Cell biology methods

10 cycles with the modification of using KAPA Hifi 2x ready mix kit. For the Nextera XT DNA Library Preparation kit 4 ng of cDNA was used as input and library preparation performed according to the manufacturer’s protocol, with the only exception of using a custom i5 primer (P5NEXTPT5). Sequencing was performed on an Illumina HiSeq 1500 on a flow cell with single end layout utilizing the standard Illumina sequencing primers and index primers. Sample reads were sequenced using 50 cycles and the UMI sequence using 16 cycles.

2.2.1.16.2 Data Processing

To obtain expression data the raw fastq files were processed by the zUMIs pipeline using default parameters²⁰⁰. Mapping to the human reference genome hg38 was performed by STAR¹⁹⁸ (version 2.5.2b) and the gene annotation GRCh38.84 was taken from Ensembl.

Differential expression analysis was performed using limma²⁰¹. For this analysis genes with a read count below 10 in all of the samples were filtered out and library sizes scaled using the package edgeR²⁰². Count data was transformed to log2-counts per million and the mean variance calculated to compute the precision weights. In order to increase statistical power empirical Bayes moderation was applied and the false discovery rate calculated by theBenjamini-Hochberg procedure.

together with cell suspension in an appropriate ratio (e.g. 3:1; 4:1; or 5:1) was placed in a new flask.

HEK293T cells were cultured in DMEM medium supplemented with 10% (v/v) FBS and 0.5% (v/v) Penicillin/Streptomycin. To passage HEK293T cells that are grown to 80-90%

confluence, cells were gently washed with PBS and incubated in trypsin-EDTA solution for 5 min at 37°C in the incubator. After detachment of cells, medium in a 1:4 ratio was added to inactivate trypsin and the cell suspension was transferred to a conical tube. Cells were centrifuged (1100 rpm, 5 min, RT), resuspended in fresh medium and placed in a TC flask.

PDX AML samples were established and recovered from mice as previously described⁵¹.PDX cells were enriched with the mouse cell depletion kit according to the supplier’s recommendation. For in vitro cultivation, PDX cells were kept in DD medium according to a previously described recommendation²⁰³ for up to 7 days. DD medium contained StemPro-34 SFM media supplemented with 2% (v/v) FBS, 1% (v/v) Penicillin/Streptomycin, 1% (v/v) L-glutamine, 10 ng/mL of rhIL3, rhTPO, rhFLT3-ligand, and rhSCF.

2.2.2.2 Cell thawing and freezing

For storage purposes, 5x10⁶ viable cells were centrifuged (1100 rpm, 5 min, RT) and the cell pellet was resuspended in 5 mL FBS containing 10% (v/v) DMSO. 1 mL aliquots of the cell suspension were each dispensed into cryogenic storage vials (Nunc^TM Cryo Tube).

Vials were placed in an isopropanol freezing container (Mr. Frosty^TM Freezing Container) and stored at -80°C overnight. For long-term storage, frozen cells were transferred to a tank of liquid nitrogen.

To thaw cells, frozen cells stored at -80°C or in liquid nitrogen were quickly thawed in a water bath at 37°C and transferred to a conical tube containing 5 mL of respective media.

After centrifugation (1100 rpm, 5 min, RT), the cell pellet was resuspended in 2-4 mL fresh medium (amount depending on the appropriate cell density for each cell line) and the cell suspension was placed in 2-4 wells of a 48-well plate. After 1-3 days, suspension cells were gradually expanded by transferring cells to bigger wells (24-well or 12-well plate).

Adherent cells were resuspended in 10 mL fresh medium and transferred to a TC flask T75.

PDX AML cell were thawed according to the protocol of Dominique Bonnet²⁰⁴ to obtain high viability. Briefly, cells were rapidly thawed in a 37°C water bath and 100 μL of DNase I (1 mg/mL) was added drop wise into the cryogenic vial. Cell suspension was gently mixed, incubated for 1 min and transferred into a 50 mL conical centrifuge tube. 1 mL FBS was added drop wise and cell suspension was gently mixed. After 1 min, 10 mL PBS with (v/v) 2% FBS was slowly added and incubated for 1 min. Volume was then slowly added up to 30 mL with PBS containing 2% (v/v) FBS. Cells were centrifuged (1100 rpm, 5 min, 4°C) and resuspended in DD medium.

