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Ex Vivo Expansion of Phenotypic and Transcriptomic Chronic Myeloid Leukemia Stem Cells

Open AccessPublished:September 14, 2022DOI:https://doi.org/10.1016/j.exphem.2022.09.001

      Highlights

      • A means to expand leukemic stem cells from murine CML where they phenocopy their primary HSCs is described.
      • Both transcriptional and functional similarities to primary LSCs exist that lead to identification of targetable pathways, replicating other's findings and suggesting additional pathways for potential therapeutic intervention.
      • This approach provides a unique means to obtain significant numbers of CML LSCs and could assist in drug screens and mapping of targetable pathways in the future.
      Despite decades of research, standard therapies remain ineffective for most leukemias, pushing toward an essential unmet need for targeted drug screens. Moreover, preclinical drug testing is an important consideration for success of clinical trials without affecting non-transformed stem cells. Using the transgenic chronic myeloid leukemia (CML) mouse model, we determine that leukemic stem cells (LSCs) are transcriptionally heterogenous with a preexistent drug-insensitive signature. To test targeting of potentially important pathways, we establish ex vivo expanded LSCs that have long-term engraftment and give rise to multilineage hematopoiesis. Expanded LSCs share transcriptomic signatures with primary LSCs including enrichment in Wnt, JAK-STAT, MAPK, mTOR and transforming growth factor β signaling pathways. Drug testing on expanded LSCs show that transforming growth factor β and Wnt inhibitors had significant effects on the viability of LSCs, but not leukemia-exposed healthy HSCs. This platform allows testing of multiple drugs at the same time to identify vulnerabilities of LSCs.
      Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm, a clonal malignancy, identified by a translocation of chromosomes 9 and 22 forming a constitutively active tyrosine kinase fusion protein, BCR-AB [
      • Vainchenker W
      • Kralovics R.
      Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms.
      ,
      • Ben-Neriah Y
      • Daley GQ
      • Mes-Masson AM
      • Witte ON
      • Baltimore D.
      The chronic myelogenous leukemia-specific P210 protein is the product of the bcr/abl hybrid gene.
      ]. The leukemia stem cells (LSCs) in CML share phenotypic hematopoietic stem cell properties [
      • Holyoake T
      • Jiang X
      • Eaves C
      • Eaves A.
      Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia.
      ,
      • Jamieson CH
      • Gotlib J
      • Durocher JA
      • et al.
      The JAK2 V617F mutation occurs in hematopoietic stem cells in polycythemia vera and predisposes toward erythroid differentiation.
      ,
      • Zhang B
      • Ho YW
      • Huang Q
      • et al.
      Altered microenvironmental regulation of leukemic and normal stem cells in chronic myelogenous leukemia.
      ]. CML is treated primarily with tyrosine kinase inhibitors (TKIs) that eliminate the bulk of the disease; however, these fail to eradicate LSCs, leading to disease relapse or progression to a more aggressive blast phase leukemia [
      • Vannucchi AM
      • Harrison CN.
      Emerging treatments for classical myeloproliferative neoplasms.
      ,
      • Bhatia R
      • Holtz M
      • Niu N
      • et al.
      Persistence of malignant hematopoietic progenitors in chronic myelogenous leukemia patients in complete cytogenetic remission following imatinib mesylate treatment.
      ,
      • Patel SB
      • Nemkov T
      • Stefanoni D
      • et al.
      Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
      ,
      • Koppikar P
      • Bhagwat N
      • Kilpivaara O
      • et al.
      Heterodimeric JAK-STAT activation as a mechanism of persistence to JAK2 inhibitor therapy.
      ]. Therefore, it is essential to identify mechanisms of LSC survival to target LSCs without affecting healthy hematopoiesis.
      One of the reasons for the TKI insensitivity of LSCs is inherent heterogeneity and clonal selection [
      • Zhang B
      • Li L
      • Ho Y
      • et al.
      Heterogeneity of leukemia-initiating capacity of chronic myelogenous leukemia stem cells.
      ,
      • Warfvinge R
      • Geironson L
      • Sommarin MNE
      • et al.
      Single-cell molecular analysis defines therapy response and immunophenotype of stem cell subpopulations in CML.
      ,
      • Giustacchini A
      • Thongjuea S
      • Barkas N
      • et al.
      Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia.
      ,
      • Tong J
      • Sun T
      • Ma S
      • et al.
      Hematopoietic stem cell heterogeneity is linked to the initiation and therapeutic response of myeloproliferative neoplasms.
      ]. CML is a model disorder in which to study these heterogeneous LSCs, and the mouse model resembles many of the clinical manifestations seen with the human disease [
      • Zhang B
      • Ho YW
      • Huang Q
      • et al.
      Altered microenvironmental regulation of leukemic and normal stem cells in chronic myelogenous leukemia.
      ,
      • Koschmieder S
      • Gottgens B
      • Zhang P
      • et al.
      Inducible chronic phase of myeloid leukemia with expansion of hematopoietic stem cells in a transgenic model of BCR-ABL leukemogenesis.
      ,
      • Welner RS
      • Amabile G
      • Bararia D
      • et al.
      Treatment of chronic myelogenous leukemia by blocking cytokine alterations found in normal stem and progenitor cells.
      ]. Importantly, LSCs within this preclinical model are a well-defined population that can be identified using cell surface markers, making it possible to determine the best therapeutic regimen targeting LSCs that can be translatable to human patients [
      • Zhang B
      • Ho YW
      • Huang Q
      • et al.
      Altered microenvironmental regulation of leukemic and normal stem cells in chronic myelogenous leukemia.
      ,
      • Patel SB
      • Nemkov T
      • Stefanoni D
      • et al.
      Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
      ]. Additionally, it is known that TKIs eliminate the bulk of the disease, but in most cases, not the quiescent LSCs because of the activation of alternative signaling pathways [
      • Patel SB
      • Nemkov T
      • Stefanoni D
      • et al.
      Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
      ,
      • Zhang B
      • Strauss AC
      • Chu S
      • et al.
      Effective targeting of quiescent chronic myelogenous leukemia stem cells by histone deacetylase inhibitors in combination with imatinib mesylate.
      ,
      • Kuepper MK
      • Butow M
      • Herrmann O
      • et al.
      Stem cell persistence in CML is mediated by extrinsically activated JAK1-STAT3 signaling.
      ]. Thus, even though a molecular response to treatment is seen in most patients, less than half can discontinue treatment [
      • Mahon FX
      • Rea D
      • Guilhot J
      • et al.
      Discontinuation of imatinib in patients with chronic myeloid leukaemia who have maintained complete molecular remission for at least 2 years: the prospective, multicentre Stop Imatinib (STIM) trial.
      ,
      • Hochhaus A
      • Larson RA
      • Guilhot F
      • et al.
      Long-term outcomes of imatinib treatment for chronic myeloid leukemia.
      ,
      • Diral E
      • Mori S
      • Antolini L
      • et al.
      Increased tumor burden in patients with chronic myeloid leukemia after 36 months of imatinib discontinuation.
      ]. These well-established phenotypes, hierarchies, and observations make CML an ideal disorder to study and identify better methodologies to target LSCs.
      Many pathways have been identified to target and eliminate CML LSCs, including transforming growth factor β (TGF), tumor necrosis factor α (TNFα), and JAK–STAT [
      • Patel SB
      • Nemkov T
      • Stefanoni D
      • et al.
      Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
      ,
      • Welner RS
      • Amabile G
      • Bararia D
      • et al.
      Treatment of chronic myelogenous leukemia by blocking cytokine alterations found in normal stem and progenitor cells.
      ,
      • Kuepper MK
      • Butow M
      • Herrmann O
      • et al.
      Stem cell persistence in CML is mediated by extrinsically activated JAK1-STAT3 signaling.
      ,
      • Naka K
      • Hoshii T
      • Muraguchi T
      • et al.
      TGF-β-FOXO signalling maintains leukaemia-initiating cells in chronic myeloid leukaemia.
      ,
      • Gallipoli P
      • Pellicano F
      • Morrison H
      • et al.
      Autocrine TNF-α production supports CML stem and progenitor cell survival and enhances their proliferation.
      ,
      • Reynaud D
      • Pietras E
      • Barry-Holson K
      • et al.
      IL-6 controls leukemic multipotent progenitor cell fate and contributes to chronic myelogenous leukemia development.
      ,
      • Eiring AM
      • Page BD
      • Kraft IL
      • et al.
      Combined STAT3 and BCR-ABL1 inhibition induces synthetic lethality in therapy-resistant chronic myeloid leukemia.
      ,
      • Hoelbl A
      • Schuster C
      • Kovacic B
      • et al.
      Stat5 is indispensable for the maintenance of bcr/abl-positive leukaemia.
      ]. These signaling pathways are enriched in human CML CD34+ stem and progenitor cells (HSPCs) obtained from patients responding poorly to TKI [
      • Giustacchini A
      • Thongjuea S
      • Barkas N
      • et al.
      Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia.
      ]. However, these studies were conducted using HSPCs because of the limiting numbers of phenotypic LSCs, making it challenging to characterize and identify LSC-specific targets. Surface markers specific for CML LSCs, including CD33, interleukin-1 receptor accessory protein (IL1-RAP), CD93, CD25, and CD26, have been discovered to circumvent this [
      • Warfvinge R
      • Geironson L
      • Sommarin MNE
      • et al.
      Single-cell molecular analysis defines therapy response and immunophenotype of stem cell subpopulations in CML.
      ,
      • Herrmann H
      • Cerny-Reiterer S
      • Gleixner KV
      • et al.
      CD34+/CD38- stem cells in chronic myeloid leukemia express Siglec-3 (CD33) and are responsive to the CD33-targeting drug gemtuzumab/ozogamicin.
      ,
      • Zhao K
      • Yin LL
      • Zhao DM
      • et al.
      IL1RAP as a surface marker for leukemia stem cells is related to clinical phase of chronic myeloid leukemia patients.
      ,
      • Sadovnik I
      • Herrmann H
      • Eisenwort G
      • et al.
      Expression of CD25 on leukemic stem cells in BCR-ABL1+ CML: Potential diagnostic value and functional implications.
      ,
      • Herrmann H
      • Sadovnik I
      • Cerny-Reiterer S
      • et al.
      Dipeptidylpeptidase IV (CD26) defines leukemic stem cells (LSC) in chronic myeloid leukemia.
      ,
      • Bonardi F
      • Fusetti F
      • Deelen P
      • van Gosliga D
      • Vellenga E
      • Schuringa JJ.
      A proteomics and transcriptomics approach to identify leukemic stem cell (LSC) markers.
      ,
      • Kinstrie R
      • Horne GA
      • Morrison H
      • et al.
      CD93 is expressed on chronic myeloid leukemia stem cells and identifies a quiescent population which persists after tyrosine kinase inhibitor therapy.
      ]. However, not all LSCs express these markers owing to their heterogeneity [
      • Houshmand M
      • Simonetti G
      • Circosta P
      • et al.
      Chronic myeloid leukemia stem cells.
      ]. Thus, the challenge of obtaining adequate numbers of LSCs for functional characterization and subsequent identification of novel targets and response to treatment persists.
      Ex vivo expansion of CML LSCs is beneficial for identifying and testing drug targets that eliminate LSCs without affecting non-transformed stem cells [
      • Kitaeva KV
      • Rutland CS
      • Rizvanov AA
      • Solovyeva VV.
      Cell culture based in vitro test systems for anticancer drug screening.
      ,
      • Cucchi DGJ
      • Groen RWJ
      • Janssen J
      • Cloos J.
      Ex vivo cultures and drug testing of primary acute myeloid leukemia samples: Current techniques and implications for experimental design and outcome.
      ]. Although different ex vivo stem cell culture techniques have been established, they promote differentiation, require technical expertise, and are labor intensive [
      • McKee C
      • Chaudhry GR.
      Advances and challenges in stem cell culture.
      ,
      • Ribeiro-Filho AC
      • Levy D
      • Ruiz JLM
      • Mantovani MDC
      • Bydlowski SP.
      Traditional and advanced cell cultures in hematopoietic stem cell studies.
      ,
      • Tajer P
      • Pike-Overzet K
      • Arias S
      • Havenga M
      • Staal FJT.
      Ex vivo expansion of hematopoietic stem cells for therapeutic purposes: Lessons from development and the niche.
      ,
      • Chou DB
      • Frismantas V
      • Milton Y
      • et al.
      On-chip recapitulation of clinical bone marrow toxicities and patient-specific pathophysiology.
      ]. Moreover, these culture methods have been developed to expand stem cells for bone marrow transplants as a therapeutic option [
      • Kobayashi H
      • Morikawa T
      • Okinaga A
      • et al.
      Environmental optimization enables maintenance of quiescent hematopoietic stem cells ex vivo.
      ,
      • Wilkinson AC
      • Ishida R
      • Kikuchi M
      • et al.
      Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.
      ,
      • Boitano AE
      • Wang J
      • Romeo R
      • et al.
      Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells.
      ]. We have adapted a recently established hematopoietic stem cell (HSC) ex vivo expansion assay for CML LSCs that share the same phenotype as HSCs [
      • Wilkinson AC
      • Ishida R
      • Kikuchi M
      • et al.
      Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.
      ]. These cultures significantly expand phenotypic LSCs that share transcriptional similarities with freshly isolated CML LSCs. We found that the ex vivo expansion of LSCs from the CML transgenic mouse serves as a promising preclinical tool with which to carry out drug screens. These cultures consider cellular heterogeneity and identify targets specifically for eliminating phenotypic LSCs, bringing the field a step closer to achieving complete remission.

