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PreprintCited by 0Acute Myeloid Leukemia ResearchHematopoietic Stem Cell TransplantationCancer Genomics and Diagnostics

The DOI matches its Crossref record; 91 of the 91 references it cites were found in a registry or an index; 1 of the 41 sources its claims cite were checked for support.

A scalable in vitro cell competition model identifies preleukemic super competitor clones

Open access
Cabrera-Cosme, Lilia, Rubio-Lara, Juan A., Bennett, Ellie, Miłek, Joanna, Baldreki, Chloë, Dowle, Adam, Vijayabaskar, M.S., Davies, Sophie J., Taylor-Smith, Ewan, Bain, Fiona M., MacDonald, Sandy, James, Sally, Hogg, Karen, Jassinskaja, Maria, Gottgens, Berthold, Wilson, Nicola K., Kent, David G.·2026·bioRxiv (Cold Spring Harbor Laboratory)
DOI 10.64898/2026.10.21.760611
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Abstract

Preleukemic and leukemic conditions are frequently characterized by the acquisition of recurrent genetic mutations in hematopoietic stem cells (HSCs). One of the most frequently mutated genes in both clonal hematopoiesis and myeloid malignancies is the ten-eleven translocation 2 (TET2) gene, which encodes an epigenetic enzyme with a critical role in the regulation of gene transcription. While substantial work has shown that TET2 loss-of-function mutations lead to a clonal advantage, the underpinning cellular mechanism remains unclear, especially with respect to the early interactions with other cells. To study these early cell interactions of mutant and non-mutant cells, we developed an in vitro clonal competition platform where single-cell derived cultures are split into subclonal fractions which can be simultaneously cross-competed against multiple different clones of an opposing genotype. We identify a novel preleukemic cell phenotype that parallels the developmental biology phenomenon of super-competition, whereby ∼10% of TET2-mutant clones actively and selectively suppress wild type (WT) cell expansion and, in some cases, completely eliminate WT clones. Molecular profiling and functional assays point to RAC1-GEF signalling inhibition as a specific WT cell vulnerability that is promoted, and tolerated, by neighbouring TET2-mutated clones. In addition to pinpointing selective drivers of non-mutant cell suppression, this clonal competition platform offers a broadly applicable new approach to study cell competition dynamics at an unprecedented scale. Abstract Figure: Key points: Split clonal culture system allows for the modelling of clonal competition in vitro between a single clone against multiple clones of an opposing genotype A subset of TET2-mutant HSCs are “super competitors”, actively suppressing wild-type cells. TET2-super competitors increase phenotypic HSCs and tolerate Rac1-GEF signalling disruption.

Overview

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TL;DR

The paper develops a scalable split-clone in vitro competition assay and uses it to identify a subset of TET2-mutant HSCs as “super competitors” that actively suppress wild-type cells. Molecular profiling implicates inflammatory/cell-adhesion programs and Rac1/GEF signaling, with Rac1 inhibition selectively impairing WT while TET2-mutant cells tolerate it.

Key findings

  • Split HSC clones into four sister wells were phenotypically, functionally, and molecularly equivalent: ICC for ELSK frequencies was 0.90, ELSK counts 0.80, and viable cell counts 0.89 across 38 parent HSC clones; ANOVA attributed 89.88%, 80.03%, and 89.13% of variation, respectively, to between-culture differences.
  • In 140 competition matrices comprising 420 WT and 420 TET2 individual HSC-initiated clones over 1,260 competition wells, 68.67% of TET2-/- clones preferentially outcompeted WT clones. About 10% of clones (83 of 840) outcompeted all three opposing-genotype wells; most were TET2-/- (~70%), and a subset (n=27) outgrew all three competitors by >70:30.
  • TET2-super competitor clones actively suppressed WT cells: WT sister-well cell production was >20-fold reduced versus a standard competition well, and WT cell number was approximately 10-fold lower when co-cultured with a TET2-super competitor. ELSK numbers showed WT cells outnumbered ~20-fold by TET2 competitors and ~80-fold by TET2-super competitors.
  • In vivo, only TET2-super competitor recipients had 100% repopulation at 16 weeks, versus 62.5% for WT competitors and 71.42% for TET2 competitors. TET2-super competitors also showed elevated inflammatory/cell-adhesion molecules, and the Rac1/GEF inhibitor NSC-23766 at 200 μM produced 4.42-fold fewer WT cells than TET2-/- cultures and reduced phenotypic HSCs 4.45-fold.

