Abstract PR17: Single-cell analysis reveals an adaptive, slowly-dividing, de-differentiated, drug-resistant cell state selectively inhibitable by drug combinations
Authors: Fallahi-Sichani Mohammad , Becker Verena , Izar Benjamin , Baker Gregory J. , Lin Jia-Ren , Boswell Sarah A. , Garraway Levi A. , Sorger Peter K. 
Published: December 2017

Abstract
Partial responsiveness of tumor cells due to drug adaptation (or tolerance) during the early phase of treatment with targeted therapeutics seems to be essential for creating a population of viable tumor cells from which resistant clones eventually arise. Thus, understanding transient drug adaptation is likely to be important for both improving the initial effectiveness of treatment and delaying or controlling acquired resistance. Despite the wealth of information available about the molecular events and feedback mechanisms leading to drug tolerance or adaptation, most of our knowledge in this area comes from studying drug response in bulk tumor cell populations. Furthermore, the phenotypic consequences of drug adaptation have been studied most frequently at a few fixed time-points, when drug-adapted cells exhibit a high population-average activity in multiple pro-growth or pro-survival signaling cascades. Therefore, it remains unclear how uniform or heterogeneous the early drug adaptation is across individual cells within a tumor cell population, and how the fate of drug-adapted cells is determined by a diversity of early drug-induced adaptive signaling responses. Uncovering the evolution of biochemical and phenotypic heterogeneity in drug-adapted cell populations is key to designing optimal combinations of drugs to overcome resistance and to achieve durable therapy.
In this study, we monitor the responses of BRAFV600E melanoma cells to RAF/MEK inhibitors at the single-cell level in real time using time-lapse live-cell imaging, and then analyze the resulting cell states using transcriptional, biochemical and phenotypic profiling. We found that exposure of tumor cells to RAF/MEK inhibitors elicits heterogeneous and time-variable responses in which some cells die, some arrest and a fraction of slowly-cycling cells adapts to drug, adopting a reversible drug-resistance phenotype characterized by up-regulation of markers of neural crest, a melanocyte precursor, including NGFR (the low affinity nerve growth factor receptor, also known as p75NTR or CD271). The slowly-cycling NFGRHigh state induced by RAF/MEK inhibitors is only transiently stable: after 1-2 weeks of outgrowth in drug-free medium, such cells reset to their initial state as measured by the restoration of RAF/MEK inhibitor sensitivity, accelerated rate of cell division and reduced expression of NGFR. Transcriptional and biochemical profiling of cell lines and human tumors implicates a role for the c-Jun/ECM/FAK/Src cascade in driving the de-differentiated (NGFRHigh) resistance program. We identify multiple drugs targeting this cascade as well as BET bromodomain inhibitors that block the slowly-cycling NGFRHigh state in cell lines and in a BRAFV600E melanoma xenograft model and increase sensitivity to RAF/MEK inhibitors. Our study reveals directly how drug adaptation happens in individual tumor cells leading to emergence of heterogeneous cell sub-populations with reduced drug-sensitivity that may be selectively targeted by drug combinations.
Citation Format: Mohammad Fallahi-Sichani, Verena Becker, Benjamin Izar, Gregory J. Baker, Jia-Ren Lin, Sarah A. Boswell, Levi A. Garraway, Peter K. Sorger. Single-cell analysis reveals an adaptive, slowly-dividing, de-differentiated, drug-resistant cell state selectively inhibitable by drug combinations [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr PR17.