PLX8394, a new generation BRAF inhibitor, selectively inhibits BRAF in colonic adenocarcinoma cells and prevents paradoxical MAPK pathway activation

Abstract

BRAF inhibitors (BRAFi) are standard of care for the treatment of BRAF V600 mutation-driven metastatic melanoma, but can lead to paradoxical activation of the mitogen-activated protein kinase (MAPK) signalling pathway. This can result in the promotion of precancerous lesions and secondary neoplasms, mainly (but not exclusively) associated with pre-existing mutations in RAS genes. We previously reported a patient with synchronous BRAF-mutated metastatic melanoma and BRAF ^wt /KRAS ^G12D-metastatic colorectal cancer (CRC), whose CRC relapsed and progressed when treated with the BRAF inhibitor dabrafenib (GSK2118436). We used tissue from the resected CRC metastasis to derive a cell line, LM-COL-1, which directly and reliably mimicked the clinical scenario including paradoxical activation of the MAPK signalling pathway resulting in increased cell proliferation upon dabrafenib treatment. Novel BRAF inhibitors (PLX8394 and PLX7904), dubbed as "paradox breakers", were developed to inhibit V600 mutated oncogenic BRAF without causing paradoxical MAPK pathway activation. In this study we used our LM-COL-1 model alongside multiple other CRC cell lines with varying mutational backgrounds to demonstrate and confirm that the paradox breaker PLX8394 retains on-target inhibition of mutated BRAF V600 without paradoxically promoting MAPK signalling.

Keywords: BRAF; Colorectal cancer; MAPK pathway; Melanoma; Paradoxical activation.

Fig. 1

Effect of the BRAF inhibitors vemurafenib and PLX8394 on the MAPK pathway in colorectal cancer cell lines. Cells were treated with DMSO, vemurafenib at 1 μM, or PLX8394 at 1 μM for 6 h. a, b Representative Western blot of a panel of BRAF ^wt /KRAS ^G12D (LM-COL-1, ALA, and LS513) and BRAF ^V600E /KRAS ^wt (LIM2405 and COLO 201) colorectal cancer cell lines after treatment with DMSO control or BRAF inhibitors. Western blots were probed for total and phosphorylated MEK1/2 and ERK1/2. The blots are representative of three independent experiments. Total ERK served as a loading control. Western blot signal intensity was quantified and used to measure protein level relative to control. c, d Densitometry of MEK1/2 phosphorylation demonstrating paradoxical activation by vemurafenib in KRAS-mutated cell lines and BRAFi sensitivity in BRAF ^V600E mutated cell lines LIM2405 and COLO 201. e, f Densitometry of ERK1/2 phosphorylation in the same cell lines as shown in c and d. In panels c–f the total protein:phosphorylated ratio is expressed as the mean ± SD of three independent replicates relative to DMSO-treated control

Fig. 2

The effect of vemurafenib and PLX8394 on proliferation and survival of BRAF ^wt / KRAS ^G12D and BRAF ^V600E / KRAS ^wt colorectal cancer cell lines. Inhibitors were used at 0 (DMSO control), 0.1, 0.5, and 1 μM. Cell proliferation was measured after 72 h of BRAFi treatment. a–c Proliferation of BRAF ^wt /KRAS ^G12D colorectal cancer cell lines after treatment with vemurafenib or PLX8394 at the indicated concentrations. Relative cell numbers are normalized to DMSO-treated control and differences shown as %. The tinted area indicates increased proliferation after treatment with vemurafenib. The Western blot inlay demonstrates the amount of ERK1/2 phosphorylation relative to the DMSO control at the concentration of vemurafenib that resulted in the biggest increase in proliferation. Lines between lanes denote non-adjacent lanes from the same membrane. d–e Inhibition of proliferation in BRAF ^V600E / KRAS ^wt colorectal cancer cell lines LIM2405 and COLO 201 after treatment with the indicated concentrations of vemurafenib or PLX8394. All data are shown as mean ± SD of independent triplicates relative to DMSO-treated controls