Function of STAT5 Isoforms in Bcr-Abl Positive Cells
Authors: Schaller-Schoenitz Michael , Dallmann Iris , Ganser Arnold , Scherr Michaela , Eder Matthias 
Published: October 2019

Abstract
Abstract 1313
Chronic myeloid leukemia (CML) is caused by expression of the BCR-ABL oncogene product which exerts constitutive tyrosine kinase activity. Specific inhibition of the BCR-ABL tyrosine kinase activity by small tyrosine kinase inhibitors (TKIs) is now well established as initial therapy for newly diagnosed chronic phase CML. However, TKI treatment is not curative in most cases since CML stem cells have been reported to be largely insensitive to this approach and may be not oncogene-addicted. Therefore, additional targets for therapeutic intervention are under investigation to further improve pharmacotherapy for CML. For example, IFNa, inhibition of the transcription factor FOXO3, or that of JAK2 may represent new options for combinatorial therapy among others. To functionally characterize genes involved in BCR-ABL signalling we earlier used gene-specific RNA interference (RNAi) and identified enhanced sensitivity to inhibition of STAT5 in BCR-ABL+ cells (Scherr et al. 2006). STAT5 which comprises two highly conserved isoforms with over 90% sequence homology (Stat5A and Stat5B) is translocated into the nucleus upon tyrosine phosphorylation and di(oligo-)merisation. To further characterize the molecular mechanisms involved in the observed differential sensitivity against anti-STAT5 RNAi we first designed isoform-specific shRNAs targeting either STAT5A or STAT5B individually. TonB cells - a murine IL-3 dependent cell line with doxycycline (dox)-inducible BCR-ABL expression - were lentivirally transduced in the presence of IL-3 before induction of BCR-ABL-expression by addition of dox. Anti STAT5A- and STAT5B-RNAi revealed isoform-specific reduction of STAT5A and STAT5B expression by over 90% as determined by immunoblotting. There is only very modest cross-reactivity with about 5% and 6% reduction of STAT5A and STAT5B protein-expression in the presence of the complementary shRNAs, respectively. Four days after lentiviral transduction cells were further cultivated with IL-3 or switched to BCR-ABL expression by addition of dox, and cell proliferation was determined by Trypan Blue staining and cell counting. In IL-3 cultures, anti-STAT5A and anti-STAT5B shRNAs reduced the cell number by 47% and 36% as compared to control shRNAs whereas in BCR-ABL cultures, RNAi targeting STAT5A and STAT5B reduced the number of viable cells by 43% and 73%, respectively. Cell viability was not affected in IL-3 cultures, but was reduced by 5% and 38% in BCR-ABL cultures by anti-STAT5A and anti-STAT5B RNAi as compared to control shRNA-transduced cells, respectively. To study whether IL-3 can enhance BCR-ABL function, wildtype TonB cells were cultured with IL-3 and dox. Cell proliferation was maximal with IL-3 alone (100%), followed by dox + IL-3 (65%), and dox (20%), respectively, suggesting that BCR-ABL and IL-3 signalling may compete for some overlapping components. Next, we studied hetero- and homodi(oligo)merisation using endogeneous or epitope-tagged STAT5A and STAT5B isoforms over-expressed in TonB cells. STAT5A was precipitated from cellular lysates with anti-STAT5A as well as epitope-specific antibodies and separated by SDS-PAGE. Immunoblotting revealed an inducible heterodi(oligo) merisation of STAT5A:STAT5B complexes by either IL-3 or BCR-ABL containing tyrosine phosphorylated STAT5. In contrast, STAT5A forms constitutive homodimers (or multimers) independent of stimulation (and tyrosine phosphorylation) by IL-3 or BCR-ABL in the presence or absence of imatinib. Finally, cell survival and proliferation was independent of the presence of STAT5A homodimers suggesting that this variant of STAT5 dimers is not functional in TonB cells. In summary, our data suggest unique roles for STAT5A and STAT5B for BCR-ABL mediated cell proliferation and survival in TonB cells. Whereas STAT5A can constitutively homodi(oligo-)merize, STAT5A:STAT5B heterodi(oligo)merisation is inducible, depends on tyrosine phosphorylation and corresponds to cell survival and proliferation. These data suggest the evaluation of STAT5B-specific therapeutic intervention in BCR-ABL expressing cells in preclinical models.

Disclosures:
No relevant conflicts of interest to declare.