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 that HA tagged proteins, and display that the two BRAF and CRAF homodimers had been formed in D cells Figures F and G . To check immediately if dimer formation was driven by drug binding to BRAF or CRAF, we utilised mutant versions of BRAF and CRAF through which the so known as gatekeeper residues PLX4032 structure had been substituted with asparagine BRAFTN and CRAFTN, respectively . We now have previously proven that this mutation blocks drug binding to BRAF Whittaker et al and verify here that both BRAFTN and CRAFTN have been resistant to imatinib, nilotinib, and dasatinib Figure A . Critically, BRAFTN and CRAFTN have been severely impaired in their potential to kind 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 data 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 inside the presence of activated RAS. Earlier research 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 tested if this was also driven via paradoxical activation of RAF. For this we employed 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 . In addition, imatinib, nilotinib, and dasatinib blocked CRAF activity in these cells Figure B , and constant with this, Acetylcysteine 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 . Additional importantly, in these cells all three medication induced CRAF phosphorylation on S Figure C and activated CRAF Figure D , MEK, and ERK Figure C . Critically, we present that whereas imatinib, nilotinib, and dasatinib didn’t impact BRAF binding to CRAF while in the BCR ABL cells, they improved BRAF binding to CRAF in BCR ABLTI Ba F cells Figures A and C . We also compared responses in BV and BVR cells. BV cells had been derived from a blast crisis CML patient and express BCR ABL endogenously, whereas BVR cells have been chosen 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 .