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Although Aurora A, B, and C kinases share high sequence similarity, especially within the kinase domain, they function distinctly in cell-cycle progression. Aurora A depletion primarily leads to mitotic spindle formation defects and consequently prometaphase arrest, whereas Aurora B/C inactivation primarily induces polyploidy from cytokinesis failure. Aurora B/C inactivation phenotypes are also epistatic to those of Aurora A, such that the concomitant inactivation of Aurora A and B, or all Aurora isoforms by nonisoform–selective Aurora inhibitors, demonstrates the Aurora B/C-dominant cytokinesis failure and polyploidy phenotypes. Several Aurora inhibitors are in clinical trials for T/B-cell lymphoma, multiple myeloma, leukemia, lung, and breast cancers. Here, we describe an Aurora A–selective inhibitor, LY3295668, which potently inhibits Aurora autophosphorylation and its kinase activity in vitro and in vivo, persistently arrests cancer cells in mitosis, and induces more profound apoptosis than Aurora B or Aurora A/B dual inhibitors without Aurora B inhibition–associated cytokinesis failure and aneuploidy. LY3295668 inhibits the growth of a broad panel of cancer cell lines, including small-cell lung and breast cancer cells. It demonstrates significant efficacy in small-cell lung cancer xenograft and patient-derived tumor preclinical models as a single agent and in combination with standard-of-care agents. LY3295668, as a highly Aurora A–selective inhibitor, may represent a preferred approach to the current pan-Aurora inhibitors as a cancer therapeutic agent.

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.

Organoid cultures derived from colorectal cancer (CRC) samples are increasingly used as preclinical models for studying tumor biology and the effects of targeted therapies under conditions capturing in vitro the genetic make-up of heterogeneous and even individual neoplasms. While 3D cultures are initiated from surgical specimens comprising multiple cell populations, the impact of tumor heterogeneity on drug effects in organoid cultures has not been addressed systematically. Here we have used a cohort of well-characterized CRC organoids to study the influence of tumor heterogeneity on the activity of the KRAS/MAPK-signaling pathway and the consequences of treatment by inhibitors targeting EGFR and downstream effectors. MAPK signaling, analyzed by targeted proteomics, shows unexpected heterogeneity irrespective of RAS mutations and is associated with variable responses to EGFR inhibition. In addition, we obtained evidence for intratumoral heterogeneity in drug response among parallel "sibling" 3D cultures established from a single KRAS-mutant CRC. Our results imply that separate testing of drug effects in multiple subpopulations may help to elucidate molecular correlates of tumor heterogeneity and to improve therapy response prediction in patients.

The application of patient-derived three-dimensional culture systems as disease-specific drug sensitivity models has enormous potential to connect compound screening and clinical trials. However, the implementation of complex cell-based assay systems in drug discovery requires reliable and robust screening platforms. Here we describe the establishment of an automated platform in 384-well format for three-dimensional organoid cultures derived from colon cancer patients. Single cells were embedded in an extracellular matrix by an automated workflow and subsequently self-organized into organoid structures within 4 days of culture before being exposed to compound treatment. We performed validation of assay robustness and reproducibility via plate uniformity and replicate-experiment studies. After assay optimization, the patient-derived organoid platform passed all relevant validation criteria. In addition, we introduced a streamlined plate uniformity study to evaluate patient-derived colon cancer samples from different donors. Our results demonstrate the feasibility of using patient-derived tumor samples for high-throughput assays and their integration as disease-specific models in drug discovery.

For decades of molecular cancer research, immortal cancer cell lines have served as an easily accessible source for basic cancer biology research and preclinical testing of anticancer drugs. However, numerous studies have suggested that these cell lines poorly recapitulate the diversity, heterogeneity, and resulting drug resistance or relapse in patients. The derivation and (short-term) culture of primary cells from solid tumors have thus gained significant importance in personalized cancer therapy. This chapter focuses on our current understanding and the pros and cons of different preclinical and prospective clinical models of three-dimensional primary tumor cultures. We will discuss cell culture approaches, such as biomimetic scaffolds and growth factor supplemented, chemically defined media for various forms of solid tumors. Complex culture models of primary tumor cells could finally provide a key missing link between compound screening and clinical trials and ultimately will help redefining therapeutic intervention with high translational relevance at the level of the individual patient.