Methods

  • Single ESLAM HSCs (CD45+EPCR+CD150+CD48−) from WT (CD45.1) or TET2−/− (CD45.2) mice were expanded for 14 days, split into four sister wells, and expanded for a further 14 days. One sister well remained a control; three were co-cultured 1:1 with sister wells from the opposing genotype in a 3×3 competition matrix.
  • Readouts included flow cytometry, single-cell RNA-sequencing, bulk RNA-sequencing, data-independent acquisition mass spectrometry of supernatants, in vivo HSC transplantation, and in vitro validation with Rho-GTPase inhibitors or recombinant proteins.
  • Analyses included intraclass correlation coefficients, one-way/two-way ANOVA, DESeq2 differential expression, KEGG/GO enrichment, DeepLoc protein localization, and linear regression.

Limitations

  • Paper-stated: IFN-γ treatment was toxic to both WT and TET2−/− cells and therefore not selective enough to drive the supercompetitor phenotype; functional studies did not reveal genotype-specific differences in expansion cultures, which the authors say warrants further testing. The authors also note that NSC-23766 has reported off-target effects on platelets in a Rac1−/− setting, which might affect interpretation.
  • Evident from the text: the model is mouse TET2−/− HSC culture, not human clonal hematopoiesis; in vivo supercompetitor validation used a small recipient number (n=4 for TET2-super), and the supercompetitor phenotype was heterogeneous, with six of eight clones changing phenotype over time and classification depending on >70:30 dominance plus active suppression.

