The role of altered calcium signaling and handling as an underlying mechanism of drug tolerance in BRAF mutant melanoma

Abstract

Molecularly‐targeted therapies in patients harboring well‐defined driver mutations have proven to be initially effective for many patients; however, tumor progression is nearly universal several months after initial regression. While it is common in cancer biology to frame treatment failure in terms of genetic mutations that either pre‐exist or are acquired during treatment, a growing body of literature is increasing awareness of non‐genetic processes that play a key role. For example, cancer stem cells, or stem‐like cells that can reversibly shift phenotypes are now a regular topic of investigation within the cancer research community. Recently, we reported that BRAF mutant melanoma cell lines exposed to prolonged treatment (>100 hours) with targeted BRAF inhibitors enter a non‐genetic, reversible state of balanced death and division. It is postulated that this state – that we term idling – acts as a haven for cancer cells to escape the stressful conditions of BRAF inhibition, while allowing for occasional cell division and, hence, DNA replication. Moreover, molecular signatures of error prone DNA replication and repair indicates that this state favors the acquisition of resistance conferring mutations, which lead to recurrence, metastasis, and ultimately death. Intriguingly, because cells are under stress from the initial treatment, it is possible that residual tumors composed of idling cancer cells may be vulnerable to a well‐targeted secondary treatment. However, developing such a treatment will require a detailed understanding of the molecular drivers of the drug tolerant phenotype. Accordingly, we have molecularly profiled cell lines transitioning into idling by RNAseq, ATACseq, and Seahorse metabolic analysis, revealing globally dysregulated ion channel expression and dramatically reduced activity of glycolysis and oxidative phosphorylation. Notably, expression of key calcium signaling and handling genes (i.e. Inositol tri‐phosphate receptors (ITPRs)) were observed to fall significantly during treatment, directing the exploration of calcium signaling and handling in the idling state. This exploration revealed reduced endoplasmic reticulum (ER) calcium content, and reduced store operated calcium entry (SOCE) in the idling state – both of which are well described to be regulators of signaling, metabolism, cell cycle progression, and programmed cell death. However, it is unclear to what extent differential calcium handling properties are responsible for the drug tolerant behavior in the idling state. Therefor this project will explore the role of calcium handling and signaling as an underlying mechanism of the idling state, and how these properties may differ between cells with different fates (i.e. death, division, or neither). In line with observations that death and division rates are low in the idling state, I hypothesize that reduced calcium handling allows cells to avoid programmed cell death, at the expense of reduced proliferative capacity. In contrast, I hypothesize that the rare dividing cells rescue their calcium handling and signaling capabilities, thereby increasing their metabolism and ability to progress through the cell cycle – a hypothesis that is consistent with the preliminary data. Increasing our understanding of the molecular mechanism behind the idling phenotype will shed new light on how BRAF mutant cancer cells survive targeted treatment and potentially provide new treatment strategies for addressing the ultimate failure in the clinic.