Survivin Selectively Modulates Genes Deregulated in Human Leukemia Stem Cells

ITD-Flt3 mutations are detected in leukemia stem cells (LSCs) in acute myeloid leukemia (AML) patients. While antagonizing Survivin normalizes ITD-Flt3-induced acute leukemia, it also impairs hematopoietic stem cell (HSC) function, indicating that identification of differences in signaling pathways downstream of Survivin between LSC and HSC are crucial to develop selective Survivin-based therapeutic strategies for AML. Using a Survivin-deletion model, we identified 1,096 genes regulated by Survivin in ITD-Flt3-transformed c-kit+, Sca-1+, and lineageneg (KSL) cells, of which 137 are deregulated in human LSC. Of the 137, 124 genes were regulated by Survivin exclusively in ITD-Flt3+ KSL cells but not in normal CD34neg KSL cells. Survivin-regulated genes in LSC connect through a network associated with the epidermal growth factor receptor signaling pathway and falls into various functional categories independent of effects on apoptosis. Pathways downstream of Survivin in LSC that are distinct from HSC can be potentially targeted for selective anti-LSC therapy.


Introduction
Survivin has been implicated in regulation of apoptosis, cell division, and cell cycle both in cancer cells and normal tissues, through caspase-dependent and -independent mechanisms [1][2][3]. We previously showed that Survivin is expressed and growth factor regulated in human CD34 + cells [4][5][6]. Antagonizing Survivin impairs production of mouse bone marrow hematopoietic progenitor cells in vitro [4,7] and conditional Survivin gene deletion in vivo in mice leads to bone marrow ablation as a result of loss of hematopoietic stem and progenitor cells (HSPC) [8]. In contrast to tight regulation of Survivin by hematopoietic growth factors in normal CD34 + cells, deregulated expression of Survivin is frequently observed in hematological diseases, particularly those associated with hematopoietic stem cell (HSC) expansion. For instance, Survivin is aberrantly overexpressed in acute myeloid leukemia [9,10] but downregulated in marrow cells of patients with aplastic anemia where HSPC are significantly reduced [11]. These findings suggest that Survivin regulates HSPC fate under normal and pathological conditions.
We previously reported that ITD-Flt3 mutations found in ∼25-30% of patients with acute myeloid leukemia (AML) and strongly associated with poor prognosis [12][13][14], increase expression of Survivin. Survivin mediates aberrant hematopoietic cell proliferation induced by ITD-Flt3 and regulates development of ITD-Flt3 + acute leukemia, suggesting that antagonizing Survivin may provide therapeutic benefit for patients with AML expressing ITD-Flt3 [14]. Survivin is the fourth most highly expressed transcript in cancer [15] and is commonly associated with a higher proliferative index, reduced apoptosis, resistance to chemotherapy and increased rate of tumor recurrence in cancer cells, making anti-Survivin therapy an attractive strategy in cancer [3]. Several anti-Survivin preclinical trials in solid tumor models 2 Journal of Oncology show that disrupting Survivin can reduce tumor growth [1][2][3]. However, studies from our group and others indicate that Survivin regulates normal HSPC [4,7,8], suggesting that targeting Survivin will likely result in hematopoietic toxicity. Therefore, identification of differences in signaling cascades downstream of Survivin between normal HSPC and cancer stem cells (CSC) or leukemia stem cells (LSC) are required to pinpoint targets that can effectively eradicate CSC/LSC with little toxicity on HSC. Previous reports show that ITD-Flt3 mutations are present in human LSC [13] and genes expressed in AML stem cells are deregulated [16]. The primary purpose of this study was to identify downstream Survivin signaling pathways in LSC that are distinct from normal HSC. Using ITD-Flt3 transformed ckit + , Sca-1 + , and lineage neg (KSL) cells from conditional Survivin knockout mice as surrogates for AML stem cells and littermate controls, we identified a panel of genes that are specifically regulated by Survivin in ITD-Flt3 transformed KSL cells and known to be deregulated in human LSC. The data identify selective signaling pathways downstream of Survivin in LSC that are distinct from normal HSC that can be potentially targeted for selective anti-LSC therapy.

