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3310 Regulation of BCMA By the Ubiquitin Proteasome System Enables Optimization of BCMA-Targeting Therapies in Multiple Myeloma

Program: Oral and Poster Abstracts
Session: 651. Multiple Myeloma and Plasma Cell Dyscrasias: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Research, Biological therapies, Translational Research, Plasma Cell Disorders, Chimeric Antigen Receptor (CAR)-T Cell Therapies, Combination therapy, Diseases, Therapies, Lymphoid Malignancies, Biological Processes, molecular biology
Sunday, December 10, 2023, 6:00 PM-8:00 PM

Leonie Rieger1,2*, Kilian Irlinger1,2*, Franziska Fuechsl1,2*, Nicolas Barbian1,2*, Marlene Tietje1,2*, Melanie Faber1,2*, Tobias Schulze1,2*, Piero Giansanti2,3*, Lenka Besse4*, Christoph Driessen, MD4, Angela Krackhardt, MD1,2,5*, Bernhard Kuster2,5,6* and Florian Bassermann, MD1,2,5*

1Department of Hematology and Oncology, Technical University of Munich, Munich, Germany
2TranslaTUM, Center for Translational Cancer Research, Technical University of Munich, Munich, Germany
3Bavarian Center for Biomolecular Mass Spectrometry, Technical University of Munich, Munich, Germany
4Division of Oncology and Hematology, Cantonal Hospital St. Gallen, St. Gallen, Switzerland
5German Cancer Consortium of Translational Cancer Research (DKTK), Heidelberg, Germany
6Chair of Proteomics and Bioanalytics, Technical University of Munich, Freising, Germany

Background: The exclusive expression of the B-cell maturation antigen (BCMA), in normal and malignant plasma cells, currently makes it a prominent target for immune cell therapies in multiple myeloma (MM) including chimeric antigen receptor T-cell (CART) and bi-specific T cell engaging antibody (BiTE) therapies. Despite initial high response rates, most patients eventually relapse highlighting the need to enhance therapy efficacy. Although loss of BCMA-antigen expression marks one reason for therapy failure, little is known about the mechanistic means of antigen escape. The ubiquitin proteasome system (UPS) controls abundance and protein degradation of about 80-90% of all cellular proteins. Protein degradation is initiated by substrate ubiquitination, which is orchestrated by the sequential action of enzymes whereby only E3-ligases possesses substrate specificity. Deubiquitinating enzymes (DUBs) can reverse substrate ubiquitination. Against this background, we raised the question whether BCMA antigen presentation, expression, and activity is regulated by the UPS and whether the UPS can be utilized to enhance BCMA directed CART therapies in MM.

Methods: Protein turnover and regulation by the UPS was assayed using cycloheximide and proteasomal inhibitors (PI) followed by immunoblotting. Direct substrate ubiquitylation was confirmed by ubiquitylation assays based on immunoprecipitation and image-based analysis. Changes in antigen surface presentation were determined using FACS analysis. Changes in cellular signaling and downstream effects were assessed by immunoblotting and quantitative PCR (qPCR). Primary CD138+ cells were obtained from bone marrow aspirations followed by MACS® cell separation. To assess the effect of PI on CART killing capacity, a classic co-culture was set up in vitro. Using a proximity-based protein purification method (TurboID), UPS-related interactors of BCMA were identified.

Results: For the first time, we show that BCMA is a short-lived protein with a half-life of approximately 30 min in MM cells. Degradation of BCMA is abrogated upon PI treatment. Inhibition of γ-secretase, an established protease of the extracellular domain of BCMA does not impair BCMA turnover. Furthermore, we show that BCMA is indeed ubiquitylated in MM cells and that ubiquitylation is induced by the ligand APRIL. These results support the hypothesis that the UPS regulates BCMA abundance. In both MM cell line models and primary CD138+ MM cells of PI refractory/ relapsed MM (RRMM) patients, inhibition of the proteasome leads to a significant increase of BCMA on the cell surface without promoting oncogenic NF-κB signaling in MM cells. Subsequently, we show that the increase in BCMA surface expression upon PI treatment substantially increases the efficacy of BCMA directed CART cells in co‑cultures with MM cells and primary CD138+ MM patient cells. Specific members of the UPS (E3 ligases and DUBs) identified from our proteome wide BCMA interactome analysis are currently being validated.

Conclusion: Here we show, for the first time, that BCMA is ubiquitylated and degraded by the ubiquitin proteasome system in multiple myeloma. The increase in BCMA expression upon proteasomal inhibition enhanced efficacy of BCMA-targeting CART cells. These insights provide a rationale for combining BCMA CART cells with PI in patients with BCMA CART relapsed/refractory disease and persistence. The identification of BCMA-specific E3-ligases and DUBs may represent novel therapeutic targets to specifically prevent BCMA degradation and antigen escape.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH