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RAS nucleotide cycling underlies the SHP2 phosphatase dependence of mutant BRAF-, NF1- and RAS-driven cancers

Nature Cell Biology volume 20, pages 1064–1073 (2018)Cite this article

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Abstract

Oncogenic alterations in the RAS/RAF/MEK/ERK pathway drive the growth of a wide spectrum of cancers. While BRAF and MEK inhibitors are efficacious against BRAFV600E-driven cancers, effective targeted therapies are lacking for most cancers driven by other pathway alterations, including non-V600E oncogenic BRAF, RAS GTPase-activating protein (GAP) NF1 (neurofibromin 1) loss and oncogenic KRAS. Here, we show that targeting the SHP2 phosphatase (encoded by PTPN11) with RMC-4550, a small-molecule allosteric inhibitor, is effective in human cancer models bearing RAS–GTP-dependent oncogenic BRAF (for example, class 3 BRAF mutants), NF1 loss or nucleotide-cycling oncogenic RAS (for example, KRASG12C). SHP2 inhibitor treatment decreases oncogenic RAS/RAF/MEK/ERK signalling and cancer growth by disrupting SOS1-mediated RAS–GTP loading. Our findings illuminate a critical function for SHP2 in promoting oncogenic RAS/MAPK pathway activation in cancers with RAS–GTP-dependent oncogenic BRAF, NF1 loss and nucleotide-cycling oncogenic KRAS. SHP2 inhibition is a promising molecular therapeutic strategy for patients with cancers bearing these oncogenic drivers.

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Fig. 1: RMC-4550 is a potent, selective, allosteric inhibitor of SHP2.
Fig. 2: SHP2 inhibition suppresses growth and RAS/MAPK signalling in cancer cell lines with class 3 BRAF mutations.
Fig. 3: SHP2 inhibition suppresses growth and RAS/MAPK signalling in cancer cell lines driven by NF1LOF mutation.
Fig. 4: SHP2 inhibition suppresses growth and RAS/MAPK signalling in a range of cancer cell lines driven by KRASG12 mutations.
Fig. 5: The mechanism of action of SHP2 inhibition is dependent on SOS1.
Fig. 6: SHP2 inhibition suppresses tumour growth and RAS/MAPK signalling in xenograft models of NF1LOF- and KRASG12C-driven cancer.
Fig. 7: SHP2 inhibition suppresses tumour growth in PDX models of class 3 BRAF-, NF1LOF- and KRASG12C-driven cancer.

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Acknowledgements

Revolution Medicines thanks S. Kelsey for scientific guidance and critical review of the manuscript, K. Shokat for scientific guidance, N. Tibrewal for development of the phosphatase selectivity panel and J. Jasper for scientific input. F. Haderk was supported by the Mildred Scheel postdoctoral fellowship from the German Cancer Aid. Funding support from NIH/NCI U54CA224081, NIH/NCI R01CA204302, NIH/NCI R01CA211052, NIH/NCI R01CA169338 and Pew Trust and Stewart Foundation (T.G.B.) is acknowledged. We are grateful to researchers at MI Bioresearch, WuXi Apptec and Genendesign who support our in vivo pharmacology efforts.

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Authors and Affiliations

  1. Department of Biology, Revolution Medicines, Inc., Redwood City, CA, USA

    Robert J. Nichols, Carlos Stahlhut, Christopher J. Schulze, David Wildes, Christos Tzitzilonis, Kasia Mordec, Abby Marquez, Jason Romero, Tientien Hsieh, Gert Kiss, Mallika Singh, Mark A. Goldsmith & Jacqueline A. M. Smith

  2. Department of Medicine, University of California, San Francisco, San Francisco, CA, USA

    Franziska Haderk, Golzar Hemmati, Aubhishek Zaman, Victor Olivas, Caroline McCoach, Collin M. Blakely & Trever G. Bivona

  3. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA

    Franziska Haderk, Golzar Hemmati, Aubhishek Zaman, Victor Olivas & Trever G. Bivona

  4. Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA

    Franziska Haderk, Golzar Hemmati, Aubhishek Zaman, Victor Olivas & Trever G. Bivona

