Abstract In the preclinical space of cancer drug discovery, multi-omics characterization of cancer models is essential for model selection, understanding mechanisms of action, and early biomarker discovery. We launched a panel of 160 cell lines (CL Proliferation Panel) to study drug responses across a large variety of cancer types and validated their molecular characteristics at both the transcriptomic and genomic levels. Recently, we characterized this set of models at the proteomic level using mass spectrometry, identifying a total of more than 15.000 proteins. In the present work, we aim to investigate the relevance of this new dataset in the daily practice of cancer model utilization. First, we demonstrated the robustness of the established proteomic profiles, showing high concordance among duplicate samples. Using a multiomics comparison and dimensionality reduction approaches, we curated the proteomic dataset to remove outlier and irrelevant protein profiles, ensuring high data quality. Next, unsupervised hierarchical clustering of the proteomic profiles revealed accurate classification of models according to their tumor type of origin, confirming the relevance of the data. Given the growing importance of proteomic information in the context of antibody-drug conjugate (ADC) development, we further assessed the relevance of our dataset for evaluating the expression of top 20 ADC targets such as ERBB2 and TACSTD2, among others. We also established a multi-omics strategy integrating transcriptomic and proteomic data to enhance target characterization. Finally, we explored the relationship between protein expression of the targets and drug sensitivity by analyzing ADC targets in the context of responses to clinically approved ADCs, including trastuzumab emtansine (Kadcyla), and sacituzumab govitecan. Citation Format: Nadine Obier, Vincent Vuaroqueqeaux, Johannes Krumm, Anne-Lise Peille, Daniel Feger, Sarah Ulrich, Johanna Wallner, Hannes Hahne, Jan E. Ehlert. Integrative analysis of proteomic, transcriptomic, and FACS-based surface marker data for ADC target discovery in cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6343.

Abstract The serine/threonine phosphatase WIP1 (PPM1D) is a key negative regulator of the DNA damage response (DDR) and a recognized oncogene, frequently amplified or truncated in various cancers including breast, ovarian, neuroblastoma, and glioblastoma. Its inhibition restores DDR signaling, offering a promising therapeutic strategy. Despite initial efforts, including the development of the allosteric inhibitor GSK2830371, clinical translation has been limited. We report the establishment of a comprehensive assay platform to support drug development targeting WIP1. Biochemical assays were optimized using fluorescein diphosphate (FDP), malachite green with phosphopeptides (p53, H2AX, p38 MAPK), and TR-FRET formats, enabling robust activity profiling. Cellular mechanism-of-action assays were developed to monitor phosphorylation changes in WIP1 substrates, with pS15-p53 ELISA in U2OS cells selected for medium-throughput iterative compound profiling. Phenotypic assays demonstrated compound efficacy in 2D and 3D proliferation models, with leukemic cell lines showing pronounced sensitivity. Combination studies revealed strong synergy between WIP1 inhibitors and MDM2 antagonists (e.g., Nutlin3a), supporting a dual-targeting approach for p53 pathway reactivation. In vivo efficacy was confirmed in MV4-11 xenograft models, with dose-dependent tumor growth inhibition and favorable tolerability profiles. Our integrated assay suite enables iterative screening and lead optimization, culminating in the identification of a potent allosteric WIP1 inhibitor with nanomolar activity and promising preclinical efficacy. These findings support WIP1 as a viable target in oncology and provide a foundation for future clinical development. Citation Format: Daniel Feger, Daniel Müller, Lena Pilgermayer, Carolin Heidemann-Dinger, Sarah Ulrich, Michael Kubbutat, Holger Weber, Joe Lewis, Birgit Zech, Jan E. Ehlert. Establishment of assay systems for drug development against the oncogenic phosphatase WIP1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 328.

Gene therapeutical intervention using viral vectors is becoming increasingly relevant for the treatment of various diseases, including cancer. A critical factor for a successful viral therapy is that the patient has not previously developed neutralizing antibodies to the respective viral serotype in the context of a naturally occurring anti-infection immune response. Such antibodies would impose the risk to neutralize and thus jeopardize the intended therapeutic response. This is especially relevant in therapies with adeno-associated virus (AAV), as immunity against AAV-serotypes is very common. We here report on the development and validation of a straightforward method to functionally detect neutralizing antibodies in primate sera, employing a transduction method on a human cell line using AAVs of different serotype coding for an easily detectable transgene. The reported methodology will be helpful for the stratification of primates and patients exploiting their sera for the prevalence of neutralizing antibodies prior to the initiation of the AAV treatment. In addition, our assay has the potential to be applied to other viral treatment strategies also. Gregor Sommerkamp, Eva M. Egenter, Katharina Schaich, Daniel Feger, Sebastian Dempe, Jan E. Ehlert. Development of a rapid method for the detection of neutralizing antibodies in sera of primates for viral gene therapy stratification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 481.

