Abstract
Resistance to RAF- and MEK-targeted therapy is a major clinical challenge1,2,3,4. RAF and MEK inhibitors are initially but only transiently effective in some but not all patients with BRAF gene mutation and are largely ineffective in those with RAS gene mutation because of resistance5,6,7,8,9,10,11,12,13,14. Through a genetic screen in BRAF-mutant tumor cells, we show that the Hippo pathway effector YAP (encoded by YAP1) acts as a parallel survival input to promote resistance to RAF and MEK inhibitor therapy. Combined YAP and RAF or MEK inhibition was synthetically lethal not only in several BRAF-mutant tumor types but also in RAS-mutant tumors. Increased YAP in tumors harboring BRAF V600E was a biomarker of worse initial response to RAF and MEK inhibition in patients, establishing the clinical relevance of our findings. Our data identify YAP as a new mechanism of resistance to RAF- and MEK-targeted therapy. The findings unveil the synthetic lethality of combined suppression of YAP and RAF or MEK as a promising strategy to enhance treatment response and patient survival.
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Acknowledgements
We thank G. Bollag and P. Lin (Plexxikon) for providing PLX4720. We thank J. Weissman and members of the Bivona laboratory for critical review of the manuscript. The authors acknowledge funding support (to T.G.B.) from the following sources: US National Institutes of Health (NIH) Director's New Innovator Award (DP2CA174497), the Howard Hughes Medical Institute (Collaborative Innovation Award), the Doris Duke Charitable Foundation, the American Lung Association, the National Lung Cancer Partnership, the Addario and Van Auken Private Foundations, the Sidney Kimmel Foundation for Cancer Research and the Searle Scholars Program. The authors also acknowledge funding from La Caixa Foundation (to R.R.) and from US NIH grant R01CA131261 (to M.M.).
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L.L., E.C., A.J.S., V.O., D.N., J.J.Y., E.P., X.L., M.M.W., L.P. and E.A.C. conducted all experiments and/or analyzed data. L.C. and S.A. conducted the analysis of the shRNA screening data. M.M. provided cell lines and analyzed data. N.K., M.G.C., J.L.M., J.L.R., J.M.S.T., F.B., C.M.R., A.A., E.R., I.O., E.C., E.M.-C., J.C., A.M., R.R., D.T.F., Z.A.C., K.T.F. and J.A.W. provided clinical samples and data. All authors contributed to the design of experiments and to data analysis and interpretation. T.G.B., L.L. and A.J.S. wrote the manuscript with input from all coauthors.
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T.G.B. is a consultant for Driver Group as well as Novartis, Clovis Oncology and Cleave Biosciences and is the recipient of a research grant from Servier.
Integrated supplementary information
Supplementary Figure 1 Effects of YAP1 inhibition on cell growth.
(a) Primary screen data showing that the YAP1 gene was not depleted over 10 d of vehicle (DMSO) treatment in HCC364 cells (DMSO versus T0). (b) Primary screening data showing that shRNAs targeting YAP1 were not depleted over 10 d of vehicle (DMSO) treatment in HCC364 cells (DMSO versus T0). (c) Effect of YAP1 knockdown using two independent shRNAs on cell growth in HCC364, A2058, HT29, WiDr, KHM-5M, HTC/C3, A549, H2347, SW1573, MOR/CPR, SK-MEL-2, MM415, HPAF-II and PANC02.03 cells, measured at 3 d by CellTiter-Glo assay.
Supplementary Figure 2 The effects of YAP silencing were specific to targeted inhibition of RAF-MEK signaling and are through YAP transcriptional activity.
(a) Effects of YAP1 knockdown using two independent shRNAs on pemetrexed sensitivity in HCC364 lung cancer cells. (b) Effects of YAP1 knockdown using two independent shRNAs on cisplatin sensitivity in HCC364 lung cancer cells. (c) Effects of TEAD2 knockdown using two independent shRNAs on vemurafenib sensitivity in HCC364 lung cancer cells (shown are the IC50 and relative cell viability). (d) Validation of the effects of TEAD2 knockdown on trametinib sensitivity in HCC364 BRAF-mutant lung cancer cells (shown are the IC50 and cell viability). (e) Effects of TEAD4 knockdown using two independent shRNAs on vemurafenib sensitivity in HCC364 lung cancer cells (shown are the IC50 and relative cell viability). (f) Validation of the effects of TEAD4 knockdown on trametinib sensitivity in HCC364 BRAF-mutant lung cancer cells (shown are the IC50 and cell viability). (g) mRNA expression of TEAD2 and TEAD4 in cells expressing scrambled control shRNA or shRNA to TEAD2 or TEAD4. (h) Effects of TEAD2 and TEAD4 knockdown on vemurafenib and trametinib sensitivity in HCC364 BRAF-mutant lung cancer cells (shown is 7-d cell growth assessed by crystal violet staining assays, with quantification of the effects on viability under each condition).
Supplementary Figure 3 Nuclear YAP expression in BRAF- and RAS-mutant cancer cell lines.
