To assess the effect of MEK1 2 inhibition on ERK1 2 ac tivation state, mel anom

Our molecular docking analysis revealed that ponatinib could target native or T674I FIP1L1 PDGFR in the DFG out binding mode, similar to ponatinib docking in T315I Bcr Abl. This characteristic of ponati nib may be related to its imidazo pyridazine core that occupies the pocket for adenine in the enzyme, whereas the methylphenyl group occupies the hydropho bic pocket JAK 阻害剤 behind the gatekeeper residue of the enzyme. Encouraged by the in silico simulation results, we evaluated the efficacy of ponatinib against imatinib resistant CEL cells both in vitro and in vivo. Ponatinib potently inhibit the viability of EOL 1 cells expressing WT FIP1L1 PDGFR, with an IC50 value of 0. 004 nM. This efficacy agrees with recent results showing an inhibitory effect in EOL 1 cells, with an IC50 of 0. 5 nM.

In the same study, ponatinib inhibited malignant cells expressing Bcr Abl, Flt3, KIT, FGFR1, with IC50 values from 2 to 36 nM. We showed that ponatinib had an inhibitory effect on imatinib resistant leukemic BaF3 T674I FIP1L1 PDGFR cells, with an IC50 purchase LDE225 of 2. 5 nM, which is comparable to the po tency in BaF3 T315I Bcr Abl cells, with an IC50 of 11 nM. Clonogenicity assay confirmed that ponatinib restrained the proliferation of BaF3 WT or T674I FIP1L1 PDGFR cells at low nanomolar concentra tions. Further, our in vivo data revealed that ponatinib, at an oral dose of 30 mg kg day, potently abrogated the growth of xenografted imatinib resistant BaF3 T674I FIP1L1 PDGFR cells, with PDGFR signaling highly sup pressed.

A pharmacokinetics study in mice indi cated that orally administrated ponatinib as a single oral dose of 30 mg kg, which was well tolerated, resulted in mean plasma concentrations of 782 and 561 nM at LY2109761 臨床試験 2 and 6 h post dosing, respectively. Such plasma levels highly exceed the in vitro IC50 values for all 3 lines of FIP1L1 PDGFR expressing cells, so ponatinib may effi ciently inhibit the growth of FIP1L1 PDGFR positive cells with clinically achievable doses. Ponatinib induced remarkable apoptosis in both imatinib sensitive and resistant CEL cells, as evidenced by Annexin V binding, activation of caspase 3, and spe cific cleavage of PARP. The apoptosis was triggered by the mitochondrial dependent pathway because of release of AIF and cytochrome c to the cytosol after treatment with ponatinib. The levels of survivin, Bcl XL and Mcl 1were decreased in ponatinib mediated apoptotic CEL cells.

The transcription of survivin and Bcl XL is regulated by Stat3, Stat5 and Erk1 2. The decreased expression of sur vivin and Bcl XL caused by ponatinib treatment is likely associated with the inhibition of Stat3, Stat5 and Erk1 2. However, future experiments can further define the pre cise mechanisms. Mcl 1, a pro survival and anti apoptotic protein with relatively short half life in the Bcl 2 family, is expressed in malignant hematological cells and protects cells against apoptosis in response to chemotherapeutic agents includ ing TKIs. The decrease in Mcl 1 by ponatinib in CEL cells may facilitate apoptosis, because silencing Mcl 1 with siRNA significantly potentiated the ponatinib mediated apoptosis in EOL 1 cells, which is in line with the finding that decreased Mcl 1 level sensitizes leukemia cells to tyrosine kinase inhibitors.