Abstract Metastatic triple-negative breast cancer (mTNBC) has high mortality rates and limited therapeutic options. Chemotherapy, the basis of standard approved therapy for mTNBC, targets rapidly dividing cells but fails to eliminate the stem-like cells that drive metastasis. While kinases are promising drug targets, single-kinase inhibitors have been largely ineffective in mTNBC due to tumor heterogeneity, drug resistance, and the absence of predictive biomarkers. Our strategy is to identify and target kinase networks in pro-metastatic tumor cells that are upregulated in drug-resistant cells or specifically targeted by physiological regulators of metastasis. We initially utilized this approach to overcome AKT resistance in mTNBC. Employing a kinase activity assay (MIB-MS profiling), we demonstrated upregulation of Src family and ATM/ATR kinase networks following treatment of patient-derived, drug-resistant mTNBC organoids with the AKT inhibitor capivasertib. Sequential administration of capivasertib and dasatinib to target the Src family did not reduce organoid viability but did disrupt organoid formation and self-renewal, suggesting effective inhibition of metastatic potential. Building on our previous work, we then optimized a multi-kinase inhibitor regimen that mimics a physiological metastasis suppressor by targeting key stress-responsive kinase networks (ERK1/2, p38, JNK, and MLK). This low-dose drug combination (4MAPKi) prevented both TNBC metastasis and activation of compensatory pathways. We further refined this to a three-drug regimen (Ralimetinib, JNK-in-8, and Trametinib) that inhibits stress kinase networks, pro-motility gene expression and TNBC cell invasion in organoid assays. Importantly, combining this antimetastatic regimen with standard chemotherapy sensitized both TNBC cells and organoids to treatment. Ongoing studies focus on optimizing treatment efficacy with antibody-drug conjugates, characterizing kinase network targets specific for other metastasis regulators such as transcription factors, and exploring kinase networks as biomarkers for treatment resistance. Our long-term goal is to develop a low-toxicity, multi-kinase inhibitor strategy that reprograms metastatic TNBC cells, enhancing the efficacy and durability of their response to chemotherapy and improving clinical outcomes. Citation Format: Margarite Matossian, Madeline Henn Bungert, Kent Schechter, Long Chi Nguyen, Michael East, Denis O. Okumu, Rita Nanda, Gary L. Johnson, Marsha R. Rosner. Pro-metastatic kinase network inhibition to treat drug-resistant triple-negative breast 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 2244.

Supplementary Data Figure 1. PDX show high correlation within the same patient line across different runs compared to different patient lines on the same run. Supplementary Data Figure 2. Mouse kinase expression is not line specific and does not associate with tumor molecular subtype. Supplementary Data Figure 3. Molecular subtyping in PDX model recapitulates basal-like and classical tumor specific subtypes. Supplementary Data Figure 4. Subtype specific kinase expression is associated with gene expression in PDX tumors. Supplementary Data Figure 5. Kinase expression from patients reflects the PDAC PDX kinome. Supplementary Data Figure 6. Overall survival (OS) and treatment response of patients who received EGFR inhibitors.

Supplementary Table 3.ttest_basalclassical

The development and validation of prognostic and predictive biomarkers in breast cancer is limited by the availability of well-annotated datasets linking tumor molecular features to treatment response and survival outcomes. To address this need, we generated an extensive mouse models dataset comprised of 26 immunocompetent mammary tumor models spanning diverse genetic backgrounds, epithelial-mesenchymal states, the basal-luminal axis, and distinct immune microenvironments. For each model, survival was measured under no treatment, immune checkpoint inhibition (ICI), and carboplatin/paclitaxel chemotherapy, and RNA-sequencing was performed on baseline tumors and on 7-day on-treatment samples for both regimens. Baseline murine tumor gene expression features were used to train a machine learning Elastic Net model that predicted survival outcomes on multiple human breast cancer datasets with performance comparable to that of existing prognostic assays. Models trained for ICI benefit, using either the untreated or 7-day ICI treated samples, predicted ICI benefit on human ICI treated datasets, with the 7-day treated tumor model showing better performance. A predictor of carboplatin/paclitaxel response developed from the murine mammary tumor data performed well in mice but did not generalize to human chemotherapy cohorts. Finally, comparison of multiple computational approaches, including XGBoost, random forests, and support vector regression, showed that all methods successfully predicted survival outcomes, with Elastic Net offering the best performance and interpretability. These results indicate conserved cancer biology between mouse and human tumors for prognosis and ICI response and establish a large preclinical dataset with linked phenotypic and genomic data as a resource for biomarker discovery.

