Ex vivo drug response profiling is emerging as a valuable tool for identifying drug resistance mechanisms and advancing precision medicine in hematological cancers. However, the functional impact of dysregulation of the epigenome and transcriptome in this context remains poorly understood. In this study, we combined ex vivo drug sensitivity profiling with transcriptomic and epigenomic analyses in diagnostic samples from 597 pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients. Ex vivo resistance to antimetabolites (e.g., cytarabine, thioguanine), glucocorticoids (e.g., dexamethasone, prednisolone), and doxorubicin was independently associated with reduced relapse-free survival (RFS, p < 0.05). Molecular profiling identified pre-treatment DNA methylation and gene expression patterns distinguishing resistant from sensitive cases, revealing key drug resistance signatures. These included aberrant expression of genes related to heme metabolism (e.g. ATPV06A) and KRAS signaling (e.g. ABCB1). Notably, we also observed atypical expression of genes usually restricted to T-cells and other immune cells (e.g., ITK) in resistant BCP-ALL cells. Our findings demonstrate that ex vivo drug response patterns are predictive of clinical outcomes and reflect intrinsic molecular states associated with drug tolerance. This integrative multi-omics approach highlights potential therapeutic targets and underscores the value of functional precision medicine in identifying treatment vulnerabilities in pediatric ALL.

Key points

Ex vivo drug response profiling provides predictors of outcome that complement standard clinical risk stratification, identifying high-risk phenotypes not captured by traditional methods.

Pre-treatment transcriptional and epigenetic profiles reveal resistance-primed molecular states detectable at diagnosis, mechanisms driving treatment resistance and offer potential therapeutic targets to counteract drug tolerance.

The authors have declared no competing interest.

This work was supported by grants from the Swedish Research Council (2019-01976 to JN), the Swedish Cancer Society (CAN2022-2395 to JN), the Swedish Childhood Cancer Fund (PR2022-0082 and HFT2023-0011 to JN, TJ2020-0039 to AH), and the Göran Gustafssons Foundation (to JN).

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The study was approved by the Regional Ethical Review Board in Uppsala, Sweden, and was conducted according to the guidelines of the Declaration of Helsinki.

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Email addresses of co-authors: Anna Pia Enblad: anna_pia.enbladuu.se, Olga Krali: olga.kralimedsci.uu.se, Henrik Gezelius: henrik.gezeliusmedsci.uu.se, Anders Lundmark: anders.lundmarkmedsci.uu.se, Kristin Blom: kristin.blommedsci.uu.se, Claes Andersson: claes.anderssonmedsci.uu.se, Josefine Palle: josefine.palleuu.se, Britt-Marie Frost: britt_marie.frostuu.se, Samppa Ryhänen: samppa.ryhanenhelsinki.fi, Trond Flægstad: trond.flaegstadunn.no, Ólafur G Jónsson: olafurgilandspitali.is, Kjeld Schmiegelow: kjeld.Schmiegelowregionh.dk, Mats Heyman: mats.heymanki.se, Arja Harila: arja.harilauu.se, Peter Nygren: peter.nygrenigp.uu.se, Rolf Larsson: rolf.larssonmedsci.uu.se, Gudmar Lönnerholm: gudmar.lonnerholmuu.se

The DNA methylation data were available from their original studies and the metadata are available at the Gene Expression Omnibus (GEO) under accession number GSE49031, and under controlled access via 10.17044/scilifelab.22303531 and 10.17044/scilifelab.26096371 (https://figshare.scilifelab.se/). The gene expression data are available under GSE227832. Data requests may be submitted to Jessica Nordlund (jessica.nordlundmedsci.uu.se).