Anticipation of the in vivo activity of an investigational new drug (IND) is challenging. In the context of the living body, compounds are subjected to degradation, metabolic modification or binding to plasma proteins - mechanisms that are well described to potentially compromise the in vivo activity seen in the patient compared to its predicted potential to inhibit its target in specific in vitro assays. As such assays are performed in artificial cell-culture media, we had previously modified our proprietary cellular FLT3 kinase phosphorylation assay to measure the activity of FLT3 inhibitors directly in and from human plasma, similar as 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 assay, PIA) in ELISA-based cellular kinase assays. We have successfully applied this methodology to determine the pharmaco-kinetics of a newly developed inhibitor of FLT3 in the context of a clinical trial targeting FLT3. We have now expanded this methodology for the detection of further kinase targets of potential clinical relevance. We were able to compile in vitro PIA assay data for further kinases such as VEGFR2 and BCR-Abl which will allow us to better predict the in vivo effect of specific inhibitors of these kinases and to determine the amount of freely available active test substance in the blood/sera of treated cancer patients. 1: Levis et al. Blood. 2006; 108 (10):3477-3483 Lena Pilgermayer, Eva M. Egenter, Katharina Schaich, Daniel Feger, Jan E. Ehlert. Establishment of plasma inhibitory kinase assays (PIA) to detect functional drug levels in patient plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4363.

Abstract Cell surface proteins are increasingly considered in the development of novel anticancer agents, as demonstrated by recent clinical successes in the field of immuno-oncology and antibody-drug conjugates (ADCs). These conjugates specifically target antigens present on the surface of tumor cells, enabling the delivery of cytotoxic agents to specific tumor sites. During the preclinical development of such compounds, for example, when they are tested on a wide panel of cell lines, it is essential to have a rapid and reliable method for monitoring target expression and distribution. Complementing our 160 Cell line panel ProLiFiler™, we report here the development of a novel method named oncoFLOW-Profiler™ which includes steps of cell fixation, micronic storage, staining, and measurement by flow cytometry. This method is applied in a 96-well format, enabling screening of all cell lines in parallel and analysis of the expression of different targets in just one day. In this proof-of-concept study, we investigated the level of ERBB2 expression across the ProLiFiler panel of 160-cell lines in order to validate this methodology. We first compared the impact of fixation methods and sample storage on knowingly positive and negative control cell lines over a period of several months. ERBB2 measurements from the oncoFLOW-Profiler™ will be analyzed and validated by comparison with various OMICs datasets from the Cancer DataMiner platform (4HF Biotec), including RNA expression, mutational status, and protein expression levels. To determine the expression cutoff of ERBB2 amplified models, results will be analyzed according to gene copy number variation. Finally, the flow cytometry data generated will be investigated for use as a predictor of response to clinically approved ADCs targeting ERBB2: Kadcyla and Enhertu. Here we present a method that we have demonstrated is feasible for investigating cell surface receptor/antigens during preclinical phase of drug development. Our method has enabled us to successfully assess ERBB2 in biological samples and to identify models sensitive to ADCs targeting ERBB2. We will also discuss the results of ongoing experiments analyzing the applicability of this screen to intracellular targets and carbohydrate-based surface antigens, which cannot be analyzed either by transcriptomics or proteomics. Citation Format: Kaja Holtorf, Anne-Lise Peille, Daniel Feger, Vincent Vuaroqueaux, Jan Erik Ehlert, Nadine Obier. Development and evaluation of a high-throughput method for rapid detection of surface antigen expression in fixed cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5157.

