Depression of oncogenecity by dephosphorylating and degrading BCR-ABL

Abstract

Aberrant phosphorylation and overexpression of BCR-ABL fusion protein are responsible for the main pathogenesis in chronic myeloid leukemia (CML). Phosphorylated BCR-ABL Y177 recruits GRB2 adaptor and triggers leukemic RAS-MAPK and PI3K-AKT signals. In this study, we engineered a SPOA system to dephosphorylate and degrade BCR-ABL by targeting BCR-ABL Y177. We tested its effect on BCR-ABL phosphorylation and expression, as well as cell proliferation and apoptosis in CML cells. We found that SPOA remarkably dephosphorylated BCR-ABL Y177, prevented GRB2 recruitment, and uncoupled RAS-MAPK and PI3K-AKT signals. Meanwhile, SPOA degraded BCR-ABL oncoprotein in ubiquitin-independent manner and depressed the signal transduction of STAT5 and CRKL by BCR-ABL. Furthermore, SPOA inhibited proliferation and induced apoptosis in CML cells and depressed the oncogenecity of K562 cells in mice. These results provide evidence that dephosphorylating and degrading oncogenic BCR-ABL offer an alternative CML therapy.

Keywords: BCR-ABL; Y177; chronic myeloid leukemia; ornithine decarboxylase; protein tyrosine phosphatase.

Figure 1. The dephosphorylation activity of SPOA system on BCR-ABL Y177

A. Schematic illustration of recombinant fusion peptides used in this study. B. The schematic diagram of SPOA system mediated dephosphorylation of BCR-ABL Y177 and degradation of BCR-ABL protein. C. Cells were treated with the indicated adenovirus or 3 μmol/L imatinib for 48 h. The phosphorylation of BCR-ABL Y177 and c-ABL Y245 were analyzed by western blot. D. The intracellular interaction between SPOA, BCR-ABL, and GRB2 was determined by co-immunoprecipitation assay. E. The effect of SPOA system on the GRB2 coupled signal cascade was analyzed by western blot. Figure shows the representative results of three replications.

Figure 2. The degradative activity of SPOA system on BCR-ABL

A. Cells were infected with the indicated adenovirus for 48 h, the expression of BCR-ABL and c-ABL were analyzed by western blot. B. Pretreated by SPOA for 48 h, the cells were treated with 20 mg/L cycloheximide (CHX) for another indicated time to inhibit BCR-ABL synthesis. BCR-ABL expression at the indicated time was determined by western blot (left). Gray values of protein bands were scanned with Quantity One® Version 4.5. The ratio of BCR-ABL to actin at 0 h was normalized to 1. The results were showed as fold change to 0 h (right). Data were represented as mean ± SD (n = 3). *p < 0.05 as compared to control. C. Pretreated with the indicated adenovirus for 36 h, the cells were treated with 20 μmol/L ubiquitin inhibitor PYR-41 or 20 μmol/L proteasome inhibitor MG132 for another 36 h. BCR-ABL expression was quantified by western blot. D. The effect of SPOA system on STAT5 and CRKL signal pathways were analyzed by western blot.

Figure 3. SPOA inhibits proliferation of imatinib sensitive and resistant CML cells

A. Cells were plated in 96-well plates and infected with the indicated adenovirus. Cell viability was evaluated by the ability to transform MTT into purple formazan. Optical density at the wavelength of 492 nm (OD₄₉₂) was measured at the indicated time. B. Cells were treated with the indicated adenovirus for 48 h, and plated in 24-well plates with methylcellulose for assessing the colony formation ability. Colonies were counted two weeks later using an inverted microscope. C. Cells were infected with the indicated adenovirus for 72 h, fixed in 70% pre-cooling ethanol, and incubated with propidium iodide for cell cycle analysis by a flow cytometer. All tests were carried out in triplicates. Data were represented as mean ± SD (n = 3). *p < 0.05 as compared to control.

Figure 4. SPOA promotes apoptosis of imatinib sensitive and resistant CML cells

Cells were infected with the indicated adenovirus for 72 h. Morphologic changes of apoptotic cells were visualized by Wright's staining A. (1000× magnification) and DAPI staining B. (400× magnification). For apoptotic rate analysis, cells were stained with AnnexinV-PE and 7-ADD, and determined by a flow cytometer C. All tests were carried out in triplicates. Data were represented as mean ± SD (n = 3). *p < 0.05 as compared to control.

Figure 5. The anti-leukemia activity of SPOA in mice

A. Comparison of the maximum of WBC count in SPOA group and null group. Data were represented as mean ± SD (n = 5). *p < 0.05 as compared to control. B. Leukemic blasts in bone marrow of diseased mice were analyzed by Wright's staining (1000× magnification). The arrows indicate the immature or blast cells. C. Human CD45⁺ cells in murine bone marrow were counted by flow cytometry. Data were represented as mean ± SD (n = 5). *p < 0.05 as compared to control. D. Comparison of the size (left) and weight (right) of liver (L), spleen (S), and solid tumor (T) in SPOA group (S) and null group (N). Without solid tumor was regarded as weight=0 g. E. The leukemic infiltration of liver and spleen was analyzed by HE staining (1000× magnification). The arrows indicate the infiltrated leukemic cell clusters. F. Kaplan-Meier survival analysis of mice.