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J. Baeten et al.
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Figure 5. Sorted side population cell gene expression is enriched for stem cell differentiation and canonical Wnt signaling. (A) Gene set enrichment analysis (GSEA) with the Gene Ontology (GO): biological processes gene sets for stem cell differentiation and canonical Wnt signaling. NES: normalized enrichment score; FDR: false discovery rate. (B) Quantitative polymerase chain reaction validation of selected stem/Wnt-associated genes identified in the side population (SP) versus non-SP dif- ferentially expressed genes and GSEA. Normalized to ef1a. Statistical significance determined by multiple t-tests: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 (n = 4).
nature and imply a likely role for Wnt/β-catenin signaling in stem cell pathways across a wide array of ALL. Further studies are needed to evaluate the impact of this signature on functional LSC and in human leukemia.
The identification of the Wnt signaling pathway associ- ated with LSC has been reported previously, further vali- dating our model system. Along with the observed enrich- ment of putative stem cell genes, the increased expression of Wnt pathway members in the zebrafish ALL SP is fur- ther evidence of the overlap between LSC and the SP. The Wnt pathway has been tied to normal stem cell and CSC function in many tissues and cancers35 including acute myelogenous leukemia36 and T-ALL.38 In a mouse T-ALL model, Giambra et al. found that leukemias with condi- tional loss of β-catenin1 had greatly impaired leukemia- propagating ability, but loss of β-catenin1 after the tumor had already been established did not affect disease progres- sion. This coincides with our data highlighting a role for
Wnt signaling in ALL specific to LSC. Our results also high- light a potential target for therapeutic intervention in Wnt16. While complete inhibition of the Wnt pathway could produce significant off-target effects, targeting an individual ligand isoform with a specific role in LSC may provide clinical benefit without substantial side effects. Wnt16 was significantly increased in the SP of all four tumors measured, and has been previously associated with transformation in a subset of ALL.49 While additional work will still be needed to validate Wnt16 as a potential thera- peutic target, this and other targets identified through this study will be pursued to examine new treatments for ALL.
Acknowledgments
The authors would like to thank David Langenau and Sowmya Iyer for their help with the gene set enrichment analy- sis. We also thank Wilfredo Marin, Paula Herbst and Isabel Scharf for excellent fish care and technical support.
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haematologica | 2019; 104(7)