Abstract
FGFR1 was recently shown to be activated as part of a compensatory response to prolonged treatment with the MEK inhibitor trametinib in several KRAS-mutant lung and pancreatic cancer cell lines. We hypothesize that other receptor tyrosine kinases (RTK) are also feedback-activated in this context. Herein, we profile a large panel of KRAS-mutant cancer cell lines for the contribution of RTKs to the feedback activation of phospho-MEK following MEK inhibition, using an SHP2 inhibitor (SHP099) that blocks RAS activation mediated by multiple RTKs. We find that RTK-driven feedback activation widely exists in KRAS-mutant cancer cells, to a less extent in those harboring the G13D variant, and involves several RTKs, including EGFR, FGFR, and MET. We further demonstrate that this pathway feedback activation is mediated through mutant KRAS, at least for the G12C, G12D, and G12V variants, and wild-type KRAS can also contribute significantly to the feedback activation. Finally, SHP099 and MEK inhibitors exhibit combination benefits inhibiting KRAS-mutant cancer cell proliferation in vitro and in vivo. These findings provide a rationale for exploration of combining SHP2 and MAPK pathway inhibitors for treating KRAS-mutant cancers in the clinic.
Introduction
KRAS mutations occur in about 30% of all cancers and account for approximately one million cancer-related deaths per year worldwide (1). Direct inhibition of mutant KRAS largely remains a pharmacologic challenge and inhibiting the downstream mitogen-activated protein kinase (MAPK) signaling pathway with MEK inhibitors (MEKi) had limited clinical efficacy in KRAS-mutant tumors (2–4). One possible explanation is reactivation of the MAPK pathway due to the alleviation of the negative feedback modulation (5, 6), which is commonly observed in KRAS-mutant cells (7–9). In such a scenario, prevention of pathway reactivation would maintain pathway suppression thereby improving antitumor efficacy. Strategies to block pathway reactivation have focused on cotargeting other MAPK pathway components such as RAF and ERK (10, 11), as cotargeting upstream RTKs was generally considered likely to be ineffective based on the dogma that mutant KRAS is constitutively active and does not require further upstream signaling input (12, 13). However, this dogma was challenged by the recent development of KRASG12C-specific inhibitors (G12Ci) that bind and stabilize KRASG12C in its guanosine diphosphate (GDP)-bound inactive conformation (14–16). These findings revealed previously unappreciated biology that KRASG12C maintains intrinsic guanosine triphosphatase (GTPase) activity and cycles between the guanosine triphosphate (GTP)-bound conformation and the GDP-bound conformation, a process regulated by RTKs (17). These observations suggest that KRASG12C may require RTK activity for pathway reactivation. Moreover, FGFR1 was recently found to be activated as a compensatory response to prolonged treatment with MEK inhibitor trametinib in KRAS-mutant lung and pancreatic cancer cells, and FGFR1 inhibition in combination with trametinib enhanced tumor growth inhibition (7, 18). However, it remains inconclusive whether other KRAS variants (e.g., non-cysteine G12, G13, or Q61 mutations) are similarly susceptible to RTK regulation and whether other RTKs besides FGFR1 contribute to the MAPK pathway reactivation.
The nonreceptor protein tyrosine phosphatase SHP2, encoded by the PTPN11 gene, is part of the signaling adaptor complex that mediates RAS activation downstream of RTK as well as signaling downstream of immune inhibitory coreceptors such as T-cell–programed cell death (PD-1; refs. 19–22). The recently described allosteric SHP2 inhibitor SHP099 enables a specific way to interrogate the collective effects of RTKs in RAS-driven processes (23, 24). Herein, we used SHP099, as well as individual RTK inhibitors, to determine how often, and in what capacity, RTKs mediate pathway reactivation in a large panel (n = 34) of KRAS-mutant cell lines, by measuring the reduction of phospho-MEK adaptive induction after cotreatment with MEKi as a surrogate readout for RAS activation. We also found that combining SHP2 and MEK inhibitors led to enhanced antiproliferative activity and MAPK pathway suppression in KRAS-mutant cells with RTK/SHP2–mediated feedback activation in vitro and in vivo.
Materials and Methods
Cell lines and drugs
Human cancer cell lines originated from the CCLE (25) authenticated by single-nucleotide polymorphism analysis and tested for Mycoplasma infection using a PCR-based detection technology (https://www.idexxbioanalytics.com/) when CCLE was established in 2012. All cell lines used were directly thawed from the CCLE collection stock. All cell lines were cultured in RPMI Medium (Thermo Fisher Scientific) except MIA PaCa-2 (DMEM) and LS 180/LS 123 (MEMα), supplemented with 10% FBS (VWR). Mouse pancreatic tumor organoid–derived cells from a genetically engineered mouse model (KrasG12D/+; Trp53−/−; Apc−/−) were obtained from Zhao Chen lab at Novartis and cultured in DMEM medium with 10% FBS. Cell lines were used within 15 passages of thawing and continuously cultured for less than 6 months.
All small-molecule inhibitors used were synthesized and structurally verified by NMR and LC/MS according to cited references at Novartis except LY3009120, which was purchased from Selleck Chemicals. The structure of LFW527 was disclosed as example 40 in patent WO 2010/002655A2 (26). Onartuzumab was produced in Chinese hamster ovary cells at Novartis in a manner similar to those in patent US 2011/0262436.
Cell line engineering
Knock-out clone was generated by CRISPR technology. Sequences encoding guide RNAs targeting NRAS, HRAS, and PTPN11 were cloned into pNGx vector (sgRNA NRAS_1 CGCCAATTAACCCTGATTACTGG, sgRNA NRAS_2 TGAATATGATCCCACCATAGAGG, sgRNA HRAS_1 ATTCCGTCATCGCTCCTC, sgRNA HRAS_2 AATACGACCCCACTATAG, sgRNA PTPN11_1 GACCACGGCGTGCCCAGCGA, sgRNA PTPN11_2 TGCGCACTGGTGATGACAAA). Cells were transfected with respective sgRNA vectors and treated with media containing 1–2 μg/mL of Puromycin (Thermo Fisher Scientific) 24 hours after transfection. NRAS/HRAS double knockout NCI-H358 and MIA PaCa-2 cells were generated sequentially. Individual clones were generated by plating puromycin-resistant cells at low density in 10-cm plates. Surviving clones were isolated using cloning cylinders (Sigma-Aldrich, #C-1059). Cas9-induced mutations in NRAS and HRAS were detected by PCR and further confirmed by Sanger sequencing following TOPO TA cloning of PCR-amplified target regions (Thermo Fisher Scientific, #450641). For PTPN11 knockout, single clones were picked by paper cloning disks (Scienceware, #F37847-0001) and their SHP2 levels were determined by immunoblotting.
