Deregulated inflammatory signaling via STAT family transcription factors, particularly STAT1, underlies a variety of immune-related diseases, including inflammatory bowel disease. Whereas activation of STATs by JAKs via canonical receptor-driven JAK-STAT signaling is well understood, little is known about JAK-independent mechanisms of STAT activation. Here, we identify the understudied nonreceptor tyrosine kinase TNK1 as a therapeutically targetable, JAK-independent activator of STAT signaling. Using a multiomics approach, we mapped a network of TNK1 substrates associated with protein condensates and proinflammatory signaling, including STAT1. We found that TNK1, but not its sister kinase ACK1, directly phosphorylates STATs at well described STAT-activating JAK sites. In cells, TNK1-mediated STAT1 phosphorylation and activation occurs independently of JAKs. Imaging and interactomics data suggest that TNK1 interacts with STAT1 in cytosolic condensates, which likely compartmentalize TNK1-substrate interactions. We show that an intrinsically disordered proline-rich region in TNK1, which includes a 14-3-3 docking phosphorylation site, is required for the formation of kinase-active TNK1 condensates and STAT1 phosphorylation. Mutations within the proline-rich region that eliminate 14-3-3 binding increase formation of TNK1 condensates, suggesting a model in which 14-3-3 acts as a clamp that constrains the flexible PRR to inhibit condensate formation and STAT1 activation. Finally, we show that TNK1 is a targetable driver of STAT1-mediated inflammation in the gut as inhibition of TNK1 reduces active STAT1 in the colon and ameliorates colitis symptoms in mice.

Autophagy is an essential cellular recycling process that maintains protein and organelle homeostasis. ATG9A vesicle recruitment is a critical early step in autophagy to initiate autophagosome biogenesis. The mechanisms of ATG9A vesicle recruitment are best understood in the context of starvation-induced nonselective autophagy, whereas less is known about the signals driving ATG9A vesicle recruitment to autophagy initiation sites in the absence of nutrient stress. Here we demonstrate that loss of ATG9A, or the lipid transfer protein ATG2, leads to the accumulation of phosphorylated p62 aggregates in nutrient replete conditions. Furthermore, we show that p62 degradation requires the lipid scramblase activity of ATG9A. Last, we present evidence that polyubiquitin is an essential signal that recruits ATG9A and mediates autophagy foci assembly in nutrient replete cells. Together, our data support a ubiquitin-driven model of ATG9A recruitment and autophagosome formation during basal autophagy.

DriverMutPTM application predicts potential PTM disrupting driver mutations.

Mass spectrometry-based proteome profiling of trace analytes including single cells benefits from liquid chromatography separations operated at low flow rates (e.g., <50 nl/min). However, high-pressure binary pumps needed to achieve such flow rates are not commercially available, and instead require splitting of the gradient flow to achieve low-nanoliter-per-minute flow rates. Gradient flow splitting can waste solvent and lead to flow inconsistencies. To address this, we have developed a method for creating gradients by combining segments of mobile phase having increasing solvent strength together in an open capillary, and then relying on Taylor dispersion to form the desired smooth gradient profile. Our method dramatically reduces costs, as only a single isocratic high-pressure pump is required. Following development of gradient profiles for both 10- and 20-min active gradients, we measured 200 pg injections of HeLa digest using a timsTOF mass spectrometer. Finally, we investigated differences in protein expression between single cells originating from two different colonies of ATG-KO HeLa cells. Thousands of proteins were quantified, and a potential mechanism explaining differential immune responses of these two colonies upon exposure to viral DNA treatment was determined.

ACK1 P633* and UBA truncation is stabilizes ACK1.

Supplementary data set 4. PTMs disrupting potential missense driver mutations.

Supplementary dataset 2. Driver mutations identified using ML method that are not included in the training data set.

