TNK1 is a non-receptor tyrosine kinase with poorly understood biological function and regulation. Here, we identify TNK1 dependencies in primary human cancers. We also discover a MARK-mediated phosphorylation on TNK1 at S502 that promotes an interaction between TNK1 and 14-3-3, which sequesters TNK1 and inhibits its kinase activity. Conversely, the release of TNK1 from 14-3-3 allows TNK1 to cluster in ubiquitin-rich puncta and become active. Active TNK1 induces growth factor-independent proliferation of lymphoid cells in cell culture and mouse models. One unusual feature of TNK1 is a ubiquitin-association domain (UBA) on its C-terminus. Here, we characterize the TNK1 UBA, which has high affinity for poly-ubiquitin. Point mutations that disrupt ubiquitin binding inhibit TNK1 activity. These data suggest a mechanism in which TNK1 toggles between 14-3-3-bound (inactive) and ubiquitin-bound (active) states. Finally, we identify a TNK1 inhibitor, TP-5801, which shows nanomolar potency against TNK1-transformed cells and suppresses tumor growth in vivo.

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Thirty‐eight‐negative kinase 1 (TNK1) is a poorly characterized non‐receptor tyrosine kinase (NRTK) first isolated from umbilical cord blood. TNK1 differs from most NRTKs in that it possesses a functional ubiquitin association (UBA) domain at its C‐terminus. We previously observed that the TNK1 UBA domain interacts with a variety of poly‐ubiquitin chains, is essential for full TNK1 activation, and facilitates the clustering of active TNK1 into ubiquitin‐rich condensates.1 Paradoxically, a genetic inversion that truncates the TNK1 C‐terminus (including the UBA domain) in human lymphoma hyperactivates the kinase and coverts it into an oncogenic driver. Our current research focuses on how deletion of the UBA, which is important for native TNK1 function, helps activate the kinase. Here we show that immediately adjacent to the UBA domain on TNK1 is an inhibitory 14‐3‐3 binding site, which is also truncated in lymphoma. Furthermore, point mutations that disrupt the 14‐3‐3 binding site are sufficient to convert TNK1 into a hyperactive oncogenic kinase. In addition, we show that although truncation of the UBA perturbs the normal function of TNK1, it also stabilizes TNK1 protein levels. Thus, the lymphoma‐associated truncations in TNK1 activate and stabilize the kinase, resulting in a highly expressed and active TNK1. Finally, we show that truncation of the 14‐3‐3 binding site and UBA domain of TNK1 is sufficient to transform pro‐B cells to growth factor‐independence and grow tumors in vivo. Thus, our data explain how genetic inversions convert TNK1 into an oncogenic driver in human cancers. 1. Chan, TY., Egbert, C.M., Maxson, J.E. et al. TNK1 is a ubiquitin‐binding and 14‐3‐3‐regulated kinase that can be targeted to block tumor growth. Nat Commun 12, 5337 (2021). https://doi.org/10.1038/s41467-021-25622-3

Thirty‐eight‐negative kinase 1 (TNK1) is a poorly understood member of the ACK family of non‐receptor tyrosine kinases. TNK1 has been linked to oncogenic activity. However, the biological function of TNK1 remains unclear. Recently, we discovered the presence of a functional ubiquitin association (UBA) domain on the C‐terminus of TNK1, which is an unusual feature on a kinase. Previously, we demonstrated that the TNK1 UBA domain binds to poly‐ubiquitin with high affinity and with no preference for length or linkage type (1). Interestingly, deletion of the UBA domain (TNK1 ΔUBA) affects TNK1 intracellular localization and decreases its phospho‐substrates network. Based on these data, we hypothesized that the UBA domain homes TNK1 to substrates. To test this hypothesis, we used quantitative phospho‐tyrosine proteomics in pro‐B cells transformed with either TNK1 or TNK1 ΔUBA to identify UBA‐dependent TNK1 substrates. Among the putative UBA‐dependent substrates, we identified phospho‐Y235 of TANK‐binding kinase 1 (TBK1). TBK1 is an important serine/threonine protein kinase involved in the regulation of inflammation, autophagy, and NF‐kB signaling. Through confocal microscopy, we demonstrated a colocalization between TNK1 and TBK1 at p62‐positive phase‐separated clusters of ubiquitin—referred to here as ubiquitin condensates. We found that TNK1 localizes to ubiquitin condensates in a UBA‐dependent manner. Furthermore, our data suggest that TBK1 is inhibited by phosphorylation at Y235, as phospho‐null mutation at Y235 (Y235F) significantly increases phosphorylation of the TBK1 substrates p62 and OPTN. We also found that CRISPR/Cas9 deletion of TNK1 increased TBK1 activity and phosphorylation of p62 and OPTN. These data suggest that TNK1 negatively regulates TBK1 activity. Intriguingly, we also identified an enrichment of other putative UBA‐dependent TNK1 substrates in the NF‐kB signaling pathway, suggesting that the UBA domain tethers TNK1 to multiple substrates in this pathway, perhaps as a means to dampen NF‐kB signaling. Our current work focuses on the biological impact of these phosphorylations on NF‐kB activity, inflammatory signaling, and autophagy. 1. Chan, TY., Egbert, C.M. et al. TNK1 is a ubiquitin‐binding and 14‐3‐3‐regulated kinase that can be targeted to block tumor growth. Nat Commun 12, 5337 (2021).

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.