RAS-GTP pull-down assay and immunoblotting
RAS-GTP pull-down was performed by using the RAS Activation Assay Kit (Millipore Sigma) following the manufacturer's instructions. Immunoblotting was performed as described previously (24). The following antibodies were used: p-MEK (CST #9154), MEK1 (CST #2352), p-ERK (CST #4370), ERK1/2 (CST #4695), p-RSK3 (CST #9348), p-AKT (CST #4060), p-SHP2 (Abcam #ab62322), SHP2 (CST #3397), Tubulin (CST #3873), KRAS (Sigma-Aldrich #WH0003845M1), NRAS (Calbiochem #OP25), HRAS (Novus Biologicals # NB110-57027), and RAS (Millipore #05-516). Band signal intensities were quantified with Odyssey Software (LI-COR). p-MEK levels were normalized to tubulin levels. Percentages of p-MEK reduction in combo = (Normalized p-MEK levels in selumetinib-treated group – Normalized p-MEK levels in selumetinib and SHP099 combination-treated group)/Normalized p-MEK levels in selumetinib-treated group, expressed as percentage values.
Phospho-ERK/total ERK MSD assay
Twenty thousand MIA PaCa-2 cells were cultured in 96-well plates per well overnight and treated with selumetinib alone or in combination with 5 μmol/L SHP099 at 3-fold serial dilutions from 10 μmol/L for 24 hours and 48 hours. Cell were lysed with 50 μL complete lysis buffer in the MSD phospho-ERK/total ERK Assay Kit (Meso Scale Discovery #K15107D-1) and processed according to the manufacturer's instructions. p-ERK inhibition was normalized by the total ERK signal and compared with the DMSO control.
Cell proliferation assay and colony formation assay
Two to three thousand cells were seeded in 96-well plates per well the night before they were treated with compounds. Cell viability was then measured by the CellTiter-Glo Assay (Promega) according to the supplier's instructions after 72 hours of treatment. For colony formation assay, 10,000 cells were seeded in 6-well plates per well the night before they were treated with compounds. Media with compounds were replaced every 7 days. After 10–14 days of culture, cell colonies were stained with crystal violet.
Tumor xenograft experiments
All animal studies were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and the Novartis Institutes for Biomedical Research Animal Care and Use Committee guidelines. Female athymic nude mice (8∼12 weeks of age) were inoculated with MIA PaCA-2 and T3M-4 cells (5 × 106 cells in 200 μL suspension containing 50% Matrigel (BD Biosciences) in Hank balanced salt solution) or human-derived colon tumor xenograft (HCOX) model HCOX1402 (25–50 mg tumor slurry in 200 μL suspension containing 50% Matrigel in PBS) subcutaneously into the right superciliary region. Mice were monitored twice a week and tumors were measured and calculated by length × width2/2. Once tumors reached roughly 250 mm3 for MIA PaCa-2 and 200 mm3 for T3M-4 and HCOX1402, mice were randomly assigned to receive either vehicle, SHP099 (25 or 50 mg/kg every other day for MIA PaCA-2, 25 mg/kg daily for T3M-4, or 50 mg/kg daily for HCOX1402), trametinib (0.3 mg/kg daily for all three models), or the combination of trametinib and SHP099 by oral gavage. At the end of the study, mice were euthanized at 3, 8, and 24 hours following the last dose. Tumor fragments were collected for immunoblotting.
Results
RTK/SHP2–mediated feedback activation is frequent in KRAS-mutant cell lines
The MAPK signaling cascade is an important effector pathway of RTK activation and is tightly regulated at multiple levels. ERK activation turns on various negative regulators of the pathway, such as dual-specificity phosphatase (DUSP) and Sprouty (SPRY) family proteins, imposing feedback inhibition at several nodes in the pathway (6). As illustrated in Supplementary Fig. S1A, inhibiting ERK signaling using MEKi relieves the feedback inhibition, which causes pathway reactivation and increased phosphorylation of MEK at Ser217/221 (p-MEK) despite its catalytic activity still being inhibited. We took advantage of this quick and robust adaptive induction of p-MEK caused by MEKi and the extent of p-MEK reduction by cotreatment with SHP099 or RTK inhibitor(s) to measure the contribution of RTKs and SHP2 to the pathway feedback activation.
As exemplified in Fig. 1A, when CCK-81, an EGFR-dependent and RAS/RAF wild-type (WT) colorectal cancer cell line, was treated with MEKi selumetinib (27) for 24 hours, a dramatic increase in RAS-GTP and p-MEK levels was observed, which could be diminished or blocked by cotreatment with SHP099 or an EGFR inhibitor, erlotinib, indicating that the feedback activation of MEK was mediated through EGFR and SHP2. Five micromolar SHP099 was chosen based on previous characterization (24). Interestingly, although SHP099 did not completely block RAS reactivation, the residual RAS activity was not sufficient to reactivate MEK, possibly due to the existence of a threshold of RAS activity for MEK reactivation (Fig. 1A). Phospho-ERK1/2 (p-ERK) and downstream phospho-RSK3 (p-RSK3) were still effectively inhibited by selumetinib despite the adaptive p-MEK induction and the combination of selumetinib with SHP2i or EGFRi further inhibited p-ERK and p-RSK3 (Fig. 1A). These data indicate that the SHP2i-sensitive p-MEK–adaptive induction by MEKi can be a convenient readout of RAS feedback activation, compared with measuring RAS-GTP levels, which is often not sensitive or monitoring the p-ERK rebound that is often delayed (48 hours∼96 hours) with varying degrees among cell lines.
RTK/SHP2-mediated feedback activation is frequent in KRAS-mutant cell lines. A, CCK-81 cells were treated with DMSO, selumetinib (0.5 μmol/L), or the combination of selumetinib with SHP099 (5 μmol/L) or erlotinib (0.5 μmol/L) for 24 hours. The effect on the levels of GTP-bound RAS was determined by a RAS activation assay and immunoblotting with a RAS antibody. The effect on the MAPK signaling was determined by immunoblotting with the indicated antibodies. Immunoblot of phospho-MEK1/2 (Ser217/221) and tubulin in 11 KRASG12C-mutant cell lines (B), 5 KRASG12D, 5 KRASG12V, and 4 KRASG12R/S/A-mutant cell lines (C), 1 KRASQ61L and 3 KRASQ61H-mutant cell lines (D), 5 melanoma cell lines with NRASQ61 mutations (E), and 5 KRASG13D-mutant cell lines (F), treated with DMSO, selumetinib (0.5 μmol/L), SHP099 (5 μmol/L), or the combination for 24 hours. Band signal intensities were quantified with Odyssey Software (LI-COR) and the percentages of reduction of p-MEK after normalization to tubulin expression in the combination groups compared with the selumetinib-treated groups for each cell line were reported.
RTK/SHP2-mediated feedback activation is frequent in KRAS-mutant cell lines. A, CCK-81 cells were treated with DMSO, selumetinib (0.5 μmol/L), or the combination of selumetinib with SHP099 (5 μmol/L) or erlotinib (0.5 μmol/L) for 24 hours. The effect on the levels of GTP-bound RAS was determined by a RAS activation assay and immunoblotting with a RAS antibody. The effect on the MAPK signaling was determined by immunoblotting with the indicated antibodies. Immunoblot of phospho-MEK1/2 (Ser217/221) and tubulin in 11 KRASG12C-mutant cell lines (B), 5 KRASG12D, 5 KRASG12V, and 4 KRASG12R/S/A-mutant cell lines (C), 1 KRASQ61L and 3 KRASQ61H-mutant cell lines (D), 5 melanoma cell lines with NRASQ61 mutations (E), and 5 KRASG13D-mutant cell lines (F), treated with DMSO, selumetinib (0.5 μmol/L), SHP099 (5 μmol/L), or the combination for 24 hours. Band signal intensities were quantified with Odyssey Software (LI-COR) and the percentages of reduction of p-MEK after normalization to tubulin expression in the combination groups compared with the selumetinib-treated groups for each cell line were reported.
