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ACUTE MYELOID LEUKEMIA

Resolving inter- and intra-patient heterogeneity in NPM1-mutated AML at single-cell resolution

Leukemia (2025)Cite this article

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Abstract

NPM1-mutated AML is one of the largest entities in international classification systems of myeloid neoplasms, which are based on integrating morphologic and clinical data with genomic data. Previous research, however, indicates that bulk transcriptomics-based subtyping may improve prognostication and therapy guidance. Here, we characterized the heterogeneity in NPM1-mutated AML by performing single-cell RNA-sequencing and spectral flow cytometry on 16 AML belonging to three distinct subtypes previously identified by bulk transcriptomics. Using single-cell expression profiling we generated a comprehensive atlas of NPM1-mutated AML, collectively reconstituting complete myelopoiesis. The three NPM1-mutated transcriptional subtypes showed consistent differences in the proportions of myeloid cell clusters with distinct patterns in lineage commitment and maturational arrest. In all samples, leukemic cells were detected across different myeloid cell clusters, indicating that NPM1-mutated AML are heavily skewed but not fully arrested in myelopoiesis. Same-sample multi-color spectral flow cytometry recapitulated these skewing patterns, indicating that the three NPM1-mutated subtypes can be consistently identified across platforms. Moreover, our analyses highlighted differences in the abundance of rare hematopoietic stem cells suggesting that skewing occurs early in myelopoiesis. To conclude, by harnessing single-cell RNA-sequencing and spectral flow cytometry, we provide a detailed description of three distinct and reproducible patterns in lineage skewing in NPM1-mutated AML that may have potential relevance for prognosis and treatment of patients with NPM1-mutated AML.

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Fig. 1: AML heterogeneity between NPM1 subtypes by single-cell RNA-sequencing.
Fig. 2: AML in NPM1(2) subtype resemble hematopoietic stem cells or early progenitor cells.
Fig. 3: AML heterogeneity between NPM1 subtypes by spectral flow cytometry.
Fig. 4: AML in NPM1(1) subtype contain healthy or preleukemic HSCs.

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Data availability

All single-cell RNA-sequencing data was submitted to EGA with the accession number EGAS50000000332, and they are accessible upon request (due to privacy rules). Processed version of the scRNA-seq data for all 16 NPM1-mutated AML samples were uploaded to Figshare under the https://doi.org/10.6084/m9.figshare.26189771.

Code availability

All source codes are available at https://github.com/eonurk/scNPM1. Our AML variant calling pipeline for single-cell RNA-seq data is also open-sourced and available under the same repository.

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Acknowledgements

Authors would like to thank Peter van Balen for his help on data acquisition and transfer, and Leon Mei and Davy Cats for their help on data upload. This study received financial support from a strategic investment from the Leiden University Medical Center, integrated within the Leiden Oncology Center and conducted within the Leiden Center for Computational Oncology, and partly by the Dutch Cancer Society (project number 15152). EBA was supported by a personal grant from the Dutch Research Council (NWO; VENI: 09150161810095). The funding entities played no part in determining the study’s design, data collection, analysis, interpretation, manuscript composition, or the decision to submit it for publication. BioRender icons were used in graphical abstract, Figs. 1a and 3a.

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Authors and Affiliations

  1. Department of Biomedical Data Sciences, Leiden University Medical Center, Leiden, The Netherlands

    E. Onur Karakaslar, Ramin Shirali HZ, Jeppe F. Severens, Marcel JT Reinders & Erik B. van den Akker

  2. Pattern Recognition & Bioinformatics, Delft University of Technology, Delft, The Netherlands

    E. Onur Karakaslar, Ramin Shirali HZ, Jeppe F. Severens, Marcel JT Reinders & Erik B. van den Akker

  3. Leiden Center for Computational Oncology, Leiden University Medical Center, Leiden, The Netherlands

    E. Onur Karakaslar, Ramin Shirali HZ, Jeppe F. Severens, Marcel JT Reinders & Erik B. van den Akker

  4. Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands

    Eva M. Argiro, Nadine E. Struckman, M. Willy Honders, Hendrik Veelken & Marieke Griffioen

  5. Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands

    Susan L. Kloet

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Contributions

EBA, MG, MJTR designed the project; EBA acquired funding; EMA, NES, MG, SK and HV helped with data generation and transfer; EOK, EMA performed computational and statistical analyses; EOK created all figures; EOK, RSHZ implemented variant calling pipeline; EOK, EMA, RSHZ, JFS, EBA, MG, and MJTR supported data exploration and interpretation; MJTR, MG and EBA provided supervision and scientific direction; EOK, EBA and MG wrote the manuscript; and all authors critically reviewed the manuscript and figures.

Corresponding authors

Correspondence to Marieke Griffioen or Erik B. van den Akker.

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The authors declare no competing interests.

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All methods were performed in accordance with the relevant guidelines and regulations, including the Declaration of Helsinki. Approval to use NPM1-mutated AML patient samples for this research was obtained from the Leiden University Medical Center (LUMC) Institutional Review Board (protocol no. RP24.056). Written informed consent to participate was obtained from all participants prior to sample collection.

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Karakaslar, E.O., Argiro, E.M., Struckman, N.E. et al. Resolving inter- and intra-patient heterogeneity in NPM1-mutated AML at single-cell resolution. Leukemia (2025). https://doi.org/10.1038/s41375-025-02745-w

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