Abstract Previously we have reported on the relevance of the joint ProLiFiler and Cancer DataMiner platforms when studying the antitumor efficacy of novel antibody-drug conjugates (ADCs), such as Sacituzumab govitecan. In this study, we use our platforms to investigate and compare the selectivity of two FDA-approved Trastuzumab-based ADCs targeting ERBB2: Kadcyla, the first-in-class ADC employing a microtubule inhibitor bound via a non-cleavable linker, and Enhertu, which combines a topoisomerase inhibitor with a protease-cleavable linker. We performed a cell proliferation and survival assay using the ProLiFilerTM (Reaction Biology) to characterize the in vitro antitumor effects of Enhertu, Kadcyla, and their individual cytotoxins, deruxtecan & mertansine on 160 human cancer cell lines (CLs). The resulting data were uploaded to 4HF´s Cancer DataMiner platform for data analysis. First, we confirmed high potency and limited selectivity of mertansine, as it displayed an IC50 < 250 nM (mean: 27.3 nM) in 95% of cell lines (CLs). A COMPARE analysis confirmed that mertansine data correlated best with those of other microtubule inhibitors in our drug databases (e.g., MMAE, Spearman rho = 0.79, p=9.6E-27). Conversely, a paired analysis showed that CLs were less sensitive to Kadcyla than to mertansine except for CLs overexpressing ERBB2. The 160-CL panel included a total of nine CLs (six mammary, two gastric, one ovarian) with strong ERBB2 overexpression (Affymetrix data >10), mostly associated with ERBB2 gene amplification; seven of them were the most sensitive CLs to Kadcyla (IC50 < 6 nM). Focusing solely on breast cancer cell lines (breast cancer is the approved indication for Kadcyla), we observed a strong correlation between sensitivity to Kadcyla and ERBB2 expression (Spearman r = -0.78, p = 0.003). Interestingly, in line with published data, the ERBB2-overexpressing cell lines SNU-216 (stomach cancer) and JIM-T1 (breast cancer) were less sensitive to Kadcyla. In non-ERBB2-amplified CLs, Kadcyla sensitivity did not correlate with ERBB2 expression levels. Focusing on ERBB2-negative CLs such as hematological CLs, we unexpectedly observed consistent cell inhibition (mean IC50 =33 nM) with Kadcyla, suggesting that the ADC was cleaved, and the payload released into the medium. Overall, this study has demonstrated the relevance of our platforms to study and compare newly developed ADCs. Cytotoxic activities recorded for conjugates and free payloads, along with molecular data for the test cell lines, provides insight in the mode of action of conjugates and their target cell populations and hence can support the development of ADCs. Investigations are underway with Enhertu and its payload deruxtecan, and results will be compared with those obtained for Kadcyla and mertansine. Citation Format: Anne-Lise Peille, Kaja Holtorf, Pablo Anton Garcia, Nadine Obier, Daniel Feger, Thomas Metz, Sebastian Dempe, Heinz-Herbert Fiebig, Jan Ehlert, Vincent Vuaroqueaux. Potency and selectivity of ERBB2-targeting antibody drug conjugates in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4708.

Abstract Background Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with poor prognosis. GMB are highly recurrent mainly because of radio- and chemoresistance. Radiotherapy with Temozolomide (TMZ) is until today the golden standard adjuvant therapy, however, the optimal treatment of recurrent glioblastoma remains controversial. Ouabain belongs to the Cardiotonic Steroids (CTS) the natural ligands of the Na/K-ATPase (NKA). It is established that the NKA represents a signal transducer with either stimulating or inhibiting cell growth, apoptosis, migration and angiogenesis. Over the last decade evidence grew that CTS have anti-tumor properties especially in GBM. Aim Proceeding from recent studies we wanted to further demonstrate a divergent effect of Ouabain on a TMZ-resistant (T98G) as compared to a TMZ-sensitive (LN229) GBM cell line. Methods We analyzed the effect of Ouabain on cell migration and plasma cell membrane potential (PCMP) in the LN229 and T98G GBM cell line as well as underlying mechanisms (Bcl-2 and p-Akt/pan-Akt expression). Moreover, we analyzed the anti-angiogenic effect of Ouabain on human umbilical vein endothelial cells (HUVECs). Results T98G cells showed a significant inhibition of cell migration and a significant depolarization of the PCMP at similar Ouabain concentrations (IC50 = 1.67 × 10–7 M) resp. (IC50 = 2.72 × 10–7 M) with a strong inverse correlation (R2 = 0.95). In contrast, LN229 cells did not respond to Ouabain in these assays at all. Similarly, only T98G but not LN229 cells revealed Bcl-2 down-regulation at nanomolar Ouabain concentrations. This unique response to Ouabain is associated with a down-regulation of pan-Akt in T98G cells 24 h after Ouabain (1.0 × 10–6 M) treatment. For the first time, the anti-angiogenic effect of Ouabain on HUVEC cells (IC50 = 5.49 × 10–8 M) was demonstrated which correlated strongly with the anti-migratory effect (R2 = 0.85). Conclusion The TMZ-resistant T98G cell line as compared to the TMZ-sensitive LN229 cell line shows a high sensitivity towards Ouabain. We consider it as a promising new compound especially in recurrent GBM to overcome the resistance to TMZ and irradiation.

