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Open Access 01-12-2018 | Research article

Pan-RAF and MEK vertical inhibition enhances therapeutic response in non-V600 BRAF mutant cells

Authors: Eszter Molnár, Dominika Rittler, Marcell Baranyi, Michael Grusch, Walter Berger, Balázs Döme, József Tóvári, Clemens Aigner, József Tímár, Tamás Garay, Balázs Hegedűs

Published in: BMC Cancer | Issue 1/2018

Abstract

Background

Currently, there are no available targeted therapy options for non-V600 BRAF mutated tumors. The aim of this study was to investigate the effects of RAF and MEK concurrent inhibition on tumor growth, migration, signaling and apoptosis induction in preclinical models of non-V600 BRAF mutant tumor cell lines.

Methods

Six BRAF mutated human tumor cell lines CRL5885 (G466 V), WM3629 (D594G), WM3670 (G469E), MDAMB231 (G464 V), CRL5922 (L597 V) and A375 (V600E as control) were investigated. Pan-RAF inhibitor (sorafenib or AZ628) and MEK inhibitor (selumetinib) or their combination were used in in vitro viability, video microscopy, immunoblot, cell cycle and TUNEL assays. The in vivo effects of the drugs were assessed in an orthotopic NSG mouse breast cancer model.

Results

All cell lines showed a significant growth inhibition with synergism in the sorafenib/AZ628 and selumetinib combination. Combination treatment resulted in higher Erk1/2 inhibition and in increased induction of apoptosis when compared to single agent treatments. However, single selumetinib treatment could cause adverse therapeutic effects, like increased cell migration in certain cells, selumetinib and sorafenib combination treatment lowered migratory capacity in all the cell lines. Importantly, combination resulted in significantly increased tumor growth inhibition in orthotropic xenografts of MDAMB231 cells when compared to sorafenib - but not to selumetinib – treatment.

Conclusions

Our data suggests that combined blocking of RAF and MEK may achieve increased therapeutic response in non-V600 BRAF mutant tumors.

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Santarpia L, Lippman SM, El-Naggar AK. Targeting the MAPK-RAS-RAF signaling pathway in cancer therapy. Expert Opin Ther Targets. 2012;16(1):103–19.CrossRefPubMedPubMedCentral

Garnett MJ, Rana S, Paterson H, Barford D, Marais R. Wild-type and mutant B-RAF activate C-RAF through distinct mechanisms involving heterodimerization. Mol Cell. 2005;20(6):963–9.CrossRefPubMed

Turski ML, Vidwans SJ, Janku F, Garrido-Laguna I, Munoz J, Schwab R, Subbiah V, Rodon J, Kurzrock R. Genomically driven tumors and Actionability across Histologies: BRAF-mutant cancers as a paradigm. Mol Cancer Ther. 2016;15(4):533–47.CrossRefPubMed

Carter J, Tseng L-H, Zheng G, Dudley J, Illei P, Gocke CD, Eshleman JR, Lin M-T. Non-p.V600E BRAF mutations are common using a more sensitive and broad detection tool. Am J Clin Pathol. 2015;144(4):620–8.CrossRefPubMed

Tissot C, Couraud S, Tanguy R, Bringuier PP, Girard N, Souquet PJ. Clinical characteristics and outcome of patients with lung cancer harboring BRAF mutations. Lung Cancer. 2016;91:23–8.CrossRefPubMed

Zheng G, Tseng LH, Chen G, Haley L, Illei P, Gocke CD, Eshleman JR, Lin MT. Clinical detection and categorization of uncommon and concomitant mutations involving BRAF. BMC Cancer. 2015;15:779.CrossRefPubMedPubMedCentral

Long GV, Stroyakovskiy D, Gogas H, Levchenko E, de Braud F, Larkin J, Garbe C, Jouary T, Hauschild A, Grob JJ, et al. Combined BRAF and MEK inhibition versus BRAF inhibition alone in melanoma. N Engl J Med. 2014;371(20):1877–88.CrossRefPubMed

Robert C, Karaszewska B, Schachter J, Rutkowski P, Mackiewicz A, Stroiakovski D, Lichinitser M, Dummer R, Grange F, Mortier L, et al. Improved overall survival in melanoma with combined dabrafenib and trametinib. N Engl J Med. 2015;372(1):30–9.CrossRefPubMed

Atefi M, Titz B, Avramis E, Ng C, Wong DJ, Lassen A, Cerniglia M, Escuin-Ordinas H, Foulad D, Comin-Anduix B, et al. Combination of pan-RAF and MEK inhibitors in NRAS mutant melanoma. Mol Cancer. 2015;14:27.CrossRefPubMedPubMedCentral

10.

