Multiple roles of cyclin-dependent kinase 4/6 inhibitors in cancer therapy

Abstract

Background: Cyclin-dependent kinases (CDKs) regulate cell proliferation and coordinate the cell cycle checkpoint response to DNA damage. Although inhibitors with varying selectivity to specific CDK family members have been developed, selective CDK4/6 inhibitors have emerged as the most attractive antineoplastic agents because of the importance of CDK4/6 activity in regulating cell proliferation and the toxic effects associated with inhibition of other CDKs (eg, CDK1 and CDK2).

Methods: FVB/N wild-type mice (n = 13) were used to evaluate carboplatin-induced myelosuppression in bone marrow by complete blood cell counts after treatment with the CDK4/6 inhibitor PD0332991. Genetically engineered murine models of retinoblastoma (Rb)-competent (MMTV-c-neu) and Rb-incompetent (C3-TAg) breast cancer (n = 16 MMTV-c-neu mice in the carboplatin plus vehicle control group, n = 17 MMTV-c-neu mice in the carboplatin plus PD0332991 group, n = 17 C3-TAg mice in the carboplatin plus vehicle control group, and n = 14 C3-TAg mice in the carboplatin plus PD0332991 group) were used to investigate the antitumor activity of PD0332991 alone or in combination with chemotherapy. All statistical tests were two-sided.

Results: Coadministration of PD0332991 with carboplatin compared with carboplatin alone in FVB/N wild-type mice increased hematocrit (51.2% vs 33.5%, difference = 17.7%, 95% confidence interval [CI] = -26.7% to -8.6%, P < .001), platelet counts (1321 vs 758.5 thousand cells per μL, difference = 562.5 thousand cells per μL, 95% CI = -902.8 to -222.6, P = .002), myeloid cells (granulocytes and monocytes; 3.1 vs 1.6 thousand cells per μL, difference = 1.5 thousand cells per μL, 95% CI = -2.23 to -0.67, P < .001), and lymphocytes (7.9 vs 5.4 thousand cells per μL, difference = 2.5 thousand cells per μL, 95% CI = -4.75 to -0.18, P = .02). Daily administration of PD0332991 exhibited antitumor activity in MMTV-c-neu mice as a single agent. However, the combination of carboplatin plus PD0332991 decreased antitumor activity compared with carboplatin alone in Rb-competent mice (mean percent change in tumor volume at day 21 = -52.6% vs 3.7% for carboplatin and carboplatin plus PD0332991, respectively, difference = 56.3%, 95% CI = -109.0% to -3.6%, P = .04). In contrast, Rb-deficient tumors in C3-Tag mice were resistant to PD0332991, and coadministration of PD0332991 plus carboplatin had no effect on in vivo tumor growth (mean percent change in tumor volume at day 21 = 118.8% and 109.1% for carboplatin and carboplatin plus PD0332991, respectively, difference = 9.7%, 95% CI = -183.5% to 202.9%, P = .92). Finally, in tumor-bearing mice, coadministration of PD0332991 with carboplatin provided statistically significant protection of platelets (P = .04).

Conclusion: We believe that the present data support a possible role for CDK4/6 inhibitors in a majority of patients with advanced cancer: to either inhibit tumor growth in CDK4/6-dependent tumors or ameliorate the dose-limiting toxicities of chemotherapy in CDK4/6-indepdendent tumors. Our data also suggest CDK4/6 inhibitors should not be combined with DNA-damaging therapies, such as carboplatin, to treat tumors that require CDK4/6 activity for proliferation.

Figure 1

Ability of pharmacological quiescence to protect against chemotherapy-induced γ-H2AX formation and apoptosis. The tHDF cells were treated with varying concentrations of PD0332991 (PD) before treatment with 100 μM carboplatin, 1 μM doxorubicin, 5 μM etoposide, 156 nM camptothecin, 250 nM paclitaxel, or 1.5 nM staurosporine. The tHDF cells were then assayed for A) γ-H2AX formation and B) caspase 3/7 activation. γ-H2AX formation data measured by flow cytometry represent at least 20 000 gated events. Caspase 3/7 data are presented as the average ratio from a single experiment of 1000 cells per well in quadruplicate. P values were calculated by a two-sided χ² test (γ-H2AX formation data) or one-way analysis of variance (caspase 3/7 data) with Bonferroni correction for multiple comparisons. All data are representative of three independent experiments. *P < .05, **P < .01, ***P < .001. RLU = relative light units; tHDF = telomerized human diploid fibroblast.