Cell Culture, Plasmid Transfection, Retrovirus Transduction, and In Vitro Survivin Gene Deletion.
Mice with the Survivin gene flanked by loxP sites and a Tamoxifeninducible form of Cre (Cre-ER) were reported previously [14]. All mice were housed in microisolators with continuous access to rodent chow and acidified water. The Indiana University School of Medicine IACUC approved all experimental procedures. Retrovirus transduction of ITD-Flt3 into mouse bone marrow cells was carried out as described [14]. Briefly, bone marrow cells from littermate control Survivin flox/flox or CreER-Survivin flox/flox mice were transduced with ITD-Flt3 (N51) in MSCV-IRES-EGFP vector [17]. Seventy-two hours after sequential transduction, the medium was replaced with IMDM containing 10% FBS and the cells were incubated for 14 days without growth factors in the presence of 1uM of 4OH-Tamoxifen to induce Survivin gene deletion. All cells were cultured in triplicates in two independent experiments. Viable cells were enumerated using trypan blue exclusion and GFP + , c-kit + , Sca-1 + , and lineage negative (KSL) cells were sorted on day 14 by FACSAria (BD Biosciences). Sorted cells were immediately lysed and subjected to differential mRNA microarray analysis.

In Vivo Survivin
Deletion. Survivin flox/flox and CreER-Survivin flox/flox mice were treated with 5 mg Tamoxifen (5 mg/mouse i.p.) for 3 consecutive days and allowed to rest for 3 days, and Tamoxifen was administered for 3 additional days. Fourteen days after the final Tamoxifen injection, marrow cells were harvested, stained with anti-CD34, c-kit, Sca-1, and lineage markers and CD34 neg KSL cells isolated by FACSAria for mRNA microarray analysis.

mRNA Microarray.
Sorted cells were immediately lysed and subjected to differential mRNA microarray analysis. Microarray analysis was performed on a fee basis by Miltenyi Biotec (Auburn, CA). Briefly, 250 nanograms of each cDNA were used as template for Cy3 and Cy5 labeling which was performed according to Miltenyi Biotec's proprietary protocol. Equal amounts of labeled cDNAs from Tamoxifentreated Survivin flox/flox and CreER-Survivin flox/flox mice or those cells expressing ITD-Flt3 were hybridized overnight (17 hours, 65 • C) to Agilent Whole Mouse Genome Oligo Microarrays (44 K) according to the manufacturers' protocol. Fluorescence signals of the hybridized microarrays were detected using Agilent's DNA microarray scanner (Agilent, Palo Alto, USA). The Agilent Feature Extraction Software (FES) was used to read out and process the microarray image files. The software determines feature intensities and ratios (including background subtraction and normalization), rejects outliers, and calculates statistical confidences (P-values). For determination of differential gene expression, FES derived output data files were further analyzed using the Rosetta Resolverâ gene expression data analysis system (Rosetta Biosoftware, Seattle, USA). Significantly regulated genes were annotated and assigned to functional categories using the DAVID 2008 (the Database for Annotation, Visualization and Integrated Discovery; http://david.abcc.ncifcrf.gov/home.jsp) program [18]. Functional networks of the genes regulated by Survivin was visualized using Cytoscape software: http://www.cytoscape.org/ [19].

