Figure 5 from INX-315, a Selective CDK2 Inhibitor, Induces Cell Cycle Arrest and Senescence in Solid Tumors

INX-315 treatment of CDK4/6i–resistant breast cancer reinstates a senescence phenotype. A, Representative images (top) and quantification (bottom) of staining MCF7 and T47D cells for beta-galactosidase activity after treatment with agents shown (500 nmol/L abemaciclib, INX-315: 300 nmol/L for MCF7 and 100 nmol/L for T47D) for 7 days (scale bar = 100 μm). Resistant cells were cultured continuously in drugs to which they were resistant (500 nmol/L abemaciclib ± 100 nmol/L fulvestrant). Experiments performed with three technical replicates. Quantification is for integrated beta-galactosidase signal per cell. Dashed line indicates cutoff used to define beta-galactosidase positivity. B, Representative images (top) and quantification of nuclear size (bottom) upon staining MCF7 and T47D cells with DAPI and phalloidin after treatment as in A (scale bar = 100 μm). Experiments performed with three technical replicates. C, Heat map shows z-scores for ssGSEA scores for four senescence-associated gene signatures, calculated from RNA-sequencing of MCF7 and T47D cells treated as in A. Bar plot above shows mean z-score for each sample across all signatures. Three technical replicates for all conditions except abemaciclib-resistant MCF7 in abemaciclib, which had two technical replicates. D, Heat map as in C, but for RNA-sequencing from abemaciclib-resistant MMTV-rtTA/tetO-HER2 tumors treated as in Fig. 4E. Six samples for vehicle and INX-315, five samples for abemaciclib and combination. E, Violin plots showing log₂-transformed normalized ATAC-seq counts for genomic regions that significantly increased chromatin accessibility after abemaciclib treatment of parental cells. Cells treated as in A, two technical replicates. F, Scatter plots showing significant enrichment of AP-1 motifs in the ATAC-seq up peaks after treatment with drugs as in A. G, Heat map showing z-scores for individual SASP genes (RNA-sequencing) in MCF7 cells treated as in A. Bar plot above shows mean z-score for all genes in each sample. Three technical replicates for all conditions except abemaciclib-resistant MCF7 in abemaciclib, which had two technical replicates. H, Representative ATAC-seq tracks at regions near SASP genes (IGFBP3 and VEGFA) in cells treated as in A (all P values calculated using unpaired t tests except F, determined using HOMER package).