Ubiquitin pathway-mediated downregulation of MYC protein level on compound treatment correlates consistently with cell growth sensitivity toward PIM inhibition. (A) Evaluation of putative PIM substrates by siRNA knockdown. KG1 cells were treated with siRNAs against PIM1, PIM2, or PIM3, either alone or as combinations. Modulations of the indicated phosphorylation events and protein abundance were assessed by western blot. (B) A summary of changes of specified intracellular markers on treatment with PIM inhibitors in a panel of responders and nonresponders. (C) MYC protein levels in responders and nonresponders on treatment of 3 diverse PIM inhibitors. (D) Half-life of endogenous MYC on treatment of PIM inhibitors. Endogenous MYC in KG1 cells has an average half-life of between 1 and 2 hours. In the presence of a PIM inhibitor, the half-life is shortened to at least less than 30 minutes (upper). Similar results were obtained using another PIM inhibitor with a different chemical structure (lower). (E) Ubiquitination of endogenous MYC in the presence of a PIM inhibitor. The position of MYC is specified with an arrow. Upward smearing streaks are indicative of poly-ubiquitinated MYC, which are intensified at the 20- and 30-minute points. Concurrent reduction of MYC protein levels is observed. (F) PIM kinases and MYC are present in the same complex. Cellular extracts were immunoprecipitated with antibodies against PIM1, PIM2, and PIM3 in radioimmunoprecipitation assay buffer. Presence of MYC was detected by western blot. (G) Differential perturbation of MYC phosphorylation sites by PIM inhibitor. Of all detectable MYC phosphorylation sites identified by quantitative phosphoproteomic mass spectrometry, S293 is the only site demonstrated to be significantly suppressed by compound C. (H) Direct phosphorylation of MYC S293 by PIM kinases. Biochemical reaction was carried out in the presence of recombinant MYC and PIM kinases. Quantitative phosphoproteomic mass spectrometry was then used to determine the level of phospho-S293.