ALK Activates a Non-Canonical Beta-catenin Pathway in Anaplastic Large Cell Lymphoma

Molecular profiling of Anaplastic Lymphoma Kinase (ALK)-positive versus negative cell lines and patients’ samples has unravelled critical roles for transcriptional factors (TF) (C/EBPbeta, Bcl-6) in Anaplastic Large Cell Lymphomas (ALCL), suggesting that targeting these TF with ALK tyrosine kinase inhibitors should yield better results (Lamant L et al., Blood 2008). In this latter work of our group, beta-catenin and its transcriptional partner T Cell Factor 4 (TCF4) were also found upregulated at a transcriptional level in ALK+ vs ALK− patients’ samples and cell lines. We sought to determine the relevance of such a finding. At the protein level, ALK+ cell lines (SU-DHL-1, Karpas299 and COST) and ALK− cell line (FEPD) express detectable beta-catenin. TCF-4 is also expressed at similar levels among cell lines, whatever ALK status. Since beta-catenin is mainly regulated through post-transcriptional mechanism, we assessed its phosphorylation status. ALK+ (SU-DHL-1 and Karpas299), but not ALK− cell lines displayed increased tyrosine phosphorylation at both Tyr142 and Tyr654 residues, as well as association between beta-catenin and ALK (in immuno-precipitation assays). Moreover, both total beta-catenin and phosphoTyr-beta-catenin were found associated with TCF4, and therefore transcriptionally active. Using the tetracycline system to allow conditional expression of NPM-ALK in MEF cell line (murine embryonic fibroblasts), we found that beta-catenin phosphorylation became barely detectable upon loss of NPM-ALK expression. Based on these findings, we investigated the functional consequences of the disruption of beta-catenin/TCF4 complexes using small molecules such as PKF115–584 and CGP049090 (a generous gift from Novartis). Interestingly, both compounds induced dissociation of beta-catenin/TCF4 complexes at 0.5μM for 24h, and also induced apoptosis in SU-DHL-1 cells. But, since compounds could not dissociate beta-catenin/ALK complexes, they should be combined to ALK inhibitors to fully exert their anti-lymphoma effects. Moreover, the pool of beta-catenin linked to Glycogene Synthase Kinase 3beta (regulated by external Wnt-dependant signals) is neither affected by small compounds, indicating a specific beta-catenin/TCF4 disruption with these drugs. To conclude, this study shows that, in ALCL, ALK activates a Wnt-independent pathway, which appears to be critical for cell survival. This study offers a rationale for investigating the potential of molecules designed to interfere with beta-catenin/TCF/LEF proteins and currently evaluated in colon carcinomas, alone or in combination with ALK tyrosine kinase inhibitors.