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. 2022 Feb;12(2):356-371.
doi: 10.1158/2159-8290.CD-20-1726. Epub 2021 Sep 20.

INK4 Tumor Suppressor Proteins Mediate Resistance to CDK4/6 Kinase Inhibitors

Qing Li #  1 , Baishan Jiang #  2   3 , Jiaye Guo #  4 , Hong Shao  1 , Isabella S Del Priore  1 , Qing Chang  5 , Rei Kudo  1 , Zhiqiang Li  1 , Pedram Razavi  6   7 , Bo Liu  1 , Andrew S Boghossian  8 , Matthew G Rees  8 , Melissa M Ronan  8 , Jennifer A Roth  8 , Katherine A Donovan  2   3 , Marta Palafox  9 , Jorge S Reis-Filho  10 , Elisa de Stanchina  5 , Eric S Fischer  2   3 , Neal Rosen  11 , Violeta Serra  9 , Andrew Koff  12 , John D Chodera  4 , Nathanael S Gray  2   3 , Sarat Chandarlapaty  13   6   7
Affiliations

INK4 Tumor Suppressor Proteins Mediate Resistance to CDK4/6 Kinase Inhibitors

Qing Li et al. Cancer Discov. 2022 Feb.

Abstract

Cyclin-dependent kinases 4 and 6 (CDK4/6) represent a major therapeutic vulnerability for breast cancer. The kinases are clinically targeted via ATP competitive inhibitors (CDK4/6i); however, drug resistance commonly emerges over time. To understand CDK4/6i resistance, we surveyed over 1,300 breast cancers and identified several genetic alterations (e.g., FAT1, PTEN, or ARID1A loss) converging on upregulation of CDK6. Mechanistically, we demonstrate CDK6 causes resistance by inducing and binding CDK inhibitor INK4 proteins (e.g., p18INK4C). In vitro binding and kinase assays together with physical modeling reveal that the p18INK4C-cyclin D-CDK6 complex occludes CDK4/6i binding while only weakly suppressing ATP binding. Suppression of INK4 expression or its binding to CDK6 restores CDK4/6i sensitivity. To overcome this constraint, we developed bifunctional degraders conjugating palbociclib with E3 ligands. Two resulting lead compounds potently degraded CDK4/6, leading to substantial antitumor effects in vivo, demonstrating the promising therapeutic potential for retargeting CDK4/6 despite CDK4/6i resistance. SIGNIFICANCE: CDK4/6 kinase activation represents a common mechanism by which oncogenic signaling induces proliferation and is potentially targetable by ATP competitive inhibitors. We identify a CDK6-INK4 complex that is resilient to current-generation inhibitors and develop a new strategy for more effective inhibition of CDK4/6 kinases.This article is highlighted in the In This Issue feature, p. 275.

