Tandutinib FLT inhibitor were resistant to IM

It is unknown whether phosphorylation of Bcr on Tyr177 would contribute to activation of Ras and PI 3 kinase pathways in CML cells. CML 34t cells respond to Jak2 inhibition by reduction of pTyr177 Bcr Abl and Bcr Abl Cells were harvested from the peripheral blood of a CML blast crisis patient, having 98% blasts. Tandutinib FLT inhibitor After CD34 selection, sufficient cells were available for western blotting. Flow cytometry results indicated that 87% of the cells bound to the CD34 beads were CD34t. Western blotting revealed that Jak2 inhibition by TG reduced levels of pTyr177 and Bcr Abl in CD34t cells. We note that CD34tcells from this patient  but sensitive to TG. Cord blood CD34t cells showed loss of Bcr Abl and pTyr177 Bcr Abl caused by Jak2 inhibition We examined the effects of Jak2 inhibition in CD34t cord blood cells transduced with BCR ABL.
12 The cells were treated for 6 h with 2.5 and 10 mM TG. pTyr177 Bcr Abl and Bcr Abl were drastically reduced as was Jak2 and activated Jak2 following treatment of CD34t cells with TG. These results with CD34t cord blood cells, which were recently transduced with Bcr Abl, would be consistent with Jak2 inhibiting SKI-606 early progenitor cells in CML patients. Inhibition of the Bcr Abl kinase by IM had little effect on the phosphorylation of Tyr177 of Bcr Abl in short term experiments Importantly, IM, a well known Abl kinase inhibitor, did not inhibit tyrosine phosphorylation of Tyr177 of Bcr Abl for up to 3 h within Bcr Ablt cells nor was the level of the Bcr Abl protein affected during 3 h treatment with IM.
These results provide further evidence that Tyr177 of Bcr Abl is a site phosphorylated by Jak2 and does not result from a Bcr Abl autophosphorylation reaction. However, treatment of Bcr Ablt cells with IM for longer times, as expected, drastically decreased Bcr Abl protein levels in a dose dependent manner. Jak2 inhibition reduced activation of the Ras, PI 3 kinase and STAT5 pathways We determined whether binding of Grb2 to Bcr Abl was similarly decreased by Jak2 inhibition. It is known that Grb2 binds to pTyr177 of Bcr Abl and pTyr Shc, Shc also contains the Grb2 binding sequence YvnV sequence. Shc associates with Bcr Abl and like pTyr177, drives the Ras and PI 3 kinase pathways. Grb2 levels in the Bcr Abl immunocomplex was reduced by B60% within 120 min of TG treatment compared with the control.
Jak2 inhibition by TG decreased levels of RAS GTP within 60 min in 32Dp210 cells, which is consistent with the observed decrease in Grb2 binding to Bcr Abl. We also determined whether Jak2 inhibition decreased PI 3 kinase activation. It is known that pTyr177 binds Grb2 which in turn binds Gab2.6 Our previous studies20 indicate that Jak2 activation leads to phosphorylation of Tyr 452 of Gab2, which is involved in binding to the regulatory subunit of the PI 3 kinase leading to its activation.6 In our current studies, pTyr 452 Gab2 was rapidly decreased in Bcr Ablt cells after treatment with TG. We note that PI 3 kinase activation as measured by a commercial kit was also decreased within 60 120 min of Jak2 inhibition. These results indicate that Jak2 inhibition rapidly inhibited both Ras and PI 3 kinase activation. We determined whether STAT5 activation would also be downregulated by this more potent Jak2 inh

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