Cell lines and culture conditions
Cell lines were purchased from DSMZ (Braunschweig, Germany). Cells were cultured according to standard protocols and tested negative for mycoplasma. For proliferation assays, the number of cells was counted following trypan blue exclusion. Apoptosis was measured by flow cytometry using Annexin V/SYTOX® Blue staining at day 6 post-infection. Cell cycle analysis was performed at day 14 post-infection using the Click-iT EdU Kit (Life Technologies, Darmstadt, Germany) following the manufacturer’s instruction.
Methylcellulose colony-forming assays
Primary human cells isolated from peripheral blood of AML patients were seeded in methylcellulose according to standard protocols and as published previously [10].
Animal models
All experiments were conducted after approval by the Landesverwaltungsamt Sachsen-Anhalt (42502-2-1052 UniMD) and the TLV Thüringen (02-035/16). FLT3ITD/ITD mice were a gift of Prof Benjamin L. Ebert (Harvard Medical School, Boston, MA, USA) [11]. MLL-AF9 knock-in mice were a gift from Prof Terrence Rabbitts (Institute of Cancer Research, London, UK) [12]. Mx1-Cre (Strain 03556) mice were obtained from Jackson Laboratories. C57BL/6J mice (6–8 weeks old) were purchased from Janvier Labs (Le Genest-Saint-Isle, France) and housed in a pathogen-free animal facility. Mice harboring a ‘floxed’ (flanked with loxP sites) allele of Llgl1 have been generated as previously described [13]. Exon 2 (the exon downstream from the exon with the first ATG codon) was flanked by LoxP sequences, and the β-geo selectable marker was removed by transient expression of Cre-recombinase in the ES-cells. These mice have been backcrossed more than 8 generations into a C57BL/6J background. For transplantation, mice were irradiated as indicated and transplanted via tail intravenous (IV) injection with 1 × 105 to 106 bone marrow as indicated. Mice were sacrificed and analyzed at a defined time-point or when signs of disease became evident. Disease burden was assessed by complete blood counts, flow cytometry of peripheral blood (PB), bone marrow (BM) and spleen cells or histopathological stainings. To activate Mx1-Cre in vivo, poly(I):poly(C) (Cytiva, Marlborough, MA) was injected two times every second day intraperitoneally. Injections were halted if mice showed signs of illness prior to completion of treatment. Spontaneous Mx1-Cre activation was noted as previously described consistent with spontaneous activation of Mx1-Cre in an inflammatory milieu [14]. NOD-Prkdcscid-IL2rgTm1/Rj (NXG) mice were obtained from Janvier Labs (Le Genest-Saint-Isle, France) and NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3, CSF2, KITLG)1 Eav/MloySzJ (NSGS) mice were obtained from The Jackson Laboratory (Bar Harbor, USA). AML cell lines (as indicated) or primary human xenografts were genetically modified by RNAi and subsequently injected at equal distribution into recipient mice. Therefore, no randomization was necessary. Due to the analysis in paired samples (cells transduced with either shRNA or non-targeting control), no blinding was necessary. Sample size and experimental schedule were calculated assuming a relevant difference in means of survival. We used a one-sided t-test at a = 0.05 and a power of >80% with an expected difference in means of 1.75 SD (standard deviations) based on previous experience with xenotransplantation. Equal numbers of 8–12-week-old male and female mice were used for experiments in all groups.
Blood analysis and bone marrow cytospins
Blood was collected into EDTA-coated tubes and investigated using a BC-5000Vet (Mindray, China). To analyze cell morphology, 1 × 105 bone marrow cells were centrifuged onto glass slides. Peripheral blood smears and bone marrow cytospins were stained with Wright-Giemsa (BioScientific).
Histological imaging of mouse organs
Spleen, liver and lung were fixed and embedded according to standard protocols. Slides were automatically processed for hematoxylin and eosin staining (Leica AutoStainer XL, Leica Biosystems, Wetzlar, Germany). Images were acquired at 10× magnification on an AxioImager A.2 (Carl Zeiss Microscopy, Jena, Germany). Images were processed and analyzed using the ZEN software (blue edition, version 2.3, Carl Zeiss Microscopy GmbH, Jena, Germany).
Flow cytometry
For immunophenotype analysis, peripheral blood cells, bone marrow or spleen cells were resuspended in PBS/1% FBS after erythrocyte lysis (PharmLyseTM, BD Pharmingen, San Diego, CA). Unless otherwise stated, the following antibodies were used: Sorting and analysis of LSK cells (Lin-Sca-1+cKit+) were performed as previously described [10, 15]. Biotinylated antibodies against Gr-1 (RB6-8C5), B220 (RA3-6B2), CD19 (6D5), CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.7), TER119 and IL7Ra (A7R34) (all Biolegend, SanDiego, CA) were used for lineage staining. An APC-Cy7- or BV421-labeled streptavidin-antibody (BioLegend) was used for secondary staining together with an APC-anti-cKit (clone 2B8) and a PE-Cy7- or PE-anti-Sca-1 antibody (clone E13-161.7). Cells were analyzed using an BD-Fortessa, LSRIITM or FACSCantoIITM (Becton-Dickinson) cytometer. Analysis was performed using FlowJoTM software (Treestar, Ashland, OR). Cell sorting was performed on a BD FACSAria™ II (Becton-Dickinson).