We profiled a diverse panel of 34 KRAS-mutant and 5 NRAS-mutant cancer cell lines across different lineages with this p-MEK assay (Supplementary Table S1) and quantified the “% p-MEK of reduction in combo” as described in Materials and Methods (Supplementary Table S2). As shown in Fig. 1B–F, p-MEK induction after treatment with selumetinib was observed in all cell lines examined. Cotreatment with 5 μmol/L SHP099 reduced p-MEK induction in the majority of KRAS-mutant cell lines by varying degrees, with only five cell lines (NCI-H2122, NCI-H1792, COR-L23, PSN1, and HCT 116) having less than 10% reduction, suggesting SHP2-mediated feedback activation is frequent in KRAS-mutant cell lines. Moreover, in several cell lines, SHP2 is likely the major contributor to the pathway feedback activation, with more than 50% p-MEK reduction by SHP099 (Fig. 1B–D).
On the basis of structural and biochemical studies (28, 29), KRASG12C has the highest intrinsic GTP hydrolysis activity among all KRAS mutants, suggesting it may undergo significant nucleotide cycling and depend on RTK/SHP2 for GTP loading. Our profiling indicates there is a wide spectrum of SHP2 dependency of feedback activation among KRASG12C cell lines (70%∼6%; Fig. 1B). Similar spectrum of SHP2 dependency was observed with cell lines harboring KRASG12D and KRASG12V (Fig. 1C). Unexpectedly, in cell lines harboring KRASQ61L/H, which have the lowest intrinsic GTP hydrolysis activity, we observed substantial reduction in selumetinib-induced p-MEK by SHP099 (SW948, 74% reduction, KRASQ61L/+; T3M-4, 43% reduction, KRASQ61H/+; Fig. 1D). In the two lung cancer cell lines with homozygous KRASQ61H mutation, NCI-H460 and HCC2108, SHP2 activity is associated with 27% and 14% of the feedback activation, respectively (Fig. 1D). Because we have limited number of cell lines harboring KRASQ61 mutants, we then examined five NRASQ61-mutant melanoma cell lines (Supplementary Table S1) and found little effect (0%∼11%) of SHP099 on reducing p-MEK feedback activation (Fig. 1E). Similarly, SHP099 only had modest effect reducing p-MEK–adaptive induction in the five cell lines harboring KRASG13D (Fig. 1F).
To understand the maximal contribution of SHP2 to the feedback activation, we performed the p-MEK assay with up to 100 μmol/L SHP099 and generated SHP2 knock-out clones using the CRISPR/Cas9 technology in two cell lines with abundant SHP2-dependent feedback activation, MIA PaCa-2 and HUP-T3 (Supplementary Fig. S1B). In MIA PaCa-2 cells (KRASG12C/G12C), 5 μmol/L SHP099 was sufficient to achieve maximal inhibition on selumetinib-induced p-MEK–adaptive induction, comparable with the effect of 10 μmol/L ARS853, a KRASG12C inhibitor (14), or SHP2 deletion (Supplementary Fig. S1C). In HUP-T3 cells (KRASG12R/+), higher SHP099 concentrations further inhibited the selumetinib-induced p-MEK and 100 μmol/L SHP099 as well as SHP2 deletion blocked most of the p-MEK–adaptive induction (Supplementary Fig. S1C). We then performed the SHP099 dose-dependent p-MEK assay in two cell lines with little or no p-MEK reduction by 5 μmol/L SHP099, NCI-H2122, and PSN1. Similar to HUP-T3 cells, in NCI-H2122 cells (KRASG12C/G12C), higher SHP099 concentrations also further suppressed the p-MEK–adaptive induction (Supplementary Fig. S1D). Moreover, the effect by 100 μmol/L SHP099 exceeded that seen by 10 μmol/L ARS853, suggesting HRAS/NRAS may be involved in the p-MEK–adaptive induction. In contrast, in PSN1 cells (KRASG12R/G12R), p-MEK feedback activation was unaffected by coadministration of 100 μmol/L SHP099 but was fully inhibited by 3 μmol/L LY3009120, a pan-RAF inhibitor (Supplementary Fig. S1D; ref. 30), indicating that the p-MEK feedback activation in PSN1 cells is indeed SHP2 independent. Notably, 24-hour treatment with high SHP099 concentrations did not cause obvious cytotoxicity in any cell line tested.
We also performed the p-MEK assay with a mouse cell line derived from pancreatic tumor organoids of a genetically engineered mouse model (KrasG12D/+; Trp53−/−; APC−/−) and SHP099 dramatically reduced adaptive p-MEK induction by selumetinib (Supplementary Fig. S1E). Pan-RAF inhibitor LY3009120 achieved near complete suppression of p-MEK–adaptive induction, suggesting that the residual SHP099-insensitive p-MEK–adaptive induction was likely due to feedback activation directly on RAF. Taken together, we speculate that the pathway reactivation in KRAS-mutant cell lines may be broadly and dominantly mediated by SHP2 though they may exhibit differential sensitivity to SHP099.
RTKs driving feedback activation in KRAS-mutant cell lines are diverse
Next, we used individual RTK inhibitors to demonstrate that SHP2-dependent pathway reactivation is due to RTK activity and to identify which RTKs were responsible in KRAS-mutant cell lines (Fig. 2A). In HUP-T3, the FGFR1/2/3 inhibitor BGJ398 (31, 32) reduced p-MEK induction to a comparable level as SHP099, suggesting that pathway reactivation primarily depends on FGFR1/2/3. In NCI-H358 (KRASG12C/+), LS 180 (KRASG12D/+), Panc 03.27 (KRASG12V/+), and Capan-2 (KRASG12V/+), EGFR was the major feedback-activated RTK because the p-MEK induction was decreased by both erlotinib and EGFR/HER2 dual inhibitor lapatinib. In KP-4, the p-MEK induction was reduced by crizotinib, a MET and ALK dual inhibitor. The expression of ALK is low, whereas the expression of MET ligand hepatocyte growth factor (HGF) is high in KP4 (Supplementary Table S3) and further analysis using a selective MET inhibitor INC280 (33) indicated that MET is the feedback-activated RTK in KP4 (Fig. 2D). In contrast, no single RTK inhibitor reduced p-MEK induction to the same extent as SHP099 in MIA PaCa-2 or NCI-H2030, suggesting that more than one RTK could be involved. Indeed, a combinatorial treatment with lapatinib and BGJ398 reduced p-MEK–adaptive induction to a similar level as SHP099 in both cell lines, suggesting both ERBBs and FGFRs were feedback-activated (Fig. 2B). LFW527, an IGF-1R inhibitor (34), failed to inhibit p-MEK induction in all cell lines tested. Taken together, these data indicate that RTKs that mediate selumetinib-induced pathway reactivation in KRAS-mutant cells, at least, include one or more RTKs including ERBBs, FGFRs, and MET.