Reduction in Proliferation ITD-Flt3 Transformed KSL Cells by Survivin Deletion Is Associated with Alteration of Expression of 1,096 Genes Classified into Various Biological
Functions. While incubation of marrow cells transduced with wild-type Flt3 in IMDM containing 10% FBS without hematopoietic any growth factors failed to support KSL proliferation, overexpression of ITD-Flt3-EGFP in primary mouse bone marrow cells results in the ability of KSL cells to proliferate in the same culture condition without additional any hematopoietic growth factors as we reported, indicating that ITD-Flt3 is sufficient to transform primitive HSPC [14]. Incubation of ITD-Flt3 transduced marrow cells derived from CreER-Survivin flox/flox mice with 1uM 4OH-Tamoxifen significantly reduced factor-independent KSL cells on day 14 compared to Survivin flox/flox cells treated in exactly the same Journal of Oncology   (Figure 1), confirming that Survivin is required for factor independent growth of ITD-Flt3 transformed KSL cells.
To investigate the mechanism by which Survivin regulates aberrant proliferation of ITD-Flt3 transformed KSL cells, mRNA expression was compared between ITD-Flt3 transformed Survivin flox/flox and CreER-Survivin flox/flox KSL using mRNA microarrays. Although inhibition of Survivin mRNA expression in CreER-Survivin flox/flox KSL cells by 4OH-Tamoxifen was only 50% compared to control, the mRNA microarray identified 1,096 transcripts differentially regulated by Survivin. These genes were classified based on biological process and molecular function defined by Gene Ontology Term (http://www.geneontology.org/) using DAVID 2008 program ( Figure 2). Representative groups for biological process include phosphate metabolic process, cell cycle, cell division, response to DNA damage stimulus, RNA biosynthetic process and transcription (Figure 2

Survivin Modulates Expression of Genes Deregulated in
Human AML LSC. The requirement of Survivin for the selfrenewing capability of ITD-Flt3 transformed KSL cells or CFU [14] and the presence of ITD-Flt3 in LSC [13] strongly suggests that Survivin is important for LSC fate decisions resulting from deregulated gene expression. We compared 1,096 differentially expressed genes in Survivin deleted ITD-Flt3 + KSL cells with the existing deregulated gene expression database for human AML stem cells [16]. Out of 1,096 genes regulated by Survivin in KSL cells transformed by ITD-Flt3, 137 genes are also listed in the deregulated molecules in human AML stem cells (Tables 1(a)-1(d)) [16]. The relationship between Survivin and LSC on the modulation   of the gene expression is illustrated in Figure 3(a). Of the 137 genes identified, 79 genes were downregulated by Survivin deletion while 58 genes were upregulated, implying that these genes are conversely increased and decreased by the presence of Survivin in LSC (Figure 3(a)). In contrast, 92 genes were upregulated and 45 genes were downregulated by LSC. Among the 79 genes downregulated genes by Survivin deletion, 55 transcripts were upregulated (Figure 3(a) and Table 1(a)) whereas 24 genes were downregulated by LSC (Figure 3(a) and Table 1(c)). Similarly, out of 58 upregulated transcripts by Survivin deletion, 21 genes were downregulated ( Figure 3(a) and Table 1(b)) and 37 genes were upregulated in LSC (Figure 3(a) and Table 1(d)) ( Figure 3(a)). Next, we classified the 137 genes into functional annotation groups using the DAVID program (P < .05, Figure 3(b)). Significantly enriched functional groups were annotated as phosphoprotein, nucleus, acetylation, cell cycle, ATP binding, regulation of EGF signaling pathway, cell adhesion and others. The size of each circle in Figure 3(b) represents the number of genes involved in each functional category and the thickness of the line indicates the number of genes shared with any function, suggesting enrichment of the gene groups with functional redundancy. Among the molecules most frequently shared within the functional groups include BGLAP, Chrac1, Hmgb1 and Smarce1. Similarly, 76 genes upregulated or downregulated both by Survivin and LSC (55 + 21 genes shown in Figure 1(a)) were functionally classified (Figure 3(c)). Phosphoprotein, acetylation, DNA binding, ATP binding and DNA replication were significantly enriched.
The 1,096 differentially Survivin-regulated genes were also searched against the list of genes expressed in mouse HSC [20]. We identified 94 differentially expressed genes in the list of genes that include HSC. There were 45 genes whose expression was selectively enriched in HSC compared to other populations (Table 2). Crem, Emp1, Hmga2, Lrrn1, Maff, Myef2, Rps4x, and Sos1 listed in HSC database are also deregulated in LSC. We next compared the human LSC associated 137 genes regulated by Survivin in ITD-Flt3 + KSL cells with differentially regulated genes in normal bone marrow CD34 neg KSL cells obtained from CreER-Survivin flox/flox and control Survivin flox/flox mice following Survivin deletion. Tamoxifen reduced Survivin expression by 10-fold in CD34 neg KSL cells from CreER-Survivin flox/flox mice compared to control Survivin flox/flox 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 CD34 neg KLS cells by Survivin deletion. In contrast, Cenpa, Cpd, Myef2, Nmt2, Taf1b and Tmpo were downregulated in ITD-Flt3 + KSL cells while they were upregulated in normal CD34 neg KSL cells by Survivin deletion. These findings suggest that the 124 genes are regulated by Survivin exclusively in ITD-Flt3 + KSL cells but not in CD34 neg KSL cells. The complete list of the genes regulated by Survivin in CD34 neg KSL cells will be reported elsewhere (manuscript in preparation). Fold change: ITD-Flt3 + KLS cells derived from CreER-Survivin fx/fx compared to control Survivin fx/fx in two experiments.

