Abstract Human cancers can exhibit phenotype switching, resulting in cells that are more metastatic or that are more tolerant to treatment. However, the relationship between these aggressive states is not well understood. We investigated the dynamics of phenotypic switching in human melanoma cells by monitoring the fluorescent activity from a transgenic reporter of BRN2 promoter activation. Melanoma cells exhibit heterogeneous BRN2 expression patterns that are reestablished upon isolation and clonal outgrowth. Specifically, stable BRN2 expression was generally inherited over multiple generations while undergoing occasional bidirectional interconversion. We found that BRN2 low cells were required for tumor initiation and metastasis in animal engraftment assays but were more sensitive to targeted BRAF inhibition. In contrast, the BRN2 high state was not tumorigenic, but entry into this state was uniform and persistent among cells tolerant to targeted BRAF therapy. Single-cell RNA sequencing analyses revealed core programs exclusive to either the BRN2 high or low cells, each of which is present in ex vivo tumors, demonstrating the physiological relevance of these states. Our findings emphasize that one challenge of effectively targeting phenotype switching in melanoma as a therapeutic strategy could be balancing distinct aggressive phenotypes so that sensitizing tumors to BRAF inhibition does not inadvertently lead to further dissemination. Teaser Unraveling melanoma’s shape-shifting behavior: insights into how cancer cells swap between metastasis and drug evasion.

Bidirectional interconversion between mutually exclusive tumorigenic and drug-tolerant melanoma cell phenotypes