A central role for RAF→MEK→ERK signaling in the genesis of pancreatic ductal adenocarcinoma

Abstract

KRAS mutation is a hallmark of pancreatic ductal adenocarcinoma (PDA) but remains an intractable pharmacologic target. Consequently, defining RAS effector pathway(s) required for PDA initiation and maintenance is critical to improve treatment of this disease. Here, we show that expression of BRAF(V600E), but not PIK3CA(H1047R), in the mouse pancreas leads to pancreatic intraepithelial neoplasia (PanIN) lesions. Moreover, concomitant expression of BRAF(V600E) and TP53(R270H) result in lethal PDA. We tested pharmacologic inhibitors of RAS effectors against multiple human PDA cell lines. Mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK) inhibition was highly effective both in vivo and in vitro and was synergistic with AKT inhibition in most cell lines tested. We show that RAF→MEK→ERK signaling is central to the initiation and maintenance of PDA and to rational combination strategies in this disease. These results emphasize the value of leveraging multiple complementary experimental systems to prioritize pathways for effective intervention strategies in PDA.

Significance: PDA is diffi cult to treat, in large part, due to recurrent mutations in the KRAS gene. Here, we defi ne rational treatment approaches for the disease achievable today with existing drug combinations by thorough genetic and pharmacologic dissection of the major KRAS effector pathways, RAF→MEK→ERK and phosphoinositide 3′-kinase (PI3'K)→AKT.

Figure 1

Braf^V600E is Sufficient to Induce PanIN Lesions in the Mouse. H&E staining of tamoxifen induced A) Pdx1::CreER^T2 (C) mice, B) Braf^CA/+, Pdx1::CreER^T2 (BC) mice C) Kras^LSL-G12D/+, Pdx1::CreER^T2 (KC) mice. PanIns in BC mice express ductal markers: D), CK19, are proliferative: E), Ki67, and show activation of the MAPK pathway F), phospho-ERK). Braf^CA/+-induced PanIns lack primary cilia. G) Pdx1::CreER^T2 (C) mice (red:acetylated tubulin, blue:DNA, green:CK19): normal islet (red arrow) and duct (green arrow) with cilia. H) BC mice (red:acetylated tubulin, blue:DNA, green:CK19): PanIn (green arrow) without cilia.

Figure 2

Braf^V600E and Trp53^R270H cooperate to form lethal PDA resembling the human disease. (A) a six month old Pdx1::CreER^T2; BRaf^CA/+; Trp53^lox-R270H/+ (BCP) mouse with ascites. (B) Gross images of primary pancreatic tumor (black arrow) and omental metastases (blue arrows). (C) H&E of primary PDA arising in the pancreas of a BCP mouse. H&E staining of (D) liver metastases (black arrows) or (E) lung metastases (black arrow) from same. PDA arising in BCP mice are proliferative (F, Ki67), heterogeneously express ductal markers (G, CK19), and display high levels of MAPK activation (H, phosphoERK).

Figure 3

MEK Inhibition in vivo. H & E (A and B) or phosphoERK (C and D) staining of KPC mice treated with either vehicle (A and C) or MEK1/2 inhibitor PD3258901 (B and D). Day seven (E, F) post-implantation, pre-treatment bioluminescent images of FVBn mice after orthotopic injection of syngenic Kras^LSL-G12D/+, Cdnk2a^F/+ cells and subsequent treatement with vehicle (G) or MEK1/2 inhibitor PD0325891 (H) for two weeks.

Figure 4

Combined Inhibition of MEK and AKT leads to synergistic effects across a large panel of PDA cell lines. (A) IC₅₀ measurements of human PDA cell lines treated with MEK inhibitor GSK1120212. Cell lines are on the X axis and IC₅₀ (M) is on the Y axis. Representative dose response curves of (B) 3.27 or (C) Sw1990 treated with either GSK690693 (triangles), GSK1120212 (diamonds), or a 5:1M fixed dose combination ratio of GSK1120212:GSK690693 (squares) plotted as the dose of GSK1120212 in the combination. X axis is drug concentration in M. Y axis is percent growth inhibition at 72 hours. Error bars are +/- standard deviation.