      Methods

      Mouse models

      All mice are housed in the University of Alabama at Birmingham (UAB)’s animal facility and experiments were performed under Institutional Animal Care and Use Committee-approved protocol. Double transgenic BCR–ABL × SCL–tTA mice were used as a CML model at ∼60% myeloid cells in peripheral blood, tested by flow cytometry as well as HemaVet [
      • Patel SB
      • Nemkov T
      • Stefanoni D
      • et al.
      Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
      ,
      • Koschmieder S
      • Gottgens B
      • Zhang P
      • et al.
      Inducible chronic phase of myeloid leukemia with expansion of hematopoietic stem cells in a transgenic model of BCR-ABL leukemogenesis.
      ,
      • Welner RS
      • Amabile G
      • Bararia D
      • et al.
      Treatment of chronic myelogenous leukemia by blocking cytokine alterations found in normal stem and progenitor cells.
      ]. B6-GFP mice were used for competition assays. Males and females were equally distributed throughout the study [
      • Schaefer BC
      • Schaefer ML
      • Kappler JW
      • Marrack P
      • Kedl RM.
      Observation of antigen-dependent CD8+ T-cell/dendritic cell interactions in vivo.
      ].

      Bone marrow transplant

      To test the engraftment potential of expanded stem cells, 8-week-old CD45.1 mice were sublethally irradiated (450 rad), and retroorbitally transplanted with one well of expanded CD45.2 HSCs or CML LSCs along with 200,00 CD45.1 whole bone marrow competitor cells, each. Peripheral blood was obtained from mice every 4 weeks for up to 6 months to test chimerism as previously described. At the end of 6 months, spleen and bone marrow were isolated to test for chimerism, stem and progenitor cells, and myeloid and lymphoid lineages. For single-cell RNA sequencing, 8-week-old CD45.1 mice were sublethally irradiated (450 rad), and retroorbitally transplanted with 5 × 106 bone marrow cells from CD45.2 CML or control mice. Imatinib treatment was started ∼4 weeks posttransplant as previously described [
      • Patel SB
      • Nemkov T
      • Stefanoni D
      • et al.
      Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
      ,
      • Welner RS
      • Amabile G
      • Bararia D
      • et al.
      Treatment of chronic myelogenous leukemia by blocking cytokine alterations found in normal stem and progenitor cells.
      ].

      FACS staining and single-cell sorting

      For fluorescence-activated cell sorting (FACS) profiles of expanded stem cells, whole bone marrow, spleen, and peripheral blood, a single-cell suspension was stained with a cocktail of monoclonal antibodies conjugated to fluorophores: Lineage markers included CD3 (17A2, phycoerythrin [PE]–Cy5), CD19 (6D5, PE–Cy5), B220 (RA36B2), CD11b (ICRF44, PE–Cy5), Gr-1 (RB6-8C5, PE–Cy5), and Ter-119 (TER-119, PE-Cy5), F4-80 (BM8, allophycocyanin [APC]–Cy7), Ly6G (1A8, fluorescein isothiocyanate [FITC]), Ly6C (HK1.4, PE–Cy7), CD115 (AFS98, PE). Stem and progenitor markers included cKit (2B8, APC–Cy7), Sca1 (D7, APC), CD135 (A2F10, PE), CD48 (HM48-1, Pacific Blue), CD150 (SLAM, PE–Cy7), ESAM (1G8, PE), endothelial protein C receptor (EPCR) (1560, APC), FcγRII/III (93, PE), and CD41 (HIP8, FITC), CD45.2 (104), and CD45.1 (A20) for chimerism. 4,6-Diamidino-2-phenylindole (DAPI) was used as a viability marker. Apoptosis was assessed with Annexin V (ThermoFisher Scientific, Waltham, MA) and DAPI as a counter stain using standard protocol. BD Fortessa with Diva Software was used to acquire data, and FlowJo 10.7.2 (Tree Star) was used for analysis. For cell sorting, the whole bone marrow was depleted for lineage marker-positive cells using biotin and streptavidin beads on the autoMACS sorter. The lineage-negative fraction was then stained; stem cells were sorted using a FACSAria II (BD Biosciences, San Diego, CA).

      Cell cycle and proliferation assay

      Fourteen-day expanded HSCs and CML LSCs were pulsed with 10 µmol/L EdU for 6 hours. The cells were then harvested, fixed, stained, and analyzed according to the manufacturer's protocol for the Click-iT EdU flow cytometric assay kit (Invitrogen, Waltham, MA).

      Ex vivo expansion

      We adapted the previously described polyvinyl alcohol (PVA)-based ex vivo HSC culture to expand LSCs [
      • Wilkinson AC
      • Ishida R
      • Kikuchi M
      • et al.
      Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.
      ,
      • Wilkinson AC
      • Ishida R
      • Nakauchi H
      • Yamazaki S.
      Long-term ex vivo expansion of mouse hematopoietic stem cells.
      ]. Fifty LSCs or HSCs (Lineage-cKit+Sca1+Flt3CD48CD150+) were directly sorted per well of a fibronectin-coated 96-well plate. The medium was supplemented with 10 ng/mL stem cell factor (SCF), 100 ng/mL thrombopoietin (TPO), 0.1% PVA, 1 × HEPES, 1 × insulin–transferrin–selenium–ethanolamine (ITS-X) and 1 × L-glutamine. The first medium change was carried out 4 days after sorting, with subsequent medium changes carried out every 2 days. For competition assays, 50 HSCs from B6-UBC-GFP and 50 LSCs from the CML mouse model were sorted into each well, and the culture was maintained as described above.

      Bulk RNA sequencing

      Twenty thousand expanded HSCs and CML LSCs in triplicates were lysed using Trizol. RNA was isolated using the RNeasy Plus mini kit (Qiagen, Hilden, Dusseldorf, Germany). Libraries were prepared with NexteraXT library construction (Illumina, San Diego, CA) according to the manufacturer's protocol. The quality and size of indexed libraries were determined using BioAnalyzer (Agilent, La Jolla, CA) and then sequenced. Analysis was performed using Partek Flow software. Briefly, double-ended sequencing reads were aligned to the mouse (mm10) using Spliced Transcripts Alignment to a Reference (STAR 2.7.3a). Aligned reads were then quantified to the transcriptome (Ensembl Transcripts release 101) and normalized based on counts per million. Identified differentially expressed genes were used for hierarchal clustering and pathway analysis. Pathway analysis was carried out using Enrichr [
      • Kuleshov MV
      • Jones MR
      • Rouillard AD
      • et al.
      Enrichr: A comprehensive gene set enrichment analysis web server 2016 update.
      ].

      Single-cell RNA sequencing

      Small conditional (sc) RNA sequencing experiments were conducted using inDrop, a 3′ biased droplet-based microfluidic platform on stem and progenitor cells (LSK, Lineage-Sca1+cKit+) from the CML chimeric mouse model [
      • Klein AM
      • Mazutis L
      • Akartuna I
      • et al.
      Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells.
      ]. In brief, we encapsulated 3,000–9,000 cells per sample. The protocol for library prep was followed as described before [
      • Wolock SL
      • Krishnan I
      • Tenen DE
      • et al.
      Mapping distinct bone marrow niche populations and their differentiation paths.
      ]. The quality and size distribution were determined on the BioAnalyzer (Agilent High Sensitivity DNA kit).

      Analysis of scRNA sequencing

      Bowtie Version 1.1.1 was used with parameter -e 80, and reads were aligned to Ensembl release 85 Mus musculus GRCm38 reference. Each library was filtered to include only abundant barcodes (>500 total counts). Next, we excluded putatively stressed or dying cells based on mitochondrial genes, and normalization of data was carried out [
      • Wolock SL
      • Krishnan I
      • Tenen DE
      • et al.
      Mapping distinct bone marrow niche populations and their differentiation paths.
      ]. We also used Scrublet to identify clusters of cell doublets that coexpressed marker genes of distinct cell types [
      • Wolock SL
      • Lopez R
      • Klein AM.
      Scrublet: Computational identification of cell doublets in single-cell transcriptomic data.
      ]. Sample independent graph-based clustering to remove cells expressing transcripts for committed cells [
      • Rodriguez-Fraticelli AE
      • Weinreb C
      • Wang SW
      • et al.
      Single-cell lineage tracing unveils a role for TCF15 in haematopoiesis.
      ] followed by differentially expressed gene identification and pathway analysis was done using Partek Flow software (Partek Inc., Chesterfield, MO).

      Statistical analysis

      Analyses were performed depending on the spread of the variable and reported as SEMs. Outliers were removed using the interquartile range with data points 1.5 below Q1 or 1.5 above Q3 eliminated from the data set. A Shapiro–Wilk test was used to determine normal versus abnormal distributions, and all continuous variables were tested for mean differences. Depending on the spread of variable, both nonparametric (Mann–Whitney U test, analysis of variance (ANOVA) Kruskal–Wallis test, Wilcoxon test) and parametric (Student's t test and ANOVA) tests were used. For ANOVA, Tukey's or Sidak's posttest was used to compare groups (Version 7.0, GraphPad Prism, San Diego, CA).

