Early Adaptation and Acquired Resistance to CDK4/6 Inhibition in Estrogen Receptor-Positive Breast Cancer

Abstract

Small-molecule inhibitors of the CDK4/6 cell-cycle kinases have shown clinical efficacy in estrogen receptor (ER)-positive metastatic breast cancer, although their cytostatic effects are limited by primary and acquired resistance. Here we report that ER-positive breast cancer cells can adapt quickly to CDK4/6 inhibition and evade cytostasis, in part, via noncanonical cyclin D1-CDK2-mediated S-phase entry. This adaptation was prevented by cotreatment with hormone therapies or PI3K inhibitors, which reduced the levels of cyclin D1 (CCND1) and other G1-S cyclins, abolished pRb phosphorylation, and inhibited activation of S-phase transcriptional programs. Combined targeting of both CDK4/6 and PI3K triggered cancer cell apoptosis in vitro and in patient-derived tumor xenograft (PDX) models, resulting in tumor regression and improved disease control. Furthermore, a triple combination of endocrine therapy, CDK4/6, and PI3K inhibition was more effective than paired combinations, provoking rapid tumor regressions in a PDX model. Mechanistic investigations showed that acquired resistance to CDK4/6 inhibition resulted from bypass of cyclin D1-CDK4/6 dependency through selection of CCNE1 amplification or RB1 loss. Notably, although PI3K inhibitors could prevent resistance to CDK4/6 inhibitors, they failed to resensitize cells once resistance had been acquired. However, we found that cells acquiring resistance to CDK4/6 inhibitors due to CCNE1 amplification could be resensitized by targeting CDK2. Overall, our results illustrate convergent mechanisms of early adaptation and acquired resistance to CDK4/6 inhibitors that enable alternate means of S-phase entry, highlighting strategies to prevent the acquisition of therapeutic resistance to these agents. Cancer Res; 76(8); 2301-13. ©2016 AACR.

Figure 1

Early adaptive resistance limits the efficacy of CDK4/6 inhibition. A and B, MCF-7 cells treated with 500 nmol/L palbociclib for 24 or 72 hours as indicated followed by 2 hours exposure to BrdUrd. A, images show BrdUrd-positive cells (green) and BrdUrd-negative cells (red). Graphs show number of nuclei (DAPI staining), percentage of BrdUrd–positive cells, and cell area (^***, P < 0.001, one-way ANOVA with Tukey multiple comparisons test). B, BrdUrd staining (green) and tubulin (red) illustrate that BrdUrd-negative cells correlate with extended cell area (senescence like phenotype; ^*, P < 0.05, Student t test). C and D, Western blot analysis of lysates from MCF-7 cells treated for 24 to 72 hours with palbociclib (Palbo). ^*, addition of fresh vehicle or drug every 24 hours over a 72-hour period. E, MCF-7 cells treated with palbociclib for 72 hours followed by 0.5 hours exposure to EdU, then washed and exposed to BrdUrd for another 24 hours. Graph shows percentage of BrdUrd-positive–EdU-negative cells. F, effect of the drug library on the relative sensitivity to palbociclib expressed as a z score, with z scores below −2 indicating increased sensitivity to palbociclib. Main hits are indicated with arrows. H, clonogenic survival assays in MCF-7 cells treated continuously for 14 days with vehicle, 500 nmol/L palbociclib, 250 nmol/L GDC-0941 (0941; PI3K inhibitor), or 100 nmol/L MK2206 (AKT inhibitor) or 100 nmol/L everolimus (Ever; mTOR inhibitor), and the indicated combinations with palbociclib. I, BrdUrd incorporation measured by ELISA and corrected for viable cell number. Cells treated with vehicle, palbociclib, GDC-0941, or the combination for 24 or 72 hours (^*, P < 0.05 palbociclib vs. combination at 72 hours, two-way ANOVA with Tukey multiple comparisons test; ^**, P < 0.01 palbociclib 24 hours vs. 72 hours; ^***, P < 0.001 GDC0941 24 hours vs. 72 hours, two-way ANOVA with Bonferroni posttest).

Figure 2

Early adaptation to CDK4/6 inhibition is mediated by noncanonical cyclin D1–CDK2 interaction. The addition of PI3K inhibitors reduces expression of G₁–S-phase regulators and induces apoptosis. A and H, Western blot analysis of cell lysates from MCF-7 and T47D cell treated for 96 hours with vehicle, 500 nmol/L palbociclib (Palbo), 250 nmol/L GDC-0941 or combination and blotted with the indicated antibodies. B and C, cells transfected for 4 days with individual siRNAs or SMARTpool targeting CDK2, cyclin D1, or cyclin E2 and treated with vehicle or palbociclib for 72 hours. B, BrdUrd staining and cell area quantified with Columbus software (^***, P < 0.001; ^*, P < 0.05, Student t test). C, BrdUrd incorporation-ELISA assay (^***, P < 0.001, one-way ANOVA with Tukey multiple comparisons test). D–G, Western blot analysis of MCF-7 cells lysates. D and F, cells transfected for 5 days with individual siRNAs or pools siRNAs targeting CDK2 or cyclin D1 and treated with vehicle or palbociclib for 96 hours. E, cells treated with vehicle, 500 nmol/L palbociclib, 10 μmol/L CYC202, or the combination for 72 hours. G, CDK2 immunoprecipitation (left) and cyclin D1 immunoprecipitation (right), or control normal IgG, blotted for CDK2 and cyclin D1. I, relative caspase-3/7 activation and survival fraction in MCF-7 cells treated as in A (^*, P < 0.05; ^***, P < 0.001, one-way ANOVA with Tukey multiple comparisons test).

