We expressed myc epitope or HA epitope tagged versions of BRAF or CRAF in D cells, immunoprecipitated the myc tagged proteins and western blotted for your HA tagged proteins, and demonstrate that each BRAF and CRAF homodimers had been formed in D cells Figures F and G . To check straight if dimer formation was driven by drug binding to BRAF or CRAF, we utilized mutant versions of BRAF and CRAF by which the so identified as gatekeeper residues kinase inhibitors were substituted with asparagine BRAFTN and CRAFTN, respectively . We’ve previously proven that this mutation blocks drug binding to BRAF Whittaker et al and verify right here that each BRAFTN and CRAFTN have been resistant to imatinib, nilotinib, and dasatinib Figure A . Critically, BRAFTN and CRAFTN were severely impaired in their skill to type BRAF:CRAF heterodimers and BRAF:BRAF or CRAF:CRAF homodimers Figures H J; Figure SB . Imatinib, Nilotinib, and Dasatinib Induce Paradoxical MEK ERK Pathway Activation in Leukemia Cells Expressing BCR ABLTI The information over display that imatinib, nilotinib, and dasatinib are weak RAF inhibitors that drive formation of RAF hetero and homodimers, and stimulate paradoxical activation of BRAF and CRAF from the presence of activated RAS. Past studies have shown that imatinib activates ERK in leukemia cells expressing imatinib resistant BCR ABL Yu et al ; Suzuki et al ; Mohi et al ; Chu et al.
so we examined if this was also driven by means of paradoxical activation of RAF. For this we used isogenic clones of murine Ba F professional B cells whose growth was driven by either BCR ABL or BCR ABLTI Golub et al. We confirmed that imatinib, nilotinib, and dasatinib blocked BCR ABL phosphorylation on tyrosine Y and CRKL phosphorylation on tyrosine Y in BCR ABL Ba F cells Figure A . Additionally, imatinib, nilotinib, and dasatinib blocked CRAF activity in these cells Figure B , and constant with this particular, Lapatinib they suppressed CRAF phosphorylation on S and blocked MEK and ERK activity Figure A . In contrast, in BCR ABLTI Ba F cells imatinib, nilotinib, and dasatinib did not inhibit BCR ABL or CRKL phosphorylation Figure C . A lot more importantly, in these cells all a few medicines induced CRAF phosphorylation on S Figure C and activated CRAF Figure D , MEK, and ERK Figure C . Critically, we demonstrate that whereas imatinib, nilotinib, and dasatinib didn’t have an impact on BRAF binding to CRAF while in the BCR ABL cells, they enhanced BRAF binding to CRAF in BCR ABLTI Ba F cells Figures A and C . We also in contrast responses in BV and BVR cells. BV cells were derived from a blast crisis CML patient and express BCR ABL endogenously, whereas BVR cells have been picked for imatinib resistance and express BCR ABLTI Pegoraro et al ; Bartholomeusz et al. Imatinib, nilotinib, and dasatinib inhibited BCR ABL and CRKL phosphorylation in BV, but not BVR, cells Figure E .
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