The proto-oncogene KRas is a well-described small GTPase that functions as a molecular switch for major physiological signaling pathways involved in cell proliferation, differentiation and survival. It has been shown that activating mutations in KRas are among the most common oncogenic drivers of tumorigenesis. Missense mutations of KRas result in constitutive activation due to impaired hydrolysis of GTP which enhances tumor-promoting downstream signaling pathways. Most KRas mutations are located in exon 2 or 3 including the most frequently changed glycine 12 which is present in most pancreatic cancers as well as in colorectal cancers and lung adenocarcinomas. Although direct inhibition of KRas is challenging due to its small size, smooth surface and limited druggable pockets on its surface, first promising drug candidates for selected KRas mutants have been developed in recent years (e.g. Sotorasib, MRTX1133, BI-2852). It was the aim of this study to characterize the selective inhibitory effects of KRas inhibitors in a panel of cancer cell lines harboring different KRas mutations like G12C, G12V or G12D. So, we applied different cellular techniques including phosphorylation assays as well as 2D proliferation and 3D spheroid assays to screen KRas inhibitors for their effect on distinct KRas mutants. Firstly, inhibitors were tested in a cellular assay monitoring phosphorylation of the downstream target ERK at residues Thr202/Tyr204. Analysis applying the cellular pERK AlphaLisa assay showed a high selectivity of several inhibitors for specific KRas mutants, while RAF and MEK inhibitors showed no significant selectivity in the panel of cancer cell lines tested here. Next, KRas inhibitors were tested in a cellular 2D proliferation and a 3D spheroid assay to determine the inhibitory effect on cell growth in both settings. In line with published data, for some inhibitors we could observe a high selectivity for a specific KRas mutant which was even more pronounced in the 3D format as compared to the 2D setup. Most likely due to the fact that 3D growth assays mimic physiological conditions more closely than 2D settings. Interestingly, in the 2D and 3D assays we could not find such a clear inhibitory effect of the MEK and RAF inhibitors as compared to the cellular pERK assay. These results indicate that the described cellular pERK AlphaLisa assay established for selected cancer cell lines could be a useful tool to screen newly developed KRas inhibitors for their selective inhibition of specific KRas mutants. Additionally, the application of not only 2D proliferation but also 3D growth studies can further characterize the inhibitory potential of these KRas inhibitors. Citation Format: Franziska Fimm-Todt, Ezgi Dikici, Katharina Schaich, Oliver Siedentopf, Joachim Lauterwasser, Daniel Feger, Jan Erik Ehlert. Characterization of the selective inhibitory effect of KRas inhibitors in different cellular assay formats [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 549.

Abstract With the development and approval of cutting-edge immune-based approaches for cancer treatment in recent years, the global immune-oncological (IO) pipelines have dramatically increased. To overcome the challenges of rising treatment resistance, new strategies are being developed pointing towards enhancing and sustaining T cell-mediated cytotoxicity, exploiting NK, monocytes and γδT cell killing capacity, and combinatorial therapies, among others. Examples of such strategies are the bispecific antibodies, which are mainly T-cell (BiTE) engagers, but also NK-cell engagers. In addition to the FDA approved immune checkpoint inhibitors, monoclonal antibodies against tumor antigens are being developed to enhance the cytotoxic response of NK and macrophages/monocytes through antibody-induced cell cytotoxicity (ADCC) To facilitate the efficacy analysis of BiTEs, we developed a 384-well format assay system based on luciferase-labeled tumor cells (advantages: high-throughput capability, non-radioactive, non-toxic, relatively low cost, and easily adaptable to automation). As only the tumor cells are labeled, the specific detection of luciferase exclusively correlates to the number of viable tumor cells, allowing for co-cultures with high excess of effector cells without the effector cells causing interference. Due to high sensitivity, few tumor cells are required for a sufficient signal/noise ratio, keeping the overall need for primary effector cells to a minimum, even at high effector/target cell ratios. This is of great advantage when working with rare cytotoxic subpopulations (e.g. NK cells), precious samples from patients, or simply saves resources when performing large exploratory studies. In addition, combination of low 384-well format volumes with nanodrop agent dispensing technology minimizes the amounts of expensive antibodies and other agents. Applying this technology, we evaluated the combinatorial effects of Blinatumomab with other clinically relevant agents on the luciferase-labeled GCB-like DLBCL cell line OCI-LY1. Depending on compound combination, we observe synergistic (e.g. Lenalidomide) but also antagonistic effects (e.g. MEK-inhibitor, Selumetinib, and MCL-1-inhibitor, S63845). We also developed CD3-stimulated T cell killer assays either using target cells grown in monolayer or spheroids, with the intent to study checkpoint inhibitors or other T cell activation modulators. In contrast to target cells grown in monolayers, immune cytotoxicity of spheroids by anti-CD3 stimulated T cells was incomplete, showing the additional challenge for effector cells when using 3D cultures as targets. Moreover, we show an ADCC example using NK as cytotoxic cells. Our data supports the outstanding usefulness of this methodological approach for the exploration of the overall efficiency of a therapy to enhance tumor cell killing by immune effector cells. Citation Format: Carla Castro, Philipp Metzger, Daniel Feger, Sarah Ulrich, Oliver Siedentopf, Jan E. Ehlert, Holger Weber. Use of luciferase-labeled target cells to explore immune cell killing in high throughput format in 2D and 3D co-cultures. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4419.