2.2.2.3 Mycoplasma testing

To test for mycoplasma contamination in cell cultures, the MycoAlert® Mycoplasma Detection kit, which detects the enzymatic activity of mycoplasma, was used according to the supplier’s recommendation. An aliquot (0.2 - 0.5 mL) was taken from each cell line grown in culture and transferred to a micro tube 4-6 days after thawing and subsequently every 7 days or before the start of an experiment. After centrifugation (1500 rpm, 10 min, RT), 25 µL of supernatant was transferred to a white bottom 96-well plate and mixed with 25 µL of MycoAlert^TM Reagent. The plate was incubated for 5 min at RT to lyse mycoplasma cells and luminescence was measured with a Microplate reader GloMax^® Discover (read A). After the addition of 25 µL of MycoAlert^TM Substrate, reaction was incubated for 10 min at RT in the dark. During this time, mycoplasma enzymes react with the MycoAlert^TM Substrate and generate ATP, which is then transferred into a light signal via the luciferase enzyme. Luminescence was measured a second time (read B). The ratio of B/A indicates the absence (<0.9) or presence (>1.1) of mycoplasma contamination.

2.2.2.4 Transient transfection

HEK293T cells were transiently transfected using the calcium-phosphate precipitation method. To have a confluency of 80-90%, 7x10⁶ HEK293T cells were seeded in a 10 cm dish. On the next day, medium was removed and 10 mL of fresh medium was slowly added. After 4-5 h, 13 µg endotoxin-free plasmid DNA diluted in 450 µL sterile H2O was mixed with 50 µL of 2M CaCl2. The resulting mixture was slowly added to 500 µL HBS buffer to form Ca3(PO4)2 - DNA complexes. After incubation for 3 to 4 min, the solution was added drop wise to the cells. Cells were incubated in the incubator and medium was changed after 13-15h. Cells were lysed 48h after transfection. For siRNA mediating

silencing of KDM6A expression, Lipofectamine 3000 reagent was used to transiently transfect HEK293T cells using 50 or 100 nM siRNA/well according to the supplier’s recommendation.

Suspension cells were transfected with 100 nM siRNA or 1-2 µg endotoxin-free plasmid DNA using the Nucleofector Kit V on the Amaxa Nucleofector II device according to the supplier’s recommendation. The following preprogrammed settings were used, K562: T-016; MM-1: T-030/T-036. For siRNA mediated silencing of KDM6A expression, K562 cells were nucleofected two times with siRNA against KDM6A or negative control siRNA.

After 72h, cells were nucleofected for a second time and incubated again for 72h, after which cells were used for subsequent analysis.

2.2.2.5 Stable transfection with lentivirus

Production of lentiviral particles expressing shRNA’s against KDM6A or non-target control (Table 6) was performed as previously described^205,206. For transduction, 1x10⁶ K562 cells in 1mL medium were placed in a 24-well plate and lentiviral particles together with 8 μg/mL polybrene were added. After 24h, cells were washed three times with PBS and resuspended in fresh medium. After a few days, transgene positive cells were enriched in two consecutive rounds with one week in between by flow cytometry with the red fluorochrome gate using FACSVantage SE. Knockdown efficiency was determined by immunoblotting with the appropriate antibodies (Table 4).

2.2.2.6 CRISPR/Cas9-mediated gene editing

For generation of KDM6A knockout clones, K562 or MM-1 cells were transfected with 1 µg (MM-1) or 2 µg (K562) pSpCas9(BB)-2A-GFP-gRNA-KDM6A plasmid by nucleofection. After 48h, GFP positive cells were enriched by single-cell sorting into 96-well plates (V-bottom) with the FACSVantage SE. Cells were cultured until colonies were readily visible (10 to 30 days). To screen for KDM6A KO clones, cell lysis, PCR on lysates, and restriction digest were performed as previously described with minor modifications²⁰⁷. Briefly, for gDNA isolation cells in 96-well plate were washed two times with PBS, resuspended in 50 μL/well lysis buffer SC, frozen at -80°C for 30 min, incubated at 56°C for 3 h, and finally Proteinase K heat inactivated at 85°C for 30 min. 2.5 μL/well of the resulting crude cell lysate were directly subjected to PCR (25 μL/rxn, 0.1 μL MyTaq™ DNA Polymerase) using external screening primers (KDM6A_KO_FOR

