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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE252781 | GEO | 2024/03/25

REPOSITORIES: GEO

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Project description:Recent data demonstrate that extracellular signals are transmitted through a network of proteins rather than hierarchical signaling pathways. This network model suggests why inhibition of a single component of a canonical pathway, even when targeting a mutationally activated driver of cancer, has insufficiently dramatic effects on the treatment of cancer. The biological outcome of signals propagated through a network is inherently more robust and resistant to inhibition of a single network component due to compensatory and redundant signaling events. In this study, we performed a functional chemical genetic screen analogous to synthetic lethal screening in yeast genetics to identify novel interactions between signaling inhibitors that would not be predicted based on our current understanding of signaling networks. We screened over 300 drug combinations in nine melanoma cell lines and have identified pairs of compounds that show synergistic cytotoxicity. Among the most robust and surprising results was synergy between sorafenib, a multi-kinase inhibitor with activity against Raf, and diclofenac, a non-steroidal anti-inflammatory drug (NSAID). This synergy did not correlate with the known RAS and BRAF mutational status of the melanoma cell lines. The NSAIDs celecoxib and ibuprofen could qualitatively substitute for diclofenac. Similarly, the MEK inhibitor PD325901 and the Raf inhibitor RAF265 could qualitatively substitute for sorafenib. These drug substitution experiments suggest that inhibition of cyclo-oxygenase and MAP kinase signaling are components of the observed synergistic cytotoxicity. Genome-wide expression profiling demonstrates synergy-specific down-regulation of survival-related genes. This study provides proof of principle that synthetic lethal screening can uncover novel functional drug combinations and suggests that the underlying signaling networks that control responses to targeted agents can vary substantially depending on unexplored components of the cell genotype. RNA from VMM39, DM331, and SLM2 cells with/without mutations in Ras and/or Braf, treated with Sorafenib and/or Diclofenac.