The feedback-activated RTKs in KRAS-mutant cell lines are diverse. A, Immunoblot of phospho-MEK1/2 in the indicated KRAS-mutant cell lines treated with DMSO, selumetinib (0.5 μmol/L), or selumetinib in combination with SHP099 (5 μmol/L) or the indicated RTK inhibitors (all at 0.5 μmol/L) for 24 hours. B, Immunoblot of phospho-MEK1/2 in MIA PaCa-2 and NCI-H2030 cells treated with DMSO, selumetinib (0.5 μmol/L), or selumetinib in combination with SHP099 (5 μmol/L) or with lapatinib plus BGJ398 (0.5 μmol/L each) for 24 hours. C, Immunoblot of phospho-MEK1/2 in the indicated cells that were cultured in complete or serum-free medium and treated with DMSO, selumetinib (0.5 μmol/L), or selumetinib in combination with SHP099 (5 μmol/L) for 24 hours. The samples from cells cultured with serum and serum starved for 24 hours were analyzed on the same gel and the same exposure were shown. D, Immunoblot of phospho-MET and phospho-MEK1/2 in KP4 cells treated with DMSO, selumetinib (0.5 μmol/L), or selumetinib in combination with SHP099 (5 μmol/L) or the indicated inhibitors (0.5 μmol/L for each except onartuzumab at 1 μmol/L) for 24 hours. Tubulin served as a loading control.
The feedback-activated RTKs in KRAS-mutant cell lines are diverse. A, Immunoblot of phospho-MEK1/2 in the indicated KRAS-mutant cell lines treated with DMSO, selumetinib (0.5 μmol/L), or selumetinib in combination with SHP099 (5 μmol/L) or the indicated RTK inhibitors (all at 0.5 μmol/L) for 24 hours. B, Immunoblot of phospho-MEK1/2 in MIA PaCa-2 and NCI-H2030 cells treated with DMSO, selumetinib (0.5 μmol/L), or selumetinib in combination with SHP099 (5 μmol/L) or with lapatinib plus BGJ398 (0.5 μmol/L each) for 24 hours. C, Immunoblot of phospho-MEK1/2 in the indicated cells that were cultured in complete or serum-free medium and treated with DMSO, selumetinib (0.5 μmol/L), or selumetinib in combination with SHP099 (5 μmol/L) for 24 hours. The samples from cells cultured with serum and serum starved for 24 hours were analyzed on the same gel and the same exposure were shown. D, Immunoblot of phospho-MET and phospho-MEK1/2 in KP4 cells treated with DMSO, selumetinib (0.5 μmol/L), or selumetinib in combination with SHP099 (5 μmol/L) or the indicated inhibitors (0.5 μmol/L for each except onartuzumab at 1 μmol/L) for 24 hours. Tubulin served as a loading control.
To test whether the RTK feedback activation depends on RTK ligands from the serum in media, we performed the p-MEK assay under serum-free conditions in four cell lines with RTK-driven feedback activation (Fig. 2C). Twenty-four hours after serum starvation, selumetinib treatment still led to robust p-MEK–adaptive induction, albeit to a smaller magnitude than the condition with serum, except in NCI-H2030 cells (Fig. 2C, identical exposure from the same immunoblot for each cell line). Moreover, the p-MEK feedback activation still largely depended on SHP2 in all four cell lines. These data demonstrate that the RTK feedback activation occurs in the absence of exogenous RTK ligands and depends on either autocrine RTK ligands or relief of negative regulators of RTKs such as SPRY proteins in a ligand-independent manner (6, 35). Next, we utilized KP4 cells with MET-driven feedback activation to differentiate these two possibilities. Onartuzumab (36), a monovalent mAb that binds to the extracellular domain of MET and prevents HGF binding, as well as the selective MET inhibitor INC280, but not the selective ALK inhibitor ceritinib (37), significantly reduced both baseline phospho-MET and the induced p-MEK by selumetinib treatment (Fig. 2D). These data suggest that the feedback activation of RTK may require RTK ligands in certain cell lines such as KP4.
RTKs may also activate the PI3K/AKT pathway (38, 39); we therefore tested whether MEKi treatment can induce AKT activation in KRAS-mutant cells with RTK-mediated feedback activation. In 6 of 8 cell lines tested, there was no significant increase of p-AKT levels, suggesting that the RAS/MAPK pathway is often the major effector pathway of feedback-activated RTKs, at least within the first 24-hour treatment with MEKi (Supplementary Fig. S1F). In MIA PaCa-2 cells where p-AKT was induced by selumetinib treatment, we examined the effect of SHP2 and KRASG12C inhibitors on the induced p-AKT (Supplementary Fig. S1G). The p-AKT induction was not blocked by SHP2 or KRASG12C inhibitors, suggesting the p-AKT induction is likely a result of RTK feedback activation instead of a secondary effect of RAS feedback activation and cross-talk with PI3K/AKT signaling (40). Interestingly, the increased p-AKT levels did not translate to activation of all downstream effectors such as phosho-S6 (p-S6; Supplementary Fig. S1G).
G12C/D/V–mutant KRAS can mediate the RTK/SHP2–dependent feedback activation
RTK-dependent feedback activation of p-MEK in KRAS-mutant cell lines could be mediated through mutant KRAS, WT KRAS (in KRAS heterozygotes), other RAS paralogs (e.g., NRAS and HRAS), or a combination of those possibilities. To determine the contribution of mutant KRAS, we knocked out both HRAS and NRAS using CRISPR/Cas9 technology in NCI-H358 (KRASG12C/+) and MIA PaCa-2 (KRASG12C/G12C) cells, where the feedback activation depends largely on RTK/SHP2 (Fig. 1B), and validated the loss of NRAS and HRAS in the double knock-out (DKO) clones by immunoblots (Supplementary Fig. S2A and S2B). In all DKO clones from both cell lines, the p-MEK-adaptive induction by selumetinib was comparable to that of parental cells (Supplementary Fig. S2C and S2D). Moreover, the p-MEK-adaptive induction by selumetinib was efficiently reduced by SHP099, in two selected DKO clones of both cell lines (Supplementary Fig. S2E and Fig. 3A). Because MIA PaCa-2 cells harbor a homozygous KRASG12C mutation, these data suggest that KRASG12C can mediate RTK-driven p-MEK feedback activation.
G12C/D/V–mutant KRAS can mediate the p-MEK feedback activation from RTK/SHP2. A, Immunoblot of phospho-MEK1/2 in HRAS/NRAS double knockout (DKO) clones of MIA PaCa-2 (KRASG12C/G12C) cells treated with DMSO, selumetinib (0.5 μmol/L), SHP099 (5 μmol/L), or the combination for 24 hours. B, MIA PaCa-2 cells were treated with DMSO, selumetinib (0.5 μmol/L), or the combination of selumetinib with ARS853 (10 μmol/L) or SHP099 (10 μmol/L) for 24 hours, and a SHP2 knockout clone of MIA PaCa-2 was treated with DMSO or selumetinib (0.5 μmol/L) for 24 hours. RAS-GTP levels were determined by a RAS activation assay and immunoblotting with a pan-RAS antibody. C, NCI-H2122 cells were treated with DMSO, selumetinib (0.5 μmol/L), or the combination of selumetinib with SHP099 (10 μmol/L or 100 μmol/L) or ARS853 (10 μmol/L) for 24 hours. The effect on the levels of GTP-bound KRAS was determined by a RAS activation assay and immunoblotting with a KRAS-specific antibody. D, LS 180 cells were treated with DMSO, selumetinib (0.5 μmol/L), SHP099 (5 μmol/L), or the combination for 24 hours. KRASG12D-GTP and RAS-GTP levels were determined by a RAS activation assay and immunoblotting with RASG12D-specific or pan-RAS antibodies, respectively. COR-L23 (E), NCI-H647 (F), NCI-H460, and T3M-4 (G) cells were treated with DMSO, selumetinib (0.5 μmol/L), or the combination of selumetinib with SHP099 (10 μmol/L, 30 μmol/L for T3M-4 only, or 100 μmol/L), or RAFi (LY3009120, 3 μmol/L) for COR-L23 only for 24 hours. The effect on the levels of GTP-bound KRAS was determined by a RAS activation assay and immunoblotting with a KRAS-specific antibody. Phospho-MEK1/2, phospho-ERK1/2, and phospho-RSK3 levels were determined by immunoblotting. Tubulin served as a loading control.
