Intermittent dietary methionine deprivation facilitates tumoral ferroptosis and synergizes with checkpoint blockade

The study addresses how dietary methionine deprivation modulates tumor ferroptosis and interacts with cancer immunotherapy. Methionine, an essential amino acid central to one‑carbon metabolism, supports glutathione (GSH) production, nucleotide and polyamine synthesis, and methyl donation via S‑adenosylmethionine, and also controls translation initiation. Many cancers exhibit methionine dependence, motivating dietary methionine restriction as a therapeutic strategy that has previously enhanced responses to chemotherapy and radiotherapy. Ferroptosis is a non‑apoptotic cell death driven by lethal lipid peroxidation when GPX4 function is compromised or intracellular GSH is depleted. Cystine uptake via system x_c⁻ supplies cysteine for GSH synthesis; blocking cystine uptake or cystine deprivation induces ferroptosis. Methionine can contribute cysteine via transsulfuration, and prior reports showed methionine deprivation can either promote or suppress ferroptosis depending on context. The interplay between ferroptosis and antitumor immunity is increasingly appreciated: CD8⁺ T cells can sensitize tumor ferroptosis, and ferroptosis can be immunogenic, synergizing with checkpoint blockade. The authors hypothesize that the timing of methionine deprivation differentially regulates ferroptosis, potentially via CHAC1‑mediated GSH degradation, and that intermittent dietary methionine deprivation may potentiate ferroptosis and immunotherapy in vivo.

- Methionine restriction has shown tumor growth suppression and sensitization to apoptosis‑inducing chemotherapy and radiation across models. - Ferroptosis is prevented by GPX4 using GSH; blocking cystine uptake (system x_c⁻) or GPX4 induces ferroptosis. Cystine deprivation causes GSH depletion; methionine contributes to GSH via transsulfuration. - Reports conflict on methionine deprivation’s effect on ferroptosis: some show promotion (via BTG1 induction), others suppression (potentially via proliferation arrest). - CHAC1 is a γ‑glutamyl cyclotransferase that specifically degrades intracellular GSH to 5‑oxoproline and Cys‑Gly. It is transcriptionally induced through the eIF2α/ATF4 pathway under amino acid starvation and ER stress and has been implicated in ferroptosis/necroptosis. - CD8⁺ T cells can enhance tumor ferroptosis (e.g., via IFNγ signaling and lipid metabolism changes), and ferroptosis can be immunogenic, synergizing with immune checkpoint blockade (ICB) and various ferroptosis‑inducing agents. These prior findings set the stage for testing whether methionine deprivation timing modulates CHAC1, GSH homeostasis, ferroptosis, and ICB synergy.

