Figure 1.

INK4–CDK6 complex promotes resistance to CDK4/6i in cells. A, Schematic for analysis of CDK4 and CDK6 interactions and activity via coimmunoprecipitation (co-IP) followed by ADP-Glo kinase assays and mass spectrometry. B, ADP-Glo kinase assay showing immunoprecipitated CDK4 and CDK6 (IP-CDK4 and IP-CDK6) kinase activity from MCF7 parental and CDK6-high cells [cells with FAT1 CRISPR knockout (CR) that have high CDK6 expression, previously shown to have resistance to CDK4/6i; ref. 8], with or without 100 nmol/L abemaciclib treatment. Data are shown as mean + SD of three biologically independent samples. P values were determined by unpaired two-sided Student t test. RLU, relative luminescence units. C, Venn diagram showing the number of unique proteins identified by mass spectrometry coimmunoprecipitated from IP-CDK4 and IP-CDK6 in FAT1-loss cells. Percentages were calculated by number of proteins identified in each subgroup divided by total proteins identified by IP of either CDK4 or CDK6. Data are shown as means of three replicates. D, Pathway analysis by Gene Ontology of proteins interacting with CDK6 but not CDK4 in the FAT1-loss cells. The proteins were grouped by their putative biological functions. E, Unique peptide counts of cyclin-dependent kinases and their endogenous inhibitor proteins identified in the co-IP/mass spectrometry associated with CDK4 or CDK6 in the FAT1-loss cells. N = 2. F, Co-IP and immunoblotting reveal association of p15^INK4B and p18^INK4C with CDK6, but not CDK4, in CDK6-high cells. G, Cell line screening results showing that models with high CDKN2A or low RB1 mRNA expression are correlated with poor response to palbociclib. H, Interface residues in CDK6 in close proximity with INK4 isoforms based on previous INK4-bound CDK6 structures in the Protein Data Bank (ref. 65; no available structure for p15^INK4B). CDK6-HA was immunoprecipitated using HA beads in parental MCF7 cells and MCF7 cells expressing HA-WT-CDK6-, HA-V16D-, and R31C-mutant CDK6 (disrupted INK4–CDK6 interaction) or HA-K43M/D163N-mutant CDK6 (kinase dead), and interaction with INK4 proteins was determined by immunoblotting. I, Disruption of INK4s and CDK6 binding or impairment of CDK6 kinase activity restores the sensitivity of CDK6-overexpressing cells to CDK4/6i. Cells were treated with DMSO or 100 nmol/L abemaciclib for 24 hours prior to collection. J, Percentage of cell viability of cells overexpressing WT CDK6 or R31C- or D163N-mutant CDK6 treated with increasing concentrations of abemaciclib compared with parental cells. IC₅₀ values were recorded on day 5 following treatment. Data are shown as mean ± SD; n = 6. K, Knockdown of p15^INK4B and p18^INK4C in FAT1-loss cells promotes suppression of RB phosphorylation in response to abemaciclib to a similar extent as in parental cells. Cells were collected 24 hours after 100 nmol/L abemaciclib treatment. Representative blots are shown, which were repeated independently three times. L, The growth rate of p15^INK4B and p18^INK4C knockout in FAT1-loss cells was inhibited by 100 nmol/L abemaciclib. The cell viability was recorded at day 14 and day 21. ****, P < 0.0001. Data are shown as mean ± SD; n = 6. See also Supplementary Fig. S1.