Long-term breast cancer response to CDK4/6 inhibition defined by TP53-mediated geroconversion

Abstract

Inhibition of CDK4/6 kinases has led to improved outcomes in breast cancer. Nevertheless, only a minority of patients experience long-term disease control. Using a large, clinically annotated cohort of patients with metastatic hormone receptor-positive (HR+) breast cancer, we identify TP53 loss (27.6%) and MDM2 amplification (6.4%) to be associated with lack of long-term disease control. Human breast cancer models reveal that p53 loss does not alter CDK4/6 activity or G1 blockade but instead promotes drug-insensitive p130 phosphorylation by CDK2. The persistence of phospho-p130 prevents DREAM complex assembly, enabling cell-cycle re-entry and tumor progression. Inhibitors of CDK2 can overcome p53 loss, leading to geroconversion and manifestation of senescence phenotypes. Complete inhibition of both CDK4/6 and CDK2 kinases appears to be necessary to facilitate long-term response across genomically diverse HR+ breast cancers.

Keywords: CDK2; CDK4/6; breast cancer; cell cycle; cyclin dependent kinase; drug resistance; p53; quiescence; senescence.

Figure 1.. Somatic variants enriched in patients failing to achieve long-term response to CDK4/6i

(A and B) The association of individual genes with long term disease control (progression free survival, PFS) as compared to (A) inter-mediate and (B) short-term disease control with CDK4/6i and endocrine therapy (ET), based on even tertiles of treatment duration. The colors indicate statistical significance (q < 0.1) and the circles’ size reflects the frequency of alteration in the cohort. All q values are calculated based on the Benjamini and Hochberg method correction of log rank p values. (C) The results of an elastic net Cox proportional hazard model, whereby risk groups are stratified based on K-means clustering. Here, the optimal number of clusters (n = 3) was selected automatically by employing an Akaike information criterion and again separated patients into short, intermediate, and prolonged time on treatment. (D–F) The variable importance of the elastic-net Cox proportional hazard model, after 50 runs and 5-fold cross-validation. Selection frequency is defined as the proportion of runs in which each gene achieves a coefficient greater than zero, and the mean hazard ratio is the mean coefficient across all runs. (E and F) The PFS of patients receiving first-line CDK4/6i and ET, harboring tumors with pre-treatment functional alterations in TP53 (E) and MDM2 (F). (G) The PFS of patients receiving first and second-line CDK4/6i and ET in the Tempus cohort. Survival analyses were performed with univariate and multivariate Cox proportional hazard models, adjusted by treatment class (CDK4/6i + AI or CDK4/6i + SERD). See also Tables S1–S3.

Figure 2.. p53 loss promotes long-term cell outgrowth

(A) Immunoblotting of indicated proteins in MCF7 parental, p53KO, MDM2OE, and FAT1KO cells treated for 24 h. (B) Cell-cycle distribution after 24 h. Student’s t test was performed to compare G1 fraction. The mean value of three replicates (top). Changes in the proportion of cells in G1 phase. Student’s t test (bottom). Data are means ± SD of three biological replicates. (C) IC50s of abemaciclib on day 4. (D) Cell viability assay with 50 nM abemaciclib. Data are means ± SEM of four replicates. Two-way ANOVA, Tukey’s. (E) Relative tumor volume at 5 weeks compared to that on day 0 after treatment with vehicle or ribociclib (25 mg/kg) (n = 5). Data are represented as means of five replicates ±SEM. Student’s t test. (F) Immunoblotting of indicated proteins in V5-WT, V5-R273H, and V5-R280K treated for 24 h. (G) Cell-cycle distribution after 24 h. Data are means of three replicates. Student’s t test. (H) Cell viability with 50 nM abemaciclib. Data are means ± SEM of four replicates. Two-way ANOVA, Tukey’s test. (I) Tumor volume ratio of V5-WT or V5-R280K cells xenograft tumors treated with 25 mg/kg ribociclib for 29 days compared to day 0. Data are means ± SD of four replicates. Two-tailed unpaired t test. (J) Patient-derived organoids PDO #6 were originally derived from patient tumors with HR+/HER− and WT-TP53. Scale bar: 400 μM. (K) Representative images of PDO #6 NT sgRNA or p53sgRNA treated with DMSO, 12.5 nM abemaciclib and combination of abemaciclib and 10 nM fulvestrant on indicated days. Scale bar: 400 μM. See also Figure S1.