- Cell lines and culture: Human tumor cell lines (e.g., HT‑1080, OS‑RC‑2, Caki‑1, 786‑O, ACHN, SNU‑182, SNU‑387, HT‑29; Hep3B) and mouse lines (Hepa1‑6, MC38, Panc02, B16F10, NIH‑3T3). Media prepared to selectively deplete cystine and/or methionine; short‑term methionine starvation (St‑Met) defined as ≤8 h in methionine‑free medium followed by cystine deprivation or drug treatment. - Ferroptosis perturbations: Cystine withdrawal; pharmacologic system x_c⁻ inhibition with erastin or IKE; GPX4 inhibition with RSL3; ferroptosis inhibition with ferrostatin‑1 or liproxstatin‑1. - Genetic manipulations: CRISPR/Cas9 knockout (sgRNAs) or shRNA knockdown of CHAC1, ATF4, and GCN2; generation of stable, low‑level CHAC1‑Flag overexpression lines; Tet‑On doxycycline‑inducible CHAC1 lines. - Assays: - Cell death by PI or 7‑AAD flow cytometry. - Lipid peroxidation with BODIPY 581/591 C11 flow assay; MDA tissue assay; 4‑HNE IHC. - GSH/GSSG quantification (luminescent assay in cells; colorimetric assay in tissues). - LC‑MS metabolomics (148 metabolites across 32 classes) under amino acid deprivation conditions; isotopic tracing with 13C‑cystine and 34S‑methionine (and 2H8‑methionine) to assess contributions to cysteine/GSH pools and degradation dynamics. - Western blot for CHAC1, p‑eIF2α, eIF2α, ATF4, Flag; qPCR for CHAC1, PTGS2, HMOX1, TFRC and controls; CHX chase to assess CHAC1 protein stability; ActD/CHX tests for transcription/translation requirement. - Mouse models: - Hepa1‑6 in C57BL/6 with sustained methionine‑free vs control diet; IKE ± liproxstatin‑1 to probe in vivo ferroptosis requirement. - B16F10 melanoma in C57BL/6 with intermittent dietary methionine deprivation (methionine‑free periods with methionine re‑supplementation), with/without IKE, anti‑PD‑1, and liproxstatin‑1; serum methionine measured to verify dietary modulation. - CHAC1‑deficient vs WT B16F10 tumors for dependence of therapy on tumoral CHAC1. - Triple‑combination regimens: intermittent methionine deprivation + low‑dose IKE + anti‑PD‑1; tumor growth and survival monitored. - CTL co‑culture assays: OVA/Luc B16F10 co‑cultured with activated OT‑I CD8⁺ T cells; tumor viability by luciferase readout, tumor death by CFSE/7‑AAD flow, lipid ROS in tumor cells; effects of St‑Met, CHAC1 loss/gain tested; OT‑I supernatant applied to tumor cells to assess CHAC1 induction. - Immune profiling: Flow cytometry of TILs for CD8⁺/CD4⁺ frequencies and IFNγ/TNFα production after treatments. - Clinical data analyses: Public melanoma cohorts receiving anti‑PD‑1 or anti‑PD‑1+anti‑CTLA‑4; baseline tumoral CHAC1 expression vs response and survival (violin plots, Kaplan–Meier). - Statistics: t‑tests/Mann–Whitney, ANOVA with corrections, Kaplan–Meier/log‑rank; GraphPad Prism.

- Prolonged methionine deprivation suppresses ferroptosis induced by cystine depletion/system x_c⁻ inhibition in vitro and in vivo, whereas short‑term methionine starvation (<8 h) enhances ferroptosis. - Mechanism: Cystine deprivation triggers CHAC1 induction via the GCN2–eIF2α–ATF4 pathway, accelerating GSH degradation and ensuring ferroptosis onset. CHAC1 knockout/knockdown attenuates GSH depletion and ferroptotic death; CHAC1 overexpression lowers basal GSH, further depletes GSH even under double deprivation, and can be sufficient to induce ferroptosis. - GSH threshold: Time‑course matching showed ferroptotic death occurs when intracellular GSH falls to ≤6% of baseline in HT‑1080 or ≤17% in Hepa1‑6; modest GSH restoration above threshold rescues cell viability. - Metabolomics and isotope tracing: Cystine is the dominant source for intracellular cysteine and GSH; methionine contributes minimally via transsulfuration in HT‑1080. Cystine deprivation reduces 13C‑labeled cysteine and GSH and decreases 34S‑labeled GSH without lowering 34S‑cysteine, indicating enhanced GSH degradation contributes to GSH loss. - Prolonged methionine deprivation blocks CHAC1 at the translational level (despite increased CHAC1 mRNA), preventing GSH depletion beyond death threshold; CHX recapitulates ferroptosis inhibition by blocking CHAC1 synthesis. - Short‑term methionine starvation increases CHAC1 transcription/protein (via eIF2α/ATF4), augments GSH depletion and lipid peroxidation, and sensitizes multiple tumor lines to cystine deprivation/IKE in vitro. - In vivo: - Sustained methionine‑free diet abrogates IKE antitumor efficacy and reduces tumoral lipid peroxidation and ferroptosis markers; intermittent methionine deprivation reduces serum methionine rapidly and, combined with IKE, nearly halts B16F10 tumor growth, increases MDA, PTGS2/HMOX1/TFRC, elevates CHAC1 mRNA, and lowers tumoral GSH. - Intermittent methionine deprivation synergizes with anti‑PD‑1 to reduce tumor burden; effects are attenuated by liproxstatin‑1, implicating ferroptosis. Combination increases CD8⁺ T cell infiltration and IFNγ/TNFα production. - CHAC1 mediates CTL sensitivity: St‑Met sensitizes B16F10 to OT‑I killing and lipid peroxidation; CHAC1 loss confers resistance to CTL cytotoxicity, whereas CHAC1 overexpression (including doxycycline‑inducible) enhances CTL‑induced lipid ROS and death. OT‑I supernatant induces tumoral CHAC1 mRNA. - Tumor CHAC1 is required for therapeutic synergy: CHAC1‑deficient tumors show impaired response to St‑Met + anti‑PD‑1 and resist anti‑PD‑1 monotherapy. - Clinical correlation: Higher baseline tumoral CHAC1 associates with response to PD‑1/PD‑1+CTLA‑4 blockade and with improved overall and progression‑free survival in melanoma cohorts. - Triple combination (intermittent methionine deprivation + low‑dose IKE + anti‑PD‑1) provides superior tumor control, higher lipid peroxidation, and improved survival compared to doublet regimens.

