The Bcr Abl kinase whereas IM, as expected, inhibited the VX-680 639089-54-6 Bcr Abl kinase. Tyr177 Y to F mutant behaves as wild type Bcr Abl with respect to Jak2 inhibition We compared the disappearance of Y177F Bcr Abl mutant with wild type Bcr Abl in 32D cells transduced with either wild type or mutant BCR ABL. The results indicate that Jak2 inhibition of Bcr Abl disappearance in both mutant and wild type forms. Moreover, as expected, Tyr177 phosphorylation was not detected in the Y177F mutant. These results support the concept that Tyr177 is just one of possibly several Jak2 phosphorylation sites, and that phosphorylation of these sites is necessary to maintain Bcr Abl in a functional state.
Jak2 inhibition reduced tumorgenicity in mouse models As WP1193 was a more potent Jak2 inhibitor than TG, we tested the effects of WP1193 on the growth of tumors induced by IM resistant K562 R cells. K562 R cells16 contain activated Lyn kinase, which maintains the leukemic state of the K562 R cells despite the presence of IM. Therefore, we tested the inhibitory effects of WP1193 on the growth of solid tumors induced by K562 R in a nude mouse model. Solid tumors were allowed to form for 12 days following injection of K562 R cells, and treatment with WP1193 was initiated at 12 days through day 22. The volume of solid tumors was determined following injection of WP1193 at 30 mg/kg of mouse body weight every 48 h. Solid tumor growth was significantly reduced over this time period. Injection of these mice with WP1193 by intraperitoneal had little effect on the weights of spleen and liver tissue.
We next compared the effects of WP1193 at 30 mg/kg on the oncogenic effects of IM resistant T315I Bcr Ablt 32D cells injected intravenous in the nude mouse model. The mice developed large intestinal tumors over a 2 week period, which were largely prevented by WP1193 treatment. Spleen and liver weights were reduced, and spleens were enlarged in the diseased mice compared with WP1193 treated mice. Importantly, spleens and livers of mice not injected with leukemia cells were not significantly affected in weight by injection of WP1193 over a 3 week period, all of these drug treated mice survived and showed little evidence of acute toxicity as the mice appeared healthy.
Jak2 inhibition induced apoptosis in CD34t cells from blast crisis IM resistant CML patients and cells from chronic phase IM resistant CML patients We harvested CD34t progenitor cells from a patient with blast crisis CML. Fractionated cells were treated with either IM or TG. After 48 h, cells were analyzed by annexin/PI flow cytometry. TG at 5 mM concentration induced 80% apoptosis whereas IM at 5 10 mM concentration had little apoptotic inducing activity, All of our previous studies had shown that Jak2 inhibition20,21 induced apoptosis in cells from blast crisis CML patients. We also examined cells from chronic phase CML patients who were resistant to IM. Treatment of the chronic phase cells with 2.5 10 mM TG induced high levels of apoptosis whereas IM treatment had little effect in these samples. Cells from one chronic phase patient were partially sensitive to IM but these cells were almost completely killed by 5 mM TG.