Fig 2: Overexpression of KDM5D was associated with poor platinum responses in HNSCC patients. (A) Representative images of KDM5D staining in tissue specimens of TMU-SHH HNSCC cohort in respective order: normal adjacent epithelium, well-differentiated tumor, and poorly differentiated tumors. Higher KDM5D expression was noted in HNSCC tissues than in adjacent normal epithelial tissues. (B) The highest KDM5D expression was observed in poorly differentiated squamous cell carcinoma tissues. (C) Representative images of KDM5D staining in tissue specimens of TMU-SHH HNSCC cohort according to cisplatin response and recurrence disease. (D) Among the patients with HNSCC, KDM5D expression was higher in the platinum non-responders than in the responders. (E) Among HNSCC patients who responded to platinum-based chemotherapy, higher KDM5D expression was observed in those with early disease recurrence than those with no/late-recurrence. Significance level: * p < 0.05; *** p < 0.001; **** p < 0.0001. Scale bar: 200 μm.

Fig 3: KDM5D and AURKB co-expression delineates cluster of HNSCC persister cells. (A) Representative tSNE plots of single-cell profiling in GSE103322 dataset showed eight distinct clusters of cells. (B) Array of tSNE plots portrayed expression level of interest genes such as HNSCC tumor markers (KRT6A, KRT14, CDH1), CSCs and persister marker (ALDH1A3), putative main targets of this study (KDM5D and AURKB), and diapause-related genes (CCND1, FAS, ALDH6A1). (C) Dot plot described level of expression of each gene (KDM5D, ALDH1A3, AURKB, CCND1, FAS, ALDH6A1) in eight distinct clusters. (D) Scatter plot depicted co-association of each interest gene; Pearson’s coefficient and p-value was provided in the top margin. The KDM5D expression was significantly correlated to AURKB (r = 0.75, p = 0.012) and ALDH1A3 (r = 0.38, p = 0.035). (E) Scatter plot portrayed correlation between several diapause-related genes and KDM5D, such as CCND1 (r = 0.21, p = 0.037), FAS (r = 0.24, p = 0.021), and ALDH6A1 (r = 0.39, p = 0.018). (F) tSNE plot illustrates Diapause signature scores in each individual tumor cell. The Diapause_UP module score consisted of gene scores that are overexpressed during diapause, while Diapause_DOWN module score comprised genes that are downregulated at diapause stage. Persister cells were enriched among the clusters within the circle marker (red dash line). Predominant ALDH1A3 and KDM5D expression were noted in cell clusters no. 1, 3, and 5 which was consistent with diapause state activation. (G) The clusters, which were speculated to activate diapause state (clusters no. 1 and 3) exhibited several common features of diapause state, including the activation of NRF2, glutathione, drug metabolism, glycolysis, and epithelial-mesenchymal transition pathways.

Fig 4: KDM5D promotes the generation of platinum-tolerant persister cells. (A) Brief schematic figure shows the steps to generate cisplatin-tolerant persister cells. Parental HNSCC cells were treated with a short-course of cisplatin treatment followed by a ‘drug-holiday’ or recovery stage and a final cisplatin course. At the end of this stage, the HNSCC cells were relatively viable and exhibited increased platinum tolerance. (B) KDM5D knockdown significantly reduced the expression levels of AURKB and ALDH1A3 in both SAS and FaDu persister cells, indicating that KDM5D regulates AURKB and ALDH1A3 expression. (C) KDM5D knockdown significantly reduced tumor sphere formation in both PT-SAS and PT-FaDu cells. (D) Both platinum-tolerant persister HNSCC cells exhibited cell cycle arrest, as indicated by a significant increase in the G0/G1 subpopulation and a decrease in S and G2/M subpopulations. (E,F) KDM5D silencing re-sensitized platinum-tolerant persister cells upon cisplatin treatment, indicating that KDM5D plays a crucial role in promoting platinum tolerance in HNSCCs. Significance level: * p < 0.05; ** p < 0.01, *** p < 0.001. Scale bar: 100 μm.

Fig 5: Schematic illustration of KDM5D contribution to affect the clinical outcome and biological development of platinum-tolerant persister cells in HNSCC. Left panel schema shows association between high KDM5D expression and poor clinical outcome in HNSCC patients encompassing poor response to platinum treatment or early recurrence disease. Right panel schema illustrates development of platinum-tolerant persister cells characterized by high expression of KDM5D/AURKB axis which disrupts AURKB by barasertib treatment deregulated tolerance mechanism and promoted mitotic catastrophe in platinum-tolerant cells.