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. 2017 Feb 7;8(6):9251-9266.
doi: 10.18632/oncotarget.14002.

LY3009120, a panRAF inhibitor, has significant anti-tumor activity in BRAF and KRAS mutant preclinical models of colorectal cancer

Eliza Vakana  1 , Susan Pratt  1 , Wayne Blosser  1 , Michele Dowless  1 , Nicholas Simpson  2 , Xiu-Juan Yuan  3 , Susan Jaken  3 , Jason Manro  4 , Jennifer Stephens  1 , Youyan Zhang  1 , Lysiane Huber  1 , Sheng-Bin Peng  1 , Louis F Stancato  1
Affiliations

LY3009120, a panRAF inhibitor, has significant anti-tumor activity in BRAF and KRAS mutant preclinical models of colorectal cancer

Eliza Vakana et al. Oncotarget. .

Abstract

Activating mutations in the KRAS and BRAF genes, leading to hyperactivation of the RAS/RAF/MAPK oncogenic signaling cascade, are common in patients with colorectal cancer (CRC). While selective BRAF inhibitors are efficacious in BRAFmut melanoma, they have limited efficacy in BRAFmut CRC patients. In a RASmut background, selective BRAF inhibitors are contraindicated due to paradoxical activation of the MAPK pathway through potentiation of CRAF kinase activity. A way to overcome such paradoxical activation is through concurrent inhibition of the kinase activity of both RAF isoforms. Here, we further examined the effects of LY3009120, a panRAF and RAF dimer inhibitor, in human models of CRC with various mutational backgrounds. We demonstrate that LY3009120 induced anti-proliferative effects in BRAFmut and KRASmut CRC cell lines through G1-cell cycle arrest. The anti-proliferative effects of LY3009120 in KRASmut CRC cell lines phenocopied molecular inhibition of RAF isoforms by simultaneous siRNA-mediated knockdown of ARAF, BRAF and CRAF. Additionally, LY3009120 displayed significant activity in in vivo BRAFmut and KRASmut CRC xenograft models. Examination of potential resistance to LY3009120 demonstrated RAF-independent ERK and AKT activation in the KRASmut CRC cell line HCT 116. These findings describe the preclinical activity of a panRAF inhibitor in a BRAFmut and KRASmut CRC setting.

Keywords: RAF; RAS; colorectal cancer; signaling pathways; xenograft models.

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Conflict of interest statement

CONFLICTS OF INTEREST

Authors affiliated with Eli Lilly and Company have Eli Lilly and Company shares received via 401(k) and bonus plans.

