Factors regulating the nuclear localization of β-catenin in acute myeloid leukaemia

Acute myeloid leukaemia (AML) is a highly heterogenous haematological malignancy that accounts for approximately 34% of newly diagnosed leukaemia cases in the United Kingdom. It is a disease that primarily affects the elderly, and the prognosis for AML is generally poor, with a 5-year survival rate of 25% in adults. Identifying factors that contribute to the pathogenesis of AML is important in developing new targeted treatments and improving patient outcome. The Wnt signalling pathway is one of the most commonly dysregulated signalling pathways in AML, and overexpression of its principal effector, β-catenin, is associated with a poor prognosis. β-catenin is a transcription factor that regulates key cellular processes, including proliferation and cell survival, by binding in complex with T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) proteins to Wnt target gene promoters. Nuclear localization of β-catenin is fundamental for its role as a transcription factor. Despite this, little is known about the mechanisms regulating this process in AML; previous work has shown AML blasts and cell lines are highly variable in their ability to translocate β-catenin from the cytosol to the nucleus. This study used a mass spectrometric approach to identify candidate β-catenin nuclear localization factors in AML. This approach was based on comparisons of β-catenin binding partners in the nucleus and cytoplasm of leukaemia cell lines that i) freely translocate β-catenin to the nucleus (K562 and HEL), or ii) resist the nuclear localization of β-catenin (THP-1, U937 and NOMO-1). Cytoplasmic/nuclear fractionation and immunoprecipitation of β-catenin were optimized in K562 cells, before being used to prepare samples of each cell line for mass spectrometry. Following mass spectrometric analysis, eight candidate β-catenin nuclear localization factors were identified, of which two proteins, RUNX1 and LEF-1, were verified by western blotting. Since LEF1 has been previously implicated as a β-catenin nuclear import factor in other contexts and because of its known role in AML, LEF-1 was chosen for further analysis. To determine the relevance of LEF-1 mediated β-catenin nuclear localization in AML pathogenesis, knockdown and overexpression studies were conducted. Knockdown of LEF-1 was successful in leukaemia lines. In cells stimulated with the Wnt agonists (Wnt3a and BIO), LEF-1 knockdown decreased the nuclear translocation of β-catenin and impacted β-catenin mediated transcription. Additionally, knockdown of LEF-1 appeared to reduce proliferation of leukaemia cells, but did not impact their migration or survival. Ectopic nuclear overexpression of LEF1 proved difficult to achieve in myeloid cells due to protein instability; making reciprocal demonstration of the role of LEF1 difficult to demonstrate. Overall this study has identified candidate β-catenin nuclear localization factors and has validated the role of one of these proteins (LEF1). This work provides insight into the potential mechanisms governing β-catenin nuclear localization in AML, and in the longer term, may lead to novel approaches for the treatment of AML

The forkhead transcription factor FOXK2 acts as a chromatin targeting factor for the BAP1-containing histone deubiquitinase complex.

There are numerous forkhead transcription factors in mammalian cells but we know little about the molecular functions of the majority of these. FOXK2 is a ubiquitously expressed family member suggesting an important function across multiple cell types. Here, we show that FOXK2 binds to the SIN3A and PR-DUB complexes. The PR-DUB complex contains the important tumour suppressor protein, the deubiquitinase BAP1. FOXK2 recruits BAP1 to DNA, promotes local histone deubiquitination and causes changes in target gene activity. Our results therefore provide an important link between BAP1 and the transcription factor FOXK2 and demonstrate how BAP1 can be recruited to specific regulatory loci

