Memory T cell inflation is a distinctive immunological phenomenon observed during persistent viral infections, such as Cytomegalovirus (CMV). Unlike conventional memory T cell responses, which contract after infection resolution, a subset of CMV-specific T cells undergoes a progressive and sustained expansion, termed "inflation", which is thought to be critical for long-term immune surveillance. The molecular mechanisms that govern memory T cell inflation remain incompletely understood, yet they are pivotal for understanding immune persistence and designing strategies against chronic viral infections. In this study, we investigate the role of MAP kinase-activated protein kinase 2 (MK2), a key downstream effector of p38 MAPK signaling, in regulating T cell responses during murine CMV (MCMV) infection. Using MK2 knockout (MK2-KO) mice, we demonstrate that MK2 deficiency alters the dynamics of MCMV-specific CD8+ T cell responses without impairing viral control or tissue replication. MK2 deficiency led to a reduction in non-inflationary MCMV-specific CD8+ T cells during the acute phase, followed by enhanced expansion of inflationary CD8+ T cell subsets during persistence. Furthermore, MK2-KO mice exhibited impaired effector differentiation, as evidenced by decreased expression of the terminal differentiation marker KLRG1 on MCMV-specific CD8+ T cells. Collectively, these findings identify MK2 as a pivotal regulator of CD8+ T cell magnitude, kinetics, and phenotype during both acute and chronic MCMV infection. By elucidating the role of MK2 in the regulation of memory T cell inflation, this study provides new mechanistic insight into immune regulation with implications for vaccination, chronic infection, and immune aging.

Oncolytic virotherapy represents a promising yet under-explored approach for precision cancer treatment, particularly when tailored to tumor-specific molecular profiles. Patients with high-grade isocitrate dehydrogenase (IDH) mutant astrocytomas have limited treatment options and poor prognoses. Here, we investigate the therapeutic efficacy of rQNestin34.5 v.2 (CAN-3110), an engineered oncolytic herpes simplex virus 1 (oHSV-1), in IDH1-R132H-mutant diffuse gliomas. We demonstrate that the IDH1-R132H mutation enhances glioma susceptibility to viral infection through upregulation of Nectin-1, the main HSV-1 entry receptor. Concurrently, IDH1-R132H-driven DNA hypermethylation suppresses interferon (IFN) signaling, creating a permissive microenvironment that facilitates viral replication and tumor cell apoptosis. In immunocompetent murine glioma models, intratumoral administration of rQNestin34.5 v.2 induces robust antitumor immune activation, including increased immune infiltration and systemic IFN-γ release. However, elevated expression of poliovirus receptor (PVR) and the immune checkpoint T-cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) on tumor-infiltrating leukocytes suggests a potential resistance mechanism to virotherapy. Combining rQNestin34.5 v.2 with TIGIT blockade enhances therapeutic efficacy compared to monotherapy, identifying IDH1-R132H as a potential predictive biomarker for oncolytic virotherapy response.

Abstract Mass-spectrometry-based phosphoproteomics now profiles phosphorylation at proteome scale, yet converting site-level measurements into coherent, kinase-centered biology remains a persistent barrier to interpretation and action. The Kinase Library addresses this gap with the first-in-class, unbiased, experimentally characterized motif atlas of the human kinome, coupled to enrichment frameworks that translate phosphoproteomics data into quantitative maps of kinase activity. Rather than relying on heterogeneous annotations or heuristic rules, KL grounds inference in experimentally derived kinase-substrate relationships, providing a principled basis for comparative signaling analysis. The Kinase Library has broad utility across discovery and translational applications. It enables mechanism-of-action profiling for small molecules and combinations; delineates adaptive signaling and resistance trajectories; supports time-course and dose-response studies to resolve pathway dynamics; and stratifies models and patients in low-N-high-D (few samples with high dimensionality of data) settings where conventional statistics underperform. In clinical and preclinical contexts alike — cell lines, organoids, xenografts, and patient specimens — the Kinase Library delivers harmonized, interpretable kinase signatures that are readily integrated with genomic, transcriptomic, and phenotypic readouts to generate and prioritize actionable hypotheses. The novelty of the Kinase Library is twofold. First, scope and provenance: an experimental, unbaised atlas spanning the entire kinome, with comprehensive inclusion of the dark kinome. Second, operationalization: a unified enrichment paradigm that yields robust, rank-ordered kinase programs suitable for decisionmaking — whether the objective is target nomination, combination design, biomarker discovery, or comparative benchmarking across cohorts and studies. Looking forward, the Kinase Library is positioned to empower emerging frontiers in proteomics: single-cell and spatial phosphoproteomics; longitudinal “N-of-1” monitoring to guide therapy; cross-species translation for model selection; and cloudnative workflows that interoperate with community pipelines and public datasets. By elevating kinases from disparate lists of regulated sites to coherent, testable signaling hypotheses, the Kinase Library reframes what phosphoproteomics can deliver — shifting the field from descriptive measurement toward predictive, mechanism-guided intervention. Citation Format: Tomer M. Yaron-Barir, Jared L. Johnson, Benjamin E. Turk, Michael B. Yaffe, Lewis C. Cantley. The Kinase Library: A global atlas of the human protein kinome and its applications in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1331.

