Um729

HEK293T cells (ATCC CRL-3216) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) plus GlutaMAX (Gibco) supplemented with 10% (v/v) fetal bovine serum (Gibco, qualified) and 1% (v/v) penicillin-streptomycin (Gibco). K562 (ATCC CCL-243) and Jurkat (ATCC TIB-152) cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium plus GlutaMAX (Gibco) supplemented with 10% (v/v) fetal bovine serum (Gibco, qualified) and 1% (v/v) penicillin-streptomycin (Gibco). Human cord-blood-derived primary CD34+ hematopoietic stem and progenitor cells (HSPC) (70008.2, StemCell Technologies) were cultured in StemSpan SFEM II (StemCell Technologies) supplemented with 1 x StemSpan CD34 + expansion supplement (StemCell Technologies), 1 μM UM729 (StemCell Technologies) and 1% (v/v) penicillin-streptomycin (Gibco). Cells were authenticated by the supplier using STR (short tandem repeat) analysis. All described cells were grown at 37 °C in 5% CO2 incubators and passaged upon reaching 80% confluency. Cell culture media was tested for mycoplasma contamination every 2 months using the Myco-Blue Mycoplasma Detector (Vazyme) and all tests were negative throughout the experiments.

De-identified cord blood samples were purchased from the State University of New York Upstate Medical Center’s Upstate Cord Blood Bank Program. Mononuclear cells were isolated using Ficoll-Paque Plus (Cytiva). CD34 cells were then purified using human CD34 MicroBead kit (Miltenyi Biotec) following the manufacturer’s protocol and cultured in CD34 expansion medium (StemSpan SFEMII (Stemcell Technology), 50 ng/ml SCF, IL-3, IL-6, TPO, FLT3L (Peprotech) and 500 nM UM729 (Stemcell Technology)).

C/EBPA^N/C-8 cells were cultured at the concentration of 1 × 10⁶ cell/mL in StemSpanTM Serum-Free Expansion Medium II (SFEMII) plus 10% StemSpanTM CD34+ Expansion Supplement and 175 nM UM171 (STEMCELL Technologies). All other patient cells were grown in SFEMII supplemented with 100 ng/mL thrombopoietin, 10 ng/mL FMS-like tyrosine kinase 3 ligand, 750 nM Stem Regenin 1 (SRI), 10 ng/mL Interleukin 3, 10 ng/mL human granulocyte/macrophage colony stimulating factor, 150 ng/mL Stem Cell Factor (all Peprotech) and UM729 (STEMCELL Technologies). Patient CD34+ or CD117+ progenitors were isolated with CD34 MicroBead Kit, human or CD117 MicroBead Kit, human (Miltenyi-Biotech) as described in [4 (link)].

MAT2A inhibitor PF-9366 (Carbosynth, Compton, UK), DOT1L inhibitor EPZ004777 (Tocris, Bristol, UK), and PRMT5 inhibitor EPZ015666 (Sigma-Aldrich) were prepared in stock solutions with DMSO [18 (link),57 (link),58 (link),59 (link)]. Cytarabine (Ara-C, Stadapharm, Bad Vilbel, Germany) was diluted in PBS for stock solutions. MLLr cells and culture-expanded CD34⁺ huCB control cells seeded with 7.5 × 10⁵ cells/mL were subjected to inhibitor treatment in StemMACS HSC Expansion Media XF (Miltenyi, Bergisch Gladbach, Germany) supplemented with 1% penicillin-streptomycin (Lonza), 10% FBS, and 50ng/mL G-CSF, TPO, SCF. FLT3L, IL-3, and IL-6 (Peprotech, Rocky Hill, NJ, USA), and 0.75 µM SR-1 and UM-729 (Stem Cell Technologies, Vancouver, BC, Canada) for a total of 6 days or otherwise as indicated. Cells were retreated and reseeded at the original density every second day. For long-term experiments, treatment was prolonged to a maximum of 2 weeks. For sequential experiments, cells were preincubated for 6 days, compound was washed out, and cells were reseeded at 7.5 × 10⁵ cells/mL followed by respective treatment.
SKM-1 and SEM cells were seeded at 0.5 × 10⁶/mL and THP-1 cells at 0.2 × 10⁶/mL. Cells were subjected to inhibitor treatment for 2, 4, or 6 days in the respective medium described above and reseeded at the original density after 4 days.

