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R.G. Morgan et al.
to play a key role in the initiation of AML and chronic myeloid leukemia (CML).6,7 Furthermore, frequent chro- mosomal aberrations driving AML and CML are known to co-operate with β-catenin.8,9 Key to the activation of Wnt signaling is the movement of β-catenin into the nucleus and this is frequently observed in AML.10 We have previ- ously demonstrated that approximately 10% of primary AML patient blast samples exhibit little nuclear β-catenin expression, despite substantial cytosolic levels, a phenom- enon replicated in 10-20% of myeloid leukemia cell lines upon Wnt stimulation.3,11 In fact, this is characteristic of normal human hematopoietic stem/progenitor cells (HSPC) which similarly limit β-catenin nuclear-localiza- tion, possibly to protect normal HSC from detrimental levels of Wnt signaling.12 The permissive nuclear-localiza- tion of β-catenin observed in myeloid leukemias is there- fore aberrant and warrants further investigation.
Mini Protease-Inhibitor Cocktail (PIC; Sigma-Aldrich) for 10 min- utes (min) at 4°C. The supernatant (cytosolic fraction) was recov- ered following centrifugation at 800 g for 5 min, and the nuclear pellet washed twice with PBS. Nuclear pellets were resuspended in lysis buffer (Cell Signaling Technology, Leiden, the Netherlands) containing PIC and incubated for 45 min with sonication to max- imize nuclear lysis. Insoluble material was removed at 21,000 g for 10 min and solubilized nuclear fractions stored at -80°C.
Lentiviral transduction
K562 and HEL cells were lentivirally-transduced with the β-catenin-activated reporter (BAR) or mutant ‘found unresponsive’ control (fuBAR) system as previously.11 For LEF-1 knockdown/overexpression, cells were lentivirally-transduced with human LEF-1 shRNA (TRCN0000-020163, -413476, -418104, -428178 and -428355, MISSION® Sigma), or LEF-1 overexpression vector (pLV-EGFP:T2A:Puro-EF1A>hLEF-1 VectorBuilder, Neu- Isenburg, Germany). Cells transduced with scrambled shRNA/empty vector served as controls.
β-catenin co-immunoprecipitation and immunoblotting For co-immunoprecipitation (co-IP), 8 μg of crosslinked β-catenin (Clone-14) or IgG (Clone MOPC-31C) antibody (Becton Dickinson, Oxford, UK) were incubated with 1 mg of either pre- cleared cytoplasmic or nuclear lysate overnight at 4°C (Online Supplementary Methods). Subsequently beads were washed five times prior to proteomic analyses or boiled for 95°C for 5 min fol- lowing washes for immunoblotting. Immunoblotting was per- formed as previously described13 using antibodies to total β-Catenin (as above), phosphorylated β-catenin (Ser33/37/Thr41), β-Catenin (Clone-5), E-Cadherin (Clone-36), GSK3β (Clone-7; BD), a-tubulin (DM1A), lamin A/C (4C11; Sigma), Axin1 (C76H11), Axin2 (76G6), TCF-4 (C48H11), LEF-1 (C12A5), Survivin (71G4B7), CyclinD1 (92G2; Cell Signaling Technology), active β-catenin (8E7, Merck-Millipore, Watford, UK) and c-MYC (9E10; Santa Cruz, Heidelberg, Germany). β-Catenin and LEF-1 densitometry were performed as described in the Online
Supplementary Methods.
To better understand β-catenin nuclear-localization mechanisms in myeloid leukemia cells, we generated the first β-catenin interactomes in hematopoietic cells. These analyses have shown that LEF-1, a β-catenin-dependent transcription factor, can also regulate the level of nuclear β-catenin in myeloid leukemia cells. The relative level of nuclear LEF-1 expression correlates with relative nuclear levels of β-catenin in primary AML patient blasts indicat- ing this axis has clinical relevance. Furthermore, the nuclear-localization of β-catenin can be promoted by LEF-1 overexpression and conversely is reduced by LEF-1 knockdown. Finally, we demonstrate LEF-1 expression is suppressed in Wnt-unresponsive cells through rapid prote- olytic degradation that is not observed in Wnt-responsive cells. Overall, this study characterizes β-catenin interac- tions within a hematopoietic context and identifies LEF-1 as a regulator of nuclear β-catenin localization in human leukemia.
Methods
Patient samples, cell culture and β-catenin stabilization
Bone marrow, peripheral blood or leukapheresis samples from patients diagnosed with AML/myelodysplastic syndromes (MDS) (for clinical information see Online Supplementary Table S1) were collected in accordance with the Declaration of Helsinki and with approval of University Hospitals Bristol NHS Trust and London Brent Research Ethics Committee. Mononuclear cells were sepa- rated using Ficoll-Hypaque (Sigma-Aldrich, Poole, UK) and sam- ples with ≥80% viability included in the study. K562, HEL, ML-1, U937, THP1 and PLB-985 cell lines (ECACC, Salisbury, UK) were cultured as previously described.11 For proliferation assays, cell lines were seeded in triplicate at 1x105/mL into 24-well plates within medium containing 10, 5, 1 or 0.5% fetal bovine serum (Labtech, East Sussex, UK) and cellular density counted using a hemocytometer at 24, 48 and 72 hours (h). For Wnt signaling acti- vation, cell lines were treated with 5 μM of the GSK-3β inhibitor CHIR99021 (Sigma-Aldrich) or 1 μg/mL recombinant murine Wnt3a (Peprotech, London, UK) for 16 h (unless otherwise stated) at 37°C.
Mass spectrometry and data analyses
Nuclear/cytoplasmic fractionation
Assessment of T-cell factor reporter and flow cytometry
Activity of BAR lentiviral construct was performed as previous- ly described.11 Multi-parameter flow cytometric measurements were acquired using a MACSQuant® Analyzer 10 in conjunction with MACSQuantifyTM v.2.8 (Miltenyi Biotec, Bisley, UK) or an Accuri C6 in conjunction with C sampler software v.1.0.264.21 (BD). Post-acquisition analyses were performed using FlowJo
2-8x106 cells were washed in PBS and resuspended in 250 μL cytoplasmic lysis buffer (10 mM Tris-HCl (pH8), 10 mM NaCl, 1.5 mM MgCl2, 0.5% Igepal-CA630/NP40) containing completeTM
Cytosolic or nuclear β-Catenin/IgG co-IPs were prepared and analyzed by mass spectrometry (MS) as detailed in the Online Supplementary Methods. Post-acquisition, duplicate values and pro- teins detected by only a single peptide were first removed. Tandem Mass Tag (TMT) ratios were imported into Perseus v.1.5.6.0 (Max Planck Institute of Biochemistry, Munich, Germany) followed by logarithmic transformation, normalization (through median subtraction) and removal of proteins not present in at least two of three replicates. A one-sample t-test was per- formed with significance of protein binding (-Log10 P-value) plot- ted versus fold change in protein binding (Log2). The MS pro- teomics data have been deposited with the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD009305. Interaction specificity was assessed using the publicly available CRAPome database (Contaminant Repository for Affinity Purification: http://www.crapome.org).
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haematologica | 2019; 104(7)