      Data sharing statement

      These data are deposited in the National Center for Biotechnology Information (NCBI)'s Gene Expression Omnibus and accessible through GEO Series Accession Nos. GSE189368 and GSE189369.

      Results

      Heterogenous clusters of CML stem and progenitor cells activate distinct pathways

      Chronic myeloid leukemia is a clonal disorder with favorable treatment outcomes; however, patients often relapse and fail therapy. Various targets have been identified and advanced for clinical trials with limited success. It was essential to consider LSC heterogeneity to identify unique pathways to target LSCs without affecting the healthy HSCs [
      • Zhang B
      • Li L
      • Ho Y
      • et al.
      Heterogeneity of leukemia-initiating capacity of chronic myelogenous leukemia stem cells.
      ,
      • Warfvinge R
      • Geironson L
      • Sommarin MNE
      • et al.
      Single-cell molecular analysis defines therapy response and immunophenotype of stem cell subpopulations in CML.
      ,
      • Giustacchini A
      • Thongjuea S
      • Barkas N
      • et al.
      Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia.
      ,
      • Holyoake TL
      • Vetrie D.
      The chronic myeloid leukemia stem cell: Stemming the tide of persistence.
      ]. Thus, we performed scRNA sequencing on freshly isolated HSPCs obtained from transplanted CML mice (Figure 1A). The control cells (WT) were recipient HSPCs (CD45.1+) present in the same inflammatory environment as the donor CML HSPCs, in other words, leukemia exposed. Using this approach, we were able to identify unique signatures masked by inflammation and mimic human patient samples (Figure 1A) [
      • Welner RS
      • Amabile G
      • Bararia D
      • et al.
      Treatment of chronic myelogenous leukemia by blocking cytokine alterations found in normal stem and progenitor cells.
      ]. Differential gene expression in CML HSPCs compared with bystander WT HSPCs (Supplementary Figure E1A,) revealed that the top 25 differentially expressed genes in CML HSPCs are mostly AP1 transcription factors; one of their regulators includes STAT3, which plays a vital role in CML drug persistence [
      • Patel SB
      • Nemkov T
      • Stefanoni D
      • et al.
      Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
      ]. Of these, Fos and Dusp1 are essential for TKI insensitivity in CML [
      • Kesarwani M
      • Kincaid Z
      • Gomaa A
      • et al.
      Targeting c-FOS and DUSP1 abrogates intrinsic resistance to tyrosine-kinase inhibitor therapy in BCR-ABL-induced leukemia.
      ]. Pathway analysis of these differentially expressed genes revealed activation of MAPK and IL-17 signaling along with enrichment of the citric acid cycle (Supplementary Figure E1B). These are consistent with the previous publications that reported that BCR–ABL activates the MAPK pathway and that CML stem and progenitor cells rely on oxidative phosphorylation (OxPhos) over glycolysis [
      • Patel SB
      • Nemkov T
      • Stefanoni D
      • et al.
      Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
      ,
      • Chu S
      • Holtz M
      • Gupta M
      • Bhatia R.
      BCR/ABL kinase inhibition by imatinib mesylate enhances MAP kinase activity in chronic myelogenous leukemia CD34+ cells.
      ,
      • Ma L
      • Xu Z
      • Wang J
      • et al.
      Matrine inhibits BCR/ABL mediated ERK/MAPK pathway in human leukemia cells.
      ,
      • Kuntz EM
      • Baquero P
      • Michie AM
      • et al.
      Targeting mitochondrial oxidative phosphorylation eradicates therapy-resistant chronic myeloid leukemia stem cells.
      ].
      Figure 1
      Figure 1Heterogenous chronic myeloid leukemia (CML) hematopoietic stem and progenitor cells (HSPCs) upregulate distinct pathways compared with leukemia-exposed HSPCs. (A) Experimental design for small conditional (sc)RNA sequencing on LincKit+Sca1+ (LSK) stem and progenitor cells (HSPCs) obtained from transplanted CML mice. (B) k-Means clustering of CML HSPCs (top) and uniform manifold approximation and projection (UMAP) depicting expression of differentially upregulated CML transcriptional signatures compared with leukemia-exposed HSPCs (bottom). See . (C) UMAP depicting expression of differentially upregulated bulk RNA sequencing transcriptional signature of CML leukemic stem cells (LSCs) applied to CML HSPC scRNA sequencing k-means clusters. (D) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis for genes uniquely expressed in clusters C2, C3, and C4 obtained by nonparametric analysis of variance analysis. See .
      To identify different subsets of stem and progenitor populations, namely, HSCs and multipotent progenitors 2–4 (MPPs 2–4), we used published transcriptomic data sets for HSCs, MPP2, MPP3, and MPP4 from healthy mice to demarcate these populations and applied it to our non-transformed and CML HSPCs (Supplementary Figure E1C–F, Supplementary Table E1) [
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      ]. We observed that the HSC transcriptional signature is overrepresented (39%) in CML HSPCs compared with WT, even though there are fewer in phenotypic LSCs (Figure E1C, D; Supplementary Table E1) [
      • Patel SB
      • Nemkov T
      • Stefanoni D
      • et al.
      Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
      ]. Consequently, K-means clustering was applied to CML HSPCs, and four unique clusters were identified. The unique CML HSPC signature was differentially expressed compared with leukemia-exposed non-transformed HSPCs comprised of clusters 3 and 4 (Figure 1B; Supplementary Figure E1A; Supplementary Table E1). We found that MPP2 and MPP4 were overrepresented in cluster (C) 1, whereas MPP3 was found primarily in C2. Conversely, C3 comprises 70% transcriptional HSCs while cluster 4 (C4) contained only (100%) HSCs, where C4 represents 4% of all cells and C3 represents 42.7% (Figure 1B; Supplementary Figure E1E,F). This indicates that targeting clusters C3 and C4 would be a good therapeutic option as these are HSC-like clusters.
      To determine whether the HSC-like clusters (C3 and C4) share a transcriptional signature with the CML LSCs, we applied bulk RNA sequencing data obtained from CML LSCs (LSK CD48CD150+) [
      • Patel SB
      • Nemkov T
      • Stefanoni D
      • et al.
      Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
      ]. We observed that our recently published CML LSC signatures, correlated with both C3 and C4, suggesting the heterogeneity of LSCs distributed over two clusters (Figure 1C; Supplementary Table E1) [
      • Patel SB
      • Nemkov T
      • Stefanoni D
      • et al.
      Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
      ]. To determine differential pathways activated in these clusters, we performed nonparametric ANOVA differential gene expression analysis followed by KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis. We observed enrichment in the IL-17 signaling pathway, specifically in C3 driven by mitogen-activated protein kinase (MAPK), while hypoxia-inducible factor 1 (HIF-1) signaling was enriched in both C3 and C4 (Figure 1D). Furthermore, we observed significant enrichment of the Wnt signaling pathway and oxidative phosphorylation in C4 (the cluster that is mostly LSCs), an important source of energy for CML HSPCs. These pathway analyses point toward activation of different pathways in heterogenous LSC clones, one of the probable reasons for the failure of prior attempts targeting a single signaling pathway. Overall, these data suggest heterogeneity of LSCs, with each cluster having a distinct gene expression profile and pathway activation compared with bystander healthy cells that can be exploited to eliminate CML LSCs.

      Imatinib treatment transcriptionally rewires a preexistent TKI-persistent signature

