Ewing's sarcoma (EWS) is the second most common primary malignant bone cancer in children. Advances in the treatment of 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 examine the repeatability of collateral sensitivity in EWS cell lines over time as evolutionary replicates evolve resistance to standard treatment. In doing so, we produced a temporal collateral sensitivity map that allows us to examine the evolution of collateral sensitivity and resistance in EWS. We found that the evolution of collateral sensitivity and resistance was predictable with some drugs, but had significant variation in response to other drugs. Samples that were most sensitive and most resistant to all drugs were compared using differential gene expression. Using this map of temporal collateral sensitivity in EWS, we can see that the path towards collateral sensitivity is not always repeatable, nor is there always a clear trajectory towards resistance or sensitivity. Identifying transcriptomic changes that accompany these states of transient collateral sensitivity could improve treatment planning for EWS patients. 2 50-70% 5-year survival rate, and metastatic disease has a devastating 18-30% 5-year survival rate. [2][3][4] Advances in the treatment 3 of EWS are desperately needed, particularly in the case of metastatic disease. Unfortunately, all recent attempts to improve 4 the chemotherapy regimen for EWS have only yielded modest results for non-metastatic cancer with little-to-no impact on 5 the course of metastatic disease. 3, 5 Researchers have tried adding ifosfamide and etoposide to standard EWS chemotherapy, 6 increasing the drug doses administered, and decreasing the interval between doses, all without meaningful improvement to 7 metastatic disease outcomes. 3, 5, 6 Even when treatment is initially successful, EWS often evolves therapeutic resistance, which 8 ultimately leads to disease relapse. 7 A deeper understanding of the evolutionary dynamics at play as EWS develops therapeutic 9 resistance may improve our ability to effectively target this disease.
10During the evolution of therapeutic resistance, both bacteria and cancer can exhibit a phenomenon termed collateral 11 sensitivity, where resistance to one drug aligns with sensitivity to another drug. 8-10 Likewise, collateral resistance occurs when 12 resistance to one drug aligns with resistance to another drug. The relationship between genotype (e.g. gene expression, somatic 13 mutations, etc.) and fitness of a cell line can be represented by a fitness landscape. In the case of drug response, we define 14 fitness as the EC50 of a cell line to a given drug, where increasing EC50 denotes higher fitness in the presence of this drug. Of 15 importance, a cell line with the same genotype may have varying...