Molecular landscape of acquired resistance to targeted therapy combinations in BRAF mutant colorectal cancer

Summary

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. In order 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.

Introduction

Activating mutations in the BRAF oncogene occur in approximately 7% of human malignancies, including 50–60% of melanomas and 5–8% of colorectal cancers (CRCs) (1). The most frequent BRAF mutation (V600E) affects the kinase domain, mimics BRAF phosphorylated state, and leads to constitutive activation of the protein (1). In CRC, BRAF mutations are associated with hypermethylated tumor subtypes and are linked with aggressive, less-differentiated and therapy-resistant disease (2). Metastatic CRC (mCRC) patients with BRAF V600E mutant tumors show poor sensitivity to the EGFR-targeted monoclonal antibodies panitumumab and cetuximab and display poor prognosis with a median overall survival of only about 6–9 months (3).

BRAF V600E mutant tumor types do not respond uniformly to BRAF-targeted therapy (4). Targeted inhibitors of mutant BRAF alone, or in combination with inhibitors of its downstream effector MEK, induce high response rates in BRAF mutant melanoma (5,6); by contrast, a phase I study of mCRC patients has shown that the BRAF inhibitor (BRAFi) vemurafenib has no clinical benefit when given as monotherapy (7). The molecular basis of this discrepancy has been partly explained by dissimilar EGFR expression levels between these two malignancies. Intrinsic resistance of CRC cells to BRAF or MEK targeted agents is mediated by the release of a feedback loop which activates EGFR signaling, leading to reactivation of MAPK signaling and often to upregulation of parallel PI3K-AKT pathways, triggering proliferation and survival (8–10). Melanomas are sensitive to BRAFi as they originate from the neural crest and do not express EGFR, making this feedback loop ineffective. On the other hand, CRCs arise from epithelial cells in which EGFR is generally constitutively expressed.

These preclinical studies have provided the rationale for testing dual/triple vertical blockade of the MAPK pathway by targeting EGFR, BRAF, and MEK in BRAF mutant mCRC patients. Combinations targeting EGFR, BRAF, and the pro-survival PI3K pathways are also being explored. Clinical objective responses have been seen in 20–40% of patients treated with doublet or triplet combinatorial regimens (11–13).

Nevertheless, preliminary clinical evidence from phase Ib trials shows that responses are limited in duration (4,11–16). The molecular basis underlying intrinsic or acquired resistance to these drug combinations in BRAF mutant mCRC has not been comprehensively defined. The mechanisms by which cancer cells evade targeted therapies are usually molecularly heterogeneous, but they often converge downstream in the pathway which was originally blocked by the targeted agent. For instance, cell lines and mCRC patients that become resistant to single-agent cetuximab or panitumumab show a variety of molecular mechanisms that converge in reactivating the MAPK pathway, including mutations in the drug binding sites of EGFR, RAS/RAF amplification or mutations, or genetic alterations leading to activation of alternative receptor tyrosine kinases (RTKs) such as MET or HER2 (reviewed in (17)). Similarly, BRAF mutant melanomas that become refractory to BRAF and/or MEK inhibitors (MEKi) also show a variety of molecular mechanisms leading to reactivation of MAPK and/or AKT signaling. These include increased expression of RTKs such as PDGFRβ, IGF-1R and EGFR; overexpression of the COT kinase; mutation of MEK1 (MAP2K1) and MEK2 (MAP2K2) kinase; MITF or NRAS mutations; amplification, or alternative splicing of the BRAF gene; CDKN2A loss; or genetic alterations in the PI3K-PTEN-AKT pathway (reviewed in (18)).

On these premises, we hypothesized that heterogeneous genetic alterations leading to reactivation of the MAPK pathway could be responsible for acquired resistance to regimens co-targeting EGFR, BRAF, MEK, and PI3K in CRC patients, despite vertical pathway suppression at multiple key nodes. To perform a comprehensive assessment of the landscape of potential acquired resistance mechanisms, we cultured BRAF mutant CRC cell lines in the presence of seven distinct clinically-relevant combinatorial regimens until the emergence of resistant derivatives. These cell lines were subjected to genetic, biochemical, and functional analyses to identify molecular alterations underlying drug resistance. Since in vitro modeling of acquired resistance in cancer cell models has proven effective in identifying resistance mechanisms that occur clinically (19–21), these findings may predict those mechanisms of resistance likely to arise in patients. These preclinical models also represent valuable tools for key functional studies aimed at identifying effective strategies to overcome drug resistance.

