Fig. 1: Palbociclib enhances Rt3D cytotoxicity and virus defence response.

A A375 were treated with Rt3D (MOI 1) and 80 compounds at various doses. Low Z scores (below −2, dashed line) indicate sensitisers (palbociclib is shown in red, with relative Z scores that represent PD-0332991 doses 1 μM (−2.041) and PF-332991 doses at 1 μM (−5.076) and 0.5 μM (−5.570)). B Cell viability in A375 cells treated with Rt3D ± palbociclib (1 μM) by MTT (left) and crystal violet (right) 72 h after treatment (data are presented as mean values ± SEM, n = 3 biologically independent experiments). C Cell viability in melanoma cells MeWo (BRAF/RAS wild-type), and DO4 (Ras mutant) treated with Rt3D ± palbociclib (1 μM) by MTT (72 h). D Cell viability measured at 72 h by MTT assay in 4434 cells treated ∓ palbociclib (1 μM) (data are presented in (C, D) as mean values ± SEM, n = 3 biologically independent experiments). E Volcano plot of RNA sequencing showing upregulated and downregulated genes for A375 cells treated with palbociclib (1 μM), Rt3D (MOI 0.1) or the combination compared to basal at 48 h. Highlighted genes in red belong to the GSEA set ‘reactome interferon signalling’. F RT-qPCR of IFNa, IFNb, IL-28, IL-29, ISG15 and OASL in A375 cells treated with Rt3D ± palbociclib (1 μM) at 48 h. Data presented are mean values ± SEM, n = 3 biologically independent experiments. P values were determined by one-way ANOVA corrected for multiple comparisons. G RT-qPCR of mouse IFNα and IFNβ in murine BRAF-mutant melanoma 4434 cells treated with Rt3D ± palbociclib (1 μM) at 48 h. Data are presented as mean values ± SEM, n = 3 biologically independent experiments. P values were determined by one-way ANOVA corrected for multiple comparisons. H Proteomic analysis of A375 cells treated with Rt3D (0.1) in combination with palbociclib (1 μM) reveal upregulated (red) and downregulated (blue) proteins categorised under the GO term ‘defence response to virus’ (24 h, data are from 1 independent experiment). I IFNα and IFNβ, measured in cell-free supernatant from Rt3D-treated samples (MOI 0.05-0.5) ± palbociclib (1 μM) by ELISA. Results from 2 independent experiments. The lines joining the open and closed circles indicate the data points derived from the same experiment. J Tumour volumes for animals bearing A375 tumours treated with either a sham injection with vehicle (n = 13), an intra-tumoural injection of Rt3D (1 × 10⁶ pfu, n = 11), palbociclib (n = 10), or the combination (n = 9). Tumour volumes for C57BL/6 mice bearing murine BRAF-mutant 4434 tumours were treated with a sham injection with vehicle (n = 6), an intra-tumoural injection of Rt3D (1 × 10⁶ pfu, n = 9), palbociclib (n = 6), or the combination (n = 8). Data are presented as mean values ± SEM, and p values were derived from one-way ANOVA adjusted for multiple comparisons from area under curve values from individual mice, where p > 0.05 ns, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Source data are provided as a Source Data file.