Abstract B019: Does order matter? Dissecting the single-cell differences underlying resistance to sequential drug treatment
Authors: Fasse Aria J. , Schaff Dylan L. , Shaffer Sydney M. 
Published: May 2022

Most cancer patients are given multiple different drugs during the course of their cancer treatment. An important but outstanding question is the role that drug order plays in the development of resistance to sequential drug treatment– that is, if cells that develop resistance to a certain first and second-line therapy are similar to cells that develop resistance to the same drugs given in the opposite order. During treatment with a first drug, both selection and transcriptional reprogramming events alter the cells within a tumor. As such, surviving cells may exhibit increased sensitivity or increased resistance to future drug therapies. Here, we zoom in to the single-cell level using cell barcoding and sequencing and explicitly test whether the order of treatment leads to different populations of cells developing resistance to a panel of different therapies. As the initial application of drug therapy changes a tumor in multiple ways, it is currently unclear which changes are most influential in the overall modification of a tumor’s sensitivity to a second-line therapy. One hypothesis is that changes in sensitivity are due to specific drug-induced modifications in the characteristics of cells that survived the first-line therapy, in which case order may matter. An alternative hypothesis is that changes in sensitivity are simply the result of a decrease in clonal diversity within the tumor, where the ability of individual subpopulations to survive the second-line therapy is unchanged by the first-line treatment, in which case order may not matter. To distinguish between these hypotheses, we have combined single-cell RNA sequencing with cellular barcoding to assess both the survival and transcriptome of cells from over 100,000 cells over the course of treatment with multiple stressors. These stressors include a combination of targeted inhibitors (Dabrafenib and Trametinib), chemotherapy (Cisplatin), and hypoxic stress (CoCl2). Through analysis of the transcriptome of cells within the same lineage after surviving each therapy in the sequential treatment scheme, we are now working to determine whether development of resistance to a first-line therapy causes individual cells within a lineage to change in ways that alter their sensitivity to subsequent therapies. Should we find that developing resistance to first-line therapy does increase resistance to specific second-line therapies, we will use our sequencing data to identify specific transcriptomic changes which lead to this induced resistance. We will then look to block this induced resistance through either small-molecule inhibitors (if available) or CRISPR-mediated knockout of genes that promote resistance to second-line therapies. Overall, this study will provide two methods for increasing the efficacy of sequential drug treatment through both the identification of ideal drug ordering and the targeting of changes that promote induced resistance, thus leading to resistance-free treatment strategies.

Citation Format: Aria J. Fasse, Dylan L. Schaff, Sydney M. Shaffer. Does order matter? Dissecting the single-cell differences underlying resistance to sequential drug treatment [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr B019.