Lymphoprep

PBMC and serum samples were collected at a single time point for each patient (Supplementary Table S2).
PBMCs were isolated from the whole blood using either Cellular Preparation Tubes (CPT) tubes (BD vacutainer, # 362782) or Lymphoprep™ (Serumwerk Berburg, # 1858). CPT tubes were spun at 1,600g for 25 min at RT and plasma collected and stored at −20°C for future analysis. Lymphoprep™ isolation was performed according to the manufacturer’s instructions. Briefly, a 1:1 mix of blood and PBS (35 mL) was layered onto 10–15 mL Lymphoprep™ solution in a 50-mL Falcon tube. Tubes were then centrifuged for 25 min at 800g and PBMC collected, washed in PBS (Gibco, # 10010-015), and spun three more times (400g, 7 min, 4°C) before being counted and resuspended in Fetal bovine serum (FBS) (Gibco, # 10270-106) complemented with 10% Dimethyl sulfoxide (DMSO). After overnight pre-chilling at −80°C in Mr. Frosty (Nalgene™, # 5100-0001), cells were transferred to liquid nitrogen for long-term storage.

PBMCs were separated from 15 mL of peripheral blood using Lymphoprep (Stemcell 29283-PIS) following manufacturer recommendations. Briefly, 15 mL of Lymphoprep at room temperature was added to 50 mL Falcon tubes. 15 mL of peripheral blood was mixed with 15 mL of RPMI-1640 media, and then layered on top of Lymphoprep prior to centrifugation at 800 × g for 30 min at RT without brake. Opaque layer containing Bone Marrow Mononuclear Cells (BMMCs) was retained and washed with D-PBS, followed by Red Blood Cell Lysis. B-cells were isolated using Easysep Human B-cell Isolation Kit (STEMCELL cat# 17954) following manufacturer recommendations prior to CSC Micro Proteomics and TMT labeling.

Mononuclear cells were isolated from peripheral blood samples of healthy donors for hematopoietic stem cell transplantation (n=8) and bone marrow samples of newly diagnosed patients with AML (n=17), which were obtained with the informed consent for research purposes only at Department of Hematology, Nanfang Hospital, Southern Medical University and Department of Hematology, First Affiliated Hospital of Xiamen University. This study is approved by the Ethics Review Board of Nanfang Hospital and First Affiliated Hospital of Xiamen University, and performed in accordance with the Declaration of Helsinki. Clinical characteristics are summarized in Table 1. Mononuclear cells were isolated by density gradient centrifugation using Lymphoprep (BD, Franklin Lakes, NJ, USA) and cultured in IMDM (HyClone, Thermo Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Life Technologies, Grand Island, NY, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (1 × P/S) after enrichment with a CD34 selection MACS kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol.

MV4–11 and MOLM-13 (TP53 wild type, FLT3-ITD positive) and U937, THP-1, and KG-1a (TP53 mutated or null), as well as MV4–11-luciferase-GEP cells were kindly provided by professor PT Liu (Wellcome Trust Sanger Institute, UK). Cells were cultured in RPMI-1640 medium or Iscove’s modified Dulbecco’s medium (HyClone, Thermo Scientific, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Life Technologies, NY, USA) at 37 °C in a humidified CO₂ incubator. Bone marrow samples were obtained with the informed consent from healthy donors of hematopoietic stem cell transplantation (n = 10) and AML patients (n = 40) at Department of Hematology, the First Affiliated Hospital of Xiamen University (Xiamen, China). Bone marrow mononuclear cells (BMMC) were isolated by density gradient centrifugation using Lymphoprep (BD, Franklin Lakes, NJ, USA) and cultured in RPMI-1640 medium supplemented with 10% FBS. This study was approved by the Ethics Committee of the First Affiliated Hospital of Xiamen University and conducted in agreement with the guidelines of the Declaration of Helsinki and.

B-ALL patient samples were obtained after informed consent following the tenets of the Declaration of Helsinki. The study was approved by the Ethical Committee board of the University of Padova, the Padova Academic Hospital and the Italian Association of Pediatric Onco-Hematology (AIEOP). Diagnosis was made according to standard cytomorphology, cytochemistry and immunophenotypic criteria [46 ]. All analyzed B-ALL samples were obtained at the time of diagnosis before treatment, after Lymphoprep (Fresenius KABI, Norge AS) separation of mononuclear cells. The percentage of CD19⁺ cells ranged from 80% to 95%.
Peripheral blood mononuclear cells (PBMC) and bone marrow cells from healthy donors were obtained by separation on Lymphoprep (Fresenius KABI, Norge AS) gradient. After extensive washing, cells were resuspended (1.0 × 10⁶ cells/ml) in RPMI1640 with 10% fetal bovine serum and incubated overnight in 96-well tissue culture microtiter plate. For cytotoxicity evaluations in proliferating PBMC cultures, non-adherent cells were resuspended in growth medium, containing 2.5 μg/ml phytohematoglutinin (PHA) (Irvine Scientific). To isolate B-lymphocyte, PBMC obtained by Lymphoprep (Fresenius KABI, Norge AS) separation were labeled with anti-CD19-APC (BD Biosciences, Italy) and collected by cell sorting.