PMC:3940921 / 5702-7276 JSONTXT

{"project":"2_test","denotations":[{"id":"24590311-11003655-79065513","span":{"begin":608,"end":610},"obj":"11003655"},{"id":"24590311-9585502-79065514","span":{"begin":759,"end":761},"obj":"9585502"},{"id":"24590311-18632609-79065515","span":{"begin":1306,"end":1308},"obj":"18632609"}],"text":"Effect of carfilzomib on key signaling pathways in CML\nCell lines and primary cells were pulsed with carfilzomib at IC50 doses for 1 h and returned to fresh medium for 24 h before protein lysates were prepared and immunoblot analysis was performed to determine the effect of carfilzomib on Bcr-Abl signaling pathways. Carfilzomib treatment resulted in a decrease of p-ERK by 52±11% (P\u003c0.01), with no effect on STAT5 or PI3K signaling pathways (Figure 1a). We next looked at the effect of carfilzomib treatment on ABI 1/2 proteins, which have been reported to inhibit ERK activation that was induced by v-Abl.22 ABI 1/2 proteins have been shown to be stable in normal cells but rapidly degraded via the ubiquitin–proteasome pathway in Bcr-Abl expressing cells.23 We observed increased expression of ABI 1/2 in cells in which BCR-ABL was knocked down using siRNA and also in NBM CD34+38−-enriched cells compared with CML counterparts (Supplementary Figure 1). Treatment with carfilzomib at IC50 levels increased the expression of ABI 1/2 in cell lines and primary CD34+38−-enriched cells (range 2.2–6.8 fold, Figure 1b) and this was associated with an accumulation of polyubiquitinated ABI 1/2 (Figure 1c). ABI 2 has been reported to be tagged for degradation by the E3 ligase TRIM32 in head and neck cancer.24 Figure 1d demonstrates an association of ABI 2 with TRIM32 in the CML cell lines, which appears to be enhanced following treatment with carfilzomib, suggesting that TRIM32 also acts as an E3 ligase to target ABI proteins for degradation in Bcr-Abl expressing cells."}