**8. Survivin 3***γ* **demonstrates antiapoptotic activity**

To evaluate the function of the novel Survivin 3 splice variants, we stably expressed human (Hu) wild-type Survivin with or without an N-terminal Flag-tag or Survivin 3 with an Nterminal HA-tag in murine YAC-1 lymphoma cells. Wild-type HuSurvivin, wild-type Flag-HuSurvivin and HA-Survivin 3 were all expressed, although their levels of expression varied, with wild-type HuSurvivin expressing at 31-fold, whereas wild-type Flag-HuSurvivin and HA-Survivin 3 were expressed at 76- and 63-fold higher levels, respectively than the empty vector control cells at the RNA level. Western blot analysis using a rabbit anti-Survivin polyclonal antibody that recognizes both the human and the murine Survivin showed background levels of murine Survivin as well as the presence of each of the transduced Survivins, indicating that each of the splice variants was translated. Analysis of band intensity indicated that wild-type HuSurvivin was expressed at the highest level followed by wild-type Flag-HuSurvivin with HA-Survivin 3 expressed at a lower level. The same pattern of expression was observed with a mouse anti-Survivin monoclonal antibody that recognizes predominantly human Survivin.

As a measure of anti-apoptotic function we evaluated resistance of YAC-1 cells transduced with wild-type Survivin, wild-type Flag-Survivin and HA-Survivin 3 to the chemotherapeutic drugs Etoposide and Taxol at their optimal therapeutic dose. Treatment of cells with Taxol showed that, as expected, over expression of either wild-type Survivin or wild-type Flag-Survivin provided 2.7- and 2.8-fold resistance to Taxol-induced cell death, respectively, compared to YAC-1 cells transduced with vector control (Figure 6). Ectopic expression of HA-Survivin 3 provided 2.6-fold resistance. In multiple experiments, the IC50's for wild-type HuSurvivin, wild-type Flag-HuSurvivin and HA-Survivin 3 were significantly higher than control (2.85 ± 0.32; P<0.05). YAC-1 cells expressing wild-type

Survivin: Identification of Selective Functional Signaling Pathways

be potentially targeted for selective anti-leukemia therapy.

utilized for development of anti-leukemia therapies.

*Oncogene*, Vol.22:8581-8589.

myeloid leukaemia. *Br J Haematol*, Vol.111:196-203.

**10. References** 

**9. Summary** 

in Transformed Cells and Identification of a New Splice Variant with Growth Survival Activity 197

We have shown that ITD-Flt3 mutations increase expression of Survivin, which regulates development of ITD-Flt3+ acute leukemia, clearly indicating a potential therapeutic benefit for antagonizing Survivin in patients with ITD-Flt3+ AML (Fukuda et al., 2009; Fukuda et al., 2011; Sampath & Pelus, 2007). However, we have shown that Survivin also regulates normal HSPC function (Fukuda et al., 2004; Fukuda & Pelus, 2002; Leung et al., 2007; Sampath & Pelus, 2007); raising caution that targeting Survivin can have hematopoietic toxicity. Therefore, identification of differential signaling cascades downstream of Survivin between normal hematopoietic cells versus cancer and leukemia stem cells is required to pinpoint targets that can lead to effective anti-malignant therapies with acceptable normal stem cell toxicity. Our genetic profiling using normal and ITD-Flt3 transformed cells from conditional Survivin knockout mice has identified a panel of genes that are specifically regulated by Survivin in ITD-Flt3 transformed cells and known to be deregulated in human LSC that can

Inherited and acquired changes in pre-mRNA splicing have been documented to play a significant role in human disease development and loss of fidelity or variation of the splicing process may play a major role in carcinogenesis (Fackenthal & Godley, 2008; Matlin et al., 2005; Skotheim & Nees, 2007) . Splice variants that are found predominantly in tumors have clear diagnostic value (Brinkman, 2004; Caballero et al., 2001) and may provide potential drug targets. Splice variants of Survivin have been reported but their prognostic value is not clear, partially based upon detection methods that are not sufficiently specific (Sampath & Pelus, 2007), or due to lack of information on biological function. We have identified a novel Survivin splice variant with anti-apoptotic activity similar to wild-type Survivin, at least with respect to sensitivity to chemotherapeutic drugs. We characterized its expression in several transformed cell lines, and similar to other Survivin splice variants, wild-type Survivin is the predominant transcript. However, in contrast to wild-type Survivin, Survivin 3 expression does not change with increasing stage/severity of primary prostate and breast cancers. In addition, unlike other Survivin splice variants, Survivin 3 is expressed at higher levels than wild-type Survivin in primary HSPC, and their lack of increase with cell proliferation suggests that they may play a role in stem cell maintenance. Since Survivin 3 was cloned from and found to be highly expressed in hematopoietic cells, it will be particularly interesting to evaluate its expression in hematopoietic malignancies and their correlation with disease/stage/survival. Furthermore, since ITD-Flt3 mutations increase Survivin expression, it will be interesting to determine if they also modulate Survivin 3 expression and how this relates to disease progression and prognostic value. Given the specific genes regulated by Survivin in leukemic stem cells, it will also be of interest whether Survivin 3 elicits overlapping or divergent gene products that can be

Adida, C. et.al (2000). Expression and prognostic significance of survivin in de novo acute

Altieri, D.C. (2003a). Survivin, versatile modulation of cell division and apoptosis in cancer.

Survivin also showed significant resistance to Etoposide. Wild-type Flag-Survivin and HA-Survivin 3 conferred resistance to Etoposide in YAC-1 cells to a similar degree as wild-type Survivin. These studies suggest that expression of Survivin 3 could potentially contribute to drug resistance. Expression levels of murine wild-type Survivin and the only known murine Survivin splice variant, Survivin-121 (Conway et al., 2000) were unchanged (not shown), indicating that changes in endogenous murine Survivin levels were not involved in the increase in resistance to chemotherapeutic compounds. These findings suggest that wildtype and Survivin 3 proteins possess equivalent anti-apoptotic function. Since crystal structure of wild-type Survivin indicates that the C-terminal helix could be involved in protein-protein interaction (Chantalat et al., 2000) and required for interaction with the chromosomal passenger protein, INCEP (Wheatley et al., 2004), whereas the N-terminus of wild-type Survivin was unordered (Chantalat et al., 2000), we hypothesized that addition of a C-terminal tag would hinder protein-protein interactions, therefore we engineered Nterminal tags into all the Survivin cDNAs. The presence of HA-tag at the N-terminus of the Survivin coding region did not affect activity since they were equivalent to N-terminal Flagtagged wild-type Survivin, which itself behaved similar to wild-type Survivin at least for drug sensitivity. The facts that Survivin 3confers drug resistance/anti-apoptotic function to two different classes of chemotherapeutic drugs Taxol, a microtubule destabilizing agent, and Etoposide, a topoisomerase II inhibitor, as well as growth factor independence similar to wild-type Survivin, and that it may be preferentially associated with maintenance of stem cell quiescence, makes it an interesting potential therapeutic target .

Fig. 6. Response of YAC1 cells over expressing wild-type human Survivin (SurWT), wild-type human Flag-Survivin (Flag-Sur WT) and HA Survivin 3 (HA-Sur3) to chemotherapeutic drugs. Fold resistance of stable transduced YAC-1 cells treated with 50 nM Taxol or 0.25 µg/ml Etoposide.