Trametinib (Trm) is a highly selective mitogen-activated protein kinase kinase (MEK) inhibitor that potently and persistently abrogates extracellular signal-regulated kinase 1/2 activation. Trm initially was used to treat BRAF Val600→Glu (V600E)-mutated melanoma, but its Food and Drug Administration-approved indications are expanding rapidly. Trm generally is well tolerated, but it can cause dose-limiting cardiomyopathy and heart failure. Here, we characterize a mouse model of Trm cardiotoxicity using complementary in vitro approaches to show that Trm induces mitochondrial dysfunction in cardiomyocytes and some cancer cell types. In vivo, Trm caused contractile dysfunction within 3 days and heart failure within 2 weeks. High-resolution respirometry using isolated cardiac mitochondria revealed that Trm compromises oxidative metabolism, in part, through blunted activity of electron transport system complexes. Trm-mediated mitochondrial injury led to the release of mitochondrial damage-associated molecular patterns including mitochondrial DNA in both mice and humans, triggering activation of canonical innate immune pathways including cGAS-STING. In multiple rodent and human cardiomyocyte platforms, Trm diminished mitochondrial respiratory capacity at nanomolar concentrations, but this lesion was reversed by expression of a phosphomimetic signal transducer and activator of transcription 3-S727 construct. We also found that Trm induced mitochondrial dysfunction in some but not all cancer cell lines, identifying a previously unrecognized effect that could contribute to Trm's anticancer efficacy.

Supplementary Table 6.overall_survival

Abstract Triple Negative Breast Cancers (TNBC) are a biologically heterogeneous clinical subtype of breast cancer with limited treatment options and generally poor prognosis. Basal-like breast cancer (BLBC), which accounts for ∼75% of TNBCs by gene expression profiling, is particularly aggressive, highlighting an urgent need to develop targeted therapies for this subtype. Our lab previously identified an enrichment of pyrimidine synthesis metabolites in tumors from BLBC patients. Further, an analysis of the Chinese Breast Cancer Genome Atlas (PMID: 38347143), a multi-omics dataset including 443 breast cancer metabolomics samples, also revealed a similar feature of pyrimidine-enriched metabolites in both BLBC and TNBC tumors. Therefore, we hypothesize that pyrimidine biosynthesis is essential for BLBC survival and may represent a therapeutic vulnerability. To investigate this unique metabolic phenotype, we utilized a panel of syngeneic, serially transplanted, genetically engineered mouse (GEM) model mammary tumors representing multiple breast cancer subtypes (luminal, basal-like, mesenchymal). Metabolomic mass spectrometry of 20 GEM models, including 6 BLBC models and normal mammary glands, identified 2 distinct metabolic subtypes, namely a Nucleotide-enriched and Fatty Acid-enriched groups, mirroring the patterns observed in human breast tumors. To evaluate the dependency of these murine tumors on pyrimidine biosynthesis, we used leflunomide, an FDA-approved dihydroorotate dehydrogenase (DHODH) inhibitor. In vivo testing across 4 tumor models, including the basal-like p53-null 2225L model, showed that leflunomide slowed tumor growth and extended survival in 3 models. However, 1 model exhibited resistance, suggesting that combination therapies may still be needed. To identify potential combination therapies based upon DHODH inhibitors, we conducted a drug combination screen using both the murine 2225L and human SUM102 basal-like cell lines. Notably, we found that kinase inhibitors with off-target activity against ENT1 sensitized cells to DHODH inhibition. These findings are currently being validated in vivo, which may support a strategy of co-targeting pyrimidine biosynthesis and the nucleotide salvage pathways to overcome resistance. Citation Format: Amanda Linke, Kevin Mott, Felix Olivares, Michael East, Chengheng Liao, Qing Zhang, Gary L. Johnson, Charles M. Perou. Targeting metabolic vulnerabilities in basal-like breast 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 2009.