Materials and Methods

Generation of drug resistant cell lines

WiDr parental cells were a gift from Dr René Bernards (Amsterdam, The Netherlands) in July 2011. HROC87 parental cells were shared by Michael Linnebacher (Rostock, Germany) in September 2011. VACO432 parental cells were obtained from Horizon Discovery (Cambridge, United Kingdom) in March 2011. The genetic identity of parental cell lines and their resistant derivatives was confirmed by short tandem repeat profiling (Cell ID System; Promega) not fewer than 2 months before drug profiling experiments. BRAF mutant HROC87, VACO432 and WiDr cells were seeded in 100-mm dishes at a density of 5x10⁶/plate and treated with drug combinations as indicated in Supplementary Table S1. Additional information is provided in Supplementary Materials and Methods.

Drug sensitivity assay

Cell proliferation and cytoxicity were determined by cellular ATP levels (CellTiter-Glo Luminescent Assay; Promega) and DNA incorporation of a fluorescent cyanine dye (CellTox Green; Promega) after 72 hours’ drug treatment, respectively. Additional information is provided in Supplementary Materials and Methods and Supplementary Table S2.

Western Blot analysis

Protein quantification, SDS-PAGE, western blotting and chemiluminescent detection were performed as previously described (19). Detailed information is provided in Supplementary Materials and Methods.

Gene copy number analysis qPCR

Cell line DNA (10 ng) was amplified by quantitative PCR using the GoTaq QPCR Master Mix (Promega) with an ABI PRISM 7900HT apparatus (Applied Biosytems). HER2, MET, EGFR, KRAS and BRAF gene copy number was assessed as previously described (19). Data were normalized to a control diploid cell line, HCEC (22) and expressed as the ratio between resistant and the corresponding parental cells. Primer sequences are reported in Supplementary Table S3.

Fluorescence In Situ Hybridization (FISH)

Dual color FISH analysis was performed using: Chr7q (7q11.21) / BRAF (7q34) probes; Chr7q / EGFR (7p12) probes; Chr12q (12q12) / KRAS (12p12.1) probes (Abnova); all probe pairs labelled with FITC and Texas Red, respectively. Details are provided in Supplementary Materials and Methods.

Candidate-gene mutational analysis

Cell line DNA was extracted by Wizard® SV Genomic DNA Purification System (Promega) according to manufacturer’s directions. The following genes and exons were analyzed by automated Sanger sequencing by ABI PRISM 3730 (Applied Biosystems): KRAS (exons 2, 3 and 4), NRAS (exons 2 and 3), BRAF (exon 15), EGFR (exon12), MAP2K1 (exons 2 and 3), MAP2K2 (exon 2). Primer sequences are listed in Supplementary Table S3.

Droplet Digital PCR (ddPCR)

Genomic DNA from CRC cells was amplified using ddPCR™ Supermix for Probes (Bio-Rad) using BRAF V600E assay (PrimePCR™ ddPCR™ Mutation Assay, Bio-Rad). ddPCR was then performed according to manufacturer’s protocol and the results reported as percentage or fractional abundance of mutant DNA alleles to total (mutant plus wild type) DNA alleles, as previously described (23).

Viral Infection

The lentivirus production, cell infection, and transduction procedures were performed as previously described (24). WiDr cells were transduced with a lenti-control vector or a lentiviral vector carrying a mutated hBRAF V600E cDNA (a gift of Maria S. Soengas, CNIO, Madrid, Spain) or EGFR WT cDNA (a gift from Dr. C. Sun and Prof R. Bernards, NKI, Amsterdam). VACO432 cells were transduced with a lentiviral vector carrying EGFR G465R mutant cDNA (25).