Drug-conjugates are an emerging class of anticancer agents combining the cytotoxic activity of highly potent chemotherapeutic agents with the target specificity of an antibody, a small molecule or a peptide ligand directed against a cancer-associated protein. With nearly half a century of development, profound advancements have been made in the development of these therapeutics. The most advanced are antibody-drug conjugates (ADCs) with currently >100 in development and 11 approved by the FDA. To facilitate preclinical evaluation of novel drug conjugates, Reaction Biology and 4HF Biotec have developed a dedicated platform allowing specific in vitro and in silico analyses for this class of therapeutics. The platform is intended to provide information on the potency of the drug conjugate over the corresponding stand-alone cytotoxin, the cancer entities to be treated, the target engagement and the determinant(s) of cancer cell sensitivity. Here, we present our platform to refine antitumor potential of Sacituzumab govitecan (ScG), a recently approved TROP-2 targeted ADC used for the treatment of triple-negative breast cancer. First, we used the ProLiFiler™ (Reaction Biology), a cell proliferation and survival assay with 140 human cancer cell lines, to establish the antitumor profile of ScG, its payload SN-38 and three other cytotoxins (camptothecin, MMAE and mertansine) to validate the results of our assay. The IC50 obtained for the cytotoxins were next validated using the MoAFinder (4HF Biotec), a tool derived from the NIH COMPARE algorithm that ranks drugs based on the similarity of their growth inhibitory profiles. The analysis confirmed that SN-38 and camptothecin antitumor profiles, as established with the ProLiFiler, correlated best with other topoisomerase-1 inhibitors from our database of more than 1,000 compounds. Mertansine and MMAE correlated best with tubulin inhibitors and were more potent than camptothecin and SN-38. Overall, the response to these cytotoxins was stronger in cell lines from hyperproliferative cancers such as hematological malignancies than in those from solid cancers. Next, by using OMICS data (internal and publicly available), qPCR and flow cytometry connected to drug sensitivity profiles, we will present a differential biomarker screen of ScG vs. SN-38 to identify molecular determinants of sensitivity toward the ADC and its cytotoxin. We will address the expression level of TROP2 and its predictivity for tumor cell sensitivity, screen for predictors of SN-38 response and investigate possible off-target effects in cell lines sensitive to ScG but lacking TROP2 expression. Overall, the study demonstrates the potential of our platform to investigate drug conjugates. The comparative profiling of ScG and its payload allows to identify candidate cancer entities for ScG treatment and to improve patient enrollment into clinical trials using biomarkers. Citation Format: Vincent Vuaroqueaux, Nadine Obier, Anne-Lise Peille, Daniel Feger, Katharina Schaich, Thomas Metz, Sebastian Dempe, Heinz-Herbert Fiebig, Jan Erik Ehlert. Introduction of a platform for preclinical profiling of drug conjugates: a case study with sacituzumab govitecan. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4895.

