Abemaciclib is an ATP-competitive, reversible kinase inhibitor selective for CDK4 and CDK6 that has shown antitumor activity as a single agent in hormone receptor positive (HR+) metastatic breast cancer in clinical trials. Here, we examined the mechanistic effects of abemaciclib treatment using in vitro and in vivo breast cancer models. Treatment of estrogen receptor positive (ER+) breast cancer cells with abemaciclib alone led to a decrease in phosphorylation of Rb, arrest at G1, and a decrease in cell proliferation. Moreover, abemaciclib exposure led to durable inhibition of pRb, TopoIIα expression and DNA synthesis, which were maintained after drug removal. Treatment of ER+ breast cancer cells also led to a senescence response as indicated by accumulation of β-galactosidase, formation of senescence-associated heterochromatin foci, and a decrease in FOXM1 positive cells. Continuous exposure to abemaciclib altered breast cancer cell metabolism and induced apoptosis. In a xenograft model of ER+ breast cancer, abemaciclib monotherapy caused regression of tumor growth. Overall these data indicate that abemaciclib is a CDK4 and CDK6 inhibitor that, as a single agent, blocks breast cancer cell progression, and upon longer treatment can lead to sustained antitumor effects through the induction of senescence, apoptosis, and alteration of cellular metabolism.

Appropriate therapeutic modulation of endothelial proliferation and sprouting is essential for the effective inhibition of angiogenesis in cancer or its induction in cardiovascular disease. The current view is that an increase in growth factor concentration, and the resulting mitogenic activity, increases both endothelial proliferation and sprouting. Here, we modulate mitogenic stimuli in different vascular contexts by interfering with the function of the VEGF and Notch signalling pathways at high spatiotemporal resolution in vivo. Contrary to the prevailing view, our results indicate that high mitogenic stimulation induced by VEGF, or Notch inhibition, arrests the proliferation of angiogenic vessels. This is due to the existence of a bell-shaped dose-response to VEGF and MAPK activity that is counteracted by Notch and p21, determining whether endothelial cells sprout, proliferate, or become quiescent. The identified mechanism should be considered to achieve optimal therapeutic modulation of angiogenesis.

Improved methods for manipulating and analyzing gene function have provided a better understanding of how genes work during organ development and disease. Inducible functional genetic mosaics can be extraordinarily useful in the study of biological systems; however, this experimental approach is still rarely used in vertebrates. This is mainly due to technical difficulties in the assembly of large DNA constructs carrying multiple genes and regulatory elements and their targeting to the genome. In addition, mosaic phenotypic analysis, unlike classical single gene-function analysis, requires clear labeling and detection of multiple cell clones in the same tissue. Here, we describe several methods for the rapid generation of transgenic or gene-targeted mice and embryonic stem (ES) cell lines containing all the necessary elements for inducible, fluorescent, and functional genetic mosaic (ifgMosaic) analysis. This technology enables the interrogation of multiple and combinatorial gene function with high temporal and cellular resolution.

The original version of this Article contained errors in Fig. 8. In panel a, the labels 'VEGF', 'Notch', 'p21', and 'P-ERK' were inadvertently omitted. This has been corrected in the PDF and HTML versions of the Article.

Dysregulation of the cell-cycle is a hallmark of cancer and genetic alterations in its regulatory machinery (or checkpoints) occur in most human tumors. The majority these defects are found in genes encoding for proteins regulating G1 phase progression, such as Rb, E2F1, CyclinD1, CDK4 and CDK6. Aberrant regulation of the G1 kinases CDK4 and CDK6, as well as overexpression or gene amplification of CyclinD, lead to inhibition of tumor suppressors such as Rb resulting in an accelerated cell cycle progression. Alterations in the CyclinD-CDK4/6-Rb pathway are common in breast cancer. Amplification of CCND1 gene encoding CyclinD1, occurs in 15% to 20% of breast cancers, and CyclinD1 overexpression is even more common (up to 50% of breast cancers). Abemaciclib is a reversible, ATP competitive, kinase inhibitor selective for CDK4 and CDK6 that has been shown to prevent growth of malignant cells in-vitro and in-vivo. This antitumor activity is mediated by inhibiting the phosphorylation of Rb and subsequent blockade of tumor cell cycle progression through G1/S. CDK4/6 inhibitors in general have shown significant potential for the treatment of metastatic breast cancer and Abemaciclib, in particular, is currently being evaluated in advanced clinical trials (Phase II as single agent and Phase III in combination with anti-hormone therapy) in hormone receptor positive metastatic breast cancer patients. The goal of this study was to investigate the mechanism of action of Abemaciclib in ER+ luminal breast cancer. We have evaluated the response of the drug in a diversity of breast cancer cell lines. Phenotypic characterization of sensitive cell lines was carried out by monitoring proliferation, cell cycle progression and phosphorylation of Rb using High Content Imaging. Senescence markers were included in the study to monitor the final outcome of the cells upon sustained exposure to the drug. Luminal ER+ breast cancer cells showed a marked sensitivity to treatment with Abemaciclib with IC50 values ranging from 5nM to 2uM. Simultaneous decrease in Rb phosphorylation with sustained accumulation of the 2N subpopulation was observed. Associated to the G1S arrest phenotype, Abemaciclib treatment resulted in a decrease of cell proliferation markers (Ki67 and BrdU). Additionally, a marked hyper-methylation profile (Histone H3K9met3) and a decrease of FOXM1 expression were observed, as well as an accumulation of endogenous beta-galactosidase and p21. Taken together this profile suggests that Abemaciclib acts through promotion of senescence in breast cancer cells. Abemaciclib prevents proliferation of breast cancer cell lines expressing D-types cyclins by promoting cell cycle arrest mediated by inhibition of Rb phosphorylation. Abemaciclib is a CDK4/6 inhibitor with potential to treat breast cancer by blocking cell proliferation leading to induction of senescence. Citation Format: Maria Jose Lallena, Karsten Boehnke, Raquel Torres, Ana Hermoso, Joaquin Amat, Bruna Calsina, Alfonso De Dios, Sean Buchanan, Jian Du, Richard Paul Beckmann, Xueqian Gong, Ann Mcnulty. In-vitro characterization of Abemaciclib pharmacology in ER+ breast cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3101. doi:10.1158/1538-7445.AM2015-3101

