Juan A. Velasco

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.

Characterization of LY2228820 Dimesylate, a Potent and Selective Inhibitor of p38 MAPK with Antitumor Activity

p38α mitogen-activated protein kinase (MAPK) is activated in cancer cells in response to environmental factors, oncogenic stress, radiation, and chemotherapy. p38α MAPK phosphorylates a number of substrates, including MAPKAP-K2 (MK2), and regulates the production of cytokines in the tumor microenvironment, such as TNF-α, interleukin-1β (IL-1β), IL-6, and CXCL8 (IL-8). p38α MAPK is highly expressed in human cancers and may play a role in tumor growth, invasion, metastasis, and drug resistance. LY2228820 dimesylate (hereafter LY2228820), a trisubstituted imidazole derivative, is a potent and selective, ATP-competitive inhibitor of the α- and β-isoforms of p38 MAPK in vitro (IC50 = 5.3 and 3.2 nmol/L, respectively). In cell-based assays, LY2228820 potently and selectively inhibited phosphorylation of MK2 (Thr334) in anisomycin-stimulated HeLa cells (at 9.8 nmol/L by Western blot analysis) and anisomycin-induced mouse RAW264.7 macrophages (IC50 = 35.3 nmol/L) with no changes in phosphorylation of p38α MAPK, JNK, ERK1/2, c-Jun, ATF2, or c-Myc ≤ 10 μmol/L. LY2228820 also reduced TNF-α secretion by lipopolysaccharide/IFN-γ–stimulated macrophages (IC50 = 6.3 nmol/L). In mice transplanted with B16-F10 melanoma, tumor phospho-MK2 (p-MK2) was inhibited by LY2228820 in a dose-dependent manner [threshold effective dose (TED)70 = 11.2 mg/kg]. Significant target inhibition (>40% reduction in p-MK2) was maintained for 4 to 8 hours following a single 10 mg/kg oral dose. LY2228820 produced significant tumor growth delay in multiple in vivo cancer models (melanoma, non–small cell lung cancer, ovarian, glioma, myeloma, breast). In summary, LY2228820 is a p38 MAPK inhibitor, which has been optimized for potency, selectivity, drug-like properties (such as oral bioavailability), and efficacy in animal models of human cancer. Mol Cancer Ther; 13(2); 364–74. ©2013 AACR.

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.

The product of the cph oncogene is a truncated, nucleotide-binding protein that enhances cellular survival to stress

Cph was isolated from neoplastic Syrian hamster embryo fibroblasts initiated by 3-methylcholanthrene (MCA), and was shown to be a single copy gene in the hamster genome, conserved from yeast to human cells, expressed in fetal cells and most adult tissues, and acting synergistically with H-ras in the transformation of murine NIH3T3 fibroblasts. We have now isolated Syrian hamster full-length cDNAs for the cph oncogene and proto-oncogene. Nucleotide sequence analysis revealed that cph was activated in MCA-treated cells by a point-mutational deletion at codon 214, which caused a shift in the normal open reading frame (ORF) and brought a translation termination codon 33 amino acids downstream. While proto-cph encodes a protein (pcph) of 469 amino acids, cph encodes a truncated protein (cph) of 246 amino acids with a new, hydrophobic C-terminus. Similar mechanisms activated cph in other MCA-treated Syrian hamster cells. The cph and proto-cph proteins have partial sequence homology with two protein families: GDP/GTP exchange factors and nucleotide phosphohydrolases. In vitro translated, gel-purified cph proteins did not catalyze nucleotide exchange for H-ras, but were able to bind nucleotide phosphates, in particular ribonucleotide diphosphates such as UDP and GDP. Steady-state levels of cph mRNA increased 6.7-fold in hamster neoplastic cells, relative to a 2.2-fold increase in normal cells, when they were subjected to a nutritional stress such as serum deprivation. Moreover, cph-transformed NIH3T3 cells showed increased survival to various forms of stress (serum starvation, hyperthermia, ionizing radiation), strongly suggesting that cph participates in cellular mechanisms of response to stress.

