Jak2 Regulates Lyn Tyrosine Kinase through Interference with the SET/PP2A/Shp1 Pathway and Inhibition of Jak2 Induced Apoptosis in BCR-ABL+ Imatinib-Sensitive and Resistant CML Cells.

Chronic myelogenous leukemia (CML) patients treated with imatinib mesylate (IM) results in drug resistance in accelerated and blast crisis stages predominantly involving either mutation of the kinase domain of Bcr-Abl or increased expression of the Lyn tyrosine kinase, whose regulation by Bcr-Abl is not yet understood. Treatment of CML patients with IM can at times cause a decline in levels of wild-type Bcr-Abl protein followed in some cases by activation of Lyn/Hck tyrosine kinases. Lyn is reported to be involved in Bcr-Abl signaling pathway and the Bcr-Abl oncoprotein activates Lyn. Although increased expression of Lyn with the progression of the disease is known and Lyn is involved in the survival of CML cells, its exact position in the Bcr-Abl driven signal transduction pathway, its down-stream targets, its regulation by upstream regulators and the mechanism of continuous activation of Lyn in accelerated and blast crisis stages of CML are still unclear. Our previous reports suggested that Bcr-Abl activates the Jak2 tyrosine kinase, by phosphorylation of the Tyr 1007 which involves the enhanced expression of c-Myc. Recently, we have shown that Jak2 and the Bcr-Abl tyrosine kinase are part of a large Network of signaling proteins (Samanta et al Cancer Research, 2006). Here we report that inhibition of Jak2, a target of Bcr-Abl, by either Jak2-specific siRNA or by the Jak2 inhibitor II (1,2,3,4,5,6-hexabromocyclohexan, Sandberg. et al J.Med. Chem. 2005) or AG490 reduced the level of activated Lyn, pGab2 (YxxM), pAkt, pGSK3 and c-Myc. Inhibition of Lyn by knocking down by Lyn specific siRNA or by Lyn inhibitor resulted the similar results further supporting that Lyn is a downstream target of Jak2. We further explored the possible regulatory mechanism of Lyn by Jak2. Jak2 inhibition decreased the level of the SET protein, which strongly inhibits activity of PP2A. Activation of PP2A combined with Jak2 inhibition enhanced the reduction of activated Lyn compared to Jak2 inhibition alone in BCR-ABL+ cells. In contrast suppression of both PP2A and the tyrosine phosphatase Shp1 interfered with the loss of activated Lyn kinase caused by Jak2 inhibition, indicating the involvement of PP2A and Shp1 in the inactivation of the Lyn kinase by Jak2 inhibition. Inhibition of either Jak2 or Lyn kinase induced apoptosis in BCR-ABL+ cell lines, including imatinib-resistant (IM) T315I Bcr-Abl+ cells. Jak2 inhibition induced apoptosis in CML patient cells accelerated and blast crisis stages but not normal cells including cells resistant to IM. In conclusion, our findings indicate that Lyn is downstream of Jak2 and is regulated by Jak2 in Bcr-Abl+ cells. We have shown that activated Jak2 prevents the dephosphorylation of Tyr 396 of Lyn (the active form of Lyn) in Bcr-Abl+ cells through a SET-dependent inactivation of the PP2A tumor suppressor/Shp1 pathway. Our studies in Bcr-Abl driven CML cells indicate that therapeutic strategies to inhibit Jak2 and its downstream target Lyn may be a powerful approaches for the management of IM-resistant CML specifically T315I where the dual kinase inhibitor -dasatanib fails to kill CML cells.