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 xc− 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 and A375 parental and persister cells treated with and without 5 μM FSP1 inhibitor. (H and I) PC9 and A375 persister cells co-treated with 1 μM FSP1 inhibitor, 50 nM RSL3, or both for 24 hours. (B, C, F to I) n = 3 biological replicates; mean ± s.d. is shown; P values calculated with two-tailed Student’s t-test.

Fig 3: 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 xc− 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 4: 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.