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4682 Lipid Handling Induces Ferroptosis and Conveys Sensitivity to Anti-BCMA Immunotherapy in Multiple Myeloma

Program: Oral and Poster Abstracts
Session: 651. Multiple Myeloma and Plasma Cell Dyscrasias: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Research, Translational Research, Plasma Cell Disorders, Diseases, Lymphoid Malignancies, metabolism, Biological Processes
Monday, December 11, 2023, 6:00 PM-8:00 PM

Enrico La Spina1*, Salvatore Cortellino2*, Cesarina Giallongo, PhD3*, Marisabel Mecca4*, Simona Picerno4*, Angela Amoresano5*, Gabriella Pinto6*, Stefania Serpico5*, Alessandro Sgambato7*, Grazia Scandura1*, Daniela Cambria1*, Daniele Tibullo8*, Antonino Neri, MD9*, Francesco Di Raimondo, MD10 and Alessandra Romano, MD, PhD11*

1Divisione di Ematologia, Dipartimento di Chirurgia e Specialità Medico Chirurgiche, Università degli Studi di Catania, Catania, Italy
2Laboratory of Pre-Clinical and Translational Research, IRCCS-CROB, Referral Cancer Center of Basilicata, Rionero in Vulture, Italy
3Università degli Studi di Catania, Catania, CT, ITA
4IRCCS-CROB, Referral Cancer Center of Basilicata, Laboratory of Pre-Clinical and Translational Research, Rionero in Vulture, Italy
5Department of Chemical Sciences, University of Naples Federico II, Napoli, Italy
6Department of Chemical Science, University of Naples, Napoli, Italy
7Catholic University of Rome, Roma, ITA
8Università degli Studi di Catania, Catania, ITA
9Azienda USL-IRCCS di Reggio Emilia, Reggio Emilia, Italy
10Haematology, A.O.U. Policlinico “G. Rodolico-San Marco”, Catania, Italy
11Divisione di Ematologia, Dipartimento di Chirurgia e Specialità Medico Chirurgiche, Università degli Studi di Catania, Tremestieri Etneo, Cantania, Italy

Lipid metabolic changes in cancer are attracting increased attention. BAFF/APRIL axis is a multifaceted immune regulator of adipose tissue function and an emerging target for anti-BCMA therapy in multiple myeloma (MM). However, lipid analyses are scarce in MM onset and response to treatment.

First, we measured metabolites and proteins in sera obtained from matched bone marrow and peripheral blood of 17 newly diagnosed and 9 relapsed/refractory MM patients, by LC-MS/MS. String analysis identified crucial pathways including fibrinogen regulation, inflammatory response, structural integrity of lipoproteins and lipid transport, with a significant increase of apoliproteins APO-A1 (key component of HDL particle and cofactor for lecithin—cholesterol acyltransferase (LCAT) which is responsible for the formation of most plasma cholesteryl esters), A2,C2 (which hydrolyzes triglycerides to provide free fatty acids for cells), C3, components of HDL and LDL, confirming a deranged lipid handling in RR-MM patients.

Second, we studied the lipid metabolic changes upon anti-BCMA exposure with Elranatamab (EL), an investigational, off-the-shelf, humanized BCMA-CD3-directed bispecific antibody engineered to elicit potent T-cell-mediated anti-MM activity. Combining in vitro assays, FACS, qRT-PCR, immunofluorescence, Western Blot and RNA-seq we evaluated the response to EL treatment in 3 human myeloma cell lines U266, NCI-H929 and OPM2 (3 cell lines chosen for their different basal bioenergetics profile). MM cells were co-incubated with various concentrations of EL in presence of CD3+T cells isolated from healthy donors in 96- well U-bottom plates for 4 h and 24 h at an E:T ratio of 1:5 and 1:10, then stained with anti-CD138-PE antibody . CD138 + cells were detected by flow cytometry and counted as surviving MM cells. BODIPY™ 581/591 C11 (Lipid Peroxidation Sensor) and BODIPY 493/503 for Neutral Lipid Droplets Staining for FACS and immunofluorescence were used to quantify lipid peroxidation and cytoplasmatic LDs on surviving CD138 + cells.

Despite the BCMA target was equally expressed on U266, NCI-H929 and OPM2, the sensitivity to EL was different, inducing cell cycle arrest in the G2/M phase in U266 and NCI-H929 cell lines but not in OPM2, in E:T ratio independent-manner. The expression of the activation markers CD69 and CD25 on T cells and the release of cytokines by T cells were target-dependent and occurred only in the presence of BCMA-positive cells.

Oil-Red-O staining revealed an increased lipid content after EL exposure in all tested MM cell lines, with OPM2 the richest in basal lipids content. To dissect the contribution of lipophagy versus lipolysis we performed an autophagy flux analysis by western blot and RNA-seq essay, showing that lipolysis was the emerging response to EL treatment. In EL-sensitive U266 we found an increase of lipid peroxides, hallmark of ferroptosis, a form of cell death due to missed control of membrane lipid peroxidation, a phenomenon negligible in EL-resistant OPM2. Lipidomics demonstrated higher levels of monounsaturated fatty acids (MUFA) than polyunsaturated fatty acids (PUFA) in OPM2, associated to an increased expression of Stearoyl CoA desaturase 1 (SCD1), the enzyme that catalyzes the rate-limiting step in the production of MUFA.

While the PUFA oxidation is pro-ferroptotic, MUFA activation promotes a ferroptosis-resistant cell state. Indeed, a ferroptosis-primed resistant phenotype in EL-sensitive U266 was associated to downregulation of lipid peroxides scavengers GPX4 and SLC7A11 and up-regulation of pro-ferroptotic acyl-CoA synthetase long-chain family member 4 (ACSL4). Exogenous MUFA (via supplementation with palmitic acid 125 µM conjugated to BSA 1:6) alleviated ferroptosis induction in U266 making them resistant to EL, a phenotype associated to overexpression of CD36, the main free fatty acids (FFA) transporter. Conversely, in OPM2, inducing iron-dependent lipid peroxidation through RSL3 (3 µM) treatment rescued EL resistance, resulting in a time-dependent increase in cytosolic lipid peroxidation beginning at 6 hours, and overt cell death via ferroptosis at 18 hours.

Taken together, our findings suggest that MUFA/PUFA ratio drives ferroptosis and mediates EL sensitivity, addressing lipid handling as a novel means to tailor immunotherapy in RRMM.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH