14-3-3 Binding and Sumoylation Concur to the Down-Modulation of ß-catenin Antagonist chibby 1 in Chronic Myeloid Leukemia.

The down-modulation of the ß-catenin antagonist Chibby 1 (CBY1) associated with the BCR-ABL1 fusion gene of chronic myeloid leukemia (CML) contributes to the aberrant activation of ß-catenin, particularly in leukemic stem cells (LSC) resistant to tyrosine kinase (TK) inhibitors. It is, at least partly, driven by transcriptional events and gene promoter hyper-methylation. Here we demonstrate that it also arises from reduced protein stability upon binding to 14-3-3σ adapter protein. CBY1/14-3-3σ interaction in BCR-ABL1+ cells is mediated by the fusion protein TK and AKT phosphorylation of CBY1 at critical serine 20, and encompasses the 14-3-3σ binding modes I and II involved in the binding with client proteins. Moreover, it is impaired by c-Jun N-terminal kinase (JNK) phosphorylation of 14-3-3σ at serine 186, which promotes dissociation of client proteins. The ubiquitin proteasome system UPS participates in reducing stability of CBY1 bound with 14-3-3σ through enhanced SUMOylation. Our results open new routes towards the research on molecular pathways promoting the proliferative advantage of leukemic hematopoiesis over the normal counterpart.

A calpain-cleaved fragment of ß-catenin promotes BCRABL1+ cell survival evoked by autophagy induction in response to imatinib.

Autophagy protects chronic myeloid leukemia stem cells from tyrosine kinase inhibitors hence supporting the disease persistence under therapy. However, the signals involved in autophagy regulation relative to BCR-ABL1 are still elusive. The autophagic flux proceeding from the inhibition of BCR-ABL1 tyrosine kinase represents a regulatory mechanism of ß-catenin stability through events encompassing the activation of calpain, which targets ß-catenin for proteasome-independent degradation. Accordingly, its inactivation may contribute to induce autophagy and autophagy induction may, in turn, promote ß-catenin autolysosomal degradation to originate a regulatory loop where ß-catenin plays a central role in cell decision between life and death. Here we proved that the cytoplasmic accumulation of ß-catenin driven by up-regulation of its antagonist Chibby1 is a component of autophagy induction in response to imatinib in BCR-ABL1+ cells opposing the apoptotic death. It is contingent upon ER stress and elevation of free Ca(2+) cytosolic concentration and results in the calpain cleavage into a 28kDa fragment implicated in ß-catenin proteasome-independent degradation. More important for BCR-ABL1+ cell survival and proliferation following IM treatment, might be the calpain-mediated cleavage of ß-catenin accumulated within the cytoplasmic compartment into a 75kDa fragment, still owning TCF-dependent transcriptional activity. Such a ß-catenin fragment might be crucial for BCR-ABL1+ cell survival following the fusion protein TK inhibition.

Chibby drives ß catenin cytoplasmic accumulation leading to activation of the unfolded protein response in BCR-ABL1+ cells.

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the constitutive tyrosine kinase (TK) activity of the BCR-ABL fusion protein. However, the phenotype of leukemic stem cells (LSC) is sustained by ß catenin rather than by the BCR-ABL TK. ß catenin activity in CML is contingent upon its stabilization proceeding from the BCR-ABL-induced phosphorylation at critical residues for interaction with the Adenomatous polyposis coli (APC)/Axin/glycogen synthase kinase 3 (GSK3) destruction complex or GSK3 inactivating mutations. Here we studied the impact of ß catenin antagonist Chibby (CBY) on ß catenin signaling in BCR-ABL1+ cells. CBY is a small conserved protein which interacts with ß catenin and impairs ß catenin-mediated transcriptional activation through two distinct molecular mechanisms: 1) competition with T cell factor (TCF) or lymphoid enhancer factor (LEF) for ß catenin binding; and 2) nuclear export of ß catenin via interaction with 14-3-3. We found that its enforced expression in K562 cell line promoted ß catenin cytoplasmic translocation resulting in inhibition of target gene transcription. Moreover, cytoplasmic accumulation of ß catenin activated the endoplasmic reticulum (ER) stress-associated pathway known as unfolded protein response (UPR). CBY-driven cytoplasmic accumulation of ß catenin is also a component of BCR-ABL1+ cell response to the TK inhibitor Imatinib (IM). It evoked the UPR activation leading to the induction of BCL2-interacting mediator of cell death (BIM) by UPR sensors. BIM, in turn, contributed to the execution phase of apoptosis in the activation of ER resident caspase 12 and mobilization of Ca(2+) stores.