Targeting cell cycle phase-specific drug sensitivity for melanoma therapy

The development of small molecule MAPK pathway inhibitors (MAPKi) and antagonists of the immune checkpoints has revolutionized melanoma therapy. However, MAPKi only work in approximately 40% of cases as a BRAFV600 mutation must be present; moreover, rapid development of resistance is common. Immune checkpoint inhibitors (ICi) show response rates up to 60%, depending on drug or combination, with durable effects but resistance can still occur. Thus there is a clear need to develop combination therapies to delay the onset of resistance. Many anti‐cancer drugs impact the cell cycle but are also dependent on specific cell cycle phases, which results in cell cycle phase‐specific drug insensitivity. The tumor microenvironment is characterized by cancer cell subpopulations with heterogeneous cell cycle profiles. For example, hypoxic tumor zones contain clusters of cancer cells that arrest in G1‐phase whereas actively cycling cells cluster around blood vessels. Importantly, neoplastic cells exhibit differential drug sensitivity based on their residence in specific cell cycle phases. We have established a model to study the effects of the cell cycle on drug sensitivity in real‐time: Utilizing two‐ and three‐dimensional melanoma culture models in combination with fluorescent cell cycle indicators (FUCCI), we investigated the effect of G1‐arrest on drug sensitivity. G1‐arrested melanoma cells were more resistant to apoptosis induced by agents that selectively target S/G2/M phase cells, such as the proteasome inhibitor bortezomib or the alkylating agent temozolomide. In contrast, G1‐arrested cells were more sensitive to MAPKi‐induced cell death as this pathway is essential for G1‐phase progression. Of major clinical relevance, pretreatment of melanoma cells with sub‐lethal but G1‐arresting doses of a MAPKi resulted in resistance to temozolomide or bortezomib. We also found that changing environmental conditions, such as applying hypoxia, resulted in protective G1‐ arrest. However, hypoxia can also result in endoplasmic reticulum (ER) stress. If the level of ER stress is persistent or excessive it switches to an apoptotic response. We show that the protective effect of G1‐arrest can be overridden by the apoptotic effect of ER stress. This was then used as a therapeutic approach: Using the F‐XBP1ΔDBD‐venus reporter construct, to visualize ER stress, we showed that single agent low dose ER stress‐inducing agents, bortezomib and fenretinide, induced ER‐stress and cell cycle arrest but only limited cell death, while the combination resulted in synergistic apoptosis. Taken together, our data demonstrate that cell cycle‐tailored targeting of metastatic melanoma can improve therapy outcomes and that additional induction of ER stress is a strategy worth investigating to further improve melanoma therapy. Furthermore, we propose that our real‐time cell cycle imaging 3D melanoma spheroid model could be utilized as a tool to measure and design drug scheduling approaches.