Figure 3.. p53 loss enables cells re-enter cell cycle and prevent geroconversion

(A) Cells were treated with 50 nM abemaciclib for seven days. Cell viability was measured with continuous drug treatment (red, blue, and green) or after drug withdrawal (pink, light blue, and light green). Data are means ± SEM of four replicates. Linear regression analysis. (B) Colony formation assay. Cells were treated for 10 days, re-seeded and cultured without drug. (C) MCF7 and p53KO cell expressing IncuCyte-Cell-Cycle-Green/Red-Lentivirus were treated with 100 nM abemaciclib for six days. After withdrawal of drug, cells were subjected to time-lapse fluorescence microscopy. Representative images at 27 h after drug withdrawal. Scale bar: 100μM (top). Time course change of each cell-cycle phase proportion. Data were analyzed using IncuCyte starting after drug withdrawal (bottom). (D) The histogram shows counts of SA-β-Gal-positive cells under 50 nM abemaciclib treatment for six days (top). Bars represent the percentage of SA-β-Gal positive cells (bottom). Data are means ± SD of three replicates. Two-way ANOVA, Sidak’s. (E) Bars show the percentage of SA-β-Gal positive cells on day 5. Data are means ± SD of three replicates. Two-way ANOVA, Sidak’s. (F) Bars represent the percentage of SA-β-Gal positive cells of each PDO #6 after 16 days. Data are means ± SD of three replicates. Two-way ANOVA, Sidak’s. (G) Representative images of each cell line treated with 100 nM abemaciclib for eight days and the colocalization of HP1γ foci and DAPI (yellow arrows). Scale bar: 5 μM (left), 25 μM (right). (H) The percentage of HP1γ foci-positive cells was quantified in three different fields. Data are represented as means ± SD. Two-way ANOVA, Sidak’s. (I) The percentage of HP1γ foci-positive cells treated for 8 days was quantified in three different fields. Data are represented as represented as mean ± SD. Two-way ANOVA, Sidak’s. (J) qPCR of indicated genes expression in xenograft tumors with vehicle or 25 mg/kg ribociclib after 5 weeks. The ratios are represented as means ± SD from two tumors. n = 6. Student’s t test. (K) Representative images of SA-β-Gal staining of xenograft tumors in J. Scale bar: 100 μM. n = 5. See also Figures S2 and S3 and Video S1.

Figure 4.. Loss of p53 suppresses DREAM complex assembly

(A) GESA plot with normalized enrichment score (NES) of REACTOME gene sets. NES was analyzed between MCF7 and p53KO treated with 50 nM abemaciclib at day21. REACTOME-E2F-mediated-regulation-of-DNA-replication (top), REACTOME-Transcription-of-E2F-targets-under-negative-control-by-DREAM-complex (middle) and REACTOME-Transcription-of-E2F-targets-under-negative-control-byp107 RBL1-and-p130 RBL2-in-complex-with-HDAC1 (bottom). (B) Immunoblotting of DREAM complex components with 50 nM abemaciclib. (C) Cells were treated with 100 nM abemaciclib for 48 h. Lysates were immunoprecipitated with p130 and IgG antibodies. (D) Cells were treated with 7 days of abamacilib and tested by ChIP followed by real-time PCR. Bar showed technical duplicate. Unpaired, Student’s t test. (E) Western blot results of MCF7 cells and p53KO transduced with doxycycline (dox)-inducible HA-tagged p21. The cells were treated with 50 nM abemaciclib or DMSO +/− dox for 48 h. (F) Dox-inducible HA-p21 MCF7 and p53KO cells were treated with 100 nM abemaciclib and +/− dox for 72 h. Lysates were immunoprecipitated with E2F4 and IgG antibodies. (G) Time course change of the proportion of G1 phase after drug withdrawal. FUCCI labeled MCF7 were treated with 50 nM abemaciclib and FUCCI labeled p53KO cells were treated with or without dox and with 50 nM abemaciclib for 4 days. The cells were cultured without abemaciclib for 62 h under time-lapse imaging. Two-way ANOVA, Tukey’s. (H) Cell viability treated with 50 nM abemaciclib or DMSO +/− dox. Data are means ± SEM of four replicates. Two-way ANOVA, Tukey’s. (I) Flow cytometry analysis showing the percentage of SA-β-Gal positive cells. Data are means ± SD of three replicates. Two-way ANOVA, Sidak’s. (J) Colony formation assay. Cells were treated with 50 nM abemaciclib ± dox for 11 days and reseeded without drug. (K) Cell viability of p130KO in dox inducible HA-p21 cells. The cells were treated with 50 nM abemaciclib or DMSO +/− dox. Data are means ± SEM of six replicates. Two-way ANOVA, Tukey’s. See also Figure S4 and Video S2.

