Polyunsaturated fatty acids control lipid membrane dynamics and ferroptosis sensitivity in B-cell lymphoma Free

Background and Significance: Induction of ferroptosis, a form of cell death driven by iron-dependent peroxidation of polyunsaturated fatty acid (PUFA)-containing phospholipids (PUFA-PL), has shown potential in therapy-resistant tumors. However, the factors determining sensitivity of cancer cells to this type of death are not well understood, and no potent ferroptosis inducers are available clinically. Our comparative analysis of publicly availablegene dependency and drug sensitivity data (lymphoblasts.org) identified B-cell lymphoma as one of the most ferroptosis-sensitive tumor types. We uncovered that B-cell lymphomas are highly enriched in PUFA and selectively dependent on pro-ferroptotic PUFA metabolism to maintain competitive fitness and lipid membrane properties, which endows them with an intrinsic vulnerability to ferroptotic cell death.

Results: Our comparative analyses of public drug screening data (CTD, GDSC) and a validation screen we performed uncovered that B-cell lymphomas are the most sensitive type of tumor to all evaluated ferroptosis inducers, including GPX4 inhibitors (RSL3, ML210), the inhibitor of the cystine/glutamate antiporter system X_c^- erastin, and the iron oxidizer FINO₂. To further understand the factors contributing to this ferroptosis vulnerability, we performed comparative analyses of CRISPR dependency screens from the Cancer Dependency map. Unexpectedly, this approach uncovered ACSL4, a key enzyme in PUFA-PL production, as a selective B-cell dependency. This was unexpected because ACSL4 induces sensitivity to ferroptosis through production of long-chain PUFA-PL, especially containing arachidonic acid. In line with this surprising dependency, comparative analysis of Cancer Cell Line Encyclopedia metabolomics data showed a marked enrichment in multiple types of PUFA in B-cell lymphomas. Similarly, data from the Immunological Proteomic Resource showed that ACSL4 is one of the most upregulated proteins upon B-cell activation, suggesting a key role of PUFA metabolism during increased energetic demands. Furthermore, high expression of ACSL4 was associated with decreased overall survival in the MMMLNP diffuse large B-cell lymphoma clinical trial cohort, while the opposite trend was observed with expression of ACSL3, which counteracts PUFA-PL by driving metabolism of anti-ferroptotic monounsaturated fatty acids. A PUFA-rich gene expression signature strongly predicted prognosis, as indicated by a hazard ratio for mortality of 2.484 in ACSL4_high/ACSL3_low individuals (95% confidence interval: 1.709 – 3.609; measured by log-rank test). In line with the key role of PUFA in ferroptosis regulation, our whole-genome CRISPR knockout screen performed under the selective pressure of RSL3 showed that ACSL4 is one of the key genes promoting ferroptosis sensitivity in B-cell malignancies, while the opposite was seen with ACSL3. These findings suggest that, while B-cell lymphomas are dependent on PUFA metabolism, this dependence might represent a vulnerability making them highly sensitive to ferroptosis.

To evaluate the roles of ACSL4 and PUFA-PL in B-cell lymphomas, we utilized CRISPR-mediated homology-directed repair to knock-in an ACSL4 degradation tag (dTAG) in B-cell lymphoma cell lines. dTAG induction led to complete loss of ACSL4 within one hour and led to progressive increase in resistance to lipid peroxidation, consistent with loss of PUFA-PL at cell membranes. While loss of ACSL4 promoted ferroptosis resistance, it also led to cell depletion in competitive growth assays, indicating loss of competitive fitness upon PUFA-PL depletion. Molecular biophysics experiments showed that ACSL4 loss and resulting PUFA-PL depletion increases membrane flow resistance, as measured by an increase in membrane cytoskeleton attachment (9.246 ± 1.43 x 10⁵ vs 5.141 ± 1.57 x 10⁵ pN³s/μM for ACSL4 loss vs negative control, p = 0.00015). PUFA-PL loss thus compromises membrane properties of B-cell lymphomas, which might affect key membrane-dependent functions.

Conclusions: We show that B-cell lymphomas are selectively dependent on PUFA metabolism to maintain membrane properties and competitive fitness. However, this intrinsic metabolic dependency is a key factor making them highly vulnerable to ferroptosis. Our findings also provide insight into B-cell lymphoma metabolism and lipid membrane dynamics, and how it could be leveraged as a therapeutic strategy to potently induce ferroptosis in B-cell lymphomas.