Adavosertib (AZD1775, MK-1775), a clinical stage inhibitor of the tyrosine kinase WEE1, was investigated in a cell proliferation and survival assay with 140 human cancer cell lines (CLs) representing all major tumor types (ProLiFiler platform of Reaction Biology) followed by mechanism of action (MoA) and biomarker analyses using 4HF Biotec’s Cancer Data Miner in silico platform. Adavosertib exhibited a broad anti-cancer activity across all hematological and solid tumor types with IC50 values ranging from 0.06 to 10 µM (median: 0.38 µM), matching the consistently high expression of the WEE1 gene. Among 900 reference compounds, the activity profile of adavosertib correlated best with the profiles of compounds targeting the replication stress response including other WEE1 inhibitors but also inhibitors of checkpoint kinase 1 and 2 (CHK1/2) or ataxia telangiectasia-mutated (ATM). Significant correlations were also seen with compounds blocking mitosis, DNA replication and DNA repair. Interestingly, we observed a subset of cell lines that were resistant to both DNA synthesis and PARP inhibitors but were sensitive to WEE1 inhibition. Moreover, by using multiple datasets of WEE1 inhibitors connected to the molecular annotations of CLs for a data driven biomarker screening, we identified MYC mutations as a predictive marker of sensitivity and PIK3CA or ERBB2 gene amplifications as predictors of resistance. Transcriptome analysis identified up to 900 genes for which higher expression in CLs was associated with CL sensitivity to the compound. Preliminary pathway analysis indicated that these genes are well represented among nuclear factor and Myc-regulated genes. In conclusion, our studies demonstrate broad anticancer activity of adavosertib and confirm its proposed MoA. The biomarkers we identified will facilitate the selection of pre-clinical in vivo tumor models and, if confirmed, even patient selection for clinical trials. The combined use of the ProLiFiler and Cancer Data Miner Platforms has the potential to accelerate and de-risk the development of anti-cancer agents. Citation Format: Vincent Vuaroqueaux, Daniel Feger, Anne-Lise Peille, Oliver Siedentopf, Sadhana Panzade, Sarah Ulrich, Sebastian Dempe, Heinz-Herbert Fiebig, Jan Erik Ehlert. A systems biology approach combining ProLiFiler and Cancer Data Miner for an enhanced preclinical characterization of the WEE-1 inhibitor Adavosertib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1132.

Bispecific antibodies are being explored as a means to modulate responses of the immune system to tumor infiltration. They are emerging as a growing class of immunotherapies with potential to further improve clinical efficacy and safety. Besides tumor-targeted and dual immunomodulators, the most important class of therapeutically relevant bispecific antibodies are engagers of cytotoxic immune cells to kill tumor cells. These are mainly engager of T-cells (BiTE´s), but also NK-cell engager several of which have entered clinics with Blinatumomab (anti-CD3/anti-CD19) being FDA-approved for the treatment of Acute Lymphoblastic Leukemia. Several studies are under way to address potential combinatorial effects with other compounds in clinics. In order to facilitate analysis of the cytotoxic impact of BiTE´s in high throughput, we developed a 384-well format assay system based on Luciferase-labeled tumor cells. This detection technology has many advantages. As only the tumor cells are labeled, the specific detection of luciferase exclusively correlates to the number of viable tumor cells, allowing for co-cultures with high excess of effector cells without them interfering in the detection. Due to high sensitivity, only few tumor cells are required for a decent signal/noise ratio, keeping the overall need for primary effector cells to the lowest, even at high effector/target cell ratios. That can be of great advantage when working with rare cytotoxic subpopulations (e.g. NK-cells), precious samples from patients, or simply saves resources when performing large exploratory studies. In addition, combination of low 384-well format volumes with nanodrop agent dispensing technology minimizes amounts of expensive antibodies and other agents. Applying this technology, we evaluated the combinatorial effects of Blinatumomab with immune modulators (e.g. Lenalidomide), kinase inhibitors (e.g. MEK-inhibitor, Selumetinib) and other clinically relevant agents (e.g. MCL1-inhibitor, S63845) on the Luciferase-labeled GCB-like DLBCL cell line OCI-LY1. Depending on compound combination, we observe synergistic but also antagonistic effects. Our data support the outstanding usefulness of this methodological approach for the exploration of bispecific cytotoxic immune cell engager agents. Citation Format: Carla N. Castro, Daniel Feger, Sarah Ulrich, Oliver Siedentopf, Jan E. Ehlert. Exploring the combinatorial potential of bispecific T-cell engagers in high throughput format [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2893.

