Abstract 2417: Detection and quantification of site-specific DNA methylation from liquid biopsies as a pharmacodynamic biomarker of OTX-2002, a novel MYC-targeting epigenomic mRNA therapeutic
Authors: Chen Justin , Senapedis William , Siecinski Stephen , Figueroa Elmer , Katz Adam , Mildrum Samuel , Wang Yaoyu E. , Belaghzal Houda , Gallagher Kayleigh , Hodgson Graeme , O'Donnell Charles W. , McCauley Thomas G. 
Published: March 2024

Abstract
Omega Therapeutics has developed a novel platform of programmable epigenomic mRNA medicines capable of modifying chromatin state to specifically tune gene expression at the pre-transcriptional level. Epigenomic controllers (ECs) unlock targets that have been historically considered “undruggable,” with one of the most elusive being the MYC oncogene. A direct MYC-targeting anti-cancer agent has previously remained intangible, largely due to the absence of a drug binding pocket and tight autoregulation. A clinical trial is underway (MYCHELANGELO, NCT05497453) to investigate pre-transcriptional inhibition of MYC with OTX-2002 in patients with hepatocellular carcinoma (HCC). OTX-2002, a first-in-class mRNA therapeutic delivered via lipid nanoparticles (LNP), encodes two proteins that durably modify chromatin, in part, through CpG DNA methylation at the MYC locus. Using both liquid and solid biopsy sampling from in vivo studies, we investigated whether target engagement for OTX-2002 could be assessed by MYC methylation.
Detection of DNA methylation has long held promise as an oncology biomarker given its functional roles in various cancer types and the potential signal afforded by methylated CpGs. Indeed, DNA methylation has been shown to serve as a robust analyte in liquid biopsy-derived multi-cancer early detection (MCED) and minimal residual disease (MRD) tests that have recently been utilized in the clinic. Many of these platforms are founded on complex models that leverage methylation signals across &gt;1 million CpGs that can span tens of megabases of genomic space. We faced a distinct challenge when compared to these MCED/MRD tests in developing a pharmacodynamic methylation assay for OTX-2002 as the target region consists of just a few kilobases. With a tissue specific LNP delivery system, ultra-high sensitivity was required to identify rare ctDNA events from the larger cfDNA population.
To this end, we designed a minimal hybridization/capture panel that targeted ~50 kb of genomic space, effectively allowing for ultra-deep methylation sequencing of MYC. When this technique was paired with enzymatic (EM) conversion for methylation detection and supported by an analysis pipeline focused on epiallele identification, this assay was able to detect methylation down to the theoretical limit in a dilution series of control genomic DNA, 1 in 104 copies of MYC. This degree of sensitivity translated to successful preclinical detection of on-target methylation by OTX-2002 from DNA extracted from plasma samples collected from mice-bearing human HCC xenografts. Overall, we present a non-invasive and exquisitely sensitive method of assessing target engagement and site-specific pharmacodynamic activity of a novel epigenomic medicine that can be directly translated to the clinical setting.
Citation Format: Justin Chen, William Senapedis, Stephen Siecinski, Elmer Figueroa, Adam Katz, Samuel Mildrum, Yaoyu E. Wang, Houda Belaghzal, Kayleigh Gallagher, Graeme Hodgson, Charles W. O'Donnell, Thomas G. McCauley. Detection and quantification of site-specific DNA methylation from liquid biopsies as a pharmacodynamic biomarker of OTX-2002, a novel MYC-targeting epigenomic mRNA therapeutic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2417.