**3. Survivin regulated genes**

188 Advances in Cancer Therapy

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

G0/G1 cells

wild-type Flt3

ITD-Flt3

W51 W73 W78

leukemia (AML) expressing ITD-Flt3.

whole cells

Survivin Survivin

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).

events

events

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,

Survivin: Identification of Selective Functional Signaling Pathways

selective anti-Survivin anti-cancer therapies.

acetylation

AEBP2 ACAA2 SNAPC4 MYEF2 LARS2 WTAP TRPM2 NRIP1 PANK4 UBE2N SLC1A5 PGLS ANKRD28 PSMD11 DPP9 RPS20

TMPO

phosphoprotein

GAS2L3 OTUD5 TDRD3 SOLH FGR UBE3B CREM SYNJ1 POLA2 WTAP SENP7 RNF126 APP GIT2 NDRG1 RPS20 GFI1 AEBP2 ACAA2 CARS ANP32B TRPM7 ARHGEF18 SNAPC4 NSFL1C ATP11A SPIRE1 NRIP1 ANKRD28 PSMD11 ETS2 TRPS1 MAPK8IP3 TMPO

LRRK1

functional category are shown in the text box.

in Transformed Cells and Identification of a New Splice Variant with Growth Survival Activity 191

Rps4x and Sos1 are known to be deregulated in LSC. Comparison of the human LSC associated 137 genes regulated by Survivin in ITD-Flt3+KSL cells with differentially regulated genes in normal CD34neg KSL cells from control and Survivin deleted mice indicated that coincident with reduction of Survivin expression (10-fold in CD34neg KSL cells from CreERTM-Survivin fx/fx mice compared to control Survivin fx/fx in two independent experiments) out of 137 genes, Arg2, Med25, Pmaip1, Pola2, Ube3b, Ephb2 and Rab18 were differentially regulated in both ITD-Flt3+ KSL cells and normal CD34neg KLS cells. In contrast, Cenpa, Cpd, Myef2, Nmt2, Taf1b and Tmpo were down regulated in ITD-Flt3+ KSL cells while they were upregulated in normal CD34neg KSL cells. These findings suggest that 124 genes are regulated by Survivin exclusively in ITD-Flt3+ KSL cells but not in normal CD34neg KSL cells (The complete list of the genes regulated by Survivin in CD34neg KSL cells will be reported elsewhere). However, these data clearly indicate that Survivin contributes to deregulation of gene expression in AML stem cells via effects on selective signaling pathways that are distinct from normal HSC that can be potentially targeted for more

ATP binding DNA binding

AEBP2 MCM8 POLG SNAPC4 *CREM* TRPS1 ETS2 MYEF2 GFI1 TMPO TRIM21 RTEL1

DNA replication

MCM8 POLG POLA2

UBE2N PANK4 MCM8 CARS FGR TRPM7 PHKG2 ATP11A LARS2 LRRK1 RTEL1

Fig. 3. Genes regulated in common by Survivin and LSC. The 76 genes up-regulated or down regulated by Survivin and LSC were functionally classified by DAVID software. The size of each circle represents the number of genes involved in each functional categories and the thickness of the line indicates the number of genes shared with any function. Functional classification was performed based on Swissprot Keywords. Genes annotated in each