Kinases are central regulators of multiple signaling cascades, controlling processes such as cellular growth, proliferation, and differentiation. Given their vital role within the cell, dysregulated kinase activity contributes to several skin diseases, including melanoma and dermatitis. Poor disease response or resistance to targeted inhibitors can be driven by adaptive kinase responses. Genomic assays are highly informative but do not accurately capture kinase abundance and activity at the protein level. In this paper, we review 2 complementary mass spectrometry-based proteomics methods for functional kinome analysis that are readily applicable to dermatology research. Multiplexed inhibitor beads coupled with mass spectrometry (MIB-MS) uses broad-spectrum, immobilized kinase inhibitors to enrich for kinases in active conformation, providing an unbiased, pathway-level readout of kinase network dynamics, adaptive rewiring, and drug specificity. Internal standard triggered-parallel reaction monitoring (IS-PRM)-targeted proteomics, including the Thermo SureQuant acquisition method, leverages heavy peptide triggers to deliver sensitive, consistent quantification of predefined kinase peptides from limited input clinical specimens, including formalin fixed, paraffin embedded. We summarize optimized workflows, instrument set-up, sample requirements, technical considerations, and limitations. Together, MIB-MS and IS-PRM SureQuant offer orthogonal, scalable strategies to profile kinase networks in skin biology and to inform target discovery, biomarker development, and rational therapeutic strategies.

Supplementary Table 5.demographic_molecular_data

Supplementary Table 2.limma_kinomeclusters

Supplementary Table 1.MIB_experimental_setup

Supplementary Table 4.RASH_ACCEPT_stats

Resistance to endocrine therapy (ET) is common in estrogen receptor-positive (ER+) breast cancer. Multiple studies have demonstrated that upregulation of MAPK signaling pathways contributes to ET resistance. Herein we show that vandetanib treatment suppresses MAPK signaling and enhances sensitivity to ET across ET-sensitive and ET-resistant ER+ cell lines and patient derived organoids. Vandetanib treatment reprograms transcription toward a less proliferative, more estrogen responsive, Luminal-A like state by enriching ERα chromatin binding at canonical estrogen response elements. Multiplexed kinase inhibitor beads-mass spectrometry (MIB/MS) revealed kinase network reprogramming, including upregulation of PI3K and HER2 activity, as shared adaptive resistance mechanisms to vandetanib treatment. Co-treatment with the HER2 inhibitor lapatinib, further enhanced sensitivity to vandetanib. Using an operating room-to-laboratory short-term ex-vivo assay coupled to single-cell RNA sequencing, we demonstrate conserved gene expression changes in primary tumor cells, including increased HER2 activity signatures, following vandetanib treatment. Vandetanib sensitivity signatures were generated from cell line and primary human tumor cells which correlate with vandetanib sensitivity in ER+ patient-derived organoid and xenograft models. Future studies should evaluate if signatures predict responses in patients with ER+ breast tumors, and determine the role of co-targeting HER2 signaling with vandetanib therapy.

Supplementary Figure S1 shows kinome remodeling in response to ALK inhibition is robust and heterogenous.

Metabolic stress in the tumor microenvironment (TME) promotes T cell dysfunction and immune checkpoint inhibitor (ICI) resistance. We examined the contribution of activating transcription factor 4 (ATF4), the central node of the integrated stress response (ISR), to T cell dysfunction in tumors. CD8+ tumor-infiltrating lymphocytes (TILs) in patient samples exhibited chronic ATF4 activity, which was reflected across various tumor models. Hypoxia in the TME imposed chronic ATF4 activity via the ISR kinases. ATF4 overexpression in CD8+ T cells induced metabolic polarity, mitochondrial oxidative stress, and cell death, impairing antitumor immunity. Chronic ATF4 transcriptional activity replicated the terminal exhaustion CD8+ T cell state independent of T cell receptor (TCR) stimulation. Genetic or pharmacologic attenuation of ATF4 reduced mitochondrial oxidative stress and promoted CD8+ TIL viability, enabling response to programmed cell death protein-1 (PD-1) inhibitor therapy and conferring protection from re-emergent disease. Thus, the ISR converges on chronic ATF4 activity in CD8+ TILs as a barrier to ICI response, positioning ISR therapeutics as candidates for immunotherapy.

Supplementary Figure S16 shows chromatin immunoprecipitation sequencing assay whereby c-Myc binds to promoters of several G2/M kinases.

Supplementary Figure S3 shows immunoblot validation of MIB/MS data.

