Distinct Functions of Stat5A and Stat5B in Chronic Myeloid Leukemia (CML): Stat5B Is Implicated in Survival and Self-Renewal and Stat5A in Imatinib Resistance.

Abstract 1214

The use of tyrosine kinase inhibitors (TKI) has dramatically changed the prognosis and the natural history of CML. However the occurrence of drug resistances, especially at the stem cell level, requires the identification of novel functional targets. The signal transducer and activator of transcription 5 (Stat5), constitutively phosphorylated by BCR-ABL, is thought to be involved in the pathophysiology of CML, especially by its contribution to the antiapoptotic phenotype observed in CML cells via activation of Bcl-xL. There is currently no data linking clearly the involvement of Stat5 activation to TKI resistance in CML. Moreover, it has not been established, if the two closely related Stat5 factors, Stat5A and Stat5B, fulfill the same or distinct roles in leukemic cells. In order to study these potentially differential functions, we have specifically inhibited Stat5A and Stat5B through lentivirus-mediated RNA interference in human Ph+ CML cell lines K562, LAMA84 and Meg01 and their Imatinib mesylate (IM)-resistant counterparts. We have also transduced with the same lentiviral vectors, primary human CML CD34+ cells and evaluated the effects of Stat5A or B invalidation in terms of hematopoietic stem cell self-renewal in long-term culture initiating cell (LTC-IC) assays. In preliminary experiments, the successful inhibition of Stat5A or B was demonstrated using Western blotting with specific inhibition of each factor with the corresponding shRNA. In CML cell lines, Stat5B inhibition led to a massive apoptosis with growth arrest in all three cell lines whereas Stat5A inhibition had no effect. Surprisingly, Stat5B was active in the absence of canonical tyrosine phosphorylation and its absence strongly enhanced the apoptosis-inducing effects of TKI (Imatinib and Nilotinib). We then evaluated the role of Stat5A or B in the context of CML cell lines resistant to IM, in which there was no detectable ABL-kinase domain mutations neither MDR amplification. In this context, the Stat5A and B protein levels were identical but there was a strong over-activation of Stat5, as detected by Tyr_694/699 phosphorylation status. In the two resistant sublines LAMA84-R and Meg01-R, anti-Stat5A and anti Stat5B selective immunoprecipitation assays further revealed that only Stat5A, and not Stat5B, was Tyr-phosphorylated. Therefore, in two independent IM-resistant sublines, LAMA84-R and Meg01-R, resistance correlated with over-activation of Stat5A, in the absence of canonical activation of Stat5B.

We next investigated the role of Stat5A or B in primary CML stem cell self-renewal, using CD34+ cells from CML patients in chronic phase (CP-CML) at diagnosis. CD34⁺ cells were transduced with control (luciferase)-, Stat5A- or Stat5B-shRNA encoding lentiviral vectors and Long Term Colony Initiating Cell (LTC-IC) assays were performed. In four out of five patient samples, Stat5B-shRNA induced a massive inhibition of the LTC-IC activity. Stat5A-shRNA, on the reverse, had highly variable impact depending on the patients, either enhancing or somehow decreasing LTC-IC activity. We also performed similar assays with CD34⁺ progenitor cells collected from healthy donors. Except for one sample, Stat5B knock down had rather weak consequences on LTC-IC activity. Finally, to extend the potential roles of Stat5A and B in Imatinib resistance, we have transduced CD34+ cells of an IM-resistant CML patient whose cells had also been shown to be resistant to IM in vitro. GFP+ Stat5A-shRNA transduced cells were cell-sorted by FACS and their IM-resistance was tested in clonogenic assays. As compared to the control Luciferase shRNA vector, Stat5A inactivation reduced the colony numbers by 75%, with development of small sized colonies.

Thus, these data strongly suggest that Stat5b plays a major role in the survival of CML cells, especially in the primitive hematopoietic cells, extending the results obtained in CML cell lines. Our data further show for the first time that this effect takes place in the absence of Tyrosine phosphorylation of Stat5B. Conversely, CML cells express tyrosine-phosphorylated Stat5A, whose levels strongly increased in TKI-resistant cells and its inhibition by an shRNA strategy leads to the reversal of TKI resistance of phenotype. Thus, Stat5A and Stat5B exhibit entirely distinct - not interchangeable- oncogenic activities in CML pathophysiology and represent novel therapeutic targets.