Clinical samples

A chemorefractory metastatic CRC patient was enrolled in the CMEK162X2110 clinical trial (Trial registration ID: NCT01543698) at Niguarda Cancer Center, Milan, Italy. The patient was treated with the BRAFi encorafenib (LGX818) in combination with the MEKi binimetinib (MEK162) from September 2013 to March 2014, obtaining a partial response in January 2014, followed by radiological progression in March 2014. Blood samples from this patient were obtained at baseline (September 2013) and at progression (March 2014) through a separate liquid biopsy research protocol approved by the Ethics Committee at Ospedale Niguarda, Milan, Italy. The study was conducted according to the provisions of the Declaration of Helsinki, and the patient signed and provided informed consent before sample collection.

Next Generation Sequencing (NGS) analysis

Germline DNA was obtained from PBMC (Promega, ReliaPrep Tissue Kit), while cell free circulating DNA of tumor origin (ctDNA) was extracted from 2 ml plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen) according to the manufacturer's instructions. Libraries were prepared with Nextera Rapid Capture Custom Enrichment Kit (Illumina Inc., San Diego, CA, USA), according to the manufacturer’s protocol, as previously described (23). The custom-panel included the coding region of 226 genes, as previously detailed (23). Further details are provided in Supplementary Materials and Methods.

Bioinformatics analysis

NGS bioinformatics analysis was performed as previously described (23). Mutational analyses were the result of comparison between pre- and post-treatment samples. Details are provided in Supplementary Materials and Methods.

Results

Generation of models of acquired resistance to combinatorial therapies targeting EGFR-BRAF-MEK-PI3K

We selected three BRAF V600E mutant CRC cell lines, HROC87, WiDr and VACO432, that are resistant to single-agent BRAFi or MEKi, but sensitive to combined BRAFi/MEKi or their combinations with cetuximab (Supplementary Fig. S1). To gain a comprehensive understanding of potential therapeutic resistance mechanisms in BRAF mutant CRC, cell lines were cultured until resistant derivatives emerged in the presence of seven different drug combinations currently being explored in clinical trials. The drugs included the BRAFi dabrafenib, encorafenib, and vemurafenib; the MEKi selumetinib and trametinib; the EGFR-targeted antibody cetuximab; and the selective PI3K-α inhibitor (PI3Ki) alpelisib (Fig. 1A). A total of eleven resistant cell line models were generated. Two independent resistant cell populations were obtained by growing VACO432 cells with vemurafenib and cetuximab (V+C) and these were therefore indicated as resistant A (R.A) and resistant B (R.B). Resistance to drug treatment was confirmed by cell viability assay comparing parental and resistant cell derivatives. All resistant cell models were clearly refractory at all drug concentrations tested (Fig. 1B).

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.

Cells with acquired resistance to BRAF inhibitor combinations display biochemical reactivation of MAPK signaling

Prior studies indicate that tumors with acquired resistance to BRAF or EGFR targeted agents in monotherapy maintain sustained levels of MEK/ERK or (occasionally) AKT phosphorylation even in the presence of drug (19,26–29). We tested whether the same biochemical rewiring could occur in cells made resistant to combinations of therapies targeting EGFR-BRAF-MEK-PI3K. Amounts of total MEK, ERK, or AKT proteins were not substantially different between parental cells and their resistant counterparts. However, variation of their phosphorylation levels (pMEK, pERK, or pAKT) was evident after drug treatment. Some, but not all, resistant models displayed increased phosphorylation of AKT at Ser473 upon drug treatment. However, every resistant model showed sustained levels of ERK phosphorylation despite drug treatment, in stark contrast to parental cells in which robust inhibition of ERK phosphorylation was observed with all treatments (Fig. 2).

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.

Overall, these analyses indicate that combinatorial EGFRi/BRAFi/MEKi/PI3Ki treatments abrogate ERK phosphorylation in parental sensitive cells, but that their resistant counterparts can sustain MAPK signaling in the presence of these therapeutic combinations (Fig. 2).

