Figure 4. Fitness threshold model.

(a) The BRAF mRNA expression as a function of CN in 145 untreated BRAF^V600E-mutant melanomas. The data were obtained from TCGA. The dotted area represents tumors at risk for selective propagation of BRAF^amp 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 BRAF^amp. In contrast, combination therapy is predicted to maximally elevate the fitness threshold, thus suppressing the selection and propagation of BRAF^amp 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 patients⁹. PR: partial response, SD: stable disease, PD: progressive disease.