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

Abstract

Dysregulation of cytokines in the bone marrow (BM) microenvironment promotes acute myeloid leukemia (AML) cell growth. Due to the complexity and low throughput of in vivo stem-cell based assays, studying the role of cytokines in the BM niche in a screening setting is challenging. Here, 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 AML 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 BM 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 AML-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.

Figure 1.

A barcoded cytokine screen identifies TNFSF13 as a positive regulator of acute myeloid leukemia (AML)-initiating cells. (A) Schematic flowchart depicting the arrayed barcoded ex vivo cytokine screen with AML cells. In total, 12 pools in triplicate were used to screen the entire cytokine library. After seven (screen I) or 12 (screen II) days, mice were sacrificed. Data were normalized to the input representation for each barcode. (B) Pie chart displaying the contribution of each barcoded cell population in vivo following ex vivo cultures with stem cell factor (SCF) only for all barcoded cell populations. (C) Scatter plot showing fold-change in vivo versus input of barcoded cell populations for the two screens. Dotted lines represent a fold-change threshold of 2 to identify cytokines that promote leukemia-initiating cells. Red dots: barcoded cell populations stimulated ex vivo with SCF as the positive control within each pool; blue dots: cells stimulated with TNFSF13. NGS: next-generation sequencing.

Figure 2.

TNFSF13 stimulation promotes leukemia-initiating cells. (A) Output cell number from a total of 10,000 seeded c-Kit⁺ MLL-AF9 leukemia cells, following dose titration with TNFSF13 for three days (n=3). (B) Output cell number from 10,000 seeded normal Lin⁻Sca-1⁺+c-Kit⁺(LSK) cells stimulated with TNFSF13 or no cytokine (Control) for three days (n=3). (C-E) A total of 10,000 c-Kit⁺ MLL-AF9 acute myeloid leukemia cells were cultured ex vivo with SCF, TNFSF13, or no cytokine (Control) for three days and then transplanted into sublethally irradiated mice (10 mice per group). Pooled data from two independent experiments. (D) Percentage of leukemic (dsRed+) cells in the peripheral blood (PB) 19 days after transplantation. (E) Kaplan-Meier curves showing the survival of the mice. Values are means±Standard Deviation. ^**P<0.01; ^***P<0.001; ^****P<0.0001.

Figure 3.

TNFSF13 is present in the bone marrow and peripheral blood and is secreted by myeloid cells. ELISA quantification of TNFSF13 in (A) blood plasma samples from healthy (10 mice) and leukemic mice (12 mice) and (B) bone marrow samples from healthy (12 mice) and leukemic mice (13 mice). (C-E) c-Kit⁺ normal (control) and c-Kit⁺ leukemic bone marrow cells were cultured for three days under conditions favoring myeloid cell growth. (C) Representative dot plots showing expression of CD11b and Gr-1 on control and leukemic bone marrow cells after three days of culture. (D) Tnfsf13 mRNA expression in control and leukemic bone marrow cells determined by real-time polymerase chain reaction and normalized to a control sample after three days of culture (n=3 per group). (E) ELISA quantification of TNFSF13 in supernatants of normal or leukemic c-Kit⁺ bone marrow cells cultured for three days (n=8 per group). Values are means±Standard Deviation. ^*P<0.05; ^**P<0.01; ^***P<0.001.

Figure 4.

Tnfsf13^−/− mice have myelopoiesis defects and acute myeloid leukemia cells express TNFRSF17. Frequency of (A) long-term hematopoietic stem cells (LT-HSC), (B) granulocyte and macrophage progenitor (GMP) cells, (C) monocytes, and (D) granulocytes in the bone marrow of Tnfsf13^+/+ (n=14) and Tnfsf13^−/− (n=13) mice. Flow cytometric analysis showing (E) TNFRSF17 (purple) and (F) TNFRSF13B (green) expression on c-Kit⁺ MLL-AF9 leukemic cells. Isotype control is shown in light gray. TNFRSF17 geometric mean fluorescent intensity (gMFI) expression within (G) hematopoietic stem and progenitor cells (HSPC) (n=5) and (H) lineage populations (n=10) in the bone marrow of normal mice, after subtracting the signal using matching isotype control antibodies. Values are means±Standard Deviation. ^*P<0.05; ^****P<0.0001. WBC: white blood cells.

