Single-cell analysis resolves the cell state transition and signaling dynamics associated with melanoma drug-induced resistance

Abstract

Continuous BRAF inhibition of BRAF mutant melanomas triggers a series of cell state changes that lead to therapy resistance and escape from immune control before establishing acquired resistance genetically. We used genome-wide transcriptomics and single-cell phenotyping to explore the response kinetics to BRAF inhibition for a panel of patient-derived BRAFV600-mutant melanoma cell lines. A subset of plastic cell lines, which followed a trajectory covering multiple known cell state transitions, provided models for more detailed biophysical investigations. Markov modeling revealed that the cell state transitions were reversible and mediated by both Lamarckian induction and nongenetic Darwinian selection of drug-tolerant states. Single-cell functional proteomics revealed activation of certain signaling networks shortly after BRAF inhibition, and before the appearance of drug-resistant phenotypes. Drug targeting those networks, in combination with BRAF inhibition, halted the adaptive transition and led to prolonged growth inhibition in multiple patient-derived cell lines.

Drug persistence – From antibiotics to cancer therapies

“…The first hint stems from the observation that slow-growing cancer cells also tend to be more drug-tolerant in a wide range of cell types and model systems [12,30–35]. Such drug-tolerance has been reported during exposure to chemo-[35] and targeted [30] therapy, for adherent [32] and suspension [31] cells in vitro , as well as in vivo in mouse models [12], suggesting a general phenomenon.…”

“…The first process is epithelial-to-mesenchymal transition (EMT), which causes cells to gradually lose their differentiation status and become more stem-cell like [44]. Expression of EMT/stem-cell markers is a frequent hallmark of persister subpopulations, which can be exploited to isolate persister cells within isogenic populations [30,31,35,36,45]. Second, several lines of evidence point towards chromatin remodeling as a key step in persister formation [2,32,46,47].…”

“…However, as we discussed above, persisters not only emerge spontaneously in untreated populations, but can also be induced by various environmental stresses. Recent in vitro studies have begun to elucidate the importance of induced persistence, also termed ‘type I persistence’ [9], in cancer cell populations [31,45]. Pisco et al showed in clonally derived leukemia cells that the rapid emergence of multidrug resistance 1 (MDR1) mediated persisters upon chemotherapeutic treatment is largely driven by induced persistence [45].…”

“…Whether such ‘Larmarckian induction’ [45] is the exception or the norm in the emergence of cancer drug persisters remains an open question. Nevertheless, these works suggest that inhibition of the mechanisms that mediate the transition to a persister state during drug treatment may help to prevent the emergence of drug resistance in cancer [31,45,58–60]. …”

“…It is tempting to speculate that in cancer cells similar mechanisms could potentially induce an EMT-type transcriptional program if growth is impaired. Such a mechanism might explain the rapid increase in persister fraction that has been observed in drug exposed populations [31,45]. Another intriguing question is whether metabolism can directly induce persistence in cancer cells, similar to the nutrient-shift induced persisters in bacteria [21–24].…”