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IRE1α–XBP1 safeguards hematopoietic stem and progenitor cells by restricting pro-leukemogenic gene programs

Nature Immunology volume 26, pages 200–214 (2025)Cite this article

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Abstract

Hematopoietic stem cells must mitigate myriad stressors throughout their lifetime to ensure normal blood cell generation. Here, we uncover unfolded protein response stress sensor inositol-requiring enzyme-1α (IRE1α) signaling in hematopoietic stem and progenitor cells (HSPCs) as a safeguard against myeloid leukemogenesis. Activated in part by an NADPH oxidase-2 mechanism, IRE1α-induced X-box binding protein-1 (XBP1) mediated repression of pro-leukemogenic programs exemplified by the Wnt–β-catenin pathway. Transcriptome analysis and genome-wide mapping of XBP1 targets in HSPCs identified an ‘18-gene signature’ of XBP1-repressed β-catenin targets that were highly expressed in acute myeloid leukemia (AML) cases with worse prognosis. Accordingly, IRE1α deficiency cooperated with a myeloproliferative oncogene in HSPCs to cause a lethal AML in mice, while genetic induction of XBP1 suppressed the leukemia stem cell program and activity of patient-derived AML cells. Thus, IRE1α–XBP1 signaling safeguards the integrity of the blood system by restricting pro-leukemogenic programs in HSPCs.

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Fig. 1: Dynamics of IRE1α–XBP1 activity in adult BM progenitors.
Fig. 2: Genetic inactivation of IRE1α in the hematopoietic system cooperates with Flt3ITD to cause a lethal AML in mice.
Fig. 3: Hematopoietic cell-intrinsic activity of IRE1α in adult mice.
Fig. 4: IRE1α deficiency disrupts Flt3ITD BM hematopoiesis and confers AML-initiating potential to restricted Flt3ITD-expressing HSPCs.
Fig. 5: IRE1α deficiency enriches a pro-leukemogenic gene program in HSPCs.
Fig. 6: XBP1 directly binds to TCF/LEF proteins and represses β-catenin transcriptional activity.
Fig. 7: Significance of IRE1α–XBP1 signaling in AML pathogenesis.
Fig. 8: XBP1 represses pro-LSC gene programs and activity of patient-derived AML cells.

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Data availability

All sequencing data from RNA-seq and ChIP–seq have been deposited in the Gene Expression Omnibus database under accession numbers GSE163136 and GSE163137 (superseries accession number GSE163192) and are publicly available without restriction. All requests for resources and reagents reported in this study should be directed to and will be fulfilled by S.A. upon completion of the appropriate material transfer agreement. Source data are provided with this paper.

Code availability

All original codes for XBP1 splicing detection can be found at https://github.com/akv3001/RNASeq-Based-XBP1s-Detection-.

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Acknowledgements

We thank M. Carroll for critical reading of the manuscript and suggestions, D. Wald for primary human AML samples, T. Iwawaki for ERAI transgenic and Ern1fl/fl mice and I. Aifantis for HPC-7 cells. We thank the Cytometry & Imaging Microscopy Shared Resource of the Case Comprehensive Cancer Center for cell sorting and the Epigenomics Core and the Genomics Resources Core at Weill Cornell Medicine for RNA and DNA sequencing services. We acknowledge use of the Leica TCS SP8 confocal microscope at the Case Western Reserve University Microscopy Imaging Core funded by the National Institutes of Health (NIH) Office of Research Infrastructure Shared Instrumentation grant S10OD024996. This work was funded in part by a NCI Career Development Award (K22 CA 218467), an American Cancer Society Research Scholar Grant (RSG-19-025-01-DDC); the Intramural Research Program of the NIH, NCI, Center for Cancer Research (ZIA BC 012135) to S.A.; and Institutional Funding from The Dana-Farber Cancer Institute (to L.H.G.). B.M.B. was supported by Medical Scientist Training Program NIGMS T32 GM007250 and Immunology Training Program NIAID T32 AI089474 grants. K.U.-W. was supported by a Cell and Molecular Biology Training grant NIGMS T32 GM008056. A.M.M. is funded by NCI UG1 CA233332, NCI R01 CA198089 and LLS SCOR 7013-17. X.C. is funded by NIH R01HL146642 and R37CA228304 and a DOD/CDMRP award W81XWH1910524. Primary human AML samples were from the Hematopoietic Biorepository and Cellular Therapy Shared Resource of the Case Comprehensive Cancer Center supported by NCI P30CA043703.