      The first line of therapy for CML patients is TKIs (imatinib); however, the LSCs have been reported to persist following this treatment [
      • Bhatia R
      • Holtz M
      • Niu N
      • et al.
      Persistence of malignant hematopoietic progenitors in chronic myelogenous leukemia patients in complete cytogenetic remission following imatinib mesylate treatment.
      ,
      • Patel SB
      • Nemkov T
      • Stefanoni D
      • et al.
      Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
      ,
      • Chu S
      • McDonald T
      • Lin A
      • et al.
      Persistence of leukemia stem cells in chronic myelogenous leukemia patients in prolonged remission with imatinib treatment.
      ,
      • Hamilton A
      • Helgason GV
      • Schemionek M
      • et al.
      Chronic myeloid leukemia stem cells are not dependent on Bcr-Abl kinase activity for their survival.
      ]. To identify signatures unique to these persistent LSCs, we performed scRNA sequencing on CML HSPCs obtained from CML mice treated with TKIs (Figure 2A). On exposure to TKI for 4 weeks, we found that transcriptional patterns of the transformed stem and progenitor cells were altered, consisting of several genes involved in myeloid differentiation, cellular structure, and cell–cell interaction (Supplementary Figure E2A. Compared with CML HSPCs, the top genes in the CML-TKI cluster were S100A8–S100A9. By applying published transcriptomic data sets for HSCs, MPP2, MPP3, and MPP4 from healthy mice, as in Supplementary Figure E1C, we observed that the HSC transcriptional signature is underrepresented (1%) in CML-TKI HSPCs compared with WT and CML (Supplementary Figure E2B,C; Supplementary Table E1) [
      • Patel SB
      • Nemkov T
      • Stefanoni D
      • et al.
      Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
      ]. On the contrary, CML-TKI HSPCs are overrepresented by MPP3 transcriptional signature (37%) compared with WT and CML (Supplementary Figure E2B,C; Supplementary Table E1). k-Means clustering of CML-TKI HSPCs identified two clusters, suggesting less heterogeneity of CML HSPCs post-imatinib treatment (Figure 2B). Cluster 1 (T1) was transcriptionally composed (46%) of steady-state MPP3 signature while cluster 2 (T2) comprised a 39% MPP2 and ∼5% HSC signature (Supplementary Figure E2D; Supplementary Table E1). Applying the CML-TKI LSC bulk RNA sequencing gene signature to the CML–TKI HSPCs revealed a strong correlation with T1 (mean = 1.05); meanwhile, the primary and expanded CML LSC signature was found enriched in only a few cells of T2 (mean = 0.46) (Figure 2C, Supplementary Figure E2, Supplementary Table E1) [
      • Patel SB
      • Nemkov T
      • Stefanoni D
      • et al.
      Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
      ]. This suggests that the LSCs, on exposure to imatinib, transcriptionally rewire [
      • Patel SB
      • Nemkov T
      • Stefanoni D
      • et al.
      Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
      ]. Pathway analysis on the differentially upregulated genes in TKI-resistant clusters revealed that cluster T1 was enriched in metabolic pathways such as carbon metabolism, NF-κB signaling, and HIF-1 signaling pathways (Supplementary Figure E2F). This corroborates with CD34+ stem and progenitor cells obtained from TKI-treated CML patients [
      • Zhao F
      • Mancuso A
      • Bui TV
      • et al.
      Imatinib resistance associated with BCR-ABL upregulation is dependent on HIF-1alpha-induced metabolic reprograming.
      ,
      • Ng KP
      • Manjeri A
      • Lee KL
      • et al.
      Physiologic hypoxia promotes maintenance of CML stem cells despite effective BCR-ABL1 inhibition.
      ,
      • Cheloni G
      • Tanturli M
      • Tusa I
      • et al.
      Targeting chronic myeloid leukemia stem cells with the hypoxia-inducible factor inhibitor acriflavine.
      ,
      • Cokic VP
      • Mojsilovic S
      • Jaukovic A
      • et al.
      Gene expression profile of circulating CD34+ cells and granulocytes in chronic myeloid leukemia.
      ]. Moreover, cluster T1 was also enriched in genes regulating drug metabolism, which implies reduced TKI treatment efficacy in these cells and a potential mechanism of TKI persistence in CML LSCs (Supplementary Figure E2F). These pathways provide a window of finding targets without affecting the healthy cells and suggest transcriptional reprogramming of CML LSCs on exposure to TKI [
      • Patel SB
      • Nemkov T
      • Stefanoni D
      • et al.
      Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
      ].
      Figure 2
      Figure 2A tyrosine kinase inhibitor (TKI)-persistent transcriptional leukemic stem cell (LSC) signature preexists in chronic myeloid leukemia (CML) hematopoietic stem and progenitor cells (HSPCs). (A) Experimental design for scRNA sequencing on HSPCs obtained from transplanted CML mice treated with 200 mg/kg imatinib for 4 weeks. (B) k-Means clustering of CML–TKI HSPCs. (C) Uniform manifold approximation and projection (UMAP) depicting expression of differentially upregulated bulk RNA sequencing transcriptional signature of CML–TKI LSCs applied to CML-TKI HSPC small conditional (sc) RNA sequencing k-means clusters. See . (D) UMAP depicting expression of differentially upregulated bulk RNA sequencing transcriptional signature of CML–TKI LSCs applied to CML HSPC scRNA sequencing k-means clusters. See also . (E) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis for genes uniquely expressed in CML–TKI clusters T1 and T2 and CML cluster C4. See .
      To determine if a transcriptional drug-persistent clone of LSCs preexists or a clone of cells reprograms on drug exposure [
      • Dagogo-Jack I
      • Shaw AT.
      Tumour heterogeneity and resistance to cancer therapies.
      ], we applied the CML–TKI LSC signature to the CML HSPCs and found that it was associated with C2 and C4 (Figures 1B and 2D; Supplementary Table E1) [
      • Patel SB
      • Nemkov T
      • Stefanoni D
      • et al.
      Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
      ]. The LSC signature also marked C4, indicating that this HSC-like population is shared in both CML and CML–TKI. This also suggests that the TKI-persistent signature potentially preexists in the LSCs before drug treatment. Interestingly, we observed that the JAK–STAT, HIF-1, Wnt, and mTOR signaling pathways are shared among the CML-TKI LSCs as well as cluster C4 from the LSCs (Figure 2E). The JAK–STAT signaling pathway in this instance is attributed to the upregulation of AP1 transcription factors. Targeting STAT3, one of the regulators of AP1 transcription factors and the Wnt signaling pathway, has been reported to induce apoptosis of the TKI-persistent CML stem and progenitor cells [
      • Patel SB
      • Nemkov T
      • Stefanoni D
      • et al.
      Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
      ,
      • Eiring AM
      • Page BD
      • Kraft IL
      • et al.
      Combined STAT3 and BCR-ABL1 inhibition induces synthetic lethality in therapy-resistant chronic myeloid leukemia.
      ,
      • Bewry NN
      • Nair RR
      • Emmons MF
      • Boulware D
      • Pinilla-Ibarz J
      • Hazlehurst LA.
      Stat3 contributes to resistance toward BCR-ABL inhibitors in a bone marrow microenvironment model of drug resistance.
      ,
      • Zhao C
      • Blum J
      • Chen A
      • et al.
      Loss of beta-catenin impairs the renewal of normal and CML stem cells in vivo.
      ,
      • Schurch C
      • Riether C
      • Matter MS
      • Tzankov A
      • Ochsenbein AF.
      CD27 signaling on chronic myelogenous leukemia stem cells activates Wnt target genes and promotes disease progression.
      ,
      • Zhang B
      • Li M
      • McDonald T
      • et al.
      Microenvironmental protection of CML stem and progenitor cells from tyrosine kinase inhibitors through N-cadherin and Wnt-beta-catenin signaling.
      ]. Moreover, we also observed upregulation of cell adhesion molecules known to support and protect the CML cells from TKI treatment (Figure 2E) [
      • Zhang B
      • Li M
      • McDonald T
      • et al.
      Microenvironmental protection of CML stem and progenitor cells from tyrosine kinase inhibitors through N-cadherin and Wnt-beta-catenin signaling.
      ,
      • Rothe K
      • Babaian A
      • Nakamichi N
      • et al.
      Integrin-linked kinase mediates therapeutic resistance of quiescent CML stem cells to tyrosine kinase inhibitors.
      ,
      • Krenn PW
      • Koschmieder S
      • Fassler R.
      Kindlin-3 loss curbs chronic myeloid leukemia in mice by mobilizing leukemic stem cells from protective bone marrow niches.
      ]. This indicates that the preexistent TKI-persistent signature seen in the LSCs becomes the predominant clone on exposure to TKI. These pathways can hence be targeted in patients who have been treated with imatinib, potentially leading to treatment-free remission. However, preclinical drug testing on LSCs specifically has been technically challenging, which is essential to identify specific targets with limited effect on healthy cells.

      Ex vivo expansion of leukemic stem cells

      To study LSCs with the goal of pre-clinical drug testing, we worked on expanding LSCs ex vivo since in CML, LSCs comprise ∼0.01% of the whole bone marrow [
      • Zhang B
      • Ho YW
      • Huang Q
      • et al.
      Altered microenvironmental regulation of leukemic and normal stem cells in chronic myelogenous leukemia.
      ,
      • Patel SB
      • Nemkov T
      • Stefanoni D
      • et al.
      Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
      ]. The rarity of these cells makes it difficult to obtain adequate numbers of LSCs for characterization using assays that require more than 100,000 cells and identify targetable vulnerabilities that do not affect the non-transformed hematopoietic stem cells. We adapted the PVA-based ex vivo HSC culture to expand phenotypically well-characterized CML LSCs (Figure 3A) [
      • Zhang B
      • Ho YW
      • Huang Q
      • et al.
      Altered microenvironmental regulation of leukemic and normal stem cells in chronic myelogenous leukemia.
      ,
      • Patel SB
      • Nemkov T
      • Stefanoni D
      • et al.
      Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
      ,
      • Koschmieder S
      • Gottgens B
      • Zhang P
      • et al.
      Inducible chronic phase of myeloid leukemia with expansion of hematopoietic stem cells in a transgenic model of BCR-ABL leukemogenesis.
      ,
      • Welner RS
      • Amabile G
      • Bararia D
      • et al.
      Treatment of chronic myelogenous leukemia by blocking cytokine alterations found in normal stem and progenitor cells.
      ,
      • Wilkinson AC
      • Ishida R
      • Kikuchi M
      • et al.
      Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.
      ]. Flow cytometry analysis revealed exponential growth of both healthy HSCs and LSCs. After day 12, HSC growth began to plateau while LSC growth declined (Figure 3B; Supplementary E3A,B. Based on these analyses, we decided to use days 12–14 of phenotypic LSC expansion as the time point for further experiments. Moreover, we found that CD48 does not delineate progenitors from HSCs in vivo; thus, we used CD150 to define phenotypic stem cells (Figure 3C). Flow cytometry of the expanded LSCs on day 12 revealed that 22.39 ± 3.09%) of the well was LineageSca1+cKit+ (LSK) leukemic cells, and 71.29 ± 6.4% of these were CD150+ LSCs (Figure 3C; Supplementary Figure E3C). Meanwhile, in freshly isolated CML whole bone marrow, 14.82 ± 0.62% of viable cells are LSK cells, of which 2.81 ± 0.35% are LSCs (CD150+CD48) (Supplementary Figure E3D). Importantly, we observed a 460 ± 125-fold expansion of LSCs by day 12 (Figure 3D). Expansion of LSCs was significantly less than that of HSCs; however, the proliferation rates of HSCs and LSCs were similar (Figure 3E). Meanwhile, apoptosis revealed more deaths of LSCs than HSCs (Supplementary Figure E3E,), which could account for the reduced output. To further characterize the expanded LSCs, we ran flow cytometry for human and murine markers known to be expressed in CML [
      • Zhang B
      • Li L
      • Ho Y
      • et al.
      Heterogeneity of leukemia-initiating capacity of chronic myelogenous leukemia stem cells.
      ,
      • Sadovnik I
      • Herrmann H
      • Eisenwort G
      • et al.
      Expression of CD25 on leukemic stem cells in BCR-ABL1+ CML: Potential diagnostic value and functional implications.
      ,
      • Herrmann H
      • Sadovnik I
      • Cerny-Reiterer S
      • et al.
      Dipeptidylpeptidase IV (CD26) defines leukemic stem cells (LSC) in chronic myeloid leukemia.
      ]. ESAM+, EPCR+, and CD34 describe more primitive murine stem cells, and we see conservation of this phenotype in our cultures of both HSCs and LSCs (Figure 3F) [
      • Bonardi F
      • Fusetti F
      • Deelen P
      • van Gosliga D
      • Vellenga E
      • Schuringa JJ.
      A proteomics and transcriptomics approach to identify leukemic stem cell (LSC) markers.
      ,
      • Rabe JL
      • Hernandez G
      • Chavez JS
      • Mills TS
      • Nerlov C
      • Pietras EM.
      CD34 and EPCR coordinately enrich functional murine hematopoietic stem cells under normal and inflammatory conditions.
      ]. Although not significantly different from the HSCs, the expanded LSCs express CD41, CD90, FcgRII/III, TNFR II, and PDL1, the former four receptors previously identified in CML, while the latter is an immune checkpoint (Figure 3F; Supplementary Figure E3F) [
      • Zhang B
      • Li L
      • Ho Y
      • et al.
      Heterogeneity of leukemia-initiating capacity of chronic myelogenous leukemia stem cells.
      ,
      • Warfvinge R
      • Geironson L
      • Sommarin MNE
      • et al.
      Single-cell molecular analysis defines therapy response and immunophenotype of stem cell subpopulations in CML.
      ,
      • Giustacchini A
      • Thongjuea S
      • Barkas N
      • et al.
      Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia.
      ,
      • Schurch C
      • Riether C
      • Matter MS
      • Tzankov A
      • Ochsenbein AF.
      CD27 signaling on chronic myelogenous leukemia stem cells activates Wnt target genes and promotes disease progression.
      ,
      • Hsieh YC
      • Kirschner K
      • Copland M.
      Improving outcomes in chronic myeloid leukemia through harnessing the immunological landscape.
      ,
      • Herrmann O
      • Kuepper MK
      • Butow M
      • et al.
      Infliximab therapy together with tyrosine kinase inhibition targets leukemic stem cells in chronic myeloid leukemia.
      ,
      • Parting O
      • Langer S
      • Kuepper MK
      • et al.
      Therapeutic inhibition of FcγRIIb signaling targets leukemic stem cells in chronic myeloid leukemia.
      ]. On the other hand, we did not see marked expression of CD25, CD26, and CD105 receptors and other immune checkpoint markers such as Lag3, CTLA4, and Tim3 (Figure 3F; Supplementary Figure E3F) [
      • Sadovnik I
      • Herrmann H
      • Eisenwort G
      • et al.
      Expression of CD25 on leukemic stem cells in BCR-ABL1+ CML: Potential diagnostic value and functional implications.
      ,
      • Herrmann H
      • Sadovnik I
      • Cerny-Reiterer S
      • et al.
      Dipeptidylpeptidase IV (CD26) defines leukemic stem cells (LSC) in chronic myeloid leukemia.
      ,
      • Krenn PW
      • Koschmieder S
      • Fassler R.
      Kindlin-3 loss curbs chronic myeloid leukemia in mice by mobilizing leukemic stem cells from protective bone marrow niches.
      ,
      • Hsieh YC
      • Kirschner K
      • Copland M.
      Improving outcomes in chronic myeloid leukemia through harnessing the immunological landscape.
      ]. Apart from surface marker expression, we also observed transcript expression of genes important for stemness such as Ctnna1, Fzd3, Gata1, Ikfz1, Itgam, Myc, and Mycn was maintained in the expanded LSCs, similar to HSCs (Supplementary Figure E3G) [
      • Rodriguez-Fraticelli AE
      • Weinreb C
      • Wang SW
      • et al.
      Single-cell lineage tracing unveils a role for TCF15 in haematopoiesis.
      ,
      • Pietras EM
      • Reynaud D
      • Kang YA
      • et al.
      Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
      ,
      • Gazit R
      • Mandal PK
      • Ebina W
      • et al.
      Fgd5 identifies hematopoietic stem cells in the murine bone marrow.
      ]. In summary, we found that phenotypic LSCs can be expanded ex vivo.
      Figure 3
      Figure 3. Leukemic stem cells (LSCs) maintain stem cell phenotype on ex vivo expansion. (A) Experimental layout for ex vivo expansion of normal hematopoietic stem cells (HSCs) and chronic myeloid leukemia (CML) LSCs. (B) Growth curve of HSCs and LSCs over 2 weeks. Cultures were initiated with 50 cells, and LincKit+Sca1+ (LSK) CD150+ cells were counted using flow cytometry-based counting beads every 4 days. (C) Representative flow cytometry dotplot of LSK and LSK CD150+ cells from ex vivo expanded HSC and LSC cultures. The values represent percentages of parent. (D) Total number of CD150+ LSK cells obtained after 12 days of ex vivo expansion of 50 HSCs and LSCs. (E) Percentage EdU incorporation over 4 hours in LSK CD150+ cells after 12 days of expansion of 50 HSCs and LSCs. (F) Median expression of stem cell surface markers on ex vivo expanded HSCs and LSCs after day 12. The data are means ± SEM representative of three independent experiments. An unpaired Student t test or two-way analysis of variance was used to determine statistical significance with Tukey's multiple comparison. p Values < 0.05 were considered to indicate statistical significance. *p < 0.05, **p <0.01, ***p < 0.001, ****p < 0.0001. ns = not significant. See also .

