Bioinformatic database mining
We performed differential gene analysis using RNA-sequence data in the GEO database (GSE63270 and GSE24797; https://www.ncbi.nlm.nih.gov/gds). The GEPIA database (http://gepia. cancer-pku.cn/) and The cBioPortal database (http://www.cbioportal.org) were applied to analyse the level of HCK mRNA expression in tumour tissues and normal tissues of AML and the correlation between the level of HCK mRNA expression and patient overall survival[21,22]. The Cancer Cell Line Encyclopedia (CCLE) database (www.portals.broadinstitute.org/ccle) was mined to analyse the level of HCK mRNA transcripts in kinds of cell lines.
Cell isolation
To sort human CD34+ CD38-cells, antibodies against the CD34+ (BD Pharmigen, 550761) and CD38- (BD Pharmigen, 562444) markers were used. Red blood cells were lysed using RBC Lysis Buffer (Solarbio, China) before staining. Stained cells were then sorted by the BD FACSAria II instrument. AML patient sample information is in Supplementary Table 1. All human samples were conducted with approval from the Experimental Animal Committee of Shanghai Tongji Hospital.
Mice
HCK knockout mice (denoted as HCK-/-) were provided by Shaoguang Li at the University of Massachusetts Medical School (Worcester, MA, USA). Conditional knockout mice (exon-3 floxed; HCK fl/fl) were successively mated with H11-CAG-FLPO mice (Nanjing Biomedical Research Institute of Nanjing University, China) and Rosa26-CreERT2 mice (Nanjing Biomedical Research Institute of Nanjing University, China) to generate HCK fl/fl-Rosa26-CreER mice (Supplemental Fig. 1a, b). The C57BL/6-CD45.1 mice used as transplant recipients were provided by Caiwen Duan at Shanghai Jiao Tong University School of Medicine (Shanghai, China). The C57BL/6-CD45.2 mice were ordered from LARC, Tongji University (Shanghai, China). All animal experiments were supported by our institution and conducted under the Guideline for Animal Care at Tongji University School of Medicine.
Generation of the murine AML model
Bone marrow (BM) lineage-negative (Lin–) cells were sorted from mice and cultured overnight in 1640 medium supplemented with 20% foetal bovine serum (FBS), 100 ng/mL stem cell factor (SCF), 10 ng/mL granulocyte colony-stimulating factor (G-CSF), 10 ng/mL interleukin-3 (IL-3), and 10 ng/mL interleukin-6 (IL-6). An MSCV-MLL-AF9-IRES-YFP-encoding plasmid and a PCL-ECO packaging plasmid were transfected into 293T cells to produce retroviruses. The above Lin– BM cells were infected by two rounds of spin-occultation in the presence of 10 μg/ml polybrene. The infected bone marrow cells were transplanted into lethally irradiated (10.0 Gy) C57BL/6 mice by tail vein injection. Serial transplantations were performed with 2,000 sorted YFP+ c-Kit+ BM leukaemia cells from primary or secondary recipient mice and were transplanted into lethally irradiated (8.0 Gy) C57BL/6 mice by tail vein injection. Following transplantation, the recipients were provided acidified water (pH 1.3 to 2.0) for two weeks[23]. Where indicated, mice were administered tamoxifen (Sigma) in corn oil (20 mg/mL) daily by intraperitoneal injection for five consecutive days (150 mg per gram of body weight). For homing assays, lethally irradiated (10.0 Gy) recipients were transplanted with 106 HCK+/+ or HCK-/- BM leukaemia cells from the primary recipient mice, and the BM was analysed 16 hours after transplant.
Haematopoietic reconstruction and chimaerism assessment
Non-competitive transplants were performed with 106 BM cells from HCK+/+ or HCK-/-mice, injected into lethally irradiated (10.0 Gy) B6-CD45.1 recipients. Chimaerism was assessed eight weeks after transplantation. To assess the chimaerism of mature cells in BM, antibodies against the following markers were used: Mac1-APC, Gr1- PeCy7, Ter119-APC, B220- PeCy7, CD3-APC, CD45.2-BV421, and CD45.1-BV421. To assess the chimaerism of stem cells and progenitor cells after transplantation, the following panel was used: lineage (CD3, CD4, CD8, Gr1, B220, CD19, and TER119)-APC, Sca1-PeCy7, CD34-FITC, c-KIT-PE, CD135-BV421, CD127-APC/BV421, CD16/32-BV421. All antibodies were purchased from BD Pharmigen and BioLegend. Stained cells were then analysed by the BD FACSVerse instrument, and the data were analysed by FlowJo 10. The following markers were used to define different cell populations: Long-term HSCs (LT-HSCs, Lin-C-kit+Sca-1+Flk2-CD34-), Short-term HSCs (ST-HSCs, Lin-C-kit+Sca-1+Flk2-CD34+), Multipotent progenitors (MPPs, Lin-C-kit+Sca-1+Flk2+CD34+), Common myeloid progenitors (CMPs, Lin-C-kit+Sca-1-CD16/32lowCD34+IL7R-), Granulocyte-monocyte progenitors (GMPs, Lin-C-kit+Sca-1-CD16/32+CD34+IL7R-), Multiple myeloid progenitors (MEMEPs, Lin-C-kit+Sca-1-CD16/32-CD34-IL7R-), Common lymphoid progenitors (CLPs, Lin-C-kitlowSca-1lowIL7R+), B cells (B220+), T cells (CD3+) and early erythroid cells (Ter119+). For homing assays, lethally irradiated (10.0 Gy) C57BL/6-CD45.1 recipients were transplanted with 106 C57BL/6-CD45.2 HCK+/+ or HCK-/- BM cells from the primary recipient mice, and the BM was analysed 16 hours after transplant.
