Karsten Boehnke

Molecular dissection of colorectal cancer in pre-clinical models identifies biomarkers predicting sensitivity to EGFR inhibitors

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.

Assay Establishment and Validation of a High-Throughput Screening Platform for Three-Dimensional Patient-Derived Colon Cancer Organoid Cultures

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.

Preclinical characterization of abemaciclib in hormone receptor positive breast cancer

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.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.

In Vitro Three-Dimensional Cell Cultures as Tool for Precision Medicine

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.

Abstract 3101: In-vitro characterization of Abemaciclib pharmacology in ER+ breast cancer cell lines

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

Abstract LB-318: Characterization of the mechanism of action of abemaciclib in NSCLC cell lines harboring KRAS mutation

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-Guzman, Carmen Baquero, Carlos Marugan, Cecilia Mur, Severine I. Gharbi, Sandra Gomez, Joaquin Amat, Karsten Boehnke, Philip W. Iversen, Alfonso deDios, Xueqian Gong, Sean Buchanan, Richard P. Beckman, Maria Jose 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

Abstract A07: The identification of combinations for the CDK4 and CDK6 inhibitor, abemaciclib

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.

Abstract 2829: Preclinical analysis and characterization of abemaciclib using three-dimensional patient-derived colorectal cancer organoid cultures

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, Joaquin 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, Maria Jose 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.

Abstract 2412: Reconstructing intratumor heterogeneity: lessons from therapeutic intervention in patient-specific models

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.

Abstract 2818: 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

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.