Colorectal carcinoma represents a heterogeneous entity, with only a fraction of the tumours responding to available therapies, requiring a better molecular understanding of the disease in precision oncology. To address this challenge, the OncoTrack consortium recruited 106 CRC patients (stages I–IV) and developed a pre-clinical platform generating a compendium of drug sensitivity data totalling >4,000 assays testing 16 clinical drugs on patient-derived in vivo and in vitro models. This large biobank of 106 tumours, 35 organoids and 59 xenografts, with extensive omics data comparing donor tumours and derived models provides a resource for advancing our understanding of CRC. Models recapitulate many of the genetic and transcriptomic features of the donors, but defined less complex molecular sub-groups because of the loss of human stroma. Linking molecular profiles with drug sensitivity patterns identifies novel biomarkers, including a signature outperforming RAS/RAF mutations in predicting sensitivity to the EGFR inhibitor cetuximab., The heterogeneity of colorectal cancer has important clinical and therapeutic implications. Here the authors analysed the responses of a large biobank of organoids and xenografts derived from colorectal patients to a panel of clinically relevant therapeutic agents to identify genes signatures associated with drug response.

TNBC, the most aggressive subtype of breast cancer, is characterized by high level of genomic instability, defects in DNA damage response (DDR) and increased replication stress (RS). The current treatment options for TNBC are limited and new approaches are needed. Checkpoint kinase 1 (Chk1) is a key protein kinase that regulates the cell cycle, DNA damage and RS response, and has emerged as an attractive target for anticancer therapy. Prexasertib, an ATP-competitive inhibitor of Chk1 has demonstrated activity in vitro and in vivo across a variety of tumor histologies. Prexasertib is being evaluated in patients with TNBC in a Phase II study sponsored by NCI (NCT02203513). The preliminary results reported in 2016 showed modest single agent activity. PI3K/AKT/mTOR is a critical pathway with key roles on cancer cell survival, homologous recombination repair and drug resistance. Our previous study indicated the expression level of genes related to PI3K/AKT signaling is elevated in prexasertib resistant TNBC patient-derived xenograft (PDX) models. It is hypothesized that inhibiting both the Chk1 and PI3K/AKT pathway may improve prexasertib efficacy. In this study, we evaluated the combination effect of prexasertib with a PI3K/mTOR inhibitor (LY3023414, a selective dual inhibitor of PI3K and mTOR, which is being investigated in Phase 2 clinical trials) in TNBC in vitro and in vivo. The prexasertib/LY3023414 combination induced the synergistic or additive inhibition on cell proliferation in 10 of 12 TNBC cell lines. The prexasertib/LY3023414 combination enhanced DNA damage (γH2AX), replication stress (phospho-RPA32) and proliferation inhibition in MDA-MB-231 cells when compare with single agent treatment. In three TNBC xenograft models (HCC1806, HCC1187 and MX-1) LY3023414 induced 0%, 62% and 24% tumor inhibition; prexasertib induced 94%, 78% and 96% tumor inhibition; and the combination led to 35%, 61% and 21% tumor regression, respectively. The prexasertib/LY3023414 combination also increased the inhibition of both primary tumor growth and spontaneous lung metastasis in a MDA-MB-231 mammary fat pad orthotopic model when compared with the respective single agent activity. The efficacy of prexasertib/LY3023414 combination was further assessed in 38 TNBC PDX models, and the combination showed a synergistic effect in 8 of 38 models and an additive effect in 22 of 38 models. Overall, the combined inhibition of Chk1 and PI3K/mTOR pathway enhanced antitumor activity in TNBC models. Taken together, these data will inform the clinical development of potential combination of Chk1 inhibitor prexasertib with a PI3K/mTOR inhibitor in the treatment of TNBC patients. The safety of this combination is being assessed in an ongoing Phase 1b clinical trial (NCT02124148), which includes an expansion cohort focused on patients with TNBC. Citation Format: Wenjuan Wu, Greg Donoho, Philip Iversen, Jack Dempsey, Andrew Capen, Mark Castanares, Jennifer Stephens, Karsten Boehnke, Christoph Reinhard Reinhard, Aimee Lin. Combination of the Chk1 inhibitor (prexasertib) with a PI3K/mTOR inhibitor (LY3023414) induces synergistic anti-tumor activity in triple negative breast cancer (TNBC) models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3508.