nucleotide binding, DNA binding and protein kinase activity were the functions most significantly affected by Survivin deletion. Although Survivin is known to inhibit caspases 3, 7 and 10 that mediate apoptosis (Altieri, 2003a; Altieri, 2003b; Fukuda & Pelus, 2006), we did not observe significant changes in genes that mediate apoptosis, suggesting that Survivin can regulate ITD-Flt3 transformed KSL cell fate independent of its ability to inhibit caspase activity. However, microarray analysis was performed on lineage marker negative viable cells that express GFP and stem cell markers and survived in culture, perhaps explaining lack of effects on genes involved in apoptotic pathways. The finding that Survivin deletion affected transcription was surprising since there is no direct evidence that Survivin regulates gene transcription. However, this is consistent with the fact that the *Caenorhabditis elegans* Bir1 Survivin homologue regulates transcription, most likely through effects on histone phosphorylation by Aurora kinase (Kostrouchova et al., 2003). Survivin is essential for activation of Aurora kinase that phosphorylates histone H3 (Bolton et al., 2002; Chen et al., 2003), an event required for transcriptional regulation (Cheung et al., 2000; Li et al., 2002; Nowak & Corces, 2000) and cytokinesis (Wei et al., 1999). Furthermore, modulation of Survivin affects transcription in cancer cells (Asanuma et al., 2004; Balkhi et al., 2008; Takizawa et al., 2007) and transgenic Survivin expression alters gene expression in bladder cells (Salz et al., 2005). Overexpression of Survivin also leads to phosphorylation of the Sp1 transcription factor (Asanuma et al., 2004).

#### **4. Survivin modulates expression of genes deregulated in human AML LSC**

The facts that Survivin is required for the self-renewal of ITD-Flt3 transformed KSL cells and ITD-Flt3 is present in leukemic stem cells (LSC) suggests that Survivin is important for LSC fate decisions. When the 1,096 differentially expressed genes in Survivin deleted ITD-Flt3+ KSL cells was compared with the deregulated gene expression database for human AML stem cells (Majeti et al., 2009), 137 genes were identified in common. Survivin deletion resulted in down-regulation of 79 genes, while 58 genes were up-regulated, implying that these genes are conversely increased and decreased by the presence of Survivin. In contrast, 92 genes were upregulated and 45 genes were downregulated in LSC. Among the 79 genes downregulated by Survivin deletion, 55 were upregulated while 24 genes were downregulated in LSC. Among the 58 transcripts upregulated by Survivin deletion, 21 were downregulated and 37 upregulated in LSC.

Classification of these genes into functional annotation groups using DAVID program identified significantly (p<0.05) enriched functional groups annotated as phosphoprotein, nucleus, acetylation, cell cycle, ATP binding, regulation of EGF signaling pathway, and cell adhesion, with a number of genes showing shared function, suggesting enrichment of gene groups with redundant function. Among the molecules most frequently shared within the functional groups included BGLAP [bone gamma carboxyglutamate protein 1(∆ -10.7)], Chrac1 [chromatin accessibility complex 1 (∆ -4.3), Hmgb1 [high mobility group box 1 (∆ - 3.4)] and Smarce1 [SWI ∆ -2.9)]. Phosphoprotein, acetylation, DNA binding, ATP binding and DNA replication were significantly enriched when the 76 genes upregulated or downregulated both by Survivin deletion and LSC were classified (Figure 3).

Comparison of the 1,096 differentially Survivin-regulated genes against the mouse HSC database (Ivanova et al., 2002) identified 94 differentially expressed genes. This included 45 genes regulated by Survivin in ITD-Flt3+ HSC whose expression was selectively enriched in HSC compared to other populations. Notably, Crem, Emp1, Hmga2, Lrrn1, Maff, Myef2,

nucleotide binding, DNA binding and protein kinase activity were the functions most significantly affected by Survivin deletion. Although Survivin is known to inhibit caspases 3, 7 and 10 that mediate apoptosis (Altieri, 2003a; Altieri, 2003b; Fukuda & Pelus, 2006), we did not observe significant changes in genes that mediate apoptosis, suggesting that Survivin can regulate ITD-Flt3 transformed KSL cell fate independent of its ability to inhibit caspase activity. However, microarray analysis was performed on lineage marker negative viable cells that express GFP and stem cell markers and survived in culture, perhaps explaining lack of effects on genes involved in apoptotic pathways. The finding that Survivin deletion affected transcription was surprising since there is no direct evidence that Survivin regulates gene transcription. However, this is consistent with the fact that the *Caenorhabditis elegans* Bir1 Survivin homologue regulates transcription, most likely through effects on histone phosphorylation by Aurora kinase (Kostrouchova et al., 2003). Survivin is essential for activation of Aurora kinase that phosphorylates histone H3 (Bolton et al., 2002; Chen et al., 2003), an event required for transcriptional regulation (Cheung et al., 2000; Li et al., 2002; Nowak & Corces, 2000) and cytokinesis (Wei et al., 1999). Furthermore, modulation of Survivin affects transcription in cancer cells (Asanuma et al., 2004; Balkhi et al., 2008; Takizawa et al., 2007) and transgenic Survivin expression alters gene expression in bladder cells (Salz et al., 2005). Overexpression of Survivin also leads to phosphorylation of the Sp1

