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TNFSF13 is a positive regulator of AML cells
observed in plasma (Figure 3A and Online Supplementary Figure S7B). The c-Kit+ murine leukemia cells did not show a significant response to TNFSF13B stimulation in vitro (Online Supplementary Figure S7C), but in human Mono- Mac-6 cells, TNFSF13B stimulation had similar effect as TNFSF13, resulting in increased cell number (Online Supplementary Figure S7D).
TNFSF13 supports acute myeloid leukemia cells in an NF-kB-dependent manner
To explore the molecular mechanism by which TNFSF13 promotes AML cell growth and survival, we generated RNA sequencing data of Mono-Mac-6 cells that had been stimulated with TNFSF13 for 24 hours. Consistent with TNFSF13 being a member of the tumor necrosis factor (TNF) superfamily, gene set enrichment analysis (GSEA) demonstrated an enriched TNF receptor activation signature upon TNFSF13 stimulation [false dis- covery rate (FDR) <0.10] (Figure 7F and Online Supplementary Table S6). Furthermore, an enriched NF-kB signature was evident in TNFSF13-stimulated cells (FDR<0.05) (Figure 7G and Online Supplementary Table S6) and phospho-flow cytometric analysis confirmed NF-kB activation upon TNFSF13 stimulation (Figure 7H). Consistent with this data, TNFSF13 stimulation resulted
in a significant upregulation of the NF-kB target genes AGT, IRF1, IRF7, PTGDS, and VIM (Online Supplementary Figure S8A). By using a TNFRSF17 blocking antibody, TNFSF13-induced cell proliferation and NF-kB activation was hindered (Figure 7I and J), demonstrating that TNFSF13 activates NF-kB by binding to TNFRSF17. To assess whether TNFSF13 promotes cell proliferation by activating NF-kB, we inhibited NF-kB activation using TPCA1 and IKK-16, two selective inhibitors of IkB kinase (IKK).24 Both TPCA1 and IKK-16 treatment inhibited NF-kB activation and reversed the effects of TNFSF13 in Mono-Mac-6 cells (Figure 7K and L and Online Supplementary Figure S8B and C). These data demonstrate that TNFSF13 supports the growth of human AML cells in an NF-kB-dependent manner.
Discussion
Although in vivo assays are critical to assess stem cell function, performing screens in vivo is challenging due to the large number of experimental animals needed to pro- vide meaningful data. In this study, we developed arrayed molecular barcodes in lentiviral vectors, which enabled us to assess the effects of 114 cytokines on leukemia-initiat-
AB
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Figure 6. TNFSF13 maintains acute myeloid leukemia (AML) cells by suppressing apoptosis and promoting cell cycle progression. c-Kit+ MLL-AF9 AML cells were cultured for 3 days with stem cell factor (SCF) as a baseline and stimulated with TNFSF13 or no TNFSF13 (Control) prior to apoptosis and cell cycle analysis. (A) Representative contour plots showing Annexin-V and 7AAD staining. (B) Percentage of early (Annexin-V+7AAD–) and late (Annexin-V+7AAD+) apoptotic cells (n=3). (C) Representative contour plots showing Ki67 and DAPI staining. (D) Percentage of cells in G0, G1, or S/G2/M phase of the cell cycle (n=3) determined by Ki67 and DAPI staining. Values are means±Standard Deviation. *P<0.05; **P<0.01.
haematologica | 2019; 104(10)
2013