FSP1 and histone deacetylases suppress cancer persister cell ferroptosis

Abstract

Cancer persister cells which survive oncogene targeted therapies are sensitized to ferroptosis, but mechanistic understanding of this vulnerability remains limited. Here, we found that while levels of iron, glutathione, and various ferroptosis-suppressing enzymes vary among persister cell types, ferroptosis suppressor protein 1 (FSP1) is down-regulated in multiple persister cell types, and persister cells which survive glutathione peroxidase 4 (GPX4) inhibition rely on residual FSP1 to survive. Furthermore, persister cells which survive GPX4 inhibition down-regulate oxidative phosphorylation, a key source of mitochondrial reactive oxygen species which are required for persister cell ferroptosis. We also found that persister cell treatment with histone deacetylase inhibitors induces reactive oxygen species and sensitizes multiple persister cell types to GPX4 inhibition. Together, these findings reveal that FSP1 and histone deacetylases suppress persister cell ferroptosis.

Fig. 1.. OXPHOS contributes to ferroptosis sensitization in cancer persister cells.

(A) UMAP of PC9 parental and persister cells treated with or without 1 μM RSL3 for 24 hours. (B) Enriched Hallmarks gene sets in persister cells treated with or without RSL3. Positive normalized enrichment score (NES) values indicate enrichment in RSL3-treated persister cells. TNFα, tumor necrosis factor–α; NF-κB, nuclear factor κB; IL-6, interleukin-6; JAK, Janus kinase; STAT3, signal transducer and activator of transcription 3; UV, ultraviolet. (C) UMAP of PC9 persister cells treated with and without RSL3. (D) Pseudotime analysis of PC9 persister cells treated with and without RSL3. Solid black line indicates the estimated trajectory across cell states. (E) UMAP of PC9 persister cells treated with and without RSL3 colored by cluster. (F) Ferroptosis driver gene set signature score across clusters in (E). (G) Ferroptosis suppressor gene set signature score across clusters in (E). [(F) and (G)] P values calculated with Mann-Whitney test. (H) PC9 parental, persister, and persister cells treated with 500 nM RSL3 for 24 hours were analyzed for oxygen consumption rate (OCR). A total of 1.25 μM oligomycin (Oligo), 1 μM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and 1 μM rotenone plus 1 μM antimycin A (R + A). n = 3 biological replicates; mean ± SEM is shown; P values calculated between parental and persister cell conditions (stars) or between persister cells and RSL3-treated persister cells (crosses) using two-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ††P < 0.01, and †††P < 0.001. (I) PC9 parental and persister cells derived from 2.5 μM erlotinib analyzed for mitochondrial ROS. Par, parental; pers, persister. (J and K) PC9 persister cells derived from 2.5 μM erlotinib (J) and A375 persister cells derived from 250 nM dabrafenib and 25 nM trametinib (K) in combination with mitoTEMPO were treated with 500 nM RSL3 for 24 hours. Viability was normalized to the respective mitoTEMPO-treated persister cells without RSL3 treatment. [(I) to (K)] n = 3 biological replicates; mean ± SD is shown; P values calculated with two-tailed Student’s t test.

Fig. 2.. Persister cells have variable antioxidant deficiencies and depend on FSP1 to survive GPX4 inhibition.

(A) Parental and persister cells analyzed for NRF2, KEAP1, and system x_c⁻ components SLC7A11 and SLC3A2 expression. (B and C) A375 and PC9 parental and persister cells analyzed for reduced GSH or total GSH (GSSG) levels. (D) Protein expression of ferroptosis suppressor genes in parental and persister cells. (E) FSP1 mRNA expression in PC9 parental and persister cells treated with and without RSL3. P values calculated with the Wilcoxon rank sum test with Bonferroni correction. (F and G) PC9 (F) and A375 (G) persister cells cotreated with 1 μM FSP1 inhibitor, iFSP1, 50 nM RSL3, or both for 24 hours. (H and I) PC9 (H) and A375 (I) viability of parental cells and persister cells after treatment with the combination of 1 μM iFSP1 with 50 nM RSL3 for 24 hours. [(B), (C), and (F) to (I)] n = 3 biological replicates; mean ± SD is shown; P values calculated with two-tailed Student’s t test. ns, not significant.

