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Chemotherapy is curative for most children with acute lymphoblastic leukemia (ALL). Here we provide direct evidence that thiopurine chemotherapeutics can also directly induce drug resistance mutations leading to relapse. Using a large relapsed ALL cohort assembled from Chinese, US and German patients, we found that TP53 R248Q mutations were highly enriched at relapse compared to diagnosis. Relapse-specific TP53 R248Q was associated with the acquisition of MMR deficiency mutations in MSH2, MSH6, or PMS2 and a novel relapse-specific mutational signature. Using isogenic MCF10A cells with or without engineered MSH2 knockout, and the Nalm6 ALL cell line which has native MMR deficiency, we found that this novel signature was caused by a synergistic mutagenic interaction between thiopurine treatment and mismatch repair (MMR) deficiency (called the thio-dMMR signature) that contributes to a hypermutator phenotype and acquisition of TP53 R248Q in residual ALL during remission. Treatment-induced TP53-mutant clones then expand due to broad chemoresistance, leading to eventual relapse. Indeed, thiopurines preferentially induced C>T mutations at the center of NCG trinucleotides, which can lead to TP53 R248Q, and the thiopurine mutation rate was accelerated 2- to 10-fold in MMR-deficient ALL and cell lines. Thiopurine treatment induced C>T mutations preferentially on the transcribed strand, rather than the untranscribed strand, of mRNAs, which further increased the likelihood of TP53 R248Q induction. Further, experimental thiopurine treatment was able to directly induce TP53 R248Q variants in MMR-deficient cultured cells, including Nalm6 and MCF10A MSH2-/-, by activating the thio-dMMR mutational signature, while MMR-proficient MCF10A cells did not experience R248Q induction. The sequential acquisition of MMR deficiency mutations, followed by TP53 mutations, during post-diagnosis ALL evolution was supported by clonal evolution analysis of serial patient samples. p53 R248Q promoted resistance to multiple ALL chemotherapeutic agents, and was associated with on-treatment relapse and poor relapse-treatment response. Our findings indicate that the enrichment of TP53 R248Q in relapsed ALL is due to synergistic mutagenesis from thiopurine treatment and MMR deficiency, followed by selection for TP53 R248Q's chemoresistance phenotype. This suggests that cancer drug resistance mutations may not always pre-exist subclonally at diagnosis, but may be therapy-induced in some patients. Additionally, the qualitative and quantitative mutational signature output of a mutagen (e.g., thiopurines) can vary based on the genetic background. Finally, our findings suggest potential therapeutic strategies, including avoiding thiopurine treatment in MMR-deficient relapses, and therapeutic p53 mutant reactivation, to deal with this genetically-unstable, chemoresistant disease. Citation Format: Fan Yang, Samuel W. Brady, Huiying Sun, Chao Tang, Lijuan Du, Malwine Barz, Xiaotu Ma, Yao Chen, Houshun Fang, Xiaomeng Li, Pandurang Kolekar, Omkar Pathak, Jiaoyang Cai, Lixia Ding, Tianyi Wang, Arend von Stackelberg, Shuhong Shen, Caiwen Duan, Cornelia Eckert, Hongzhuan Chen, Yu Liu, Jeffery M. Klco, Hui Li, Benshang Li, Jinghui Zhang, Renate Kirschner-Schwabe, Bin-Bing S. Zhou. Thiopurines and mismatch repair deficiency cooperate to fuel TP53 mutagenesis and ALL relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 633.