Nuclear/cytoplasmic fractionation and immunoblot analysis of the indicated proteins in (a) BRAF V600E–mutant cancer cell lines and (b) RAS-mutant cancer cell lines. Data represent three independent experiments.
Supplementary Figure 4 Exogenous expression of YAP or TAZ promotes resistance to RAF-MEK inhibition.
(a) Effects of YAP1 or TAZ overexpression on vemurafenib sensitivity in HCC364 lung cancer cells (shown are the IC50 and relative cell viability). (b) Effects of YAP1 or TAZ overexpression on trametinib sensitivity in HCC364 lung cancer cells (shown are the IC50 and relative cell viability).
Supplementary Figure 5 Increase in maximal growth inhibition upon trametinib treatment in YAP1-depleted cells.
(a) Effects of YAP1 knockdown on trametinib sensitivity in the indicated BRAF-mutant cell lines, shown as percentage of maximal growth inhibition (n = 4, +s.e.m. for all cell viability data shown). (b) Effects of YAP1 knockdown on trametinib sensitivity in the indicated RAS-mutant cell lines, shown as percentage of maximal growth inhibition (n = 4, +s.e.m. for all cell viability data shown).
Supplementary Figure 6 YAP1 knockdown sensitizes cancer cells to RAF-MEK inhibition across multiple tumor types.
(a,b) Effects of YAP1 knockdown using two independent shRNAs on vemurafenib and trametinib sensitivity in (a) A2058 and (b) WM793 BRAF V600E–mutant melanoma cancer cells. (c,d) Effects of YAP1 knockdown on vemurafenib and trametinib sensitivity in (c) A2058 and (d) WM793 BRAF V600E–mutant melanoma cancer cells (shown is 7-d cell growth assessed by crystal violet staining assays, with quantification of the effects on viability under each condition). (e,f) Effects of YAP1 knockdown using two independent shRNAs on vemurafenib and trametinib sensitivity in (e) HT29 and (f) WiDr BRAF V600E–mutant colon cancer cells. (g,h) Effects of YAP1 knockdown using two independent shRNAs on vemurafenib and trametinib sensitivity in (g) KHM-5M and (h) HTC/C3 BRAF V600E–mutant thyroid cancer cells.
Supplementary Figure 7 YAP1 inhibition sensitizes tumors to RAF-MEK inhibition in vivo.
(a) Average tumor volume of xenografts generated from A2058 cells infected with either scrambled control shRNA or YAP1 shRNA (n = 10/group, +s.e.m.). (b) Waterfall plot indicating fold change in tumor volume upon vemurafenib or trametinib treatment of A2058 xenografts infected with either scrambled control shRNA or YAP1 shRNA (n = 8–12/group, ±s.e.m.). (c) Immunoblot analysis for each indicated protein in lysates from representative A2058 xenograft tumors. d, Average tumor volume of xenografts generated from HT29 cells infected with either scrambled control shRNA or YAP1 shRNA (n = 10/group, +s.e.m.). (e) Waterfall plot indicating fold change in tumor volume upon vemurafenib or trametinib treatment of HT29 xenograft model infected with either scrambled control shRNA or YAP1 shRNA (n = 8–12/group, ±s.e.m.). (f) Immunoblot analysis for each indicated protein in lysates from representative HT29 xenograft tumors. (g) Waterfall plot indicating fold change in tumor volume upon trametinib treatment of MOR/CPR xenograft model infected with either scrambled control shRNA or YAP1 shRNA (n = 8–12/group, ±s.e.m.). (h) Immunoblot analysis for each indicated protein in lysates from representative MOR/CPR xenograft tumors.
Supplementary Figure 8 Effect of treatment with the EGFR kinase inhibitor erlotinib on RAF-MEK inhibitor sensitivity.
(a,b) Effect of treatment with the EGFR kinase inhibitor erlotinib on (a) vemurafenib and (b) trametinib sensitivity in A2058 BRAF V600E–mutant melanoma cells. (c,d) Effect of EGFR kinase inhibitor erlotinib on (c) vemurafenib and (d) trametinib sensitivity in KHM-5M BRAF V600E–mutant thyroid cancer cells.
Supplementary Figure 9 YAP1 knockdown sensitizes RAS-mutant melanoma and pancreatic cancer cells to MEK inhibition.
(a,b) Effects of YAP1 knockdown using two independent shRNAs on trametinib sensitivity in (a) MM415 NRAS Q61L–mutant, (b) SK-MEL-2 NRAS Q61R–mutant melanoma cells. (c,d) Effects of YAP1 knockdown using two independent shRNAs on trametinib sensitivity in (c) HPAF-II KRAS G12D–mutant and (d) Panc 02.03 KRAS G12D–mutant pancreatic cancer cells. (e,f) Effects of YAP1 knockdown using two independent shRNAs on trametinib sensitivity in (e) MM415 NRAS Q61L–mutant and (f) SK-MEL-2 NRAS Q61R–mutant melanoma cells (shown is 7-d cell growth assessed by crystal violet staining assays, with quantification of the effects on viability under each condition).
