The role of survivin splice variants in hypoxia-induced therapy resistance to the survivin targeting agent YM155
Acute myeloid leukemia (AML) accounts for 20% of childhood leukemia. The 5-year survival rate is 60%. The most common causes of death are relapse and chemotherapeutic resistance. AML cells reside in the hypoxic bone marrow. Hypoxia contributes to therapy resistance in AML. Previous experiments in the Aleem lab demonstrated that survivin is implicated in hypoxia-induced drug resistance. Survivin, encoded by the BIRC5 gene, is an inhibitor of apoptosis and overexpressed in various cancers. Survivin have both anti- and pro-apoptotic functions dependent on the expression of splice variants and on subcellular localization.
Our hypothesis is that survivin splice variants play a role in hypoxia-induced resistance to YM155 in AML cells. The specific aims of this project were: (1) to identify survivin splice variants in two AML cell lines, MV4-11 and THP-1, and (2) to determine the subcellular localization of survivin wild type (WT) and splice variants. To accomplish these aims; proliferation, viability, and BIRC5 mRNA expression were determined in MV411 and THP1 cells grown in culture under normoxia (21% O2), hypoxia (1% O2) and normoxia with CoCl2 (hypoxia mimic). Two splice variants (2B and del3) were identified, cloned, and reintroduced into HEK 293A cells to assess variant influence on YM155 cytotoxicity. While treatment with YM155 resulted in >95% death of HEK293A cells transfected with vector only, survivin WT, or variants in normoxia, cells transfected with 2B and del3 were more resistant to YM155 in hypoxia compared to cells transfected with either vector control or WT. Survivin protein expression in 293A co-transfected with WT-HA and 2B His6 plasmid constructs detected by fluorescence microscopy revealed that 2B is localized in the nucleus while WT is both nuclear and cytoplasmic.
Our results suggest that survivin splice variants play a role in hypoxia-induced YM155 resistance and this may be regulated by their subcellular concentration.