LEF-1 drives aberrant β-catenin nuclear localization in myeloid leukemia cells

Canonical Wnt/β-catenin signaling is frequently dysregulated in myeloid leukemias and is implicated in leukemogenesis. Nuclear-localized β-catenin is indicative of active Wnt signaling and is frequently observed in acute myeloid leukemia patients; however, some patients exhibit little or no nuclear β-catenin even where cytosolic β-catenin is abundant. Control of the subcellular localization of β-catenin therefore represents an additional mechanism regulating Wnt signaling in hematopoietic cells. To investigate the factors mediating the nuclear-localization of β-catenin we carried out the first nuclear/cytoplasmic proteomic analysis of the β-catenin interactome in myeloid leukemia cells and identified putative novel β-catenin interactors. Comparison of interacting factors between Wnt-responsive cells (high nuclear β-catenin) versus Wnt-unresponsive cells (low nuclear β-catenin) suggested the transcriptional partner, LEF-1, could direct the nuclear-localization of β-catenin. The relative levels of nuclear LEF-1 and β-catenin were tightly correlated in both cell lines and in primary AML blasts. Furthermore, LEF-1 knockdown perturbed β-catenin nuclear-localization and transcriptional activation in Wnt-responsive cells. Conversely, LEF-1 overexpression was able to promote both nuclear-localization and β-catenin-dependent transcriptional responses in previously Wnt-unresponsive cells. This is the first β-catenin interactome study in hematopoietic cells and reveals LEF-1 as a mediator of nuclear β-catenin level human myeloid leukemia

LEF-1 drives aberrant β-catenin nuclear localization in myeloid leukemia cells.

Canonical Wnt/β-catenin signaling is frequently dysregulated in myeloid leukemias and is implicated in leukemogenesis. Nuclear-localized β-catenin is indicative of active Wnt signaling and is frequently observed in acute myeloid leukemia (AML) patients; however, some patients exhibit little or no nuclear β-catenin even where cytosolic β-catenin is abundant. Control of the subcellular localization of β-catenin therefore represents an additional mechanism regulating Wnt signaling in hematopoietic cells. To investigate the factors mediating the nuclear-localization of β-catenin, we carried out the first nuclear/cytoplasmic proteomic analysis of the β-catenin interactome in myeloid leukemia cells and identified putative novel β-catenin interactors. Comparison of interacting factors between Wnt-responsive cells (high nuclear β-catenin) versus Wnt-unresponsive cells (low nuclear β-catenin) suggested the transcriptional partner, LEF-1, could direct the nuclear-localization of β-catenin. The relative levels of nuclear LEF-1 and β-catenin were tightly correlated in both cell lines and in primary AML blasts. Furthermore, LEF-1 knockdown perturbed β-catenin nuclear-localization and transcriptional activation in Wnt-responsive cells. Conversely, LEF-1 overexpression was able to promote both nuclear-localization and β-catenin-dependent transcriptional responses in previously Wnt-unresponsive cells. This is the first β-catenin interactome study in hematopoietic cells and reveals LEF-1 as a mediator of nuclear β- catenin level in human myeloid leukemia

LEF-1 drives aberrant ß-catenin nuclear localization in myeloid leukemia cells

Canonical Wnt/ß-catenin signaling is frequently dysregulated in myeloid leukemias and is implicated in leukemogenesis. Nuclear-localized ß-catenin is indicative of active Wnt signaling and is frequently observed in acute myeloid leukemia patients; however, some patients exhibit little or no nuclear ß-catenin even where cytosolic ß-catenin is abundant. Control of the subcellular localization of ß-catenin therefore represents an additional mechanism regulating Wnt signaling in hematopoietic cells. To investigate the factors mediating the nuclear-localization of ß-catenin we carried out the first nuclear/cytoplasmic proteomic analysis of the ß-catenin interactome in myeloid leukemia cells and identified putative novel ß-catenin interactors. Comparison of interacting factors between Wnt-responsive cells (high nuclear ß-catenin) versus Wnt-unresponsive cells (low nuclear ß-catenin) suggested the transcriptional partner, LEF-1, could direct the nuclear-localization of ß-catenin. The relative levels of nuclear LEF-1 and ß-catenin were tightly correlated in both cell lines and in primary AML blasts. Furthermore, LEF-1 knockdown perturbed ß-catenin nuclear-localization and transcriptional activation in Wnt-responsive cells. Conversely, LEF-1 overexpression was able to promote both nuclear-localization and ß-catenin-dependent transcriptional responses in previously Wnt-unresponsive cells. This is the first ß-catenin interactome study in hematopoietic cells and reveals LEF-1 as a mediator of nuclear ß-catenin level human myeloid leukemia