Figure 3

The triplet combination of ER degradation, CDK4/6 inhibition, and PI3K inhibition is more active than doublet combinations. A–D, MCF-7 or T47D cells exposed to 500 nmol/L palbociclib (Palbo), 250 nmol/L GDC-0941 (0941), 100 nmol/L fulvestrant (Fulv), or the indicated drug combinations. A, clonogenic survival assays for 14 days. B, clonogenic survival assay in MCF-7 cells treated for 10 days before drugs were washed and colonies were allowed to grow for a further 7 days. C and D, Western blot analysis of lysates from T47D cells blotted with the indicated antibodies.

Figure 4

Duplet and triplet combination is efficacious in human-derived xenografts. A, relative tumor growth of PDX191 following vehicle, BYL719 (35 mg/kg, once daily, 6IW), LEE011 (75 mg/kg, once daily, 6IW), or the combination (^**, P < 0.01;^***, P < 0.001, two-way ANOVA). The total number of tumors in each arm (n) and SEM of each point are indicated. B, Western blots with the indicated antibodies in PDX191 after 42 days of vehicle, BYL719, LEE011, or the combination. Each lane belongs to one individual tumor. C, relative tumor growth of PDX244 following vehicle, fulvestrant (5 mg/kg, i.p., 1IW), fulvestrant plus BYL719, fulvestrant plus LEE011, and fulvestrant plus BYL719 plus LEE011 treatment.

Figure 5

Acquisition of resistance to CDK4/6 inhibition through RB1 loss or cyclin E1 amplification. A, BrdUrd incorporation-ELISA assay in parental (MCF-7, T47D) and palbociclib-resistant (MCF-7pR and T47DpR) cells treated with vehicle or palbociclib for 72 hours (^*, P < 0.001 vehicle vs. palbociclib; NS, not significant, two-way ANOVA with Sidak multiple comparisons test). B, cells treated for 96 hours with vehicle or 500 nmol/L palbociclib, and lysates blotted with the indicated antibodies. C, comparison of copy number for MCF-7 parental versus MCF-7pR–resistant cell line. There is an amplification of chromosome 19q12 region encompassing the CCNE1 gene. D, CCNE1 relative copy number change assessment by ddPCR against two different reference genes, RNase P and TERT. E, BrdUrd incorporation-ELISA assay in cells transfected 4 days earlier with control siCON2 or SMARTpool siRNA targeting CDK4 or cyclin D1. Western blot analysis of the same experiment showing knockdowns in MCF-7 cells. F, Western blots of MCF-7 and MCF-7pR cells treated for 24, 48, and 72 hours with palbociclib (Palbo) and blotted phospho-CDK2 Thr160 and total CDK2. G and H, cells transfected for 4 days with control siCON2 or the indicated SMARTpool siRNAs and treated with vehicle or palbociclib for 72 hours. G, BrdUrd incorporation-ELISA assay adjusted with viable cell number. H, cell number after vehicle or palbociclib for 72 hours (^**, P < 0.001; ^***, P < 0.0001, two-way ANOVA with Tukey multiple comparisons test).

Figure 6

Acquisition of resistance to CDK4/6 inhibition through RB1 mutation in PDX. A, tumor growth of PDX244 following vehicle (top graphic) or LEE011 (75 mg/kg, once daily, 6IW; bottom graphic) treatment. Tumor volume (mm³) is shown over the days of treatment. The total number of mice in each arm (n) and the relevant genetic alterations of PDX244 are indicated. B, Western blots with the indicated antibodies in PDX244 after 23 days of vehicle treatment or 89 days of LEE011 treatment. Each lane belongs to one individual tumor. C, relative tumor growth of PDX244LR1 following vehicle or LEE011 treatment (^***, P < 0.001, Student t test). The relevant genetic alterations of PDX244LR1 are indicated. D, Western blots with the indicated antibodies in PDX244LR1 after 16 days of vehicle or LEE011 treatment. Each lane belongs to one individual tumor. E, RB1 pM695fs^*26 fraction in vehicle or LEE011-treated samples from PDX244LR1 assessed by ddPCR against RB1 wild-type (^*, P < 0.05, Student t test).

Figure 7

Early treatment with CDK4/6 plus PI3K inhibitors prevents acquisition of resistance in PDXs, but is not efficacious in resistant cells. A, relative growth of parental (MCF-7 and T47D) and palbociclib-resistant (MCF-7pR and T47DpR) cell lines treated for 6 days with control vehicle, 500 nmol/L palbociclib, 250 nmol/L GDC0941 (0941), or the combination (^*, P < 0.05; ^***, P < 0.001; NS, not significant, two-way ANOVA with Tukey multiple comparisons test). B, clonogenic survival assays in MCF-7pR and T47DpR cells treated for 14 days. C, BrdUrd incorporation-ELISA assays in cells treated for 24 or 72 hours (^*, P < 0.05 palbociclib or GDC0941 vs. combination at 72 hours, two-way ANOVA with Tukey multiple comparisons test). D, Western blots of resistant cells treated for 96 hours as indicated. E, relative tumor growth of PDX244 following vehicle, BYL719, LEE011, or the combination (^***, P < 0.0003 Student t test for LEE011 vs. BYL719 plus LEE011). F, Western blot analysis with the indicated antibodies in PDX244 after 103 days of vehicle, BYL719, LEE011, or the combination. Each lane belongs to one individual tumor.