Author information

Author notes

  1. Jin Cao

    Present address: MOE Key Laboratory of Biosystems Homeostasis & Protection and Zhejiang Key Laboratory of Molecular Cancer Biology, Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou, China

  2. These authors contributed equally: Brendan M. Barton, Francheska Son, Akanksha Verma.

Authors and Affiliations

  1. Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

    Brendan M. Barton, Katharine Umphred-Wilson, Josephine Trichka & Stanley Adoro

  2. Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH, USA

    Brendan M. Barton, Francheska Son, Saswat Kumar Bal, Katharine Umphred-Wilson, Rehan Khan & Josephine Trichka

  3. Institute of Computational Biomedicine, Caryl and Israel Englander Institute for Precision Medicine, Weill Cornell Medicine, New York, NY, USA

    Akanksha Verma & Olivier Elemento

  4. College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, China

    Qianzi Tang & Rui Wang

  5. Department of Cancer Immunology and Virology, Dana Farber Cancer Institute, Boston, MA, USA

    Han Dong, Claudia Lentucci & Laurie H. Glimcher

  6. Weill Cornell Graduate School of Medical Sciences, New York, NY, USA

    Xi Chen

  7. Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH, USA

    Yinghua Chen

  8. Department of Biochemistry and Chemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia

    Yuning Hong

  9. Cancer Signaling and Epigenetics Program, Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA, USA

    Cihangir Duy

  10. Department of Medicine, Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, NY, USA

    Ari M. Melnick

  11. Department of Experimental Therapeutics, James P. Allison Institute, MD Anderson Cancer Center, Houston, TX, USA

    Jin Cao & Xi Chen

  12. Department of Medicine, Harvard Medical School, Boston, MA, USA

    Laurie H. Glimcher

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Contributions

Conceptualization: B.M.B., L.H.G. and S.A. Data acquisition: S.A., B.M.B., F.S. and S.K.B. assisted by H.D., C.L., K.U.-W., R.K., J.T. and X.C. Data analysis: S.A., B.M.B., F.S., S.K.B. and L.H.G. ChIP–seq: J.C. and X.C. Bioinformatics: A.V. and O.E. AML dataset analysis: R.W. and Q.T. Surface plasmon resonance: Y.C. Patient-derived AML samples: C.D. and A.M.M. Key reagent: Y.H. Writing: B.M.B., L.H.G. and S.A. with input from all authors. Supervision: S.A. and L.H.G. Funding: S.A. and L.H.G.

Corresponding author

Correspondence to Stanley Adoro.

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Competing interests

L.H.G. is a former Director of Bristol Myers Squibb and the Waters Corporation and currently serves on the Board of Directors of GlaxoSmithKline Pharmaceuticals and Analog Devices. L.H.G. also serves on the scientific advisory boards of Repare Therapeutics, Abpro Therapeutics and Kaleido Therapeutics. A.M.M. receives research funding from Janssen, Daiichi Sankyo and Sanofi; has consulted for Epizyme, Constellation, BMI and Exo-Therapeutics; and is a scientific advisor to KDAC. A.V. is a current employee of Volastra Therapeutics. All other authors declare no competing interests.

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Peer review information

Nature Immunology thanks Maria Carolina Florian, Stephanie Xie and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: L. A. Dempsey, in collaboration with the Nature Immunology team.

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Extended data

Extended Data Fig. 1 Cell sorting gates and dynamics of IRE1α-XBP1 activity and expression in normal bone marrow hematopoietic stem and progenitor cells.

