Drug-induced expression of the RNA-binding protein HuR attenuates the adaptive response to BRAF inhibition in melanoma.

Strategies that aim to limit the adaptive response to pathway inhibition in BRAF-mutated melanoma face the inherent limit of signaling redundancy and multiplicity of possible bypass mechanisms. Drug-induced expression of selected RNA-binding proteins, like the ubiquitously expressed HuR, has the potential to differentially stabilize the expression of many genes involved in the compensatory mechanisms of adaptive response. Here, we detect in BRAF-mutated melanoma cell lines having a higher propensity for adaptive response and in non-responding melanoma tumors, a larger proportion of HuRLow cells in the expression distribution of HuR. Using knockdown experiments, we demonstrate, through expression profiling and phenotypic assays, that increasing the proportion of HuRLow cells favors the adaptive response to BRAF inhibition, provided that the HuRLow state stays reversible. The MAPK dependency of melanoma cells appears to be diminished as the proportion of HuRLow cells increases. In single-cell assays, we demonstrate that the HuRLow cells display plasticity in their growth expression profile. Importantly, the adaptive over-proliferating cells emerge in the subpopulation containing the HuRLow cells. Therapeutic concentrations of lithium salts, although they moderately increase the global expression of HuR, are sufficient to suppress the HuRLow cells, induce an overall less resistant expression profile and attenuate in a HuR-dependent manner the adaptive response of melanoma cells in ex vivo assays. The therapeutic effectiveness of this approach is also demonstrated in vivo in mice xenografts. This study has immediate clinical relevance for melanoma therapy and opens a new avenue of strategies to prevent the adaptive response to targeted cancer therapy.

A mutual information-based in vivo monitoring of adaptive response to targeted therapies in melanoma.

The mechanisms of adaptive resistance to genetic-based targeted therapies of solid malignancies have been the subject of intense research. These studies hold great promise for finding co-targetable hub/pathways which in turn would control the downstream non-genetic mechanisms of adaptive resistance. Many such mechanisms have been described in the paradigmatic BRAF-mutated melanoma model of adaptive response to BRAF inhibition. Currently, a major challenge for these mechanistic studies is to confirm in vivo, at the single-cell proteomic level, the existence of dependencies between the co-targeted hub/pathways and their downstream effectors. Moreover, the drug-induced in vivo modulation of these dependencies needs to be demonstrated. Here, we implement such single-cell-based in vivo expression dependency quantification using immunohistochemistry (IHC)-based analyses of sequential biopsies in two xenograft models. These mimic phase 2 and 3 trials in our own therapeutic strategy to prevent the adaptive response to BRAF inhibition. In this mechanistic model, the dependencies between the targeted Li2CO3-inducible hub HuR and the resistance effectors are more likely time-shifted and transient since the minority of HuRLow cells, which act as a reservoir of adaptive plasticity, switch to a HuRHigh state as they paradoxically proliferate under BRAF inhibition. Nevertheless, we show that a copula/kernel density estimator (KDE)-based quantification of mutual information (MI) efficiently captures, at the individual level, the dependencies between HuR and two relevant resistance markers pERK and EGFR, and outperforms classic expression correlation coefficients. Ultimately, the validation of MI as a predictive IHC-based metric of response to our therapeutic strategy will be carried in clinical trials.