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Registry

Crossref · 10.64898/2026.10.21.760611

Cites

91 of 91 references verified0 citation errors · 0 not checkable

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Figures

Image 1
CC-BY · From the PDF
Figure 1 Single-cell split cultures are phenotypically, functionally and molecular homogeneous. A) Experimental design for split cultures. Single HSCs were isolated from WT mice (n = 3) and expanded for 14 days and then fractioned into four equal portions across four wells of a new plate. Resulting sister wells were cultured for 14 days (28 days total) and harvested for flow cytometry, transplantations and scRNA-seq. B) Frequencies of phenotypic HSCs (defined as ELSK cells) (top), counts (middle), and viable cell counts (bottom) in sister well
Figure 1 Single-cell split cultures are phenotypically, functionally and molecular homogeneous. A) Experimental design for split cultures. Single HSCs were isolated from WT mice (n = 3) and expanded for 14 days and then fractioned into four equal portions across four wells of a new plate. Resulting sister wells were cultured for 14 days (28 days total) and harvested for flow cytometry, transplantations and scRNA-seq. B) Frequencies of phenotypic HSCs (defined as ELSK cells) (top), counts (middle), and viable cell counts (bottom) in sister wellCC-BY · From the PDF
Figure 2 Design of a clonal competition model and the identification of TET2-super competitors. A) Experimental design of an in vitro model of cell competition. Single HSCs from wild-type and TET2 -/- mice were sorted and expanded independently for 14 days. Clones from each genotype were visually assessed and split four ways. Resulting sister wells were evenly distributed across a competition matrix, a 3x3 arrangement consisting of three control sister wells from each corresponding genotype on each side of the matrix. At the centre of the matrix, sister wells from WT are mixed with sister wells of TET2 -/- at a 1:1 ratio (50% of WT and 50% of TET2 by volume).
Figure 2 Design of a clonal competition model and the identification of TET2-super competitors. A) Experimental design of an in vitro model of cell competition. Single HSCs from wild-type and TET2 -/- mice were sorted and expanded independently for 14 days. Clones from each genotype were visually assessed and split four ways. Resulting sister wells were evenly distributed across a competition matrix, a 3x3 arrangement consisting of three control sister wells from each corresponding genotype on each side of the matrix. At the centre of the matrix, sister wells from WT are mixed with sister wells of TET2 -/- at a 1:1 ratio (50% of WT and 50% of TET2 by volume).CC-BY · From the PDF
Image 4
CC-BY · From the PDF
Figure 4 In vivo functional characterization of competitor types. A-B) Analysis of donor contribution to total WBC fraction in recipients of WT- (n = 8), TET2- (n = 8), and TET2-super- (n = 4) competitor clones, from 4-24 weeks post-transplant. A) Donor-derived chimerism at 16 weeks post-transplant. For in vivo transplantations, 30% of corresponding, non-competed controls was harvested, and injected into recipients together with bone marrow supportive cells (see Supplemental Methods). WT-competitors had repopulated 62.5% of recipients, TET2competitors 71.42% of recipients. In contrast, all TET2-super competitor clone recipients had been repopulated by 16 weeks. Bars represent mean and error bars represent standard error of the mean (SEM). B) Donor-derived chimerism throughout the experiment. TET2 competitors attained the highest chimaerism levels, followed by TET2super competitors, and lastly, by WT competitors. Two of the TET2-competitor recipients died before the completion of the experiment due to reasons unrelated to the transplant. C) Linear regression analysis of ELSK absolute numbers in relation to donor-derived chimerism in TET2- (left) and TET2-super competitors. ELSK counts in vitro significantly correlated with donor-derived chimerism in vivo for TET2-competitors (r 2 = 0.6048, p = 0.00396). Average ELSK number in TET2-competitor donors (μ = 1.993 x 10 3 ) was ten-fold reduced relative to TET2-super competitors (μ = 2.664 x 10 4 ). D) Competitor clone overall contribution to the live CD45+ fraction in the bone marrow (left) and spleen (right) of recipients. A) One-way ANOVA; B), Two-way ANOVA; C) Simple linear regression model; D) One-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Error bars represent the standard error of the mean (SEM).
Figure 4 In vivo functional characterization of competitor types. A-B) Analysis of donor contribution to total WBC fraction in recipients of WT- (n = 8), TET2- (n = 8), and TET2-super- (n = 4) competitor clones, from 4-24 weeks post-transplant. A) Donor-derived chimerism at 16 weeks post-transplant. For in vivo transplantations, 30% of corresponding, non-competed controls was harvested, and injected into recipients together with bone marrow supportive cells (see Supplemental Methods). WT-competitors had repopulated 62.5% of recipients, TET2competitors 71.42% of recipients. In contrast, all TET2-super competitor clone recipients had been repopulated by 16 weeks. Bars represent mean and error bars represent standard error of the mean (SEM). B) Donor-derived chimerism throughout the experiment. TET2 competitors attained the highest chimaerism levels, followed by TET2super competitors, and lastly, by WT competitors. Two of the TET2-competitor recipients died before the completion of the experiment due to reasons unrelated to the transplant. C) Linear regression analysis of ELSK absolute numbers in relation to donor-derived chimerism in TET2- (left) and TET2-super competitors. ELSK counts in vitro significantly correlated with donor-derived chimerism in vivo for TET2-competitors (r 2 = 0.6048, p = 0.00396). Average ELSK number in TET2-competitor donors (μ = 1.993 x 10 3 ) was ten-fold reduced relative to TET2-super competitors (μ = 2.664 x 10 4 ). D) Competitor clone overall contribution to the live CD45+ fraction in the bone marrow (left) and spleen (right) of recipients. A) One-way ANOVA; B), Two-way ANOVA; C) Simple linear regression model; D) One-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Error bars represent the standard error of the mean (SEM).CC-BY · From the PDF
Image 6
CC-BY · From the PDF
