PD-0332991 induces cell cycle arrest and senescence of GBM cells dependent on Rb status. A, Flow cytometry analysis of three Rb-proficient GBM cell lines (DBTRG-05MG, LN229, and U87MG) and one Rb-deficient cell line (42MGBA) following culture in the presence of vehicle or 1 µM PD-0332991 for 48 hrs and then pulsed with BrdU for 1 hr. Cell cycle distribution plots of BrdU vs. propidium iodide (PI) intensity are shown. B, Quantification of reduction in S phase cells depicted in A. C, Photomicrographs of the Rb-proficient cell lines after culture in the presence of 1 µM PD-0332991 for 7 days reveal growth inhibition and morphological changes resembling cellular senescence, whereas the Rb-deficient cell line (42MGBA) shows no growth inhibition or morphological changes. D, Staining for senescence-associated β-galactosidase activity in two Rb-proficient cell lines and one Rb-deficient cell line after culture in the presence of vehicle or 1 µM PD-0332991 for 2 weeks.

Depletion of Rb expression alleviates growth arrest induced by PD-0332991. A, Five unique shRNAs to RB1 were transduced into U87MG cells, and total protein from stably expressing clones was resolved by SDS-PAGE and immunoblotted with an Rb antibody. Clones 19 and 63 show greater than 99% knockdown of Rb expression relative to the empty pLKO.1-infected clone. B, Flow cytometry analysis following a 1 hr BrdU pulse of U87MG Rb shRNA clones 19 and 63 after 48 hrs of treatment with vehicle or 1 µM PD-0332991. C, Photomicrographs of clones 19 and 63 following incubation with 1 µM PD-0332991 for 3 weeks demonstrates their failure to arrest and eventual growth to confluence in spite of sustained incubation with the cdk4/6 inhibitor.

PD-0332991 crosses the blood-brain barrier and potently suppresses the growth of intracranial human GBM xenografts. U87MG cells (A–C) and primary xenograft GBM 39 (D) were modified to express luciferase and injected into the brains of nude mice to establish intracranial tumors. Mice were randomized to control (vehicle treated) and PD-0332991 treatment groups, with PD-0332991 administered daily by oral gavage at 150 mg/kg (4 weeks for U87MG; 12 weeks for GBM 39: see gray horizontal arrows in A,D). Mice were imaged 1–2× weekly for bioluminescence intensity (see examples in A and D), with luminescence values of individual mice normalized to their corresponding luminescence at the start of therapy, and mean normalized values plotted (left panels of A,D). B, One mouse from the U87MG treatment group was euthanized and its brain was dissected for the isolation of pure tumor, brain adjacent to tumor (BAT), and normal tissue from the opposite hemisphere (OH). These specimens were analyzed by LC-MS/MS for determination o f P D-0332991 content which demonstrated dissemination of PD-0332991 into the brain and accumulation in tumor tissue. C, One mouse from the U87MG treatment group and one mouse from the control group were euthanized on the same day post tumor cell injection, and their brains dissected, fixed, and embedded in paraffin. Immunohistochemistry with MIB-1 antibody revealed >8-fold reduction in MIB-1 index in the tumor from the PD-0332991 treated mouse relative to the control. A,D (right panels) Kaplan-Meier survival plots for the U87MG and GBM 39 experiments demonstrating significant survival benefit from PD-0332991 treatment (p < 0.001 in each case). See for statistical analysis of mean survival.

PD-0332991 combined with radiation therapy shows enhanced anti-tumor activity and extends survival. Luciferase-modified U87MG cells were injected into the brains of nude mice to establish intracranial tumors. Mice were randomized to four treatment groups: untreated (Control), radiation only (RT, 2 Gy/day ×5 days, horizontal gray arrow), PD-0332991 only (150 mg/kg/day × 14 days, horizontal black arrow), PD-0332991 and concurrent radiation (RTc), and PD-0332991 with subsequent radiation (RTs). A, Bioluminescence monitoring was conducted 1–2× weekly throughout the course of the experiment, with mean normalized values plotted for each treatment group. B, Kaplan-Meier survival plots demonstrating survival benefit from PD-0332991 and radiation combination therapy. See for statistical analysis of mean survival.

Evaluation of PD-0332991 activity against intracranial GBM xenografts that have recurred following initial treatment with temozolomide. A, Luciferase-modified U87MG cells were injected into the brains of athymic mice. Mice with proliferating tumors were randomized into untreated (Control) and temozolomide treated groups (TMZ, 10 mg/kg/day for 5 days). B, When successive mean normalized luminescence values indicated tumor recurrence of TMZ treated mice (day 35), these mice were randomized to 4 treatment groups: no additional treatment, repeat treatment with the same TMZ regimen, treatment with PD-0332991 (150 mg/kg/day for 14 days), or combined TMZ + PD-0332991 treatment, and then monitored for bioluminescent intensity 1–2× weekly during therapy. C, Kaplan-Meier survival plots demonstrating significant survival benefit from PD-0332991 therapy of recurrent tumors. See for statistical analysis of mean survival.