ACK1 is an oncogene in the ACK family of non-receptor tyrosine kinases (NRTK). In humans, ACK1 is located on chromosome 3q29, a region that is frequently amplified in a variety of cancers including prostate, lung and breast. The ACK kinase family has a unique domain arrangement with, most notably, a putative ubiquitin association (UBA) domain at their C-termini. In the present study, we focus on understanding the ubiquitin-binding nature of the ACK1 UBA and its role in kinase regulation. Structural modeling and sequence analysis suggest that the ACK1 UBA diverges from many other UBAs but shares similarities with the ACK family kinase, TNK1. We found that the ACK family UBA has a remarkably high affinity for diverse poly-ubiquitin linkages with dissociation constants in the low nanomolar range. Our preliminary data suggest that a non-covalent interaction between the UBA and ubiquitin is necessary for ACK kinase activation in cell culture systems. Furthermore, our preliminary data suggest this interaction is sufficient to induce ACK1 activation in vitro. In support of the idea that the UBA is important for ACK1 activation, we identified a variety of cancer patient mutations that disrupt the ACK1 UBA, which we are currently testing for oncogenic activity. Lastly, we have identified small molecule inhibitors of ACK with low nanomolar IC50 values in ACK1-driven cells. Altogether, our data suggest a model of ACK1 activation that involves direct, non-covalent interaction with ubiquitin and potential therapeutic approaches to inhibit ACK1 in cancer. Citation Format: Eranga Roshan Balasooriya Loku Balasooriyage, Jacob Owen, Jack Gashler, Colin Muir, Katie Pennington, James Moody, Joshua L. Andersen. Regulation of the oncogenic tyrosine kinase ACK1 through ubiquitin-dependent mechanism [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 2304.