Abstract The KRas/Raf/MEK signalling axis has been identified to play a critical role in the formation of cancer, resulting in several investigational new drugs targeting this pathway. Identification of appropriate drugs is hampered, however, by the assumption that a three-dimensional setting may be required to observe significant inhibitory effects, possibly due to alternate gene expression and protein activity in such a “closer-to-physiology” setting. In order to explore that topic, in the current study we have made use of our 140 cell lines (CL)-2D proliferation panel (3 days incubation) and our 100CL-3D soft agar panel (> 7 days incubation). On these platforms, we have compared inhibitors targeting the KRas/Raf/MEK pathway via either KRas:SOS interaction (e.g. BI3406), the KRas mutation at G12C (e.g. AMG510), or Raf-kinase (e.g. AZ628) and MEK1/2-kinase activity (e.g. AZD6244). It showed that especially the Raf- and MEK kinase inhibitors exhibited significantly enhanced potency in the 3D soft agar setting in the majority of the cell lines tested. The other inhibitors also showed an increased potency under soft agar conditions, however, on clearly fewer cell lines and usually not as strongly. For all kinds of inhibitors tested, the most significant changes in potency were associated with enhanced efficacy as observed by stronger cell death induction. Differences may be attributable to three-dimensional growth, but also to the elongated duration of the soft agar assay. For selected conditions, we addressed this topic by analyzing the inhibitor impact on 3D spheroid growth after shorter incubation times. Our results show that 3D growth analysis either in the soft agar or spheroid setting clearly supports the development of KRas/Raf/MEK pathway inhibitors. Citation Format: Katharina Schaich, Daniel Feger, Oliver Siedentopf, Sarah Ulrich, Jan Erik Ehlert. Characterization of KRas pathway inhibitors in 2D and 3D screening formats [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 357.

With the development and approval of cutting-edge immune-based approaches for cancer treatment in the latest years, the global immune-oncological (IO) pipelines have dramatically increased. To overcome the challenges of growing treatment resistance many new strategies are being developed pointing towards enhancing and sustaining T cell- mediated cytotoxicity, exploiting NK, monocytes and γδT cell killing capacity, and combinatorial therapies, among others. Examples of such strategies are the bispecific antibodies, which are mainly engagers of T-cells (BiTE´s), but also NK-cell engagers, several of which have entered clinics, such as Blinatumomab (anti-CD3/anti-CD19). Also, apart from the classical anti-PD1/PDL1 and anti-CTLA4, novel immune checkpoint inhibitors are being discovered, studied, and used in combination. Monoclonal antibodies against tumor antigens are being developed to enhance the cytotoxic response of NK and macrophages/monocytes through antibody-induced cell cytotoxicity (ADCC). To facilitate the efficacy analysis of BiTE´s, and other biologicals in high throughput, we developed a 384-well format assay system based on Luciferase-labeled tumor cells. This detection technology has many advantages. As only the tumor cells are labeled, the specific detection of luciferase exclusively correlates to the number of viable tumor cells, allowing for co-cultures with high excess of effector cells without them interfering in the detection. Due to high sensitivity, only few tumor cells are required for a decent signal/noise ratio, keeping the overall need for primary effector cells to the lowest, even at high effector/target cell ratios. That can be of great advantage when working with rare cytotoxic subpopulations (e.g. NK cells), precious samples from patients, or simply saves resources when performing large exploratory studies. In addition, combination of low 384-well format volumes with nanodrop agent dispensing technology minimizes amounts of expensive antibodies and other agents. Applying this technology, we evaluated the combinatorial effects of Blinatumomab with immune modulators (e.g. Lenalidomide), kinase inhibitors (e.g. MEK-inhibitor, Selumetinib) and other clinically relevant agents (e.g. MCL1-inhibitor, S63845) on the Luciferase-labeled GCB-like DLBCL cell line OCI-LY1. Depending on compound combination, we observe synergistic but also antagonistic effects. We developed as well, CD3-stimulated T cell killer assays, specially thought to study checkpoint inhibitors or other T cell activation modulators. And finally, we show an ADCC example using NK as cytotoxic cells. Our data supports the outstanding usefulness of this methodological approach for the exploration of bispecific cytotoxic immune cell engager agents. Disclosure Information C. Castro: A. Employment (full or part-time); Significant; Reaction Biology Europe GmbH.

