Single-cell trajectory analysis reveals a CD9 positive state to contribute to exit from stem cell-like and embryonic diapause states and transit to drug-resistant states

Bromo- and extra-terminal domain (BET) inhibitors (BETi) have been shown to decrease tumor growth in preclinical models and clinical trials. However, toxicity and rapid emergence of resistance have limited their clinical implementation. To identify state changes underlying acquisition of resistance to the JQ1 BETi, we reanalyzed single-cell RNAseq data from JQ1 sensitive and resistant SUM149 and SUM159 triple-negative breast cancer cell lines. Parental and JQ1-resistant SUM149 and SUM159 exhibited a stem cell-like and embryonic diapause (SCLED) cell state as well as a transitional cell state between the SCLED state that is present in both treatment naïve and JQ1 treated cells, and a number of JQ1 resistant cell states. A transitional cell state transcriptional signature but not a SCLED state transcriptional signature predicted worsened outcomes in basal-like breast cancer patients suggesting that transit from the SCLED state to drug-resistant states contributes to patient outcomes. Entry of SUM149 and SUM159 into the transitional cell state was characterized by elevated expression of the CD9 tetraspanin. Knockdown or inhibition of CD9-sensitized cells to multiple targeted and cytotoxic drugs in vitro. Importantly, CD9 knockdown or blockade sensitized SUM149 to JQ1 in vivo by trapping cells in the SCLED state and limiting transit to resistant cell states. Thus, CD9 appears to be critical for the transition from a SCLED state into treatment-resistant cell states and warrants exploration as a therapeutic target in basal-like breast cancer.