Rebecca VW, Alicea GM, Paraiso KH, Lawrence H, Gibney GT, Smalley KS. Vertical inhibition of the MAPK pathway enhances therapeutic responses in NRAS-mutant melanoma. Pigment Cell Melanoma Res. 2014;27(6):1154–8.CrossRefPubMedPubMedCentral

11.

Lamba S, Russo M, Sun C, Lazzari L, Cancelliere C, Grernrum W, Lieftink C, Bernards R, Di Nicolantonio F, Bardelli A. RAF suppression synergizes with MEK inhibition in KRAS mutant cancer cells. Cell Rep. 2014;8(5):1475–83.CrossRefPubMed

12.

Huynh H, Ngo VC, Koong HN, Poon D, Choo SP, Toh HC, Thng CH, Chow P, Ong HS, Chung A, et al. AZD6244 enhances the anti-tumor activity of sorafenib in ectopic and orthotopic models of human hepatocellular carcinoma (HCC). J Hepatol. 2010;52(1):79–87.CrossRefPubMed

13.

Koh YW, Shah MH, Agarwal K, McCarty SK, Koo BS, Brendel VJ, Wang C, Porter K, Jarjoura D, Saji M, et al. Sorafenib and Mek inhibition is synergistic in medullary thyroid carcinoma in vitro. Endocr Relat Cancer. 2012;19(1):29–38.CrossRefPubMedPubMedCentral

14.

Nguyen TK, Jordan N, Friedberg J, Fisher RI, Dent P, Grant S. Inhibition of MEK/ERK1/2 sensitizes lymphoma cells to sorafenib-induced apoptosis. Leuk Res. 2010;34(3):379–86.CrossRefPubMedPubMedCentral

15.

Yuen JS, Sim MY, Sim HG, Chong TW, Lau WK, Cheng CW, Ong RW, Huynh H. Combination of the ERK inhibitor AZD6244 and low-dose sorafenib in a xenograft model of human renal cell carcinoma. Int J Oncol. 2012;41(2):712–20.CrossRefPubMed

16.

Cichowski K, Jänne PA. Drug discovery: inhibitors that activate. Nature. 2010;464(7287):358–9.CrossRefPubMed

17.

Heidorn SJ, Milagre C, Whittaker S, Nourry A, Niculescu-Duvas I, Dhomen N, Hussain J, Reis-Filho JS, Springer CJ, Pritchard C, et al. Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell. 2010;140(2):209–21.CrossRefPubMedPubMedCentral

18.

Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good VM, Jones CM, Marshall CJ, Springer CJ, Barford D, et al. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell. 2004;116(6):855–67.CrossRefPubMed

19.

Gadaleta-Caldarola G, Divella R, Mazzocca A, Infusino S, Ferraro E, Filippelli G, Daniele A, Sabbà C, Abbate I, Brandi M. Sorafenib: the gold standard therapy in advanced hepatocellular carcinoma and beyond. Future Oncol. 2015;11(16):2263–6.CrossRefPubMed

20.

Wilhelm SM, Adnane L, Newell P, Villanueva A, Llovet JM, Lynch M. Preclinical overview of sorafenib, a multikinase inhibitor that targets both Raf and VEGF and PDGF receptor tyrosine kinase signaling. Mol Cancer Ther. 2008;7(10):3129–40.CrossRefPubMed

21.

Montagut C, Sharma SV, Shioda T, McDermott U, Ulman M, Ulkus LE, Dias-Santagata D, Stubbs H, Lee DY, Singh A, et al. Elevated CRAF as a potential mechanism of acquired resistance to BRAF inhibition in melanoma. Cancer Res. 2008;68(12):4853–61.CrossRefPubMedPubMedCentral

22.

Akinleye A, Furqan M, Mukhi N, Ravella P, Liu D. MEK and the inhibitors: from bench to bedside. J Hematol Oncol. 2013;6:27.CrossRefPubMedPubMedCentral

23.

