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TNFSF13 is a positive regulator of AML cells
of leukemia-initiating activity are needed. Molecular bar- coding strategies, combined with next-generation sequencing (NGS), enable an in vivo readout of stem cell function in a competitive setting.5-7 By using this strategy, the in vivo cell fate of multiple hematopoietic stem cells (HSC) or leukemia clones can be monitored on a clonal level.5,8 However, because these approaches use pooled barcoded libraries, the in vivo cell fate of the genetically marked stem cell clones within mice cannot be traced to separate experimental conditions, such as cytokine stimu- lations.
In this study, we created a library of 11 arrayed molecu- lar barcodes that were used to mark leukemia cells exposed to 114 separate cytokine conditions. The 11 bar- coded leukemia cell populations were then pooled and injected into mice allowing for an in vivo competition read- out of leukemia-initiating activity. By using this method- ology, we identified the tumor necrosis factor ligand superfamily member 13 (TNFSF13; also named, A prolif- eration-inducing ligand, APRIL) as a novel positive regula- tor of leukemia-initiating cells. TNFSF13 promoted AML cell growth by suppressing apoptosis and activating nuclear factor kappa B (NF-kB).
Methods
Murine leukemia model
MLL-AF9 (KMT2A-MLLT3) leukemias were generated on a dsRed+ C57BL/6 transgenic background (6051; Jackson Laboratory, Bar Harbor, ME, USA), as previously described.9,10 Experiments involving murine leukemia cells were performed using tertiary or quaternary transplanted leukemia cells serially propagated in sub- lethally irradiated (600 cGy) recipient mice. All animal experi- ments were conducted according to an Animal Care and Use Committee protocol approved by the Lund/Malmö Ethical Committee. Except for the propagation of leukemia cells, all experiments involving murine leukemia cells were performed using c-Kit+ bone marrow cells. For details on the c-Kit+ cells isola- tion and cell culture conditions, see the Online Supplementary Methods.
Generation of 11 lentiviral vectors containing molecular barcodes
To generate lentiviral vectors containing non-expressed molec- ular barcodes, we used a lentiviral pLKO.1 vector (Addgene #32684) that co-expressed a short hairpin RNA (shRNA) and a green fluorescent protein (GFP) marker gene.11 The shRNA sequence, along with its promotor, was replaced by genetic bar- codes (42 to 46 nucleotides) flanked by 23mer primer sequences using the Nde1 and EcoR1 restriction sites (Online Supplementary Table S1). Viral vectors with VSV-g pseudotyping were produced using standard protocols.
Ex vivo cytokine screening using barcoded leukemia cells
Freshly isolated c-Kit+ dsRed+ leukemia cells were transduced with the barcoded lentiviral vectors and exposed to the cytokine library of 114 cytokines (Online Supplementary Table S2) in 96-well plates. After 72 hours, cells from different wells were pooled, with each pool containing leukemia cells from up to 11 barcoded pop- ulations representing specific culture conditions, and injected into sublethally irradiated (600 cGy) recipient C57BL/6 mice via tail vein injection. After 7-12 days, mice were sacrificed, BM cells were harvested, and DNA was extracted (Qiagen Blood and
Tissue DNA Extraction Kit). For details on the screen see the Online Supplementary Methods.
Sequencing of barcodes and bioinformatics analysis
Following BM cell extraction and DNA purification (DNAeasy Blood and Tissue Kit, Qiagen), the regions containing the barcodes were amplified using two-step polymerase chain reaction (PCR). Step one utilized pLKO.1-specific primers containing Nextera overhangs (Online Supplementary Table S3). The number of reads per barcode of each sample was extracted and normalized to the total read count within each sample. The mean of the three bio- logical replicates was then calculated after normalization to the input (Day 0). For further details on the sequencing and bioinfor- matics, see the Online Supplementary Methods.
Tnfsf13-/- mouse model
The C57BL/6 Tnfsf13-/- mouse (#022971; B6.Cg-
Tnfsf13tm1Pod/J)12,13 was obtained from The Jackson Laboratory (Bar Harbor, Maine, USA) and further backcrossed (>5 genera- tions) onto C57BL/6 wild-type mice. Genotyping was performed by PCR using protocols provided by the Jackson Laboratory.
Retroviral MLL-AF9 expression in c-Kit+ bone marrow cells and transplantations into sublethally irradiated mice
Murine stem cell virus gammaretroviral vectors co-expressing MLL-AF9 and GFP (MIG-MLL-AF9)14 were produced with an ecotropic envelope using standard protocols in 293T cells. c-Kit+ BM cells were pre-stimulated for two days and spinoculated. Following overnight incubation at 37°C, transduced cells were injected into sublethally irradiated (600 cGy) recipient mice via tail vein injection. Each recipient mouse received cells corresponding to 250,000 initially seeded cells. Blood samples were taken after 40 days, and mice were sacrificed when they showed signs of dis- ease. To assess leukemia development in sublethally irradiated (600 cGy) secondary recipient mice, 1,000 or 10,000 spleen leukemia cells from primary recipients were injected via the tail vein. For details on the transduction of c-Kit+ BM cells, see the Online Supplementary Methods.
Flow cytometric analysis and cell sorting
The flow cytometric analyses were performed using a FACS Canto II (BD Biosciences, San Jose, CA, USA) or a FACS LSRFortessa (BD Biosciences), and cell sorting was performed using a FACS Aria II (BD Biosciences). For detailed information on antibodies used and staining for LSK and HSPC analysis, cell cycle, apoptosis and phosphoflow, see the Online Supplementary Methods.
Statistical analysis
Prism 6 (Graphpad) was used for the statistical analyses, includ- ing Student t-test and Kaplan-Meier survival analysis. Statistical significance is shown with asterisks: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Data are presented as mean±Standard Deviations (SD).
Results
Development of an ex vivo cytokine screen with a competitive in vivo read-out of leukemia-initiating activity using molecularly barcoded leukemia cells
To identify cytokines that regulate AML stem cells using a competitive in vivo read-out of leukemia-initiating activ- ity, we generated lentiviral vectors harboring genetic bar- codes in an arrayed setting. This approach allows for
haematologica | 2019; 104(10)
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