Acquired molecular alterations in BRAF mutant CRC cell lines confer resistance to BRAF inhibitor combinations

In order to identify likely candidate drug resistance mechanisms leading to biochemical reactivation of MAPK signaling, we focused our analysis on components of the MAPK pathway by performing copy-number analyses of HER2, EGFR, MET, KRAS and BRAF and Sanger sequencing of the most pertinent exons of EGFR, KRAS, NRAS, BRAF, MAP2K2 and MAP2K1.

Quantitative PCR on genomic DNA extracted from resistant cells showed no changes in HER2 or MET gene copy number while EGFR, KRAS, or BRAF gene copy number increased in three WiDr derivatives resistant to V+S, D+C or S+C, respectively (Fig. 3A). All gene amplifications were only found in the resistant cell populations and were confirmed by fluorescence in situ hybridization (FISH) analyses (Fig. 3B). Sanger sequencing of hotspot regions of EGFR (exon 12), KRAS (exons 2, 3, and 4), NRAS (exons 2 and 3), BRAF (exon 15), MAP2K1 (exons 2 and 3) and MAP2K2 (exon 2) revealed acquired gene mutations in eight cell lines, as summarized in Table 1. All resistant cell populations retained the original BRAF V600E mutation. All other mutations found in resistant cells were not detected in their parental counterparts by conventional Sanger sequencing.

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.

Table 1.

Molecular alterations acquired upon resistance to targeted therapy combinations in BRAF mutant CRC cell lines.

Drugs	Cell line	EGFR	KRAS	NRAS	MAP2K1	MAP2K2	BRAF gene CNV
D + T	VACO432	WT	WT	WT	L115P	WT	none
V + S	WiDr	EGFR ampl.	WT	WT	WT	WT	none
E + C	HROC87
	VACO432	WT	A146T	WT	WT	WT	none
D + C	VACO432	WT	A146T	WT	WT	WT	none
	WiDr	WT	KRAS ampl.	WT	WT	WT	none
V + C	VACO432 R.A	WT	G12D	WT	WT	WT	none
	VACO432 R.B	G465R	WT	WT	WT	WT	none
E + C + A	VACO432	WT	A146V A146T	WT	WT	WT	none
S + C	HROC87	WT	WT	WT	V211D	WT	none
	WiDr	WT	WT	WT	WT	WT	600E ampl.

Alterations in KRAS were the most common resistance mechanisms observed. Acquired KRAS mutations affecting exons 2 and 4 (G12D, G13D and A146T/V) were found in five different cell line models resistant to doublet BRAFi+EGFRi or triplet E+C+A. In one case, multiple KRAS mutations were concomitantly present in the resistant cell population, suggesting polyclonality. Prior functional studies in cell models have already demonstrated a causative role of exon 2 KRAS mutations in driving resistance to BRAFi+EGFRi (30). Our data suggest that exon 4 KRAS mutations can also promote resistance. Additionally, KRAS amplification was identified in WiDr resistant to BRAFi+EGFRi (D+C). KRAS amplification was found in the post-treatment biopsy of a CRC patient with acquired resistance to the combination of encorafenib and cetuximab (30). These findings suggest that the cell models generated in this work have the potential to recapitulate clinically-relevant resistance mechanisms.

Increased BRAF gene copy number was seen in WiDr resistant to MEKi+EGFRi (S+C). Selective amplification of mutant BRAF V600E allele was previously identified in a BRAF mutant CRC patient with acquired resistance to BRAFi+EGFRi (30), in CRC cell lines with secondary resistance to the MEKi selumetinib (31,32), as well as in melanoma patients upon progression on the BRAFi vemurafenib (33), but not yet implicated in refractoriness to combined MEKi+EGFRi. To assess whether BRAF gene amplification had occurred in an allele selective manner, we performed digital PCR analyses. WiDr parental cells carried 1 mutant and 3 wild-type alleles of BRAF, while their S+C resistant derivatives displayed a 9:1 mutant/wildtype ratio (Supplementary Fig. S2A). Western blot with a diagnostic antibody specific for the V600E variant showed that the mutant protein was selectively overexpressed (Supplementary Fig. S2B). Finally, we validated that ectopic overexpression of mutant BRAF in WiDr parental cells can confer resistance to combined MEKi+EGFRi (Supplementary Fig. S2C and S2D).