Background: The proliferative and pro-survival signals driven by the insulin-like growth factor (IGF) ligands, IGF-1 and IGF-2, are transmitted through their binding to the IGF-1 receptor (IGF-1R). In addition, IGF-2 promotes proliferation through activation of the insulin receptor variant A (IR-A) that is expressed during embryonic development as well as in many cancers. IGF survival signaling has been implicated in driving resistance to cancer therapies with diverse mechanisms of action, due to cross-talk between cellular signaling networks. Recent studies[1][2] suggest that the combination of IGF-1R signaling inhibitors with CDK4/6 inhibitors can result in enhanced anti-tumor activity. The aim of this study was to explore the potential of the IGF-1/-2 ligand blocking antibody, xentuzumab (BI 836845[3]), to enhance the anti-tumor activity of the CDK4/6 inhibitor abemaciclib, alone or in combination with fulvestrant, in human breast cancer (BC) cell lines. Methods: The anti-proliferative activity of the xentuzumab/abemaciclib combination was evaluated using CellTiter-Glo and propidium iodide staining in a panel of 51 and 20 BC cell lines, respectively. Detailed studies of abemaciclib (+/- fulvestrant), xentuzumab and combinations thereof were performed in MCF7 cells. Cell cycle analysis was done by FACS and BrdU ELISA, cellular signaling was assessed by Western blotting, proliferation was evaluated by Incucyte, CellTiter-Glo and alamarBlue assay. Apoptosis was measured by detection of cleaved PARP and caspase 3. Results: Among a panel of BC cell lines, enhanced anti-proliferative activity of xentuzumab+abemaciclib vs. abemaciclib alone was observed specifically in hormone receptor positive (HR+) cell lines. Combined treatment resulted in more pronounced cell cycle arrest in MCF7 cells, associated with synergistic blockade of IGF survival signaling and suppression of cell cycle genes downstream of CDK4/6. The triple combination with fulvestrant more effectively inhibited tumor cell proliferation than the doublet abemaciclib+fulvestrant, and led to induction of apoptosis. Conclusion: The study results show that addition of the IGF-1/-2 neutralizing antibody xentuzumab to abemaciclib, in the absence or presence of fulvestrant, leads to improved anti-proliferative activity and, in the triple combination, results in cellular death in MCF7 HR+ breast cancer cells. A phase Ib trial evaluating the abemaciclib+xentuzumab combination, including triplets with endocrine therapy in HR+BC patients, is currently ongoing. References: [1] Miller ML et al. (2013). Sci Signal 6;ra85 [2] Heilmann AM et al. (2014). Cancer Res 74:3947-58 [3] Friedbichler K et al. (2014). Mol Cancer Ther 13(2):399-409 Citation Format: Ulrike Weyer-Czernilofsky, Rosa Baumgartinger, Susanne Schmittner, Xueqian Gong, Sean Buchanan, Richard P. Beckmann, Carlos Marugan, Raquel Torres, Karsten Boehnke, Maria Jose Lallena, Flavio Solca, Norbert Kraut, Thomas Bogenrieder. Combination of the CDK4/6 inhibitor abemaciclib with xentuzumab, a humanized IGF-1 and IGF-2 ligand co-neutralizing monoclonal antibody, results in synergistic antineoplastic effects in human breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1852.