Figure 5.. Combined inhibition of CDK2 and CDK4/6 enables long-term growth suppression

(A) Immunoblotting of indicated proteins in cells with DMSO, 50 nM abemaciclib, 500 nM AZD8421, or combination for 72 h. (B) MCF7 and p53KO cells with FUCCI were treated with 50 nM abemaciclib +/− 500 nM AZD8421 for 8 days. Time course change of G1 phase proportion starting after drug withdrawal. Two-tailed, unpaired t test. (C) Cell viability of the cells treated with drug. Data are means ± SEM of four replicates. Two-way ANOVA, Tukey’s. (D) Colony formation assay. Cells were treated for 10 days, re-seeded and cultured without drug. (E) Bar shows the percentage of SA-β-positive cells on day 5. Data are means ± SD of three replicates. Two-way ANOVA, Sidak’s. (F) Immunoblotting of p53 mutant BC primary cell line #5 treated with drugs for 48h. (G) Cell viability of PD BC line #5 treated with drugs. Data are means ± SEM of four replicates. Two-way ANOVA, Sidak’s. (H) The cells were treated with DMSO, 500 nM AZD8421, 50 nM abemaciclib, or combination for 6 days. Confluency of PD BC line #5 was analyzed by Incucyte for 13 days after drug washout. Data are represented as means of duplicates ± SD. Two-way ANOVA, Tukey’s. (I) Bars indicate the percentage of SA-β-Gal-positive cells in PD BC line #5 cells. Data are means ± SD of three replicates. Two-way ANOVA, Sidak’s. (J) Relative mRNA expression of MKI67. The ratios are represented as means ± SD from two tumors. n = 6. Student’s t test. (K) Immunoblotting of indicated antibodies. Lysate was extracted from PDX #5 tumors treated with drugs for 5 days. See also Figures S5 and S6.

Figure 6.. p53 loss is associated with cell-cycle re-entry in patients treated with neoadjuvant CDK4/6i

(A) Sample collection in FELINE trial. Core needle biopsy was performed over the course of treatment: screening (day 0), cycle 1 (day 14), and end of treatment (day 180). (B) Ki-67 trend is depicted for all patients in the FELINE trial for whom Ki-67 was evaluable at all time points and sequencing of the day 0 tumor was performed. The rates of Ki-67 > 10% at surgery are compared by pre-treatment TP53 status utilizing Fisher’s exact (two-sided) test. (C) Proliferation score in wild type and mutant TP53 tumors at baseline, on-treatment day14 and EOT. (D and F) GSEA of DREAM complex genes in WT compared vs. MT at day14 (left) and EOT (right). (E) GSEA of senescence ribo genes. EOT vs. baseline in WT TP53 (left) and in MT (right) (F) Model for loss of p53 resistant mechanism. In the presence of p53, CDK4/6i inhibited phosphorylation of RB and p130. The cells were arrested in quiescence through DREAM complex, leading to irreversible cell-cycle arrest. Loss-of-function p53 and insufficient p21 enabled CDK2 to retain phosphorylated p130, leading to cell-cycle re-entry and escape from quiescence. See also Figure S6.