Figure 5.

TNFSF13 supports leukemia development in vivo. A total of 250,000 c-Kit⁺ bone marrow (BM) cells from Tnfsf13^−/− mice were transduced with a MIG-MLL-AF9 retroviral vector and transplanted into sublethally irradiated Tnfsf13^+/+ and Tnfsf13^−/− primary recipient mice (22 mice per group, pooled from four independent experiments). 1,000 leukemia cells harvested from spleens of primary recipients were transplanted into secondary recipient mice with matching genetic background. (A) Schematic picture showing the experimental setup. (B) Percentage of leukemic (GFP⁺) cells in the peripheral blood of the Tnfsf13^−/− and Tnfsf13^+/+ recipient mice 40 days after transplantation, normalized to the mean of the control (Tnfsf13^+/+) group in each experiment. Horizontal lines show the mean values within groups. (C) Kaplan-Meier curves showing the survival of primary recipient mice. (D) Kaplan-Meier curves showing the survival of secondary recipient mice (6 mice per group; pooled data from 2 donors per group). ^*P<0.05; ^**P<0.01.

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.

Figure 7.

TNFSF13 promotes human acute myeloid leukemia cells in an NF-κB-dependent manner. (A) Output cell number of human myeloid leukemia cell lines cultured under serum-free conditions for three days with or without (Control) TNFSF13. A total of 1,000 cells were seeded per well (n=3). (B) Representative flow cytometric analysis showing staining of TNFSF13 on the cell surface of Mono-Mac-6 following 5-minute (min) stimulation with TNFSF13 (blue, 100 ng/mL) compared to non-stimulated cells (Control, white). Isotype control is shown in light gray. (C) Representative flow cytometric analysis showing SYNDECAN-1 (CD138) (dark gray) expression on Mono-Mac-6 cells. Isotype control is shown in light gray. (D) Representative flow cytometric analysis showing staining of TNFSF13 on the cell surface of Mono-Mac-6 following 5 min stimulation with TNFSF13 (100 ng/mL); with (red) or without (blue) heparin (4 IU/mL). Non-stimulated cells were used as control (white). (E) Percentage of early (Annexin-V⁺7AAD⁻) and late (Annexin-V⁺7AAD⁺) apoptotic Mono-Mac-6 cells following three days of stimulation with TNFSF13 (n=3). (F and G) Mono-Mac-6 cells were stimulated with TNFSF13 for 24 hours (h) prior to RNA sequencing. Gene Set Enrichment Analysis identified an enriched (F) TNF receptor signature (gene set from MsigDB, molecular signatures database) and an (G) NF-κB signature. (H) Phospho-flow cytometric analysis of Mono-Mac-6 cells stimulated with TNFSF13 for 1 h. Isotype (light gray) and pNF-κB (Control, dark gray; TNFSF13, blue) staining. (I and J) Mono-Mac-6 cells were stimulated with TNFSF13 in the presence of a TNFRSF17 blocking antibody or corresponding isotype control, and analyzed for (I) pNF-κB expression after subtracting the signal using matching isotype control antibodies, and (J) output cell number after three days. (K and L) Mono-Mac-6 cells were treated with the IKK inhibitor TPCA1 at 1 or 3 μM during TNFSF13 stimulation and analyzed after three days for (K) pNF-κB expression after subtracting the signal using matching isotype control antibodies and (L) output cell number. Values are means±Standard Deviation. ^*P<0.05; ^**P<0.01; ^****P<0.0001. FDR: false discovery rate; DMSO: dimethyl sulfoxide.