Identification of PLX4032-resistance mechanisms and implications for novel RAF inhibitors

Abstract

BRAF inhibitors improve melanoma patient survival, but resistance invariably develops. Here we report the discovery of a novel BRAF mutation that confers resistance to PLX4032 employing whole-exome sequencing of drug-resistant BRAF(V600K) melanoma cells. We further describe a new screening approach, a genome-wide piggyBac mutagenesis screen that revealed clinically relevant aberrations (N-terminal BRAF truncations and CRAF overexpression). The novel BRAF mutation, a Leu505 to His substitution (BRAF(L505H) ), is the first resistance-conferring second-site mutation identified in BRAF mutant cells. The mutation replaces a small nonpolar amino acid at the BRAF-PLX4032 interface with a larger polar residue. Moreover, we show that BRAF(L505H) , found in human prostate cancer, is itself a MAPK-activating, PLX4032-resistant oncogenic mutation. Lastly, we demonstrate that the PLX4032-resistant melanoma cells are sensitive to novel, next-generation BRAF inhibitors, especially the 'paradox-blocker' PLX8394, supporting its use in clinical trials for treatment of melanoma patients with BRAF-mutations.

Keywords: BRAF; PLX4032; drug resistance; melanoma; paradox blockers.

Figure 1

PB mutagenesis of YUMAC cell induces PLX4032 resistance. (A) Schematic of PB[Mut-tetO-KAT]. The Actin promoter (black pointed box) and Katushka red fluorescent protein (red box) couples KAT expression with ectopic expression of a downstream gene or partial gene transcript via the IRES (orange box). The tetO (blue box) allows binding of TetR-KRAB (TetR), which binds and represses expression in the absence of doxycycline (Dox). (B) FACS plots of KAT red fluorescence signal comparing the parental YUMAC cell line (YUMAC-P, green) to YUMAC-TIM cells transduced with TetR-KRAB (TIM-TetR) with (red) and without doxycycline (blue). (C) Dose–response curve of PLX4032 on TIM-TetR in the presence or absence of doxycycline. Cell numbers in increasing concentrations of PLX4032 were determined by CellTiter-Glo assays (72 h). (D) Transposon insertions cluster (red arrowheads) in introns eight and nine of BRAF and in introns five and six and exon six of CRAF. (E) Relative expression of BRAF and CRAF transcripts 5′ and 3′ to the transposon insertion sites in TIM-BRAF and TIM-CRAF clones. Transcript levels were normalized to YUMAC-P. (F) Western blot analysis of BRAF (top) and CRAF (bottom) in YUMAC-TIM. BRAF levels were assessed with an antibody targeting a C-terminal epitope. Protein levels were assessed in YUMAC-TIM and TIM-TetR in the presence or absence of doxycycline.

Figure 2

BRAF^L505H mutation is present in unmanipulated YUMAC PLX4032-resistant clones and at low prevalence in the parental unselected cells. (A) Chromatograph demonstrating AT dinucleotide change in BRAF in resistant cells (YUMAC-L505H, black arrows). (B) BRAF copy number as determined by quantitative PCR and normalized to multicopy reference control (Qiagen). (C) BRAF^V600K/L505H allele frequency in the six YUMAC PLX4032 resistant clones calculated from analysis of exome sequencing. The six clones are annotated as YUMAC-R1-R6. (D) The prevalence of dinucleotide substitution at chr 7: 140477793–140477794 (hg19) is plotted from deep sequencing of an 82 bp amplicon utilizing genomic DNA from the YUMAC-P and YUGEN8 cell lines. (E) Proposed model of YUMAC-BRAF^L505H evolution. Based on common SNV's, YUMAC- BRAF^L505H and YUMAC-P share a common progenitor that has 780 novel SNV's not found in publically available databases. YUMAC- BRAF^L505H and YUMAC-P then diverge. YUMAC-BRAF^L505H and YUMAC-P have 12 and 6 unique SNV's, respectively. (F) Proposed model for the persistent emergence of YUMAC- BRAF^L505H (red) from YUMAC-P (blue) after PLX4032 selection.