  5. Department of Development Sciences, Revolution Medicines, Inc., Redwood City, CA, USA

    Zhengping Wang

  6. Department of Chemistry, Revolution Medicines, Inc., Redwood City, CA, USA

    Elena S. Koltun, Adrian L. Gill & Mark A. Goldsmith

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Contributions

R.J.N. contributed to the design, conduct and interpretation of all experiments. F.H. contributed in vitro and in vivo experimental design, conduct and interpretation. C.S., C.J.S, G.H., D.W., C.T., K.M., A.M., J.R., T.H. and A.Z. contributed in vitro experimental design, conduct and interpretation. V.O., C.M. and Z.W. contributed design, conduct and interpretation of in vivo experiments. C.M.B. established and maintains the TH77 model. G.K., E.S.K. and A.L.G. directed the design and synthesis of RMC-4550. M.S. contributed design, conduct and interpretation of in vivo experiments. M.A.G. contributed to the interpretation of all experiments. J.A.M.S. and T.G.B. supervised the project and contributed to the design and interpretation of all experiments. R.J.N., J.A.M.S. and T.G.B. wrote the manuscript with input from all co-authors.

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Correspondence to Jacqueline A. M. Smith or Trever G. Bivona.

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Competing interests

T.G.B. is a consultant to Novartis, AstraZeneca, Array Biopharma (Scientific Advisory Board) and Inventiva, and is the recipient of research grants from Ignyta and Revolution Medicines. T.G.B. is a member of the Revolution Medicines Scientific Advisory Board. R.J.N., C.S., C.J.S., D.W., C.T., K.M., A.M., J.R., T.H., Z.W., G.K., E.S.K., A.L.G., M.S., M.A.G. and J.A.M.S. are employees of Revolution Medicines, Inc. Revolution Medicines holds the patent mentioned in the Methods section. The other authors declare no competing interests.

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Integrated supplementary information

Supplementary Figure 1 Western blot validation of a RAS-GTP ELISA.

NCI-H358 cells were grown in 2D culture and incubated with increasing concentrations of RMC-4450 for one hour. a) Cellular lysates were prepared and levels of RAS-GTP were measured by ELISA. Shown are three independent biological replicates performed in technical duplicate. b) Assessment of RAS-GTP in samples from replicate 2 by western blot. RAS-GTP was affinity purified from the cellular lysates using the GST-RAF-RBD bound to glutathione resin. Source data is provided in Supplementary Table 9.

Supplementary Figure 2 Expression of SHP2E76K rescues Class 3 BRAF, NF1LOF and KRASG12C mutant cell lines from RMC-4550.

Stable expression of SHP2-WT and SHP2-E76K was induced upon lentiviral transfection of NCI-H508 (Class 3 BRAF), NCI-H1838 (NF1LOF), and NCI-H358 (KRASG12C) cells (n = 3 biologically independent experiments; figures show mean +/− S.D.) a, c, e) Effect of RMC-4550 on proliferation of NCI-H508 (a), NCI-H1838 cells (c), and NCI-H358 cells (e) in 3D culture. Three days after seeding, cells were treated with RMC-4550 and cell viability measured on Day 8 using CTG (data are representative of n = 2 independent experiments for NCI-H508 and NCI-H1838, and n = 3 observations for H358, in technical duplicate; figures show mean +/− S.D.). RMC-4550 exhibited concentration-dependent inhibition of growth in parental and SHP2WT-expressing cell lines representing all three genotypes but failed to elicit a reduction in proliferation ≥50% of control signal in SHP2E76K expressing cells up to the maximal test concentration of 10 μM. b, d, f) Effect of RMC-4550 on pERK levels in NCI-H508 (b), NCI-H1838 cells (d), and NCI-H358 cells (f). Cells were grown in 2D culture and incubated with 1 µM RMC-4550 for 90 minutes. Cellular lysates were prepared and levels of pERK determined by immunoblot. RMC-4550 treatment resulted in reduction of pERK levels in parental and SHP2WT expressing cells across all genotypes, but failed to suppress pERK in SHP2E76K expressing cells. Shown is one representative example of three independent experiments. Source data is provided in Supplementary Table 9.

Supplementary Figure 3 Effects of SHP2 inhibition in RAS pathway-driven cancer cells.