Two different MAP2K1 mutations leading to the V211D and L115P amino acid changes were identified in HROC87 and VACO432 resistant to MEKi+EGFRi (S+C) and BRAFi+MEKi (D+T), respectively. These mutations have previously been reported to confer resistance to MEK allosteric inhibitors in melanoma and CRC by preventing drug binding (27,34), so they were not subjected to further functional validation.

Interestingly, amplification of EGFR was found in WiDr resistant to BRAFi+MEKi (V+S). Although EGFR signaling has been implicated in intrinsic resistance to BRAFi monotherapy in BRAF mutant CRC (8,9), EGFR gene amplification has not previously been established as a potential resistance mechanism in BRAF mutant CRC. This result is consistent with previous observations that induction of EGFR protein expression can drive resistance to BRAFi or MEKi in melanoma (35). Ectopic overexpression of EGFR in WiDr parental cells was able to confer resistance to combined BRAFi+MEKi or BRAFi+EGFRi (Fig. 4A and 4B). Importantly, however, the triple combination of BRAFi+EGFRi+MEKi was able to restore sensitivity in resistant cells carrying EGFR amplification (Fig. 4C).

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.

A single point mutation affecting the ectodomain of EGFR (G465R) was found in VACO432 V+C (R.B). Although this variant has previously been shown to disrupt receptor-antibody interaction, leading to cetuximab or panitumumab resistance in RAS/BRAF wild-type CRCs (25), mutations affecting the EGFR ectodomain have not been reported previously as potential resistance mechanisms in the context of BRAF mutant tumors. To investigate the role of this mutation, we induced ectopic expression of EGFR G465R in VACO432 parental cells. Analysis of transduced cells indicated that the EGFR G465R mutation is able to sustain ERK phosphorylation and cell proliferation in the presence of combined V+C treatment (Fig. 4D and 4E). Cross-resistance to the combination of vemurafenib and panitumumab was seen. However, consistent with its known role in disrupting anti-EGFR antibody binding, the ability of the EGFR G465R mutation to promote resistance was specific to BRAFi+EGFRi combinations with anti-EGFR antibodies only, and kinase inhibition of EGFR by gefitinib was able to restore sensitivity in combination with BRAFi (Fig. 4F).

Clinical acquired resistance to combined therapy with BRAF and MEK inhibitors

Identification of clinical acquired resistance mechanisms to targeted therapy combinations was performed by genotyping of liquid biopsy samples. Plasma samples taken before treatment and after disease progression were collected from a patient with BRAF V600E mutant colorectal cancer who had achieved a partial response on a RAF/MEK inhibitor combination (Trial registration ID: NCT01543698). Circulating tumor DNA (ctDNA) was extracted and subjected to molecular profiling by NGS analysis of a custom panel of 226 cancer related genes (23). The analysis revealed that the percentage of reads carrying TP53 p.R282W mutated allele were consistent between the baseline and the progression plasma (Fig. 5), indicating similar ctDNA content in both samples. By contrast, the proportion of BRAF V600E mutant reads at resistance was twice as much as those in the baseline, suggesting selective amplification of the BRAF mutant allele. NGS analysis revealed concomitantly the emergence of a KRAS G12C allele, which was undetectable in the pretreatment sample. These results indicate that the mechanisms of resistance to target inhibitors identified in cell lines could faithfully recapitulate those found in clinical samples.

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.

Overall, we observed that a diverse array of molecular mechanisms can drive acquired resistance to clinically-relevant therapeutic combinations targeting the EGFR-BRAF-MEK-PI3K pathways in BRAF mutant CRC. However, we also found that each of these heterogeneous resistance mechanisms converges on a common signaling output to promote resistance-reactivation of MAPK signaling — suggesting that it may be possible to devise a universal targeted combination strategy capable of overcoming multiple resistance mechanisms.

