Identifying States of Collateral Sensitivity during the Evolution of Therapeutic Resistance in Ewing's Sarcoma

Abstract

Advances in the treatment of Ewing's sarcoma (EWS) are desperately needed, particularly in the case of metastatic disease. A deeper understanding of collateral sensitivity, where the evolution of therapeutic resistance to one drug aligns with sensitivity to another drug, may improve our ability to effectively target this disease. For the first time in a solid tumor, we produced a temporal collateral sensitivity map that demonstrates the evolution of collateral sensitivity and resistance in EWS. We found that the evolution of collateral resistance was predictable with some drugs but had significant variation in response to other drugs. Using this map of temporal collateral sensitivity in EWS, we can see that the path toward collateral sensitivity is not always repeatable, nor is there always a clear trajectory toward resistance or sensitivity. Identifying transcriptomic changes that accompany these states of transient collateral sensitivity could improve treatment planning for patients with EWS.

Keywords: Biological Sciences; Cancer; Cancer Systems Biology; Evolutionary Biology.

Graphical abstract

Figure 1

Overview of Experimental Evolution of Resistance in Ewing’s Sarcoma Cell Lines As cells recovered from each exposure, cells were tested for their sensitivity to a panel of drugs and samples were frozen for potential use in RNA sequencing. The drug dosage was only increased once throughout the experiment, at the fifth exposure to the VDC combination, described in Transparent Methods. Although each cell line began with five experimental and three control evolutionary replicates, the A673 cell line lost one experimental replicate owing to contamination.

Figure 2

Temporal Collateral Sensitivity Map Representing EC50 Changes to a Panel of Drugs as the A673 Cell Line Develops Resistance to Standard Treatment Left: A heatmap representing how the EC50 to a panel of nine drugs changes in four A673 cell line evolutionary replicates as they are exposed to the VDC/EC drug combinations over time. Color represents the log₂ fold change of EC50 to a drug (columns) for a replicate at a given evolutionary time point (rows) compared with the average EC50 of the three control evolutionary replicates at the corresponding time point. Values above log₂(3) or below log₂(-3) are represented by log₂(3) and log₂(-3), respectively. Time points are denoted as the drug combination that a given replicate has recently recovered from. For example, the data representing dose-response models after the first application of the VDC drug combination would be labeled with VDC1. Of note, the EC50 of olaparib in Replicate 5 at the VDC5 time point is indeterminate owing to a poorly fit dose-response model. This value in the heatmap is denoted as gray, but Figure S1 remains uncensored. Right: Top, a plot of the dose-response curves for Replicate 3 and all control replicates (Replicates 6, 7, 8) in response to SP-2509 (SP) at the VDC4 time point. Bottom, a plot of the dose-response curve for Replicate 5 and all control replicates in response to dactinomycin at the VDC4 time point. Cellular activity is measured by enzymatic conversion of alamarBlue, normalized to background florescence. Estimated EC50 for each replicate is denoted with a red circle. These two dose-response plots demonstrate how the heatmap (left) values were calculated, where the control EC50 values are averaged, and the heatmap values represent the log₂ fold change between a given replicate and this mean EC50 value.

Figure 3

Point-Range Plots Demonstrating EC50 Changes in A673 Experimental and Control Replicates Over Time Bottom: Point-range plots representing the changes in drug response to a panel of nine drugs. Experimental time points (x axis) represent which step in the drug cycle the replicates have just recovered from. Points on the plot represent the average EC50 for the group, either experimental or control. Lines represent the range for the entire group. The EC50 of olaparib for Replicate 5 after the fifth exposure to VDC is indeterminate owing to a poorly fit dose-response model and has been removed from this drug's VDC5 time point experimental group EC50 average and range calculations. This value has not been censored in Figures S1 and S2. The y axis of all the point-range plots has μM units, except Cyclo, where the unit is percent of chemically activated 4-hydroxycyclophosphamide solution by volume. Top: Two plots demonstrating a more detailed view of the dose-response data represented at the EC3 and VDC5 time points in the Doxo point-range chart. Cellular activity is measured by enzymatic conversion of alamarBlue, normalized to background florescence. Comparing these two plots shows the clear divergence in drug response between experimental and control evolutionary replicates as the treatment regimen continued.

Figure 4

RNA Sequencing and Differential Gene Expression Analysis Provide Insight into States of Collateral Sensitivity and Resistance Left: The temporal collateral sensitivity map from Figure 2, where all samples that were not sequenced are overlayed with gray. Right: Two waterfall plots representing the samples ranked by their responses to the two drugs, vorinostat (SAHA, top) and SP-2509 (SP, bottom). Sample labels on the x axis are represented by darker colors the longer they have been evolved in the evolutionary experiment.