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LEF-1 regulates β-catenin nuclear localization
These data suggest that LEF-1-targeted proteases are active in Wnt-unresponsive U937 cells, but inactive/absent in Wnt-responsive K562 cells. To test this, we mixed nuclear lysates from K562 cells directly with whole cell lysates from U937 control cells (which are LEF- 1 negative). Remarkably, the previously stable full-length LEF-1 band present in K562 cells exhibited marked and rapid reduction with a concomitant increase in the short- form polypeptide, a process exacerbated by PIC removal (Figure 8D). These data confirm that constitutive LEF-1 degradation mechanisms are active in Wnt-unresponsive cells but are absent/inhibited in Wnt-responsive cells.
Discussion
Canonical Wnt signaling has emerged as one of the most frequently dysregulated signaling pathways in myeloid neoplasms which has led to considerable interest in targeting this pathway. The central mediator β-catenin represents an appealing therapeutic target because of its leukemogenic role,6,7 prognostic influence,5 and functional redundancy in normal hematopoietic development.40 The characterization of the hematopoietic interactome of β- catenin is, therefore, of considerable interest, and the experimental approach adopted in this study was validat- ed by the identification of multiple known β-catenin part- ners, such as Axin, TCF-4, a-catenin and APC. Associations with key hematopoietic transcription fac- tors, C/EBPζ and GATA-1, were identified and these are known to co-occupy genomic sites with TCF-4 (a known β-catenin partner) during hematopoietic development.41 We confirmed the previously reported interaction of β- catenin with the oncogenic fusion protein BCR-ABL in CML, which is present in K562 cells.9 In addition to known partners, we identified putative novel interactions which have known relevance to leukemia and/or Wnt signaling (MBD3,14,16 PRC1,15,17 MSI2,18-20 LIN28B,21-23 DDX10,24,27 RBM6,25 RBM15,26,28 PUM2,29 MKRN230 and WT131).
To investigate the nuclear localization mechanisms of β-catenin, we focused on LEF-1, given that it was one of the most significantly enriched interactors present in Wnt-responsive cell nuclei, and its nuclear expression was highly predictive of β-catenin nuclear-localization in both myeloid cell lines and primary AML blasts. A role for LEF- 1 in mediating nuclear-localization of β-catenin was con- firmed using both knockdown and overexpression approaches. LEF-1 contains a nuclear localization sequence and is known to shuttle between cytoplasmic and nuclear compartments.42 This suggests LEF-1 could serve as a cytosolic-nuclear chaperone for β-catenin. However, the high nuclear:cytosol ratio of LEF-1 expres- sion observed in Wnt-responsive cells (K562 and HEL) would be more consistent with LEF-1 serving as a nuclear retention factor for β-catenin, as has previously been demonstrated for other Wnt signaling components.34 This is the first evidence of LEF-1 contributing to β-catenin nuclear-localization capacity in human myeloid leukemia cells; however, our data cannot exclude the role of other factors in this process. Nuclear export mechanisms may also be influential in limiting nuclear accumulation of β-catenin in Wnt-unresponsive cell lines. In particular, RanBP1 was an interacting partner detected for β-catenin in ML-1 nuclei but absent in K562/HEL nuclei, and may
warrant further study given the documented role for RanBP3 in mediating β-catenin export.43 Two factors recently implicated in nuclear β-catenin regulation includ- ing RAPGEF544 and Twa145 were not detected in our analyses, suggesting their interaction with β-catenin is context-dependent.
A strong correlation between the relative levels of nuclear localized β-catenin and LEF-1 was identified in primary AML samples suggesting this relationship may have clinical relevance. Indeed, expression of constitu- tively active LEF-1 in HSPC induced AML in mice,38 and LEF-1 expression promotes the survival of myeloid leukemia cell lines.46 In our study, we observed signifi- cantly inhibited growth in K562 and HEL cells harboring LEF-1 knockdown (Figure 6C). Given the adverse progno- sis of nuclear β-catenin in AML,5 and recent studies demonstrating its therapeutic merit in AML models,47-49 LEF-1 represents an attractive target in myeloid leukemia since it also inhibits β-catenin nuclear localization. Small molecule inhibitors of β-catenin: TCF/LEF interaction are under development and have shown efficacy in leukemia treatment.50-52 It would be interesting to observe if this efficacy is partly driven by reducing the level of nuclear β- catenin in leukemia cells. This study focused on the role of LEF-1 driving aberrant β-catenin nuclear-localization in myeloid leukemia; however, given the frequency of Wnt/β-catenin dysregulation in human cancer, this axis could be active in other malignancies.
We also observed that LEF-1 is proteolytically degraded in Wnt-unresponsive leukemia cells resulting in the emer- gence of short-form LEF-1 proteins (25-30kDa). These were smaller than the 38kDa transcriptional isoforms pre- viously reported to derive from alternative splicing or alternative promoter usage39,53 which serve as dominant- negative inhibitors of Wnt signaling because they lack the β-catenin binding domain necessary to initiate transcrip- tion.39 In contrast, the proteolytic fragments observed in this study retained β-catenin binding capacity (Figure 7A) and did not appear to have dominant-negative function (Wnt targets were still activated) (Figure 6C and D). Under the conditions of the experiment, however, where LEF-1 is being over-expressed, the abundance of these fragments may have a dominant effect on β-catenin retention in the nucleus, whereas under normal circum- stances, it is likely that degradation serves to remove LEF- 1 and suppress the nuclear retention of β-catenin. This post-translational regulation of LEF-1 has parallels with embryonic stem cells where proteolytic cleavage of TCF- 3 (TCF7L1), a closely related family member, removes it from target genes when differentiation signals trigger the suppression of Wnt signaling.54 In leukemia cells, this pro- teolytic cleavage could be mediated by NLK (Nemo-like kinase) which binds the E3 ubiquitin-ligase NARF (NLK associated RING finger protein) and reportedly induces ubiquitylation (and proteasomal degradation) of LEF-1 in co-operation with the E2 conjugating enzyme E2-25K,55 a mechanism previously reported to be active in leukemia cells.56
In summary, our study has made three key findings: firstly, the generation of the first β-catenin interactomes in leukemia cells; secondly, the characterization of LEF-1 as a regulator of nuclear β-catenin localization in leukemia; and, finally, the demonstration of post-tran- scriptional proteolytic degradation mechanisms for con- trolling LEF-1 expression in myeloid leukemia cells.
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