Deciphering melanoma transcriptional heterogeneity: TCF4 and the mesenchymal-like switch

Non-genetic mechanisms of cancer progression have been gaining progressively more importance in melanoma. A dedifferentiated mesenchymal-like state has been shown to be implicated in invasion and resistance to targeted therapy. However, the evolution of melanoma transcriptomic landscape under immune checkpoint blocker (ICB) therapy remains an answered question. Here we show a longitudinal study including melanoma patients undergoing treatment with ICBs (nivolumab or nivolumab+ipilimumab) in which patient and lesion-matched samples were collected before and after the first cycle of immunotherapy and processed for single cell-RNA sequencing.

Critically, we identified a significant enrichment of the mesenchymal-like (MES) state on treatment in the non-responder patients. We also shown TCF4 to be the master regulator of the MES state. Moreover, by suppressing both the melanocytic (MEL) and the antigen presentation programs, TCF4 was shown to drive resistance to ICBs, both in vitro and in vivo. Consistent with these observations, silencing TCF4 in MES cells increased sensitivity to targeted therapy. We also describe a possible way of targeting TCF4, using an inhibitor against the Bromo- and Extra-Terminal domain (BET) protein family.

Furthermore, we expanded our analysis on the MES population by focusing on the dynamics at the level of the cell surface. We reasoned that a more invasive phenotype should be accompanied by massive changes in the composition of the plasma membrane and in doing so, we would increase the resolution of our downstream analysis in regards to possible future clinical targets. Therefore we isolated plasma membranes from a panel of patient-derived melanoma cultures and performed unbiased quantitative lipidomic and proteomic analyses.

An enrichment in poly-unsaturated fatty acids in MES cultures was observed when compared their melanocytic counterparts, implying an increase in sensitivity to ferroptosis, as suggested by other reports in the field. MEL cultures showed high levels of pigment biosynthesis, while MES cultures showed an enrichment in proteins related to membrane remodeling and cell adhesion.

In summary, the work presented in this thesis, revealed the importance of the mesenchymal-like state in driving resistance to ICB and described its dynamics at transcriptomic, lipidomic, proteomic and molecular level.