      Ex vivo leukemic stem cells maintain engraftment and multilineage potential

      Leukemic stem cells are defined by their potential to self-renew and engraft as well as being drivers of leukemia. Thus, to test the function of expanded LSCs, we transplanted one well of 14-day expanded LSCs per sublethally irradiated recipient mouse (Figure 4A). Of the mice transplanted with expanded HSCs and LSCs, we observed 100% of mice engrafted with HSCs, while 77.8% of mice transplanted with LSCs exhibited long-term engraftment, as measured by CD45.2 expression in peripheral blood (PB) (Figure 4B; Supplementary Figure E4A [
      • Kiel MJ
      • Yilmaz OH
      • Iwashita T
      • Yilmaz OH
      • Terhorst C
      • Morrison SJ.
      SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.
      ,
      • Purton LE
      • Scadden DT.
      Limiting factors in murine hematopoietic stem cell assays.
      ]. At week 12, HSCs exhibited 21.13 ± 7.47% chimerism while LSCs exhibited 2.80 ± 1.76% chimerism in PB (Figure 4B; Supplementary Figure E4A). Mature cell screening of the PB revealed trilineage potential, with HSCs giving rise to 7.26 ± 2.18% myeloid cells (defined by Gr1+CD11b+) and LSCs giving rise to 8.96 ± 3.77% (Figure 4C). Although we did not see a significant difference in the myeloid cell lineage between the HSCs and LSCs, we observed a significant reduction in PB CD3+ T cells and B220+ B cells from the transplanted CML mice, as is observed in primary CML mice (Figure 4C) [
      • Zhang B
      • Ho YW
      • Huang Q
      • et al.
      Altered microenvironmental regulation of leukemic and normal stem cells in chronic myelogenous leukemia.
      ,
      • Welner RS
      • Amabile G
      • Bararia D
      • et al.
      Treatment of chronic myelogenous leukemia by blocking cytokine alterations found in normal stem and progenitor cells.
      ]. Additionally, we saw 0.41 ± 0.31% engraftment of LSCs in the BM and 2.27 ± 1.84% in the spleen as compared with 3.96 ± 1.66% of HSCs in the marrow and 10.54 ± 4.51% in the spleen (Figure 4D). Although we did not see significant myeloid skewing in the bone marrow or spleens of the mice transplanted with LSCs, we did observe reduced lineage-negative cells or HSPCs in the bone marrow as previously described (Supplementary Figure E4B,C) [
      • Zhang B
      • Ho YW
      • Huang Q
      • et al.
      Altered microenvironmental regulation of leukemic and normal stem cells in chronic myelogenous leukemia.
      ,
      • Patel SB
      • Nemkov T
      • Stefanoni D
      • et al.
      Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
      ]. However, five of the seven mice engrafted with LSCs survived 6 months post-transplant; splenomegaly was observed in the two mice that had died (Figure 4E). Heterogeneity of CML LSCs has been previously reported, where even though LSCs engraft, not all mice develop leukemia after primary and secondary transplants [
      • Zhang B
      • Li L
      • Ho Y
      • et al.
      Heterogeneity of leukemia-initiating capacity of chronic myelogenous leukemia stem cells.
      ]. Consistently, we found that expanded LSCs retained part of their functional potential even after ex vivo expansion.
      Figure 4
      Figure 4Ex vivo expanded leukemic stem cells (LSCs) engraft and reconstitute hematopoiesis. (A) Schematic of the transplant experiment carried out after 14 days of expansion of HSCs and LSCs (n = 9 for the WT and n = 7 for the CML transplants). (B) Line graph tracking frequency of CD45.2+ cells in the peripheral blood obtained every 4 weeks posttransplant. (C) Frequency of myeloid, T, and B cells in the CD45.2+ fraction of peripheral blood obtained 12 weeks posttransplant. (D) Frequency of CD45.2+ cells in the bone marrow and spleen obtained 24 weeks post-transplant. (E) Survival probability of mice tracked for 6 months transplanted with ex vivo expanded HSCs and LSCs. The data are means ± SEM representative of two independent experiments. A Student t test or two-way analysis of variance was used to determine statistical significance with Tukey's multiple comparison. p values < 0.05 were considered to indicate statistical significance. *p < 0.05, **p <0.01, ***p < 0.001, ****p < 0.0001. ns = not significant. See .

      Expanded LSCs are transcriptionally similar to freshly sorted LSCs and so could be used for drug screens

      Ex vivo culture forces cells to proliferate and induce cultural artifacts, which could pose potential problems for drug screens. Thus, to validate that expanded LSCs are comparable to freshly isolated LSCs, we sought to determine if these populations share transcriptional similarity using bulk RNA sequencing. Differential gene expression comparing expanded healthy HSCs and expanded LSCs revealed that genes upregulated in LSCs were significantly enriched in TNFα, TGFβ, and STAT5 signaling pathways as well as inflammatory response (Figure 5A). These pathways have been proven important for CML, where STAT5 is activated by BCR–ABL tyrosine kinase and required for initiation and maintenance of CML [
      • Hoelbl A
      • Schuster C
      • Kovacic B
      • et al.
      Stat5 is indispensable for the maintenance of bcr/abl-positive leukaemia.
      ,
      • Ilaria Jr., RL
      • Van Etten RA
      P210 and P190(BCR/ABL) induce the tyrosine phosphorylation and DNA binding activity of multiple specific STAT family members.
      ]. TNFα and TGFβ signaling pathways are signatures of CML stem and progenitor cells correlated with poor response to TKI treatment [
      • Giustacchini A
      • Thongjuea S
      • Barkas N
      • et al.
      Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia.
      ,
      • Kesarwani M
      • Kincaid Z
      • Gomaa A
      • et al.
      Targeting c-FOS and DUSP1 abrogates intrinsic resistance to tyrosine-kinase inhibitor therapy in BCR-ABL-induced leukemia.
      ]. To identify transcriptional similarities and differences between expanded and freshly sorted LSCs, we compared independent differential gene lists of both LSCs using Venn analysis (Supplementary Figure E5A and Table E2,). One of the pathways differentially activated in the expanded LSCs, but not in the freshly isolated LSCs, was KRAS signaling, a known mediator of cell division while the latter had distinct activation of Notch signaling, a yet understudied pathway in CML (Supplementary Figure E5A and Table E2) [
      • Peeper DS
      • Upton TM
      • Ladha MH
      • et al.
      Ras signalling linked to the cell-cycle machinery by the retinoblastoma protein.
      ], whereas both LSCs were enriched for reactive oxygen species (ROS) pathways. CML HSPCs are known to accumulate reactive oxygen species (ROS), leading to oxidative stress and subsequently BCR–ABL mutation-mediated drug resistance [
      • Nieborowska-Skorska M
      • Kopinski PK
      • Ray R
      • et al.
      Rac2-MRC-cIII-generated ROS cause genomic instability in chronic myeloid leukemia stem cells and primitive progenitors.
      ]. Furthermore, we found that TNFα, TGFβ, and STAT5 signaling pathways were also enriched among the 460 genes commonly expressed between freshly isolated LSCs and expanded LSCs when each was previously compared with their respective healthy controls (Figure 5B; Supplementary Figure E5A and Table E2). This suggested that the expanded LSCs are transcriptionally comparable to freshly isolated LSCs, proving that this expansion assay can be used to characterize LSCs. Moreover, the single-cell profiles and pathways are shared with the ex vivo expansion of LSCs and hence is a promising tool to validate drug targets (Supplementary Figure E5B,C and Table E1).
      Figure 5
      Figure 5Drug screens on expanded leukemic stem cells (LSCs), which are transcriptionally similar to freshly isolated LSCs. (A) Hallmark pathway analysis of differentially upregulated bulk RNA sequencing transcriptional signature of ex vivo expanded chronic myeloid leukemia (CML) LincKit+Sca1+ (LSK) CD150+ cells compared with wild type (WT). See . (B) Hallmark pathway analysis of genes common to differentially upregulated bulk RNA sequencing transcriptional signature of ex vivo expanded CML LSK CD150+ cells and CML LSCs. See . (C) Normalized count (left) and relative frequency (right) of LSK CD150+ cells obtained by flow cytometry after 4-day treatment of 14-day expanded hematopoietic stem cells (HSCs) and LSCs with mentioned doses of imatinib. (D) Normalized count (left) and relative frequency (right) of LSK CD150+ cells obtained by flow cytometry after 4-day treatment of 14-day-expanded HSCs and LSCs with 2 μmol/L AZD148, 2 μmol/L ruxolitinib, 20 nmol/L rapamycin, 2 μmol/L SB 431542, 5 μmol/L U0126, and 20 μmol/L iCRT14. The data are means ± SEM and are representative of three independent experiments (C,D). An unpaired Student t test or two-way analysis of variance was used to determine statistical significance with Tukey's multiple comparison. p Values < 0.05 were considered to indicate statistical significance. *p < 0.05, **p <0.01, ***p < 0.001, ****p < 0.0001. ns = not significant. See .
      To test drugs with the aim of eliminating LSCs without affecting non-transformed leukemia-exposed HSCs, we established a competition assay where we sorted 50 HSCs and 50 LSCs per well of a 96-well plate (Supplementary Figure E5D). Fourteen days after expansion, we noticed that, as expected, the HSCs outcompeted CML LSCs, with ∼40% of the well comprising LSCs (Supplementary Figure E5D). We exposed our competition-based expanded stem cells with imatinib treatment for 4 days to determine if our ex vivo model is reproducible in patients and primary CML murine models. We confirmed that LSCs persisted through imatinib treatment, unless treated with a nonphysiologically high dose of 5 μmol/L (Figure 5C; Supplementary Figure E5E) as we and others had previously reported using primary CML mice as well as patient samples [
      • Patel SB
      • Nemkov T
      • Stefanoni D
      • et al.
      Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
      ,
      • Chu S
      • McDonald T
      • Lin A
      • et al.
      Persistence of leukemia stem cells in chronic myelogenous leukemia patients in prolonged remission with imatinib treatment.
      ,
      • Hamilton A
      • Helgason GV
      • Schemionek M
      • et al.
      Chronic myeloid leukemia stem cells are not dependent on Bcr-Abl kinase activity for their survival.
      ]. To validate the targets identified by our bulk and scRNA sequencing, we tested drugs for the pathways identified for LSCs. After competition-based 14-day expansion culture, we subsequently treated the cells with JAK1/2 inhibitors (AZD1480 and ruxolitinib), mTOR inhibitor (rapamycin), TGFβ inhibitor (SB 431542), MAPK inhibitor (U0126), and Wnt signaling pathway inhibitor (iCRT14) (Figure 5D, Supplementary Figure E5F) [
      • Chu S
      • Holtz M
      • Gupta M
      • Bhatia R.
      BCR/ABL kinase inhibition by imatinib mesylate enhances MAP kinase activity in chronic myelogenous leukemia CD34+ cells.
      ,
      • Li J
      • Xue L
      • Hao H
      • Han Y
      • Yang J
      • Luo J
      Rapamycin provides a therapeutic option through inhibition of mTOR signaling in chronic myelogenous leukemia.
      ,
      • Gallipoli P
      • Cook A
      • Rhodes S
      • et al.
      JAK2/STAT5 inhibition by nilotinib with ruxolitinib contributes to the elimination of CML CD34+ cells in vitro and in vivo.
      ,
      • Moller GM
      • Frost V
      • Melo JV
      • Chantry A.
      Upregulation of the TGFbeta signalling pathway by Bcr-Abl: Implications for haemopoietic cell growth and chronic myeloid leukaemia.
      ,
      • Gonsalves FC
      • Klein K
      • Carson BB
      • et al.
      An RNAi-based chemical genetic screen identifies three small-molecule inhibitors of the Wnt/wingless signaling pathway.
      ]. We observed that among all these treatments, only iCRT14, SB 431542, AZD1480, and ruxolitinib were significantly able to reduce the count of LSK CD150+ CML cells with no difference observed in relative frequency (Figure 5D; Supplementary Figure E5F). However, AZD1480 and ruxolitinib also affected the nonmutant LSK CD150+ cells, suggesting that targeting the JAK signaling pathway could potentially lead to toxic effects in patients (Figure 5D; Supplementary Figure E5F). On the other hand, inhibiting the Wnt signaling pathway, previously found to eliminate CML LSCs, would be a good target for clinical trials [
      • Zhao C
      • Blum J
      • Chen A
      • et al.
      Loss of beta-catenin impairs the renewal of normal and CML stem cells in vivo.
      ,
      • Schurch C
      • Riether C
      • Matter MS
      • Tzankov A
      • Ochsenbein AF.
      CD27 signaling on chronic myelogenous leukemia stem cells activates Wnt target genes and promotes disease progression.
      ,
      • Zhang B
      • Li M
      • McDonald T
      • et al.
      Microenvironmental protection of CML stem and progenitor cells from tyrosine kinase inhibitors through N-cadherin and Wnt-beta-catenin signaling.
      ,
      • Agarwal P
      • Zhang B
      • Ho Y
      • et al.
      Enhanced targeting of CML stem and progenitor cells by inhibition of porcupine acyltransferase in combination with TKI.
      ]. We thus found that ex vivo expanded LSCs can serve as a preclinical tool for testing drugs and dosages and validating drug targets.