and KDM6A_KO_REV) and following cycling settings: 95°C/5 min - [95°C/30s - 60°C/30s - 72°C/30s] x 45 - 72°C/40s - 4°C/∞. KDM6A KO clones were identified by restriction-fragment length polymorphism (RFLP) analysis of PCR products using HpyF10VI. Enzyme recognition site is lost after successful CRISPR/Cas9 targeting.

Clones were confirmed by immunoblotting and Sanger sequencing. Sequences were analyzed and aligned with Geneious 8.1.7 and Benchling software.

2.2.2.7 MG132 treatment

HEK293T cells were transiently transfected as described under 2.2.2.4 with pcDNA6-HA-KDM6A WT or mutant plasmids. After 42h, medium was carefully removed and cells were incubated for 6h with 10 mL of fresh medium containing 50 µM of the proteasomal inhibitor MG132. Cells were then lysed and analyzed by immunoblotting.

2.2.2.8 Proliferation assay and cell counting

For proliferation assays with chemotherapeutic agents, 0.5 mL medium containing DMSO as control or increasing concentrations of the respective drugs were added to wells of a 48-well plate. Cells were resuspended in medium at 4x10⁵ cells/mL and 0.5 mL of cell suspension corresponding to 2x10⁵ cells was each added to the wells. After incubation for 72h or 96h, viable cells were counted on a Vi-CELL^TM Cell Viability Analyzer XR. To count viable cells, cells were resuspended and 0.5 mL was transferred to a counting vial.

The vial was then placed inside the Vi-CELL^TM Cell Viability Analyzer XR and the amount of viable cells was measured by Trypan Blue exclusion.

For long-term proliferation, 3 mL of 4x10⁵ cells/mL were placed in a TC flask T25 and 3 mL of medium containing DMSO as control or a certain drug concentration was added.

The amount of viable cells was counted in duplicates every second day for 14 days. On day 4 and 8, 1 mL of cell suspension was transferred to a new TC flask T25 and 5 mL medium containing DMSO as control or a certain drug concentration was added.

2.2.2.9 Competitive growth assay

Competitive growth analysis in the presence of AraC was performed with the mixed population of lentiviral transduced K562 cells (10 days after transduction, not enriched for positive cells), containing both transduced shRNA-DsRed2(+) cells and untransduced

shRNA-DsRed2(-) cells. The mixed culture of native and transgene K562 cells (2x10⁵ cells/mL) were placed in a TC flask T25 and treated with 111 nM of AraC every three days or left untreated for a time period of 24 days. The percentage of transduced DsRed2(+) cells was determined every 3 days by flow cytometry analysis with the red fluorochrome gate using FACSVantage SE. For flow cytometry analysis, cells were washed twice with PBS and resuspended in 0.5 mL buffer containing PBS, 3% [v/v] FBS and 1 μg/mL DAPI. DAPI was used to exclude non-viable cells.

2.2.2.10 In vivo therapy trial

Patient-derived xenograft (PDX) cells expressing firefly luciferase were established as previously described⁵¹ in the laboratory of Irmela Jeremias (Helmholtz Zentrum München, Germany). 5x10⁵ AML-491 or AML-393 cells were injected i.v. into groups of NSG mice (NOD scid gamma, The Jackson Laboratory, Bar Harbour, ME, USA), and tumor growth was regularly monitored by bioluminescence imaging (BLI) as previously described⁵¹. At defined imaging signals, mice were treated with a combination of AraC (100 mg/kg, i.p., days 1-4 and 14-17) and DaunoXome (1 mg/kg, i.v., days 1, 4, 14, 17). 28 days after start of therapy, BLI was performed and increase in BLI signals relative to day 0 were calculated. All animal trials were performed in accordance with the current ethical standards of the official committee on animal experimentation (Regierung von Oberbayern, number 55.2-1-54-2531-95-2010).

2.2.3 Protein biochemistry methods