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are an uncommon and poorly understood malignancy with low mutational burden, lacking well-defined oncogenic drivers. GEP-NET mortality frequently results from extensive hepatic metastases. Accordingly, we interrogated phosphoproteomic data from GEP-NET liver metastases and patient-matched uninvolved liver to identify tumor-specific signaling and targetable tumor vulnerabilities using Kinase Motif Enrichment Analysis (KMEA), a new tool leveraging the recent Kinase Library compendium of the substrate motif specificity for nearly the entire human kinome. KMEA identified patient tumor-specific upregulation of mTOR or casein kinase 2 (CK2) activity that would be undiscoverable by standard personalized genomic and transcriptomic approaches. Striking concordance was observed between KMEA predictions for specific tumors, and their sensitivity to inhibitors of mTOR or CK2 using patient tumor-derived organoids. These findings reveal potential clinically-actionable protein kinases hyperactivated in GEP-NETs, and more broadly indicate a general method for personalized cancer treatment using phosphoproteomics and KMEA-derived kinase activity signatures.

Normal mitotic cell division

Extent of IMPACT CpG island promoter methylation in mouse cell lines (KPC, 4964-POP, and 4964-HOP), and five human pancreatic liver metastases compared to three primary PDACs, and two normal pancreas and liver tissue.

Protein tyrosine kinases activate signaling pathways by catalyzing the phosphorylation of tyrosine residues in their substrates. Mounting evidence suggests that, in addition to recognizing phosphorylated tyrosine (pTyr) residues through specific phosphobinding modules, many protein kinases selectively recognize pTyr directly adjacent to the tyrosine residue they phosphorylate and catalyze the formation of twin pTyr-pTyr sites. Here, we demonstrate the importance of this phosphopriming-driven twin pTyr signaling in promoting cell cycle progression through the cell cycle-inhibitory protein p27Kip1. We identify, structurally resolve, and tune two distinct molecular determinants driving the selective recognition of pTyr directly N- and C-terminal to the target phospho-acceptor tyrosine site. We further show structural and biochemical conservation in this recognition, and identify cancer-associated alterations to these determinants that are unable to recognize phosphoprimed substrates. Finally, using an in vivo mouse model of leukemia we show that Bcr-Abl mutants unable to recognize phosphoprimed substrates paradoxically result in enhanced tumor development and progression. These data indicate that Bcr-Abl, like other proto-oncogenes such as Ras or Myc, engages both pro- and anti-oncogenic programs - but in the case of Bcr-Abl, this is accomplished through a mechanism involving traditional and phosphoprimed substrate recognition.

Chromatin structure in abiraterone treated C4-2 cells

GCN1 cDNA sequence in 4964-POP and 4964-HOP cells.

Seven Supplementary Figures and Legends

Background Hyperfibrinolysis after trauma increases mortality, yet rapid identification remains a challenge. Prior work demonstrates liver transplantation is an idealized model of fibrinolysis. Early resource mobilization and antifibrinolytic therapy improves outcomes in trauma, but efficacy is lost with delays in care. We set out to develop a novel plasmin-activated clotting time (PACT) assay to rapidly diagnose hyperfibrinolysis. Methods Whole blood from adult liver transplant(n ​= ​40) and trauma patients(n ​= ​32) underwent PACT assay alongside traditional and tPA-challenged thromboelastography(TEG). Primary outcomes of massive transfusion and early mortality were assessed. Results PACT accurately identified hyperfibrinolysis within 3 ​min during the anhepatic phase of liver transplant (35 ​% prevalence; p ​< ​0.001). In trauma, a PACT threshold (ΔACT >18.5 ​s) predicted massive transfusion or death (AUC ​= ​0.857, p ​< ​0.001), outperforming conventional TEG metrics (AUC<0.625). Conclusion The PACT assay predicts hyperfibrinolysis, massive transfusion, and mortality in trauma patients in 3 ​min, offering potential for rapid resource triage and early targeted therapeutic administration.

