Epigenetic Modifications As a Mechanism of Resistance to BCMA-Directed Immunotherapies in Multiple Myeloma: Insights from Multi-Omic Profiling
Authors: Haertle Larissa , Buenache Cuenda Natalia , Espejo Diaz Isabel , Lázaro Del Campo Paula , Arroyo-Barea Andres , Rieger Leonie , Rosa-Rosa Juan Manuel , Alonso Fernandez Rafael , Lopez Nieves , Sanchez-Pina José , García-Ortiz Almudena , Valeri Antonio , Barrio Santiago , Al-Shahrour Fatima , Bassermann Florian , Martínez-López Joaquin , Di Domenico Tomas 
Published: December 2024

Introduction
Recent advancements in B-cell maturation antigen (BCMA)-directed immunotherapies, such as CAR T-cell therapy and bispecific T-cell engagers, have significantly improved treatment outcomes for Multiple Myeloma (MM). However, resistance to these therapies remains a substantial clinical challenge. Current research has identified large structural events on chromosome 16, focal deletions of the BCMA locus, and mutations within the extracellular domain. Yet, these alterations alone do not explain the majority of the resistant cases, suggesting the existence of other unexplored mechanisms, such as epigenetic dysregulation. Understanding the transient durability of clinical responses is crucial for improving therapeutic efficacy and treatment strategies. This study aims to comprehensively explore all potential layers of tumor-intrinsic anti-BCMA treatment escape, with a special focus on the DNA methylome, using innovative multi-omic Oxford Nanopore Technology (ONT) for profiling sequential patient samples. The technological advantage of long-read ONT sequencing over short-read NGS approaches like Illumina or Ion Torrent lies in its ability to directly and simultaneously assess whole-(epi)genome-wide structural variations (SVs), single nucleotide variants (SNVs), and DNA methylation in the same sample without bisulfite conversion. It benefits haplotype phasing, resolving complex repetitive regions and large complex SVs.
Methods
We analyzed pre- and post-treatment samples and PBMCs from three RRMM patients resistant to different BCMA-directed therapies (BCMA CART, elranatamab, and belantamab mafodotin). DNA from CD138+ MM cells with fragment lengths ≥ 22 kB (TapeStation 4200) was sequenced on the PromethION 2 Solo platform, aiming at ≥ 20x genomic coverage, even for low-concentrated samples. ONT SNP calling was compared to a targeted capture-hybridization DNA sequencing panel. RNA-Seq assessing the whole transcriptome will be available for the conference. BCMA level was also individually measured by FACS, enabling the association of genomic and epigenetic alterations detected with target gene expression.
Results
Plasma MM cells exhibited global hypomethylation compared to PBMCs. However, two out of three patients showed increased methylation in post-therapy samples. Interestingly, both gained a long-range hypermethylation (chr.16 11963020-11969160) spanning approximately 6.1 kb and encompassing 91 individual CpGs, including the entire BCMA gene and two promoter regions, one up- and one downstream. In patient one, BCMA methylation increased by 53%, rising from 36% in the pre-CART to 89% in the post-CART sample (and 89% as well at a later relapse post-cevostamab). Similarly, in patient two, BCMA methylation increased from 7% pre-elranatamab to 76% post-elranatamab. PBMCs from both patients were fully methylated, with levels of 83% and 85%. FACS analysis showed a loss of BCMA expression on the cell surface in patient one, decreasing from 80% to 1%. FACS data for patient two was unavailable. Additional epigenetic alterations were observed: patients two and three showed CRBN enhancer methylation, previously linked to IMiD resistance (Haertle, et al. 2021, Blood, PMID: 34115836), and patient one exhibited PSMD9 promoter methylation, potentially contributing to PI resistance as previously reported for PSMD5 (Haertle, et al. 2023, Clin. Cancer Res., PMID: 36282272). Screening of MM cell lines and BCMA-therapy naïve MM patients indicated no intrinsic BCMA methylation.
Conclusions
Our study identifies DNA hypermethylation of the BCMA gene as a novel universal resistance mechanism to all BCMA-directed immunotherapies. This epigenetic modification leads to gene silencing, resulting in antigenic escape. It underscores the need for integrated multi-omic approaches to elucidate therapy resistance mechanisms, improve patient outcomes, and emphasizes the importance of periodic screenings to guide personalized treatment plans and enable early detection of emerging resistant clones. The efficacy of demethylating agents to overcome drug resistance needs further investigation. Methylation marks can trace a cancer cell´s memory, reflecting its development and treatment history.