Figure 6 Disruption of Rac1/GEF interaction is selectively tolerated by TET2 -/- HSCs. Bulk HSCs were cultured and treated with different molecules at different doses. Comparisons were made against the matched untreated control for each genotype. A) Treatment with IFN-γ caused a significant reduction both WT and TET2 cells, especially at the highest dose. B) Rho-GTPase signalling was disrupted by treatment with Rhosin and led to toxicity in both WT and TET2 -/cells. C) NSC-23766, a specific inhibitor of Rac1/GEF interaction, selectively reduced WT cell viability while maintaining TET2 viability up to 200 μM. D) ELSK numbers in wild-type cultures were likewise reduced in a dose-dependent manner, in relation to the control (non-treated) conditions. TET2 ELSK number, in contrast, was not significantly affected by NSC-23766 treatment up to 200 μM concentration. Two-way ANOVA, * p < 0.05, *** p < 0.001, **** p< 0.0001. Error bars represent the SEM.
Figure 6 Disruption of Rac1/GEF interaction is selectively tolerated by TET2 -/- HSCs. Bulk HSCs were cultured and treated with different molecules at different doses. Comparisons were made against the matched untreated control for each genotype. A) Treatment with IFN-γ caused a significant reduction both WT and TET2 cells, especially at the highest dose. B) Rho-GTPase signalling was disrupted by treatment with Rhosin and led to toxicity in both WT and TET2 -/cells. C) NSC-23766, a specific inhibitor of Rac1/GEF interaction, selectively reduced WT cell viability while maintaining TET2 viability up to 200 μM. D) ELSK numbers in wild-type cultures were likewise reduced in a dose-dependent manner, in relation to the control (non-treated) conditions. TET2 ELSK number, in contrast, was not significantly affected by NSC-23766 treatment up to 200 μM concentration. Two-way ANOVA, * p < 0.05, *** p < 0.001, **** p< 0.0001. Error bars represent the SEM.CC-BY · From the PDF
Supplementary Figure 1 - Extended phenotypic and molecular analysis of split split cultures. A) ELSK frequencies calculated from live cells for TET2 split cultures (n = 41). TET2 single-cell derived clones were split
Supplementary Figure 1 - Extended phenotypic and molecular analysis of split split cultures. A) ELSK frequencies calculated from live cells for TET2 split cultures (n = 41). TET2 single-cell derived clones were splitCC-BY · From the PDF
Supplementary Figure 2 - Extended phenotypic profiling of competitor clones. A) Normalised cell count and B) frequencies of live CD45+ cells in competition wells for each competitor types. C-H) Phenotypic analysis in control (non-competed) clones corresponding to competitor clones in (A) and (B) . C) Lin- and D) LSK
Supplementary Figure 2 - Extended phenotypic profiling of competitor clones. A) Normalised cell count and B) frequencies of live CD45+ cells in competition wells for each competitor types. C-H) Phenotypic analysis in control (non-competed) clones corresponding to competitor clones in (A) and (B) . C) Lin- and D) LSKCC-BY · From the PDF
Image 10
CC-BY · From the PDF
Supplementary Figure 4. Extended functional profiling of competitor clones. A) Multilineage reconstitution analysis of competitor clones.Granulocyte-monocyte (GM) fraction percentages were higher in TET2-competitor recipients, albeit statistically non-significant, which agrees with their overall higher contribution to the total WBC fraction. Significantly elevated contributions to the B- (middle) and T-cell (right) lineages by both TET2- and TET2-super competitors provided evidence for their multilineage reconstitution capacity. B) Linear regression analysis of frequency of live CD45+ cells in competition wells relative to their donor-derived chimaerism levels. None of the correlations were statistically significant. C) Linear regression analysis of normalised ELSK counts
Supplementary Figure 4. Extended functional profiling of competitor clones. A) Multilineage reconstitution analysis of competitor clones.Granulocyte-monocyte (GM) fraction percentages were higher in TET2-competitor recipients, albeit statistically non-significant, which agrees with their overall higher contribution to the total WBC fraction. Significantly elevated contributions to the B- (middle) and T-cell (right) lineages by both TET2- and TET2-super competitors provided evidence for their multilineage reconstitution capacity. B) Linear regression analysis of frequency of live CD45+ cells in competition wells relative to their donor-derived chimaerism levels. None of the correlations were statistically significant. C) Linear regression analysis of normalised ELSK countsCC-BY · From the PDF
Supplementary Figure 5 - Extended molecular profiling of competitor clones. A) Principal component analysis (PCA) of wild-type-, TET2-, and TET2-super competitor clones. B) Heatmap of the top 200 expressed genes across competitor types. A signature of inflammatory, cell adhesion and cytoskeletal organisation molecules was detected in TET2-super competitors. Red-colour scale bar to the top right corner of the heatmap indicates relative gene expression.
Supplementary Figure 5 - Extended molecular profiling of competitor clones. A) Principal component analysis (PCA) of wild-type-, TET2-, and TET2-super competitor clones. B) Heatmap of the top 200 expressed genes across competitor types. A signature of inflammatory, cell adhesion and cytoskeletal organisation molecules was detected in TET2-super competitors. Red-colour scale bar to the top right corner of the heatmap indicates relative gene expression.CC-BY · From the PDF
Image 13
CC-BY · From the PDF
Supplementary Figure 6 - Extended phenotypic profiling of wild-type and TET2 bulk HSC cultures under different treatment conditions. Lin- frequencies calculated from live cells and LSK numbers are shown. A) IFNγ treatment had a mild impact over the HSC/HSPC pool in both wild-type and TET2 cultures. B) As CASIN was lethal, cellular proportions were obliterated. No ELSK expansion was observed under these conditions. C) Rhosin negatively regulated the expansion of HSC/HSPCs in equal proportion for both genotypes. D) NSC-23766
Supplementary Figure 6 - Extended phenotypic profiling of wild-type and TET2 bulk HSC cultures under different treatment conditions. Lin- frequencies calculated from live cells and LSK numbers are shown. A) IFNγ treatment had a mild impact over the HSC/HSPC pool in both wild-type and TET2 cultures. B) As CASIN was lethal, cellular proportions were obliterated. No ELSK expansion was observed under these conditions. C) Rhosin negatively regulated the expansion of HSC/HSPCs in equal proportion for both genotypes. D) NSC-23766CC-BY · From the PDF