(a and b) Representative flow cytometry gates used for the identification and sorting of mouse (a) and human (b) hematopoietic stem and progenitor cells (HSPC). Human HSPC populations were defined follows: HSC, Lineage (Lin)-CD34+CD38-CD45RA-CD90+CD49f+; MPP, Lin-CD34+CD38-CD45RA-CD90-CD49f-; MLP, Lin-CD34+CD38-CD90-CD45RA+; CMP, Lin-CD34+CD38+CD10-CD45RA-Flt3+; GMP, Lin-CD34+CD38+CD10-CD45RA+Flt3+; MEP, Lin-CD34+CD38+CD10-CD45RA-Flt3-. Mouse HSPCs were defined as follows: HSC, Lin-c-Kit+Sca1+CD150+CD48-; MPP, Lin-c-Kit+Sca1+CD150-CD48-; HPC, Lin-c-Kit+Sca1+CD150-CD48+; CMP, Lin-c-Kit+Sca1-CD34+CD16/32-; GMP, Lin-c-Kit+Sca1-CD34+CD16/32+; MEP, Lin-c-Kit+Sca1-CD34-CD16/32-. (c-e) Gene expression in mouse HSPC subsets and GMPs determined by qRT-PCR. Expression of Ern1, Xbp1, and Xbp1s is normalized to Actb. Data are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ns, not significant; Students t-Test. (f) Relative (±s.e.m., normalized to HSC) XBP1S/XBP1 ratio in human BM progenitor and mature CD14+ cells sorted from BM mononuclear fraction (N = 6 donors). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant; Students t-Test. (g) Flow cytometry detection of reactive oxygen species (ROS) by dihydroethidium (DHE) staining in total bone marrow (BM) c-Kit+ cells, LSK and GMP cells from Cybb+/+ and Cybb-/- mice. Unshaded histograms represent unstained control sample. Graphs show relative (average ± S.D., N = 4 mice/group) DHE mean fluorescence intensity (MFI). Mice were 3-4 months old mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001; Students t-Test. (h) Unfolded proteins were measured by staining with the thiol probe tetraphenylethene maleimide (TPE-MI). Data show average (± S.D., N = 5 mice/group) TPE-MI MFI in age-matched 3-4 months old mice. *, P < 0.05; ns, not significant; Students t-Test.

Source data

Extended Data Fig. 2 Effect of IRE1α-XBP1 inactivation on normal mouse and human bone marrow hematopoietic progenitor cell generation and function.

(a) Xbp1s/Xbp1 total gene expression (±s.e.m., N = 4 mice/group) in purified LSK and GMP cells. (b-e) Representative flow cytometry of Lin- and LSK cells (b,d) and average (± S.E.M.) proportions (c,e) of BM progenitor cell subsets in adult (3-4 months) mice with HSC-targeted (Vav-iCre) deletion of Ern1 (N = 4 mice/group) (b and c) or Xbp1 (N = 3 mice/group) (d and e) and age-matched littermate controls. Mouse genotypes (a-e): Ern1wt, Vav-iCre-Ern1fl/fl; Ern1ko, Vav-iCre+Ern1fl/fl; Xbp1wt, Vav-iCre-Xbp1fl/fl; Xbp1ko, Vav-iCre+Xbp1fl/fl. Each data point represents one mouse. ns, not significant; unpaired Students t-Test. (f-i) Effect of IRE1α inhibition by the small molecule inhibitor MKC8866 (8866) on purified human HSC activity from two healthy donors. For colony-forming unit (CFU) assay, 1 ×103 FACS-purified HSCs were cultured in MethoCult H4434 (STEMCELL Technologies) supplemented with vehicle (DMSO) or MKC8866 at a final concentration of 10 mm. Data show qRT-PCR-determined XBP1S/XBP1 gene expression ratio (f), representative pictures of CFU assay wells (g), total CFU counts in duplicates wells (h) and enumeration of progenitor cell type (i) after 14 days. Data in f,h,i are average (± S.E.M.) of duplicate wells and the results were reproducible in the two donors.

