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

Abstract

The down-modulation of the β-catenin antagonist Chibby 1 (CBY1) associated with the BCR-ABL1 fusion gene of chronic myeloid leukemia (CML) contributes to the aberrant activation of β-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.

Fig 1. IM promotes changes in CBY1 and β-catenin expression associated with the dissolution of CBY1 and β-catenin binding with 14-3-3σ.

A- Cytoplasmic and nuclear protein analysis was performed after 4 and 24 hours of exposure to IM. In first instance, BCR-ABL1 de-phosphorylation at the critical residue for constitutive activation of the fusion protein enzymatic activity (tytosine-Y-245) was assessed, hence proving IM inhibitory effect on its target at the time other protein expression and interactions were investigated; B- WB and IP/IB have been performed according to published procedures and confirmed in three independent experiments. Densitometric analysis of signal intensities shows a statistically significance difference (p<0.05; Student’s t test) in treated vs untreated cells. Actin and histone H1 were used as control for loading of cytoplasmic and nuclear proteins, respectively. Lack of IM off target effects is shown in the Supplementary section, S2 Fig

Fig 2. The dissolution of CBY/14-3-3σ complex in response to BV02, an inhibitor of 14-3-3 binding modes I and II, is associated with a CBY1 increment in the cytoplasmic and nuclear compartment.

A- CBY1 expression and interaction with 14-3-3σ and βcatenin expression and interaction with 14-3-3σ were assayed in the cytoplasmic and nuclear compartments of C22orf2 K562 cells at 24^th hour of exposure to BV02. See legend to Fig 1 for technical details.

Fig 3. AKT inactivation in response to m-TOR inhibitor RAD001 hinders CBY1 interaction with 14-3-3σ and is associated with cytoplasmic CBY1 increment.

A- RAD001 inhibits its target: AKT; B- CBY1 expression and interaction with 14-3-3σ and C- βcatenin expression and interaction with 14-3-3σ were assayed in the cytoplasmic and nuclear compartments of C22orf2 K562 cells at 24^th hour of exposure to RAD001. The absence of off-target effects was assayed (Supplementary section, S2 Fig). See legend to Fig 1 for technical details.

Fig 4. JNK and 14-3-3σ de-phosphorylation at threonine 183 and serine 186, respectively, in response to JNK inhibitor SP600125 prevents the dissolution of CBY1/14-3-3σ complex and leaves steady the cytoplasmic expression of CBY1.

A- SP600125 inhibitory effects on its targets (JNK and 14-3-3); B- CBY1 expression and interaction with 14-3-3σ and βcatenin expression and interaction with 14-3-3σ were assayed in the cytoplasmic and nuclear compartments of C22orf2 K562 cells 24^th hour of exposure to SP600125. The absence of off-target effects was assayed (Supplementary section, S2 Fig). See legend to Fig 1 for technical details.

Fig 5. Reduction of JNK and 14-3-3σ post-translational modifications is associated with CBY1 reduced expression and interaction with 14-3-3σ in CML.

Bone marrow MCF of 12 CML patients at diagnosis and 8 HDs were compared for CBY1 expression and interaction with 14-3-3σ, JNK phosphorylation at threonine 183 and 14-3-3σ phosphorylation at serine 186. Clinical details of CML patients included in our study are illustrated in the Supplementary section, S1 Table. A pool consisting of equal amounts of proteins from MCF of bone marrow samples of HDs was used as control for IP/IB to avoid individual differences. See legend to Fig 1 for technical details. The two blots shown here have been run separately and confirmed by two additional experiments.

Fig 6. Proteasome degradation through SUMOylation has a role in CBY1 reduced stability associated with BCR-ABL1 TK-mediated binding with 14-3-3σ.

A- Cytoplasmic CBY1 expression was upraised at 24th hour of treatment with Bortezomib (5nM) in C22orf2 K562 cells; B- Reduction of CBY1 SUMOylation was associated with CBY1 upraised expression in response to compounds (IM, BV02 and RAD001) which impair the interaction with 14-3-3σ. Conversely, CBY1 SUMOylation was increased in response to the JNK inhibitor, which precludes CBY1 release from 14-3-3σ and reduces CBY1 cytoplasmic levels; C- Higher CBY1 SUMOylation was seen in MCF from bone marrow samples of 12 CML patients at diagnosis compared to pooled HD. Clinical details of CML patients included in our study are illustrated in the S1 Table. The two blots shown here have been run separately and confirmed by two additional independent experiments. D- CBY1 SUMOylation in MCF from CML patients was inversely correlated with the expression of CBY1. Signal intensities of WB obtained with equal amounts of protein from CML and HD samples. CBY1 expression exhibited a decrease of more than 50% with respect to the normal control, while SUMOylation showed an almost 1,5-fold increment compared to the normal control (signal intensity of HD protein pool was set to 1 to represent the reference value). See legend to Fig 1 for technical details.