Fig. 10. Loss of senescence-inducing and amplification of senescence-inhibiting cell cycle regulator genes in melanoma metastases of patients resistant to ICB.

a–c Sequencing data from metastases of non-responder patients (N = 30) versus metastases of responder patients (N = 12). In non-responder patients disease progressed within 3 months of ICB therapy. In responder patients metastases regressed ≥1 year of ICB therapy. Tumour mutational burden (TMB, copy number variations) (a). Number of tumour-specific alterations in 19 genes of the cell cycle, JAK or MYC pathway (b). Number of genes with homozygous deletions in cycle inhibitors or amplifications ≥ 3fold in cell cycle promoters. Genes analysed: CDKN2A/B/C, CDKN1A/B, RB1, TP53, JAK1/2/3, CCND1/2/3, CDK4/6, CCNE1, MDM2/4, MYC. We performed paired panel sequencing of the tumour DNA and of normal DNA to identify tumour-specific alterations. Box plots show the median with 25th and 75th interquartile range (IQR), and whiskers indicate 1.5 × IQR (c). d, e Growth curves of patient derived melanoma lines from two metastases of non-responder patients (d) or from two metastases of responder patients (e). Cells were cultured for the senescence assay either with medium (Ctr) or with medium containing 100 ng ml⁻¹ IFN-γ and 10 ng ml⁻¹ TNF for 96 h, washed and then cultured with medium for another 4–6 days. d, e Apoptosis assay with melanoma cells derived from two non-responder (d, including gating strategy) or two responder patients (e) were exposed to either medium (Ctr) or etoposide (Eto, 100 µM) or staurosporine (Sta, 0.5 µM) for 24 h for apoptosis induction and then stained with propidium iodide. SubG₁ cells were detected by flow cytometry analysis; data show the geometric mean with geometric SD, n = 3. Significance tested by using two-tailed Mann-Whitney test (a–c).