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Extended Data Fig. 3 Transcriptome and gene expression analysis of IRE1α (Ern1)-deficient bone marrow HSPCs.

(a) Principal component analysis (PCA) of differentially expressed genes (DEG) determined by RNA-seq of Ern1wt (Vav-iCre-Ern1fl/fl) and Ern1ko (Vav-iCre+Ern1fl/fl) LSK cells. N = 3 mice/group. (b) Heatmap of canonical IRE1α-induced XBP1 target genes in the transcriptome of LSK and GMP cells within the same mice. N = 3 mice/group. (c and d) Expression of the indicated unfolded protein response (UPR) and ER-associated degradation (ERAD) pathway genes genes in LSK (c) and GMP (d) cells from Ern1wt and Ern1ko mice (N = 4 mice/group). (e) Ex vivo cell cycle analysis of LSK cells from wildtype Flt3 (that is, Flt3+/+) Ern1wt (N = 4) and Ern1ko (N = 3) determined by Ki-67 and DAPI staining. Data show average (± S.E.M.) proportion of cells in G0, G1 and S/G2/M phase. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant; unpaired, two-tailed Students t-test.

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Extended Data Fig. 4 Characterization of IRE1α-XBP1 deficient mice and HSPCs expressing Flt3ITD/ITD at 4 weeks.

(a) Representative flow cytometry of Flt3 expression on mouse HSPC subsets. (b) Semi-quantitative PCR of Xbp1 splicing in LSK and GMP cells from the indicated mice. Ratio of Xbp1s to Xbp1u is indicated below each lane. (c) Kaplan-Meier survival curve of Ern1wt (N = 14) and Ern1ko (N = 11) mice expressing Flt3ITD/ITD. P < 0.0001, Log-rank (Mantel-Cox) test. (d-g) Histopathology of experimental animals with the indicated genotypes: representative femurs showing pale bones (d), white blood cell counts (± S.D.) and hematocrit (e), and Giemsa-stained whole blood (f). Inset in panel f, zoomed-in boxed regions), images and average (± S.D.) weights of spleens. Statistics: each data point represents one mouse (N = 8-10 mice/group). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; unpaired Student’s t-Test comparison between Ern1wt and Ern1ko genotypes. (h-k) Immunophenotyping of spleens from experimental mice. Moribund Ern1koFlt3ITD/ITD mice were euthanized at median survival time-point (4-weeks) along with age-matched control animals. Data show representative flow cytometry plot of c-Kit and CD11b expression (h), CD34 expression on c-Kit+CD11b- (i) and average (± S.D., N = 9 mice/group) splenocyte numbers (j) and proportions (± S.E.M.; N = 4 mice/group) of the indicated splenocyte populations (k). (l) Representative hematoxylin and eosin-stained tissue sections. (m-q) Characterization of Xbp1wt and Xbp1ko mice expressing Flt3ITD/ITD at 4 weeks. Kaplan-Meier survival curve (m; P < 0.0072, Log-rank (Mantel-Cox) test, N = 5 mice/group), white blood cell counts (n), Giemsa-stained whole blood (o; inset: detail of boxed region), representative flow cytometry plot of Gr-1 and CD11b expression (p) and average (± S.D., N = 4 mice/group) numbers of CD11b+ splenocytes (q) in four weeks-old animals with Xbp1-deficient HSPCs. Statistics *, P < 0.05, **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant; Student’s t-test. Scale bar (f and o): 20 mm.

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Extended Data Fig. 5 IRE1α-deficiency promotes AML with a bone marrow origin via a cell autonomous mechanism.

(a) White blood cell count (WBC, ± S.D., N = 11-12 mice/group) of IRE1α-sufficient (“wt”, Mx1Cre-Ern1fl/fl.Flt3ITD/+ or Mx1Cre+Ern1+/+.Flt3ITD/+) or IRE1α-deficient (Mx1Cre+Ern1fl/fl.Flt3ITD/+) animals at six months. Data points represent individual mice; *, P < 0.05; unpaired Student’s t-test. (b-c) Validation of Mx1Cre activity in Mx1Cre.Ern1fl/fl.Flt3ITD/+ mice harboring dual reporter membrane TdTomato (mT) and membrane GFP (mGFP) proteins from the Rosa26 (R26) locus in which Cre activity activates mGFP and inactivates TdTomato (R26LSLmT/mG) allele expression. Flow cytometry analysis was used to identify Mx1Cre-expressing (GFP+) c-Kit+ BM cells (b) for cell sorting and analysis. Consistent with loss of IRE1α (Ern1), Xbp1s-to-Xbp1 total ratio is diminished in c-Kit+GFP+ BM cells from Mx1Cre+Ern1fl/fl.Flt3ITD/+R26LSLmT/mG mice. **, P < 0.01; ***, P < 0.001; ns, not significant; Student’s t-test. (d) Representative flow cytometry plots of CD11b expression on GFP- and GFP+ splenocytes from IRE1α-sufficient (Mx1Cre+Ern1+/+.Flt3ITD/+R26LSLmT/mG) and IRE1α-deficient (Mx1Cre+Ern1fl/fl.Flt3ITD/+R26LSLmT/mG) mice at six months of age. (e) Representative CD48 vs CD150 expression on LSK cells from the indicated Flt3ITD/+ mice. (f-h) Representative flow cytometry plots of c-Kit vs Sca-1 expression on Lineagenegative (Lin-) BM cells (f), CD48 vs CD150 expression on LSK cells (g) and CD16/32 vs CD34 expression on Lin-Sca-1-c-Kit+ BM cells (h) from 4-weeks old Ern1wt and Ern1ko mice expressing Flt3ITD/ITD. Average (± S.E.M., N = 8-9 mice/group) BM cell numbers are indicated in panel f. (i and j) Average (± S.E.M.) proportion of BM Lin-c-Kit+ and Lin+ cells (i) and box-whiskers plot (represents min and max values) of the indicated BM progenitor cell fractions (j). N = 6-9 mice per group. **, P < 0.01; ***, P < 0.001; ns, not significant; unpaired Student’s t-test. (k) Chromosome karyotypes of bone marrow metaphase leukemic blast cells. Data are representative of two independent experiments.

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Extended Data Fig. 6 IRE1α-deficiency enriches a pro-leukemogenic transcription program exemplified by the Wnt/b-catenin pathway in HSPCs.

(a) Principal component analysis (PCA) of differentially expressed genes in LSK cells from non-Flt3ITD/ITD and Flt3ITD/ITD-expressing Ern1wt and Ern1ko mice (N = 3 mice per group). (b) White blood cell counts in experimental mice treated after 7-days of daily treatment with 100 mg/kg of the MAPK inhibitor CI-1040 (“MAPKi”) or vehicle. Data points represent individual mice (N = 5-8 mice/group; error bar, ± S.E.M); ns, not-significant, multiple Student’s t-test. (c) Average (± S.D.) expression of indicated genes relative to Actb. Each data point represents a mouse, N = 3 to 4 mice/group. (d and e) GSEA plot (d) and heatmap (e) of KEGG annotated Wnt/b-catenin signaling pathway genes in wildtype Flt3 (that is, Flt3+/+) LSK cells. (f) Effect of XBP1 on an ER stress responsive promoter element (ERSE)-driven luciferase reporter in HEK293T cells. (g and h) Comparison of XBP1 (encoded by Xbp1s) and Xbp1u-encoded products (XBP1u and XBP1(kkk), a more stable XBP1u protein) schematized in (g) on TOPFLASH reporter induced by a constitutively stable b-cateninS33Y co-transfected into transfected into HEK293T cells. Data are average ( ± S.D.) relative luciferase units (RLU) of duplicate wells. (i) Average (± S.D.) of Ctnnb1 gene expression in BM c-Kit+ cells from two each of the indicated mice. (j) Dnajb9, Tet2 and Dnmt3a expression shown as average (± S.D., N = 3 mice/group) normalized to Actb in LSK cells from the indicated mice. Data in f and h are representative of three independent experiments. Statistics (c and j): *, P < 0.05; **, P < 0.01; ns, not-significant, unpaired, two-tailed Student’s t-test.