Summary Most cancers preserve functional retinoblastoma (Rb) and may, therefore, respond to inhibition of D-cyclin-dependent Rb kinases, CDK4 and CDK6. To date, CDK4/6 inhibitors have shown promising clinical activity in breast cancer and lymphomas, but it is not clear which additional Rb-positive cancers might benefit from these agents. No systematic survey to compare relative sensitivities across tumor types and define molecular determinants of response has been described. We report a subset of cancers highly sensitive to CDK4/6 inhibition and characterized by various genomic aberrations known to elevate D-cyclin levels and describe a recurrent CCND1 3′UTR mutation associated with increased expression in endometrial cancer. The results suggest multiple additional classes of cancer that may benefit from CDK4/6-inhibiting drugs such as abemaciclib.

Lung cancer is the most common tumor cancer worldwide and approximately 15-25% of the patients with lung adenocarcinoma have KRAS driven tumors. These malignancies involve, in the majority of cases, a constitutive activation of KRAS signaling pathway (1,2) and are associated with poor prognosis in patients with advanced disease (metastatic setting). Currently there is no specific therapy to target KRAS driven tumors approved by FDA (3), then finding alternative targeted therapies is a need to cover for this disease. Pharmacological inhibition of CDK4 was been suggested as a beneficial therapy to treat NSCLC patients carrying K-RAS oncogenes; and researchers base the potential efficacy of this approach on a synthetic lethal interaction between K-ras and CDK4 in in this type of tumors (4). Hence, CDK4/6 inhibitors appear as promising therapy to treat this type of tumors. Abemaciclib is a cell cycle inhibitor with selective activity against CDK4 and CDK6 and is being evaluated in advanced clinical trials for its potential to reduce NSCLC cancer growth. Here we describe studies towards the in-vitro mechanism of action of abemaciclib to reduce tumor cells growth in NSCLC cell lines harboring mutations in KRAS. Overall, abemaciclib reduces NSCLC cell growth as indicated by a reduction of cell number and proliferation biomarker Ki67 upon treatment. This tumor growth inhibition is mediated by arrest of cell cycle in G1 phase as a direct consequence of Rb phosphorylation blockade. In this study we are further reporting a phenotypic characterization of sensitive cell lines monitoring cell proliferation, senescence, and apoptosis markers using flow cytometry and high content imaging approaches as well as metabolic profiling. . Bibliography (1) Schubbert S, Shannon K and Bollang G (2007) Nature Rev. Cancer 7(4) 295-308. (2) Ihle NT et al (2012) J Natl Cancer Inst. 104(3): 228-239. (3) Roberts PJ et al (2010) J Clin Oncol. 28(31):4769-77 (4) Puyol M et al (2010) Cancer Cell 1(13): 63-73. Citation Format: Raquel Torres-Guzmán, Carmen Baquero, Carlos Marugan, Cecilia Mur, Severine I. Gharbi, Sandra Gomez, Joaquín Amat, Karsten Boehnke, Philip W. Iversen, Alfonso deDios, Xueqian Gong, Sean Buchanan, Richard P. Beckman, Maria José J. Lallena. Characterization of the mechanism of action of abemaciclib in NSCLC cell lines harboring KRAS mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-318. doi:10.1158/1538-7445.AM2017-LB-318

