Survivin Modulates Gene Expression That Connects through An Extensive Functional Signaling Network and Regulates Proliferation of Hematopoietic Stem Cells through Evi-1

Abstract 87

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that antagonizes caspases and has been implicated in regulation of apoptosis, cell division and cell cycle both in cancer cells and normal tissues. Survivin is essential for activation of Aurora kinase that phosphorylates Histone H3, an event required for transcriptional regulation and cytokinesis. Although there is no evidence that Survivin directly regulates gene transcription, Bir1, a C. elegans homologue of Survivin, regulates transcription, most likely through Histone phosphorylation by Aurora kinase. We previously showed that Survivin is expressed and growth factor regulated in human CD34⁺ cells (Fukuda & Pelus. Blood 2001 & 2002). Antagonizing Survivin impairs production of mouse bone marrow hematopoietic progenitor cells in vitro (Fukuda et al. Blood 2004) and conditional Survivin gene deletion in vivo in mice leads to bone marrow ablation as a result from loss of hematopoietic stem and progenitor cells (Leung et al. JEM 2007). However, little is known about signaling pathways downstream of Survivin in hematopoietic stem cells (HSC). Herein, we investigated the mechanism of action of Survivin by creating Survivin gene signature network following gene deletion in HSC and by functional rescue on impaired HSC activity induced by Survivin deletion.

Survivin deletion in Tamoxifen-Cre Survivin^flox/flox mice led to significant changes in 2,228 mRNA in marrow CD34^neg, c-kit⁺, Sca-1⁺, lineage⁻ cells. Molecular network of the genes affected by Survivin deletion in HSC were created using DAVID (Huang et al. Nature Protocol 2009) and Cytoscape (Cline et al. Nature Protocol 2007). Among the signaling pathways defined by KEGG (www.kegg.org), phosphatidylinositol signaling pathway was most significantly affected in HSC by Survivin gene deletion (P<0.05). In addition, at least 56 genes regulated by Survivin were connected through an extensive network structured by 183 genes surrounding the phosphatidylinositol signaling pathway. These include Stathmin1, p38, NFkB, Jun D and Evi-1, a transcriptional factor that regulates HSC proliferation and prevents stress-induced cell death. Furthermore, knocking down Survivin induced coincident repression of 43 HSC related genes such as Gata2, Pbx1 and Sal2 in addition to Evi-1. This is consistent with down-regulation of Gata2, Pbx1 and Sal2 by Evi-1 ablation in HSC in vivo (Goyama et al. 2008) and suggests that Evi-1 may function downstream of Survivin to maintain HSC function. To test this hypothesis, marrow cells from conditional Survivin^−/− mice were transduced with vector or Evi-1 and transplanted into lethally irradiated normal recipients. Subsequent Survivin deletion in vivo significantly impaired hematopoietic recovery (53.0±4.4% vs 1.6±0.3% donor chimerism in Survivin^+/+ and Survivin^−/− cells, respectively, P<0.01), whereas Survivin^−/− marrow cells over-expressing Evi-1 showed a marginal but significant increase in hematopoietic recovery at 6 months post transplantation (7.9±0.7% donor chimerism, P<0.01 compared to control vector in Survivin^−/−). Gata2 over-expression did not affect hematopoietic recovery (0.6±0.1% donor chimerism). None of the mice transplanted with cells harboring ectopic Evi-1 developed leukemia after 12 months, making it unlikely that recovery of donor chimerism is a consequence of clonal expansion of transduced cells by Evi-1.

These results suggest that Evi-1 lies down stream of Survivin in HSC and that repression of Evi-1 expression is a functional consequence of Survivin ablation rather than an indirect effect resulting from apoptosis and/or cell cycle alteration of HSC induced by Survivin deletion. Our data indicate that Survivin regulates HSC proliferation at least through Evi-1. Alteration of genes with multiple cellular functions as a consequence of Survivin deletion and the presence of a functional network downstream of Survivin suggest that Survivin regulates multiple effector pathways in HSC, independent of its activity as a caspase inhibitor.