Figure 2

Pharmacodynamic assessment of CDK4/6 inhibition in the bone marrow. A) FVB/N mice (n = 3 mice per group) were treated with a single dose of 150 mg/kg PD0332991 by oral gavage to assess the temporal effect of transient CDK4/6 inhibition on bone marrow arrest and was quantitated by EdU incorporation at the indicated times. Four hours before bone marrow harvest, mice were treated with 100 µg of EdU. After the mice were killed by CO₂ inhalation followed by cervical dislocation, bone marrow was harvested and sorted by expression of lineage markers (L), c-KIT (K), and Sca1 (S) to evaluate the temporal impact of CDK4/6 inhibition on specific cell populations of the bone marrow: hematopoietic stem and progenitor cells (L⁻K⁺S⁺) and myeloid progenitors (L⁻K⁺S⁻). Error bars represent 95% confidence intervals. B) To assess the effect of transient CDK4/6 inhibition on carboplatin-induced apoptosis in the bone marrow, FVB/N mice (three mice per group) were treated with vehicle control, 150 mg/kg PD0332991 by oral gavage, 90 mg/kg carboplatin by intraperitoneal injection, or 150 mg/kg PD0332991 oral gavage plus 90 mg/kg carboplatin by intraperitoneal injection. Twenty-four hours after treatment, mice were killed, bone marrow was harvested, and apoptosis was measured by caspase 3/7 activation. Results shown represent a single experiment, in which each mouse represents a unique biological replicate and four technical replicates from each mouse were analyzed. Error bars represent 95% confidence intervals. Two-sided P values were calculated by student t test. Carbo = carboplatin; CDK = Cyclin-dependent kinases; PD = PD0332991; RLU = relative light units.

Figure 3

Ability of transient pharmacological quiescence to provide quadrilineage protection from chemotherapy-induced myelosuppression. FVB/N wild-type mice (n = 13 mice per group) were treated with vehicle control, 90 mg/kg carboplatin by intraperitoneal injection, or 90 mg/kg carboplatin by intraperitoneal injection plus 150 mg/kg PD0332991 by oral gavage. Complete blood cell counts were analyzed on day 14. A) hematocrit levels and the number of B) platelets, C) myeloid cells, and D) lymphocytes were determined. Boxes represent the 5%–95% distribution, whiskers represent minimum and maximum values, and the middle bar represents the median. Student's t test was done to calculate two-sided P values. Carbo = carboplatin; PD = PD0332991.

Figure 4

The use of CDK4/6 inhibitors in a CDK4/6-dependent genetically engineered murine model of breast cancer. A) A single dose of 150 mg/kg PD0332991 (n = 4) or vehicle control (n = 3) was administered by oral gavage to tumor-bearing MMTV-c-neu mice. Twenty-four hours later, tumors were excised, and the mean tumor EdU labeling, a measure of proliferation, was assessed. Boxes represent the 5%–95% distribution, whiskers represent the minimum and maximum values, and the middle bar represents the median number of proliferating cells labeled with EdU. Nonlinear regression analysis was used to compare the change in baseline tumor volume for the two groups during the course of the study. B–C) Tumor-bearing MMTV-c-neu mice (PD0332991, n = 7; control, n = 19) were treated with either PD0332991 (100 mg/kg/d) delivered in chow or standard chow (control) for 21 days (purple line). B) Tumor volumes were recorded weekly and graphed as the percent change in baseline tumor volume. Error bars represent the 95% confidence intervals (CI). Nonlinear regression analysis was used to compare the change in baseline tumor volume for the two groups during the course of the study. C) Kaplan–Meier curves for the control and PD0332991-treated groups are shown. The error bars represent the 95% CIs. The hazard ratio (HR) and 95% CI were calculated, and the P value was calculated using a two-sided log-rank test. D) Tumor-bearing MMTV-c-neu mice were randomly assigned to each of the study groups (carboplatin plus vehicle control, n = 16, or carboplatin plus PD0332991, n = 17). Mice received carboplatin at 75 mg/kg by intraperitoneal injection with or without PD0332991 (150 mg/kg by oral gavage) once a week for 3 weeks (purple arrows). Error bars represent 95% confidence intervals. Student t test was done to calculate two-sided P values for EdU incorporation in (B) and (D). All statistical tests were two-sided.

Figure 5

The use of CDK4/6 inhibitors in a CDK4/6-independent genetically engineered murine model of breast cancer. A) A single dose of 150 mg/kg PD0332991 (n = 4) or vehicle control (n = 6) was administered by oral gavage to tumor-bearing C3-TAg mice. Twenty-four hours later, tumors were excised, and the mean tumor EdU labeling, a measure of proliferation, was assessed. Boxes represent 5%–95% distribution, whiskers represent the minimum and maximum values, and the middle bar represents the median number of proliferating cells labeled with EdU. Student t test was done to calculate two-sided P value for EdU incorporation. B–C) Tumor-bearing C3-TAg mice (PD0332991, n = 7; control, n = 11) were treated with either PD0332991 (100 mg/kg/d) delivered in chow or standard chow (control) for 21 days (purple line). B) Tumor volumes were recorded weekly and graphed as the percent change in baseline tumor volume. Error bars represent the 95% confidence intervals. Nonlinear regression analysis was used to compare the change in baseline tumor volume for the two groups during the course of the study. C) Kaplan–Meier curves for the control and PD0332991-treated groups are shown. The dashed lines indicate the 95% confidence intervals. D) Tumor-bearing MMTV-c-neu mice were randomly assigned to each of the study groups (carboplatin plus vehicle control, n = 17, or carboplatin plus PD0332991, n = 14). Mice were administered 75 mg/kg carboplatin by intraperitoneal injection with or without PD0332991 (150 mg/kg by oral gavage) once a week for 3 weeks (purple arrows). Error bars represent 95% confidence intervals. Nonlinear regression analysis was used to compare the change in baseline tumor volume for the two groups during the course of the study in (B) and (D). All statistical tests were two-sided.