G12C/D/V–mutant KRAS can mediate the p-MEK feedback activation from RTK/SHP2. A, Immunoblot of phospho-MEK1/2 in HRAS/NRAS double knockout (DKO) clones of MIA PaCa-2 (KRASG12C/G12C) cells treated with DMSO, selumetinib (0.5 μmol/L), SHP099 (5 μmol/L), or the combination for 24 hours. B, MIA PaCa-2 cells were treated with DMSO, selumetinib (0.5 μmol/L), or the combination of selumetinib with ARS853 (10 μmol/L) or SHP099 (10 μmol/L) for 24 hours, and a SHP2 knockout clone of MIA PaCa-2 was treated with DMSO or selumetinib (0.5 μmol/L) for 24 hours. RAS-GTP levels were determined by a RAS activation assay and immunoblotting with a pan-RAS antibody. C, NCI-H2122 cells were treated with DMSO, selumetinib (0.5 μmol/L), or the combination of selumetinib with SHP099 (10 μmol/L or 100 μmol/L) or ARS853 (10 μmol/L) for 24 hours. The effect on the levels of GTP-bound KRAS was determined by a RAS activation assay and immunoblotting with a KRAS-specific antibody. D, LS 180 cells were treated with DMSO, selumetinib (0.5 μmol/L), SHP099 (5 μmol/L), or the combination for 24 hours. KRASG12D-GTP and RAS-GTP levels were determined by a RAS activation assay and immunoblotting with RASG12D-specific or pan-RAS antibodies, respectively. COR-L23 (E), NCI-H647 (F), NCI-H460, and T3M-4 (G) cells were treated with DMSO, selumetinib (0.5 μmol/L), or the combination of selumetinib with SHP099 (10 μmol/L, 30 μmol/L for T3M-4 only, or 100 μmol/L), or RAFi (LY3009120, 3 μmol/L) for COR-L23 only for 24 hours. The effect on the levels of GTP-bound KRAS was determined by a RAS activation assay and immunoblotting with a KRAS-specific antibody. Phospho-MEK1/2, phospho-ERK1/2, and phospho-RSK3 levels were determined by immunoblotting. Tubulin served as a loading control.
To further determine the contribution of mutant KRAS to RTK-driven feedback activation in the presence of HRAS/NRAS, we took advantage of the KRAS G12Ci and examined the RAS activity directly in MIA PaCa-2 cells (Fig. 3B). Selumetinib treatment resulted in increased RAS-GTP levels, consistent with increased p-MEK levels and both increases were abolished by 10 μmol/L ARS853 and significantly reduced by 10 μmol/L SHP099. In addition, the combination of ARS853 or SHP099 with selumetinib further decreased p-ERK and p-RSK3 levels than selumetinib alone. Consistent with the SHP099 effect, in the SHP2 KO clone of MIA PaCa-2 cells, treatment with selumetinib induced only a slight increase of RAS-GTP and p-MEK levels. These data suggest that the feedback activation in MIA PaCa-2 cells is mainly mediated through KRASG12C which can be diminished by SHP2 inhibition. In another KRASG12C/G12C cell line NCI-H2122, KRAS-GTP induction by selumetinib was also blocked by SHP099 or ARS853 but the p-MEK induction was only partially blocked (Fig. 3C), suggesting additional feedback activation of the pathway downstream of RAS (e.g., RAF) may exist in NCI-H2122.
We further explored whether SHP2 inhibition can influence feedback activation of KRASG12D by measuring RAS-GTP levels using a RASG12D-specific antibody in LS 180 cells (KRASG12D/+; Supplementary Fig. S2F and Fig. 3D). We observed increased KRASG12D-GTP and p-MEK levels following treatment with selumetinib, which was effectively reduced by SHP099. The combination of SHP2 and MEK inhibitors also further decreased p-ERK and p-RSK3 levels than selumetinib alone. These data indicate that KRASG12D can also mediate the RTK/SHP2–driven pathway feedback activation.
For other KRAS mutants (G12V, Q61H and G13D), due to the lack of validated mutant-specific RAS antibodies, we performed the RAS activation assays with homozygous mutant lines (Fig. 3E–G) and used a KRAS-specific antibody to investigate whether those mutants can be feedback activated through SHP2. As shown with COR-L23 (KRASG12V/G12V), KRASG12V can also be feedback activated in a SHP2-dependent manner. However, the p-MEK feedback activation was not blocked by up to 100 μmol/L SHP099 while sensitive to the pan-RAF inhibitor LY3009120. Consistent with the effect on p-MEK feedback activation, the combination of SHP2 and MEK inhibitors did not further decrease p-ERK or p-RSK3 levels as the combination of RAF and MEK inhibitors did (Fig. 3E).
We also discovered in NCI-H647 (KRASG13D/G13D) and NCI-H460 (KRASQ61H/Q61H) cells, KRASG13D and KRASQ61H were feedback activated following the treatment with selumetinib but the activation was not dependent on SHP2 (Fig. 3F and G). Consistently, the p-MEK feedback activation was not impacted by SHP2 inhibition and the combination of selumetinib and SHP099 did not further inhibit p-ERK or p-RSK3 in both cell lines. The observation in NCI-H460 cells raised the possibility that the SHP099-sensitive p-MEK induction in SW948 (KRASQ61L/+) and T3M-4 (KRASQ61H/+; Fig. 1D) may be due to the effect on the WT KRAS or HRAS or NRAS. Indeed, in T3M-4 the KRAS feedback activation was strongly inhibited by SHP099 (Fig. 3G) and we speculate that WT KRAS dominantly contributed to the feedback activation in T3M-4 cells despite the presence of more active KRASQ61H.
