MSACL 2025 Abstract

Development and Validation of an LC-MS/MS Assay for Measuring KEAP1 Cys151 Target Engagement in Tumor Biopsies

Melissa Hoffman (1), Egor Vorontsov (2), Seth Negron (1), Stephanie Ballweg (2), Katrin Rottluff (2), Michael Bremang (2), Stefan Selzer (2), Antje Berfelde (2), Christie Eissler (1), Stephen McComish (1), Gabriel Simon (1), Matthew Patricelli (1), David Van Meter (2), Ian Pike (2), Marielena Mata (1)
(1) Vividion Therapeutics, San Diego, CA, (2) Proteome Sciences, Frankfurt, Germany

Melissa Hoffman, PhD (Presenter) Vividion Therapeutics

Presenter Bio: Melissa joined Vividion’s proteomics team in 2020 as a Scientist I. She has progressed to lead the proteomics platform group operations. The team supports Early Discovery Sciences hit identification efforts and compound tractability through chemoproteomics screening and later-stage programs by measuring in vivo target engagement and protein PD responses. She is also leading the clinical target engagement assay development to support Vividion’s clinical trials and building out translational proteomics workflows.

Before coming to Vividion, Melissa completed a Clinical Chemistry Fellowship at UC San Diego under Dr. Robert Fitzgerald’s mentorship where she learned clinical assay validation and toxicology. She received her PhD in Cancer Biology from Moffitt Cancer Center/University of South Florida where she studied translational proteomics in Dr. John Koomen’s lab. She received her BS in Biology from California State University, Chico and is from the SF Bay Area. Melissa is a co-author of 14 scientific publications.

Relevant Financial Disclosures (within past 24 months, reported on Jul 14, 2025) Other Potential Conflicts Vividion Therapeutics / Employee Conflict mitigation NOT REQUIRED.

INTRODUCTION:
The transcription factor NRF2 plays an important role in defense against oxidative stress, and its constitutive activation in cancer contributes to therapeutic resistance and tumor progression. KEAP1 (Kelch-like ECH-associated protein 1), a substrate adaptor for the CUL3 E3 ligase, regulates NRF2. Vividion recently developed VVD-130037, a first-in-class covalent allosteric molecular glue that selectively binds KEAP1 at cysteine 151 (C151), enhancing KEAP1-CUL3 complex formation leading to NRF2 degradation. To support clinical translation of this approach, a quantitative LC-MS/MS assay was developed to measure KEAP1 target engagement in tumor biopsies from a Phase I clinical trial (NCT05954312) following treatment with VVD-130037. Pre-clinically, a KEAP1 Target Engagement (TE) assay was developed and applied to mouse xenografts and tissues by comparing vehicle and compound treated samples.1 Building on this work, a novel approach was developed to miniaturize the chemoproteomics workflow to support needle biopsy sample analysis and to calculate TE in the absence of a pre-treatment sample.

OBJECTIVES:
This study aimed to (1) optimize the KEAP1 C151 TE assay for tumor biopsy samples without a pre-treatment sample and (2) validate assay sensitivity, linearity, precision, and accuracy at a GCLP compliant lab.

METHODS:
Tumor biopsies were homogenized in IP lysis buffer by probe sonication. A single-pot, solid-phase-enhanced sample preparation (SP3) method was implemented, which used 50 µg of tumor protein (reduced from 500 µg) mixed with 50 µg of heavy isotope-labeled (SILAC) MDA-MB-231 cell lysate. Free cysteines were covalently labeled with a desthiobiotin-iodoacetamide probe, followed by tryptic digestion, and streptavidin enrichment of labeled peptides. Resulting peptides were separated on a nanoflow C18 column using a Dionex UltiMate 3000 UPLC and analyzed on an Orbitrap Exploris 480 using parallel reaction monitoring (PRM) mass spectrometry. SILAC MDA-MB-231 cell lysates were used as internal standard and quantification was performed using an external calibration curve using a full length purified KEAP1 protein spiked into a blank matrix (insect cell lysate). The assay was successfully transferred to a GCLP compliant lab (Proteome Sciences) and validation included optimization of LC-MS parameters for KEAP1 C151, C288, C297, C319, and C434, protein extraction from small biopsies, calibration curve generation to establish the linear range and lower limit of quantification (LLOQ), assessment of inter- and intra-day precision and accuracy for low, mid, and high spiked samples, and evaluation of TE in xenograft tumor models comparing TE calculated with and without pre-treatment samples. Inter-operator and inter-instrument performance was also evaluated.

RESULTS:
The assay achieved a LLOQ of 0.06 fmol/µg for the KEAP1 C151 peptide and 0.03–0.12 fmol/µg for other KEAP1 peptides. Linearity (R² ≥ 0.986) was confirmed over the 0.06–1.50 fmol/µg range. Protein extraction yielded sufficient material from 5–19 mg tumor biopsies from 10 donors with consistent peptide concentrations across replicates. KEAP1 C151 intra-day precision ranged from 9–12% across the low, mid, and high spiked samples and inter-day precision was 22% for the low spike and 15% for the mid and high spiked samples. Assay precision for the other KEAP1 peptides was also within the acceptability criteria of ≤ 20% for mid and high spiked samples and ≤ 30% for low spike samples. TE values calculated using vehicle-treated baselines were comparable to those using concentrations of C297, C319, and C434 in the same treated sample. The validated assay was successfully applied in a clinical trial (data not shown).

CONCLUSION:
The KEAP1 C151 TE assay was successfully optimized for low protein input samples and TE calculated using non-C151 KEAP1 peptides. The assay was successfully transferred to a GCLP compliant lab, validated for use with human tumor biopsies, and proof-of-concept demonstrated by a reduction in C151 signal after VVD-130037 treatment. Covalent TE from patient tumor biopsies will support clinical evaluation of VVD-130037.

REFERENCES:
1. Roy N, et al., Suppression of NRF2-dependent cancer growth by a covalent allosteric molecular glue. bioRxiv 2024.10.04.616592.