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Figures

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 p15INK4B and p18INK4C 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 p15INK4B). 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 p15INK4B and p18INK4C 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 p15INK4B and p18INK4C 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.
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 p15INK4B and p18INK4C 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. ; no available structure for p15INK4B). 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 p15INK4B and p18INK4C 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 p15INK4B and p18INK4C 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.
Figure 2. INK4–CDK6 complexes are insensitive to CDK4/6i. A, In vitro kinase assay using recombinant CDK6–cyclin D3 and RB substrate demonstrates that preincubation of the complex with p18 (purple) prevents complete inhibition of kinase activity by abemaciclib. Data are shown as mean ± SD of two biological replicates. RLU, relative luminescence units. B, Effect of preincubation of p18 on CDK6–cyclin D3 in vitro kinase activity. Data are shown as mean ± SD of two biological replicates. C, Assay of CDK6–cyclin D3 kinase activity and response to p18 by immunoblotting demonstrating that p18 impairs the ability of abemaciclib to inhibit CDK6 phosphorylation of RB. D, Computational modeling of the effect of p18 binding to the CDK6 binding pocket expressed as volume change for abemaciclib (top) or AMP-PNP (bottom). Structures of CDK6–cyclin complex before and after p18 binding are represented by crystallographic structures with PDB IDs 2EUF and 1G3N (shown in ribbons). The binding pockets were approximated by spheres (shown in green and indicated by red arrows; shown in purple and indicated by green arrows). The volume of each binding pocket was quantified using the total volume of the corresponding set of spheres, and percentage of changes was calculated. E, The table summarizes the changes of binding pocket volume for two CDK4/6i (palbociclib and abemaciclib) and AMP-PNP upon binding of INK4s (p16, p18, p19). F, MST assay of CDK6 binding to abemaciclib showing the change in Kd as a result of p18 binding (red). Data are shown as mean ± SD of two independent measurements. See also Supplementary Fig. S2.
Figure 2.
INK4–CDK6 complexes are insensitive to CDK4/6i. A,In vitro kinase assay using recombinant CDK6–cyclin D3 and RB substrate demonstrates that preincubation of the complex with p18 (purple) prevents complete inhibition of kinase activity by abemaciclib. Data are shown as mean ± SD of two biological replicates. RLU, relative luminescence units. B, Effect of preincubation of p18 on CDK6–cyclin D3 in vitro kinase activity. Data are shown as mean ± SD of two biological replicates. C, Assay of CDK6–cyclin D3 kinase activity and response to p18 by immunoblotting demonstrating that p18 impairs the ability of abemaciclib to inhibit CDK6 phosphorylation of RB. D, Computational modeling of the effect of p18 binding to the CDK6 binding pocket expressed as volume change for abemaciclib (top) or AMP-PNP (bottom). Structures of CDK6–cyclin complex before and after p18 binding are represented by crystallographic structures with PDB IDs 2EUF and 1G3N (shown in ribbons). The binding pockets were approximated by spheres (shown in green and indicated by red arrows; shown in purple and indicated by green arrows). The volume of each binding pocket was quantified using the total volume of the corresponding set of spheres, and percentage of changes was calculated. E, The table summarizes the changes of binding pocket volume for two CDK4/6i (palbociclib and abemaciclib) and AMP-PNP upon binding of INK4s (p16, p18, p19). F, MST assay of CDK6 binding to abemaciclib showing the change in Kd as a result of p18 binding (red). Data are shown as mean ± SD of two independent measurements. See also Supplementary Fig. S2.
Figure 3. Multiple genetic alterations promote CDK6-mediated resistance in patients. A, IHC of FAT1, CDK6, YAP, p15INK4B, and p18INK4C in representative patient-derived xenograft (PDX) models that are sensitive or resistant to CDK4/6i. B, Number of cases that show high or low CDK6 in PDX models that are sensitive or resistant to CDK4/6i. Immunoreactive score (IRS) >2 is recorded as high CDK6 expression. C, IRS of CDK6, nuclear YAP, FAT1, p15, and p18 staining in sensitive and resistant PDX models. D and E, Immunoblotting demonstrating that knockdown of PTEN or ARID1A in MCF7 cells promotes upregulation of CDK6 and resistance to 100 nmol/L abemaciclib (abema) treatment. Cells were treated for 24 hours prior to collection. ns, not significant. F, Cell viability (percentage of control cells) plots showing that both PTEN knockdown cells have decreased sensitivity to abemaciclib compared with parental cells. IC50 values were recorded on day 5. Data are shown as mean ± SD; n = 6. G, Cell viability (percentage of control cells) plots showing that ARID1A knockdown cells have decreased sensitivity to abemaciclib compared with parental cells. Knockdown of YAP1 in shARID1A cells restores its sensitivity to abemaciclib. IC50 values were recorded on day 7. Data are shown as mean ± SD; n = 6. H, Immunoblotting showing inhibition of AKT (2 μmol/L MK-2206) suppresses induction of CDK6 expression in PTEN knockdown cells. I, Immunoblotting showing that knockdown of YAP1 in shARID1A cells decreases CDK6 expression. All blots were repeated at least three times, and representative blots are shown. J, The pattern, frequency, and type of genomic alterations in CDK6-associated genes in 1,366 metastatic tumors from 1,115 patients with HR+/HER2− metastatic breast cancer. A total of 190 cases show at least one of the genetic alterations associated with CDK6 upregulation. See also Supplementary Fig. S3.
Figure 3.
