An approach to suppress the evolution of resistance in BRAFV600E-mutant cancer

Abstract

The principles that govern the evolution of tumors exposed to targeted therapy are poorly understood. Here we modeled the selection and propagation of an amplification in the BRAF oncogene (BRAFamp) in patient-derived tumor xenografts (PDXs) that were treated with a direct inhibitor of the kinase ERK, either alone or in combination with other ERK signaling inhibitors. Single-cell sequencing and multiplex fluorescence in situ hybridization analyses mapped the emergence of extra-chromosomal amplification in parallel evolutionary trajectories that arose in the same tumor shortly after treatment. The evolutionary selection of BRAFamp was determined by the fitness threshold, the barrier that subclonal populations need to overcome to regain fitness in the presence of therapy. This differed for inhibitors of ERK signaling, suggesting that sequential monotherapy is ineffective and selects for a progressively higher BRAF copy number. Concurrent targeting of the RAF, MEK and ERK kinases, however, imposed a sufficiently high fitness threshold to prevent the propagation of subclones with high-level BRAFamp. When administered on an intermittent schedule, this treatment inhibited tumor growth in 11/11 PDXs of lung cancer or melanoma without apparent toxicity in mice. Thus, gene amplification can be acquired and expanded through parallel evolution, enabling tumors to adapt while maintaining their intratumoral heterogeneity. Treatments that impose the highest fitness threshold will likely prevent the evolution of resistance-causing alterations and, thus, merit testing in patients.

Figures

Figure 1. ERK inhibitor-resistant populations with extrachromosomal BRAF amplification

(a) Patient-derived xenograft (PDX) models from patients with BRAFV600E-mutant lung cancer or melanoma were treated with ERK inhibitor (ERKi) SCH984 over time (n = 5 mice, mean ± s.e.m). (b) H&E stained sections of the PDX models before and after ERKi treatment. (c) Single nuclei extracted from PDX1D tumors were analyzed by FACS to determine the distribution of cells according to their DNA content. A human diploid cell line was used as a control. (d) Copy number (CN) profiles of 69 single cells derived from parental (Par) and ERK inhibitor-resistant (EiR) PDX-1D tumors. (e), Projection of single cells into the top three principal components. (f) Subclonal distribution of parental and resistant tumors. (g) Segment values spanning the BRAF locus in tumor and stromal cells. For stromal cells, sequenced reads were mapped to the mouse genome (see Supplementary Fig. 1e). (h) Representative images of fluorescence in situ hybridization (FISH) analysis with probes spanning BRAF or chr7 centromere in red or green, respectively (a representative of five different fields is shown). (i) Probes were quantified by manual counting (n = 100 cells, all data are shown). (j) Representative image of extra-chromosomal localization of the BRAF gene (arrows) in an 1D-EiR cell undergoing metaphase. (k) The expression of BRAFV600E protein in matched PDX1D and 1E tumor sets was determined by mass spectrometry (n = 3, mean ± s.e.m). Actin and tubulin were used as controls.

Figure 2. BRAFamp emerges in parallel evolutionary tracts

(a) The clonal relationship of single cells, as inferred by Manhattan-Ward clustering of integer CN. The bar graph shows BRAF CN. (b, c) Single cells derived from parental (b) or resistant (c) tumors were subjected to hierarchical clustering and subclonal analysis independent of each other. Phylogenetic inference was established using a heuristic maximal parsimony approach. Subclones A and B were subdivided on the basis of additional CN alterations and their inferred phylogeny. (d) Single cells with high-level BRAFamp were found in three distinct resistant clones. (e) The CN state of select chromosomal regions with heterogeneous profiles. Note the emergence of BRAFamp cells in three distinct evolutionary trajectories depending on co-occurring losses in chr2p, 11q and/or 13 (arrows). (f, g) Multiplex FISH with probes targeting the indicated chromosomal regions in PDX1D-EiR. Manual quantification (f) and representative images (g) are shown.

Figure 3. BRAFamp is sufficient to confer a selective advantage in the presence of ERKi treatment

(a) Immunoblot analysis of cell lines derived from parental (1D) or ERK inhibitor-resistant (1D-EiR) PDX. (b) Immunoblot analysis of signaling intermediates in 1D and 1D-EiR cells treated for 1h with SCH984. (c) Cell viability at 72h after treatment. (d) Cell viability of 1D-EiR cells transfected with BRAF specific or control siRNAs followed by drug treatment as in c. (e & f) A375 cells, engineered to express BRAFV600E under a doxycycline (dox)-induced promoter, were treated as shown (dox, 2μg/mL; SCH984, 500 nM) to determine the effect on signaling by immunoblotting (e) or viability (f). Withdrawal of dox after a 6-week stimulation restored sensitivity to the ERKi. A representative of at least two independent experiments is shown for the immunoblots in this figure. In viability experiments, n = 3, mean ± s.e.m.