The work demonstrates that the timing of methionine deprivation dictates ferroptosis outcomes through differential CHAC1 regulation. Cystine deprivation alone induces CHAC1 via GCN2–eIF2α–ATF4, promoting GSH degradation to push GSH below a death threshold and trigger ferroptosis. Prolonged methionine deprivation inhibits translation, blocking CHAC1 protein accumulation despite elevated mRNA, thereby preventing excessive GSH depletion and ferroptosis. Conversely, short‑term methionine starvation stimulates CHAC1 transcription without impairing translation, enhancing GSH degradation and ferroptosis sensitivity. Translating this to vivo, intermittent but not sustained dietary methionine deprivation increases tumoral ferroptosis and synergizes with IKE. Importantly, ferroptosis contributes to antitumor immunity: intermittent methionine deprivation augments PD‑1 blockade efficacy, increases T cell infiltration and function, and these benefits are reversed by ferroptosis inhibition. CHAC1 in tumor cells is a critical node linking metabolic stress, ferroptosis, and immunity; its loss confers CTL and immunotherapy resistance, whereas higher expression correlates with clinical benefit to checkpoint blockade. Thus, dietary modulation offers a non‑pharmacologic lever to sensitize tumors to ferroptosis and to potentiate immune checkpoint therapy.

This study identifies CHAC1‑mediated GSH degradation as a key determinant of ferroptosis onset under cystine deprivation and reveals that methionine deprivation exerts opposing effects on ferroptosis depending on duration: prolonged deprivation inhibits CHAC1 translation and ferroptosis, while short‑term starvation induces CHAC1 and sensitizes ferroptosis. Intermittent dietary methionine deprivation enhances tumoral ferroptosis, improves CTL‑mediated cytotoxicity, synergizes with PD‑1 blockade, and in combination with a system x_c⁻ inhibitor yields superior antitumor efficacy. Tumoral CHAC1 emerges as both a mechanistic driver and a potential biomarker of response to immunotherapy. Future work should optimize timing/dosing regimens of methionine deprivation in humans, validate CHAC1 as a predictive biomarker across cancers, and assess safety and efficacy of triple‑combination strategies in clinical settings.

- Predominant reliance on murine models (Hepa1‑6, B16F10) and subcutaneous tumors may limit generalizability to human cancers and spontaneous tumor microenvironments. - Dietary regimens (duration, intensity, re‑supplementation schedules) optimized in mice may not directly translate to humans; systemic tolerability and long‑term safety were not fully evaluated. - Effects on immune cells: while short intermittent deprivation appeared compatible with T cell function, prolonged methionine deprivation can impair effector T cells; precise therapeutic windows need definition. - Mechanistic focus on CHAC1 does not exclude contributions from other GSH metabolism or stress‑response pathways; comprehensive omics under different deprivation timings could uncover additional mediators. - Clinical correlations for CHAC1 are retrospective and limited to melanoma cohorts; prospective validation across tumor types receiving checkpoint blockade is needed.