ABSTRACT Due to the vast number of potential drug combinations, “testing them all” in clinical trials is impossible, putting pressure on preclinical research to find the most promising compounds to advance to human studies. While certain drug combination effects can now be modeled in silico, this approach is generally limited to the best- studied biological pathways and may not work for analyzing complex disease mechanisms that involve multiple interconnected pathways. Protein immunoassays detect activated biological pathways by analyzing site-specific phosphorylation of kinases and/or their substrates, normalized against the abundance of unmodified proteins. Recently, an interest in detecting and quantifying protein mutations has emerged, due to the development of novel therapeutic options based on covalent binding of a drug to a mutant protein or targeted protein degradation. Currently, there are no immunoassays for measuring multiple protein targets (total and phospho-) across multiple major cancer pathways, which can resolve differentially phosphorylated kinases and kinase substrates. While several platforms for building multiplexed protein-based assays exist they either a) have a limited ability to detect phosphorylation (immunoassay-based technologies); or b) are low-throughput, expensive and often difficult to standardize (LC-MS), making them suitable for discovery proteomics but not for routine screening of tens, hundreds or thousands of samples. Importantly, none of these analytical technologies are ideal for quantifying protein targets containing multiple phosphorylated sites within a specific protein region. Therefore, the existing assays cannot accurately measure kinases that are activated by sequential phosphorylation events. We propose to initiate development of Path10™ - a multiplexed, protein-based, multi-pathway profiling assay for the Bead-Assisted Mass Spectrometry (BAMS™) platform - which will simultaneously measure protein targets across 10 major cell signaling pathways: RAF-MEK-ERK, cell cycle, Hippo, Myc, Notch, Nrf2, PI3K/Akt, TGFβ, p53 and β-catenin/Wnt. It will achieve broad, multi-pathway coverage by including targets unique to each pathway and also common targets shared by different pathways, e.g. protein substrates of multiple kinases. By selecting relevant proteins and modification sites, future assays can be configured to extend coverage within selected pathways or to profile disease pathways other than cancer, such as diabetes. In the proposed Phase I we will start the product development, focusing initially on the RAF-MEK-ERK pathway, which has the highest frequency of genomic alterations. In the follow-up Phase II, we will rapidly expand assay coverage to the remaining 9 pathways using the methods developed in this Phase I. Post-Phase II, we will focus on assay automation and applying Path10™ to human tissue and animal models, e.g., patient-derived xenografts (PDXs).

NARRATIVE This project aims to initiate development of a protein immunoassay for measuring molecular markers of multiple cancer pathways in cell culture-based assays. The proposed assay will be marketed to researchers in cancer biology, pre-clinical drug development and oncology biomarker discovery as it will replace dozens of currently used individual assays with a single, broad coverage multiplexed assay, which is fast, inexpensive and easy to use.