**4. Survivin modulates expression of genes deregulated in human AML LSC**  The facts that Survivin is required for the self-renewal of ITD-Flt3 transformed KSL cells and ITD-Flt3 is present in leukemic stem cells (LSC) suggests that Survivin is important for LSC fate decisions. When the 1,096 differentially expressed genes in Survivin deleted ITD-Flt3+ KSL cells was compared with the deregulated gene expression database for human AML stem cells (Majeti et al., 2009), 137 genes were identified in common. Survivin deletion resulted in down-regulation of 79 genes, while 58 genes were up-regulated, implying that these genes are conversely increased and decreased by the presence of Survivin. In contrast, 92 genes were upregulated and 45 genes were downregulated in LSC. Among the 79 genes downregulated by Survivin deletion, 55 were upregulated while 24 genes were downregulated in LSC. Among the 58 transcripts upregulated by Survivin deletion, 21 were

Classification of these genes into functional annotation groups using DAVID program identified significantly (p<0.05) enriched functional groups annotated as phosphoprotein, nucleus, acetylation, cell cycle, ATP binding, regulation of EGF signaling pathway, and cell adhesion, with a number of genes showing shared function, suggesting enrichment of gene groups with redundant function. Among the molecules most frequently shared within the functional groups included BGLAP [bone gamma carboxyglutamate protein 1(∆ -10.7)], Chrac1 [chromatin accessibility complex 1 (∆ -4.3), Hmgb1 [high mobility group box 1 (∆ - 3.4)] and Smarce1 [SWI ∆ -2.9)]. Phosphoprotein, acetylation, DNA binding, ATP binding and DNA replication were significantly enriched when the 76 genes upregulated or

Comparison of the 1,096 differentially Survivin-regulated genes against the mouse HSC database (Ivanova et al., 2002) identified 94 differentially expressed genes. This included 45 genes regulated by Survivin in ITD-Flt3+ HSC whose expression was selectively enriched in HSC compared to other populations. Notably, Crem, Emp1, Hmga2, Lrrn1, Maff, Myef2,

downregulated both by Survivin deletion and LSC were classified (Figure 3).

transcription factor (Asanuma et al., 2004).

downregulated and 37 upregulated in LSC.

Rps4x and Sos1 are known to be deregulated in LSC. Comparison of the human LSC associated 137 genes regulated by Survivin in ITD-Flt3+KSL cells with differentially regulated genes in normal CD34neg KSL cells from control and Survivin deleted mice indicated that coincident with reduction of Survivin expression (10-fold in CD34neg KSL cells from CreERTM-Survivin fx/fx mice compared to control Survivin fx/fx in two independent experiments) out of 137 genes, Arg2, Med25, Pmaip1, Pola2, Ube3b, Ephb2 and Rab18 were differentially regulated in both ITD-Flt3+ KSL cells and normal CD34neg KLS cells. In contrast, Cenpa, Cpd, Myef2, Nmt2, Taf1b and Tmpo were down regulated in ITD-Flt3+ KSL cells while they were upregulated in normal CD34neg KSL cells. These findings suggest that 124 genes are regulated by Survivin exclusively in ITD-Flt3+ KSL cells but not in normal CD34neg KSL cells (The complete list of the genes regulated by Survivin in CD34neg KSL cells will be reported elsewhere). However, these data clearly indicate that Survivin contributes to deregulation of gene expression in AML stem cells via effects on selective signaling pathways that are distinct from normal HSC that can be potentially targeted for more selective anti-Survivin anti-cancer therapies.