Vertical combined suppression of the MAPK pathway has residual activity on drug resistant cells

Based on our observations that all resistant cell models show persistent MAPK signaling activation (Fig. 2), we postulated that they could retain sensitivity to suppression of the pathway downstream. In this regard, previous data indicate that some melanomas with acquired resistance to BRAFi monotherapy can benefit from additional treatment based on combined BRAFi and MEKi blockade (36). Additionally, vertical triple blockade of EGFR+BRAF+MEK displayed the highest ability to suppress ERK phosphorylation in BRAF V600E CRC cells (37) and this combination has been shown to induce response rates of up to 40% in BRAF mutant CRC patients (11). Similarly, previously published reports have documented promising preclinical activity of ERK inhibition in BRAFi or MEKi resistant melanoma models (27,38,39) as well as in MEKi+BRAFi or BRAFi+EGFRi resistant BRAF mutant CRC cells (30). However, it has not yet been established whether ERK inhibitors might exhibit improved ability to overcome resistance when given as monotherapy, or in combination with BRAFi and/or EGFRi. Accordingly, we hypothesized that acquired resistance to BRAFi combinations could be overcome by more profound MAPK pathway suppression achieved by triplet combinations or by the incorporation of ERK inhibitor-based combinations. In order to test these hypotheses, the effect on viability was systematically tested across all resistant cell line models for all drug combinations used to generate resistant derivatives, as well as combinations incorporating the ERK inhibitor (ERKi) SCH772984 and the vertical cetuximab+dabrafenib+trametinib (BRAFi+MEKi+EGFRi) triplet combination (Fig. 6).

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.

As expected, parental cell lines were highly sensitive to all drug treatments (Fig. 6). In general, resistant cell lines derived from one BRAFi+MEKi combination (D+T or V+S) showed cross-resistance to the other BRAFi+MEKi combination; and cell lines resistant to cetuximab in combination with encorafenib, dabrafenib or vemurafenib were cross-resistant to other BRAFi+EGFRi combinations, irrespective of the specific drug used in the selection protocol. This suggests that resistance mechanisms emerging under the selective pressure of these specific drug combinations are capable of conferring resistance to that class of inhibitors, and are unlikely to be related to any unique properties of the specific drugs used.

Interestingly, the addition of PI3Ki to BRAFi+EGFRi treatment did not robustly affect viability in any of the resistant cells relative to BRAFi+EGFRi alone. This finding is consistent with initial results of a clinical trial comparing encorafenib and cetuximab to encorafenib, cetuximab, and alpelisib, which have not demonstrated a clear benefit in response rate or progression-free survival with the addition of the PI3K inhibitor alpelisib (12,40). In marked contrast, the triple combination of BRAFi+EGFRi+MEKi showed improved efficacy in many models relative to either BRAFi+EGFRi, BRAFi+MEKi, or MEKi+EGFRi alone. Finally, the addition of BRAFi and/or EGFRi to ERKi appeared to improve efficacy in some resistant models relative to ERKi alone, suggesting that ERKi may best be administered as part of therapeutic combinations in future clinical trials for BRAF mutant CRC. Indeed, analysis of resistant cell lines indicated that ERK inhibition could induce cytotoxicity, which was further enhanced when combined with BRAFi and/or EGFRi (Supplementary Figures 3A and 3B).

Discussion

Over the past few years, BRAF inhibitors have demonstrated striking clinical efficacy in patients with BRAF mutant melanoma. However, BRAF inhibitors are not equally effective in other BRAF mutant cancer histologies (4). Preclinical studies defining EGFR and MAPK pathway reactivation as key drivers of BRAF inhibitor resistance in BRAF mutant CRC have provided the rationale for testing double or triple combinations of therapies targeting EGFR/BRAF/MEK/PI3K in this disease (11–13,15,16).

