Preclinical characterization of the CDK4/6 inhibitor LY2835219: in-vivo cell cycle-dependent/independent anti-tumor activities alone/in combination with gemcitabine

Abstract

The G1 restriction point is critical for regulating the cell cycle and is controlled by the Rb pathway (CDK4/6-cyclin D1-Rb-p16/ink4a). This pathway is important because of its inactivation in a majority of human tumors. Transition through the restriction point requires phosphorylation of retinoblastoma protein (Rb) by CDK4/6, which are highly validated cancer drug targets. We present the identification and characterization of a potent CDK4/6 inhibitor, LY2835219. LY2835219 inhibits CDK4 and CDK6 with low nanomolar potency, inhibits Rb phosphorylation resulting in a G1 arrest and inhibition of proliferation, and its activity is specific for Rb-proficient cells. In vivo target inhibition studies show LY2835219 is a potent inhibitor of Rb phosphorylation, induces a complete cell cycle arrest and suppresses expression of several Rb-E2F-regulated proteins 24 hours after a single dose. Oral administration of LY2835219 inhibits tumor growth in human tumor xenografts representing different histologies in tumor-bearing mice. LY2835219 is effective and well tolerated when administered up to 56 days in immunodeficient mice without significant loss of body weight or tumor outgrowth. In calu-6 xenografts, LY2835219 in combination with gemcitabine enhanced in vivo antitumor activity without a G1 cell cycle arrest, but was associated with a reduction of ribonucleotide reductase expression. These results suggest LY2835219 may be used alone or in combination with standard-of-care cytotoxic therapy. In summary, we have identified a potent, orally active small-molecule inhibitor of CDK4/6 that is active in xenograft tumors. LY2835219 is currently in clinical development.

Trial registration: ClinicalTrials.gov NCT01394016 NCT01739309 NCT02014129 NCT02057133 NCT02079636 NCT02117648.

Figures

Fig. 1

a Structure of LY2835219. b KINOMEscan dendogram for biochemical kinase selectivity profile against 456 kinases. single point binding at 200 nM (left) and 2 μM (right) LY2835219

Fig. 2

a Activity of LY2835219 in colo-205 and MCF10A cells using a multiplexed assay measuring p-Rb serine 780 (green), cells in G1 (2 N DNA content, red) and total cell number (black). after 24-hour exposure LY2835219 shows a dose-dependent inhibition of p-Rb (ser780) and a corresponding sustained G1 arrest. b MV4-11 acute myelocytic leukemia (AML) cells treated with increasing concentrations of LY2835219 for 24 h and cell cycle activity profiled by flow cytometry. c cell cycle activity of LY2835219 in cells with and without functional Rb. LY2835219 gives a G1 cell cycle arrest in Rb-proficient MDA-MB-231 breast cancer cells, but not in Rb-deficient MDA-MB-468 breast cancer cells

Fig. 3

In vivo target inhibition by LY2835219. a tumors harvested 24 h after dosing for analysis with the markers topoIIα (S phase-specific), p-Rb (pRb ser 780, G1-specific), pHH3 (M-specific), and the Rb/E2F regulated proteins DHFR, RRM1 and RRM2. GAPDH was used as a loading control. statistical significance (p <0.05) is indicated above each dose by an asterisk for pRb, Topollα, and pHH3, respectively. b time course of LY2835219. tumor-bearing animals were dosed orally with 50 mg/kg of LY2835219 and tumors harvested at the indicated times (n = 5 per treatment, n = 8 for vehicle). the percent change in band intensity was calculated using vehicle-treated bands as 100 %. mean (±SD) plasma concentrations of the compound are shown in the lower right panel

Fig. 4

In vivo antitumor activity of LY2835219 in subcutaneous human tumor xenografts. Tumors were implanted in the rear flank of athymic mice and randomized for treatment when the mean tumor volume reached 150 to 200 mm3. LY2835219 and PD0332991 were administered at the indicated dose and schedule. treatment period is indicated by the horizontal black bar along the X-axis, body weight shown for each experiment is shown in the upper left corner. a effect of 25, 50, or 100 mg/kg of LY2835219 and 100 mg/kg PD0332991 on colo-205 xenografts. b inhibition of cell cycle markers in colo-205 xenografts at the end of treatment with 100 mg/kg LY2835219. c effect of 25, 50, or 100 mg/kg LY2835219 and 50 mg/kg PD0332991 in MV4-11 xenografts. d LY2835219 inhibits tumor growth and is well tolerated in mice bearing colo-205 xenografts when dosed 56 days with 50 mg/kg continuously or intermittently

Fig. 5

In vivo antitumor activity of LY2835219 and gemcitabine. a Calu-6 lung xenografts were treated with LY2835219 orally, with gemcitabine by intraperitoneal injection, LY2835219 followed by gemcitabine, or both compounds together. treatment period is indicated by horizontal black bar along the X-axis and body weight is shown in the upper left corner. b results of target inhibition studies