Fig. 3. Genes regulated in common by Survivin and LSC. The 76 genes up-regulated or down regulated by Survivin and LSC were functionally classified by DAVID software. The size of each circle represents the number of genes involved in each functional categories and the thickness of the line indicates the number of genes shared with any function. Functional classification was performed based on Swissprot Keywords. Genes annotated in each functional category are shown in the text box.

Survivin: Identification of Selective Functional Signaling Pathways

signaling is small.

**6. Survivin splice variants** 

**7. Expression of Survivin 3***γ*

in Transformed Cells and Identification of a New Splice Variant with Growth Survival Activity 193

activates signaling cascades involved in cell proliferation, such as Src, Sos and MAP-kinases and is known to be dysregulated in a number of solid tumors (Alvarez et al., 2010; Harris & McCormick, 2010). These findings suggest a potential role of EGFR signaling downstream of Survivin in AML stem cells, despite the fact that EGFR is not usually up regulated in AML cells (Hahn et al., 2009). However, recent studies indicate an anti-neoplastic effect of an EGFR inhibitor in AML via off-target effects (Boehrer et al., 2008; Hahn et al., 2009). Thus, our data would support EGFR signaling as a candidate pathway for treatment of patients with AML, even though the number of molecules detected that associates with EGFR

Survivin, like many genes, is alternatively spliced and variants having both similar or opposite activities have been identified, some of which are of prognostic significance (Fukuda & Pelus, 2006; Sampath & Pelus, 2007) . Spliced variants identified to date include Survivin 2 (Caldas et al., 2005; Mahotka et al., 2002) , 2B, Ex3 (Ling et al., 2005; Mahotka et al., 1999; Mahotka et al., 2002; Song & Wu, 2005) and 3B (Badran et al., 2004), and some of these variants have been evaluated for their anti- or pro-apoptotic function and ability to interact with wild-type Survivin and other chromosomal passenger proteins (CPC) in some model systems. More recently, several new Survivin splice variants were found in the expressed sequence tag (est) database or described based on RT-PCR using RNA from Molt-4 cells (Li & Ling, 2006; Mola et al., 2007), however the biological functions of these new variants are not known. Overall, with most all the variants reported to date, there is still a considerable lack of understanding of their biology and their function and prognostic value is only now being realized (Sampath & Pelus, 2007). In order to better understand the biology of human Survivin splice variants, we cloned each variant and evaluated their antiapoptotic functions. In the process, we identified a new splice variant we termed Survivin 3 that is similar to Survivin 3B (Figure 5). To determine if this was truly a novel variant, a forward primer specific for all splice variants and a splice variant specific reverse primer were designed and used to amplify cDNA using a polydT reverse transcribed cDNA library from HL60, K562 and Mo7e-Bcr-abl cells, the products resolved on an agarose gel and ~400bp bands excised, cloned and sequenced. Sequence analysis confirmed the presence of the novel splice variant. Survivin 3 contains 12 new amino acids at the Cterminus compared to wild-type Survivin and differs from all of the previously reported Survivin splice variants. Secondary structure prediction using Jpred suggests that Survivin 3 is structurally similar to wild-type Survivin, with the new amino acids predicted to be capable of forming a C-terminal helix that could potentially form the coiled coil domain,

even though the length of this helix would be smaller than wild-type Survivin.

Survivin 3 expression in primary cancer tissues was determined by quantitative RT-PCR on primary breast and prostate tumor samples obtained in an array format. In contrast to most cancers where wild-type Survivin is the predominant transcript, Survivin 3 was expressed at higher levels in benign prostate tissue and at equivalent levels in breast cancer cells. In primary breast cancer samples a statistically significant increase in wild-type Survivin expression at stages 1, 2 and 3 compared to control normal breast tissue was observed, with
