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R.G. Morgan et al.
binding site showed no induction (Figure 1B). In contrast, ML-1 and U937 cells had a highly restricted capacity for nuclear β-catenin localization (vs. K562 and HEL cells) despite substantial cytosolic stabilization of the protein (Figure 1B and C). Consistent with this, CHIR99021 treat- ment of ML-1 or U937 cells caused no detectable activa- tion of the TCF reporter (Figure 1B and 1D). The same pat- terns of Wnt-responsiveness were observed when cell lines were treated with rWnt3a (Online Supplementary Figure S2). These findings demonstrate that the ability of some leukemia cells to drive a transcriptional response to Wnt agonist is limited by their capacity to permit the nuclear accumulation of β-catenin; we termed these cells Wnt-unresponsive.
β-catenin interactome analyses reveal contrasting protein interactions between Wnt-responsive and Wnt-unresponsive leukemia cell lines
Given the wealth of factors previously reported to regu- late the nuclear localization of β-catenin,3 we designed an interactome screen of cytosolic and nuclear β-catenin interacting partners from representative Wnt-responsive (K562 and HEL), and Wnt-unresponsive (ML1) cell lines so that we could shortlist candidate factors involved in this process (Figure 2A). Prior to mass spectrometry, we vali- dated the efficiency of β-catenin co-IP (Figure 2B) from both a positive control for high β-catenin expression (SW620 colorectal cells containing mutated APC: demon- strating a 4.0- and 4.3-fold enrichment of β-catenin in cytosol and nuclear fractions, respectively) and in the con- text of agonist-stabilized β-catenin (HEL: showing a 6.3- and 9.1-fold enrichment). We also confirmed co-IP of a known interactor, TCF-4 (TCF7L2), from the nuclear com- partment.
Following MS, raw tandem mass tag (TMT) ratios were processed to generate a set of statistically ranked interac- tions based on significance of fold-change in protein bind- ing (raw and processed MS data available in Online Supplementary MS data sheets). An extensive profile of β-catenin interactions were observed in K562 cells (225 significantly enriched cytosolic interactions, 118 signifi- cantly enriched nuclear interactions) (Figure 3A and B). In contrast, a comparatively sparse interaction profile was observed in ML1 cells (38 significantly enriched cytosolic interactions, 26 significantly enriched nuclear interactions) (Figure 3C and D). An extensive repertoire of β-catenin interactions was also detected in the other Wnt-respon- sive cell line analyzed, (HEL; 154 significantly enriched cytosolic interactions, 138 significantly enriched nuclear interactions) (Online Supplementary Figure S3). The sparse nuclear β-catenin interaction network observed in ML1 cells is perhaps unsurprising given the low levels of nuclear β-catenin; however, the relative paucity of interac- tors observed in the cytosol of this line was more surpris- ing given the comparable abundance of β-catenin in this fraction compared with K562/HEL cells.
Our experimental and bioinformatics strategy was vali- dated by the identification of known β-catenin interac- tions (highlighted in green), which were also more abun- dant in the K562 cells (23 in cytosol, 26 in nucleus) versus the ML-1 cells (7 in cytosol, 8 in nucleus). From our signif- icantly enriched interactions (Figure 3, red dots), we iden- tified several putative novel partners for β-catenin as sum- marized for K562 (Figure 4A and B and Online Supplementary Table S2), ML1 (Figure 4C and D and Online
Supplementary Table S2), and for HEL (Online Supplementary
Table S3 and Online Supplementary Figure S4). Within these
significant interactions we identified a number of novel
associations of particular interest to myeloid leukemias
and/or Wnt signaling which appeared in one or more cell
lines (red asterisks), but were outside the remit of this
study to investigate further. MBD3 and PRC1 have been
found to co-operate with the oncogenic fusion proteins
PML/RARa14 or PLZF/RARa15 in acute promyelocytic
leukemia (APL), and regulate stemness through Wnt/β-
catenin signaling.16,17 The RNA binding protein MSI2 pre-
dicts poor prognosis in AML,18 more aggressive CML,19
and can promote cancer via Wnt signaling.20 LIN28B, a
microRNA-binding protein, is over-expressed in multiple
leukemias including AML,21 where it promotes prolifera-
22
tion, and co-operates with Wnt signaling to drive malig-
nancy.23 DDX10, RBM6 and RBM15 are known to form oncogenic fusion proteins in myeloid leukemias,24-26 and DDX10 and RBM15 also have roles in promoting Wnt sig- naling.27,28 PUM2 and MKRN2 are two further proteins reported to promote the growth of both normal and malignant hematopoietic cells.29,30 We also confirmed the first reported β-catenin interaction with Wilms Tumor-1 (WT1) by MS and immunoblotting (Online Supplementary Figure S5), which is of considerable interest in leukemia biology given its frequent dysregulation in AML and asso- ciation with adverse patient survival.31
Nuclear LEF-1 expression correlates with nuclear β-catenin localization in cell lines and primary acute myeloid leukemia patient cells
We next examined the data from the fractions of Wnt- responsive cells for candidate proteins that could promote the nuclear-localization of β-catenin. Several had previ- ously been implicated in negative regulation of β-catenin nuclear localization, GSK3β,32 a-catenin,33 Axin1/2,34 and APC,34 whilst TCF-434 and LEF-135 are nuclear localized transcription factors that bind β-catenin. To validate the MS data, we examined the protein expression of these candidates in a panel of myeloid cell lines by immunoblot- ting. With the exception of APC, which is problematic to blot,36 immunoblotting confirmed MS analysis in that expression of the negative regulators was mostly limited to Wnt-responsive cells making it unlikely they were responsible for restricted nuclear β-catenin in Wnt-unre- sponsive cells (Figure 5A).
We next examined the expression of two known nuclear β-catenin interactors, TCF-4 and LEF-1, which are ubiquitously expressed in multiple tissues. Both of these proteins are predominantly nuclear in Wnt-responsive cell lines and were absent from the Wnt-unresponsive lines (Figure 5A; matching the proteomics data in Figure 3 and Online Supplementary Figure S3). Of these two proteins, LEF-1 bound β-catenin with higher significance than TCF4 in both K562 and HEL nuclei. This, together with our pre- vious observation that overexpression of TCF-4 actually suppressed β-catenin-dependent transcription37 (and thus is unlikely to promote nuclear β-catenin level), led us to focus our investigation on LEF-1. This protein is known to be dysregulated in AML38 so we examined the clinical rel- evance of this by correlating relative LEF-1 nuclear local- ization with that of β-catenin in primary AML blasts. In our cohort of 23 nuclear/cytosol fractionated AML patient samples, we observed a highly significant degree of corre- lation (Spearman Rank R=0.63, P<0.005) between the rel-
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haematologica | 2019; 104(7)