Figure 4. Fitness threshold model

(a) The BRAF mRNA expression as a function of CN in 145 untreated BRAFV600E-mutant melanomas. The data were obtained from TCGA. The dotted area represents tumors at risk for selective propagation of BRAFamp during drug treatment. (b, c) A375 cells were stimulated with increasing concentrations of dox (24h), followed by treatment with ERK signaling inhibitors (RAFi: vemurafenib, 1 μM; MEKi: trametinib, 25 nM; or ERKi: SCH984, 500 nM) for 1h (b) or 72h (c), to determine the effect on signaling (b, quantification of representative immunoblots of 2 independent experiments) or relative fitness (c, n = 3, mean ± s.e.m.). The effect on signaling was adjusted for the effect of dox alone. Relative fitness is the change in log(IC50) with increasing concentrations of dox. (d) A schematic representation of the fitness threshold model. Sequential monotherapy is predicted to impose a selective gradient for the propagation of high level BRAFamp. In contrast, combination therapy is predicted to maximally elevate the fitness threshold, thus suppressing the selection and propagation of BRAFamp subclones. (e) Genomic DNA extracted from the patient biopsies before and after RAFi treatment (pre, post) or their derivative PDX models, before and after exposure to the ERKi, were sequenced to determine the BRAF CN. Diploid A375 cells were used as a control. (f) The duration of ERKi treatment response in patients who were either targeted therapy-naïve (pt. A and B) or pretreated with a RAF/MEKi combination (pt. C, D, E). See also Supplementary Table II. (g) The duration of treatment response in lung cancer patients treated first with a RAFi (left) followed by the addition of a MEKi (right). By comparison, the RAF/MEKi combination therapy has nearly a 60% response rate in treatment-native lung cancer patients. PR: partial response, SD: stable disease, PD: progressive disease.

Figure 5. Identification of a treatment to suppress the evolution of BRAF amplified clones

(a) Immunoblot analysis (n = 2 independent experiments) of dox-induced A375 cells treated as shown (doses as in Fig. 4b) for 24h to determine the effect on ERK signaling intermediates. (b) As in a, but cells were treated for 72h to determine the effect on viability (n = 3, mean ± s.e.m). (c, d) Mice bearing PDX1D (c) or PDX1E (d) were treated with dabrafenib (RAFi), trametinib and/or SCH984 daily for 14 days followed by discontinuation of treatment to determine the effect on tumor growth (n = 5 mice, mean ± s.e.m). A vemurafenib analogue (PLX4720), alone or in combination, had a similar effect to dabrafenib (see below). Mice treated with the MEK/ERKi combination experienced significant toxicity leading to discontinuation of the experiment in (d). (e) Tumors that regrew after discontinuation of drug treatment from (c) were analyzed to determine BRAF CN by sequencing and BRAF protein expression by immunoblot analysis.

Figure 6. An intermittent combination treatment inhibits tumor growth in lung cancer and melanoma BRAFV600E PDX models

(a, b) A schematic representation (a) of several three-drug combination treatment schedules and their effect on the growth of PDX1D tumors in athymic mice (b, n = 5 mice, mean ± s.e.m, RAFi: vemurafenib analogue PLX4270, MEKi: trametinib, ERKi: SCH984). Treatment related toxicity was determined by monitoring animal weight or mortality. Mice treated on Schedule 5 remained free of tumor for up to 180 days after drug discontinuation. (c) Additional optimization of the off-drug interval in order to minimize toxicity, while retaining maximal tumor growth inhibition. (d) The expression of total BRAF in the PDX models was determined using mass spectrometry (n = 3, mean ± s.e.m). (e) The profile of genetic alterations in the BRAFV600E PDX models utilized in this study. (f) Effect of the intermittent regimen in a model with de-novo insensitivity to ERKi treatment. (g) The effect of the intermittent three drug combination treatment (administered on a 3/7-day schedule) in lung and melanoma PDX models (n = 5 mice, for each untreated or treated arm, mean ± range; ns: p>0.05; primary data are shown in Supplementary Fig. 6).