Cell lines and cell culture
KG-1α, HEL, K562, NB4, THP-1, U937, MV4-11, and 293T cell lines were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China). The 293T cell line was maintained in DMEM; the KG-1α, HEL, and K562 cell lines were cultured in IMDM; and the rest of the cell lines were cultured in RPMI-1640 medium. The mentioned media were supplemented with 10% foetal bovine serum (FBS). The cells were cultured at 37°C in a humidified atmosphere of 5% CO2.
Lentivirus construction, infection, andphenotype analysis
Lentiviral short hairpin RNAs (shRNAs) targeting human HCK, CDK6, and a scrambled shRNA (SCR) were constructed using a lentiviral vector, pLVX-shRNA2 (Clontech Laboratories, Inc., Mountainview, CA, USA). The interference sequences were as follows: shRNA-HCK-1, 5’- GCTGTGATTTGGAAGGGAA-3’; shRNA-HCK-2, 5’-GGATAGCGAGACCACTAAA-3’; shRNA-CDK6, 5’- GAGTAGTGCATCGCGATCTAA-3‘; shRNA-c-Myc-1, 5’- CAGTTGAAACACAAACTTGAA-3’; shRNA-Scramble, 5'-GTTCTCCGAACGTGTCACGT-3'. For the rescue experiments, the retroviral plasmid MSCV-IRES-mCherry was used. Lentiviruses were produced using the calcium phosphate transfection method with the packaging plasmids pCMV-dR8.91 dvpr (Δ89) and pCMV-VSV-G (VSVG). The lentiviral supernatant was used to infect several human leukaemia cell lines, which were subjected to proliferation, colony formation and signalling pathway analyses in vitro at the indicated time points.
Colony-forming assays
A colony assay for murine LSCs was performed by plating 500 sorted YFP+c-Kit+ LSCs on methylcellulose (MethoCult M3434, Stem Cell Technologies), and colonies were counted 14 days later. A standard two-layer soft agar culture was used for the colony-forming assay of the human cell lines (0.6% agarose bottom layer and a 0.3% agarose top layer). The cells were seeded at 105/ml in 24-well plates with soft agar. Similarly, colonies were imaged and counted 14 days after plating.
Western blotting
Cell lysates of fluorescence-activated cell sorting (FACS)-purified HCK+/+ or HCK-/- YFP+ c-Kit+ LSCs, shRNAs targeting human HCK and CDK6, CDK6-overexpressing THP-1 cells, and their control cells were electrophoresed on 10% SDS polyacrylamide gels and transferred onto PVDF membranes (Millipore). The membranes were blocked with 5% non-fat milk and reacted with the indicated primary antibodies, followed by incubation with appropriate HRP-conjugated secondary antibodies. The primary antibodies were as follows: anti-HCK (Abcam, ab124245), anti-CDK6 (Cell Signaling Technology, 3136), anti-c-Myc (Cell Signaling Technology, 18583), anti-MAPK1/2 (Cell Signaling Technology, 4695), anti-p-MAPK1/2 (Cell Signaling Technology, 4376), and anti-β-Actin (Cell Signaling Technology, 3700).
RT-PCR
Total RNA was extracted from cells using a Quick-RNATM Microprep kit (Zymo). An equal amount of RNA from the samples was reverse transcribed into cDNA with a FastQuant RT Kit (TIANGEN), and qPCR was performed using an ABI 7500 sequence detection system using the primers listed in Supplemental Table 2. All primers were obtained from PrimerBank[24].
Proliferation assay for human cell lines
Cells were plated at 10,000 cells/ml in a 24-well plate for the proliferation assay. Cells were counted for six consecutive days.
Cell Counting Kit-8 (CCK-8) assay of human cell lines
Cells were seeded in 96-well plates for cell viability analysis using Cell Counting Kit-8 (CCK-8; Dojindo). CCK-8 assays were performed with three replicates, and the optical density (OD) values at 450 nm were measured using a microplate imaging system.
Apoptosis analysis
Apoptosis was analysed by staining with Annexin V and propidium iodide (BD Biosciences) according to the manufacturer’s instructions. Flow cytometry analysis was then performed within 1 hour. The cell apoptotic ratio was detected by a FACSVerse cytometer (BD Biosciences) and was analysed by FlowJo 10.
Cell cycle analysis in vivo and in vitro
We used immunofluorescent staining of incorporated BrdU (BD Biosciences) and flow cytometric analysis to determine the frequency and nature of individual cells that have synthesized DNA. In vivo, intraperitoneal injection of BrdU (2 mg at 10 mg/mL) for 4 hours. In vitro, the addition of BrdU (5 µg/ml) for 2 hours.
Statistical analysis
All quantitative data are represented as the mean ± SEM. Unpaired 2-tailed Student’s t-test was used to assess differences between two independent groups. The Mann-Whitney U test was used to assess differences between nonparametric data, and one-way analysis of variance (ANOVA) was used to assess multigroup comparisons. For the Kaplan-Meier survival analysis, the log-rank test was used. Differences with a p-value less than 0.05 were considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001). The data were analysed using GraphPad Prism 8.