**Survivin: Identification of Selective Functional Signaling Pathways in Transformed Cells and Identification of a New Splice Variant with Growth Survival Activity**

Louis M. Pelus1 and Seiji Fukuda2

*1Department of Microbiology and Immunology Indiana University School of Medicine, Indianapolis, IN 2Department of Pediatrics, Shimane University School of Medicine, Shimane 1USA 2Japan* 

### **1. Introduction**

Survivin is a member of the inhibitors of apoptosis (IAP) family of highly conserved proteins implicated in regulation of mitosis, cytokinesis, cell cycle and apoptosis (Altieri, 2003a; Altieri, 2003b; Fukuda & Pelus, 2006). It is expressed during development but down regulated in most adult tissues. However, Survivin is over-expressed in the majority of solid tumors and leukemias, and is usually associated with higher proliferative index, reduced apoptosis, resistance to chemotherapy and increased rate of tumor recurrence, making it an attractive therapeutic target. We have previously shown that Survivin is expressed and growth factor regulated in normal hematopoietic cells and regulates apoptosis and cell cycle entry (Fukuda et al., 2002; Fukuda et al., 2004; Fukuda & Pelus, 2001; Fukuda & Pelus, 2002). Antagonizing Survivin impairs mouse progenitor cell production in vitro (Fukuda et al., 2002; Fukuda et al., 2004) and loss of function upon conditional deletion in vivo leads to bone marrow ablation as a consequence of loss of stem cell function (Leung et al., 2007). While Survivin is tightly regulated in normal hematopoietic cells, deregulated expression is frequently observed in hematologic diseases particularly those characterized by stem cell expansion. Survivin is aberrantly over expressed in acute myeloid leukemia (Adida et al., 2000; Carter et al., 2001) but down regulated in aplastic anemia where hematopoietic stem and progenitor cells are reduced (Badran et al., 2003). It is now clear that Survivin can regulate cell growth under both physiological and pathological conditions. Therefore, identification of differential signaling cascades between normal and abnormal cells downstream of Survivin is required in order to identify cancer cell specific targets without toxicity to normal cells.

#### **2. Survivin mediates aberrant proliferation of hematopoietic progenitor cells transformed by ITD-Flt3**

Internal tandem duplication (ITD) mutations of the Flt3 tyrosine kinase receptor are found in many patients with acute leukemia, and are an unfavorable prognostic factor (Fukuda et al.,

Survivin: Identification of Selective Functional Signaling Pathways

4

Survivinflox/flox

8

Growth factor independent

**3. Survivin regulated genes** 

CFU-GM/104GFP+

 cells 12

in Transformed Cells and Identification of a New Splice Variant with Growth Survival Activity 189

P<0.05

Cre-ERTM Survivin flox/flox

Fig. 2. Wild type Flt3 or ITD-Flt3 were retrovirally transduced into marrow cells from Cre ER Survivin fx/fx or control Survivin fx/fx mice. Following transduction, cells were plated in a CFU assay in agar with 30% FBS and 1uM 4OH tamoxifen in the absence of hematopoietic growth factors for 2 weeks. Treatment with 4OH-TM significantly reduced growth factor independent CFU-GM induced by ITD-Flt3 in Cre-ERTM Survivin fx/fx marrow cells.

While it is clear that antagonizing Survivin may reduce the aberrant proliferation of hematopoietic progenitor cells transformed by ITD-Flt3 and that ITD-Flt3 mutations are present in human leukemic stem cells (LSC) (Levis et al., 2005) , studies from our group and others indicate that Survivin is a normal regulator of hematopoietic stem and progenitor cells (HSPC) (Fukuda et al., 2002; Fukuda et al., 2004; Leung et al., 2007). This suggests that Survivin targeted therapies will likely result in hematopoietic toxicity. Therefore, identification of differential signaling cascades downstream of Survivin between normal HSPC and cancer stem cells (CSC) or LSC are required to pinpoint targets that can effectively eradicate CSC/LSC with acceptable toxicity to normal HSPC. In order to identify Survivin regulated genes associated with ITD-Flt3 signaling but not normal Flt3 signaling, we evaluated human ITD-Flt3 transformed KSL cells from conditional Survivin knockout mice as a model of normal and leukemic stem cells. We identified 1096 transcripts differentially regulated by Survivin in ITD-Flt3 transformed stem cells [Tables of Survivin regulated genes can be found in (Fukuda et al., 2011)]. Classification of these genes based upon biological process and molecular function defined by Gene Ontology Term using David 2008 showed significant regulation of biological processes, notably phosphate metabolic processes, cell cycle, cell division response to DNA stimulus, RNA biosynthetic processes and transcription. When evaluated based upon molecular function, iron binding,

wild-type Flt3 ITD-Flt3

2009; Gilliland & Griffin, 2002; Levis et al., 2005). We reported that the combination of Flt3 ligand (FL), stem cell factor (SCF) and thrombopoietin (TPO) induces Survivin expression in human CD34+ cells (Fukuda & Pelus, 2001), suggesting that Survivin lies downstream of Flt3 signaling. We therefore evaluated the effects of constitutive ITD-Flt3 signaling on Survivin expression. Survivin is up regulated by ITD-Flt3 in whole cells, as well as cells in G0/G1 phase of the cell cycle (Figure 1) as determined by intracellular flow cytometric analyses. This suggests that up regulation of Survivin is not a consequence of cell cycle progression. Similarly, Survivin mRNA was up regulated in Ba/F3 cells by ITD-Flt3 compared to wild-type Flt3 following IL3 withdrawal. Up regulation of Survivin by ITD-Flt3 was associated with growth factor independent proliferation of Ba/F3 cells (Fukuda et al., 2009). We next evaluated whether Survivin plays a role in aberrant growth factor-independent primary HPC proliferation upon transformation by ITD-Flt3. Over expression of human wild-type Flt3 in CFU-GM from control Survivin fx/fx or Survivin fx/fx mice harboring a tamoxifen-inducible Cre recombinase (Cre-ERTM) failed to proliferate in vitro, whereas ectopic expression of ITD-Flt3 resulted in significant growth of CFU-GM in the absence of added growth factors. Treatment with 4OH-Tamoxifen to delete Survivin resulted in a significant reduction in the growth factor-independent proliferation of CFU-GM induced by ITD-Flt3 (Figure 2). Identical results were observed in the immunophenotypically defined c-kit+, Sca-1+, Lineage negative (KSL) population of cells enriched for mouse stem and progenitor cells. These findings suggest that Survivin is involved in the growth factor-independent proliferation of HPC induced by ITD-Flt3 and that antagonizing Survivin may be therapeutically beneficial for acute myeloid leukemia (AML) expressing ITD-Flt3.

Fig. 1. BaF3 cells expressing wild-type Flt3 or ITD-Flt3 were fixed and stained for Survivin and DNA in whole cells (left) and cells with 2N DNA representing G0/G1 cells (right).

Fig. 2. Wild type Flt3 or ITD-Flt3 were retrovirally transduced into marrow cells from Cre ER Survivin fx/fx or control Survivin fx/fx mice. Following transduction, cells were plated in a CFU assay in agar with 30% FBS and 1uM 4OH tamoxifen in the absence of hematopoietic growth factors for 2 weeks. Treatment with 4OH-TM significantly reduced growth factor independent CFU-GM induced by ITD-Flt3 in Cre-ERTM Survivin fx/fx marrow cells.