Supplementary Figure 10 YAP1 knockdown sensitizes RAS-mutant NSCLC cells to MEK inhibition.
(a–f) Effects of YAP1 knockdown using two independent shRNAs on trametinib sensitivity in (a) A549 KRAS G12S–mutant, (b) H23 KRAS G12C–mutant, (c) Calu-6 KRAS G12C–mutant, (d) H2347 NRAS Q61R–mutant, (e) MOR/CPR KRAS G12C–mutant and (f) SW1573 KRAS G12C–mutant NSCLC cell lines. (g–i) Effects of YAP1 knockdown using two independent shRNAs on trametinib sensitivity in (g) MOR/CPR KRAS G12C–mutant cells, (h) SW1573 KRAS G12C–mutant cells and (i) H2347 NRAS Q61R–mutant cells (shown is 7-d cell growth assessed by crystal violet staining assays, with quantification of the effects on viability under each condition).
Supplementary Figure 11 YAP1 knockdown induces apoptosis upon RAF-MEK inhibition.
(a,b) Effects of YAP1 knockdown on apoptosis induced upon (a) vemurafenib and (b) trametinib treatment in WiDr BRAF-mutant colon cancer cells as measured by caspase-3/7 activation. (c,d) Effects of YAP1 knockdown on apoptosis induced upon (c) vemurafenib and (d) trametinib treatment in A549 KRAS-mutant lung cancer cells as measured by caspase-3/7 activation. (e,f) Effects of pharmacological inhibition of BCL-xL using TW-37 on vemurafenib or trametinib sensitivity in (e) HCC364 BRAF-mutant lung cancer cells and (f) A2058 BRAF-mutant melanoma cells (n = 3, ±s.e.m. for all cell viability data shown; P values are indicated for statistical analysis). (g,h) Effects of pharmacological inhibition of BCL-xL using ABT-263 or TW-37 on trametinib sensitivity in (g) A549 KRAS G12S–mutant and (h) Calu-1 KRAS G12C–mutant lung cancer cells (n = 3, ±s.e.m. for all cell viability data shown; P values are indicated for statistical analysis).
Supplementary Figure 12 YAP inhibition plus RAF-MEK inhibition suppresses BCL-xL expression.
(a–c) Effects of YAP suppression and vemurafenib or trametinib treatment on BCL-xL levels by immunoblot analysis in BRAF V600E–mutant (a) A2058 melanoma, (b) HT29 colon and (c) KHM-5M thyroid cancer cells. Results represent three independent experiments.
Supplementary Figure 13 BCL-xL expression rescues YAP1-depleted cells from growth suppression by RAF-MEK inhibition.
(a,b) Effects of BCL-xL overexpression and YAP1 knockdown on (a) vemurafenib and (b) trametinib sensitivity in HCC364 lung cancer cells (shown are the IC50 and relative cell viability). (c) Immunoblot analysis for each indicated protein in lysates from HCC364 cells overexpressing either GFP or BCL-xL and treated with either control siRNA or YAP1 siRNA.
Supplementary Figure 14 Gene sets enriched among the genes specifically altered by YAP and MEK coinhibition.
Pathway analysis indicating enriched gene sets among those genes whose expression was specifically decreased by YAP and MEK coinhibition (Supplementary Table 5). The top six significantly represented pathways among these genes are shown (significance of pathway representation is shown as –log q, with the red line indicating q < 0.05). The genes highlighted in the inset are the significantly altered apoptosis-related genes.
Supplementary Figure 15 YAP protein expression in human tumor specimens by YAP immunohistochemistry.
(a) Representative images of immunohistochemistry staining of YAP protein in representative human tumor specimens with low, intermediate and high YAP staining (brown). (b) YAP staining by immunohistochemistry in BRAF V600E–mutant melanomas obtained before RAF or MEK inhibitor treatment and upon acquired resistance. Shown are the 4 out of 16 cases in which the pretreatment tumor did not harbor high levels of YAP (showing either low or intermediate staining by immunohistochemistry). Bars represent 50 microns.
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Supplementary Text and Figures
Supplementary Figures 1–15 and Supplementary Tables 2–4, 6 and 7 (PDF 2308 kb)
Supplementary Table 1
Gene targets included in the pooled shRNA screening library. (XLSX 101 kb)
Supplementary Table 5
Differentially expressed genes in BRAFV600E NSCLC cells (HCC364) upon either YAP knockdown alone (by shRNA), trametinib treatment alone and combined YAP knockdown and trametinib treatment. (XLSX 398 kb)
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Lin, L., Sabnis, A., Chan, E. et al. The Hippo effector YAP promotes resistance to RAF- and MEK-targeted cancer therapies. Nat Genet 47, 250–256 (2015). https://doi.org/10.1038/ng.3218
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DOI: https://doi.org/10.1038/ng.3218
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