Vectors
For RNAi, shRNAs were cloned into a lentiviral pLKO.1_puro vector system for puromycin selection. For HoxA9 overexpression (rescue) experiments, an MSCV-IRES-GFP backbone was used. Lentiviral and retroviral infections were performed as previously described (Schnoeder et al. Blood 2022). Detailed information on vectors and sequences are provided in Supplementary Tables 1 and 2.
Genome editing by CRISPR/Cas9
Genetic editing by CRISPR/Cas9 was performed as previously described [10, 16] unless otherwise stated. Guide RNAs were designed using the Broad GPP tool [17]. For cloning of sgRNA sequences, the improved-scaffold-pU6-sgRNA-EF1Alpha-PURO-T2A-RFP (ipUSEPR) vector system [18], with puromycin resistance and RFP selection marker was used. Genetic inactivation by CRISPR/Cas9 was performed as published before [15]. HEL cells were transduced with the screen library and selected for 2 days with puromycin following collection of an aliquot as the input reference. Cells were cultured in vitro and samples for sequencing were collected 2 and 3 weeks later (Fig. 1A). The average relative abundance of each sgRNA in the output compared to the input samples was determined. We calculated a depletion score for each sgRNA. The median of 3-4 sgRNAs per gene was used to represent the score of the corresponding gene. Knockdown efficiency was assessed by quantitative real-time PCR (RT-qPCR) 5-7 days post-infection as published before [10]. qPCR primer sequences are listed in the Supplementary Table 3. sgRNA sequences are provided in the Supplementary Tables 4 and 5.
CRISPR/Cas9 in vitro screen
HEL cells were transduced with the Scribble complex member library at a multiplicity of infection (MOI) of 20%, selected for 2 days with puromycin and an input reference (baseline) was taken at day 4 post-infection. The cells were cultured in vitro for 21 days and replicates 1–4 were taken at day 14 and day 21, respectively. Genomic DNA was isolated using the QIAmp DNA Blood Mini Kit (Qiagen, Hilden Germany), and amplification was performed using specific Illumina primer compatible sequences (Supplementary Table 6). Sequencing was performed at Genewiz (HiSseq, 150 bp, paired end) (Illumina, South Plainfield, NJ, USA). A depletion score for day 14 and day 21 were analyzed compared to the input reference using the MAGeCKFlute [19].
RNA-sequencing
RNA was isolated from cultured cells using the Qiagen RNeasy Mini kit or TRIZOL as previously described [15, 20]. Subsequently, mRNAs were purified using the “NEBNext® Poly(A) mRNA Magnetic Isolation Module” followed by RNAseq library preparation using the “NEBNext® Ultra™ RNA Library Prep Kit for Illumina®” according to the manufacturer’s instruction. Sequencing was performed on an Illumina NextSeq500 or an Illumina HiSeq2000 (75 bp, single end) (Illumina, South Plainfield, NJ, USA).
SC-flow cytometry
For the single-cell flow cytometry analysis, bone marrow cells were resuspended in PBS/2%FBS. The staining was performed for 20 minutes at 4 °C with the antibodies mentioned in Supplementary Table 7. After washing, samples were acquired on an Aurora spectral flow cytometer (Cytek Bioscinces, Fremont, CA) equipped with 5 lasers (355 nm, 405 nm, 561 nm, 640 nm) using SpectroFlo version 3.1.0 (Cytek Bioscinces, Fremont, CA). All flow cytometry data were analyzed and exported as compensated channel values with FlowJoTM software (Treestar, Ashland, OR; version 10.8.1).
Dimensionality reduction of flow cytometry data
Each sample was subset down to 30,000 cells LogNormalization (Seurat function; v4.3.0) was applied before downstream analysis [21]. The compensated channel value for each cell were normalized by total channel values for that cell, multiplied by 10,000 (TP10K), and then log-transformed by log10 (TP10k + 1). After scaling, the dimensionality of the flow data was set to 33 principal components that were used as input for UMAP representation.
Clustering of flow cytometry data
Following the selection of variable genes and the performance of Principal Component Analysis (PCA), Uniform Manifold Approximation and Projection (UMAP) was applied to visualize the cell population. Subsequently, the cells were clustered using the Louvain algorithm [22] based on a Shared Nearest Neighbor (SNN) graph with a resolution parameter set to 0.29. However, one particular cell cluster was excluded from further analysis as it displayed no detectable expression of any marker genes. Ultimately, these identified clusters were annotated according to their respective cluster markers. The plots were generated using the packages ggplot2 (v3.4.1) and virdis (v0.6.3) in R 4.2.3.
Statistical analysis
Kaplan–Meier curves were plotted using GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA) using the log-rank test (Mantel–Cox test). Statistical analyses were performed using ANOVA with FDR p-value correction for comparing more than two groups or t-test for comparing two groups, unless stated otherwise. Significance of p-values in figures are indicated using the following ranges: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Each dot represents an individual biological replicate.