Garay T, Kenessey I, Molnar E, Juhasz E, Reti A, Laszlo V, Rozsas A, Dobos J, Dome B, Berger W, et al. Prenylation inhibition-induced cell death in melanoma: reduced sensitivity in BRAF mutant/PTEN wild-type melanoma cells. PLoS One. 2015;10(2):e0117021.CrossRefPubMedPubMedCentral

24.

Greenman C, Stephens P, Smith R, Dalgliesh GL, Hunter C, Bignell G, Davies H, Teague J, Butler A, Stevens C, et al. Patterns of somatic mutation in human cancer genomes. Nature. 2007;446(7132):153–8.CrossRefPubMedPubMedCentral

25.

Guo A, Villen J, Kornhauser J, Lee KA, Stokes MP, Rikova K, Possemato A, Nardone J, Innocenti G, Wetzel R, et al. Signaling networks assembled by oncogenic EGFR and c-met. Proc Natl Acad Sci U S A. 2008;105(2):692–7.CrossRefPubMedPubMedCentral

26.

Hoeflich KP, O'Brien C, Boyd Z, Cavet G, Guerrero S, Jung K, Januario T, Savage H, Punnoose E, Truong T, et al. In vivo antitumor activity of MEK and phosphatidylinositol 3-kinase inhibitors in basal-like breast cancer models. Clin Cancer Res. 2009;15(14):4649–64.CrossRefPubMed

27.

Lin WM, Baker AC, Beroukhim R, Winckler W, Feng W, Marmion JM, Laine E, Greulich H, Tseng H, Gates C, et al. Modeling genomic diversity and tumor dependency in malignant melanoma. Cancer Res. 2008;68(3):664–73.CrossRefPubMed

28.

Chou TC. Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res. 2010;70(2):440–6.CrossRefPubMed

29.

Hegedus L, Padanyi R, Molnar J, Paszty K, Varga K, Kenessey I, Sarkozy E, Wolf M, Grusch M, Hegyi Z, et al. Histone deacetylase inhibitor treatment increases the expression of the plasma membrane ca(2+) pump PMCA4b and inhibits the migration of melanoma cells independent of ERK. Front Oncol. 2017;7:95.CrossRefPubMedPubMedCentral

30.

Garay T, Juhász É, Molnár E, Eisenbauer M, Czirók A, Dekan B, László V, Hoda MA, Döme B, Tímár J, et al. Cell migration or cytokinesis and proliferation?--revisiting the “go or grow” hypothesis in cancer cells in vitro. Exp Cell Res. 2013;319(20):3094–103.CrossRefPubMed

31.

Iorns E, Drews-Elger K, Ward TM, Dean S, Clarke J, Berry D, El Ashry D, Lippman M. A new mouse model for the study of human breast cancer metastasis. PLoS One. 2012;7(10):e47995.CrossRefPubMedPubMedCentral

32.

Joshi M, Rice SJ, Liu X, Miller B, Belani CP. Trametinib with or without vemurafenib in BRAF mutated non-small cell lung cancer. PLoS One. 2015;10(2):e0118210.CrossRefPubMedPubMedCentral

33.

Noeparast A, Teugels E, Giron P, Verschelden G, De Brakeleer S, Decoster L, De Grève J. Non-V600 BRAF mutations recurrently found in lung cancer predict sensitivity to the combination of Trametinib and Dabrafenib. Oncotarget. 2016;

34.

Smalley KS, Xiao M, Villanueva J, Nguyen TK, Flaherty KT, Letrero R, Van Belle P, Elder DE, Wang Y, Nathanson KL, et al. CRAF inhibition induces apoptosis in melanoma cells with non-V600E BRAF mutations. Oncogene. 2009;28(1):85–94.CrossRefPubMed

35.

Fritsche-Guenther R, Witzel F, Kempa S, Brummer T, Sers C, Blüthgen N. Effects of RAF inhibitors on PI3K/AKT signalling depend on mutational status of the RAS/RAF signalling axis. Oncotarget. 2016;7(7):7960–9.CrossRefPubMedPubMedCentral

36.

Won JK, Yang HW, Shin SY, Lee JH, Heo WD, Cho KH. The crossregulation between ERK and PI3K signaling pathways determines the tumoricidal efficacy of MEK inhibitor. J Mol Cell Biol. 2012;4(3):153–63.CrossRefPubMed

37.

