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. 2016 Nov 22;113(47):13456-13461.
doi: 10.1073/pnas.1610456113. Epub 2016 Nov 9.

Preclinical efficacy of a RAF inhibitor that evades paradoxical MAPK pathway activation in protein kinase BRAF-mutant lung cancer

Ross A Okimoto  1   2   3 , Luping Lin  1   2 , Victor Olivas  1   2   3 , Elton Chan  1   2   3 , Evan Markegard  2 , Andrey Rymar  4 , Dana Neel  1   2   3 , Xiao Chen  1   2 , Golzar Hemmati  1   2   3 , Gideon Bollag  4 , Trever G Bivona  5   2   3
Affiliations

Preclinical efficacy of a RAF inhibitor that evades paradoxical MAPK pathway activation in protein kinase BRAF-mutant lung cancer

Ross A Okimoto et al. Proc Natl Acad Sci U S A. .

Abstract

Oncogenic activation of protein kinase BRAF drives tumor growth by promoting mitogen-activated protein kinase (MAPK) pathway signaling. Because oncogenic mutations in BRAF occur in ∼2-7% of lung adenocarcinoma (LA), BRAF-mutant LA is the most frequent cause of BRAF-mutant cancer mortality worldwide. Whereas most tumor types harbor predominantly the BRAFV600E-mutant allele, the spectrum of BRAF mutations in LA includes BRAFV600E (∼60% of cases) and non-V600E mutant alleles (∼40% of cases) such as BRAFG469A and BRAFG466V The presence of BRAFV600E in LA has prompted clinical trials testing selective BRAF inhibitors such as vemurafenib in BRAFV600E-mutant patients. Despite promising clinical efficacy, both innate and acquired resistance often result from reactivation of MAPK pathway signaling, thus limiting durable responses to the current BRAF inhibitors. Further, the optimal therapeutic strategy to block non-V600E BRAF-mutant LA remains unclear. Here, we report the efficacy of the Raf proto-oncogene serine/threonine protein kinase (RAF) inhibitor, PLX8394, that evades MAPK pathway reactivation in BRAF-mutant LA models. We show that PLX8394 treatment is effective in both BRAFV600E and certain non-V600 LA models, in vitro and in vivo. PLX8394 was effective against treatment-naive BRAF-mutant LAs and those with acquired vemurafenib resistance caused by an alternatively spliced, truncated BRAFV600E that promotes vemurafenib-insensitive MAPK pathway signaling. We further show that acquired PLX8394 resistance occurs via EGFR-mediated RAS-mTOR signaling and is prevented by upfront combination therapy with PLX8394 and either an EGFR or mTOR inhibitor. Our study provides a biological rationale and potential polytherapy strategy to aid the deployment of PLX8394 in lung cancer patients.

Keywords: BRAF; cancer; lung; targeted therapy.

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Conflict of interest statement

G.B. and A.R. are employees of Plexxikon, Inc., which produced vemurafenib and PLX8394. T.G.B. is a consultant to Driver Group, Novartis, Astellas, Natera, Array Biopharma, Ariad, Teva, Astrazeneca, and a recipient of research grants from Servier and Ignyta.