Survivin Modulates Gene Expression in LSC That Connects Through a Functional Signaling Network Associated with Epidermal Growth Factor Receptor Signaling Pathway.
Functional annotation analysis indicates that genes related with dorsoventral axis formation or epidermal growth factor receptor signaling pathway (EGFR) are significantly enriched in the shared genes associated with LSC and Survivin signaling in KEGG database (http://www.genome.jp/kegg/) (P < .03). Similarly, genes associated with regulation of EGFR signaling pathway are enriched in the Gene Ontology database (P < .02). Our analysis shows that 13 molecules shared by LSC and Survivin signaling are mapped on a functional signaling network that connects through the EGFR pathway (Figure 4).

Discussion
Survivin deletion in ITD-Flt3 transformed KSL cells results in a significant reduction in growth factor-independent proliferation coincident with growth inhibition. Our data shows that Survivin deletion modulates gene expression of 1,096 genes that are associated with various cellular and metabolic functions. Comparison of the 1,096 Survivin regulated genes in ITD-Flt3 + KSL cells with 3,005 differentially regulated genes in the human AML stem cell database identified 137 shared genes. Functional classification of these 137 genes indicates they connect through a functional signaling network associated with EGFR signaling pathway and affect various biological and molecular processes. Comparison of the 137 Survivin-regulated genes in LSC with transcripts regulated by Survivin in normal CD34 neg KSL cells show that 124 genes are regulated by Survivin exclusively in ITD-Flt3 + KSL cells and not in normal HSC. These data suggest that Survivin contributes to deregulation of gene expression in AML stem cells via selective signaling pathways distinct from normal HSC that can be potentially targeted for therapeutic benefit.
There is no direct evidence that Survivin directly regulates gene transcription; however, modulation of Survivin can clearly affect transcription in cancer cells [21][22][23] and transgenic expression of Survivin alters the expression of multiple genes in the bladder [24].
The involvement of Survivin in transcriptional regulation is consistent with the fact that Bir1, a Caenorhabditis  AEBP2, MCM8,  POLG, SNAPC4,  CREM, TRPS1,  ETS2, MYEF2,  GFI1, TMPO,  TRIM21,   elegans homologue of Survivin, regulates transcription, most likely through Histone phosphorylation by Aurora kinase [25]. Survivin is essential for activation of Aurora kinase that phosphorylates Histone H3 [26,27], an event required for transcriptional regulation [28][29][30] and cytokinesis [31], suggesting that Survivin may regulate transcription via Aurora kinase mediated Histone H3 phosphorylation. Over expression of Survivin also leads to phosphorylation of the Sp1 transcription factor [21]. Since microarray analysis was performed on lineage marker negative viable cells that express GFP, c-kit, and Sca-1 and survived in culture, it is unlikely that the effects of Survivin on gene expression is a consequence of KSL apoptosis, although we cannot rule out indirect effects resulting from cell cycle arrest as a consequence of Survivin deletion.
Survivin is known to inhibit caspases 3, 7, and 10 that mediate apoptosis [1][2][3]. Our functional annotation analysis of the genes regulated by Survivin indicates that Survivin modulates a number of genes that affect multiple biological processes and molecular functions, although inhibition of Survivin mRNA expression in CreER-Survivin flox/flox KSL cells by 4OH-Tamoxifen was only 50% compared to control. While incompleteness of the gene deletion may affect the downstream signals, significant reduction of total cell number was observed. Since Survivin deletion induces cell death, the deleted cells are likely no longer present in the culture at the time of cell harvest for analyses, resulting in an underestimate of the efficiency of Survivin gene deletion and likely represents Survivin