Optimizing capivasertib response prediction in cancer: unveiling resistance mechanisms through precision proteomics

Introduction
The PI3K/AKT/mTOR pathway plays a role in more than half of breast cancers, making it a crucial focus for new treatments. Capivasertib (AZD5363), an effective inhibitor of all AKT isoforms, has shown promise in Phase III trials for both breast and prostate cancers. However, pinpointing the right patients for this treatment is challenging due to gene-based uncertainties. To address this, we're using advanced mass spectrometry (MS) techniques to explore how Capivasertib affects the proteomic landscape. Our goal is to gain insights that will improve patient selection and refine precision medicine approaches for cancer treatment.

Methods
We've developed and validated MS-based targeted assays to quantify proteins associated with Capivasertib in FFPE tumor blocks and breast cancer cell lines. These assays use synthetic peptides for calibration and stable isotope-labeled standard peptides for quantitation, ensuring accurate and reproducible results. Additionally, we've created targeted MS-based assays to investigate off-target effects, helping us understand Capivasertib"s mechanisms and resistance, and refine patient selection criteria.

Results

In a previous study using untargeted proteomic methods, distinct protein profiles were identified in PIK3CA-mutant tumors from patients receiving Capivasertib. Pathways like EIF2 signaling, eIF4/p70S6K, and the mTOR pathway were linked to these profiles and helped distinguish patients who benefited from Capivasertib. To confirm the association, targeted MRM-MS assays for 50 proteins were developed, distinguishing between Capivasertib-resistant and sensitive PIK3CA-altered breast cancer cell lines. However, in patient samples, the lower limit of quantification (LLOQ) in FFPE samples posed challenges. Thus, we developed more sensitive Evosep-MRM-MS and Evosep-PRM-MS assays using Evosep-Agilent 6495C and Evosep-timsTof-HT, respectively. To further understand Capivasertib"s effects, we are developing assays for absolute quantitation of cancer proteins like EGFR, PI3K, and KRAS, and for 1000 mutation events in these proteins. We have optimized 522 assays, enabling deeper exploration of Capivasertib"s molecular effects.

Conclusions
Our study uses advanced MS techniques to investigate how Capivasertib interacts with cellular proteins. While there are challenges in quantifying proteins from patient samples, our targeted assays offer a more sensitive approach, needing only 1 μg of protein, making them suitable for clinical use. Moving forward, our research aims to delve deeper into Capivasertib"s impact on various cancer-related proteins and pathways. By expanding our targeted assays to cover a broader range of protein networks, we hope to better understand the complex interactions between Capivasertib and cellular processes, ultimately enhancing its therapeutic potential and application.