Defining the transcriptional and biological response to CDK4/6 inhibition in relation to ER+/HER2- breast cancer

Abstract

ER positive (ER+) and HER2 negative (HER2-) breast cancers are routinely treated based on estrogen dependence. CDK4/6 inhibitors in combination with endocrine therapy have significantly improved the progression-free survival of patients with ER+/HER2- metastatic breast cancer. Gene expression profiling in ER+/HER2- models was used to define the basis for the efficacy of CDK4/6 inhibitors and develop a gene expression signature of CDK4/6 inhibition. CDK4/6 inhibition robustly suppressed cell cycle progression of ER+/HER2- models and complements the activity of limiting estrogen. Chronic treatment with CDK4/6 inhibitors results in the consistent suppression of genes involved in cell cycle, while eliciting the induction of a comparable number of genes involved in multiple processes. The CDK4/6 inhibitor treatment shifted ER+/HER2- models from a high risk (luminal B) to a low risk (luminal A) molecular-phenotype using established gene expression panels. Consonantly, genes repressed by CDK4/6 inhibition are strongly associated with clinical prognosis in ER+/HER2- cases. This gene repression program was conserved in an aggressive triple negative breast cancer xenograft, indicating that this is a common feature of CDK4/6 inhibition. Interestingly, the genes upregulated as a consequence of CDK4/6 inhibition were more variable, but associated with improved outcome in ER+/HER2- clinical cases, indicating dual and heretofore unknown consequence of CDK4/6 inhibition. Interestingly, CDK4/6 inhibition was also associated with the induction of a collection of genes associated with cell growth; but unlike suppression of cell cycle genes this signaling was antagonized by endocrine therapy. Consistent with the stimulation of a mitogenic pathway, cell size and metabolism were induced with CDK4/6 inhibition but ameliorated with endocrine therapy. Together, the data herein support the basis for profound interaction between CDK4/6 inhibitors and endocrine therapy by cooperating for the suppression of cell cycle progression and limiting compensatory pro-growth processes that could contribute to therapeutic failure.

Keywords: CDK4/6; PAM50; RB-pathway; breast cancer; molecular subtypes.

Figure 1

Distinct gene regulation by CDK4/6 inhibition and estrogen withdrawal—cooperation for suppression of cell cycle (A) Venn diagram showing the overlap in genes modified by greater than 1.5-fold and p < 0.05 in MCF7 cells treated with PD-0332991 vs. MCF7 challenged with estrogen withdrawal. B. Relative expression of select genes that are estrogen-specific, similarly repressed, or preferentially repressed with PD-0332991. C. BrdU incorporation of MCF7 and T47D cells treated with the indicated agents. CDT—charcoal dextran treated serum lacking steroid hormones, PD—the CDK4/6 inhibitor PD-0332991 (**p < 0.01).

Figure 2. Defining CDK4/6 inhibition signature in ER+/Her2- models A

Venn diagram showing the overlap in genes repressed by greater than 1.5-fold and p < 0.05 in MCF7 and T47D cells treated with PD-0332991 for 120 hours. Top gene ontologies were determined for the genes repressed in both models. B. Venn diagram showing the overlap in genes induced by greater than 1.5-fold and p < 0.05 in MCF7 and T47D cells treated with PD-0332991 for 120 hours. Top gene ontologies were determined for the genes that were induced in both the models. C. Gene set enrichment analysis of selected terms associated with transcriptional repression (e.g. cell cycle) vs. activation (e.g. pregnancy and wound healing).

Figure 3. Transcriptional repression by CDK4/6 inhibition and impact on luminal subtypes A

The levels of top repressed genes in T47D cells are presented by rank order. Expression in both T47D and MCF7 cells is shown as denoted by the legend. B. Relative level of genes that make up the OncotypeDX proliferation module were evaluated in MCF7 cells untreated (black bars) and treated with PD-0332991 (gray bars). C. Relative expression of the PAM50 genes untreated and treated with PD-0332991 in MCF7 and T47D cells. Data indicate the consistent suppression of proliferation associated genes that are associated with risk of recurrence. D. Kaplan-Meier analysis of the survival data from 967 ER+/Her2- tumors stratified based on the level of the 230 gene CDK4/6 repression signature. The quartiles associated with transcriptional repression are shown statistical significance was determined by log-rank of the quartiles. The highest expression (Q4) of gene expression is associated with worse prognosis E. Select genes that are significantly repressed by CDK4/6 inhibition in MCF7 and T47D cells and have potent prognostic activity in ER+/HER2- breast cancer are shown. The highest expression (Q4) of gene expression is associated with worse prognosis.

Figure 4. Conservation of CDK4/6 inhibitor gene repression in xenografts and different breast cancer subtypes A

Histological analysis of MDA-MB-231 orthotopic xenograft controls or treated with PD-0332991 orally for seven days. Representative hematoxylin/eosin stained sections and Ki67 staining are shown. Scale bar is 100 μm. B. Gene ontology was performed on 278 genes that were repressed 1.5-fold with a p-value < 0.05. C. Specific analysis of the expression levels of top repressed genes in cell culture models.

Figure 5. Unexpected impact of genes upregulated through CDK4/6 inhibition on prognosis A

The levels of top induced genes in MCF7 or T47D cells are presented by rank order. B. The common CDK4/6 inhibitor induced signature (336 genes) was used to stratify the expression data from 967 ER+/HER2- cases. Survival outcomes were determined by Kaplan-Meier analysis C. Select genes from the signature that are associated with prognosis are presented. Statistical analysis was determined by log-rank analysis. D. Heatmap of K-Means clustering including upregulated (orange) and downregulated (green) genes from the CDK4/6 inhibitor signature, the 5 clusters are denoted by color bar at the top of the heatmap. E. The indicated K-means clusters were used to determined the association with prognosis by Kaplan-Meier analysis. Statistical signifiace was determined by log-rank analysis.

Figure 6. CDK4/6 induction of cellular growth and metabolism are ameliorated by endocrine therapy A

The levels of the estrogen receptor (ER), cyclin D1, and cyclin A were determined by western blotting under the indicated conditions. B. The expression of the indicated genes was determined by RNA sequencing analysis from T47D cells treated with the indicated therapeutics (CDT—charcoal dextran treated serum, ICI—fulvestrant, or PD—the CDK4/6 inhibitor PD-0332991. The relative gene expression is shown in reference to standard growth conditions. C. The impact of CDK4/6 inhibition on the expression of select metabolic genes is shown for MCF7 and T47D cells. D. Impact of the indicated therapeutics on cellular complexity was determined by flow cytometry and impact on mitochondria was determined by mitotracker staining and flow cytometry. E. Representative transmission electron micrographs showing the accumulation of mitochondria in cells treated with PD-0332991. G. Impact of the indicated treatments on oxidative metabolism as determined by oxygen consumption rate (OCR) analysis. H. Relative ATP present in T47D cells following the indicated treatment (**p < 0.01).