Abstract Beyond the most common oncogenes activated by mutation (mut-drivers), there likely exists a variety of low-frequency mut-drivers, each of which is a possible frontier for targeted therapy. To identify new and understudied mut-drivers, we developed a machine learning (ML) model that integrates curated clinical cancer data and posttranslational modification (PTM) proteomics databases. We applied the approach to 62,746 patient cancers spanning 84 cancer types and predicted 3,964 oncogenic mutations across 1,148 genes, many of which disrupt PTMs of known and unknown function. The list of putative mut-drivers includes established drivers and others with poorly understood roles in cancer. This ML model is available as a web application. As a case study, we focused the approach on nonreceptor tyrosine kinases (NRTK) and found a recurrent mutation in activated CDC42 kinase-1 (ACK1) that disrupts the Mig6 homology region (MHR) and ubiquitin-association (UBA) domains on the ACK1 C-terminus. By studying these domains in cultured cells, we found that disruption of the MHR domain helps activate the kinase while disruption of the UBA increases kinase stability by blocking its lysosomal degradation. This ACK1 mutation is analogous to lymphoma-associated mutations in its sister kinase, TNK1, which also disrupt a C-terminal inhibitory motif and UBA domain. This study establishes a mut-driver discovery tool for the research community and identifies a mechanism of ACK1 hyperactivation shared among ACK family kinases. Implications: This research identifies a potentially targetable activating mutation in ACK1 and other possible oncogenic mutations, including PTM-disrupting mutations, for further study.

Machine learning model predicts cancer driver mutations.

Supplementary dataset 1. Potential driver mutations identified against cancer types.

Comparison of ACK1 WT and mutants by phospho-substrate protein array.

Supplementary data set 3. Potential dominant and co-drivers identified my the ML approach.

ACK1 frameshift mutatants have similar RNA expression levels as ACK1 WT.

AbstractWe combined efficient sample preparation and ultra‐low‐flow liquid chromatography with a newly developed data acquisition and analysis scheme termed wide window acquisition (WWA) to quantify >3,000 proteins from single cells in rapid label‐free analyses. WWA employs large isolation windows to intentionally co‐isolate and co‐fragment adjacent precursors along with the selected precursor. Optimized WWA increased the number of MS2‐identified proteins by ≈40 % relative to standard data‐dependent acquisition. For a 40‐min LC gradient operated at ≈15 nL/min, we identified an average of 3,524 proteins per single‐cell‐sized aliquot of protein digest. Reducing the active gradient to 20 min resulted in a modest 10 % decrease in proteome coverage. Using this platform, we compared protein expression between single HeLa cells having an essential autophagy gene, atg9a, knocked out, with their isogenic WT parental line. Similar proteome coverage was observed, and 268 proteins were significantly up‐ or downregulated. Protein upregulation primarily related to innate immunity, vesicle trafficking and protein degradation.

The 1,287 genes targeted by the shRNA of the leukemia library.

A20 is an anti-inflammatory protein with a dual ubiquitin-editing activity. Through its N-terminal ovarian tumor (OTU) domain, A20 negatively regulates NF-ĸB by removing the K63-linked ubiquitin chains on RIP1 that act as platforms for NF-ĸB activation downstream of tumor necrosis factor alpha (TNFα). On the other hand, A20 also catalyzes the addition of K48-linked ubiquitin chains on RIP1 via its C-terminal zinc finger domain 4 (ZnF4) to promote its proteasomal degradation. Through both mechanisms, A20 is a means of negative feedback on TNFα-induced NF-ĸB activity. Importantly, inactivation of A20 is frequently found in B-cell lymphomas, where loss-of-function A20 truncations promote cell proliferation and upregulation of NF-ĸB signaling. Among the post-translational modifications (PTMs) reported in A20, there are several phosphorylation events in its C-terminus, including within the ZnF4 domain. Besides its known E3 ubiquitin ligase activity, previous reports show that the C-terminal region of A20 is important for ubiquitin binding, having preference for K63-linked poly-ubiquitin. However, a little is known about the effects of phosphorylation at this region on A20 activity as well as its consequences in downstream signaling. Our current work suggests that phosphorylation in the C-terminus of A20 inhibits its E3 ubiquitin ligase activity and disrupts its interaction with ubiquitin, which may also affect its deubiquitinase activity by preventing its recruitment to ubiquitinated proteins in the TNFα receptor (TNFR) complex. We also propose that the C-terminal region is important for the formation of the A20 ubiquitin-editing complex, necessary to downregulate NF-ĸB-activated inflammatory signals. Based on these observations, we hypothesize that not only gene-inactivating mutations, but also post-translational mechanisms, such as phosphorylation, inhibit A20 to promote cancer development. Our research is now focused on understanding the upstream signaling that regulates A20 phosphorylation. Citation Format: Tania Lopez Palacios, Tsz-Yin Chan, Christina Egbert, Jacob Truman, Spencer Ashworth, Alec Vaughan, Joshua L. Andersen. The regulation of inflammatory signaling in cancer via A20 phosphorylation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5028.

