Elucidation Of A Unique Regulatory Mechanism For Tnk1 Provides Potential Therapeutic Targeting Opportunities In Cancer.

Abstract Sumitomo Dainippon Pharma Oncology (f/k/a Tolero Pharmaceuticals), Lehi, UTTNK1 is a poorly understood member of the ACK family of non-receptor tyrosine kinases with a unusual domain arrangement, but no known mechanism of regulation nor conclusive link to disease. We initially identified TNK1 as a mediator of cell survival in a subset of primary patient cancer samples. In an effort to understand how TNK1 is regulated, we discovered a MARK-mediated phosphorylation at S502 near the TNK1 C-terminus that mediates an interaction with 14-3-3. We found that 14-3-3 binding inhibitsTNK1 kinase activity, whereas disruption of this interaction renders TNK1 highly active and capable of driving tumor growth in vivo. In support of this idea, kinase substrate profiling at the proteome level revealed a variety of pro-growth and motility substrates for active TNK1. One unique feature of TNK1 is a ubiquitin-association domain (UBA) on its C-terminus, which sits adjacent to the 14-3-3 binding site. We found that the TNK1 UBA has a high affinity for a variety of poly-ubiquitin linkages—making TNK1the first kinase, to our knowledge, that directly interacts (non-covalently) with ubiquitin. Remarkably, point mutations within the UBA that disrupt ubiquitin binding inhibit TNK1 activation and oncogenic signaling, revealing a unique UBA-centric mechanism of tyrosine kinase regulation, in which TNK1toggles between 14-3-3-bound (inactive) and ubiquitin-bound (active) states. Finally, we used a structure-guided approach to identify novel small molecule TNK1 inhibitors with high potency and selectivity. One such compound, TP-5809, inhibits TNK1 dependent STAT5 phosphorylation in vitro and in vivo in L540, a Hodgkin lymphoma cell line with a TNK1-activating mutation. TP-5809 also potently inhibits the in vitro growth of mutant TNK1-driven pro-B cells and reduces disease burden and prolongs survival in mice with xenografted tumors. Together, our data elucidate the first mechanism of TNK1regulation and identify lead compounds for the development of a TNK1 inhibitor. Citation Format: Tsz-Yin Chan, Christina Egbert, Logan Larsen, Jeremy Tsang, Julia Maxson, Eranga Roshan, Clifford J. Whatcott, Kim Wontak, Gaelle Mercenne, Savannah Free, Adam Siddiqui, Tetyana Forostyan, Ethika Tyagi, Kenneth A. Christensen, David J. Bearss, James Moody, Jeffrey Tyner, Jason M. Foulks, Steven L. Warner, Joshua Lyon Andersen. Elucidation of a unique regulatory mechanism for TNK1 provides potential therapeutic targeting opportunities in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2307.