Fig 2: Heme-metabolism genes are regulated by KDM4C and GATA1. A Top six KDM4C-correlated pathways from GSEA analysis of KDM4C knockdown (KD) RNA-seq result. B KDM4C-KD SAS cells enrich for gene signatures characteristic of heme-metabolism loss, as identified by mRNA-seq followed by GSEA. C Enrichr analysis of key transcription factors in heme-metabolism genes derived from (A). D Global binding profile of KDM4C at transcription start sites (TSSs) within ±3 kb regions, visualized as a heatmap and peak density plot derived from KDM4C CUT&Tag-seq analysis. The heatmap highlights the binding intensity of KDM4C across TSSs, with color gradients representing varying binding levels. Data visualization was generated using the Galaxy plot heatmap tool, allowing for a clear illustration of KDM4C enrichment at promoter regions, indicative of its regulatory role in gene expression. E Identification of heme metabolism genes regulated by KDM4C. The Venn diagram depicts the overlap between KDM4C-downregulated genes and KDM4C-bound promoter genes. The intersecting set represents KDM4C-regulated genes involved in heme metabolism. An Enrichr analysis (https://maayanlab.cloud/Enrichr/) was performed on these genes to identify consensus transcription factors, with GATA1 emerging as the top-ranking transcription factor, highlighted in the enrichment bar chart to indicate its pivotal role in the KDM4C regulatory network. F KDM4C (this study), GATA1 (SRX4172742_HCT116), and H3K9me3 (ENCFF794 WNF) at proximal promoter regions of FECH and E2F2. G, H Analysis of occupancy changes at the promoter region of FECH (G) and E2F2 (H) via ChIP-qPCR. ChIP was conducted with specific antibodies (anti-GATA1 and anti-H3K9me3) in LKO and KDM4C-KD SAS cells. I, J The relative mRNA levels of heme-metabolism genes (FECH and E2F2) in the LKO and KDM4C-KD SAS cells. Data in (G–J) are represented in individual points and mean. P-values are determined by one-way ANOVA with Tukey’s multiple comparisons test (G–I), or two-way ANOVA with Tukey’s multiple comparisons test (J). *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant

Fig 3: Effect of FECH overexpression on invasion and proliferation in KDM4C-knockdown SAS and FaDu cells. A, B Analysis of FECH expression in LKO and KDM4C-knockdown (shKDM4C#1 or #2) SAS (A) and FaDu (B) cells. Cells were transfected with control or Flag-FECH expression vector, followed by Western blot analysis. C, D Invasion assay of LKO and FECH-overexpressing KDM4C-KD SAS (C) and FaDu (D) cells. E, F MTT cell proliferation assay of LKO and FECH-overexpressing KDM4C-KD SAS (E) and FaDu (F) cells at indicated time points. Data in (C, D) are represented in individual points and mean, and data in (E, F) are mean ± SD. P-values are determined by one-way ANOVA with Tukey’s multiple comparisons test (C, D) and two-way ANOVA with Tukey’s multiple comparisons test (E, F). *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant

Fig 4: Effects of KDM4 inhibitors on heme metabolism and tumor growth in HNSCC. A, B SAS-LN cells were treated with varying concentrations of myricetin (A) or 22S0 (B) for 3 days. Cell survival rates were measured using the MTT assay and are shown as dose-response curves. C, D Analysis of H3K9me3 levels in inhibitor-treated SAS-LN cells for 3 days. The H3K9me3 signals were detected by Western blot analysis. E Relative mRNA levels of heme metabolism genes (FECH and E2F2) in SAS-LN cells following 24-hour treatment with control (DMSO, 0.1%), myricetin (12.5 µM), or 22S0 (6.25 µM). F Representative images of SAS-LN cells xenografted in zebrafish and treated with drugs. Treatments include control (DMSO, 0.1%), myricetin (11.07 µM), 22S0 (6.17 µM), and docetaxel (0.38 nM). Scale bar: 200 µm. G Quantification of cell migration of SAS-LN cells under various drug treatments from (F). Each data point represents the percentage of embryos exhibiting tumor cell migration at 3 dpi in one of three independent biological experiments. The total number of embryos analyzed per group is as follows: control (n = 33), myricetin (n = 41), 22S0 (n = 32), and docetaxel (n = 31). H‒J SAS cells (1×106 cells) were subcutaneously injected into the BALB/cAnN.Cg-Foxn1nu/CrlNarl mice. When the tumors had grown to approximately 100 mm3, the mice received treatment. They were given two injections per week, with either 75 mg/kg of 22S0 or a control vehicle solution (DMSO/PEG300/PBS), administered via intra-tumor injection. Tumor volumes (H) and body weights (I) were measured at each treatment session, and tumor weights were measured at sacrificed endpoint (J). Data in (E, G, J) are represented as individual points and mean. Data in (A, B) are represented in mean ± SD, and data in (H, I) are mean ± SEM. P-values are determined by two-way ANOVA with Tukey’s multiple comparisons test (E, I), one-way ANOVA with Tukey’s multiple comparisons test (G), and two-tailed Student’s t–test (J). *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant