Imatinib and Bortezomib Induce the Expression and Distribution of Anaphase-Promoting Complex Cdh1 in Blast Crisis of Chronic Myelogenous Leukemia

CML is well-known as the best understood human malignancy and a paradigm for cancer research from bench to bedside. However, treatment options for imatinib-resistant patients are still limited. Imatinib (IM) has already established as the standard first-line therapy for patients with chronic-phase CML. Bortezomib (BOR) not only prolonged life span for relapsed multiple myeloma(MM) patients, but also induced cell apoptosis in CML. However, the efficacy of TKIs and bortezomib on imatinib-resistant CML remains obscure.

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that regulates cell cycle progression. Cdh1, the adaptor protein of APC/C, in conjunction with APC/C in G1-phase, is required for preventing unscheduled proliferation and protecting primary mammalian cells from genomic instability. Cdh1-deficient cells showed a large variety of chromosomal aberrations. On the contrary, Cdc20, the substrate of Cdh1, could contribute to genetic aberration. However, it’s marginally addressed about the relationship between interaction of Cdh1-Cdc20 with IM resistance and genetic aberrations.

We treated K562-WT and IM-resistant K562 cells with IM/nilotinib(AMN) and BOR, verified the effect of TKIs and BOR by FACS, and explored the change of the expression and the subcellular localization of Cdh1 by immunoblot and immunofluorescence microscopic analysis. Then, we enrolled ten patients with newly diagnosed CML-BC and dissected Cdh1-Skp2-p27 cascade in primary CML cells by immunoblot analysis. We explored the subcellular localization and interaction in space between Cdh1 and Skp2 by immunofluorescence in IM-sensitive or resistant primary cells and cell line. Moreover, the single siRNAs of Cdh1 and Skp2 were designed and were transiently transfected with HiPerFect Transfection Reagent. The expressions of Cdh1-Skp2-p27 pathway proteins were detected by immunoblot, the change of cell cycle and apoptosis were detected by flow cytometry.

We found that IM and BOR induced cell cycle quiescence and arrest, and changed the expression and nuclear relocation of Cdh1 in CML-BC cells. Moreover, AMN and BOR resulted in up-regulation of Cdh1 in IM-resistant cells. Our study revealed Cdh1 and Skp2 were co-localized more abundantly in the cytoplasm of IM-sensitive cells, but co-distributed to nuclears in IM-resistant cells. Furthermore, Cdh1 was lower level in IM-resistant CML-blast crisis (BC) than that of IM-sensitive patients. On the other side, Cdh1 silencing resulted in stabilization of Skp2 and Cdc20, subsequently promoting G1-S transition and formation of multinucleated cells.

Expression and dynamic distribution of Cdh1, induced by IM and BOR, provide a novel interpretation of underlying mechanism to inhibit BCR-ABL downstream cascade via Cdh1-Skp2-p27 axis. Furthermore, we demonstrated BOR still exerted the regulation effect on expression and relocation of Cdh1 even in IM-resistant CML-BC cells, which provided evidences for synergy combination of TKI and proteasome inhibitor for overcoming IM-resistance. Besides, our results revealed Cdh1 tends to be related to genomic stability in CML-BC.

No relevant conflicts of interest to declare.

Footnotes:* Asterisk with author names denotes non-ASH members.