      Discussion

      Leukemic stem cells (LSCs) are functionally similar to HSCs, defined by their ability to initiate and propagate all lineages while self-renewing [
      • Lane SW
      • Gilliland DG.
      ]. CML has a well-defined LSC population, phenotypically defined as HSCs. Even though progress has been made in CML treatment, LSCs can evade drug treatment because of their heterogeneous properties. scRNA sequencing of CML stem and progenitor cells revealed heterogeneity of LSCs with enrichment of metabolic and signaling pathways in one cluster and cytokine–chemokines in the other cluster. scRNA sequencing on imatinib-treated CML HSPCs revealed that a persistent signature preexists and undergoes transcriptional rewiring, forming a new clone after imatinib treatment. Wnt, Jak–STAT, MAPK, mTOR, and TGFβ signaling pathways are commonly upregulated in LSCs and imatinib-persistent LSCs. CML LSCs form a minor part (<0.01%) of the whole bone marrow [
      • Holyoake T
      • Jiang X
      • Eaves C
      • Eaves A.
      Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia.
      ,
      • Jamieson CH
      • Gotlib J
      • Durocher JA
      • et al.
      The JAK2 V617F mutation occurs in hematopoietic stem cells in polycythemia vera and predisposes toward erythroid differentiation.
      ,
      • Zhang B
      • Ho YW
      • Huang Q
      • et al.
      Altered microenvironmental regulation of leukemic and normal stem cells in chronic myelogenous leukemia.
      ,
      • Elias HK
      • Schinke C
      • Bhattacharyya S
      • Will B
      • Verma A
      • Steidl U
      Stem cell origin of myelodysplastic syndromes.
      ]; therefore, in this study, we adapted a protocol for long-term ex vivo expansion of phenotypical and functional LSCs [
      • Wilkinson AC
      • Ishida R
      • Kikuchi M
      • et al.
      Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.
      ]. We found that these cultures result in an ∼450- to 1000-fold expansion of LSCs and HSCs, which maintain engraftment and multilineage potential, a measure of stem cell function. Moreover, expanded LSCs share most transcriptional signatures with primary LSCs. Drug testing using our ex vivo expansion culture revealed that inhibition of Wnt and TGFβ signaling pathways is a promising target. These pathways have been previously identified in CML, thus validating our ex vivo expansion cultures. Using the primary CML transgenic mouse model as an in vivo setting and the ex vivo expansion of LSCs provides assays requiring more cells that have previously not been feasible to carry out with LSCs, such as large drug screens.
      Ex vivo cultures, such as colony assays, and liquid cultures with cytokine cocktails have been used for years as functional progenitor readouts and drug testing [
      • Purton LE
      • Scadden DT.
      Limiting factors in murine hematopoietic stem cell assays.
      ]. The most widely used systems are 2-D suspension cultures and cocultures, 3-D organoids, and, more recently, organ-on-a-chip [
      • Kitaeva KV
      • Rutland CS
      • Rizvanov AA
      • Solovyeva VV.
      Cell culture based in vitro test systems for anticancer drug screening.
      ,
      • Cucchi DGJ
      • Groen RWJ
      • Janssen J
      • Cloos J.
      Ex vivo cultures and drug testing of primary acute myeloid leukemia samples: Current techniques and implications for experimental design and outcome.
      ,
      • McKee C
      • Chaudhry GR.
      Advances and challenges in stem cell culture.
      ,
      • Ribeiro-Filho AC
      • Levy D
      • Ruiz JLM
      • Mantovani MDC
      • Bydlowski SP.
      Traditional and advanced cell cultures in hematopoietic stem cell studies.
      ,
      • Tajer P
      • Pike-Overzet K
      • Arias S
      • Havenga M
      • Staal FJT.
      Ex vivo expansion of hematopoietic stem cells for therapeutic purposes: Lessons from development and the niche.
      ,
      • Chou DB
      • Frismantas V
      • Milton Y
      • et al.
      On-chip recapitulation of clinical bone marrow toxicities and patient-specific pathophysiology.
      ]. Although the latter two culture methods mimic a complex in vivo system, they are technically challenging and labor intensive. On the other hand, suspension cultures are cost-effective; however, they sustain LSCs for a short time before they start differentiating, limiting the maintenance of a stemlike property. Additionally, all these cultures have been optimized for stem and progenitor cells, limiting our understanding of LSCs. Ex vivo expansion cultures are cost-effective for expanding LSCs for downstream assays. Importantly, we found that expanded LSCs retain engraftment potential and maintain hematopoiesis. This assay can also be used for clonal studies, initiating the culture with a single cell, thus also considering the heterogeneity of LSCs. This would be especially useful for human patient samples in which surface markers are still being studied to differentiate the mutant LSCs from the non-mutant HSCs [
      • Wilkinson AC
      • Ishida R
      • Kikuchi M
      • et al.
      Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.
      ].
      Heterogeneity of LSCs is an aspect now appreciated to be responsible for transcriptional rewiring, drug persistence, and even relapse [
      • Giustacchini A
      • Thongjuea S
      • Barkas N
      • et al.
      Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia.
      ]. CML LSCs with higher thrombopoietin receptor expression and CD93 and CD26 expression are insensitive to TKI treatment [
      • Zhang B
      • Li L
      • Ho Y
      • et al.
      Heterogeneity of leukemia-initiating capacity of chronic myelogenous leukemia stem cells.
      ,
      • Kinstrie R
      • Horne GA
      • Morrison H
      • et al.
      CD93 is expressed on chronic myeloid leukemia stem cells and identifies a quiescent population which persists after tyrosine kinase inhibitor therapy.
      ]. On the other hand, low expression of CD7 and increased expression of elastase and proteinase 3 have been considered markers of improved patient survival [
      • Yong AS
      • Szydlo RM
      • Goldman JM
      • Apperley JF
      • Melo JV.
      Molecular profiling of CD34+ cells identifies low expression of CD7, along with high expression of proteinase 3 or elastase, as predictors of longer survival in patients with CML.
      ]. Moreover, single-cell RNA sequencing analysis of human CML CD34+ cells obtained at diagnosis or after TKI treatment reveals a persistent population that is quiescent and enriched in TGFβ and TNFα inflammatory signaling pathways and JAK–STAT signaling pathways [
      • Giustacchini A
      • Thongjuea S
      • Barkas N
      • et al.
      Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia.
      ]. However, stem cell phenotype in humans is still under study, making it difficult to extrapolate the findings to LSCs. Hence, using our transgenic CML mouse model and scRNA sequencing, we consistently found that a small subset of CML LSCs have a preexistent TKI-persistent signature. Subsequently, this preexistent clone is selected with TKI treatment and is transcriptionally rewired, leading to disease relapse and even progression to blast. This can be corroborated by our recent work reporting transcriptional and metabolic reprogramming via STAT3 signaling [
      • Patel SB
      • Nemkov T
      • Stefanoni D
      • et al.
      Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
      ]. Additionally, we found that the transcriptional profiles of expanded CML LSCs are similar to those of the freshly sorted CML LSCs compared with their respective non-transformed controls. Drug screens inhibiting pathways commonly upregulated in the CML and persistent CML LSCs revealed that although these pathways are potential targets [
      • Ma L
      • Xu Z
      • Wang J
      • et al.
      Matrine inhibits BCR/ABL mediated ERK/MAPK pathway in human leukemia cells.
      ,
      • Ng KP
      • Manjeri A
      • Lee KL
      • et al.
      Physiologic hypoxia promotes maintenance of CML stem cells despite effective BCR-ABL1 inhibition.
      ,
      • Cheloni G
      • Tanturli M
      • Tusa I
      • et al.
      Targeting chronic myeloid leukemia stem cells with the hypoxia-inducible factor inhibitor acriflavine.
      ,
      • Bewry NN
      • Nair RR
      • Emmons MF
      • Boulware D
      • Pinilla-Ibarz J
      • Hazlehurst LA.
      Stat3 contributes to resistance toward BCR-ABL inhibitors in a bone marrow microenvironment model of drug resistance.
      ,
      • Li J
      • Xue L
      • Hao H
      • Han Y
      • Yang J
      • Luo J
      Rapamycin provides a therapeutic option through inhibition of mTOR signaling in chronic myelogenous leukemia.
      ,
      • Agarwal P
      • Zhang B
      • Ho Y
      • et al.
      Enhanced targeting of CML stem and progenitor cells by inhibition of porcupine acyltransferase in combination with TKI.
      ], not all can be carried forward for clinical trials because of the effect on nonmutant HSCs found in the same inflamed environment as the LSCs. In this study, we thus found that ex vivo expansion is a preclinical tool for identifying and validating drug targets before moving to more complex, time-intensive, and expensive modeling.
      These expansion cultures can be used for high-throughput proteomic screens such as Western blot and immunoprecipitation assays using LSCs as transcripts do not always correlate to the presence of functional protein. Moreover, we can now use LSCs to expand scalable cells for functional genomic studies, such as knockouts and knockins, and metabolic assays, such as seahorse and metabolite flux tracing, that require many more cells. Additionally, these expansion cultures can also be optimized for human LSCs to validate results obtained from murine LSCs. Ex vivo expansion culture can hence be used to answer questions that have been impractical to conduct with rare LSCs that share phenotypic characteristics with HSCs.

