Temporal dynamics of signaling and transcription underlying early adaptation to oncogenic BRAF inhibition in melanoma

Despite the recent remarkable progress of targeted therapies for the clinical management of many cancers, response rates remain lower than desired and long-term response durability is often poor due to drug resistance. A major contributor to treatment failure is drug-induced cellular adaptation to therapy, whereby systems-level reprogramming permits a drug-tolerant phenotype that may gradually lead to cell cycle re-entry and portend relapse. Drug adaptation is known to rapidly implicate complex signaling and transcriptional regulatory networks, but the scale and temporal dynamics of these remodeling events have yet to be fully resolved. Here, we used mass spectrometry-based phosphoproteomics and RNA sequencing to capture molecular snapshots within the first minutes, hours, and days of BRAF kinase inhibitor exposure in a drug-tolerant human BRAF-mutant melanoma model. By enriching specific phospho-motifs associated with mitogenic kinases, we were able to detect and monitor thousands of protein phosphorylation sites over a three-day period of drug treatment, followed by a six-day drug holiday to characterize the plasticity of the adaptive response. We observed early and sustained inhibition of the BRAF-ERK axis, gradual downregulation of canonical cell cycle-dependent phosphorylation events, and a clear delineation between three distinct phase transitions toward drug tolerance, which was almost completely reversible following drug removal. Through measuring the time evolution of large-scale networks, we were able to exploit phosphoproteome and transcriptome dynamics to infer kinetically-defined regulatory modules, revealing a concerted response to oncogenic BRAF inhibition involving cellular metabolism, RNA processing, and mitogenic signaling that demonstrated strong agreement with prior knowledge. The adaptive response to BRAF inhibition was dominated by a compensatory induction of SRC-family kinase (SFK) signaling, which we found to be at least partially driven by impaired redox homeostasis and accumulation of reactive oxygen species – providing a fine degree of temporal, mechanistic, and phosphorylation site-specific context to an important axis in tumor cell survival. This induction sensitized cells to co-treatment with an SFK inhibitor, and combination therapy significantly outperformed single-agent BRAF inhibition across a panel of patient-derived melanoma cell lines and in an orthotopic mouse xenograft model, underscoring the high translational potential for measuring the temporal dynamics of signaling and transcription networks under therapeutic challenge.