Page 129 - Haematologica Vol. 110 - January 2025

ARTICLE - CITE-seq analysis of HU effects on CML cells
H. Komic et al.
all SPC, disregarding cluster identity (Figure 4B). Further- more, the expression pattern of the CHEK2 gene (known to be upregulated following cell cycle arrest22) suggested that cells in the EP-Cy and EP-II clusters may be arrested in the cell cycle (Figure 4C).
To further address aspects of cell cycling in blood samples
obtained before and after HU treatment, we performed flow cytometry analysis using DAPI and Ki67 staining of CD14- CD34+ cells (Figure 4D). In agreement with the CITE-se- quencing findings, a larger fraction of CD14-CD34+ cells appeared in the S/G2/M cell cycle phases after HU treat- ment (Figure 4E). Furthermore, an increased frequency
ABC
D
E
Figure 3. Hydroxyurea stimulates erythroid maturation among chronic myeloid leukemia stem and progenitor cells. Analysis of CD14-CD34+ CITE-sequencing data from two patients with chronic phase chronic myeloid leukemia (sCML14 and sCML23) with peripheral blood samples collected before and after hydroxyurea (HU) treatment and paired bone marrow samples obtained af- ter treatment. “After HU” samples were collected after 7 and 9 days of treatment, respectively. (A) Uniform manifold approxima- tion and projection (UMAP) representation of an mRNA-based clustering analysis of cells from all six samples (21,044 cells) after integration by patient. (B, C) Scaled expression of selected marker genes (B) and proteins (C) used for annotation of each UMAP cluster. Colors represent average expression level and dot size represents the percentage of cells expressing the specific marker within the cluster. (D) Distribution of cells from each analyzed sample across clusters. In the left panel, all cell populations are subsampled to 1,286 cells. (E) Feature plots of expression of the genes HBA1, HBA2 and HBB among cells in the UMAP. Red indicates maximum expression and blue minimum expression. MP: myeloid progenitors; LSC: leukemic stem cells; EBMP: eosinophil/basophil/ mast cell progenitors; EP: erythroid progenitors; EP-Cy: cycling erythroid progenitors: MEP: megakaryocytic/erythroid progenitors; MKP: megakaryocytic progenitors; BM: bone marrow.
Haematologica | 110 January 2025
121