trametinib (25 nM), SCH772984 (500 nM), or their combination for the times shown. f, Clonogenic assay of H23 cells treated with trametinib, ERK inhibitor SCH772984, or their combination as indicated. (n = 3). g, Immunoblot of KRAS-mutant lung cancer cells treated with 25 nM trametinib for various times. For gel source data, see supplementary Fig. 1.

Figure 2. Feedback activation of FGFR1 mediates adaptive resistance to trametinib in KRAS-mutant lung cells

a, qRT-PCR for FGFR1 and FGF2 in H23 cells treated with trametinib for the indicated times (n = 3). b, Immunoblot of H23 cells treated with 25 nM of trametinib for various times. c, Immunoblot of H23 cells transduced with a doxycycline-inducible shRNA targeting FGFR1 and treated with trametinib (25 nM) and doxycycline for the times shown. d, Quantification of fluorescent cells in competitive proliferation assay in H23 and H2030 cells transduced with doxycycline-inducible non-targeting control (Ren) or FGFR1 shRNAs (n = 3). e, Clonogenic assay of H23 cells transduced with FGFR1 and non-targeting control shRNAs, and cultured with DMSO or trametinib (25 nM). Relative growth of DMSO- (grey bars) and trametinib-treated cells (blue and red bars) is shown (right) (n = 3). f, g, Quantification of fluorescent cells in competitive proliferation assays in H23 (f) and the indicated lung cancer cells (g) transduced with doxycycline-inducible non-targeting control (Ren (Renilla)) or the indicated shRNAs (n = 3). a, Paired two-tailed t-test. d, f, g, Unpaired two-tailed t-test. Data presented as mean ± s.d. ns: not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For gel source data, see supplementary Fig. 1.

Figure 3. Ponatinib synergizes with trametinib at inhibiting cell proliferation of KRAS-mutant lung cells

a, Clonogenic assay of H23 cells treated with trametinib, ponatinib, or their combination as indicated. Percent inhibition at each concentration of the drugs in H23 and H2030 cells is presented (right). Data presented as mean of three independent experiments. b, Immunoblot of H23 cells treated with trametinib (25 nM), ponatinib (750 nM), or their combination for the times shown. c, Dot plot illustrating the sensitivity increase to trametinib after treatment with ponatinib (100 nM) in a panel of KRAS mutant (n=15) and KRAS wild-type (n=15) cancer cell lines. Data presented as mean of two independent replicates. d, Quantification of the relative growth of H2030 cells transduced with PTEN and non-targeting control shRNAs, and treated with ponatinib (300 nM) in combination with trametinib (1, 5, and 25 nM). Data presented as mean of two independent replicates. e, Quantification of AnnexinV/PI double positive cells in H23 cells treated with trametinib (25 nM), ponatinib (300 nM), SCH772984 (1 µM) or their combination for the times shown (n = 3). f, Quantification of the relative growth of H23 cells treated with trametinib alone or in combination with 500 nM crizotinib, gefitinib, CP-724714, afatinib, or 300 nM ponatinib (n = 3). g, Immunoblot of H23 cells pretreated with trametinib (25 nM) for 4 days, followed by treatment with trametinib (25 nM) alone or in combination with crizotinib (1 µM), gefitinib (1 µM), CP-724714 (1 µM) and ponatinib (750 nM) for 2 days. c–f, Unpaired two-tailed t-test. Error bars represent mean ± s.d. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For gel source data, see supplementary Fig. 1. Source data for Figure 3.

Figure 4. Suppression of FGFR1 in combination with trametinib leads to tumor regression of KRAS-mutant lung tumors

a, Tumor volumes of mice bearing A549 and H2122 xenografts, and JHU-LX55a patient-derived xenograft tumors and treated with vehicle, trametinib (3 mg/kg), ponatinib (30 mg/kg), or both drugs in combination for the indicated times. Error bars represent mean ± s.e.m. (n ≥ 6 per treatment group). b, c, d, Representative µCT images of the lungs of KrasG12D; Trp53−/− genetically engineered mice treated with vehicle, trametinib (3 mg/kg), ponatinib (30 mg/kg), or both drugs in combination for 3 and 7 weeks. Lung tumors are indicated by yellow arrows and red asterisks mark the hearts (b). A waterfall representation of the response for each tumor after three weeks of treatment is shown. (n ≥ 5 per group). (c). Representative hematoxylin and eosin stains are shown. A black asterisk indicates necrosis (d). e, Kaplan-Meier survival analysis of mice bearing pancreatic tumors resulting from orthotopic transplantation of GEMM-KPCflox/+ PDAC organoids and treated as in b (n ≥ 4 per group) (log-rank test). a, c, Unpaired two-tailed t-test. ns: not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Source data for Figure 4.

Figure 5. Trametinib induces FGFR1 signaling in KRAS-mutant lung tumors

a, Tumor tissue from JHU-LX55a patient derived xenografts treated with vehicle, trametinib (3 mg/kg), ponatinib (30 mg/kg), or both drugs in combination for 18 days was evaluated by IHC for phospho-FRS2, phospho-ERK, and phospho-AKT. b, Paired tumor biopsies from patients having KRAS-mutant lung adenocarcinomas (before and after treatment with the MEK inhibitor trametinib) were evaluated by IHC for phospho-FRS2.