      Conflict of interest disclosure

      The authors have declared that no conflict of interest exists.

      Acknowledgments

      We thank Dr. Samuel L. Wolock and Dr. Virginia Camacho for their help with data collection and feedback, respectively. This project was supported by National Institutes of Health (NIH) Grant 1PO1HL131477; startup funds from the Division of Hematology/Oncology at the University of Alabama at Birmingham (UAB); American Cancer Society–Institutional Research Grant Junior Faculty Development Grant (2019); the American Society of Hematology Bridge Grant (2018); and Leukemia Research Funding (2019). Flow cytometry was supported by the UAB Center for AIDS Research (CFAR), an NIH-funded program (P30 AI027767-31).

      References

        • Vainchenker W
        • Kralovics R.
        Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms.
        Blood. 2017; 129: 667-679
        • Ben-Neriah Y
        • Daley GQ
        • Mes-Masson AM
        • Witte ON
        • Baltimore D.
        The chronic myelogenous leukemia-specific P210 protein is the product of the bcr/abl hybrid gene.
        Science. 1986; 233: 212-214
        • Holyoake T
        • Jiang X
        • Eaves C
        • Eaves A.
        Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia.
        Blood. 1999; 94: 2056-2064
        • Jamieson CH
        • Gotlib J
        • Durocher JA
        • et al.
        The JAK2 V617F mutation occurs in hematopoietic stem cells in polycythemia vera and predisposes toward erythroid differentiation.
        Proc Natl Acad Sci USA. 2006; 103: 6224-6229
        • Zhang B
        • Ho YW
        • Huang Q
        • et al.
        Altered microenvironmental regulation of leukemic and normal stem cells in chronic myelogenous leukemia.
        Cancer Cell. 2012; 21: 577-592
        • Vannucchi AM
        • Harrison CN.
        Emerging treatments for classical myeloproliferative neoplasms.
        Blood. 2017; 129: 693-703
        • Bhatia R
        • Holtz M
        • Niu N
        • et al.
        Persistence of malignant hematopoietic progenitors in chronic myelogenous leukemia patients in complete cytogenetic remission following imatinib mesylate treatment.
        Blood. 2003; 101: 4701-4707
        • Patel SB
        • Nemkov T
        • Stefanoni D
        • et al.
        Metabolic alterations mediated by STAT3 promotes drug persistence in CML.
        Leukemia. 2021; 35: 3371-3382
        • Koppikar P
        • Bhagwat N
        • Kilpivaara O
        • et al.
        Heterodimeric JAK-STAT activation as a mechanism of persistence to JAK2 inhibitor therapy.
        Nature. 2012; 489: 155-159
        • Zhang B
        • Li L
        • Ho Y
        • et al.
        Heterogeneity of leukemia-initiating capacity of chronic myelogenous leukemia stem cells.
        J Clin Invest. 2016; 126: 975-991
        • Warfvinge R
        • Geironson L
        • Sommarin MNE
        • et al.
        Single-cell molecular analysis defines therapy response and immunophenotype of stem cell subpopulations in CML.
        Blood. 2017; 129: 2384-2394
        • Giustacchini A
        • Thongjuea S
        • Barkas N
        • et al.
        Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia.
        Nat Med. 2017; 23: 692-702
        • Tong J
        • Sun T
        • Ma S
        • et al.
        Hematopoietic stem cell heterogeneity is linked to the initiation and therapeutic response of myeloproliferative neoplasms.
        Cell Stem Cell. 2021; 28 (e506): 502-513
        • Koschmieder S
        • Gottgens B
        • Zhang P
        • et al.
        Inducible chronic phase of myeloid leukemia with expansion of hematopoietic stem cells in a transgenic model of BCR-ABL leukemogenesis.
        Blood. 2005; 105: 324-334
        • Welner RS
        • Amabile G
        • Bararia D
        • et al.
        Treatment of chronic myelogenous leukemia by blocking cytokine alterations found in normal stem and progenitor cells.
        Cancer Cell. 2015; 27: 671-681
        • Zhang B
        • Strauss AC
        • Chu S
        • et al.
        Effective targeting of quiescent chronic myelogenous leukemia stem cells by histone deacetylase inhibitors in combination with imatinib mesylate.
        Cancer Cell. 2010; 17: 427-442
        • Kuepper MK
        • Butow M
        • Herrmann O
        • et al.
        Stem cell persistence in CML is mediated by extrinsically activated JAK1-STAT3 signaling.
        Leukemia. 2019; 33: 1964-1977
        • Mahon FX
        • Rea D
        • Guilhot J
        • et al.
        Discontinuation of imatinib in patients with chronic myeloid leukaemia who have maintained complete molecular remission for at least 2 years: the prospective, multicentre Stop Imatinib (STIM) trial.
        Lancet Oncol. 2010; 11: 1029-1035
        • Hochhaus A
        • Larson RA
        • Guilhot F
        • et al.
        Long-term outcomes of imatinib treatment for chronic myeloid leukemia.
        N Engl J Med. 2017; 376: 917-927
        • Diral E
        • Mori S
        • Antolini L
        • et al.
        Increased tumor burden in patients with chronic myeloid leukemia after 36 months of imatinib discontinuation.
        Blood. 2020; 136: 2237-2240
        • Naka K
        • Hoshii T
        • Muraguchi T
        • et al.
        TGF-β-FOXO signalling maintains leukaemia-initiating cells in chronic myeloid leukaemia.
        Nature. 2010; 463: 676-680
        • Gallipoli P
        • Pellicano F
        • Morrison H
        • et al.
        Autocrine TNF-α production supports CML stem and progenitor cell survival and enhances their proliferation.
        Blood. 2013; 122: 3335-3339
        • Reynaud D
        • Pietras E
        • Barry-Holson K
        • et al.
        IL-6 controls leukemic multipotent progenitor cell fate and contributes to chronic myelogenous leukemia development.
        Cancer Cell. 2011; 20: 661-673
        • Eiring AM
        • Page BD
        • Kraft IL
        • et al.
        Combined STAT3 and BCR-ABL1 inhibition induces synthetic lethality in therapy-resistant chronic myeloid leukemia.
        Leukemia. 2015; 29: 586-597
        • Hoelbl A
        • Schuster C
        • Kovacic B
        • et al.
        Stat5 is indispensable for the maintenance of bcr/abl-positive leukaemia.
        EMBO Mol Med. 2010; 2: 98-110
        • Herrmann H
        • Cerny-Reiterer S
        • Gleixner KV
        • et al.
        CD34+/CD38- stem cells in chronic myeloid leukemia express Siglec-3 (CD33) and are responsive to the CD33-targeting drug gemtuzumab/ozogamicin.
        Haematologica. 2012; 97: 219-226
        • Zhao K
        • Yin LL
        • Zhao DM
        • et al.
        IL1RAP as a surface marker for leukemia stem cells is related to clinical phase of chronic myeloid leukemia patients.
        Int J Clin Exp Med. 2014; 7: 4787-4798
        • Sadovnik I
        • Herrmann H
        • Eisenwort G
        • et al.
        Expression of CD25 on leukemic stem cells in BCR-ABL1+ CML: Potential diagnostic value and functional implications.
        Exp Hematol. 2017; 51: 17-24
        • Herrmann H
        • Sadovnik I
        • Cerny-Reiterer S
        • et al.
        Dipeptidylpeptidase IV (CD26) defines leukemic stem cells (LSC) in chronic myeloid leukemia.
        Blood. 2014; 123: 3951-3962
        • Bonardi F
        • Fusetti F
        • Deelen P
        • van Gosliga D
        • Vellenga E
        • Schuringa JJ.
        A proteomics and transcriptomics approach to identify leukemic stem cell (LSC) markers.
        Mol Cell Proteomics. 2013; 12: 626-637
        • Kinstrie R
        • Horne GA
        • Morrison H
        • et al.
        CD93 is expressed on chronic myeloid leukemia stem cells and identifies a quiescent population which persists after tyrosine kinase inhibitor therapy.
        Leukemia. 2020; 34: 1613-1625
        • Houshmand M
        • Simonetti G
        • Circosta P
        • et al.
        Chronic myeloid leukemia stem cells.
        Leukemia. 2019; 33: 1543-1556
        • Kitaeva KV
        • Rutland CS
        • Rizvanov AA
        • Solovyeva VV.
        Cell culture based in vitro test systems for anticancer drug screening.
        Front Bioeng Biotechnol. 2020; 8: 322
        • Cucchi DGJ
        • Groen RWJ
        • Janssen J
        • Cloos J.
        Ex vivo cultures and drug testing of primary acute myeloid leukemia samples: Current techniques and implications for experimental design and outcome.
        Drug Resist Updat. 2020; 53100730
        • McKee C
        • Chaudhry GR.
        Advances and challenges in stem cell culture.
        Colloids Surf B Biointerfaces. 2017; 159: 62-77
        • Ribeiro-Filho AC
        • Levy D
        • Ruiz JLM
        • Mantovani MDC
        • Bydlowski SP.
        Traditional and advanced cell cultures in hematopoietic stem cell studies.
        Cells. 2019; 8: 1628
        • Tajer P
        • Pike-Overzet K
        • Arias S
        • Havenga M
        • Staal FJT.
        Ex vivo expansion of hematopoietic stem cells for therapeutic purposes: Lessons from development and the niche.
        Cells. 2019; 8: 169
        • Chou DB
        • Frismantas V
        • Milton Y
        • et al.
        On-chip recapitulation of clinical bone marrow toxicities and patient-specific pathophysiology.
        Nat Biomed Eng. 2020; 4: 394-406
        • Kobayashi H
        • Morikawa T
        • Okinaga A
        • et al.
        Environmental optimization enables maintenance of quiescent hematopoietic stem cells ex vivo.
        Cell Rep. 2019; 28 (e149): 145-158
        • Wilkinson AC
        • Ishida R
        • Kikuchi M
        • et al.
        Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.
        Nature. 2019; 571: 117-121
        • Boitano AE
        • Wang J
        • Romeo R
        • et al.
        Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells.
        Science. 2010; 329: 1345-1348
        • Schaefer BC
        • Schaefer ML
        • Kappler JW
        • Marrack P
        • Kedl RM.
        Observation of antigen-dependent CD8+ T-cell/dendritic cell interactions in vivo.
        Cellular Immunol. 2001; 214: 110-122
        • Wilkinson AC
        • Ishida R
        • Nakauchi H
        • Yamazaki S.
        Long-term ex vivo expansion of mouse hematopoietic stem cells.
        Nat Protoc. 2020; 15: 628-648
        • Kuleshov MV
        • Jones MR
        • Rouillard AD
        • et al.
        Enrichr: A comprehensive gene set enrichment analysis web server 2016 update.
        Nucleic Acids Res. 2016; 44: W90-W97
        • Klein AM
        • Mazutis L
        • Akartuna I
        • et al.
        Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells.
        Cell. 2015; 161: 1187-1201
        • Wolock SL
        • Krishnan I
        • Tenen DE
        • et al.
        Mapping distinct bone marrow niche populations and their differentiation paths.
        Cell Rep. 2019; 28 (e305): 302-311
        • Wolock SL
        • Lopez R
        • Klein AM.
        Scrublet: Computational identification of cell doublets in single-cell transcriptomic data.
        Cell Syst. 2019; 8 (e289): 281-291
        • Rodriguez-Fraticelli AE
        • Weinreb C
        • Wang SW
        • et al.
        Single-cell lineage tracing unveils a role for TCF15 in haematopoiesis.
        Nature. 2020; 583: 585-589
        • Holyoake TL
        • Vetrie D.
        The chronic myeloid leukemia stem cell: Stemming the tide of persistence.
        Blood. 2017; 129: 1595-1606
        • Kesarwani M
        • Kincaid Z
        • Gomaa A
        • et al.
        Targeting c-FOS and DUSP1 abrogates intrinsic resistance to tyrosine-kinase inhibitor therapy in BCR-ABL-induced leukemia.
        Nature Med. 2017; 23: 472-482
        • Chu S
        • Holtz M
        • Gupta M
        • Bhatia R.
        BCR/ABL kinase inhibition by imatinib mesylate enhances MAP kinase activity in chronic myelogenous leukemia CD34+ cells.
        Blood. 2004; 103: 3167-3174
        • Ma L
        • Xu Z
        • Wang J
        • et al.
        Matrine inhibits BCR/ABL mediated ERK/MAPK pathway in human leukemia cells.
        Oncotarget. 2017; 8: 108880-108889
        • Kuntz EM
        • Baquero P
        • Michie AM
        • et al.
        Targeting mitochondrial oxidative phosphorylation eradicates therapy-resistant chronic myeloid leukemia stem cells.
        Nat Med. 2017; 23: 1234-1240
        • Pietras EM
        • Reynaud D
        • Kang YA
        • et al.
        Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.
        Cell Stem Cell. 2015; 17: 35-46
        • Chu S
        • McDonald T
        • Lin A
        • et al.
        Persistence of leukemia stem cells in chronic myelogenous leukemia patients in prolonged remission with imatinib treatment.
        Blood. 2011; 118: 5565-5572
        • Hamilton A
        • Helgason GV
        • Schemionek M
        • et al.
        Chronic myeloid leukemia stem cells are not dependent on Bcr-Abl kinase activity for their survival.
        Blood. 2012; 119: 1501-1510
        • Zhao F
        • Mancuso A
        • Bui TV
        • et al.
        Imatinib resistance associated with BCR-ABL upregulation is dependent on HIF-1alpha-induced metabolic reprograming.
        Oncogene. 2010; 29: 2962-2972
        • Ng KP
        • Manjeri A
        • Lee KL
        • et al.
        Physiologic hypoxia promotes maintenance of CML stem cells despite effective BCR-ABL1 inhibition.
        Blood. 2014; 123: 3316-3326
        • Cheloni G
        • Tanturli M
        • Tusa I
        • et al.
        Targeting chronic myeloid leukemia stem cells with the hypoxia-inducible factor inhibitor acriflavine.
        Blood. 2017; 130: 655-665
        • Cokic VP
        • Mojsilovic S
        • Jaukovic A
        • et al.
        Gene expression profile of circulating CD34+ cells and granulocytes in chronic myeloid leukemia.
        Blood Cells Mol Dis. 2015; 55: 373-381
        • Dagogo-Jack I
        • Shaw AT.
        Tumour heterogeneity and resistance to cancer therapies.
        Nat Rev Clin Oncol. 2018; 15: 81-94
        • Bewry NN
        • Nair RR
        • Emmons MF
        • Boulware D
        • Pinilla-Ibarz J
        • Hazlehurst LA.
        Stat3 contributes to resistance toward BCR-ABL inhibitors in a bone marrow microenvironment model of drug resistance.
        Mol Cancer Ther. 2008; 7: 3169-3175
        • Zhao C
        • Blum J
        • Chen A
        • et al.
        Loss of beta-catenin impairs the renewal of normal and CML stem cells in vivo.
        Cancer Cell. 2007; 12: 528-541
        • Schurch C
        • Riether C
        • Matter MS
        • Tzankov A
        • Ochsenbein AF.
        CD27 signaling on chronic myelogenous leukemia stem cells activates Wnt target genes and promotes disease progression.
        J Clin Invest. 2012; 122: 624-638
        • Zhang B
        • Li M
        • McDonald T
        • et al.
        Microenvironmental protection of CML stem and progenitor cells from tyrosine kinase inhibitors through N-cadherin and Wnt-beta-catenin signaling.
        Blood. 2013; 121: 1824-1838
        • Rothe K
        • Babaian A
        • Nakamichi N
        • et al.
        Integrin-linked kinase mediates therapeutic resistance of quiescent CML stem cells to tyrosine kinase inhibitors.
        Cell Stem Cell. 2020; 27 (e119): 110-124
        • Krenn PW
        • Koschmieder S
        • Fassler R.
        Kindlin-3 loss curbs chronic myeloid leukemia in mice by mobilizing leukemic stem cells from protective bone marrow niches.
        Proc Natl Acad Sci USA. 2020; 117: 24326-24335
        • Rabe JL
        • Hernandez G
        • Chavez JS
        • Mills TS
        • Nerlov C
        • Pietras EM.
        CD34 and EPCR coordinately enrich functional murine hematopoietic stem cells under normal and inflammatory conditions.
        Exp Hematol. 2020; 81 (e16): 1-15
        • Hsieh YC
        • Kirschner K
        • Copland M.
        Improving outcomes in chronic myeloid leukemia through harnessing the immunological landscape.
        Leukemia. 2021; 35: 1229-1242
        • Herrmann O
        • Kuepper MK
        • Butow M
        • et al.
        Infliximab therapy together with tyrosine kinase inhibition targets leukemic stem cells in chronic myeloid leukemia.
        BMC Cancer. 2019; 19: 658
        • Parting O
        • Langer S
        • Kuepper MK
        • et al.
        Therapeutic inhibition of FcγRIIb signaling targets leukemic stem cells in chronic myeloid leukemia.
        Leukemia. 2020; 34: 2635-2647
        • Gazit R
        • Mandal PK
        • Ebina W
        • et al.
        Fgd5 identifies hematopoietic stem cells in the murine bone marrow.
        J Exp Med. 2014; 211: 1315-1331
        • Kiel MJ
        • Yilmaz OH
        • Iwashita T
        • Yilmaz OH
        • Terhorst C
        • Morrison SJ.
        SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.
        Cell. 2005; 121: 1109-1121
        • Purton LE
        • Scadden DT.
        Limiting factors in murine hematopoietic stem cell assays.
        Cell Stem Cell. 2007; 1: 263-270
        • Ilaria Jr., RL
        • Van Etten RA
        P210 and P190(BCR/ABL) induce the tyrosine phosphorylation and DNA binding activity of multiple specific STAT family members.
        J Biol Chem. 1996; 271: 31704-31710
        • Peeper DS
        • Upton TM
        • Ladha MH
        • et al.
        Ras signalling linked to the cell-cycle machinery by the retinoblastoma protein.
        Nature. 1997; 386: 177-181
        • Nieborowska-Skorska M
        • Kopinski PK
        • Ray R
        • et al.
        Rac2-MRC-cIII-generated ROS cause genomic instability in chronic myeloid leukemia stem cells and primitive progenitors.
        Blood. 2012; 119: 4253-4263
        • Li J
        • Xue L
        • Hao H
        • Han Y
        • Yang J
        • Luo J
        Rapamycin provides a therapeutic option through inhibition of mTOR signaling in chronic myelogenous leukemia.
        Oncol Rep. 2012; 27: 461-466
        • Gallipoli P
        • Cook A
        • Rhodes S
        • et al.
        JAK2/STAT5 inhibition by nilotinib with ruxolitinib contributes to the elimination of CML CD34+ cells in vitro and in vivo.
        Blood. 2014; 124: 1492-1501
        • Moller GM
        • Frost V
        • Melo JV
        • Chantry A.
        Upregulation of the TGFbeta signalling pathway by Bcr-Abl: Implications for haemopoietic cell growth and chronic myeloid leukaemia.
        FEBS Lett. 2007; 581: 1329-1334
        • Gonsalves FC
        • Klein K
        • Carson BB
        • et al.
        An RNAi-based chemical genetic screen identifies three small-molecule inhibitors of the Wnt/wingless signaling pathway.
        Proc Natl Acad Sci USA. 2011; 108: 5954-5963
        • Agarwal P
        • Zhang B
        • Ho Y
        • et al.
        Enhanced targeting of CML stem and progenitor cells by inhibition of porcupine acyltransferase in combination with TKI.
        Blood. 2017; 129: 1008-1020
        • Lane SW
        • Gilliland DG.
        Leukemia stem cells. Semin Cancer Biol. 2010; 20: 71-76
        • Elias HK
        • Schinke C
        • Bhattacharyya S
        • Will B
        • Verma A
        • Steidl U
        Stem cell origin of myelodysplastic syndromes.
        Oncogene. 2014; 33: 5139-5150
        • Yong AS
        • Szydlo RM
        • Goldman JM
        • Apperley JF
        • Melo JV.
        Molecular profiling of CD34+ cells identifies low expression of CD7, along with high expression of proteinase 3 or elastase, as predictors of longer survival in patients with CML.
        Blood. 2006; 107: 205-212