(a) Effect of RMC-4550 on RAS-GTP levels in BEAS-2B cells stably transfected with scrambled (shScramble) or NF1 targeting (shNF1) shRNA. Data shows a representative of three independent experiments. (b-d) (b) Cell sensitivities to the knockdowns of the indicated genes were determined using crystal violet staining assays (n = 2). The spectrophotometric intensities of the crystal violet stains were quantified and represented as a bar plot. (c) RAS-GTP levels in cellular lysates are shown by GST-RAF-RBD affinity capture. (d) Individual western blot band intensities were quantified and shown for pERK/ERK, RAS-GTP/RAS (total) level and knock down efficiency, expressed relative to a control scrambled shRNA intensity of 1.0 and the effect of knockdown on pERK. (e-h) (e), (f) Effect of RMC-4550 and the KRASG12C-specific inhibitor ARS-853 on pERK levels in KRASG12C cancer cell lines. Cells were incubated with increasing concentrations of RMC-4550 or ARS-853 for one hour. Cellular lysates were prepared and levels of pERK determined. RMC-4550 produced a concentration-dependent reduction in pERK levels. Geometric mean IC50 values for reduction in pERK by RMC-4550 in NCI-H358 and CALU-1 were 36 nM and 7 nM, respectively. A concentration-dependent reduction in pERK levels was observed for ARS-853 (geometric mean IC50 values of 4700 nM and 430 nM for NCI-H358 and CALU-1 cells, respectively); data represent n = 4 independent experiments, performed in technical duplicate; figures show mean +/− S.D.). (g), (h) Effect of RMC-4550 and ARS-853 on proliferation of two KRASG12C cancer cell lines in 3D culture. NCI-H358 and CALU-1 cells were treated with RMC-4550 or ARS-853 and cell viability measured on Day 7 using Cell Titer Glo (data represent n = 4 independent experiments, performed in duplicate for NCI-H358, and n = 3 independent experiments, performed in duplicate for CALU-1; figures show mean +/− S.D.). RMC-4550 and ARS-853 exhibited geometric mean IC50 values of 43 nM and 1020 nM, respectively, in NCI-H358 cells. Source data is provided in Supplementary Table 9.

Supplementary Figure 4 Effects of SHP2 inhibition in KRAS-driven cancer cells.

(a) Comparison of the effect of SHP2 inhibition and KRAS knockdown on the proliferation of KRAS-mutant cancer cell lines bearing different oncogenic mutations in KRAS as indicated by color coding. The negative logarithm of RMC-4550 GI50 values for cancer cells bearing activating KRAS mutations (described in Supplementary Table 5) were plotted against the negative value of their Project Drive Sensitivity Score for KRAS shRNA knockdown. Cells with high sensitivity to KRAS knockdown have larger negative Sensitivity Scores, and cells with greater sensitivity to SHP2 inhibition have larger negative log(RMC-4550 GI50) values. (b-c) Effect of RMC-4550 on RAS-GTP and pERK levels in Hras−/−, Nras−/−, Kras−/− mouse embryonic fibroblasts (MEFs) expressing solely the indicated RAS or BRAF transcript. Cells were grown in 2D culture and incubated with 1 µM RMC-4550 for 24 h. b) Cellular lysates were prepared and levels of RAS-GTP and pERK determined by immunoblot. RAS-GTP was affinity purified from the cellular lysates using GST-tagged RAF-RBD coupled to glutathione agarose beads. (c) RAS-GTP levels were quantified in whole cell lysates via RAS-GTP ELISA. Significant reduction in RAS-GTP and pERK levels is observed for KRAS WT (WT-4B) and KRAS G12 expressing MEFs, while cells expressing KRASQ61H lack response. Values above bars represent fraction of RAS-GTP in RMC-4550 vs. DMSO treated cells. Data in (b) shows a representative example of three independent experiments; data in (c) shows a representative example of two independent experiments, and bars indicates mean of two technical replicates. (d-e) (d) Expression of the wild type SOS1 and SOS-F proteins was induced by doxycycline treatment for 24 h. Cells were treated with 50 ng/mL EGF at various time intervals, lysed, and total pERK levels were measured by AlphaLisa. The SOS-F construct showed increased and sustained levels of pERK signal in the absence of EGF stimulation. Shown is one representative example performed in technical duplicate of three independent experiments. Figure indicates mean +/− S.D. (e) NCI-H358 cells transiently expressing SOS1 and SOS-F encoding cDNA for 48 h were treated with vehicle or 1 µM RMC-4550 for 1 and 6 h. Harvested lysates from the cells were probed for the indicated proteins using western blot. Data shown represent at least 3 independent biological replicates. Source data is provided in Supplementary Table 9.

