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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 β-catenin stability through events encompassing the activation of calpain, which targets β-catenin for proteasome-independent degradation. Accordingly, its inactivation may contribute to induce autophagy and autophagy induction may, in turn, promote β-catenin autolysosomal degradation to originate a regulatory loop where β-catenin plays a central role in cell decision between life and death. Here we proved that the cytoplasmic accumulation of β-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 Ca2 + cytosolic concentration and results in the calpain cleavage into a 28 kDa fragment implicated in β-catenin proteasome-independent degradation. More important for BCR–ABL1 + cell survival and proliferation following IM treatment, might be the calpain-mediated cleavage of β-catenin accumulated within the cytoplasmic compartment into a 75 kDa fragment, still owning TCF-dependent transcriptional activity. Such a β-catenin fragment might be crucial for BCR–ABL1 + cell survival following the fusion protein TK inhibition.

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.

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 β catenin rather than by the BCR-ABL TK. β 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 β catenin antagonist Chibby (CBY) on β catenin signaling in BCR-ABL1 + cells. CBY is a small conserved protein which interacts with β catenin and impairs β catenin-mediated transcriptional activation through two distinct molecular mechanisms: 1) competition with T cell factor (TCF) or lymphoid enhancer factor (LEF) for β catenin binding; and 2) nuclear export of β catenin via interaction with 14-3-3. We found that its enforced expression in K562 cell line promoted β catenin cytoplasmic translocation resulting in inhibition of target gene transcription. Moreover, cytoplasmic accumulation of β catenin activated the endoplasmic reticulum (ER) stress-associated pathway known as unfolded protein response (UPR). CBY-driven cytoplasmic accumulation of β 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.