Highly biosynthetic cancer cells produce proteins at a rate that may exceed the capacity of protein folding machinery, resulting in an accumulation of misfolded proteins that are poly-ubiquitinated and clustered into phase-separated ‘ubiquitin-rich’ condensates. These condensates are the initiation point for a form of autophagy called aggrephagy, which maintains cancer cell homeostasis by degrading the misfolded protein condensates via the lysosome. Aggrephagy is a cancer cell vulnerability, yet its mechanisms are poorly understood, particularly the mechanisms by which autophagy machinery recognizes the ubiquitin-rich condensates to initiate aggrephagy. Among 15 core autophagy proteins, ATG9A is the only multi-pass transmembrane protein and is essential for all known forms of autophagy. Recent evidence indicates that ATG9A is a lipid scramblase, in complex with ATG2A, that channels lipids to a growing autophagosome, which ultimately engulfs the ubiquitin-rich condensate. However, the mechanism of ATG9A recruitment to these condensates is not understood. Our data indicate that genetic or chemical manipulations of cells that cause ubiquitin-rich condensates to accumulate causing a corresponding accumulation of ATG9A. Furthermore, the induction of artificial ubiquitin-rich condensates, composed of chains of M1-linked ubiquitin, is sufficient to recruit ATG9A. Using a CRISPR knockout and reconstitution approach, we show that ATG9A is essential for assembling other autophagy machinery at ubiquitin-rich condensates. Together, our data support a model in which ATG9A recruitment is the initiating step for aggrephagy. Furthermore, our data suggest a signal or molecular pattern within the condensate promotes ATG9A recruitment to initiate aggrephagy. Our current work focuses on identifying features of ATG9A and components of the ubiquitin-rich condensate that cooperate to recruit ATG9A to these sites of aggrephagy initiation. Citation Format: Deshan Madhusanka, Colton McEwan, David Broadbent, Jens Schmidt, Joshua Andersen. Mechanisms of ATG9 mediated aggrephagy initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1383.

Next generation sequencing for 52 myeloid malignancies-related genes.

Thirty-eight-negative kinase 1 (TNK1) is a poorly characterized member of the ACK family of non-receptor tyrosine kinases, which we recently identified as a driver of cell survival in a subset of primary hematological malignancies. However, the biological function of TNK1 is not well understood. We also found that TNK1 is unusual among kinases for the presence of a functional ubiquitin association (UBA) domain on its C-terminus. We discovered that the TNK1 UBA domain binds to poly-ubiquitin with high affinity and has no apparent preference for ubiquitin chain length or linkage type. Interestingly, the UBA domain is important for TNK1 function, given that deletion of the UBA domain (TNK1 ΔUBA) alters its localization and phospho-substrate network, while also weakening the oncogenic activity of TNK1 in cell transformation assays. Based on these data, we hypothesized that the UBA domain tethers TNK1 to its substrates. To test this hypothesis, we performed quantitative phospho-tyrosine proteomics using murine pro-B cells transformed with either TNK1 full length or TNK1 ΔUBA and identified TANK-binding kinase 1 (TBK1) as a putative UBA-dependent TNK1 substrate. TBK1 is a serine/threonine kinase involved in the regulation of inflammation, autophagy, and NF-ĸB signaling. During the selective autophagic degradation of misfolded proteins (aggrephagy), TBK1 phosphorylates p62 at S403 to increase its affinity to ubiquitin, and thereby increases p62-mediated recruitment of misfolded proteins into the condensate. Unchecked growth of ubiquitin condensates can overwhelm the size capacity of autophagosomes, potentially causing misfolded proteins to become proteotoxic aggregates. Our preliminary data suggest a model in which active TNK1 accumulates at ubiquitin condensates to phosphorylate and inhibit TBK1. Based on these data, we propose that TNK1 acts as a kinase sensor of poly-ubiquitin to control the TBK1-mediated growth of ubiquitin condensates. Citation Format: Emmalee Kohler, Tania Lopez-Palacios, Tsz-Yin Chan, Christina Egbert, Jacob Truman, Spencer Ashworth, Alec Vaughan, Joshua Andersen, D. Madhusanka. Determining the biological function of TNK1. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5029.

Patient sample information.