We developed a combination screening protocol to look for synergistic interactions with abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6 (CDK4 and CDK6). Abemaciclib (LY2835219), has shown cytostatic effects in some cell lines while inducing senescence and apoptosis in particularly sensitive cell lines. Abemaciclib, combined with various compounds, was screened across panels of genomically characterized tumor cells. These screens identified several synergistic interactions that improved the activity of abemaciclib in cancer cells lines that respond to abemaciclib monotherapy (e.g. mantle cell lymphoma, ER+ breast cancer) but additionally revealed certain combinations with synergy in Rb wild-type cancers that do not respond optimally to single agent abemaciclib treatment. Most interestingly, MEK inhibitors and LY3009120, a novel Raf dimer inhibitor that inhibits all three Raf isoforms (Cancer Cell 28:384-98) were found to potentiate the cytostatic effects of abemaciclib in these cell lines leading to apoptosis in vitro and tumor regression in vivo. Further analysis of the effects of combined inhibition of CDK4 and CDK6 and Raf isoforms on downstream signaling pathways provides mechanistic clues that may help explain the observed synergy. Citation Format: Gong Xueqian, Li-Chun Chio, Yue Webster, Maria Jose Lallena, Karsten Boehnke, Raquel Torres, Phil Iversen, Alfonso De Dios, Ian Smith, Christoph Reinhard, Sheng-Bin Peng, Jack Dempsey, Teresa Burke, Shih-Hsun Chen, Trent Stewart, Richard Beckmann, Wenjuan Wu, Sean G. Buchanan. The identification of combinations for the CDK4 and CDK6 inhibitor, abemaciclib. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A07.

Precision medicine approaches seeking to predict individual susceptibility to single or combinatorial drugs for advanced and/or metastasized solid cancers heavily depend on biological model systems that maintain key morphological, phenotypic and genomic features of the original tumor. Here, we present a case study of a female patient with metastasized colorectal cancer. Using tissue from five distinct areas of the primary tumor and its metastasis of the synchronously removed primary tumor and liver metastasis, we generated cell cultures. To address cell morphology and phenotypic features we performed immunohistochemistry (IHC) and immunofluorescence imaging showing that these cell cultures express colon-specific marker proteins/combinations CDX2 and CK20+/CK7-. Ultra-deep panel sequencing of original tumor tissue samples revealed damaging mutations in KRAS (G12D), PIK3CA (H1047R) and TP53 (C242F). Two cell cultures showed an additional SNP in the tumor suppressor SMAD4 (R361H), which was not detected in the original tissue, thus hinting at the existence of a small sub-population of cancer cells with a divergent mutation pattern. In parallel we established patient-derived xenografts (PDX) models from each region and investigated the response to targeted therapeutics, aiming at key molecules of the MAPK, PI3K/AKT and mTOR pathways in these genetically heterogeneous populations. drug response in single and mixed cell populations to address differential growth characteristics and response to mono- and combinatorial drug treatment. To distinguish the cells-of-origin and to calculate distributions/contribution of each population to the in vivo tumor growth in this model, sub-populations were color-coded using lentiviral constructs, expressing either a green or red tag. FACS analyses after treatment were performed and ratios of SMAD4mut(green) vs. SMAD4wt(red) were determined for each PDX tumor. These analyses, together with confirmatory IHC staining for the respective tag, showed significant effects on both tumor growth and population distributions in a treatment-dependent manner, advocating for systematic evaluation of combinatorial treatment in patients. Citation Format: Dirk Schumacher, Karsten Boehnke, Marlen Keil, Alessandra Silvestri, Maxine Silvestrov, Jens Hoffman, Iduna Fichtner, Reinhold Schaefer, Christian RA Regenbrecht. Reconstructing intratumor heterogeneity: lessons from therapeutic intervention in patient-specific models. [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 2412.

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.

