A Nonquiescent “Idling” Population State in Drug-Treated, BRAF-Mutated Melanoma

Abstract

Targeted therapy is an effective standard of care in BRAF-mutated malignant melanoma. However, the duration of tumor remission varies unpredictably among patients, and relapse is almost inevitable. Here, we examine the responses of several BRAF-mutated melanoma cell lines (including isogenic subclones) to BRAF inhibitors. We observe complex response dynamics across cell lines, with short-term responses (<100 h) varying from cell line to cell line. In the long term, however, we observe equilibration of all drug-treated populations into a nonquiescent state characterized by a balanced rate of death and division, which we term the "idling" state, and to our knowledge, this state has not been previously reported. Using mathematical modeling, we propose that the observed population-level dynamics are the result of cells transitioning between basins of attraction within a drug-modified phenotypic landscape. Each basin is associated with a drug-induced proliferation rate, a recently introduced metric of an antiproliferative drug effect. The idling population state represents a new dynamic equilibrium in which cells are distributed across the landscape such that the population achieves zero net growth. By fitting our model to experimental drug-response data, we infer the phenotypic landscapes of all considered melanoma cell lines and provide a unifying view of how BRAF-mutated melanomas respond to BRAF inhibition. We hypothesize that the residual disease observed in patients after targeted therapy is composed of a significant number of idling cells. Thus, defining molecular determinants of the phenotypic landscape that idling populations occupy may lead to "targeted landscaping" therapies based on rational modification of the landscape to favor basins with greater drug susceptibility.

Sporadic ERK pulses drive non-genetic resistance in drug-adapted BRAFV600Emelanoma cells

“…drug-resistant tumors(Ahn et al, 2017)(Fallahi-Sichani et al, 2017)(Shaffer et al, 2017)(Paudel et al, 2018). The fundamental mystery of the adapted state is how melanoma cells can grow and divide when ERK is profoundly inhibited.…”

Disruption of Redox Balance Enhances the Effects of BRAF-inhibition in Melanoma Cells

“…For combination with PLX4720, 8μM PLX4720 was used as diluant, and serially diluted for second drug from highest concentration to lowest. Direct measurements of cell counts were made using Cellavista software and ImageJ macros as previously described (Hardeman et al, 2017;Harris et al, 2016;Paudel et al, 2018) . The drug-induced proliferation (DIP) rates are calculated using the slope of the log2-normalized population curves after 24 hrs.…”

“…Single-cell derived BRAF -mutated SKMEL5 subclones were derived as previously described (Paudel et al, 2018) . BRAF -mutated melanoma cells (SKMEL5, WM88), including the SKMEL5 subclones, NRAS-mutated melanoma cells (SKMEL2), and NF1-mutated melanoma cells (MeWO) were grown and cultured in Dulbecco's modified Eagle's medium and Ham's F-12 media (DMEM:F12, 1: 1, Cat.…”

“…We recently reported that BRAF -mutated melanoma cell lines, including isogenic single-cell derived subclones, exhibit varying drug sensitivities to a small molecule BRAF kinase inhibitor (BRAFi) (Hardeman et al, 2017;Paudel et al, 2018) . Using Drug-Induced Proliferation (DIP) rates ( Table S1 ) (Harris et al, 2016) , as a measure of drug effects, we examined the molecular correlates of BRAF i sensitivity, and reported that top 200 differentially expressed genes (DEGs) in drug-insensitive cells were enriched in processes and functions related to redox metabolism (Meyer et al, 2019) .…”

“…While these therapies improve responses, treatment outcomes still vary, and benefits remain transient and unpredictable (Luke et al, 2017) . Some resistance can be attributed to genetic mutations (Greaves and Maley, 2012;Nowell, 1976) , but accumulating evidence indicates that nongenetic processes play a critical role in response of cancer cells to drug treatment (Niepel et al, 2009;Paudel et al, 2018;Shaffer et al, 2017;Smith et al, 2016) . Metabolic reprogramming, recognized as a hallmark of cancer, has recently emerged as a potential nongenetic process that contributes to the emergence of drug-tolerant cancer cells (Hanahan and Weinberg, 2011) .…”