Testing novel anti-cancer agents across large panel of tumor models covering genetic diversity of cancers is increasingly considered as a cornerstone of preclinical development. For this purpose, Reaction Biology developed “ProLiFiler” a standard panel of 140 cell lines (CLs) covering most common cancer types to evaluate anti-proliferative activity of novel drugs. Partnering has been made with 4HF Biotec and their in-silico platform, named “Cancer Data Miner”, to investigate and to understand molecular basis of drug sensitivity. Here we report the use of our platforms to realize integrative pharmacogenomic studies for three recent small molecules targeting major altered pathways in cancers. It includes SOS1::KRAS interaction inhibitor BI-3406, MDM2 inhibitor Nutlin-3a, and PI3K inhibitor Taselisib. Main goal of the study is to provide meaningful information for these three drugs regarding their efficacy and potency, the validation of their mechanism of actions (MOA), the suitable clinical indications, possible drug combinations and the predictive biomarkers of sensitivity or resistance. The three compounds are tested for anti-proliferative activity in vitro in a 2D monolayer assay using the “ProLiFiler”CLs panel. For data analytics, the resulting in vitro data are loaded on the “Cancer Data Miner” platform and connected to CL annotations including whole exome mutations, chromosomal aberrations, gene expression profiles or drug sensitivity profiles. The drug response profiles will be reported for the three compounds individually and compare between them, showing respective efficacy, potency, and CL/cancer entity selectivity. Using the MOA Finder tool, we will correlate BI-3406, Nutlin-3a, and Taselisib individual IC50 profiles to those of more than 800 compounds with known MOA that are integrated on the platform. The analyses will show the drugs most closely related to the 3 compounds and that are expected to have similar MOA. With the biomarker discovery tools, we will run high throughput statistical analyses to reveal whole exome mutations, copy number variations and expression significantly associated with drug sensitivity/resistance. For interpretation, pathway and enrichment analysis will be presented. A focus will be made on key alterations like KRAS and MAPK related genes, TP53-MDM2 and PIK3CA-PTEN to evaluate their predictivity. The work will be also completed by functional analysis, for instance, by assessing the effect of BI-3406 on ERK-MEK activation, and the impact of MDM2 inhibition on apoptotic markers. The present work will show the whole panel of analyses proposed by 140 CL-ProLiFiler and Cancer Data Miner complementary platforms, allowing to acquire key information at an early stage of drug development and helping to setup next steps such as selection of models for in vivo testing. Citation Format: Vincent Vuaroqueaux, Daniel Feger, Hoor Al-Hasani, Oliver Siedentopf, Anne-Lise Peille, Sarah Ulrich, Sebastian Dempe, Heinz-Herbert Fiebig, Jan Erik Ehlert. ProLiFiler and Cancer Data Miner, combined platforms for preclinical investigation to scrutinize impact of inhibitors on the KRAS, PI3K and MDM2 signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1027.
Cancer is a highly complex, multigenic disease with tumor cells underlying constant transition. Single drug treatments against specific targets frequently result in only partial success because mutations and redundant pathways cause drug resistance. Therefore, drug combinations that effect different synergistically acting targets in the cancer cell in parallel have become a promising strategy to improve the success in many fields of cancer therapy. One example for such an approach is the co-treatment of B-Raf driven tumors with Raf and MEK1 inhibitors. In our study, we determined the combinatorial effect of the pan-Raf inhibitor AZ-628 and the MEK1 inhibitor AZD-6244 on the viability of a large panel of more than 100 tumor cell lines. Whittaker et al. (Mol.CancerTher, 2015) could already show that this drug combination has a significant synergistic effect in several melanoma and colon cancer cell lines. The authors showed evidence that synergistic activity was correlated with enhanced mutation-triggered RAS/RAF/MAPK signaling with a special role for C-Raf in resistance due to a negative feedback mechanism. We here applied the combination of AZD-6244 and AZ-628 in a broad checkerboard pattern to a multitude of cell lines from other entities beyond melanoma and colorectal cancer. Based on these results we correlated the observed synergistic and non-synergistic effects with gene expression profiles of these cell lines and analyzed the requirement of a MAPK activating signaling. Our approach revealed that synergistic activity is not confined to melanoma and colon cancer but is observed in tumors from other entities as well. This observation may expand the usefulness of MEK/Raf inhibitor co-treatment to a larger panel of cancer types. Citation Format: Alokta Chakrabarti, Daniel Feger, Sarah Umber, Orysya Stus, Marianne Birkle, Oliver Siedentopf, Jan E. Ehlert. Analysis of the combinatorial antiproliferative effect of pan RAF inhibitor AZ-628 and MEK1-inhibitor AZD-6244 on a large panel of tumor cell lines [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 1208. doi:10.1158/1538-7445.AM2017-1208
Anticipation of the in vivo activity of new investigational drugs is challenging. In the context of the living body, compounds are subjected to degradation, modification or binding to plasma proteins - mechanisms that may compromise the activity predicted from target-specific in vitro assays. As such assays are performed in artificial cell-culture media it was our aim to measure the activity directly in human plasma, as initially described by Levis et al.1. We hence established a method to analyze the activity of kinase inhibitors in human or rodent plasma (plasma inhibitory activity, PIA) in high throughput compatible ELISA-based cellular kinase assays for e.g. FLT-3 or BCR-Abl. Testing reference compounds such as Sunitinib or Sorafenib, we could confirm the expected correlation between the plasma binding of a compound and the loss of its activity in this assay setup compared to the activity in serum free medium. Furthermore we observed a donor dependent interference of the plasma with assay performance. This issue was solved by appropriate plasma heat-inactivation which made confirmation of compound stability to heat treatment mandatory before generating pharmacokinetic data using this methodology. Our aim is to establish the PIA assay for our panel of more than 30 kinases and analyze the activity of important reference kinase inhibitors spiked in human plasma. In the next step samples from mice treated with reference compounds shall be tested. This way, we want to compile reliable in vitro PIA assays to better predict the in vivo effect of kinase-directed drugs and to determine the amount of freely available active test substance in the blood of treated patients. 1 = Levis et al.; Blood, 2006; 108:3477 Citation Format: Kira E. Boehmer, Holger Weber, Daniel Feger, Marianne Birkle, Oliver Siedentopf, Melanie Mueller, Sarah Umber, Jan E. Ehlert. Determination of the plasma inhibitory activity of drugs using cellular kinase assays. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B139.
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