A kinome‐wide screen using a NanoLuc LATS luminescent biosensor identifies ALK as a novel regulator of the Hippo pathway in tumorigenesis and immune evasion

Abstract

The Hippo pathway is an emerging signaling pathway that plays important roles in organ size control, tissue homeostasis, tumorigenesis, metastasis, drug resistance, and immune response. Although many regulators of the Hippo pathway have been reported, the extracellular stimuli and kinase regulators of the Hippo pathway remain largely unknown. To identify novel regulars of the Hippo pathway, in this study we created the first ultra‐bright NanoLuc biosensor (BS) to monitor the activity of large tumor suppressor (LATS) kinase 1, a central player of the Hippo pathway. We show that this NanoLuc BS achieves significantly advanced sensitivity and stability both in vitro using purified proteins and in vivo in living cells and mice. Using this BS, we perform the first kinome‐wide screen and identify many kinases regulating LATS and its effectors yes‐associated protein (YAP) and transcriptional co‐activator with PDZ‐ binding motif (TAZ). We also show for the first time that activation of receptor tyrosine kinase anaplastic lymphoma kinase (ALK) by its extracellular ligand family with sequence similarity (FAM)150 activates Hippo effector YAP/TAZ by increasing their nuclear translocation. Significantly, we show that constitutively active ALK induces tumorigenic phenotypes, such as increased cancer cell proliferation/colony formation via YAP/TAZ and elevated immune evasion via YAP/TAZ‐programmed death‐ligand 1 in breast and lung cancer cells. In summary, we have developed a new LATS BS for cancer biology and therapeutics research and uncovered a novel ALK‐LATS‐YAP/TAZ signaling axis that may play important roles in cancer and possibly other biologic processes.—Nouri, K., Azad, T., Lightbody, E., Khanal, P., Nicol, C. J., Yang, X. A kinome‐wide screen using a NanoLuc LATS luminescent biosensor identifies ALK as a novel regulator of the Hippo pathway in tumorigenesis and immune evasion. FASEB J. 33, 12487–12499 (2019). http://www.fasebj.org

Emerging Roles of ALK in Immunity and Insights for Immunotherapy

“…With regard to the mechanisms underlying ALK fusion-induced PD-L1 in NSCLC, several studies demonstrated the involvement of PI3K and MAPK activation [47], as well as the Hippo pathway [52]. Through a kinome-wide screen of modulators of the Hippo pathway, Nouri et al identified ALK as an inducer of the new ALK-LATS-YAP-PD-L1 axis in NSCLC cells expressing EML4-ALK.…”

Nanoluciferase complementation-based biosensor reveals the importance of N- linked glycosylation of SARS-CoV-2 Spike for viral entry

“…First we co-transfected SmBiT-ACE2 or LgBiT-RBD with LgBiT-YAP15 or SmBiT-14-3-3, respectively. While the YAP15 and 14-3-3 constructs have been previously demonstrated to interact and rescue the NanoBiT structure 11 , these proteins are not known to interact with ACE2 and RBD.…”

“…For the other component we used the soluble ectodomain of ACE2 (residues 1 to 740) as this has been shown to be su cient to interact with RBD 21 . Since RBD is the smaller protein of the two partners of interest 11 , we linked the RBD with the larger fragment of the split luciferase (LgBiT) while ACE2 was fused to the small fragment (SmBiT, Figure 1B). A glycine-serine linker was inserted between ACE2 or RBD and its respective NanoBiT component 11 to enhance folding and exibility of the fusion proteins.…”

“…Since RBD is the smaller protein of the two partners of interest 11 , we linked the RBD with the larger fragment of the split luciferase (LgBiT) while ACE2 was fused to the small fragment (SmBiT, Figure 1B). A glycine-serine linker was inserted between ACE2 or RBD and its respective NanoBiT component 11 to enhance folding and exibility of the fusion proteins. To facilitate the production of the interacting partners as secreted molecules, we carried out a series of codon optimization studies and tested different secretion signals.…”

Hippo Signaling Pathway as a Central Mediator of Receptors Tyrosine Kinases (RTKs) in Tumorigenesis

“…The aforementioned examples are not the only instances of interconnecting signaling networks between the Hippo pathway and RTKs, as numerous active studies are investigating this relationship. For instance, a recent study uses a novel biosensor [131] to investigate possible regulation of the Hippo by Anaplastic lymphoma kinase (ALK) receptor [132]. ALK is an RTK with notable mRNA expression in the human brain and some other organs [133].…”