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Extended Data Fig. 7 Structure-function analysis of XBP1 interaction with proteins of the b-catenin signaling pathway.

(a-b) Assessment of XBP1 interaction with b-catenin (a) or TLE1(L) (long isoform, L) (b) by co-immunoprecipitation (co-IP) in HEK293T cells. *, residual band from TCF4 immunoblot after membrane stripping. (c) Co-IP between XBP1 and TCF4 in HEK293T cells. HIF1a,which was previously shown to bind XBP1, was used as a positive control. d) Representative confocal microscopy images of LSK cells from Ern1+/+Flt3ITD/+ or IRE1α-deficient (Ern1koFlt3ITD/+) mice stained (red) with XBP1 antibody (clone 9D11A43; BioLegend). Nuclear DNA (green) was counter-stained with Hoechst dye. Outline of cell membrane are highlighted in the merged images. Scale bar: 25 mm. (e-g) Co-IP of XBP1 and truncation mutants of TCF4 protein (e). Whereas XBP1 co-immunoprecipitated with the HMG/DNA binding ___domain of TCF4 (residues 349-496) alone (f, lane 6), it failed to interact with a C-terminus truncated TCF4 (residues 1-356) protein (f, lane 7; g, lane 4). *, non-specific band. (h) Hypothetical model of XBP1-mediated repression of b-catenin-driven TCF/LEF transcriptional activity. TCF/LEF factors are constitutively repressed by TLE proteins (i) but become activated when b-catenin displaces TLE1 from its binding to TCF/LEF proteins downstream of canonical Wnt ligands or growth factor signaling (ii). XBP1-mediated suppression is triggered upon IRE1α activation and represses TCF/LEF even in the presence of b-catenin potentially by recruiting co-repressor proteins (iii). Data (panels a, b, c, f and g) are representative of at least three independent experiments.

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Extended Data Fig. 8 Genome-wide mapping of XBP1 gene targets in HSPCs identifies a prognostic 18-gene signature.

(a and b) Heatmap of XBP1 binding peaks (a) and normalized (average of replicate samples) read density of aligned DNA at peaks (b) in the HSPC line HPC-7 at baseline (DMSO) and after 6 hours treatment with tunicamycin. (c) Comparison of XBP1-bound genes to TCF4-target genes identified 460 overlapping genes. (d) GSEA plot of the 460 overlapping XBP1 and TCF4 genes from panel (c) in the transcriptome of LSK cells from Ern1koFlt3ITD/ITD compared to Ern1wtFlt3ITD/ITD mice. Upregulation of the 460 genes in IRE1α-deficient LSK cells suggest that these genes are repressed by XBP1 after IRE1α activation. (e and f) ENRICHR pathway analysis (e) and heatmap of expression of the leading edge 106 (among 460 total identified in panel c) XBP1-bound genes in HSPCs that are also TCF4-targets. (g) Integrated analysis of XBP-1 bound genes with significantly (Log2 fold change >1, P < 0.05) upregulated (“UP”) genes in Ern1koFlt3ITD/ITD LSK cells. The overlapping 132 genes from this integration were compared to TCF4 target genes in colorectal cancer cells, yielding an “18-gene” signature. Heatmap of expression of these 18 genes in Ern1wtFlt3ITD/ITD and Ern1koFlt3ITD/ITD LSK cells is shown in Fig. 7a. (h and i) Kaplan-Meier graphs and Multivariate Cox analysis (tables) of event-free survival of patients in the GSE14468 cohort (h) and overall survival of patients in the BEAT-AML cohort (i). P, Mantel-Cox Log-rank test.

Extended Data Fig. 9 Analysis of IRE1α and XBP1 gene expression profiles in primary human AML.

