Wnt inhibition leads to improved chemosensitivity in paediatric acute lymphoblastic leukaemia.

The prognosis for children with ALL has improved dramatically over the last five decades (Hunger, Lu et al. 2012). However up to 20% of children will experience relapse, and the prognosis for these children is poor, with documented 5-year event free survival rates (EFS) rates of 12% and 35% for early (< 36 months from diagnosis) and late relapse (≥ 36 months), respectively (Nguyen, Devidas et al. 2008, van den Berg, de Groot-Kruseman et al. 2011). Intensification of treatment has not impacted the prognosis for the majority of patients highlighting the critical need for novel approaches for the prevention and treatment of relapsed and refractory disease. Intrinsic drug resistance is a key factor in the poor response to retrieval therapy as evidenced by lower remission rates, early second relapse and by ex vivo analysis of drug sensitivity at relapse vs. diagnosis (Klumper, Pieters et al. 1995, Raetz, Borowitz et al. 2008). Aberrant Wnt signaling has been linked to cancers of the liver, colon, breast, skin (Polakis 2000, Miyoshi, Rosner et al. 2002, Miyoshi and Hennighausen 2003, Moon, Kohn et al. 2004) and more recently hematologic malignancies including acute myeloid leukemia (AML) and ALL (Reya and Clevers 2005, Gang, Hsieh et al. 2013). The transcriptional co-activator β-catenin is the key mediator of canonical Wnt signaling. Wnt activation results in increased accumulation of cytosolic β-catenin, which then translocates to the nucleus and interacts with the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of high-mobility group (HMG) transcription factors to activate transcription of target genes (Nusse 1999, Staal and Clevers 2000). Also, many of the known target genes of the signaling pathway (BIRC5, CCND1 and MYC) are involved in cellular differentiation, proliferation and survival and have been implicated in carcinogenesis (Gehrke, Gandhirajan et al. 2009), making them attractive targets for therapy. Previously, we performed an integrated genomic analysis of relapsed ALL to discover the underlying biological pathways responsible for drug resistance (Hogan, Meyer et al. 2011). We observed that many of the known negative regulators of the Wnt pathway were deleted and/or hypermethylated and/or downregulated at relapse in many patients, including the direct Wnt inhibitors: WIF1, PTPRO, SFRP2, SFRP4, SFRP5, FZD10, DKK2 and DKK3 (Nelson and Nusse 2004, Takahashi-Yanaga and Kahn 2010). Inhibitors of the β-catenin/TCF/LEF activity, including APC, WT1 and many cadherin (CDH1, CDH11 and CD13) and SOX genes (SOX2, SOX3, SOX8, SOX9, SOX11, SOX14 and SOX21), were also negatively regulated. BIRC5, a downstream target of the Wnt pathway, which encodes the anti-apoptosis protein Survivin, was upregulated by gene expression array and validated by quantitative RT-PCR. Interestingly, PTPRO, a negative feedback inhibitor of the Wnt pathway, that binds to Wnt and blocks its association with other receptors, was hypermethylated and downregulated (Kim, Kim et al. 2010). We hypothesized that overactivation of the Wnt pathway may have a critical function in mediating therapy resistance in some patients. To address this issue directly, we compared the alteration of Wnt signaling network activity (activated β-catenin) and the expression of downstream targets in 10 diagnosis-relapse paired patient samples, as well as the impact of a recently developed Wnt inhibitor, iCRT14 (Gonsalves, Klein et al. 2011) in ALL cell lines and primary patient samples. BIRC5 was chosen as readout for β-catenin/TCF transcriptional activity because we have shown it to be upregulated at relapse and because overexpression has been associated with poor outcomes in certain cancers (Adida, Haioun et al. 2000, Paik, Shak et al. 2004, Fangusaro, Caldas et al. 2006). Collectively, our results indicate that overactivation of the Wnt pathway is often observed at relapse and that inhibition of Wnt signaling restores chemosensitivity in resistant disease.