SHP2 inhibition sensitizes KRAS-mutant cells to MEK inhibitors in vitro
The observation that SHP099 blocked the pathway feedback activation induced by MEK inhibitors led us to hypothesize that SHP2 inhibition may enhance the efficacy of MEK inhibitors against KRAS-mutant cells. Therefore, we tested the effects of SHP099 or SHP2 deletion on the antiproliferative effects of selumetinib in MIA PaCa-2 and HUP-T3, two cell lines with abundant SHP2-mediated feedback activation. SHP099 at 10 μmol/L or SHP2 deletion sensitized MIA PaCa-2 cells to selumetinib, causing a decrease in the IC50 by approximately 10-fold (0.13 μmol/L vs. 1.22 μmol/L; Fig. 4A) and 4-fold (0.21 μmol/L vs. 0.87 μmol/L; Fig. 4B), respectively. Similarly, in HUP-T3 cells, SHP2 inhibition or deletion led to a roughly 3-fold (0.04 μmol/L vs. 0.14 μmol/L; Fig. 4A) and an average of 3-fold (0.02 μmol/L for KO-1 and 0.05 μmol/L for KO-2 vs. 0.11 μmol/L; Fig. 4B) decrease in the IC50 of selumetinib, respectively. More striking results were observed with another MEKi trametinib in both cell lines (Supplementary Fig. S3A and S3B). To determine whether the combination benefit of MEK and SHP2 inhibitors is additive or synergistic, we performed a cell proliferation assay using an 8 by 8 combination dose matrix of selumetinib and SHP099 in MIA PaCa-2 and HUP-T3 cells and assessed the synergy using the Loewe Excess method (41). Selumetinib and SHP099 showed synergistic effects as determined by a synergy score of greater than 2 in both cell lines (Supplementary Fig. S3C). The greatest antiproliferative synergy was observed at low concentrations of selumetinib (∼0.1 μmol/L) and high concentrations of SHP099 (>3 μmol/L). We also studied the combination synergy in six additional cells lines with both SHP2-dependent and SHP2-independent p-MEK feedback activation (Supplementary Fig. S3C) and observed a greater synergy of the combination in cell lines where the p-MEK induction largely depends on SHP2. Taken together, we conclude that SHP2 dependency of p-MEK induction could, to some extent, predict a synergistic combination benefit of MEK and SHP2 inhibitors.
SHP2 inhibition sensitizes KRAS-mutant cells to MEK inhibitors in vitro. A, Cell viability of MIA PaCa-2 cells and HUP-T3 cells treated with selumetinib in the absence or presence of 10 μmol/L SHP099 for 72 hours (mean percentage of cell viability, error bars, SD, n = 3). B, Cell viability of SHP2 knockout (SHP2 KO) clones and parental MIA PaCa-2 and HUP-T3 cells treated with selumetinib for 72 hours (mean percentage of cell viability, error bars, SD, n = 3). C, Colony formation assay of indicated cell lines treated with DMSO, selumetinib (300 nmol/L except for HUP-T3 at 50 nmol/L), SHP099 (10 μmol/L), and the combination for approximately 10 to 14 days. Cells were stained by crystal violet at the end of the assay for imaging. PSN1 cells with SHP2-independent feedback activation were included as a negative control. D, Mean phospho-ERK levels measured by Meso Scale Discovery (MSD) assays of MIA PaCa-2 cells treated with selumetinib alone or in combination with 5 μmol/L SHP099 for 24 hours or 48 hours. Error bars, SD, n = 3. P values were calculated by paired t test.
SHP2 inhibition sensitizes KRAS-mutant cells to MEK inhibitors in vitro. A, Cell viability of MIA PaCa-2 cells and HUP-T3 cells treated with selumetinib in the absence or presence of 10 μmol/L SHP099 for 72 hours (mean percentage of cell viability, error bars, SD, n = 3). B, Cell viability of SHP2 knockout (SHP2 KO) clones and parental MIA PaCa-2 and HUP-T3 cells treated with selumetinib for 72 hours (mean percentage of cell viability, error bars, SD, n = 3). C, Colony formation assay of indicated cell lines treated with DMSO, selumetinib (300 nmol/L except for HUP-T3 at 50 nmol/L), SHP099 (10 μmol/L), and the combination for approximately 10 to 14 days. Cells were stained by crystal violet at the end of the assay for imaging. PSN1 cells with SHP2-independent feedback activation were included as a negative control. D, Mean phospho-ERK levels measured by Meso Scale Discovery (MSD) assays of MIA PaCa-2 cells treated with selumetinib alone or in combination with 5 μmol/L SHP099 for 24 hours or 48 hours. Error bars, SD, n = 3. P values were calculated by paired t test.
To investigate the long-term efficacy of the SHP2i and MEKi combination, we performed colony formation assays. After 10–14 days, 10 μmol/L SHP099 did not have a significant effect on the growth of KRAS-mutant cells while selumetinib alone (50∼300 nmol/L) had a modest antiproliferative effect (Fig. 4C). The combination treatment with selumetinib and SHP099 led to enhanced inhibition of colony formation in all cell lines with SHP2-dependent feedback activation but not in PSN1 cells in which the feedback activation was independent of SHP2 (Fig. 1C; Supplementary Fig. S1D). These data demonstrate that blocking RTK-mediated MAPK pathway reactivation by inhibiting SHP2 can enhance sensitivity to MEK inhibitors in KRAS-mutant cells in vitro.
We next examined whether the combination benefit of MEKi and SHP2i was due to sustained inhibition of the MAPK pathway. p-ERK and total ERK levels in MIA PaCa-2 cells at 24 hours and 48 hours posttreatment with various doses of selumetinib in the presence or absence of 5 μmol/L SHP099 were measured using Meso Scale Discovery (MSD) MULTI-SPOT assays. As expected, while p-ERK was effectively inhibited by selumetinib at 24 hours posttreatment (94% inhibition at 0.37 μmol/L), a significant rebound was observed at 48 hours (76% inhibition at 0.37 μmol/L), which could not be inhibited by higher concentrations of selumetinib (Fig. 4D). Cotreatment with SHP099 significantly suppressed the p-ERK rebound at 48 hours (P = 0.0185, Fig. 4D). We also confirmed the enhanced inhibition of p-ERK and p-RSK3 by the combination of MEK and SHP2 inhibitors in both MIA PaCa-2 and HUP-T3 cells using immunoblot assays (Supplementary Fig. S3D).
Because we observed p-AKT feedback activation in MIA PaCa-2 and HUP-T3 cells, which was not blocked by SHP2 inhibition in MIA PaCa-2 cells (Supplementary Fig. S1G), we assessed the combination effect of trametinib and BYL719, a potent and selective PI3Kα inhibitor (42). Addition of 1 μmol/L BYL719 had no effect on trametinib efficacy in both MIA PaCa-2 and HUP-T3 cells (Supplementary Fig. S3E), consistent with the observation that downstream p-S6 was not feedback-activated in MIA PaCa-2 cells (Supplementary Fig. S1G). We next compared the MEK inhibitor combination effects of SHP099 with those of RTK inhibitors, which may block additional RTK effector pathways beyond the RAS/MAPK pathway such as PI3K/AKT and JAK/STAT pathways (38, 39, 43). In HUP-T3 cells, in which FGFRs mediate the feedback activation (Fig. 2A) and p-ATK was feedback-activated (Supplementary Fig. S1F), SHP099 at 10 μmol/L led to a similar magnitude of sensitization to trametinib compared with 1 μmol/L BGJ398 (IC50 of trametinib, DMSO = 20.64 nmol/L; with 10 μmol/L SHP099 = 3.37 nmol/L; with 1 μmol/L BGJ398 = 6.46 nmol/L; Supplementary Fig. S3F). Similar results were observed with erlotinib and SHP099 in LS 180 cells, in which EGFR mediates the feedback activation (Fig. 2A; Supplementary Fig. S3F). These data suggest that similar in vitro combination benefit with MEK inhibitors can be achieved with SHP2 inhibitors in place of RTK inhibitors.
