Targeting of GSK3ß promotes imatinib-mediated apoptosis in quiescent CD34+ chronic myeloid leukemia progenitors, preserving normal stem cells.

The targeting of BCR-ABL, a hybrid oncogenic tyrosine (Y) kinase, does not eradicate chronic myeloid leukemia (CML)-initiating cells. Activation of ß-catenin was linked to CML leukemogenesis and drug resistance through its BCR-ABL-dependent Y phosphorylation and impaired binding to GSK3ß (glycogen synthase kinase 3ß). Herein, we show that GSK3ß is constitutively Y(216) phospho-activated and predominantly relocated to the cytoplasm in primary CML stem/progenitor cells compared with its balanced active/inactive levels and cytosolic/nuclear distribution in normal cells. Under cytokine support, persistent GSK3ß activity and its altered subcellular localization were correlated with BCR-ABL-dependent and -independent activation of MAPK and p60-SRC/GSK3ß complex formation. Specifically, GSK3ß activity and nuclear import were increased by imatinib mesylate (IM), a selective ABL inhibitor, but prevented by dasatinib that targets both BCR-ABL- and cytokine-dependent MAPK/p60-SRC activity. SB216763, a specific GSK3 inhibitor, promoted an almost complete suppression of primary CML stem/progenitor cells when combined with IM, but not dasatinib, while sparing bcr-abl-negative cells. Our data indicate that GSK3 inhibition acts to prime a pro-differentiative/apoptotic transcription program in the nucleus of IM-treated CML cells by affecting the ß-catenin, cyclinD1, C-EBPα, ATF5, mTOR, and p27 levels. In conclusion, our data gain new insight in CML biology, indicating that GSK3 inhibitors may be of therapeutic value in selectively targeting leukemia-initiating cells in combination with IM but not dasatinib.

ß-catenin knockdown promotes NHERF1-mediated survival of colorectal cancer cells: implications for a double-targeted therapy.

Nuclear activated ß-catenin plays a causative role in colorectal cancers (CRC) but remains an elusive therapeutic target. Using human CRC cells harboring different Wnt/ß-catenin pathway mutations in APC/KRAS or ß-catenin/KRAS genes, and both genetic and pharmacological knockdown approaches, we show that oncogenic ß-catenin signaling negatively regulates the expression of NHERF1 (Na+/H+ exchanger 3 regulating factor 1), a PDZ-adaptor protein that is usually lost or downregulated in early dysplastic adenomas to exacerbate nuclear ß-catenin activity. Chromatin immunoprecipitation (ChIP) assays demonstrated that ß-catenin represses NHERF1 via TCF4 directly, while the association between TCF1 and the Nherf1 promoter increased upon ß-catenin knockdown. To note, the occurrence of a cytostatic survival response in settings of single ß-catenin-depleted CRC cells was abrogated by combining NHERF1 inhibition via small hairpin RNA (shRNA) or RS5517, a novel PDZ1-domain ligand of NHERF1 that prevented its ectopic nuclear entry. Mechanistically, dual NHERF1/ß-catenin targeting promoted an autophagy-to-apoptosis switch consistent with the activation of Caspase-3, the cleavage of PARP and reduced levels of phospho-ERK1/2, Beclin-1, and Rab7 autophagic proteins compared with ß-catenin knockdown alone. Collectively, our data unveil novel ß-catenin/TCF-dependent mechanisms of CRC carcinogenesis, also offering preclinical proof of concept for combining ß-catenin and NHERF1 pharmacological inhibitors as a mechanism-based strategy to augment apoptotic death of CRC cells refractory to current Wnt/ß-catenin-targeted therapeutics.