Figure S3. IMPACT limits ATF4 activation through competitive inhibition of GCN1

Example of mitotic arrest and cell death caused by abiraterone and onvansertib

PURPOSE:We observed that the tumor microenvironment (TME) in metastatic epithelial ovarian cancer (EOC) and in other solid tumors can reprogram normal neutrophils to acquire a complement-dependent suppressor phenotype characterized by inhibition of stimulated T cell activation. This study aims to evaluate whether serum markers of neutrophil activation and complement at diagnosis of EOC would be associated with clinical outcomes. EXPERIMENTAL DESIGN:We conducted a two-center prospective study of patients with newly diagnosed EOC (N = 188). Blood and ascites fluid were collected at diagnosis for biomarker analysis. Patients were evaluated for progression-free survival (PFS) and overall survival (OS). RESULTS:The median OS was 47 months (95 % CI: 34-58) and the median PFS was 12 months (95 % CI: 11-15). Pre-treatment serum levels of genomic DNA (gDNA), markers of neutrophil degranulation (myeloperoxidase [MPO]) and neutrophil extracellular traps (NETs) (citrullinated histone H3 [CitH3]), and complement activation (C3b/c) were each associated with worse OS in univariate analysis. In multivariate analyses controlling for age, stage, and optimal debulking, serum gDNA, MPO, and CitH3 remained associated with worse OS, while C3b/c levels were not. In an exploratory analysis, the largest magnitude of difference in 2-year OS occurred in patients with low C3b/c and low CitH3 compared to all other patients (87 % vs 46 % survival, respectively). In ascites fluid, increased factor H, a negative regulator of complement activation, was associated with improved OS in univariate analysis. CONCLUSIONS:These results point to serum gDNA, NETs, and complement activation as potential prognostic biomarkers in patients with newly diagnosed EOC.

Video montage comparing mitosis of vehicle control with abiraterone treated C4-2 cells

Identification of paired centromeres from sister chromatids

Cholangiocarcinoma (CCA) is a highly aggressive malignancy that arises from the biliary tree, and often presents with advanced stage disease. For patients with disease arising from intrahepatic bile ducts, genomic analyses have revealed targetable mutations. Unfortunately, these mutations are exceedingly rare in extrahepatic disease (eCCA), limiting therapeutic options. It is therefore necessary to investigate the functional crosstalk resulting from different signal transduction pathways to identify new treatment options for this patient population. IHC analysis of eCCA tumors from 170 patients demonstrated intense Exportin-7 (XPO7) cytoplasmic staining in 30% of the samples, which correlated with abbreviated survival. With the co-immunoprecipitation, proteomic analysis and molecular docking, we demonstrated that XPO7 exists in a molecular complex with the Ste-20 like kinase (SLK) and beta-tubulin in the cytoplasm. A kinome screen identified tivozanib, a potent VEGFR2 inhibitor, as a moderate inhibitor of SLK kinase (IC50 - 36 nM @ 10 µM ATP or 2.2 µM @ 1 mM ATP) and cellular target engagement (NanoBRET) of 884 nM. The X-ray crystallography results confirmed that tivozanib binds to the SLK ATP-binding pocket, with the DGF motif in the inactive “DGF-out” conformation in the N-terminal region. In in vitro experiments, shRNA-mediated knockdown of SLK and tivozanib treatment in cholangiocarcinoma cell lines showed reduced spheroid forming capacity with downregulation of PI3K-AKT-mTOR pathway. This resulted in reduction in expression of phosphorylated mTOR (S2448), AKT (T308), and S6 (S235/236). SLK KD and tivozanib treatment also resulted in upregulation of ATM-dependent DNA damage pathway in cholangiocarcinoma cell lines with resulting downregulation of beta-tubulin and G2/M cell cycle arrest confirming its potential role in impacting the cellular processes. Moreover, tivozanib regressed tumors in established eCCA patient derived xenografts with cytoplasmic XPO7 expression. We further confirmed our results in our ex vivo system by treating eCCA patient tumor slice cultures with tivozanib. The IHC and Western blotting results confirmed the significant reduction of PI3K-AKT-mTOR pathway in high XPO7/SLK-expressing tumors (N=3) compared to the lower expression pattern (N=7). Lastly, tivozanib monotherapy demonstrated in vivo efficacy in the patients with high XPO7 expression with tumor regression and arrest of tumor progression (RECIST) in an ongoing clinical trial (NCT04645160). Priyanka Prakash Desai, Surajit Sinha, Emily Smith, Reed I. Ayabe, Pedro Torres-Ayuso, Ashley Rainey, Himanshu Verma, Kirsten Remmert, Tracey Pu, Craig Thomas, John Brognard, William J. Moore, Gregory Tawa, Thorkell Andresson, David Kleiner, Michael B. Yaffe, Jonathan M. Hernandez. Cytoplasmic complex of SLK-XPO7: A targetable player in extrahepatic cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6915.