Qing Yin Zheng

Figure S3 from Cisplatin-Induced APE2 Overexpression Disrupts MYH9 Function and Causes Hearing Loss

LS/MS analyses of APE2 pull-down proteins

APE2 knockdown mitigates cisplatin-induced apoptosis in vitro.A, Knockdown of APE2 by transfection of APE2 ASO and GFP ASO as control ASO gapmers in HEI-OC1 cells. B, Flow cytometry dot plots showing Annexin V and propidium iodide staining 24 hours after transfection. The experiments shown were performed independently 3 times (n = 3) with consistent results. Representative data from one experiment are presented. C, Frequency of healthy (double negatives), early apoptotic (annexin V+/PI−), late apoptotic (annexin V+/PI+), and necrotic (annexin V−/PI+) HEI-OC1 cells 24 hours after transfection. D, Confluence of cultures in 96-well plates seeded with 24 hours–transfected HEI-OC1 cells over 72 hours. E, Frequencies, normalized to respective saline controls, of healthy, early apoptotic, late apoptotic, and necrotic cells among transfected HEI-OC1 cells treated with cisplatin at indicated concentrations for 24 hours. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure S2 from Cisplatin-Induced APE2 Overexpression Disrupts MYH9 Function and Causes Hearing Loss

APE2 ASO restores mitochondrial respiration impaired by cisplatin treatment. A, Seahorse XF analysis of the mitochondrial OCR in cells transfected with control GFP ASO or APE2 ASO. Key parameters measured include basal respiration, ATP-linked respiration, maximal respiration, reserve capacity, proton leak, and nonmitochondrial respiration. B, Seahorse XF analysis of the OCR in cells treated with cisplatin following transfection with GFP ASO or APE2 ASO. Data represent mean ± SEM. Statistical analysis was performed using the unpaired two-tailed Student t test; NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001..

Figure S4 from Cisplatin-Induced APE2 Overexpression Disrupts MYH9 Function and Causes Hearing Loss

Cisplatin treatment results in APE2 upregulation and mitochondrial dysmorphology in cochlear hair cells. A, IHC analysis of APE2 and MYH9 expression in cochlear tissues from patients with C-HL (n = 2) and normal donors (n = 1). OHCs are highlighted using blue arrow heads. Scale bar, 50 μm. B, Immunoblot analysis of APE1, APE2, and MYH9 expression in HEI-OC1 cells after 24 hours of cisplatin treatment at indicated concentrations. C, Z-stack compressed images of HEI-OC1 cells treated with 1,000 ng/mL cisplatin for 6 hours at indicated concentrations, showing 4',6-diamidino-2-phenylindole (DAPI) (blue), APE2 (green), and ATP5A/mitochondria (red). D, Immunoblot of subcellular cytosolic and mitochondrial protein fractions of HEI-OC1 cells after treatment with 1,000 ng/mL cisplatin for indicated times. ATP5A mitochondrial marker is shown. The experiments shown in B–D were performed independently 3 times (n = 3) with consistent results. Representative data from one experiment are presented. H&E, hematoxylin and eosin.

APE2 inhibits p53 signaling and provokes apoptosis. HEI-OC1 cells were transfected with indicated ASO 24 hours prior to further treatments. A, Cells were treated with 1 μg/mL or 5 μg/mL cisplatin for 6 hours prior to collection of total cellular protein for immunoblot analysis. B, Cells were treated with 5 μg/mL cisplatin for 6 hours prior to isolation of cytosolic and mitochondrial protein fractions for immunoblot analysis. Confocal microscopy analysis of HEI-OC1 cells treated with 1,000 ng/mL cisplatin for 24 hours, showing (C) DAPI (blue), p53 (green), and ATP5A/mitochondria (red) colocalization (scale bar, 5 μm), and (D) DAPI (blue), cytochrome C (green), and ATP5A/mitochondria (red) colocalization (scale bar, 3 μm). E, RT-qPCR analysis of expression of selected p53-associated genes in cells treated with 1,000 ng/mL cisplatin for 24 hours, normalized to β-actin and shown as fold change compared with untreated, anti-GFP transfected cells. The experiments shown were performed independently 3 times (n = 3) with consistent results. Representative data from one experiment are presented. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Scale bars, 10 μm.

Cisplatin remains a cornerstone chemotherapy for many solid tumors but is limited by dose-limiting toxicities, including nephrotoxicity, peripheral neuropathy, and ototoxicity—the latter of which disproportionately affects pediatric patients and lacks effective prevention strategies. Although therapeutic approaches to mitigate cisplatin-induced toxicity are urgently needed, the underlying mechanisms driving organ-specific injury remain incompletely understood. We previously identified apurinic/apyrimidinic endonuclease (APE) 2 as a critical mediator of cisplatin-induced acute kidney injury through disruption of mitochondrial integrity. In this study, we extend these findings to cisplatin-induced hearing loss (C-HL). We demonstrate that cisplatin selectively induces APE2, but not APE1, overexpression in murine and human outer hair cells. Using an inducible, outer hair cell–specific APE2 transgenic mouse model, we show that APE2 overexpression alone is sufficient to cause high-frequency hearing loss, accompanied by hair cell loss and stereocilia disorganization visualized by electron microscopy. Mechanistically, we identified a direct interaction between APE2 and MYH9, mapped the critical MYH9-binding domains, and demonstrated that APE2 knockdown preserved mitochondrial metabolism and protected cochlear cells from cisplatin-induced apoptosis. Notably, APE2 depletion activated an ATR–p53 signaling axis, promoting nuclear p53 localization and suppressing mitochondrial apoptotic pathways. Together, these findings reveal a noncanonical, APE2-dependent mechanism driving C-HL and suggest that targeting APE2 may offer a novel therapeutic strategy to prevent cisplatin-induced ototoxicity.Significance:These results reveal an unexpected role of APE2 via its interaction with MYH9, emphasizing the therapeutic promise of targeting APE2 for preventing C-HL in patients with cancer.