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Citations

Cites

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  1. 10.1182/blood.v66.2.327.bloodjournal662327 · DOI
  2. 10.1182/blood.v64.2.393.bloodjournal642393 · DOI
  3. 10.7554/elife.91826.3 · DOI

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Metadata

Authors
Cabrera-Cosme, L., Rubio-Lara, J. A., Bennett, E., Miłek, J., Baldreki, C., Dowle, A., Vijayabaskar, M. S., Davies, S. J., Taylor-Smith, E., Bain, F. M., MacDonald, S., James, S., Hogg, K., Jassinskaja, M., Gottgens, B., Wilson, N. K., & Kent, D. G.
Affiliations
  • University of York (GB)
  • Wellcome/MRC Cambridge Stem Cell Institute (GB)
  • Centre for Blood Research, York Biomedical Research Institute, Department of Biology, University of York, York, UK YO10 5DD
  • Metabolomics and Proteomics, Biosciences Technology Facility, Department of Biology, University of York, York, UK YO10 5DD
  • Lund University (SE)
  • Cambridge Stem Cell Institute & Department of Haematology, University of Cambridge, Cambridge, UK CB2 0AW
Corresponding author
Kent, David G.
ORCID
Venue
bioRxiv (Cold Spring Harbor Laboratory)
Publisher
openRxiv
Published
2026-10-22
DOI
10.64898/2026.10.21.760611
OpenAlex
W7223228908
Cited by
0
References
91
Type
posted content
Language
en
Open access
Licence
Locations
bioRxiv (Cold Spring Harbor Laboratory) (submittedVersion, cc-by)
Funders
  • Japan Agency for Medical Research and Development
  • Yorkshire Forward (EP/K039660/1, EP/M028127/1)
  • Cancer Research UK (DCRPGF∖100008, MC_PC_21043, A21762, 18002, C1163/A21762, NIHR203331)
  • University of Cambridge
  • Vetenskapsrådet (2021-00185)
  • Blood Cancer UK (24014, 18002, C1163/A21762)
  • Centre for Blood Research, University of British Columbia
  • Medical Research Council (MR/Y011945/1, 206328/Z/17/Z, MR/V005502/1, MC_PC_21043)
  • Engineering and Physical Sciences Research Council (EP/M028127/1, EP/K039660/1)
  • Leeds Biomedical Research Centre (NIHR203331)
Topics
  • Health Sciences › Medicine › Hematology › Acute Myeloid Leukemia Research
  • Health Sciences › Medicine › Hematology › Hematopoietic Stem Cell Transplantation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer Genomics and Diagnostics
Keywords
clonal competition, TET2 mutation, preleukemia, clonal hematopoiesis, hematopoietic stem cells, myeloid neoplasms, epigenetic regulation

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