Abstract Extending the successes of cancer cell-targeted kinase inhibitor therapies, we sought to identify endothelial kinases altered in cancer conditioning by combining phenotype assays, phosphoproteomics, and transcriptomics. Human umbilical vein endothelial cells (EC) cultured in conditioned media from MDA-MB-231 human breast cancer cells, display a marked and robust transformation in cellular morphology. Furthermore, scratch test assays revealed that cancer-conditioned ECs have significantly impaired wound healing responses, closing 25% less of a 750 µm-wide scratch than control ECs by 48 hours. Since regulation by phosphorylation is an important component of changes to cell shape and migration, we predicted that kinase inhibitors could affect cancerous processes in ECs. To identify specific kinases of interest, mass spectrometry-based phosphoproteomics was run to characterize changes in phosphorylation, and then combined with KinomeXplorer analysis to predict the associated kinases. To refine the predictions and overcome the limited depth of analysis inherent to the phosphoproteomic technique, we matched kinase predictions to RNA sequencing analysis of the same cultures. Based on the strongest combined predictions, a set of kinase inhibitors was chosen and tested by measuring the effects on morphology and wound healing, compared to cancer conditioning. First, inhibitors were added to control ECs: using a Src-family kinase inhibitor, for example, in control ECs resulted in a phenotype strongly resembling the cancer phenotype in both wound healing and elongation, consistent with the decreased phosphorylation of Src targets; however, a Bmx/Btk kinase inhibitor, while also predicted to have a similar impact, showed little effect in either assay. Then, a simple model of prevention and rescue was tested with inhibitors to increased predicted activity (e.g. Akt- and Pdh-family kinases and several phosphatases): inhibitors were added to EC cultures before, concurrent with, or after cancer conditioning. The inhibitors resulted in a range of phenotypic changes, depending both on the kinase as well as the prevention/rescue model. By using a combination of different assays on a well-controlled culture model we successfully developed a comprehensive library of kinase pathways in cancer-conditioned ECs, characterized by multimodal molecular data (phosphoproteomics and transcriptomics) and directly associated with functional effects of inhibitors using phenotype analysis. We hope this provides the beginning for the development of a tumor EC-targeted therapy based on kinase inhibitors, while also shedding light on the potential effects of existing cancer-targeting kinase inhibitors on the tumor endothelium. Citation Format: Or Gadish, Sabrina E. Ibarra, Elazer R. Edelman. Integrated phosphoproteomics, transcriptomics, and phenotypic analyses unveil distinct endothelial kinase pathways differentially altered by cancer conditioning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 204.