expression in cells that have escaped the Tamoxifen-induced deletion. The genes affected by Survivin deletion were classified into cell cycle, adhesion, DNA replication, transcriptional factor binding, acetylation, phosphorylation, and polymerase. We did not observe changes in genes that mediate apoptosis. These results suggest that Survivin can regulate ITD-Flt3 transformed KSL cell fate independent of its activity as a caspase inhibitor. More importantly, comparison of the 1,096 Survivin regulated genes in ITD-Flt3 + KLS cells with the  Figure 4: Genes regulated by Survivin in ITD-Flt3 + KSL cells and deregulated in LSC are mapped on functional signaling network associated with epidermal growth factor receptor signaling pathway. Epidermal growth factor receptor signaling pathway (EGFR) is significantly enriched in the shared genes associated with LSC and Survivin signaling by pathway enrichment analysis in KEGG and Gene Ontology database. Our analysis shows that 13 molecules shared by LSC and Survivin signaling are mapped on a functional signaling network associated with EGFR. The network was created based on KEGG database using Cytoscape software. Green circle and red circle represents downregulation and upregulation by Survivin gene deletion, respectively.
human AML stem cell database demonstrates that 137 genes (12.4%) modulated by Survivin are also deregulated in LSCs. Functional annotation analysis indicates that genes related to dorsoventral axis formation or epidermal growth factor receptor signaling pathway (EGFR) are significantly enriched in the shared genes associated with LSC and Survivin signaling, even though the number of molecules detected that associates with EGFR signaling is small. EGFR signaling activates signaling cascades involved in cell proliferation, such as Src, Sos, and MAP-kinases, and is known to be dysregulated in solid tumors [32,33]. Our data also indicates that Survivin modulates expression of several genes that connect through the EGFR signaling pathway (Figure 4), suggesting a potential role of EGFR signaling downstream of Survivin in AML stem cells, despite the fact that EGFR is not usually upregulated in AML cells [34]. However, recent studies indicate an antineoplastic effect of an EGFR inhibitor in AML via off-target effects [34,35]. Thus, our data would support EGFR signaling as a candidate pathway for treatment of patients with AML. While Survivin is not listed in the LSC database [16], it is expressed at higher levels in AML cells compared to normal CD34 + cells [9,10]. Our data clearly show that Survivin affects expression of 137 genes associated with LSC and reduces aberrant KSL proliferation induced by ITD-Flt3. This finding suggests that antagonizing Survivin in LSC may normalize expression of the deregulated genes, which in turn can inhibit their aberrant proliferation. Since Survivin is expressed and regulated in normal CD34 + cells [4,6] and antagonizing Survivin reduces normal HSPC in vivo [8], it is likely that Survivin deletion will affect normal HSC in the patients. Although direct comparison of gene expression in in vitro ITD-Flt3 + KSL cells with in vivo CD34 neg KSL cells may not necessarily provide definite differences in Survivin signaling between these populations in vivo, we found that 124 out of 137 Survivin associated genes shared by ITD-Flt3 + KSL cells and human LSC were not regulated in normal CD34 neg KSL cells by Survivin. This disparity of downstream signaling of Survivin between normal and transformed HSC may represent as a means of identifying novel therapeutic targets against LSC that would thereby supplant direct Survivin disruption which would otherwise toxic to normal HSC.

Conclusion
We identified genes regulated by Survivin in ITD-Flt3 transformed KSL cells that are deregulated in human AML stem cells that are distinct from normal HSC. This study will facilitate the identification of specific therapeutic targets downstream of Survivin to eradicate LSC without affecting normal HSC.