Targeting MAML for the inhibition of notch signalling, using Antp fusion proteins

Abstract

The highly conserved Notch signalling pathway plays a major role in both metazoan development and adult tissue renewal. Deregulation of this pathway is linked to several diseases, including cancer. Various strategies for Notch signalling inhibition are now being investigated as promising new approaches for cancer therapy. Notch receptor activation results in the release of the Notch cytoplasmic domain (NIC) which translocates to the nucleus to bind the transcription factor CSL. Subsequent recruitment of MAML-1 is necessary to form the active complex that initiates transcription of Notch target genes. In this study, the strategy used to inhibit Notch signalling consists of blocking the CSL-NIC-MAML tripartite complex formation by targeting MAML-1 interactions with NIC and CSL. The first tool is a fusion protein composed of the membrane translocating protein Antennapedia (Antp) and a dominant negative version of MAML-1 (DN-MAML1). A Notch activity reporter-gene assay revealed that addition of Antp-DN-MAML1 leads to a dose dependent decrease in luminescence, corresponding to an inhibition of Notch signalling. At the transcriptional level, treatment of MDA-MB-231 and bone marrow dendritic cells with Antp-DN-MAML1 resulted in a significant decrease in the mRNA levels of Notch target gene Hes1. In a cell proliferation assay, addition of Antp-DN-MAML1 had a strong growth-inhibitory effect when added to MDA-MB-231 breast cancer cells. Our in vitro data is further supported by preclinical studies where administration of Antp-DN-MAML1 halted MDA-MB-231 tumour growth. The second strategy of Notch signalling inhibition presented here attempts the intranuclear delivery of single-chain variable-fragment (scFv) antibodies fused to Antp. Suitable scFvs were selected from a phage display library for their ability to bind residues 13–74 of MAML-1. This constitutes the minimal portion of MAML-1 necessary to form the active tripartite complex, therefore we expect any scFvs that bind this portion to inhibit downstream signalling. Our study illustrates two novel strategies for inhibition of Notch signalling at the transcriptional level which represent potential future therapeutic alternatives.