Drug sensitivity profiling across genomically characterized panels of tumor cells can identify the molecular determinants of drug response. By testing compound combinations in an unbiased format, the same methodology can be used to identify the genomic context of drug-drug synergy. Based on this principle, we developed an unbiased combination screening protocol to identify synergistic interactions with LY3009120, a novel Raf dimer inhibitor that inhibits all three Raf isoforms (Peng et al. 2015, Cancer Cell 28:384-98). Inhibitors of the Ras-MAPK pathway have proven very effective in the treatment of BRAF-mutant melanoma but are, in general, only partially effective in the treatment of BRAF-mutant colorectal cancer and Ras mutant cancers. LY3009120 combined with various compounds was screened across panels of genomically characterized tumor cells. These screens identified a strong synergy with abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6 (CDK4 and CDK6). Statistical analysis of effects in over 500 cancer cell lines showed that mutations in BRAF or Ras family genes were strongly associated with sensitivity to this combination. Strong synergy was observed in skin, colorectal, lung and pancreatic cancers with Ras/Raf mutations, but was also observed in various cancer cells wild type for Ras pathway genes. This included tumor types sensitive to single agent abemaciclib, such as mantle cell lymphoma, ER+ breast cancers, certain leukemias, squamous non-small cell lung cancer, and/or lung cancer with receptor tyrosine kinase activation. In vitro and in vivo analyses of the effects of the combination treatment on signaling pathways in KRAS mutant cancers led to potential mechanistic explanations for the differing efficacy of the combination, which manifests as regression of tumor xenografts in rodent models. Citation Format: Xueqian Gong, Wenjuan Wu, Li-Chun Chio, Susan Pratt, Constance King, Yue Webster, Maria Jose Lallena, Karsten Boehnke, Raquel Torres, Philip Iversen, Christoph Reinhard, Shih-Hsun Chen, Richard Bechmann, Sheng-Bin Peng, Sean Buchanan. An unbiased tumor cell panel profiling method to identify drug-drug interactions reveals synergy between the CDK4 and CDK 6 inhibitor abemaciclib and the Raf dimer and pan-Raf inhibitor LY3009120 in Ras mutant cancers. [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 2818.

Colon cancer is a heterogeneous tumour entity. Growing evidence supports a subpopulation of cancer stem cells (CSCs) as both the drivers of tumour growth and the source of relapse following treatment. Elucidation of the cellular heterogeneity within a tumour would therefore facilitate better characterization of patient subtypes and lead to more personalized and effective treatments. Here, as part of the OncoTrack* consortium, we report the use of a Matrigel-based 3D in vitro model for the identification of genetic factors important in the regulation of CSCs. The identity and frequency of CSCs within each patient-derived culture model was determined by separating cells using fluorescence assisted cell sorting (FACS) based on expression of previously defined markers of colon CSCs (ALDH+ CD44+ CD166+). Isolated subpopulations of cells are functionally tested for CSC properties in vitro (limiting dilution serial passage and population dynamics analysis) and in vivo (limiting dilution serial xenotransplantation). These studies reveal ALDH+ colon cancer cells to be highly enriched for CSCs and demonstrate large levels of variation in CSC frequency between patients. To identify genes and pathways that could be used to specifically target CSC function, ALDH+ tumour cell subpopulations were subjected to genome-wide analyses using next-generation sequencing for detailed characterization at the level of the transcriptome and methylome. To date, genes important in the regulation of normal stem cells and CSCs, such as BMI1, EPHB2 and the WNT signalling pathway, were found to be highly expressed in CSCs. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) mediated gene technology was used to knock out genes of interest and their role in regulating CSC function was confirmed using established functional assays. These studies provide insight into the relationship between variable tumour composition and the variable response of tumours to treatment. Ultimately, these studies will facilitate the more accurate classification and personalized treatment of colon tumours. *The research leading to these results has received funding from the European Union's Seventh Framework Program (FP7/2007-2013) for the Innovative Medicine Initiative under grant agreement n°115234. Citation Format: Joseph L. Regan, Dirk Schumacher, Stephanie Staudte, Karsten Boehnke, Ulrich Keilholz, Johannes Haybaeck, Hans Lehrach, David Henderson, Reinhold Schaefer, Christian R. Regenbrecht, Dominik Mumberg, Martin Lange. 3D-models of patient-derived colon tumors for the identification of genetic factors important in the regulation of cancer stem cells. [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 977. doi:10.1158/1538-7445.AM2015-977