Combination efficacy of trametinib and SHP099 in vivo
To evaluate the in vivo efficacy of the combination of MEKi and SHP2i, we treated nude mice bearing MIA PaCa-2 xenografts with SHP099, trametinib, or the combination (Fig. 5A). Trametinib alone at a clinically relevant dose (0.3 mpk, daily) only resulted in moderate tumor growth inhibition. SHP099 alone at 50 mpk every other day dosing (reduced from the MTD at 100 mpk daily for tolerability reasons in combination with trametinib) also had significant antitumor efficacy, similar to 0.3 mpk trametinib, which coincides with inhibition of MAPK to some degree (Fig. 5B). This was not unexpected with recent studies showing SHP2 may be required for KRAS-mutant tumor growth in vivo (44, 45). The combination of trametinib and SHP099 led to significantly improved efficacy compared with either of the single agents (Fig. 5A; P = 0.0064 compared with trametinib alone and P = 0.0036 compared with SHP099 alone) and achieved tumor stasis. As expected, the combination also more effectively decreased p-ERK and p-RSK3 levels compared with either of the single agents at both 3 hours and 8 hours after the last dose (Fig. 5B). To further address the tolerability concerns of the combination in the clinic, we also tested a reduced dose of SHP099 at 25 mpk every other day and the combination remained effective in terms of both efficacy (Supplementary Fig. S4A) and p-ERK/p-RSK3 inhibition (Supplementary Fig. S4B).
MEKi and SHP2i have combination benefit in vivo. A, Mean tumor volumes of MIA PaCa-2 subcutaneous xenografts in nude mice following treatment with vehicle, trametinib (0.3 mg/kg body weight, daily), SHP099 (50 mg/kg body weight, every other day), or a combination of both drugs. Error bars, SEM. n = 9 mice per group. B, Immunoblot of p-MEK1/2, p-ERK1/2, and p-RSK3 from tumor tissues collected at 3 hours or 8 hours after last dose from mice treated as described in A. C, Mean tumor volumes of T3M-4 subcutaneous xenografts in nude mice following treatment with vehicle, trametinib (0.3 mg/kg body weight, daily), SHP099 (25 mg/kg body weight, daily), or a combination of both drugs. Error bars, SEM. n = 6 mice per group. D, Immunoblot of p-MEK1/2, p-ERK1/2, and p-RSK3 from tumor tissues collected at 3 hours after last dose from mice treated as described in C. Tubulin served as a loading control. **, P < 0.01 by paired t test.
MEKi and SHP2i have combination benefit in vivo. A, Mean tumor volumes of MIA PaCa-2 subcutaneous xenografts in nude mice following treatment with vehicle, trametinib (0.3 mg/kg body weight, daily), SHP099 (50 mg/kg body weight, every other day), or a combination of both drugs. Error bars, SEM. n = 9 mice per group. B, Immunoblot of p-MEK1/2, p-ERK1/2, and p-RSK3 from tumor tissues collected at 3 hours or 8 hours after last dose from mice treated as described in A. C, Mean tumor volumes of T3M-4 subcutaneous xenografts in nude mice following treatment with vehicle, trametinib (0.3 mg/kg body weight, daily), SHP099 (25 mg/kg body weight, daily), or a combination of both drugs. Error bars, SEM. n = 6 mice per group. D, Immunoblot of p-MEK1/2, p-ERK1/2, and p-RSK3 from tumor tissues collected at 3 hours after last dose from mice treated as described in C. Tubulin served as a loading control. **, P < 0.01 by paired t test.
We also examined the combination efficacy in a KRASQ61H tumor model, T3M-4, in which we observed selumetinib and SHP099 combination benefit in vitro (Fig. 4C).The combination resulted in tumor stasis and enhanced inhibition of p-ERK and p-RSK3 while neither of the single agents effectively inhibited the tumor growth (Fig. 5C and D). A trend of combination benefit was also observed in a KRASG12V colon cancer patient-derived xenograft HCOX1402 (Supplementary Fig. S4C), which coincided with decreased p-ERK and p-RSK3 levels (Supplementary Fig. S4D).
Discussion
We utilized an assay of adaptive induction of p-MEK following MEKi treatment as a convenient and robust readout of pathway reactivation and a SHP2 inhibitor that blocks RAS activation downstream of multiple RTKs to determine the contribution of RTK/SHP2 to the pathway feedback activation in KRAS-mutant cells. We observed a broad spectrum of dependency of feedback activation on RTK/SHP2. In some cell lines such as MIA PaCa-2 and HUP-T3, RTK/SHP2 is the major contributor to the pathway feedback activation (Fig. 1B and C; Supplementary Fig. S1C) while in other cell lines such as PSN1, the feedback activation occurs downstream of SHP2 (Supplementary Fig. S1D). This can be potentially explained by many reported MAPK pathway feedback inhibition mechanisms such as direct phosphorylation and inactivation of RAF1 (46, 47) or SOS1 (48–50) by ERK activation. For the remaining KRAS-mutant cell lines tested here, based on our further studies (Fig. 3C–G), the feedback activation likely occurs at both upstream proteins such as RTK/SHP2 and downstream proteins such as RAF/MEK. This is consistent with the notion that the MAPK pathway is tightly controlled through feedback regulation at multiple nodes (6).
We also specifically demonstrated that endogenous KRASG12C, KRASG12D, and KRASG12V can be feedback-activated by RTKs in a SHP2-dependent manner while the endogenous KRASG13D and KRASQ61H are feedback-activated in a SHP2-independent manner (Fig. 3B–G). This observation is consistent with the findings that KRASG12C and KRASG12D retain higher levels of intrinsic GTP hydrolysis activity compared with other KRAS variants and therefore may be more dependent on RTK/SHP2 for GTP-loading (28). The mechanism of SHP2-mediated feedback activation of KRASG12V with very low intrinsic GTP hydrolysis activity as KRASG13D and KRASQ61H in biochemical assays is yet to be determined but consistent with the emerging data that other KRAS G12 variants are dependent on RTK (44, 45). The SHP2-independent mechanism of GTP loading to KRASG13D and KRASQ61H also remains to be elucidated.
We have also observed a few examples of disconnections between the feedback activation of p-MEK and mutant KRAS. In NCI-H2122 (KRASG12C/G12C) and COR-L23 (KRASG12V/G12V), the reduction in p-MEK feedback activation by SHP2 inhibition is less than the reduction in mutant KRAS feedback activation (Fig. 3C and E). This disconnect is likely due to feedback activation of p-MEK downstream of RAS such as RAF, which can occur either concurrently with feedback activation of mutant KRAS or adaptively when the feedback activation of RAS is blocked by inhibition of RAS or SHP2. In this case, the p-MEK assay may underestimate the suppression of RAS activity by SHP2 inhibitors. As an imperfect surrogate assay for RAS feedback activation, our p-MEK assay coupled with SHP2 inhibition, correctly revealed that many cell lines bearing KRASG12C/D/V mutations, as well as other KRAS G12 variants, often have a large amount of feedback activation from upstream of RAS. In addition, our p-MEK assay identified a few heterozygous KRAS-mutant cell lines where WT KRAS contributed substantially to pathway feedback activation (T3M-4Q61H/+; Fig. 3G). This novel finding provides a rationale to cotarget feedback activation for various emerging therapeutics selectively targeting the mutant KRAS such as G12Ci. Overall, the p-MEK feedback activation assay may better reflect the general pathway reactivation than the RAS activation assay.
