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// Manuela Mancini 1 , Simona Soverini 1 , Gabriele Gugliotta 1 , Maria Alessandra Santucci 1 , Gianantonio Rosti 1 , Michele Cavo 1 , Giovanni Martinelli 1 and Fausto Castagnetti 1 1 Department of Experimental Diagnostic and Specialty Medicine, DIMES–Institute of Hematology “L. and A. Seragnoli”, University of Bologna Medical School, Bologna, Italy Correspondence to: Manuela Mancini, email: mancini_manu@yahoo.com Keywords: chibby1, s-catenin, ER stress, autophagy, BCR-ABL1 Received: March 31, 2017 Accepted: August 04, 2017 Published: September 22, 2017 ABSTRACT Chibby 1 (CBY1) is a small and evolutionarily conserved protein, which act as β-catenin antagonist. CBY1 is encoded by C22orf2 (22q13.1) Its antagonistic function on β-catenin involves the direct interaction with: The C-terminal activation domain of β-catenin, which hinders β-catenin binding with Tcf/Lef transcription factors hence repressing β-catenin transcriptional activation. 14-3-3 scaffolding proteins (σ or ξ), which drive CBY1 nuclear export into a stable tripartite complex with β-catenin. The relative proximity of C22orf2 gene encoding for CBY1 to the BCR breakpoint on chromosome 22q11, whose translocation and rearrangement with the c-ABL is the causative event of chronic myeloid leukemia (CML), suggested that gene haploinsufficiency may play a role in the disease pathogenesis and progression. We found CBY1 down-modulation associated with the BCR-ABL1 , promoted by transcriptional mechanisms (promoter hyper-methylation) and post-transcriptional events, addressing the protein towards proteasome-dependent degradation through SUMOylation. CBY1 reduced expression in clonal progenitors and, more importantly, in leukemic stem cells (LSC), is contingent upon the tyrosine kinase (TK) activity of BCR-ABL1 fusion protein. Accordingly, its induction by Imatinib (IM) and second generation TK inhibitors contributes to β-catenin inactivation through multiple events encompassing the activation of endoplasmic reticulum (ER) stress-associated unfolded protein response (UPR) and autophagy, eventually leading to apoptotic death. These findings support the advantage of combined regimens including drugs targeting DNA epigenetics and/or proteasome to eradicate the BCR-ABL1+ hematopoiesis.

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.

FOXM1 is a transcription factor of the Forkhead family which drives beta catenin nuclear import and transcriptional activation.Notably, it is a central component of a network which lets tumor cells bypass replicative stress and senescence and maintains the stem cell pluripotency. Accordingly, FOXM1 may have a role in the proliferative advantage and genomic instability of clonal hematopoiesis and leukemic stem cells (LSCs) of chronic myeloid leukemia (CML).Multiple events, including inactivation of protein phosphatase 2A (PP2A, which antagonizes cyclin A/Cdk-dependent activation of FOXM1), activation of Polo-like kinase 1 (PLK1, which directly promotes FOXM1 activating phosphorylation), suppression of Raf kinase inhibitor protein (RKIP, which down-regulates FOXM1 expression), depletion of APC/CDH1 E3 ligase (which degrades FOXM1 at mitotic exit) and down-regulation of micro RNA 370 (miR-370, which directly targets FOXM1 3’ UTR) may concur to BCR-ABL1-associated activation of FOXM1. Experiments carried in BCR-ABL1+ cell lines (K562 and 32D cell clones transduced with a temperature-sensitive mutant of BCR-ABL1 let establish that: 1-FOXM1 activation associated with BCR-ABL1 tyrosine kinase (TK) proceeds from post-transcriptional events encompassing the protein phosphorylation at serine-threonine residues and driving PLK1 activating phosphorylation; 2-FOXM1 phosphorylation is a key event for the interaction with beta catenin and PLK1; 3- the PLK1 inhibitor BI-2536, besides reducing the interaction of FOXM1 protein with beta catenin through events encompassing PLK1-induced phosphorylation of FOXM1, results in a significant reduction of FOXM1 transcript, suggesting a feedback mechanism involved in the regulation of FOXM1 expression in a BCR-ABL1+ cell context. Notably, a significant increment of FOXM1 transcript was found in mononuclear cells from bone marrow samples of 9 out of 15 CML patients at diagnosis compared to hematological healthy donors, which returned to control values in 4/4 patients when they reached a major molecular response to therapy with TK inhibitors. Computational analysis of FOXM1 sequences let distinguish the region encompassing a.a. 561-645 (ARM 10-12) as the one involved in FOXM1 interaction with a.a. 561-645 (ARM 10-12) of beta catenin. Site-directed mutagenesis of above mentioned FOXM1 and beta catenin regions (in progress) would let define FOXM1 role in the transformed phenotype associated with BCR-ABL1 and proceeding from FOXM1-mediated activation of beta catenin. The identification of FOXM1 as a component of BCR-ABL1-induced transformation may help in designing therapeutic strategies to specifically target beta catenin in clonal progenitors and LSCs, sparing the beta catenin signaling in other cell types, particularly, in normal hematopoietic stem cells. Acknowledgments: Umberto Veronesi Foundation, ELN, BolognaAIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project are acknowledged for financial support. MM is the recipient of a grant provided by the Umberto Veronesi Foundation. Disclosures Castagnetti: Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy. Gugliotta:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.