Multiparametric Cell-Based Assay for the Evaluation of Transcription Inhibition by High-Content Imaging

Loss of normal cell cycle regulation is a hallmark of human cancer. Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and have been actively pursued as promising therapeutic targets. Likewise, members of the CDK family are functionally related to transcriptional modulation, a molecular pathway suitable for therapeutic intervention. We used a set of 2500 compounds in the U2OS cell line to evaluate its effect in the cell division process. Interestingly, out of this analysis, we identified a subpopulation of compounds that are able to inhibit RNA polymerase activity, thus interfering with gene transcription processes. After this finding, we developed, validated, and fully automated a multiparameter high-content imaging (HCI) assay to measure phosphorylation of the RNA polymerase II carboxyl terminal domain (pCTD). Simultaneously, we measured both the DNA content and cell proliferation index in the treated cells. The linear regression analysis comparing the IC50 for pCTD and the 4N EC50 for DNA content or IC50 for cell proliferation showed an excellent agreement (r2 = 0.84 and r2 = 0.94, respectively). Our results confirm that this method allows discriminating between cell cycle and transcription inhibition and confirms HCI as a powerful technology for the identification of compounds with an effective and selective pathway phenotype.

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 2018: A pipeline within the OncoTrack project for generating Patient-tumor-derived 3D cell cultures (PT3DC) and their application for individualized, targeted drug sensitivity assays

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA OncoTrack is an international consortium funded by the Innovative Medicines Initiative (www.OncoTrack.eu) that has launched one of Europes 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

Abstract B235: Characterization of LY2228820 dimesylate, a potent and selective inhibitor of p38 MAPK with antitumor activity.

p38α mitogen-activated protein kinase (MAPK) is activated by cancer cells in response to environmental factors, such as oncogenic stress, radiation, and chemotherapy. p38α MAPK phosphorylates a number of substrates, including MAPKAP-K2 (MK2), and regulates the production/message stability of cytokines produced in the tumor microenvironment, such as TNFα, IL1 β, IL-6 and IL-8. p38α MAPK is activated and highly expressed in human cancers and may play a role in tumor growth, invasion and metastasis. LY2228820 dimesylate is a tri-substituted imidazole derivative that is a potent and ATP-competitive inhibitor of the α and β isoforms of p38 MAPK in vitro (IC50 = 5.3 nM and 3.2 nM, respectively). This compound displays > 1000-fold selectivity for p38α MAPK versus 179 other kinases tested (including p38δ and γ isoforms). In cell-based assays, LY2228820 dimesylate also potently and selectively inhibits phosphorylation of MK2 (Thr334) in TNFα-stimulated HeLa cells (IC50 = 8.1 nM) and anisomycin-induced mouse RAW264.7 macrophages (IC50 = 35.3 nM) with no changes in phosphorylation of p38α MAPK, JNK, ERK1/2, c-jun, ATF2 or cMyc at concentrations up to 10μM. LY2228820 dimesylate also reduces TNFα secretion by LPS/IFNγ-stimulated macrophages (IC50 = 6.3 nM). In mice transplanted with B16-F10 melanomas, phospho-MK2 was effectively inhibited by LY2228820 dimesylate in tumors in a dose-dependent manner (TMED70 = 19.4 mg/kg). Significant target inhibition (>40% inhibition of phospho-MK2) was maintained for approx. 4–8hrs following a single 10mg/kg oral dose. In a broad range of xenograft models (A-549 NSCLC, SK-OV-3 Ovarian, U-87MG Glioma, MDA-MB-468 Breast), LY2228820 dimesylate demonstrates significant tumor growth delay. In summary, LY2228820 dimesylate is a novel, potent and selective inhibitor of p38 MAPK with anti-tumor activity. Further studies are ongoing to determine its molecular mechanism(s) of action, potential for combination with standard-of-care agents, and potential clinical activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B235.