Molecular Landscape of Acquired Resistance to Targeted Therapy Combinations in BRAF-Mutant Colorectal Cancer

Abstract

Although recent clinical trials of BRAF inhibitor combinations have demonstrated improved efficacy in BRAF-mutant colorectal cancer, emergence of acquired resistance limits clinical benefit. Here, we undertook a comprehensive effort to define mechanisms underlying drug resistance with the goal of guiding development of therapeutic strategies to overcome this limitation. We generated a broad panel of BRAF-mutant resistant cell line models across seven different clinically relevant drug combinations. Combinatorial drug treatments were able to abrogate ERK1/2 phosphorylation in parental-sensitive cells, but not in their resistant counterparts, indicating that resistant cells escaped drug treatments through one or more mechanisms leading to biochemical reactivation of the MAPK signaling pathway. Genotyping of resistant cells identified gene amplification of EGFR, KRAS, and mutant BRAF, as well as acquired mutations in KRAS, EGFR, and MAP2K1 These mechanisms were clinically relevant, as we identified emergence of a KRAS G12C mutation and increase of mutant BRAF V600E allele frequency in the circulating tumor DNA of a patient at relapse from combined treatment with BRAF and MEK inhibitors. To identify therapeutic combinations capable of overcoming drug resistance, we performed a systematic assessment of candidate therapies across the panel of resistant cell lines. Independent of the molecular alteration acquired upon drug pressure, most resistant cells retained sensitivity to vertical MAPK pathway suppression when combinations of ERK, BRAF, and EGFR inhibitors were applied. These therapeutic combinations represent promising strategies for future clinical trials in BRAF-mutant colorectal cancer. Cancer Res; 76(15); 4504-15. ©2016 AACR.

Figure 1. Generation of BRAF mutant CRC cells resistant to EGFR targeted agent and BRAF/MEK or PI3K inhibitors

(A) Schematic representation of RAS/RAF/MEK and PI3K/AKT pathways. The orange boxes show the drugs used to generate resistant cell lines. List of the drug combinations used for generating resistant cell lines is shown on the right; all of these have been or are being evaluated in clinical trials. Drugs are abbreviated as follows: A=Alpelisib (PI3K inhibitor, PI3Ki); C= Cetuximab (EGFRi); D= Dabrafenib (BRAFi); E= Encorafenib (BRAFi); S= Selumetinib (MEKi); T= Trametinib (MEKi); V= Vemurafenib (BRAFi). (B) Parental and resistant cells were treated for 72 hours with the indicated molar drug concentrations. Cetuximab and alpelisib were given at a constant concentration of 5 μg/ml and 100 nM, respectively. In the vemurafenib and selumetinib combination, selumetinib was used at a constant concentration of 300 nM.

Figure 2. Resistant cells maintain ERK1/2 phosphorylation after treatment

WiDr, VACO432 and HROC87 parental and resistant cells were treated with different drug combinations as indicated: cetuximab (C, 5 μg/ml); dabrafenib (D, 300 nM); encorafenib (E, 400 nM); alpelisib (A, 1 μM); vemurafenib (V, 2 μM); selumetinib (S, 1 μM) and trametinib (T, 30 nM). Drug treatment was given for 5 hours prior to protein extraction.

Figure 3. EGFR, KRAS and BRAF gene amplification confer acquired resistance to BRAF combination therapies

(A) Quantitative PCR for copy number evaluation of resistant cell lines in respect to their parental counterparts. WiDr V+S, D+C and S+C resistant lines displayed gene amplification of EGFR, KRAS and BRAF, respectively. (B) FISH analysis on chromosome metaphase spreads confirmed gene amplification. Cell nuclei were colored by DAPI, FISH probes EGFR, KRAS, BRAF were labeled with texas red (red signal) and chromosome 7 (Chr7) and 12 (Chr12) with FITC (green signal). EGFR gene amplification was found extrachromosomally as double minutes, while a focal intrachromosomal amplification of KRAS and BRAF loci could be identified.

Figure 4. EGFR amplification or ectodomain mutations play a causative role in acquired resistance to BRAF combination therapies

(A) Biochemical analyses of WiDr parental and V+S resistant cell lines, and of WiDr cells transduced with either GFP cDNA or EGFR WT cDNA. Cells were treated with vemurafenib and selumetinib before protein extraction. Actin was used as a loading control. (B) Effect of vemurafenib (at the indicated molar concentrations) in combination with selumetinib (0.5 μM) on the viability of WiDr cells transduced with EGFR WT cDNA. (C) Effect on cell viability of the addition of cetuximab to V+S treatment in WiDr resistant cells carrying EGFR amplification. Cells were treated with vemurafenib (1 μM), selumetinib (0.5 μM) or cetuximab alone or in their combinations. (D) EGFR and ERK expression and phosphorylation in VACO432 parental and resistant B cells, and in cells transduced with either GFP cDNA or EGFR G465R cDNA variants. VACO432 cells were treated with vemurafenib and cetuximab for 5 hours before protein extraction. Vinculin was used as a loading control. (E) Effect of vemurafenib (at the indicated molar concentrations) in combination with cetuximab (5 μg/ml) on the viability of VACO432 cells transduced with EGFR G465R cDNA. (F) VACO432 with acquired EGFR G465R mutation upon treatment with vemurafenib and cetuximab retain sensitivity to vemurafenib and gefitinib treatment. All survival data were assessed by ATP content measurement after 72 hours of treatment. Data are expressed as average ± s.d. of two independent experiments.

Figure 5. Next generation sequencing of ctDNA of a BRAF mutant CRC patient at resistance to combined BRAF/MEK inhibition revealed an increase of BRAF V600E number of reads and the emergence of a KRAS G12C mutation

Data labels indicate number (#) of mutant reads over the total number of reads covering that position, detected by next generation sequencing in circulating tumor DNA (ctDNA) at baseline and resistance. PD, progressive disease.

Figure 6. Acquired resistance to target therapy combinations can be overcome by vertical MAPK pathway suppression

The viability of parental and resistant cell lines treated with different drug combinations targeting EGFR, BRAF, MEK, ERK and PI3K was determined by ATP assay after 72 hours incubation. Relative survival was normalized to the untreated controls. Relative cell viability is depicted as indicated in the bottom color bar. Drugs were used at the concentrations listed in Supplementary Table S2. Results represent mean of at least two independent experiments, each performed in triplicate.