CD34+ hematopoietic stem and progenitor cells (HSPCs) were isolated from fresh human umbilical cord blood (huCB) obtained from the Department of gynecology from the University Hospital Tuebingen (IRB approvals 751/2015BO2 and 461/2022BO2) using Ficoll Paque (PAN-Biotech GmbH, Aidenbach, Germany) followed by the isolation of HSPCs with the human CD34+ Microbead Kit (Miltenyi, Bergisch Gladbach, Germany). t(4;11) was induced using an RNP complex of specific self-made sgRNAs and Cas9 protein (PNA Bio Inc., Thousand Oaks, CA, USA) as previously described [8 (link), 54 (link)]. A pure culture of t(4;11) cells was defined by more than 90% of translocated cells determined by Fluorescence-In-Situ-Hybridization (FISH) as previously described [54 (link)]. CD34+ and t(4;11) cells were maintained in StemMACS™ HSC Expansion Media (Miltenyi) supplemented with 10% FCS (Gibco by Thermo Fisher Scientific, Waltham, MA, USA), 1% Penicillin/Streptomycin (Lonza, Basel, Switzerland), human cytokines (IL-3, IL-6, SCF, FLT3L, SCF, G-CSF, 50 ng/ml each, PeproTech, Rocky Hill, NJ, USA), SR-1 and UM-729 (0.75 µM each, STEMCELL Technologies, Vancouver, Canada).

Primary AML cells for scRNA-seq were cultured on hMSC feeders as described above in the following media: SFEMII (StemCell Technologies), 1 μM UM729 (StemCell Technologies), 750 nM StemReginin 1 (StemCell Technologies) supplemented with 150 ng/mL SCF, 100 ng/mL TPO, 10 ng/mL IL-3, 10 ng/mL G-CSF (Perpro tech). After 1 passage (1 week) in culture cells were treated for 24 h with 10 μM CBFβi or 0.1% DMSO in the absence of UM729 and StemReginin 1. After treatment cells were sorted for CD45 using magnetic beads (Miltenyi Biotec). Cells were loaded on a Chromium Single Cell Instrument (10X Genomics), to recover 5000 single cells. Library generation was performed using the Chromium single cell 3′ library and gel bead kit v3.1. Illumina sequencing was performed on a NovaSeq 6000 S1 run in paired-end mode for 150 cycles at a depth of 20000 reads per cell.

Umbilical cord blood units were obtained with IRB approval from the Carolinas Cord Blood Bank (Duke University). All methods were carried out in accordance with relevant guidelines and regulation. Informed consent was obtained for all subjects donating cord blood units to the bank. Mononuclear cells were extracted using Ficoll then counted via trypan blue to assess viability. CD34⁺ cells were then extracted utilizing the human UltraPure CD34 MicroBead Kit (Miltenyi, 130-100-453). Cells were expanded as described^{18 (link)} in Stemspan H3000 serum free media (StemCell Technologies, #09800) supplemented with 100 ng/mL of human thrombopoietin, IL-6, FLT3 ligand, stem cell factor (Gemini) and 1 µM StemRegenin-1 (SR-1, Cayman Chemicals, #10625).
De-identified patient samples were obtained from the Children’s Oncology Group (COG) through the AML Biology Committee (AAML15B9-Q). Bone marrow samples containing 70% blasts or greater were from patients under 21 years of age at diagnosis prior to receiving any therapy. Samples were thawed and expanded in cytokines, SR-1, and UM-729 (StemCell, #72332) as described^{19 (link)}. Samples were considered satisfactory quality for analysis if >50% viability was maintained after 5 days in culture.