Yuen HF, Abramczyk O, Montgomery G, Chan KK, Huang YH, Sasazuki T, Shirasawa S, Gopesh S, Chan KW, Fennell D, et al. Impact of oncogenic driver mutations on feedback between the PI3K and MEK pathways in cancer cells. Biosci Rep. 2012;32(4):413–22.CrossRefPubMedPubMedCentral

38.

Nagaria TS, Shi C, Leduc C, Hoskin V, Sikdar S, Sangrar W, Greer PA. Combined targeting of Raf and Mek synergistically inhibits tumorigenesis in triple negative breast cancer model systems. Oncotarget. 2017;8(46):80804–19.CrossRefPubMedPubMedCentral

39.

Kim DW, Haydu LE, Joon AY, Bassett RL Jr, Siroy AE, Tetzlaff MT, Routbort MJ, Amaria RN, Wargo JA, McQuade JL, et al. Clinicopathological features and clinical outcomes associated with TP53 and BRAFNon-V600 mutations in cutaneous melanoma patients. Cancer. 2017;123(8):1372–81.CrossRefPubMed

40.

Jones JC, Renfro LA, Al-Shamsi HO, Schrock AB, Rankin A, Zhang BY, Kasi PM, Voss JS, Leal AD, Sun J, et al. Non-V600 BRAF mutations define a clinically distinct molecular subtype of metastatic colorectal Cancer. J Clin Oncol. 2017; https://doi.org/10.1200/JCO.2016.71.4394.

41.

Gautschi O, Milia J, Cabarrou B, Bluthgen MV, Besse B, Smit EF, Wolf J, Peters S, Früh M, Koeberle D, et al. Targeted therapy for patients with BRAF-mutant lung Cancer: results from the European EURAF cohort. J Thorac Oncol. 2015;10(10):1451–7.CrossRefPubMed

42.

Litvak AM, Paik PK, Woo KM, Sima CS, Hellmann MD, Arcila ME, Ladanyi M, Rudin CM, Kris MG, Riely GJ. Clinical characteristics and course of 63 patients with BRAF mutant lung cancers. J Thorac Oncol. 2014;9(11):1669–74.CrossRefPubMedPubMedCentral

43.

Sereno M, Moreno V, Moreno Rubio J, Gómez-Raposo C, García Sánchez S, Hernández Jusdado R, Falagan S, Zambrana Tébar F, Casado Sáenz E. A significant response to sorafenib in a woman with advanced lung adenocarcinoma and a BRAF non-V600 mutation. Anti-Cancer Drugs. 2015;26(9):1004–7.CrossRefPubMed

44.

Tai WM, Yong WP, Lim C, Low LS, Tham CK, Koh TS, Ng QS, Wang WW, Wang LZ, Hartano S, et al. A phase Ib study of selumetinib (AZD6244, ARRY-142886) in combination with sorafenib in advanced hepatocellular carcinoma (HCC). Ann Oncol. 2016;27(12):2210–5.CrossRefPubMed

45.

Hoeflich KP, Herter S, Tien J, Wong L, Berry L, Chan J, O'Brien C, Modrusan Z, Seshagiri S, Lackner M, et al. Antitumor efficacy of the novel RAF inhibitor GDC-0879 is predicted by BRAFV600E mutational status and sustained extracellular signal-regulated kinase/mitogen-activated protein kinase pathway suppression. Cancer Res. 2009;69(7):3042–51.CrossRefPubMed

46.

Holderfield M, Merritt H, Chan J, Wallroth M, Tandeske L, Zhai H, Tellew J, Hardy S, Hekmat-Nejad M, Stuart DD, et al. RAF inhibitors activate the MAPK pathway by relieving inhibitory autophosphorylation. Cancer Cell. 2013;23(5):594–602.CrossRefPubMed

47.

Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ, Bottomley W, et al. Mutations of the BRAF gene in human cancer. Nature. 2002;417(6892):949–54.CrossRefPubMed

Title: Pan-RAF and MEK vertical inhibition enhances therapeutic response in non-V600 BRAF mutant cells
Authors: Eszter Molnár
Dominika Rittler
Marcell Baranyi
Michael Grusch
Walter Berger
Balázs Döme
József Tóvári
Clemens Aigner
József Tímár
Tamás Garay
Balázs Hegedűs
Publication date: 01-12-2018
Publisher: BioMed Central
Published in: BMC Cancer / Issue 1/2018
Electronic ISSN: 1471-2407
DOI: https://doi.org/10.1186/s12885-018-4455-x

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Abstract

Background

Methods

Results

Conclusions

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