We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34 + cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype agnostic and extends to RAS-and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML.SIgnIfIcAnce: Reducing SIRT5 activity is detrimental to the survival of AML cells regardless of genotype, yet well tolerated by healthy hematopoietic cells. In mouse models, disrupting SIRT5 inhibits AML progression. SIRT5 controls several metabolic pathways that are required for leukemia cell survival. These results identify SIRT5 as a therapeutic target in AML.

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.

PTOV1 is an oncogenic protein, initially identified in prostate cancer, that promotes proliferation, cell motility, and invasiveness. However, the mechanisms that regulate PTOV1 remain unclear. Here, we identify 14-3-3 as a PTOV1 interactor and show that high levels of 14-3-3 expression, like PTOV1, correlate with prostate cancer progression. We discover an SGK2-mediated phosphorylation of PTOV1 at S36, which is required for 14-3-3 binding. Disruption of the PTOV1–14–3-3 interaction results in an accumulation of PTOV1 in the nucleus and a proteasome-dependent reduction in PTOV1 protein levels. We find that loss of 14-3-3 binding leads to an increase in PTOV1 binding to the E3 ubiquitin ligase HUWE1, which promotes proteasomal degradation of PTOV1. Conversely, our data suggest that 14-3-3 stabilizes PTOV1 protein by sequestering PTOV1 in the cytosol and inhibiting its interaction with HUWE1. Finally, our data suggest that stabilization of the 14-3-3–bound form of PTOV1 promotes PTOV1-mediated expression of cJun, which drives cell-cycle progression in cancer. Together, these data provide a mechanism to understand the regulation of the oncoprotein PTOV1. Implications: These findings identify a potentially targetable mechanism that regulates the oncoprotein PTOV1.

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

<p>Next generation sequencing for 52 myeloid malignancies-related genes.</p>