Figure 3

BRAF^L505H mutation confers resistance to PLX4032. (A) YUMAC-P, YUMAC- BRAF^L505H, TIM-BRAF, and TIM-CRAF growth response to increasing concentration of PLX4032. (B) Changes in pMEK and pERK after 18 h treatment of YUMAC-P, YUMAC- BRAF^L505H, TIM-BRAF, and TIM-CRAF with DMSO or PLX4032 (0.1, 0.3, 1, 3, or 10 μM). (C) Growth response of YUMAC cells transduced with the indicated BRAF variant to increasing concentrations of PLX4032. (D) Changes in pMEK and pERK in YUMAC cells transduced with the indicated BRAF variant after 18 h of treatment with DMSO or 3 μM PLX4032. (E) Cell proliferation of parental YUGEN8-BRAF^V600E/WT (YUGEN8-P) and BRAF^V600K/L505H expressing YUGEN8 cells in response to increasing doses of PLX4032. (F) Dose response to combined treatment with MEK inhibitor (AZD6244) and PLX4032. The cells were cultured in 1 μM PLX4032 and indicated doses of AZD6244.

Figure 4

BRAF^V600K/L505H is intrinsically resistant to PLX4032. (A) Structural model of the BRAF-active site with binding to PLX4032. The amino acid L505 is highlighted in pink and PLX4032 in gray. (B) Growth responses of YUMAC-P and YUMAC-BRAF^L505H to increasing doses of sorafenib. (C) Changes in pERK in YUMAC-P and YUMAC-BRAF^L505H cells in response to sorafenib. Cells were treated with DMSO or sorafenib (0.1, 0.3, 1, or 3 μM) for 18 h. (D) Changes in pMEK and pERK of 293T cells expressing the indicated BRAF variant after 18 h of treatment with DMSO or PLX4032 (3 μM). (E) Down regulation of CRAF does not affect ERK activation in L505H expressing 293T cells. The western blot shows the level of BRAF, CRAF, pERK, and ERK, 72 h after the indicated siRNA transfection followed by 18 h of treatment with DMSO or PLX4032 (3 μM). (F) BRAF^V600E and BRAF^V600E/L505H kinase activity in the presence of DMSO or PLX4032 (0.1, 0.3, 1, 3, or 10 μM). The figure shows the total immumoprecipitated BRAF, the phosphorylated levels of the substrate MEK (pMEK), and total MEK.

Figure 5

Growth responses of PLX4032-resistant YUMAC cells to ‘paradox-blockers’. (A) The table shows the IC₅₀ in response to PLX4032, PLX7904, or PLX8394 (see also Figure S5). (B) Changes in pMEK and pERK in response to increasing concentrations of PLX8394, as compared to PLX4032 (1 μM). YUMAC-P, YUMAC-BRAF^L505H, TIM-BRAF, and TIM-CRAF were harvested after 4 h exposure to the drugs.

Figure 6

BRAF^L505H mutation induces oncogenic MAPK signaling. (A) Changes in pMEK and pERK in 293T cells expressing the indicated BRAF variant. (B) In vitro kinase activity of WT (wild-type) BRAF, BRAF^V600E, BRAF^L505H, and BRAF^V600E/L505H, employing MEK as a substrate. The figure shows the total immunoprecipitated BRAF, the phosphorylated levels of the substrate MEK (pMEK), and total MEK. (C) Changes in pMEK and pERK in 293T cells expressing the indicated BRAF variant (BRAF^V600E, BRAF^L505H, or BRAF^V600E/L505H) after 18 h treatment with DMSO or PLX4032 (1 μM, or 3 μM). (D) IP kinase assay performed on lysates from 293T cells expressing the indicated BRAF variant, treated with DMSO, or increasing concentration of PLX4032 (1, 3, or 10 μM) employing MEK as a substrate. (E) Changes in pERK in response to MEK inhibitor (AZD6244). 293T cells expressing the indicated BRAF variant were treated with DMSO, or AZD6244 (0.03, 0.1, 0.3, 1, or 3 μM) for 18 h. (F) Changes in pERK and pMEK in RWPE-1 prostate cells expressing the indicated doxycycline inducible BRAF variant. (G) Soft agar colonies of RWPE-1 prostate cells expressing the indicated doxycycline-inducible BRAF variant; numbers are averages of triplicate cultures after 3 weeks incubation. *indicates a P-value < 0.0001. (H) Representative photographs of RWPE-1 prostate cells colonies in soft agar expressing the indicated BRAF variant after 3 weeks incubation.