While a relatively limited number of known oncogenes underlie a large percentage of cancers, a variety of new genes have emerged as low-frequency cancer drivers. Each of these new oncogenes represents a frontier for targeted therapy. However, the discovery of low-frequency targetable oncogenic drivers is challenging. This study focuses on the poorly understood Tyrosine kinase non-receptor-1 (TNK1), which has been reported to have both oncogenic and tumor suppressive functions. TNK1 has been identified to promote cancer cells survival and promote chemoresistance in multiple independent studies. On the other hand, whole-body constitutive deletion of TNK1 in mice caused an increase in spontaneous carcinomas and lymphomas. All in all, with no known regulatory mechanism and substrates of TNK1, the precise biological role of TNK1 is still unclear. To understand how TNK1 is regulated, we employed a proteomic approach to identify TNK1 interactors. We found out that TNK1 interacts with the phospho-binding protein 14-3-3 and this interaction is mediated by a cluster of MARK-mediated phosphorylations within the proline-rich domain. 14-3-3 binding retains TNK1 in the cytosol and maintains TNK1 in an inactive state. Release of TNK1 from 14-3-3 binding drives TNK1 to a heavy membrane fraction, where it becomes highly active. One unique feature of TNK1 is an ubiquitin association domain (UBA) on its C-terminus. Our data suggest that the UBA domain of TNK1 binds to poly-ubiquitin chains in nondiscriminatory manner. Remarkably, point mutations within the UBA that disrupt ubiquitin binding abolish TNK1 activation and oncogenic signaling, suggesting, to our knowledge, a unique UBA-centric mechanism of tyrosine kinase regulation. Finally, we used a structure-guided approach to identify a small molecule inhibiting TNK1 with high potency and selectivity. Such compound, TP-5801, inhibits TNK1 dependent STAT3 phosphorylation. TP-5801 also prolongs the survival of mice injected via tail vein with TNK1-driven Ba/F3 cells and reduces tumor burden in a subcutaneous xenograft model. In conclusion, our data reveal a mechanism of TNK1 regulation that controls its oncogenic tyrosine kinase activity and a potential strategy for TNK1 inhibition.

Identification of novel cancer driver mutations is crucial for targeted cancer therapy, yet a difficult task especially for low frequency drivers. To identify cancer driver mutations, we developed a machine learning (ML) model to predict cancer hotspots. Here, we applied the ML program to 32 non-receptor tyrosine kinases (NRTKs) and identified 36 potential cancer driver mutations, with high probability mutations in 10 genes, including ABL1, ABL2, JAK1, JAK3, and ACK1. ACK1 is a member of the poorly understood ACK family of NRTKs that also includes TNK1. Although ACK1 is an established oncogene and high-interest therapeutic target, the exact mechanism of ACK1 regulation is largely unknown and there is still no ACK1 inhibitor in clinical use. The ACK kinase family has a unique domain arrangement with most notably, a predicted ubiquitin association (UBA) domain at its C-terminus. While the presence of a functional UBA domain on a kinase is unique to the ACK family, but the role of the UBA domain on ACK1 is unknown. Interestingly, the ML program identified the ACK1 Mig6 homology region (MHR) and UBA domains truncating mutation p633fs* as a cancer driver mutation. Our data suggest that the ACK1 UBA domain helps activate full-length ACK1 through induced proximity. It also acts as a mechanism of negative feedback by tethering ACK1 to ubiquitinated cargo that is ultimately degraded. Indeed, our preliminary data suggest that truncation of the ACK1 UBA stabilizes ACK1 protein levels, which results in spontaneous ACK1 oligomerization and activation. Furthermore, our data suggests removal of the MHR domain hyper activates ACK1. Thus, our data provide a model to explain how human mutations in ACK1 convert the kinase into an oncogenic driver. In conclusion, our data reveal a mechanism of ACK1 activation and potential strategies to target the kinase in cancer.