Figures

Fig. 1.
Fig. 1.
The effects of PLX8394 RAF inhibitor treatment in BRAFV600E and non-V600 BRAF-mutant LA cells in vitro. (A) BRAF-mutant LA cell lines are addicted to each oncoprotein. Shown is the cell growth upon shRNA-mediated knockdown, conducted and analyzed as reported (4). (B and C) The effects of RAF inhibitor treatment with each indicated agent on growth (B) and signaling (C) in each BRAF-mutant LA cell line. The experiment was conducted and analyzed as reported (4), using the indicated drug doses. Vem, vemurafenib. (D and E) The effects of RAF inhibitor treatment with each indicated agent on growth (D) and signaling (E) in BRAFV600E LA cells with acquired vemurafenib resistance that express a vemurafenib-insensitive, truncated form of BRAFV600E. The experiment was conducted and analyzed as reported (4), using the indicated drug doses. All data shown represent three independent experiments, mean ± SEM, P < 0.01.
Fig. S1.
Fig. S1.
Knockdown of BRAF expression in BRAF-mutant lung cancer cell lines. Shown is the degree of shRNA-mediated knockdown of BRAF by immunoblot analysis in select LA cell lines studied in Fig. 1, conducted and analyzed as reported (4).
Fig. 2.
Fig. 2.
The effects of PLX8394 RAF inhibitor treatment in BRAFV600E and non-V600 BRAF mutant LA cells in vivo. Representative BLI images (A) and relative photon flux (B) of HCC364 BRAFV600E-bearing mice treated with either vehicle (n = 10 mice), vemurafenib 50 mg⋅kg−1⋅d−1 (n = 10 mice), or PLX8394 150 mg⋅kg−1⋅d−1 (n = 10 mice). P value calculated with one-way ANOVA. (C) Representative IHC analysis of p-ERK and Ki-67 in tumors harvested from orthotopically implanted HCC364 mice. Data shown in the % positive p-ERK (D) and % positive Ki-67 (E) cells in explanted HCC364 tumors from mice treated with vehicle, vemurafenib, or PLX8394 for 3 wk. Data represent mean ± SEM of four tumors from each treatment cohort. P values calculated with one-way ANOVA. (F) H1755 tumor growth curve comparing vehicle (n = 10 mice) and PLX8394 (n = 10 mice) treated mice. P values calculated with Student’s t test. (G) Representative IHC analysis of p-ERK and Ki-67 in s.c.-implanted H1755 mice. Shown is the % positive p-ERK (H) and % positive Ki-67 (I) cells in explanted H1755 tumors from mice treated with either vehicle or PLX8394 for 15 d. Data represent mean ± SEM of four tumors from each treatment cohort. P values were calculated with Student’s t test. (Scale bars: 100 μm.)
Fig. S2.
Fig. S2.
Pharmacokinetics and toxicity of PLX8394 in vivo. (A) PLX8394 was given orally at 75 mg⋅kg−1⋅d−1, 150 mg⋅kg−1⋅d−1, or 300 mg⋅kg−1⋅d−1, and serum concentration (ng/mL) was measured at 0, 1, 2, 4, and 8 h after treatment (black lines). PLX8394 150 mg⋅kg−1⋅d−1 was given orally with erlotinib 12.5 mg⋅kg−1⋅d−1, 25 mg⋅kg−1⋅d−1, or 50 mg⋅kg−1⋅d−1, and serum concentration (ng/mL) was measured at 0, 1, 2, 4, and 8 h after treatment (red lines). (B) In vivo pharmacokinetics of PLX8394 monotherapy or in combination with erlonitib. AUC0-8, area under the curve 0–8 h; Cmax (μM), maximum serum concentration in μM; Cmax (ng/mL), maximum serum concentration in ng/mL; T1/2, half-life; Tmax, time to maximum concentration. (C and D) Weights derived from mice bearing either orthotopically (HCC364 cells) or s.c. (H1755 cells) implanted tumor cells and treated with either vehicle, vemurafenib, or PLX8394 for 21 (orthotopic) or 15 (s.c.) days, respectively. (E) Weights derived from mice treated with vehicle, erlotinib 12.5 mg⋅kg−1⋅d−1, PLX8394 150 mg⋅kg−1⋅d−1, or combination PLX8394 150 mg⋅kg−1⋅d−1 and erlotinib 12.5 mg⋅kg−1⋅d−1 for 14 d.
Fig. 3.
Fig. 3.
mTORC1 activation downstream of receptor kinase signaling promotes acquired PLX8394 RAF inhibitor resistance in LA cells. (A and B) BRAFV600E LA sublines with acquired PLX8394 resistance, where A shows cell growth and B shows signaling pathway activation by immunoblot analysis, both conducted and analyzed as reported (4). (C–E) The effects of inhibitor treatment with each indicated agent on growth (C and D) and signaling (E) in each indicated BRAF-mutant LA cell line. The experiment was conducted and analyzed as reported (4), using the indicated drug doses. (F) The effects of EGF stimulation on RAS-GTP levels and p-ERK and p-AKT levels in each indicated cell line, using the indicated concentration of EGF and each drug. The experiment was conducted and analyzed as reported (4). All data shown represent three independent experiments, mean ± SEM, P < 0.01. (G) HCC364 cells were treated with DMSO or PLX8394 following pretreatment with an EGF ligand (HB-EGF or TGF-α). Representative of three independent experiments.
Fig. 4.
Fig. 4.
Upfront polytherapy with PLX8394 plus either erlotinib or everolimus impairs the onset of acquired resistance in both V600E and non-V600 BRAF-mutant LA cell lines. (A–C) The effects of combined inhibition of EGFR with erlotinib or of mTORC1 with everolimus on acquired resistance in each indicated BRAF-mutant LA cell line, using the drug concentrations indicated in A. Acquired resistance was measured by using an assay we validated and published (4). The experiment was conducted and analyzed as reported (4). All data shown represent three independent experiments, mean ± SEM, P < 0.01. (D) Schematic model summarizing our findings and the therapeutic strategies informed by our study. The testing of rational polytherapy with an FDA-approved mTORC1 or EGFR inhibitor may forestall the onset of resistance to concurrent treatment with third-generation RAF inhibitors such as PLX8394, as clinical trials (for instance, NCT02428712) of these agents in lung cancer patients are initiated.
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