Unfortunately, while these approaches have led to improvements in response rate in BRAF mutant CRC patients, preliminary clinical observations have indicated that, following an initial response, acquired resistance in BRAF mutant CRC patients typically emerges after a few months of treatment (11–13,15). The mechanisms underlying acquired resistance in BRAF mutant CRC cells remain poorly characterized. In this study, we undertook a comprehensive effort to develop models of secondary resistance to a spectrum of seven clinically-relevant combinatorial therapies in order to more robustly define the landscape of molecular mechanisms leading to acquired resistance in BRAF mutant CRC. Our results indicate that the mechanisms leading to acquired resistance to these combinations can be genetically heterogeneous, but appear to converge on the reactivation of the MAPK signaling pathway at the biochemical level, suggesting that it might be possible to develop universal combination strategies capable of overcoming multiple resistance mechanisms. We acknowledge that no in vivo models were generated or assessed in this study, thus limiting our observations to cancer cell autonomous drug resistance mechanisms. However, analyses of plasma samples at baseline and at acquired resistance to BRAF combinatorial therapy, in a BRAF mutant CRC patient, revealed genetic alterations consistent with those identified in cell models, thus underscoring the clinical relevance of the broad panel of resistant lines generated in this work. Since microenvironment and non-genomic mechanisms of drug resistance may also occur, future studies based on the analysis of BRAF mutant murine models and patient samples will be needed to expand our knowledge on this aspect.

In our resistant cell line panel, we identified several novel mechanisms of acquired resistance not previously reported in BRAF mutant CRC. In particular, we identified an EGFR G465R ectodomain mutation in a cell line with acquired resistance to the combination of a BRAFi and anti-EGFR antibody. While this mutation has been established as a mechanism of acquired resistance to anti-EGFR antibody monotherapy in RAS/BRAF wild-type CRC due to disruption of antibody binding (25), this class of mutations has not previously been implicated in BRAF mutant CRC. Our observation warrants assessing for EGFR ectodomain mutations in BRAF mutant CRC patients upon acquired resistance to BRAFi and anti-EGFR antibody combinations. Importantly, we found that a resistant model harboring this mutation retained sensitivity to BRAFi and an EGFR kinase inhibitor, as well as to downstream inhibitor combinations, such as BRAFi+MEKi. We also identified EGFR amplification as a novel potential mechanism of acquired resistance in BRAF mutant CRC. Interestingly, unlike the EGFR ectodomain mutation, EGFR amplification conferred cross-resistance to BRAFi+EGFRi, BRAFi+MEKi, and MEKi+EGFRi combinations, likely as a consequence of increased EGFR signalling flux, and retained sensitivity only to the triple combinations of BRAFi+EGFRi+MEKi and ERKi+BRAFi+EGFRi. The finding that EGFR signaling leads to MAPK feedback reactivation and resistance during BRAFi monotherapy, but also can contribute to acquired resistance to MAPK combinatorial inhibition, highlights the central role of EGFR in the biology of BRAF mutant CRC.

Molecular analyses of our resistance cell line panel also identified the presence of several resistance mechanisms previously identified in the setting of acquired resistance in BRAF mutant CRC—including KRAS mutation or amplification, BRAF V600E amplification, and MAP2K1 mutation (30), thereby underscoring the likely importance of these specific mechanisms within the spectrum of acquired resistance in BRAF mutant CRC and supporting the likelihood that these specific alterations may be frequently observed in patients. This is also supported by the identification of two different genetic alterations identified at resistance to BRAF/MEK inhibition in plasma sample of a BRAF mutant CRC patient, i.e., the emergence of a KRAS mutation and a likely amplification of mutant BRAF V600E. In our resistant cell models, KRAS alterations were the most common resistance mechanism. The high prevalence of KRAS mutations in CRC and its role in resistance to anti-EGFR therapies underpin a central role for KRAS in this disease. Analysis by standard sensitivity sequencing has typically identified KRAS and BRAF mutations in a mutually exclusive fashion in CRC (41–43). In order to explain these observations, it has been suggested that concomitant oncogenic activation of KRAS and BRAF would be counter-selected during tumorigenesis, as it would result in activation of cell-cycle inhibitory proteins of the Ink4/Arf locus, leading to oncogenic stress and senescence (44). Nevertheless, the use of more sensitive techniques, such as droplet digital PCR, have recently revealed that low-allele frequency KRAS mutations could coexist with BRAF V600E in CRC samples (7). These rare subclones may be present but might possess an unfavorable fitness compared to clones with only mutant BRAF. However, the selective pressure of BRAF-directed therapy may improve the proliferation rate of the double mutant clones while reducing the viability of cells bearing only mutant BRAF, thus driving outgrowth of resistant BRAF/KRAS double mutant clones. Indeed, a recent study analyzing tumor biopsies from BRAF mutant CRC patients obtained prior to BRAF-directed therapy revealed that more than 50% bear low frequency KRAS mutations (7). This finding might be explained by the 'Big Bang' model (45), whereby tumors grow predominantly as a single expansion producing numerous intermixed subclones, where the timing of an alteration rather than clonal selection for that alteration is the primary determinant of its pervasiveness. Similarly, it is possible that some of the other common acquired resistance mechanisms we have observed in BRAF mutant CRC, such as BRAF V600E amplification and MAP2K1 mutation, may also pre-exist in rare tumor subclones. Indeed, we previously found that rare tumor cells with BRAF amplification could be identified in pre-treatment tumor biopsies from BRAF mutant CRC patients (32). Altogether, these observations suggest that KRAS as well as other resistance alterations could develop at an early stage of BRAF mutant colorectal tumorigenesis, thus laying the seeds for the eventual emergence of acquired resistance. In a resistant cell model and in our patient, BRAF combinatorial therapies have resulted in the appearance of at least two concomitant resistance mechanisms. Indeed, the lower percentage of KRAS mutant allele in comparison with to the TP53 founder mutation suggested that this variant may have been present in only a fraction of tumor cells distinct from the BRAF V600E amplified subset. These data are consistent with previous reports in melanomas resistant to BRAFi, either as monotherapy or in combination with MEKi, in which multiple resistance mechanisms have been described to co-occur in individual patients (46,47).

