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TNFSF13 is a positive regulator of AML cells
ing function using a competitive in vivo readout of leukemic cells. This approach significantly reduces the number of mice since leukemia cells from up to 11 differ- ent cytokine conditions can be pooled in each mouse. The arrayed barcoded approach is applicable to other types of ex vivo screens with in vivo readouts, as shown recently in a small-molecule screen using metastatic pancreatic cells.25
The barcoded cytokine screens identified IL9 and TNFSF13 as candidate positive regulators of leukemic-ini- tiating cells. Validation experiments confirmed that both IL9 and TNFSF13 supported AML-initiating cells, but as TNFSF13 was more potent than IL9, we selected TNFSF13, a TNF superfamily ligand,26 for further studies. The findings that Tnfsf13-/- recipient mice had lower leukemia burden and increased survival compared to con- trols following serial transplantations of leukemia cells suggest that TNFSF13 supports AML cells also under physiological condition. TNFSF13 has previously been shown to promote cancer-cell growth and survival of sev- eral types of solid tumors27,28 and B-cell malignancies, such as acute lymphoblastic leukemia, Hodgkin lymphoma, and multiple myeloma.26,29-32 In addition, elevated TNFSF13 levels have been found in multiple cancer types and are associated with a poor prognosis,33 suggesting that TNFSF13 has broad tumor-promoting activity. Whereas the first Tnfsf13-/- mouse model generated did not reveal alterations in T- and B-cell development or in vitro func- tion,34 later studies of Tnfsf13-/- mice identified increased proliferation of T cells, changes in the secretion of immuno-cytokines, and impaired antibody class switch- ing.14,34
In AML, TNFSF13 secretion has been linked to chemoresistance36 and elevated TNFSF13 serum levels have been reported in patients,36,37 but TNFSF13 has not previously been associated with AML stem cells or myelopoiesis. Given that the GMP cell stage is associated with AML initiation,38 it was interesting to note that TNFRSF17 is expressed on myeloid progenitor cell popu- lations and that Tnfsf13-/- mice have reduced numbers of GMP cells and monocytes, suggesting that TNFSF13 has a previously unrecognized role in myelopoiesis.
In agreement with previous studies showing that TNFSF13 is expressed by infiltrating neutrophils in solid tumors39,40 and by myeloid BM cells in multiple myelo- ma,41,42 we found that TNFSF13 was secreted by CD11b+Gr-1+ myeloid cells but not by the corresponding
murine AML cells. This finding suggests that mature myeloid BM cells support AML cells by secreting TNFSF13. In contrast to the findings in the murine MLL- AF9 AML model, TNFSF13 is expressed by leukemia cells in certain AML subtypes,36,37 suggesting that TNFSF13 might play a more critical role for leukemia development and progression in these subtypes.
The anti-apoptotic effect induced by TNFSF13 is consis- tent with prior findings in B-cell malignancies43-45 and glioma,46 suggesting a similar mechanism induced by TNFSF13 in various types of cancers. Moreover, TNFSF13 promoted AML cells in an NF-kB-dependent manner, which is in agreement with studies showing that TNFSF13 activates NF-kB in multiple myeloma cells,32 chronic lymphocytic leukemia,30 and non-Hodgkin lym- phoma B cells.45 NF-kB signaling is elevated and critical for AML stem cells,47,48 including MLL-rearranged AML cells, but the underlying mechanism causing NF-kB activation has not been clarified. Along with IL1-induced activation of NF-kB in primitive leukemia cells,49 our findings suggest that TNFSF13 contributes to enhanced NF-kB activity in AML cells. Because AML cells from various AML subtypes lacking MLL rearrangements were also sensitive to TNFSF13, the effects of TNFSF13 stimulation are not restricted to MLL-rearranged AML.
In summary, we have established a cytokine screen using arrayed molecular barcoding of AML cells allowing for an in vivo competitive readout of leukemia-initiating activity. The screen provides a new strategy for studying the influence of secreted factors on AML-initiating cells. This approach identified TNFSF13 as a positive regulator of AML stem cells and showed that TNFSF13 promotes AML cells in an NF-kB-dependent manner. Moreover, we identified a role for TNFSF13 in normal myelopoiesis and showed that normal myeloid cells secrete TNFSF13. This study demonstrates the utility of using arrayed molecular barcoding as a new screening tool for identifying novel stem cell regulators.
Acknowledgments
This work was supported by the Swedish Cancer Society, the Swedish Childhood Cancer Foundation, the Crafoord Foundation, the Gunnar Nilsson Cancer Foundation, the Medical Faculty of Lund University, the Royal Physiographic Society of Lund, the Swedish Research Council, BioCARE, and FP7 Marie Curie.
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