Multiple genetic alterations promote CDK6-mediated resistance in patients. A, IHC of FAT1, CDK6, YAP, p15INK4B, and p18INK4C in representative patient-derived xenograft (PDX) models that are sensitive or resistant to CDK4/6i. B, Number of cases that show high or low CDK6 in PDX models that are sensitive or resistant to CDK4/6i. Immunoreactive score (IRS) >2 is recorded as high CDK6 expression. C, IRS of CDK6, nuclear YAP, FAT1, p15, and p18 staining in sensitive and resistant PDX models. D and E, Immunoblotting demonstrating that knockdown of PTEN or ARID1A in MCF7 cells promotes upregulation of CDK6 and resistance to 100 nmol/L abemaciclib (abema) treatment. Cells were treated for 24 hours prior to collection. ns, not significant. F, Cell viability (percentage of control cells) plots showing that both PTEN knockdown cells have decreased sensitivity to abemaciclib compared with parental cells. IC50 values were recorded on day 5. Data are shown as mean ± SD; n = 6. G, Cell viability (percentage of control cells) plots showing that ARID1A knockdown cells have decreased sensitivity to abemaciclib compared with parental cells. Knockdown of YAP1 in shARID1A cells restores its sensitivity to abemaciclib. IC50 values were recorded on day 7. Data are shown as mean ± SD; n = 6. H, Immunoblotting showing inhibition of AKT (2 μmol/L MK-2206) suppresses induction of CDK6 expression in PTEN knockdown cells. I, Immunoblotting showing that knockdown of YAP1 in shARID1A cells decreases CDK6 expression. All blots were repeated at least three times, and representative blots are shown. J, The pattern, frequency, and type of genomic alterations in CDK6-associated genes in 1,366 metastatic tumors from 1,115 patients with HR+/HER2− metastatic breast cancer. A total of 190 cases show at least one of the genetic alterations associated with CDK6 upregulation. See also Supplementary Fig. S3.
Figure 4. Compounds targeting the CDK6–INK4 complex inhibit CDK4/6i-resistant tumors. A, Immunoblotting of MCF7 parental cells and cells with high CDK6 expression [CDK6-overexpressing (OE) cells and CDK6-high cells with FAT1 loss] treated for 24 hours with increasing concentrations of bifunctional degrader compound, BSJ-03-123, demonstrating dose-dependent targeting of CDK6 but not CDK4. B, Assessment of a panel of degrader compounds that target CDK4 and/or CDK6. Immunoblotting after 24-hour drug treatment (500 nmol/L) in FAT1-loss cells shows varying selectivity for CDK4 versus CDK6. Representative blots from three independent experiments are shown. Among them, BSJ-05-017 and BSJ-03-096 show the most significant degradation of both CDK4 and CDK6. C, Immunoblot depicting dose–response effects of BSJ-05-017 in both CDK4/6i-sensitive (left) and CDK4/6i-resistant (right) cells in comparison with palbociclib (500 nmol/L) after 24-hour treatment. D, Percentage of growth plot showing that BSJ-05-017 inhibits sensitive MCF7 parental and resistant CDK6-high cells with equal potency, whereas palbociclib shows only partial inhibition of resistant cells. IC50 values were recorded at day 7. Data are shown as mean ± SD; n = 6. E, Assay for drug-induced senescence (Senescence Green) demonstrating number of senescence marker–positive cells induced by 8 days of treatment with DMSO, BSJ-05-017 (500 nmol/L), abemaciclib (100 nmol/L), and palbociclib (500 nmol/L). BSJ-05-017 induced a significantly higher number of cells into senescence compared with abemaciclib or palbociclib in CDK6-high cells. F, Immunoblotting showing the degradation of CDK4/6 and decreased phospho-RB1 and E2F1 levels in CDK6-high (FAT1 loss) tumor-bearing mice administered 25 mg/kg BSJ-05-017 intraperitoneally. Tumors were collected 6 hours after 3 consecutive days of vehicle or BSJ-05-017 treatment (n = 2). G, Growth curve plots of cell-derived xenografts of MCF7 parental, CDK6-overexpressing, and PTEN-loss cells. Mice were treated with vehicle, ribociclib (25 mg/kg, orally), BSJ-05-017 (50 mg/kg, i.p.), or BSJ-03-096 (50 mg/kg, orally) daily for 25 to 35 days. Tumor volumes were recorded every 3 to 4 days. Data are shown as mean ± SD; n = 4. See also Supplementary Fig. S4.
Figure 4.
Compounds targeting the CDK6–INK4 complex inhibit CDK4/6i-resistant tumors. A, Immunoblotting of MCF7 parental cells and cells with high CDK6 expression [CDK6-overexpressing (OE) cells and CDK6-high cells with FAT1 loss] treated for 24 hours with increasing concentrations of bifunctional degrader compound, BSJ-03-123, demonstrating dose-dependent targeting of CDK6 but not CDK4. B, Assessment of a panel of degrader compounds that target CDK4 and/or CDK6. Immunoblotting after 24-hour drug treatment (500 nmol/L) in FAT1-loss cells shows varying selectivity for CDK4 versus CDK6. Representative blots from three independent experiments are shown. Among them, BSJ-05-017 and BSJ-03-096 show the most significant degradation of both CDK4 and CDK6. C, Immunoblot depicting dose–response effects of BSJ-05-017 in both CDK4/6i-sensitive (left) and CDK4/6i-resistant (right) cells in comparison with palbociclib (500 nmol/L) after 24-hour treatment. D, Percentage of growth plot showing that BSJ-05-017 inhibits sensitive MCF7 parental and resistant CDK6-high cells with equal potency, whereas palbociclib shows only partial inhibition of resistant cells. IC50 values were recorded at day 7. Data are shown as mean ± SD; n = 6. E, Assay for drug-induced senescence (Senescence Green) demonstrating number of senescence marker–positive cells induced by 8 days of treatment with DMSO, BSJ-05-017 (500 nmol/L), abemaciclib (100 nmol/L), and palbociclib (500 nmol/L). BSJ-05-017 induced a significantly higher number of cells into senescence compared with abemaciclib or palbociclib in CDK6-high cells. F, Immunoblotting showing the degradation of CDK4/6 and decreased phospho-RB1 and E2F1 levels in CDK6-high (FAT1 loss) tumor-bearing mice administered 25 mg/kg BSJ-05-017 intraperitoneally. Tumors were collected 6 hours after 3 consecutive days of vehicle or BSJ-05-017 treatment (n = 2). G, Growth curve plots of cell-derived xenografts of MCF7 parental, CDK6-overexpressing, and PTEN-loss cells. Mice were treated with vehicle, ribociclib (25 mg/kg, orally), BSJ-05-017 (50 mg/kg, i.p.), or BSJ-03-096 (50 mg/kg, orally) daily for 25 to 35 days. Tumor volumes were recorded every 3 to 4 days. Data are shown as mean ± SD; n = 4. See also Supplementary Fig. S4.
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