Supplementary Figure 5 Xenograft models of KRASG13D and KRASQ61K are insensitive to RMC-4550.

Daily oral administration of RMC-4550 (30 mg/kg) to athymic nude mice bearing (a) HCT-116 (KRASG13D) or (b) CALU-6 (KRASQ61K) xenograft tumors did not produce significant tumor growth inhibition. Treatment with RMC-4550 was well tolerated in both models, as assessed by body weight (c,d). Source data is provided in Supplementary Table 9.

Supplementary Figure 6 Tolerability assessment in animals treated with the SHP2 inhibitor.

Repeated daily oral administration of RMC-4550 of all dose groups in the MeWo (a), NCI-H1838 (b), NCI-H358 (c), and MIA PaCa-2 (d), xenograft models and LUN023 (e), LUN037 (f), TH77 (g), LUN092 (h), and LUN156 (i) PDX models was well tolerated as assessed by body weight. Source data is provided in Supplementary Table 9.

Supplementary Figure 7 Durable response to RMC-4550 in NF1LOF model.

(a) Western blot demonstrates loss of NF1 protein in TH77 PDX model. A previously published EGFRL858R, NF1wt PDX model is shown as comparator1. (b) Tumor specimen of vehicle and RMC-4550 treated groups were analyzed regarding their general tissue morphology by H&E stain (representative of n = 3 independent samples per treatment arm, 10X magnification, size bar: 100 nm). Significant areas of necrosis (arrows) were present in tumors of RMC-4550 treated animals.

Supplementary Figure 8 Unprocessed scans of raw blots.

(a) SHP2 IP from Fig. 5c, (b) GAB1 IP from Fig. 5d, and (c) membrane fractionation from Fig. 5e. (d) Effect of RMC-4550 on RAS-GTP levels from Supplementary Figure 1b, (e) Effect of SHP2 variants on pERK in H508 cells from Supplementary Figure 2b, (f) Effect of SHP2 variants on pERK in H1838 cells from Supplementary Figure 2d, (g) Effect of SHP2 variants on pERK in H358 cells from Supplementary Figure 2f. (h) Effect of NF1 shRNA knockdown on RAS-GTP levels from Supplementary Figure 3a, and (i) RAS-GTP levels from Supplementary Figure 3c, (j) Effect of SHP2 and SOS1 shRNA knockdown in RAS-GTP levels from Supplementary Figure 3d, (k) Effect of RMC-4550 on RAS-GTP and pERK levels in RAS-less MEFs from Supplementary Figure 4b, (l) Effect of SOS or SOS-F expression on pERK from Supplementary Figure 4e, (m) NF1 levels in TH77 PDX model from Supplementary Figure 7a. Note that panels (d) through (e) are located in additional pages.

Supplementary information

Supplementary Information

Supplementary Figures 1–9 and Supplementary Table legends.

Reporting Summary

Supplementary Table 1

RMC-4550 activity against a panel of 15 phosphatases.

Supplementary Table 2

RMC-4550 activity against a panel of 468 kinases at a test concentration of 10 µM.

Supplementary Table 3

RMC-4550 activity against a panel of 44 safety targets at a test concentration of 10 μM.

Supplementary Table 4

RMC-4550 activity against a panel of NF1 mutant cell lines.

Supplementary Table 5

RMC-4550 activity against a panel of 33 KRAS mutant cell lines.

Supplementary Table 6

Pharmacokinetic parameters of RMC-4550 from single dose NCI-H358 study.

Supplementary Table 7

Pharmacokinetic parameters of RMC-4550 from end of MIA PaCa-2 efficacy study.

Supplementary Table 8

Antibodies.

Supplementary Table 9

Statistics source data.

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Nichols, R.J., Haderk, F., Stahlhut, C. et al. RAS nucleotide cycling underlies the SHP2 phosphatase dependence of mutant BRAF-, NF1- and RAS-driven cancers. Nat Cell Biol 20, 1064–1073 (2018). https://doi.org/10.1038/s41556-018-0169-1

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