Figures

Figure 1
Figure 1. Effects of LY3009120 and trametinib on colorectal cancer cell lines
CRC lines were treated with increasing concentrations of LY3009120 (A) or trametinib (B) and proliferation was assessed at 72 hrs post-treatment by CTG analysis. EC50 values were calculated from at least 2 independent experiments with duplicate technical replicates (1 experiment for SW48-trametinib) and are plotted based on the sensitivity and mutational status of each assayed cell line as obtained from COSMIC, as depicted in the table below each graph. Colors denote presence of gene mutation or microsatellite (MSI) instability and KRASG13 and KRASG12 are depicted by pink and red colors respectively. Other mutations include mutations in the genes MAP2K1 (SNU-C1 and SW48), CTNNB1, EGFR (SW48) and MAP2K4 (NCI-H716). (C) Whole cell lysates of various CRC cell lines were analyzed by Western blot for baseline pathway activation using antibodies against the proteins indicated. The cell lines are oriented in decreasing sensitivity to LY3009120.
Figure 2
Figure 2. Effects of LY3009120 on signaling and gene expression
(A) CRC cell lines were treated with DMSO, 0.1 μM or 1 μM LY3009120 for 30 minutes (left panel) or 2 hrs (right panel). Whole cell lysates were analyzed by Western blot using antibodies against the proteins indicated. (B) CRC cell lines were treated with increasing concentrations of LY3009120 and fixed at the times indicated. Cells were stained with Hoechst stain and nuclei counts were obtained. At the 72 hr time point, replicate plates were assessed for proliferation by CTG analysis. (C) CRC cell lines were treated with DMSO or logarithmic concentrations of LY3009120 and fixed at 24 hrs post-treatment. Cells were stained for immunofluorescence analysis with Hoechst stain and antibodies against the proteins indicated and the average intensity of the signal for each analyte was measured by HCI. Results are plotted as percent of DMSO-treated cells and are representative of two independent experiments. The signal for pERK1/2 T202/Y204 and pS6 S240/244 was normalized to the levels of ERK1/2 and S6 respectively. (D) CRC cell lines were treated with DMSO or 1 μM LY3009120 for 24 and 48 hrs. Whole cell lysates were analyzed by Western blot using antibodies against the proteins indicated. (E) CRC cell lines were treated with DMSO or increasing concentrations of LY3009120 for 24 hrs. Expression levels of the indicated genes were assessed by Affymetrix analysis.
Figure 3
Figure 3. Effects of LY3009120 on cell cycle and apoptosis
(A) Representative experiment of CRC cell lines treated with either DMSO or LY3009120 (0.5 μM) for the times indicated, fixed and stained with propidium iodide and analyzed for cell cycle by flow cytometry. (B) CRC cell lines were treated with increasing concentrations of LY3009120, fixed at 24 or 48 hrs (left and right panels respectively) and stained for immunofluorescence with Click-iT® EdU and antibodies against pERK1/2 T202/Y204 and pHH3 S10 as indicated. The average intensity of the signal for each analyte was measured by HCI. The data are representative of two independent experiments each conducted in triplicate technical replicates, with results plotted as percent of DMSO-treated cells. (C) Cells were treated with increasing concentrations of LY3009120 and fixed at 48 hrs post-treatment. Cells were stained for immunofluorescence analysis with antibodies against the proteins indicated and the average intensity of the signal for each analyte was measured by HCI. Results are plotted as percent of DMSO-treated cells and are representative of two independent experiments.
Figure 4
Figure 4. Concomitant knockdown of A, B and CRAF induces anti-proliferative effects in KRASmut CRC lines
Upper panels: (A) KRASmut (HCT 116 and SW620) or B, BRAFmut (RKO) CRC cell lines were transfected with the indicated siRNAs. Whole cell lysates were isolated at 72 hrs post-transfection and were analyzed by Western blot using antibodies against the proteins indicated. Lower panels: A, KRASmut (HCT 116 and SW620) or (B) BRAFmut (RKO) CRC cells were transfected with the indicated siRNAs. Effects of transfection on proliferation were assessed by CTG at 96 hrs post-transfection normalized to non-targeting control. Results represent aggregated data of 2 independent experiments run in six technical replicates each.
Figure 5
Figure 5. Effects of LY3009120 on CRC xenografts
(A) Colo 205 (BRAFmut), HT-29 (BRAFmut), HCT 116 (KRASmut) or Colo 320HSR (KRASWT/BRAFWT) CRC lines were implanted subcutaneously in the right flank of nude rats and the animals were treated orally with either vehicle (•) or LY3009120 (Colo 205: 20 mg/kg; HCT 116: 30 mg/kg; HT-29: 20 mg/kg; Colo 320HSR 30mg/kg) (○) twice daily and were monitored for tumor size. Arrow: Day of dosing initiation. Blue line: Duration of dosing. p < 0.01 (**); p < 0.001 (***); n.s. (not significant). (B) HT-29 (BRAFmut) xenograft model treated with either vehicle or LY3009120 (20 mg/kg BID) or (C) Colo 320HSR xenograft model treated with either vehicle or LY3009120 (30 mg/kg BID) were assessed by Western blot for levels of phospho- and total MEK1/2, ERK1/2 and tubulin as indicated.
Figure 6
Figure 6. ERK-mediated mechanism of resistance to LY3009120
(A) HCT 116 parental (blue line) and HCT 116 LY3009120-resistant cells (red line) were generated and assessed for their sensitivity to various kinase inhibitors of the MAPK signaling cascade. LY3009120: panRAF inhibitor (top panel); trametinib: MEK1/2 inhibitor (middle panel); ERKi: VX-11e ERK2 inhibitor (lower panel). (B) RAS-GTP levels were analyzed from cell lysates of LY3009120-sensitive and LY3009120-resistant HCT 116 by an active-RAS pulldown assay as delineated in the experimental procedures. Whole cell lysates were also analyzed by Western blot using antibodies against the proteins indicated. (C) Whole cell lysates of LY3009120-sensitive and LY3009120-resistant HCT 116 were analyzed by Western blot for baseline pathway activation using antibodies against the proteins indicated.
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