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

Figure 4.

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.

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.