Interestingly, the RTK/SHP2–mediated feedback activation seems to be less common in NRASQ61 melanoma cell lines (Fig. 1E), which could be related to the low expression of RTKs in melanoma. This observation led us to hypothesize that the expression levels of SHP2, RTKs, and their ligands may potentially determine whether RTK/SHP2 is the major contributor to the pathway reactivation. However, no significant differences were found between the expression levels of those genes in cell lines that rely more on SHP2 for feedback activation and those that are less dependent (Supplementary Table S3). Phospho-FRS2 was proposed as a biomarker for FGFR1 feedback activation in KRAS-mutant lung and pancreatic cancer cells (18). Similar to FRS2, SHP2 can be phosphorylated upon RTK activation, which facilitates SHP2 activation (51, 52). It was also reported that phospho-SHP2 at Y542 (p-SHP2) may serve as a biomarker for RTK-driven acquired resistance to vemurafenib in melanoma (53). Therefore, we examined p-SHP2 changes during feedback activation. Indeed, in 3 of 4 cell lines with RTK/SHP2–driven feedback activation, selumetinib strongly increased p-SHP2 (Supplementary Fig. S5). In contrast, none of the three cell lines in which the feedback activation was less dependent on SHP2 exhibited p-SHP2 increase following treatment with selumetinib. These findings suggest that p-SHP2 induction following MEKi treatment may serve as a predictive biomarker of RTK/SHP2–mediated feedback activation to inform combination strategies with SHP2 inhibitors.
In KRAS-mutant cell lines in which feedback activation is mainly driven by RTK/SHP2, we observed both improved efficacy and enhanced pathway suppression by combined MEK and SHP2 inhibition in vitro and in vivo. However, the tolerability of this combination could be a concern. Indeed, severe body weight loss was observed in mice treated with SHP099 at its MTD (100 mpk/day) in combination with trametinib. Therefore, SHP099 was administered at a reduced dose (50 mpk or 25 mpk, every other day) when combined with trametinib (0.3 mpk/day) to improve the combination tolerability. Encouragingly, combination benefit was still achieved with the tolerated dosing regimens and intermittent dosing could also be explored in preclinical models and eventually clinical studies.
Notably, trametinib treatment caused adaptive p-MEK induction in vivo in all three xenograft models studied, consistent with the observation in vitro. However, the p-MEK reduction by cotreatment with SHP099 was not as dramatic as observed in vitro (Fig. 5B and D; Supplementary Fig. S4B). This disconnect may be due to the difference between plasma drug concentration fluctuations and the fixed drug concentration in media. Or the p-MEK–adaptive induction may not accurately reflect RAS activation in vivo due to additional feedback activation mechanisms after prolonged treatment. Nonetheless, the SHP099 and trametinib combination achieved further MAPK pathway inhibition in vivo than either inhibitor alone, which translated into enhanced antitumor efficacy in KRAS-mutant tumors. In addition, significant antitumor activity with single-agent SHP099 was observed in vivo in all three xenograft models studied, which coincided with decreased p-ERK levels to various degrees (Fig. 5B and D; Supplementary Fig. S4B). This is in contrast to the lack of SHP2i efficacy in KRAS-mutant cell lines in vitro (Fig. 4C) as previously reported (24). This may be possibly explained by our findings that some KRAS variants such as G12C/D/V can still be regulated by RTK/SHP2. This observation is consistent with recent reports that SHP2 is required for KRAS-mutant tumor growth in vivo and SHP2 inhibition may reduce the baseline GTP loading of several KRASG12 variants (44, 45).
The two recent studies (44, 45) also reported similar combination benefits of SHP2i and MEKi and observed better p-ERK inhibition in both KRAS-mutant and amplified models (54). Fedele and colleagues (55) also reported the combination benefits of SHP2 and MEK inhibition in KRAS-mutant cancer models as well as KRAS WT breast and ovarian cancer models but on the basis of time-dependent transcriptional upregulation of RTK and RTK ligand gene expression. Our findings are generally in agreement with those reports and provide a distinct rationale for the SHP2i+MEKi combination: a quick and robust (within 24 hours) feedback activation of RTK versus transcriptional upregulation of RTK and its ligands or single-agent activity of SHP2i in KRAS-mutant models in vivo. In addition, we specifically demonstrated that endogenous KRASG12C/D/V can be regulated by SHP2 using mutant-specific inhibitors and antibodies (Fig. 3B–E). Our study and the three other reports collectively demonstrated that combining SHP2 inhibitors with MEK inhibitors may achieve better suppression of mutant KRAS signaling by not only augmenting the upfront inhibition of the MAPK pathway but also preventing adaptive resistance to MEKi due to feedback activation of RTK as well as transcriptional upregulation of RTK and its ligands. With the progress of clinical development of SHP2 inhibitors such as TNO155 (ClinicalTrials.gov NCT03114319), the tolerability and effectiveness of various SHP2i and MEKi combination regimens for KRAS-mutant cancers can be further explored in clinical studies.
Disclosure of Potential Conflicts of Interest
E. Manchado has ownership interest (including stocks and patents) in Novartis. S.M. Brachmann has ownership interest (including stocks and patents) in Novartis. S.E. Moody has ownership interest (including stocks and patents) in Novartis. J.A. Engelman is global head, oncology, at Novartis Institute for Biomedical Research and has ownership interest (including stocks and patents) in Novartis. No potential conflicts of interest were disclosed by the other authors.
Authors' Contributions
Conception and design: H. Lu, R. Velazquez, S.M. Brachmann, J.A. Engelman, G. Caponigro, H.-X. Hao
Development of methodology: H. Lu, C. Liu, H. Wang, L.M. Dunkl, M. Kazic-Legueux, A. Haberkorn, S.M. Brachmann, H.-X. Hao
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H. Lu, C. Liu, R. Velazquez, E. Manchado, M. Mohseni
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H. Lu, C. Liu, R. Velazquez, S.M. Brachmann, M. Mohseni, H.-X. Hao
Writing, review, and/or revision of the manuscript: H. Lu, C. Liu, R. Velazquez, M. Kazic-Legueux, E. Manchado, S.E. Moody, P.S. Hammerman, G. Caponigro, M. Mohseni, H.-X. Hao
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): E. Billy
Study supervision: S.M. Brachmann, J.A. Engelman, P.S. Hammerman, G. Caponigro, M. Mohseni, H.-X. Hao
Acknowledgments
We thank Lisa Quinn and William Tschantz at Novartis for producing onartuzumab used in this study. We also thank the following Novartis colleagues, Yingnan Chen, Bryan Egge, Erick Morris, Tina Yuan, Mariela Jaskelioff, Matthew LaMarche, Michael Acker, Serena Silver, Zhao Chen, and Darrin Stuart, for their technical advice, discussion of data interpretation, suggestions of experiment design, and comments on the manuscript. This study was funded by Novartis Institutes for BioMedical Research.
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