Abstract 2923: Phosphoproteomic assessment of CML cells following transient potent inhibition with dasatinib is associated with durable alteration of the STAT5 and RAF/MEK/ERK pathways

Abstract

Abstract The critical reliance of CML cells upon BCR-ABL kinase activity for survival supports the concept of “oncogene addiction”, but the molecular mechanisms underlying this phenomenon are poorly understood. Consistent with clinical responses in CML patients to once daily administration of dasatinib, we have previously demonstrated that transient, but potent tyrosine kinase inhibition of BCR-ABL is sufficient to induce cytotoxicity of the CML cell line K562 in vitro. These cells display an irreversible commitment to apoptosis despite reactivation of BCR-ABL kinase activity as assessed by rephosphorylation of the substrate CRKL. We hypothesized that durable alterations in signaling occur in this in vitro model of transient kinase inhibition and that a subset of these changes are critical to eliciting cytotoxicity. To obtain an unbiased, global view of signaling before and after a high-dose pulse (HDP) of dasatinib, we optimized phosphoproteomic technology to interrogate changes in the phosphotyrosine proteome of K562 cells. Through implementation of SILAC to assess quantitative changes in tyrosine phosphorylation and a high pH C18 fractionation prior to MS/MS analysis, we compared lysates from the end-of-exposure, 3 hours, and 6 hours post-HDP, and identified 204 unique phosphosites originating from 160 different proteins. Notably, following transient exposure to dasatinib, durable alterations in phosphorylation occurred in two of the three major signaling pathways downstream of BCR-ABL. Specifically, we observed a durable loss of tyrosine phosphorylation in the SH2-domain of STAT5, as well as a loss of phosphorylation within the activation loop of ERK1/2 without alterations in the PI3K/AKT pathway. We have confirmed these findings with phospho-specific antibodies. Of particular interest is the mechanism whereby the STAT5 and RAS/MAPK signaling pathways are durably altered. Interestingly, despite durable alterations in the phosphorylation status of MEK, ERK and BIM, the overall level of activated RAS is unaltered with dasatinib treatment. We therefore hypothesize that the RAF kinases are durably altered and are presently investigating the molecular mechanism responsible. Lastly, despite durable loss of STAT5 phosphorylation we observed no activation-loop phosphorylation of JAK2 before or after a HDP, suggestive of a JAK2-independent mechanism of STAT5 activation in K562 cells. Our findings suggest that the molecular basis of oncogene addition in CML involves irreversible hijacking of the STAT5 and RAF/MEK/ERK pathways by BCR-ABL as evidenced by durable loss of signaling through these pathways following potent transient BCR-ABL inhibition. Current work is evaluating the molecular mechanisms that underlie these durable signaling alterations. MS analysis was provided by the MS Resource at UCSF (NIH NCRR P41RR001614). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2923. doi:10.1158/1538-7445.AM2011-2923