Kasumi 1

The human AML cell lines (THP1, NOMO1, Kasumi-1 and SKNO-1) were obtained from State Key Laboratory of Experimental Hematology. THP1, NOMO1 and Kasumi-1 cells were cultured at 1-2 × 10⁶ cells/mL in RPMI-1640 (C11875500BT, GIBCO, USA) containing 10% fetal bovine serum (FBS, C04001, Vivacell, China), 1% penicillin/streptomycin (SV30010, HyClone, USA). In addition to the above components, cell culture medium for SKNO-1 was supplemented with 10 ng/ml GM-CSF (300-03, PeproTech, USA). Cells were maintained at 37°C in a humidified incubator (ThermoFisher, USA).

Eight human leukemia cell lines were evaluated. Four lineages were derived from AML patients: Kasumi-1 (ACC 220) with RUNX1-RUNX1T1 (AML1-ETO; t(8;21)); OCI-AML3 (ACC 582) with mutation type A in NPM1 and DNMT3A R822C mutation; NB-4 (ACC 207), from acute promyelocytic leukemia, with PML-RARa (t(15;17)); MOLM-13 (ACC 554) secondary AML FAB M5a after initial myelodysplastic syndromes, FLT3-ITD positive. Two other cell lines were obtained from CML patients: KCL-22 (ACC 519) with BCR-ABL1, t(9;22), e13-a2 (b2a2); K562 (ACC 10) with BCR-ABL1, t(9;22), e14-a2 (b3a2). The two remaining cell lines originated from ALL patients: MOLT-4 (ACC362) T-lymphoblastic leukemia cell line; REH (ACC 22) derived from B-cell precursor leukemia with ETV6-RUNX1 (TEL-AML1; t(12;21). All cell lines were cultured in RPMI-1640 (Gibco, Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% (KCL-22, K562, REH, NB-4) or 20% (MOLT-4, OCI-AML3, MOLM-13, Kasumi-1) fetal bovine serum (FBS, Gibco) and 1% penicillin–streptomycin (Gibco) at 37 °C in a 5% CO₂ atmosphere, according to the manufacturer’s instructions (DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany). Exponentially growing cells were used in at least triplicate experiments. The cells were washed with phosphate-buffered saline (PBS), and 5 × 10⁶ cells were used for RNA extraction.

Three human AML cell lines, Kasumi-1 (ATCC, Manassas, VA) with t(8;21) and heterozygous KIT N822K mutation, SKNO-1 (JCRB, Osaka, Japan) with t(8;21) and homozygous KIT N822K mutation, and OCI-AML3 (DSMZ, Brauschweig, Germany) with NPM1 mutation and DNMT3A mutation, were used in this study. Kasumi-1 cells were maintained in RPMI 1640 medium (ThermoFisher Scientific/GIBCO, Waltham, MA) containing 20% fetal bovine serum (FBS), SKNO-1 cells in RPMI 1640 medium with 10% FBS containing 10 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF; R&D systems, Minneapolis, MN), and OCI-AML3 cells in αMEM medium (GIBCO) containing 20% FBS. Cultures were grown at 37 °C in humidified atmosphere containing 5% CO₂. These cell lines were authenticated (16-Markers STR) by the Food Industry Research and Development Institute, Hsinchu, Taiwan. Their genetic profiles were identical to reported information.
Cabozantinib-malate was purchased from Selleck Chemicals (Houston, TX) and dissolved in DMSO for storage at −20 °C. Cycloheximide (CHX), MG-132, Z-VAD-FMK, and puromycin were purchased from TargetMol (Boston, MA), Tocris Bioscience (Abingdon, UK), Abcam (Cambridge, MA), and GIBCO, respectively.

Six established human AML cell lines, as well as normal human primary bone marrow CD34+ cells, were cultured in vitro for the analysis of BSG, MCT1, and VEGF expression (as previously described [35 (link)]). The normal CD34+ cells were used as a control. AML cell lines used in the study were Kasumi-3, Kasumi-1, NB4, HT93, MUTZ-3, and U937. All the cell lines were purchased from either the American Type Culture Collection (ATCC, Rockville, MD, USA) or Leibniz Institute DSMZ (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany) and maintained at the Cell Culture Collection of the Hirszfeld Institute of Immunology and Experimental Therapy (Wrocław, Poland). The HT93, NB4, Kasumi-1, Kasumi-3, and U937 lines were grown in RPMI-1640 (Gibco, Carlsbad, CA, USA) medium supplemented with 10% (NB4, U937) or 20% (HT-93, Kasumi-1, −3) FBS (Gibco, Carlsbad, CA, USA) and antibiotics. NB4 was additionally supplemented with GlutaMax (Gibco, Carlsbad, CA, USA). Normal CD34+ cells were grown in PromoCell Hematopoietic Progenitor Medium (PromoCell GmbH, Heidelberg, Germany) and antibiotics, while MUTZ-3 was grown in MEMalfa medium (Gibco, Carlsbad, CA, USA) supplemented with 20% FBS and 20% 5637 conditioned medium (CM). All cells were incubated with 5% CO₂ at 37 °C. Cells and cell culture media were collected for further analyses.

Bone marrow samples were obtained from primary AML patients and healthy donors with informed consent in accordance with the Declaration of Helsinki. Studies were approved by the ethics committee of Zhejiang Provincial People’s Hospital, People’s Hospital of Hangzhou Medical College. KG1a, Kasumi-1, HL-60, MOLM-13, THP-1, U937 cells (human AML cell lines), HEK293 and HEK293T (human embryonic kidney cell line) were obtained from American Type Culture Collection (ATCC, Manassas, VA). KG1a, Kasumi-1, HL-60, THP-1, and U937 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS, GIBCO, Bethesda, MD, USA). MOLM-13 cells were cultured in an IMDM medium with 10% FBS. HEK293 and HEK293T cells were cultured in a DMEM medium with 10% FBS.