Arrayed molecular barcoding identifies TNFSF13 as a positive regulator of acute myeloid leukemia-initiating cells. Arrayed molecular barcoding identifies TNFSF13 as a positive regulator of acute myeloid leukemia-initiating cells

Dysregulation of cytokines in the bone marrow microenvironment promotes acute myeloid leukemia cell growth. Due to the complexity and low throughput of in vivo stem-cell based assays, studying the role of cytokines in the bone marrow niche in a screening setting is challenging. Herein, we developed an ex vivo cytokine screen using 11 arrayed molecular barcodes, allowing for a competitive in vivo readout of leukemia-initiating capacity. With this approach, we assessed the effect of 114 murine cytokines on MLL-AF9 acute myeloid leukemia mouse cells and identified the tumor necrosis factor ligand superfamily member 13 (TNFSF13) as a positive regulator of leukemia-initiating cells. By using Tnfsf13-/- recipient mice, we confirmed that TNFSF13 supports leukemia-initiation also under physiological conditions. TNFSF13 was secreted by normal myeloid cells but not by leukemia mouse cells, suggesting that mature myeloid bone marrow cells support leukemia cells by secreting TNFSF13. TNFSF13 supported leukemia cell proliferation in an NF-κB-dependent manner by binding TNFRSF17 and suppressed apoptosis. Moreover, TNFSF13 supported the growth and survival of several human myeloid leukemia cell lines, demonstrating that our findings translate to human disease. Taken together, using arrayed molecular barcoding, we identified a previously unrecognized role of TNFSF13 as a positive regulator of acute myeloid leukemia-initiating cells. The arrayed barcoded screening methodology is not limited to cytokines and leukemia, but can be extended to other types of ex vivo screens, where a multiplexed in vivo read-out of stem cell functionality is needed. Overall design: MonoMac6 cells were grown in RPMI media containing baseline cytokines with or without human APRIL (100 ng/mL) 24 hours

骨髓微环境中细胞因子的失调可促进急性髓系白血病细胞的增殖。由于基于体内干细胞的检测方法兼具复杂性与低通量的特点，在筛选情境下研究细胞因子在骨髓龛中的作用颇具挑战。本研究利用11组阵列化分子条形码（arrayed molecular barcodes）构建了离体细胞因子筛选模型，可实现对白血病起始能力的竞争性体内读值检测。借助该模型，我们评估了114种小鼠细胞因子对MLL-AF9急性髓系白血病小鼠细胞的调控作用，并鉴定出肿瘤坏死因子配体超家族成员13（TNFSF13）为白血病起始细胞的正向调控因子。通过使用Tnfsf13基因敲除受体小鼠，我们证实TNFSF13在生理条件下同样可促进白血病起始。正常髓系细胞可分泌TNFSF13，而白血病小鼠细胞则无法分泌该因子，这提示成熟骨髓髓系细胞可通过分泌TNFSF13为白血病细胞提供支持。TNFSF13可通过结合肿瘤坏死因子受体超家族成员17（TNFRSF17），以核因子κB（NF-κB）依赖的方式促进白血病细胞增殖并抑制细胞凋亡。此外，TNFSF13可支持多种人类髓系白血病细胞系的生长与存活，表明本研究结果可推广至人类疾病研究。综上，本研究通过阵列化分子条形码技术，首次揭示了TNFSF13作为急性髓系白血病起始细胞正向调控因子的全新功能。该阵列化条形码筛选方法并不局限于细胞因子与白血病研究，可拓展至其他需要实现干细胞功能多重体内读值检测的离体筛选场景。总体实验设计：将MonoMac6细胞置于含基础细胞因子的RPMI培养基中，分别添加或不添加100 ng/mL的人APRIL，培养24小时。