Fig. 3.. HDAC inhibition synergizes with GPX4 inhibition to selectively kill persister cells.

(A to F) Synergy heatmaps between GPX4 inhibitor RSL3 and HDAC inhibitors panobinostat and vorinostat following 24-hour cotreatment. Bliss synergy score calculated with SynergyFinder 3.0. Red color and positive scores indicate synergy, and green color and negative scores indicate buffering. [(A) and (B)] PC9 parental cells and persister cells derived from 50 nM erlotinib. [(C) and (D)] A375 parental cells and persister cells derived from 10 nM dabrafenib with 1 nM trametinib. [(E) and (F)] BT474 parental cells and persister cells derived from 2 μM lapatinib. (G to J) Prederived PC9 or A375 persister cells were treated for 48 hours with a nontoxic concentration of HDAC inhibitor (see fig. S7), rinsed, and then treated with RSL3 for 24 hours while maintained under targeted therapy treatment. Data normalized to untreated persister cells. Concentration and HDAC inhibitor used were as follows: (G) 7.5 nM panobinostat, (H) 5 nM panobinostat, (I) 100 nM vorinostat, and (J) 1 μM vorinostat. RSL3 concentrations used were as follows: (G) 150 nM, (H) 100 nM, (I) 150 nM, and (J) 80 nM. n = 3 biological replicates; mean ± SD is shown; P values calculated with two-tailed Student’s t test.

Fig. 4.. HDAC inhibitors induce persister cell oxidative stress to sensitize persister cells to ferroptosis.

(A) UMAP of PC9 parental and persister cells treated with or without HDAC inhibitor panobinostat for 48 hours. (B) Enriched Hallmarks gene sets between persister cells treated with and without panobinostat. Positive NES values indicate gene sets enriched in persister cells treated with panobinostat. (C and D) Treatment with panobinostat does not decrease GSH levels in PC9 (7.5 nM panobinostat) or A375 (5 nM panobinostat) persister cells. Buthionine sulfoximine (BSO; 1 mM) was used as a positive control for GSH depletion. (E) Ferroptosis sensitization of PC9 persister cells from treatment with panobinostat is not inhibited by GSH ethyl ester (GSHee, 1 mM). (F) PC9 persister cell treatment with panobinostat decreases rather than increases intracellular iron. (G and H) Panobinostat treatment of PC9 persister cells increases total cellular ROS (G) and mitochondrial ROS (H). (I and J) PC9 persister cells derived from 2.5 μM erlotinib (I) and A375 persister cells derived from 250 nM dabrafenib and 25 nM trametinib (J) were cotreated with mitoTEMPO and were treated with 7.5 and 5 nM panobinostat, respectively, for 48 hours with and without 500 nM RSL3 for 24 hours. Viability was normalized to the viability of targeted therapy with mitoTEMPO and panobinostat without RSL3. [(C) to (J)] n = 3 biological replicates; mean ± SD is shown; P values calculated with two-tailed Student’s t test.

Fig. 5.. Enhancing persister cell ferroptosis with FSP1 and HDAC inhibition.

Cancer persister cells can decrease oxidative stress to survive GPX4 inhibition (GPX4i). However, GPX4i-tolerant persister cells become dependent on the alternative ferroptosis suppressor enzyme FSP1 to survive, and addition of FSP1 inhibitor (FSP1i) increases persister cell ferroptotic death. Furthermore, persister cell oxidative stress is increased by nontoxic pre- or cotreatment with clinically available HDAC inhibitors resulting in synergistic persister cell ferroptosis in combination with GPX4 inhibitor (GPX4i). Our findings reveal previously unexplored approaches to selectively enhancing persister cell ferroptosis. HDACi, histone deacetylase inhibitor. Created in BioRender. Wang, M. (2025) https://BioRender.com/7n2cetj.