(a-b) Schematic of the bioinformatics pipeline for quantification of spliced XBP1 (XBP1S) in RNA-Seq datasets (see Methods) and pipeline validation on RNA-Seq-derived transcriptome of murine wildtype (Ern1wt, Vav-iCre-Ern1fl/fl) and IRE1α-deficient (Ern1ko, Vav-iCre+Ern1fl/fl) bone marrow (BM) LSK and GMP cells (3 mice/group). (c) Representative flow cytometry sort gates for human AML cell fractions from six representative donors. Note that Lineagenegative cells were defined as the CD3-CD20-CD19- fraction. All antibody information are reported in Supplementary Table 4. (d) Quantitative PCR analysis of gene expression and ratio of XBP1S and total XBP1 transcripts in FACS-sorted leukemia stem cells (LSC) and blast cells from primary human AML patient samples (N = 30-34 donors per gene). Wilcoxon matched-pairs signed rank test: ***, P < 0.001; ns, not significant. (e) GSVA correlation heatmap for S1PR3 (ENSG00000213694) expression was used to validate our GSVA pipeline.

Extended Data Fig. 10 Effect of XBP1 induction on patient-derived AML cells.

(a) Patient-derived AML sample characteristics. (b) Schematic diagram of bicistronic pHAGE lentiviral constructs encoding murine (m) XBP1s driven by the human EF1a promoter. IRES, internal ribosomal entry site. (c) Representative semi-quantitative qPCR of murine (m) Xbp1s and human (h) XBP1S expression in two transduced (sorted as ZsGreen+) populations of patient-derived AML cells. Note that these patient-derived AML cells expressed little or no basal human XBP1S but only the ectopic mXBP1s transgenes. (d) GSEA of differentially upregulated (“UP”) genes in Ern1koFlt3ITD/ITD versus Ern1wtFlt3ITD/ITD LSK cells in two XBP1-transduced patient-derived AML cells. (e) Representative myeloid (CFU-GM) colonies formed by patient-derived AML cells in methylcellulose cultures. *, P < 0.05; ns, not significant; Students t-Test. (f) Colony images and average (± S.D. duplicate wells) colony counts formed by MOLM-13 human AML cell line transduced with XBP1 products. (g-i) Effect of over-expression of XBP1 truncation mutants (g) on CFU activity by a patient-derived AML (h) and MOLM-13 cells (i). Graphs are average (± S.D. duplicate wells) CFU colony counts after 14 days. (j and k) Cell viability defined by percentage of Annexin-V and DAPI staining cells (j) and cell cycle populations (k) on transduced patient-derived AML cell 72 hours after transduction. Cell cycle analysis was determined by BrdU versus 7-AAD staining. (l) Wright-Giemsa-stained XBP1-transduced patient-derived AML cells. Micrographs are representative of three independent experiments with similar results. Scale bar: 5 mM. *, P < 0.05; **, P < 0.01; ns, not significant; Students t-Test. Data are representative of two (f, h, i) and three (c, e, j, k) independent experiments with similar results. (m) Model of myeloid leukemogenesis restriction by IRE1α-XBP1 signaling in HSPCs. IRE1α is activated at least in part by a NOX2-dependent mechanism to induce XBP1 mRNA splicing (XBP1 to XBP1s). XBP1, translated from the XBP1s mRNA, represses tumorigenesis-promoting genes and pathways such as Wnt/b-catenin pathway and pro-leukemia stem cells (LSC) gene signatures which ultimately facilitates AML development. Accordingly, loss of IRE1α, and consequently loss of XBP1, cooperated with Flt3-ITD to cause a lethal AML disease in mice. Conversely, overexpression of XBP1 represses pro-leukemogenic genes and restricted human AML cell activity in vitro and in vivo. Created with BioRender.com.

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Supplementary information

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Supplementary Methods.

Reporting Summary

Supplementary Tables 1–7

Tables of primers and antibodies.

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Barton, B.M., Son, F., Verma, A. et al. IRE1α–XBP1 safeguards hematopoietic stem and progenitor cells by restricting pro-leukemogenic gene programs. Nat Immunol 26, 200–214 (2025). https://doi.org/10.1038/s41590-024-02063-w

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