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

Colon cancer is a heterogeneous disease. Growing evidence supports a subpopulation of cancer stem cells (CSCs) as both the drivers of tumour growth and the source of relapse following treatment. Elucidation of the cellular heterogeneity within a tumour would therefore facilitate better characterization of patient subtypes and lead to more personalized and effective treatments. Here we report the use of a Matrigel-based 3D in vitro model for the study of tumour heterogeneity and therapeutic resistance in patient derived colon tumours. To date, this system has enabled the establishment and long term culture of more than 53 patient-specific primary and metastasis derived colon tumours. Tumour heterogeneity and the prospective isolation of CSCs within each patient-derived culture model is determined by separating cells using fluorescence assisted cell sorting (FACS) based on expression of previously defined markers of colon CSCs (e.g. ALDH+, CD133+, CD44+, CD166+, EpCAMHigh). To date, these analyses have been performed on ten of the patient derived tumour models and demonstrate both inter- and intra- tumour heterogeneity as each tumour contains varying subpopulations of cells either positive or negative for expression of CSC markers. In addition, immunofluorescence staining and 3D confocal analysis of genes important in the regulation of both normal and CSCs (e.g. Notch, Wnt and Hedgehog signalling genes) has also revealed heterogeneity of expression. Functional characterization using in vitro and in vivo assays to determine the tumorigenic and differentiation capacity of the separated subpopulations are currently underway. Validated CSC subpopulations will be subjected to genome-wide analyses using next-generation sequencing for detailed characterization at the level of the transcriptome and methylome. To study the effect of drug treatment on CSCs we have determined the IC50 values for standard of care drugs (e.g. 5-fluorouracil, oxaliplatin, irinotecan, receptor tyrosine kinase inhibitors) for each patient derived colon tumour. Tumour cells were treated for 72 - 96 hrs after which time treatment was terminated and the cells were allowed to recover. Surviving cells were then analyzed by FACS to determine the effect of treatment on the frequency of CSCs. Molecular characterization of the therapy resistant CSCs was then used to identify possible biomarkers and targets for more effective treatments. These studies will provide insight into the relationship between variable tumour composition and the variable response of tumours to treatment. Ultimately, these studies will facilitate the more accurate classification and personalized treatment of colon tumours. OncoTrack is a project funded by the Innovative Medicines Initiative Joint Undertaking (IMI JU). Citation Format: Joseph L. Regan, Dirk Schumacher, Karsten Boehnke, Cathrin Davies, Ulrich Keilholz, Johannes Haybaeck, Christoph Reinhard, Hans Lehrach, David Henderson, Reinhold Schaefer, Christian Regenbrecht, Martin Lange. Functional and molecular characterization of colon cancer stem cells in tumor heterogeneity and disease relapse using a 3D-model of patient-derived tumors. [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 3875. doi:10.1158/1538-7445.AM2014-3875

*OncoTrack is a project funded by the Innovative Medicines Initiative Joint Undertaking (IMI JU). New therapies for colon cancer barely improved cure rates over the last decade and typically only a subset of patients benefits from them. The reasons for these disappointing outcomes are likely related to the inherent genetic and cellular heterogeneity and plasticity of tumors. Therefore only comprehensive approaches that correlate data from systematic molecular tumor tissue characterization with preclinical and clinical responses could improve our understanding of this disease and help to design diagnostic procedures allowing optimal therapy for individual patients. OncoTrack is an international consortium funded by the Innovative Medicines Initiative (www.OncoTrack.eu) that has launched one of Europe's largest collaborative public-private research projects to implement novel approaches of systems biology into colon cancer therapy. Tumor tissue and circulating tumor cells from a cohort of more than 120 patients with primary or metastatic colon cancer were subjected to high-throughput protein, epigenome, and gene expression analysis as well as next generation sequencing. Three- dimensional (3D) in vitro cultures and in vivo patient-derived xenografts (PDX) in immunodeficient mice were generated from each tissue sample and used to determine response to more than 14 approved drugs, investigational drug candidates, and tool compounds. These wet-lab data, combined with clinical data were used for in silico modeling to identify new predictors for tailored therapies. Here we are presenting data from the subproject tumor models and therapy response. Tumor tissues from 118 colon cancer patients (87 primary tumors and 31 metastases) were transplanted into mice. To date, 44 permanent new PDX models have been generated from the primary tumors and 13 from the metastases; (establishment of 25 additional models is ongoing). These models are being used to determine sensitivity of the tumors to a broad range of drugs, targeting signaling pathways frequently activated in cancer. In parallel, in vitro cultures using a Matrigel-based, serum-free 3D system were established and adapted for high-throughput 384-well assay format for testing against a similar drug panel. Drug response data generated from more than 30 patient specimens using both the specific 3D in vitro assays and the in vivo PDX experiments are available and will be shown and compared. These experimental results about drug sensitivity together with the molecular signatures of the tumor tissues provide an exceptionally broad basis to analyze the relationship between tumor biology and treatment response. Such approaches may hold promise in the design of rational therapy for colon cancer in the future. Citation Format: Maria Rivera, Marlen Keil, Karsten Boehnke, Martin Lange, Dirk Schumacher, Reinhold Schäfer, Christian RA Regenbrecht, David Henderson, Ulrich Keilholz, Alexander Kuehn, Amin El-Heliebi, Tabea Hohensee, Johannes Haybäck, Christoph Reinhard, Juan A. Velasco, Hans Lehrach, Pilar Garin-Chesa, Garry Beran, Jens Hoffmann. Generation of drug response data from 57 new patient-derived colon cancer xenografts and 3D cell cultures for systematic correlation with tumor biology within the OncoTrack* project. [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 2978. doi:10.1158/1538-7445.AM2014-2978

