MIB/MS confirmed that AZD6244 altered the kinome in a different way from BEZ235, indicating that drug-induced kinome reprogramming is target-specific . MEK-ERK inhibition induces c-Myc degradation leading to RTK reprogramming ERK phosphorylates the transcription factor c-Myc at Ser62 and stabilizes c-Myc protein by avoiding its proteasomal degradation . Treatment method of both SUM159 and MDA-MB-231 cells with AZD6244 triggered rapid reduction of c-Myc protein and mRNA . This AZD6244-mediated repression of c-Myc protein and transcript, coupled with lowered phosphorylation of c-Myc at Ser62 , resulted in decreased Myc-Max heterodimerization that is definitely expected for Myc transcriptional regulation . Regardless of partial recovery of MEK-ERK activation soon after 24-72h, complete c-Myc expression remained repressed in the continued presence of AZD6244 . c-Myc binds the promoter of human PDGFR to repress PDGFR expression .
To define the position of c-Myc reduction during the AZD6244 reprogramming response, we utilized RNAi to knockdown expression of c-Myc; the result was analogous on the reprogrammed RTK and cytokine response seen with AZD6244 remedy . Comparable to NVP-BHG712 the AZD6244 response, knockdown of c-Myc induced expression of PDGFR, VEGFR2 and PDGFB, and increased Tyr phosphorylation of PDGFR, VEGFR2, HER3 and AXL. RNAi knockdown of ERK1/2 confirmed that ERK inhibition was the main signal inducing reduction of c-Myc mRNA expression in the AZD6244 reprogramming of your kinome. Dual ERK1/2 knockdown resulted in lowered c-Myc and greater PDGFR expression . Thus, reprogramming of RTKs in response to AZD6244 takes place by reduction of ERK-mediated stabilization of c-Myc along with the subsequent transcriptional derepression of RTKs and cytokines which might be negatively regulated by c-Myc.
BEZ235 inhibition of mTOR and PI3K inhibited cell growth but did not modify ERK activity, c-Myc expression or RTK reprogramming , confirming the specificity of MEK-ERK in controlling c-Myc ranges. Proteasomal degradation of c-Myc lacking Ser62 phosphorylation triggers AZD6244- induced kinome u0126 ic50 reprogramming. Expression of the non-degradable c-Myc mutant in SUM159 cells substantially blocked AZD6244-mediated induction of PDGFR, DDR1 and VEGFR2 . GSK3 promotes c-Myc degradation, and inhibition of GSK3 stabilized c-Myc protein to repress the induction of PDGFR . Similarly, treatment method of SUM159 or SUM159-R cells with the proteasome inhibitor bortezomib prevented AZD6244-mediated c-Myc degradation, blocked c-Myc mRNA repression, and inhibited the induction of PDGFR, DDR1 and VEGFR2 .
Washout of AZD6244 from SUM159 or SUM159-R cells led to greater ERK exercise, stabilization of c-Myc expression and subsequent reduction of RTK reprogramming . So, stabilizing c-Myc protein amounts prevented the onset of RTK reprogramming to AZD6244 and reversed the reprogramming in SUM159-R cells.