Abstract A040: Polyunsaturated fatty acids control lipid membrane dynamics and enable B-cell lymphomagenesis

Abstract

The lipid membrane of B-cell lymphomas is a highly dynamic environment that modulates key functions including cell migration, adhesion, and signaling. In addition, lipid membrane composition influences sensitivity to ferroptosis, a type of cell death characterized by peroxidation of polyunsaturated fatty acid (PUFA)-containing phospholipids (PUFA-PL) to which B-cell lymphomas are highly vulnerable. We show that PUFA-PL, while being key enablers of ferroptosis vulnerability, are also required for B-cell lymphoma fitness and membrane properties. ACSL4, due to its key role in generating highly oxidizable PUFA-PL, is known to promote cell death by ferroptosis. However, our analysis of publicly available data from CRISPR dependency screens, metabolomics, and clinical trials using an interactive platform we have developed (lymphoblasts.org) has uncovered ACSL4 as a selective dependency in B-cell lymphoma. This analysis also showed that high ACSL4 expression is a marker of poor prognosis in diffuse large B-cell lymphoma (DLBCL). In addition, our comparative analyses of metabolomics data showed a marked enrichment of multiple types of PUFA in B-cell lymphomas compared to solid tumors. These findings suggest that, while PUFA-PL promote sensitivity to ferroptosis, they are also required for B-cell lymphomagenesis. Accordingly, activation of murine primary B-cells with anti-IgM, IL-4, and CD40L markedly upregulated ACSL4 protein levels, suggesting an important role of PUFA-PL in states of increased metabolic demand. To evaluate the roles of ACSL4 and PUFA-PL in B-cell lymphomas, we generated DLBCL and Burkitt lymphoma (BL) cell lines with CRISPR-Cas9-mediated inducible deletion of ACSL4. While loss of ACSL4 led to ferroptosis resistance, it also compromised B-cell lymphoma fitness in competitive growth assays, suggesting an important role of ACSL4 and PUFA-PL in B-cell lymphoma fitness. To dynamically monitor lipid membrane changes in B-cell lymphoma membranes, we utilized CRISPR-mediated homology-directed repair to knock-in an ACSL4 degradation tag (dTAG) in the BL cell line DG75. Induction of the dTAG led to complete loss of ACSL4 protein within one hour and led to a progressive increase in resistance to lipid peroxidation, consistent with loss of PUFA-PL at cell membranes. In addition, molecular biophysics experiments showed that PUFA-PL contribute to lipid membrane fluidity, as ACSL4 deletion increased membrane flow resistance (membrane cytoskeleton attachment: 9.246 ± 1.43 x 105 vs 5.141 ± 1.57 x 105 pN3s/mM for ACSL4 loss vs negative control, p = 0.00015). These findings suggest that a low flow resistance of the B-cell membrane, promoted by ACSL4 and PUFA-PL, contributes to key oncogenic functions in B-cell lymphoma. Overall, our findings show that B-cell lymphomas are selectively dependent on PUFA-PL metabolism to maintain membrane properties and competitive fitness. This dependency on pro-ferroptotic lipids might however make B-cell lymphomas inherently highly vulnerable to ferroptosis, which could potentially be targeted therapeutically.