Abstract 858: Development of ultrasensitive split luciferase biosensors monitoring activity of the merlin tumor suppressor

Abstract

Abstract Understanding features common to all forms of cancer is a vital component of treatment and research into malignant disease. All solid tumours share the ability to overcome contact inhibition of proliferation (CIP), the process by which intercellular contacts engage signalling to stop proliferation. Merlin, a tumour suppressor protein that is inactivated in a wide variety of cancers, plays a crucial role in CIP; merlin-deficient cells lose the ability to be contact-inhibited and subsequently form tumours. Although the role of merlin in cancer has been investigated extensively over the past 28 years, this protein has been notoriously difficult to study. To address this issue, we develop and utilize 2 split-luciferase biosensor systems that enable accurate quantification of merlin activity in real time. Merlin undergoes a conformational change that is functionally important in tumour suppression. Phosphorylation at a key C-terminal residue promotes transition of the protein from an open, active conformation to a closed, N-to-C terminal autoinhibited conformation. Therefore, monitoring this conformation change in real time could provide unique insights into merlin function. To do this, we apply NanoBiT split-luciferase technology to develop an intramolecular merlin biosensor (intra-Mer-BS). In brief, 2 split-luciferase components, LgBiT and SmBiT, are fused to the N- and C-terminus of merlin, respectively. Upon open-to-closed conformation change of merlin, LgBiT and SmBiT complement to reconstitute a functional luciferase and emit light. This enables accurate quantification of merlin’s functionally relevant conformation changes in real time. Importantly, cotransfection of the intra-Mer-BS alongside PAK1, an upstream merlin regulator that promotes transition to the closed conformation, significantly increases luminescent activity of the intra-Mer-BS, indicating that the biosensor faithfully reports on merlin’s conformation. Moreover, merlin has been shown to exert its tumour suppressive function through activation of LATS, the central mediator of the Hippo signalling pathway. In addition to the intra-Mer-BS, we develop and validate a NanoBiT biosensor to monitor the interaction between merlin and LATS (Mer-LATS-BS). This Mer-LATS-BS is used to quantify the effect of merlin activators and inhibitors on merlin/LATS tumour suppressive activity in cancer cells. In summary, we develop and validate 2 novel bioluminescent biosensors to monitor merlin’s conformation changes and activity in cancer cells. The intra-Mer-BS and Mer-LATS-BS provide real time information with high sensitivity and excellent reproducibility. Ultimately, these biosensors enable high throughput screening to discover novel upstream regulators of merlin in cancer and provide mechanistic insight into how contact inhibitive signalling is propagated through merlin and the Hippo pathway. Citation Format: Alexander J. Pipchuk, Xiaolong Yang. Development of ultrasensitive split luciferase biosensors monitoring activity of the merlin tumor suppressor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 858.