Abstract PR003: Ferritin-dependent iron storage and ferritinophagy impact ferroptosis resistance in chromophobe renal cell carcinoma

Abstract Introduction: Chromophobe renal cell carcinoma (ChRCC) is the second most common non-clear cell RCC, with metastatic disease associated with a median overall survival of ∼2 years. ChRCC is characterized by high basal glutathione levels and marked susceptibility to ferroptosis, an iron-dependent form of cell death driven by lipid peroxidation. Nuclear protein 1 (NUPR1), a transcription factor induced by metabolic and genomic stress, has recently emerged as a suppressor of ferroptosis. Ferritin, composed of heavy (FTH1) and light (FTL) chains, is the major intracellular iron-storage complex and regulates intracellular iron availability. The contribution of these iron-regulatory pathways to ferroptosis vulnerability have not been investigated in ChRCC. Methods: TCGA RNA-seq data and published single-cell RNA-Seq were analyzed. Cell viability was evaluated using crystal violet. Results: Across 33 cancer types in TCGA with matched tumor-normal RNA-seq data, ChRCC shows the highest expression level and greatest fold increase (5-fold) in NUPR1 relative to matched normal kidney. In a published single-cell RNA-seq dataset (Labaki et al. JCO 2025), ChRCC cells have significant upregulation of NUPR1 (11-fold), FTL (4.8-fold), and FTH1 (4-fold) compared to renal intercalated cells, the cell of origin of ChRCC. NUPR1 protein was detectable in most ChRCC cell lines and expressed at the highest level in RCJ-M, the only cell line derived from a metastatic ChRCC. siRNA-mediated NUPR1 knockdown reduced ferritin protein expression in RCJ-M cells, suggesting that NUPR1 contributes to ferritin regulation in ChRCC. Although no NUPR1-ferritin link is known in ChRCC, NUPR1 overexpression in hepatocellular carcinoma increases the expression of iron-handling proteins including FTH1. To evaluate how ferritin turnover influences ferroptosis sensitivity in ChRCC cells, lysosome-dependent ferritinophagy was inhibited using 10 μM chloroquine. UOK276 cells were treated for 24 hours, followed by assessment of ferritin accumulation and ferroptotic responses. Chloroquine effectively inhibited autophagy, as indicated by increased p62, and ferritinophagy, as indicated by increased FTH1 protein levels. Notably, chloroquine treatment also increased the protein expression of the ferroptosis-protective proteins GPX4 and SLC7A11. To assess ferroptotic cell death induced by the GPX4 inhibitor RSL3, UOK276 were treated with either 10μM chloroquine + 15nM RSL3 or 2nM bafilomycin A1 + 15nM RSL3. Both lysosomal inhibitors completely rescued UOK276 cells from RSL3-induced ferroptosis (p < 0.0001). Conclusion: These data reveal a role for NUPR1 in regulating ferritin-mediated iron storage and ferroptosis resistance in ChRCC. Since NUPR1 suppresses ferroptosis and ferritin limits iron-catalyzed lipid peroxidation, NUPR1 may influence ferroptosis resistance in part through effects on ferritin expression. In the ChRCC TCGA, high FTH1 expression is associated with worse disease-free and overall survival, supporting the potential translational relevance of the NUPR1-ferritin pathway in ChRCC. Citation Format: Wafaa Bzeih, Tiegang Han, Michel Alchoueiry, Damir Khabibullin, Jessalyn M. Ubellacker, Carmen Priolo, Elizabeth P. Henske. Ferritin-dependent iron storage and ferritinophagy impact ferroptosis resistance in chromophobe renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Kidney Cancer Research: From Molecular Insights to Therapeutic Breakthroughs; 2026 Mar 13-16; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_2):Abstract nr PR003.