The observation that all resistance mechanisms identified in our cell panel converge to reactivate MAPK signaling has important clinical implications. Since it may not be practical to design specific therapeutic strategies against each of the individual acquired resistance mechanisms observed in BRAF mutant CRC, there would be clear clinical advantages to developing a more “universal” therapeutic strategy targeting a common signalling output that would be capable of overcoming a spectrum of potential resistance mechanisms. By systematically comparing multiple drug combinations designed to achieve more optimal MAPK pathway suppression across the molecular landscape of acquired resistance mechanisms in BRAF mutant CRC, we were able to identify the most promising therapeutic candidates to overcome resistance. Although a few resistant cell lines showed only modest sensitivity to these combinations, suggesting the possibility that these models might harbor additional MAPK-independent resistance mechanisms, overall we observed that the combination of BRAFi+EGFRi+MEKi or ERKi in combination with BRAFi and/or EGFRi displayed superior activity across the vast majority of resistant models. Therefore, these combinations may represent the most promising strategies for evaluation in clinical trials for patients with BRAF mutant CRC. Notably, the triple combination of BRAFi+EGFRi+MEKi is currently being evaluated in clinical trials, and preliminary results suggest improved response rate and progression-free survival compared to the individual doublet combinations (48), which is consistent with our findings, and suggests that improved activity against the common resistance mechanisms in BRAF mutant CRC may account in part for the improved clinical efficacy observed.

Consistent with our findings, previously published reports have documented promising preclinical activity of ERK inhibition in BRAFi or MEKi resistant cell line models (27,38,39) and in MEKi+BRAFi and BRAFi+EGFRi resistant BRAF mutant CRC cells (30), supporting ERKi as key potential components of future clinical trial strategies for this disease. While it is likely that secondary mutations in ERK1/2 may limit the long-term efficacy of ERKi (49), it remains an important and unanswered question as to whether it is best to administer ERKi as monotherapy or whether ERKi might be more effective as part of drug combinations in BRAF mutant CRC. Indeed, it is possible that ERK inhibition alone might trigger survival-promoting feedback loops through alternative pathways that might be optimally suppressed with therapeutic combinations. In order to help guide future clinical trial strategies, our study begins to address this critical question, and suggests that ERKi appear to be more effective against the spectrum of acquired resistance mutations in BRAF mutant CRC when administered in combination with BRAFi and/or EGFRi inhibitors. In fact, the triplet combination of ERKi+BRAFi+EGFRi appeared to be the most effective combination strategy overall across our panel of resistant cell line models. Thus, our study suggests that initial clinical trials of ERKi in BRAF mutant CRC patients should prioritize therapeutic combinations with BRAFi and EGFR inhibitors.