Page 126 - Haematologica Vol. 110 - January 2025

ARTICLE - CITE-seq analysis of HU effects on CML cells
while awaiting definitive diagnosis, as advised by European LeukemiaNet (ELN) recommendations.7 Although frequently used, recently published data suggest that HU pretreatment does not add value in terms of meeting ELN CML treatment response milestones in the TKI era, and that early-phase HU should be restricted to patients with symptomatic hy- perleukocytosis or splenomegaly.8
HU inhibits ribonucleoside diphosphate reductase, thus limiting the available deoxyribonucleotides to target rapidly proliferating cells.9 In addition to direct effects on cell cycle proliferation, HU has been shown to generate nitric oxide (NO) radicals.10-12 While the role of NO in the antileukemic effects of HU is unclear, NO-induced elevation of cyclic GMP reportedly promotes g-globin and fetal hemoglobin production and contributes to the favorable effects of HU in sickle-cell anemia.10,13 In vitro studies suggest that NO may exert pro-proliferative, anti-proliferative, or differentiating effects on hematopoietic stem and progenitor cells (SPC) depending on concentration.14
CML is maintained and propagated by a rare population of BCR::ABL1-expressing leukemic stem cells (LSC).15,16 These cells are considered partly resistant to TKI, which is why many patients require lifelong treatment with ensuing toxicity and risk of the development of resistance to TKI.17 Considerable efforts have been devoted to developing strategies to target the therapy-resistant LSC population.18 In many studies, CML patients have received HU prior to TKI, but to our knowledge no studies have evaluated to what extent HU affects the phenotype of the LSC and pro- genitors. In the present study, we aimed to address this issue through targeted proteo-transcriptomic single-cell profiling of >60,000 CML SPC from peripheral blood and bone marrow (BM) samples obtained before and after HU treatment. Our results revealed (i) enhanced erythroid maturation and (ii) increased fractions of cells in S/G2/M phase among CML SPC after HU treatment.
Methods
Samples from patients with chronic myeloid leukemia
Peripheral blood and/or BM (from the posterior iliac crest) diagnostic samples were collected from 17 chronic phase CML patients eligible for short-term treatment with HU (4-19 days) at Sahlgrenska University Hospital (Gothenburg, Sweden) and Uddevalla Hospital (Uddevalla, Sweden). From seven patients only pre-HU samples were collected, from seven patients only post-HU samples were collected, and from three patients samples were collected both before and after HU treatment. The characteristics of the patients and samples, as well as HU dosage information, are sum- marized in Online Supplementary Table S1. The study was approved by the Regional Ethics Review Board in Gothen- burg (approval number 011-17) and conducted according to the principles of the Declaration of Helsinki. All patients
H. Komic et al. gave written informed consent prior to sample collection.
Isolation and phenotypic analyses of peripheral blood and bone marrow mononuclear cells
Patients’ peripheral blood and BM mononuclear cells (MNC) were isolated by density gradient centrifugation on Lymph- oprep (STEMCELL Technologies). Samples were enriched for SPC using fluorescence activated cell sorting (FACS) of MNC, CD15 depletion, or CD34+ cell selection using MACS Microbeads (Miltenyi Biotec) and were thereafter cryo- preserved. Cells were subjected to immunophenotypic analysis and cell cycle analysis as detailed in the Online Supplementary Methods.
CITE-sequencing library preparation
Peripheral blood MNC obtained before and after HU treat- ment and BM MNC obtained after HU treatment from two patients (sCML14 and sCML23) were subjected to sin- gle-cell proteo-transcriptomic CITE (cellular indexing of transcriptomes and epitopes)-sequencing analysis, during which the expression of 596 genes and 51 surface proteins was assessed (Online Supplementary Tables S2 and S3). Samples were barcoded and pooled, after which cells were labeled with AbSeq, fluorescent and unconjugated antibodies (Online Supplementary Tables S3 and S4), and CD14-CD34+ MNC FACS-sorted. CITE-sequencing libraries were generated from the isolated CD14-CD34+ cells using the BD Rhapsody Single-Cell Analysis System (BD Biosciences) as described in Nilsson et al.19 Further details regarding the CITE-sequencing library preparation and sequencing are provided in the Online Supplementary Methods.
Data analysis
Fastq files were processed using the BD Rhapsody Target- ed Analysis Pipeline (v. 1.10.1; BD Biosciences) on the Seven Bridges Genomics platform (https://www.sevenbridges.com). Low-quality cells were removed from the UMI count files using genes expressed versus library size dot plots in SeqGeq (v. 1.8.0; BD Biosciences).19 The mRNA and AbSeq RSEC UMI count files were analyzed in Rstudio (v. 2022.07.1+554; R [v. 4.2.1]) using the Seurat package (v. 4.2.0)20 and in Python (v. 3.1.0) using the pySCENIC package (v. 0.12.1) as described in the Online Supplementary Methods.
Analyses were also performed on a previously published dataset of CD14-CD34+ CML BM cells that were subjected to CITE-sequencing using the same set of genes and surface proteins. Details on the generation of the uniform manifold approximation and projections (UMAP) in this analysis can be found in the original publication by Nilsson et al.19
Statistics
All statistical analyses were performed in GraphPad Prism (v. 9.5.1) and Rstudio (v. 2022.07.1+554; R [v. 4.2.1]). Differ- ences in cell type proportions were evaluated in GraphPad Prism using the unpaired Mann-Whitney test. Differentially
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