Breast cancer is the second most common cancer worldwide after lung cancer. About 70% of breast cancers express estrogen receptor α (ER+) and/or progesterone receptor (PR+), and these biomarkers are indicative of hormone dependence. However up to 50% acquire resistance to hormone therapy [1, 2]. Estrogen independent ER+ breast cancer depends on CDK4 for tumor growth and CDK4 inhibitors have emerged as a promising approach to treat this type of tumors [3]. Abemaciclib is a cell cycle inhibitor with selective activity against CDK4 and CDK6 and it is being evaluated in advanced clinical trials for its potential to reduce metastatic ER+ breast cancer growth. We have evaluated combination of abemaciclib with an anti-estrogen therapy in an in vitro breast cancer panel. Phenotypic characterization of sensitive cell lines was carried out by monitoring cell proliferation, senescence, and apoptosis markers using flow cytometry and high content imaging approaches. Using an in vitro panel with a diversity of breast cancer cell lines, a synergistic effect of abemaciclib in combination with the ER down-regulating drug fulvestrant was observed based on Bliss score. This combination treatment demonstrated effective growth inhibition in ER+ cells and exhibited synergism in MCF-7, T47D and ZR-75-1. The mechanistic analyses revealed that the combination of abemaciclib with fulvestrant promoted a decrease in cancer cell proliferation due to G1 phase arrest at doses tested. This growth inhibition was accompanied by increased hallmarks for cell senescence as observed by markers such as SA-β-galactosidase staining or morphological changes. Subsequently, an increase in biomarkers for apoptosis was also observed. These changes occurred in a time dependent manner and were significantly greater with the combination than fulvestrant single agent treatment. We conclude the combination of abemaciclib with fulvestrant better prevented proliferation of breast cancer cell lines by blocking cell proliferation and lead to induction of senescence and apoptosis as compared to fulvestrant treatment alone in ER+ cells. Bibliography [1] American Cancer Society, Cancer Facts & Figures 2014. [2] Dixon J.M. (2014) New Journal of Science. Volume 2014, Article ID 390618. [3] Miller TW et al. (2011) Cancer Discov. Volume 1 (4): 338-51. Citation Format: Raquel Torres, Bruna Calsina, Ana Hermoso, Carmen Baquero, Cecilia Mur, Karsten Boehnke, Joaquín Amat, Alfonso De Dios, Xueqian Gong, Sean Buchanan, Richard Paul Beckmann, Maria Jose Lallena. Characterization of the mechanism of action for abemaciclib with antiestrogen combined therapy in human breast cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2836.

Proper patient-tailoring strategy and the validation of novel therapeutic targets remain enormous challenges during drug discovery processes. Patient-derived three-dimensional organoid cell culture models possess great potential to associate compound sensitivity and disease complexity in order to provide a key missing link between compound screening and clinical trials. Abemaciclib is a reversible, ATP competitive, selective inhibitor of the kinase activity of both CDK4 and CDK6 and is currently undergoing advanced clinical testing. In this study, we established and characterized three-dimensional organoid cultures from primary colorectal cancer patients and validated their use as drug sensitivity models. We aimed to explore the antitumor activity of abemaciclib in colon cancer organoid cultures by assessing markers for cell viability, proliferation, cell cycle, senescence and apoptosis. Single cell suspension of patient-derived samples were precultured for four days to allow for complete morphogenesis of three-dimensional organoid structures. Subsequently, the cultures were treated for at least two population doubling times and analyzed by luminescent cell viability, immunohistochemistry and flow cytometry assays. Our data suggest that abemaciclib treatment decreased the cell viability of patient-derived colorectal cancer organoid cultures characterized by G1 cell cycle arrest and reduced Ki-67-positive cells. Furthermore, treated cultures showed elevated levels of reactive oxygen species and increased markers for early and late apoptosis. In summary, complex organoid models have the potential to further evaluate the antitumor activity of abemaciclib in various tumor types by enabling mechanistic studies in a patient-specific preclinical setting. Citation Format: Karsten Boehnke, Bruna Calsina, Joaquín Amat, Ana Hermoso, Raquel Torres, Christoph Reinhard, Juan A. Velasco, Philip W. Iversen, Alfonso De Dios, Sean Buchanan, Richard P. Beckmann, Dirk Schumacher, Christian RA Regenbrecht, Marie-Laure Yaspo, Hans Lehrach, María José Lallena. Preclinical analysis and characterization of abemaciclib using three-dimensional patient-derived colorectal cancer organoid cultures. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2829.