To investigate the landscape of the activated kinome in MM, we applied a proteomic approach that interrogates the activation status of more than half of the entire kinome (∼300), termed multiplexed kinase inhibitor bead affinity chromatography coupled with mass spectrometry (MIB/MS) on 45 MM patient (pt) tumors (BRAFV600, n=25; NRASQ61, n=5, NF1 loss-of-function, n=4; Triple wild-type n=11) in conjunction with whole exome sequencing and RNA sequencing (RNAseq). We performed exploratory analyses (Wilcoxon rank sum test for each kinase) on the untreated tumors to identify differentially activated kinases in BRAFV600-mutant MM vs. all others. While no activated kinases were differentially expressed in BRAFV600-mutant MM after adjustment for multiple testing, AKT1, PDGFRA, and MAP3K1 kinases exhibited higher MIB binding (i.e., more activated) in BRAFV600E-mutant pts (unadjusted p-value < 0.05). Spearman’s rank correlation tests between the activated kinase and its gene expression by RNAseq and adjusted for multiple testing did not identify a significant correlation for any kinase, suggesting a complex mechanism of kinase activation other than sole gene expression. K-means clustering analysis to classify melanomas into 4 subtypes using a 6-gene discriminant expression signature showed that BRAFV600-mutant tumors were more frequently classified as melanocytic (9/24, MLANA/MITF/SOX10high, AXLlow). In contrast, triple wild-type tumors were more frequently classified as transitory (5/10, MITF/SOX10/ETV4high, AXLlow). We then performed similar exploratory analyses on the baseline (untreated) tumors from the subset of the 20 BRAFV600E-mutant pts who received D+T as part of a prospective clinical trial (NCT01726738; median follow-up 30.5 months, range 2.0-116.1 months) to identify differentially activated kinases that would predict shorter (<12 months) vs. longer progression-free survival. Again, while no kinases survived multiple comparison testing, the bromodomain protein TRIM28, known to regulate melanoma plasticity, exhibited a more consistent loss of MIB binding in BRAFV600E-mutant pts who progressed shorter than in pts who progressed longer than 12 months on D+T (unadjusted p-value < 0.05). 7 baseline-progression tumor pairs were evaluated for MIB/MS, and 6 were for RNAseq. Although diverse resistance mechanisms were identified, as previously described, we also identified melanoma subtype switch in 4/6 pt tumors and a consistent loss of MIB binding of the non-receptor tyrosine kinase PTK6 in response to D+T, an off-target of D and vemurafenib but not encorafenib. Our in vitro findings revealed that off-target PTK6 inhibition by D could activate beta-catenin signaling and contribute to the development of resistance. Steve P. Angus, Naim U. Rashid, Andrew J. Walther, David L. Corcoran, Steven D. Rhodes, Frances A. Collichio, C. Paige Jones, Mikaela J. Bauer, Joel S. Parker, Noah Sciaky, 1Alisha R. Coffey, Samantha M. Bevill, Brian T. Golitz, v Timothy J. Stulhmiller, Nancy E. Thomas, David W. Ollila, Norman E. Sharpless, Carrie B. Lee, Gary L. Johnson, Stergios J. Moschos. Baseline and adaptive activated kinome profiling identifies PTK6 as a mediator of resistance to vemurafenib and dabrafenib but not encorafenib in BRAFV600-mutant metastatic melanoma [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 2051.

Abstract Effective therapies for pancreatic ductal adenocarcinoma (PDAC) have been largely elusive. In this study, we perform multiplexed kinase inhibitor bead mass spectrometry on 102 patient-derived xenografts derived from 14 unique primary PDACs to define the tumor-intrinsic kinome landscape. Our findings uncover three kinome subgroups making up two tumor-intrinsic kinome subtypes that we call kinotypes. The kinotypes show enrichment of different kinase classes and recapitulate previously described molecular subtypes, basal-like and classical. The kinotype characterizing basal-like tumors shows enrichment of receptor tyrosine kinases, whereas the kinotype characterizing classical tumors is enriched in understudied kinases involved in Wnt signaling and immune pathways. We validate our findings in two clinical trials and show that only patients with basal-like tumors derive significant benefit from EGFR inhibitors. Our results provide a comprehensive tumor-intrinsic kinome landscape of PDAC that strongly supports actionable kinotype-specific kinase targets and provides a roadmap for kinase inhibitor therapy in PDAC. Significance: We provide a comprehensive tumor-intrinsic kinome landscape that provides a roadmap for the use of kinase inhibitors in PDAC treatment approaches.