Abstract SY34-03: Braking the cycle: Inhibition of the cyclin D-Cdk4/6 pathway in breast cancer
Authors: Abraham Robert T. , VanArsdale Todd , Shields David V. , Lee Nathan V. , Koehler Maria , Arndt Kim 
Published: May 2015

Abstract
In spite of the dramatically improved outlook for patients with ER+ breast cancer, a subset of patients are intrinsically non-responsive to anti-hormonal therapy alone, and even the responders invariably acquire resistance to this treatment modality. In the setting of established hormone resistance, most of these patients will receive chemotherapy, with limited benefit and considerable impact on quality of life due to toxicity. Given the sheer numbers of patients affected (approximately two-thirds of all breast cancers are ER+), the unmet need for well-tolerated therapies that overcome resistance to anti-hormonal agents remains very high.
Strikingly, the vast majority (&gt;90%) of ER+ breast cancers express a functional retinoblastoma protein (Rb), a tumor suppressor that represents a critical regulator of the G1 to S phase transition in mammalian cells. In its hypo-phosphorylated state, Rb suppresses the expression of proteins that are essential for commitment to S-phase entry and progression through the remainder of the eukaryotic cell cycle. The G1 cyclin-dependent kinases 4 and 6 (cdk4 and cdk6) which function in complexes with the D-type cyclins (collectively termed cyclin D) initiate the phosphorylation of Rb and override the repressive effects of Rb on cell-cycle progression. In ER-driven breast cancers, oncogenic signaling through the ER stimulates the cyclin D-cdk4/6 -dependent phosphorylation of Rb, and this proliferative stimulus is frequently augmented by amplification of cyclin D or loss of expression of the cyclin D-cdk4/6 inhibitor, p16 (encoded by the CDKN2A gene), two alterations that lead to elevated and dysregulated cyclin D-cdk4/6 activity. The requirement for cyclin D-cdk4/6 activity to circumvent the cell-cycle braking action of Rb suggested that ER+ breast cancer cells might be vulnerable to drug-induced inhibition of cdk4/6 activity.
PD-0332991 is an orally administered, selective inhibitor of cyclin D-cdk4/6 kinase activities. In preclinical models, this drug showed significant anti-proliferative activity in Rb+ breast cancer cell lines, the majority of which also expressed the ER. The most well established substrate for the cyclin D-cdk4/6 is Rb, which is modified at multiple phosphoacceptor sites (including Ser-780, Ser-795, Ser-807, and Ser-811) by these cdks. The phosphorylation status of the Ser-807 and Ser-811 residues in Rb proved to be the most robust pharmacodynamic readouts for PD-0332991 activity in vivo, and serve as appropriate biomarkers for target modulation by this drug under in vitro and in vivo study conditions. The IC50 for reduction of Rb phosphorylation at these sites in two breast cancer cell lines (MCF7 and T47D) was approximately 20 nM. Similarly, we observed that PD-0332991 inhibits S-phase entry (measured by incorporation of thymidine into newly synthesized DNA) and cellular proliferation in panel of Rb+ cancer cell lines, with IC50 values ranging from 40-170 nM. The antiproliferative effects of PD-0332991 were not limited to transformed cells, reinforcing the idea that this drug was capable of suppressing the proliferation of any human cell type that requires cyclin D-cdk4/cdk6 activity to bypass the G1 restriction point imposed by Rb. Conversely, cancer cells that have inactivated Rb through genetic or epigenetic mechanisms are highly resistant (IC50 &gt; 3 uM) to the inhibitory effects of PD-0332991 on cell-cycle progression and cellular proliferation.
Xenograft experiments confirmed the favorable in vivo safety profile and antitumor activity of PD-0332991. The maximum tolerated dose (MTD; defined as the dose that induced &lt;10% lethality) in SCID mice was 150 mg/kg/day when administered orally, once per day, for 14 days. Daily dosing at levels equal to or less than this MTD resulted in significant growth inhibition in many but not all Rb+ human tumor xenograft models. These studies in xenograft-bearing mice also confirmed that PD-0332991 was inactive at therapeutically obtainable concentrations against Rb-negative tumors.
These preclinical studies provided a launching pad for a clinical trial in post-menopausal women with locally advanced or metastatic ER+ (HER2-) breast cancer. In this Phase 2 study, PD-0332991 was given in combination with letrozole (an inhibitor of estrogen synthesis), with the control arm receiving letrozole only. An interim analysis showed that the PD-0332991 plus letrozole combination yielded a dramatic improvement in treatment outcomes, more than tripling progression-free survival relative to the letrozole-only treatment arm. The drug was generally well-tolerated, due in part to its high degree of selectivity for cdk4/6 relative to other members of the cyclin-dependent kinase family, as well as other members of the extended kinase superfamily. These positive results led to a Breakthrough Therapy designation in 2013 for PD-0332991 in the treatment of patients with ER+ breast cancer. This presentation will provide information on the mechanisms of antitumor action of PD-0332991, together with updates on the current status of clinical development of this new addition to the armentarium of breast cancer treatments.
Citation Format: Robert T. Abraham, Todd VanArsdale, David V. Shields, Nathan V. Lee, Maria Koehler, Kim Arndt. Braking the cycle: Inhibition of the cyclin D-Cdk4/6 pathway in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY34-03. doi:10.1158/1538-7445.AM2014-SY34-03