OncoTrack is an international consortium funded by the Innovative Medicines Initiative (www.OncoTrack.eu) that has launched one of Europe's largest collaborative public-private research projects to implement novel approaches of systems biology in colon cancer therapy. Within OncoTrack, tumor tissue and circulating tumor cells from a cohort of more than 120 patients with primary or metastatic colon cancer are subjected to whole genome-, exome-, epigenome- and transcriptome sequencing, as well as high-throughput protein analysis. Alongside the systematic analysis of colon cancer tissues, in vivo xenografts in immunodeficient mice (patient-derived xenografts, PDX) and in vitro 3D cell cultures are generated from each tissue sample and used to determine response to more than 14 approved drugs, investigational drug candidates and tool compounds. These wet-lab data, combined with clinical data, will serve as a basis for in silico modeling to identify new predictors for tailored therapies. Although early diagnosis and molecular characterization of colon cancer has improved significantly, rapid and cost-effective means to address molecular genotyping and therapeutic options on the level of the individual patient are still in high demand. Here, we present an experimental pipeline within the OncoTrack project starting from Matrigel-based Patient-tumor-derived 3D cell cultures (PT3DC). To date, we were able to establish 50 long-term 3D cell culture strains originating from 35 primary tumors, 9 metastases and 6 PDX-derived tissues as suitable models for basic and translational research. On the basis of immunohistochemistry, we show that our in vitro cultures preserve an in vivo like architecture, preventing tumor cells from differentiating and allowing the investigation of intra-tumor heterogeneity and cancer stem cell like sub-populations. To interrogate the mutation status of selected clinically relevant oncogenes and tumor suppressors in PT3DC cultures, we applied cost-efficient benchtop sequencing and show the preservation of putative driver mutations found in the original tumor. IC50 data generated by automated 384-well based dose-response experiments of approved drugs are then used to link individual genotypes with drug sensitivity phenotypes and serve as a source of comparison and complementation to drug response data of the corresponding in vivo PDX models, where applicable. Citation Format: Dirk Schumacher, Karsten Boehnke, Martin Lange, Yvonne Welte, Cathrin Davies, Maria Rivera, Marlen Keil, Ulrich Keilholz, Johannes Haybaeck, Juan Angel Velasco, Marie-Laure Yaspo, Hans Lehrach, David Henderson, Christoph Reinhard, Jens Hoffmann, Reinhold Schaefer, Christian Regenbrecht. A pipeline within the OncoTrack project for generating Patient-tumor-derived 3D cell cultures (PT3DC) and their application for individualized, targeted drug sensitivity assays. [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 2018. doi:10.1158/1538-7445.AM2014-2018