Leucin-rich repeat kinase 2 (LRRK2) plays an important role in the onset of sporadic as well as familial Parkinson’s disease. Pathogenic gain-of-function mutations of LRRK2 are associated with aberrant LRRK2 hyperactivity which results in neurotoxicity and protein aggregation caused by dysfunctional autophagy and vesicle trafficking. Thus, the development of LRRK2 inhibitors represents a promising strategy for the treatment of Parkinson’s disease. Interestingly LRRK2 mutations have also been reported to increase the risk for the onset of different types of cancer (e.g. breast, thyroid, lung). Several studies suggest that LRRK2 is involved in the regulation of different cancer-related pathways (e.g. ATM-p53-p21-pathway, JNK pathway). Deregulation of LRRK2 activity caused by mutations has been shown to interfere with these pathways thereby increasing the risk to develop certain types of cancer. Since anti-cancer treatments mostly target the same pathways, we hypothesized that LRRK2 inhibitors may affect anti-cancer treatments in specific cancer cell types. In this study we report on the analyses of various inhibitors (e.g. MLi-2 and PF-06447475) on LRRK2 autophosphorylation at S935 in a cellular phosphorylation assay using the non-small cell lung cancer cell line A549 in comparison to biochemical LRRK2 activity assays. Furthermore, we compare the direct LRRK2 inhibitor effect on the proliferation of 140 cell lines, as well as their potential combinatorial impact on the potency of chemotherapeutic agents (e.g. Adriamycin). The observed effects can help to understand the implications of pharmaceutical LRRK2 inhibition in the treatment of both Parkinson’s disease and cancer. Citation Format: Franziska Fimm-Todt, Joachim Lauterwasser, Eva-Maria Egenter, Christian Weber, Daniel Feger, Katharina Schaich, Sarah Ulrich, Oliver Siedentopf, Frank Totzke, Michael Kubbutat, Jan Erik Ehlert. Cytotoxic effects of LRRK2 inhibitors in combined treatment with chemotherapeutic agents on a large panel of cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4072.

Abstract 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.

Detailed structure activity relationship of two series of quinazoline EHMT1/EHMT2 inhibitors (UNC0224 and UNC0638) have been elaborated. New and active alternatives are presented for the ubiquitous substitution patterns found in literature for the linker to the lysine mimicking region and the lysine mimic itself. These findings could allow for advancing EHMT1/EHMT2 inhibitors of that type beyond tool compounds by fine-tuning physicochemical properties making these inhibitors more drug-like.

Abstract TAM kinases, a relatively recent group of RTKs, have gained interest as therapeutic targets in cancer, chronic inflammatory, and autoimmune diseases. Comprising Tyro-3, Axl, and MerTK, TAMs need an extracellular lipid-protein complex for their activation, which is unique to this family of RTKs. For example, they bind to phosphatidyl serine bound gamma-carboxylated proteins as Gas-6 on apoptotic cells as ligands. Normally, TAMs function as regulators of the immune system such as controlling innate immunity or differentiation of natural killer cells, and lately a significant role in hematopoiesis has been deciphered. Though not potent oncogenic drivers, deregulated TAM signaling is often strongly associated with cancer progression, metastasis, and resistance to targeted therapies. They promote tumor growth by activation of prosurvival signals in tumor cells and reduction of antitumor immune responses. Several TAM kinase inhibitors are under (pre-)clinical investigation, e.g., BGB324 (R428), an Axl inhibitor is currently in Phase 1 trials as mono- or combination therapy. While for dual MerTK-Flt3 inhibitors MRX-2843 has received FDA Investigational New Drug (IND) status, UNC2025 is restricted to certain cancers like acute myeloid leukemia since inhibition of Flt3 has been related to severe hematopoietic toxicity. With the rapid increase in TAM-selective targeting strategies, there is an unmet need of assay systems to identify and categorize specific inhibitors. Due to their unique activation mechanism, set-up of cellular assay systems especially poses a challenge. Here, we report on the progress of set-up of various cell-based assays for all TAM kinases. To this end, we successfully developed a MefToff system for Axl and full-length expression in one cell background (e.g. Rat1) for Axl and Tyro-3 to measure cellular kinase activity. Additionally, we compared the data and feasibility of our cellular systems with biochemical tests using recently published commercially available inhibitors including UNC2025. To identify off-targets, we also tested 320 kinases in our ProQinase biochemical wild-type profiler assays. Furthermore, phenotypic screens were run to substantiate these outcomes. Currently, we try to set up assays for MerTK using various transmembrane domain mutants and chimeric systems. Assays with primary as well as tumor cells are in progress that would allow us to validate results from the cell-based artificial systems in to a more physiologic context. We believe such assays will allow a detailed molecular characterization of TAM kinases and will open opportunities for development of more advanced molecules as well for many new areas of interest. Citation Format: Alokta Chakrabarti, Daniel Feger, Frank Totzke, Sarah Umber, Orysya Stus, Marianne Birkle, Oliver Siedentopf, Marcel Pathe, Thomas Weber, Michael H.G. Kubbutat, Jan E. Ehlert. Exploring the functions of TAM kinases: assay development and characterization of potential therapeutics [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B159.

Nat. Med. 24, 282–291 (2018); published online 12 February 2018; corrected after print 6 March 2018 In the version of this article initially published, Omid Shah's name was misspelled as Omid Sha. The error has been corrected in the PDF and HTML versions of this article.