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TNFSF13 is a positive regulator of AML cells
showed a relative increase within all pools in vivo, thus val- idating the new method (Figure 1C). Although a number of cytokines, such as interleukin-3 (IL3) and granulocyte- macrophage colony-stimulating factor (GM-CSF), that greatly expand (>20-fold over 3 days) AML cells in culture were included in the screen,4 none of them expanded the leukemia-initiating cell population as assessed with in vivo read-out (Online Supplementary Table S3). This finding highlights the need for in vivo readout to assess the impact of cytokines on the leukemia-initiating capacity of the cells. Setting a threshold for 2-fold enrichment, TNFSF13 was the only cytokine, other than SCF, that scored in both screens (on average a 3.1-fold relative increase). In addi- tion, the screens identified Interleukin 9 (IL9) as a candi- date positive regulator (1.8 relative increase) of leukemia- initiating cells (Online Supplementary Table S2). Moreover, several negative regulators of leukemia-initiating cells were identified that were not further investigated in the present study (Online Supplementary Table S2).
Ex vivo stimulation with TNFSF13 or IL9 promotes leukemia-initiating cells
To validate the findings from the screen and first explore TNFSF13 as a regulator of primitive AML cells, we cul-
AB
tured c-Kit+ MLL-AF9 leukemia cells with increasing doses of TNFSF13. TNFSF13 supported the growth and survival of c-Kit+ leukemic cells in culture (Figure 2A and Online Supplementary Figure S2A), but did not affect normal HSPC, as assessed by analyzing the effect of TNFSF13 on Lin-Sca- 1+ c-Kit+ (LSK) BM cells from healthy mice (Figure 2B). Moreover, ex vivo stimulation of c-Kit+ leukemia cells with TNFSF13, followed by transplantation into sublethally irradiated mice, resulted in significantly higher levels of circulating leukemia cells in peripheral blood (mean 25.6% vs. 1.3% for non-stimulated cells; P<0.0001) (Figure 2C and D). Notably, the numbers of circulating leukemia cells were even higher than for mice that had received leukemia cells that were stimulated with SCF as a positive control (25.6% vs. 7.8%; P<0.01) (Figure 2D). Consistent with this finding, the TNFSF13-treated group exhibited reduced survival (median survival 29 vs. 38 days for the non-stimulated group; P<0.0001) (Figure 2E) and survival was even slightly shorter than for the SCF-treated group (Figure 2E). At the time of sacrifice, all except for one con- trol mouse had a high leukemic burden, as confirmed by enlarged spleens and a high percentage of leukemia cells (>95% dsRed+ cells) in their BM (Online Supplementary Figure S2B and C). Consistent with the findings in the
C
DE
Figure 2. TNFSF13 stimulation pro- motes leukemia-initiating cells. (A) Output cell number from a total of
10,000 seeded c-Kit+
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 inde- pendent 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.
MLL-AF9
haematologica | 2019; 104(10)
2009