Page 131 - Haematologica Vol. 110 - January 2025

ARTICLE - CITE-seq analysis of HU effects on CML cells
H. Komic et al.
(Online Supplementary Figure S4A). This identified 4,222 immature CD34+CD38- cells that were included in subse- quent analysis. The positions of the gated cells within the larger CD14-CD34+ UMAP confirmed that the selected cells mainly derived from the immature LSC and MP clusters, with additional contribution from MEP and MKP clusters (Online Supplementary Figure S4B). In line with the flow cytometry results shown in Online Supplementary Figure S1B, the CITE-sequencing data showed marginally reduced frequencies of CD38- out of CD34+ cells following HU treat- ment (Online Supplementary Figure S4C).
Re-clustering of the selected cells yielded nine clusters (Figure 6A), without major patient-specific clustering (Online
Supplementary Figure S4D). Based on expressional patterns (Online Supplementary Figure S4E, F, Online Supplementary Tables S8 and S9), the UMAP largely divided into stem cells/ multipotent progenitors (SC/MPP; e.g., characterized by CD90, HLF, CD25, CD26), cells with myeloid lineage com- mitment (MP; e.g. CD371, CD45RA, SPINK2, CSF3R, CEBPA) and cells showing signs of megakaryocytic/erythroid/eo- sinophil/basophil/mast cell lineage bias (MEP/EBMP; e.g., GATA1, MPL, CD35, HDC).
Assessment of the relative proportions of cells in G0/G1 versus S/G2/M phase across samples revealed an HU-in- duced shift towards S/G2/M phase-related gene expression also among the immature CD34+CD38- cells (Figure 6B). The
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Figure 5. CITE-sequencing analysis of unpaired chronic myeloid leukemia bone marrow samples confirms enhanced frequencies of stem and progenitor cells with features of erythroid maturation and S/G2/M cell cycle phases after hydroxyurea treatment. Analysis of CITE-sequencing data from a previously published dataset of CD14-CD34+ bone marrow cells collected from patients with chronic phase chronic myeloid leukemia before (N=5; 17,968 cells) or after (N=7; 29,736 cells) a median of 9 days (range, 4-19) of hydroxyurea (HU) treatment (unpaired samples; Nilsson et al.19). (A) Visualization of the distribution of cells obtained before and after HU treatment across the CD14-CD34+ uniform manifold approximation and projection (UMAP) from Nilsson et al.19 (B) Percentage of cells in each erythroid UMAP cluster in samples obtained before and after HU. Statistics by Mann-Whitney. *P<0.05. (C) Feature plots of HBA1, HBA2, HBB and CHEK2 gene expression. Red indicates maximum expression and blue minimum expres- sion. (D) Proportion of cells in G0/G1 versus S/G2/M phase in each erythroid cluster. CP-CML: chronic phase chronic myeloid leukemia; BM: bone marrow; BCP: B-cell progenitors; EBMP: eosinophil/basophil/mast cell progenitors; EP-Cy: cycling erythroid progenitors; EP: erythroid progenitors; HSC: hematopoietic stem cells; LMP: lympho-myeloid progenitors; LSC: leukemic stem cells; MDP: monocyte/dendritic cell progenitors; MEP: megakaryocytic/erythroid progenitors; MEP-Cy: cycling megakaryocytic/ erythroid progenitors; MKP: megakaryocytic progenitors; NP: neutrophil progenitors.
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