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Background & Aims In a phase 3 trial (RESORCE), regorafenib increased overall survival compared with placebo in patients with hepatocellular carcinoma (HCC) previously treated with sorafenib. In an exploratory study, we analyzed plasma and tumor samples from study participants to identify genetic, microRNA (miRNA), and protein biomarkers associated with response to regorafenib. Methods We obtained archived tumor tissues and baseline plasma samples from patients with HCC given regorafenib in the RESORCE trial. Baseline plasma samples from 499 patients were analyzed for expression of 294 proteins (DiscoveryMAP) and plasma samples from 349 patients were analyzed for levels of 750 miRNAs (miRCURY miRNA PCR). Tumor tissues from 7 responders and 10 patients who did not respond (progressors) were analyzed by next-generation sequencing (FoundationOne). Forty-six tumor tissues were analyzed for expression patterns of 770 genes involved in oncogenic and inflammatory pathways (PanCancer Immune Profiling). Associations between plasma levels of proteins and miRNAs and response to treatment (overall survival and time to progression) were evaluated using a Cox proportional hazards model. Results Decreased baseline plasma concentrations of 5 of 266 evaluable proteins (angiopoietin 1, cystatin B, the latency-associated peptide of transforming growth factor beta 1, oxidized low-density lipoprotein receptor 1, and C-C motif chemokine ligand 3; adjusted P ≤ .05) were significantly associated with increased overall survival time after regorafenib treatment. Levels of these 5 proteins, which have roles in inflammation and/or HCC pathogenesis, were not associated with survival independently of treatment. Only 20 of 499 patients had high levels and a reduced survival time. Plasma levels of α-fetoprotein and c-MET were associated with poor outcome (overall survival) independently of regorafenib treatment only. We identified 9 plasma miRNAs (MIR30A, MIR122, MIR125B, MIR200A, MIR374B, MIR15B, MIR107, MIR320, and MIR645) whose levels significantly associated with overall survival time with regorafenib (adjusted P ≤ .05). Functional analyses of these miRNAs indicated that their expression level associated with increased overall survival of patients with tumors of the Hoshida S3 subtype. Next-generation sequencing analyses of tumor tissues revealed 49 variants in 27 oncogenes or tumor suppressor genes. Mutations in CTNNB1 were detected in 3 of 10 progressors and VEGFA amplification in 1 of 7 responders. Conclusion We identified expression patterns of plasma proteins and miRNAs that associated with increased overall survival times of patients with HCC following treatment with regorafenib in the RESORCE trial. Levels of these circulating biomarkers and genetic features of tumors might be used to identify patients with HCC most likely to respond to regorafenib. ClinicalTrials.gov number NCT01774344 . NCBI GEO accession numbers: mRNA data (NanoString): GSE119220 ; miRNA data (Exiqon): GSE119221

Velcrin compounds are a class of small molecules that induce complex formation between PDE3A and SLFN12, killing cancer cells that express elevated levels of these two proteins by a mechanism independent of PDE3A enzymatic inhibition. Instead, PDE3A binding stimulates the RNase activity of SLFN12, resulting in cleavage of the specific SLFN12 substrate, tRNA-Leu-TAA. Cleavage of tRNA-Leu-TAA in turn causes ribosomal pausing, inhibition of protein synthesis, and cancer cell death. Unlike traditional targeted therapies that leverage dependencies created in cancer cells by genomic alterations, velcrins instead kill cancer cells by a gain-of-function mechanism dependent on the RNase activity of SLFN12. In a collaboration between the Broad Institute and Bayer Pharmaceuticals, we developed the first velcrin, BAY 2666605, to enter Phase I clinical trials. BAY 2666605 is active in cell line and patient-derived xenografts of several tumor types, specifically where elevated levels of the two biomarkers, PDE3A and SLFN12, are expressed. Biomarker-positive tumors are especially enriched among melanomas, and we have consistently observed tumor regression in biomarker-positive melanoma tumor models in vivo. BAY 2666605 furthermore shows drug-like properties, excellent brain penetration, increased stimulation of SLFN12 RNase activity, and reduced inhibition of PDE3A enzymatic activity compared with most other velcrins and approved PDE3A inhibitors. BAY 2666605 has recently entered a First-in-Human study (NCT04809805) in patients with advanced solid tumors that co-express PDE3A and SLFN12, including melanoma, ovarian cancer, and sarcoma. Citation Format: Stefan Gradl, Sooncheol Lee, Martin Lange, Xiaoyun Wu, Silvia Goldoni, Timothy Lewis, Charlotte Kopitz, Colin Garvie, Philip Lienau, Stephanie Hoyt, Henrik Seidel, Stephan Kaulfuss, Manuel Ellermann, Luc de Waal, Adrian Tersteegen, Sven Golfier, Detlev Suelzle, Christa Hegele-Hartung, James Carr, Frederick Brookfield, Michael Bruening, Melanie Berthold, Thibaud Jourdan, Monica Schenone, Galen Gao, Joseph McGaunn, Antje Wengner, Elisa Aquilanti, Franziska Siegel, Marine Garrido, Annette Walter, Isabelle Genvresse, Andrew Cherniack, Stuart Schreiber, Knut Eis, Ashley Eheim, Matthew Meyerson, Heidi Greulich. BAY 2666605: The first PDE3A-SLFN12 complex inducer for cancer therapy [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 ND04.

BAY 2666605, co-developed by the Broad Institute and Bayer Pharmaceuticals, is a selective and potent molecular glue, part of a family of small molecules recently baptized as ‘velcrins’, that induces complex formation between phosphodiesterase 3A (PDE3A) and SLFN12. BAY 2666605 has recently entered a First-in-Human study (NCT04809805) in patients with advanced solid tumors and here we describe its pre-clinical pharmacology profile. DNMDP, the precursor to BAY 2666605, was discovered in a phenotypic screen of genomically annotated cancer cell lines and sensitivity to treatment correlated to high expression of PDE3A (1). Upon treatment, SLFN12 is recruited into a stable complex with PDE3A where its RNase activity is enhanced and required for response (2). BAY 2666605 is a potent complex inducer (EC50 = 7 nM) and cytotoxic in vitro with nanomolar potency (IC50 = 1nM, in the most sensitive cell lines). Cancer cells with high expression of PDE3A and co-expression of SLFN12 are killed by a mechanism independent of PDE3A enzymatic inhibition. PDE3A-SLFN12 binding is required for cytotoxicity. Biomarker-positive lines are enriched in the melanoma lineage and show dose-dependent sensitivity to BAY 2666605 both in vitro and in vivo. Notably, we have consistently observed tumor regression in biomarker-positive melanoma models, including in PDX models (10mg/kg po BID). Based on target expression data from TCGA and tumor arrays, various other tumor types also co-express PDE3A and SLFN12, such as sarcomas and ovarian cancer. To this end, we show that BAY 2666605 inhibits tumor growth of PDX models of sarcoma and ovarian cancer in vivo. BAY 2666605 has excellent brain penetration, making glioblastoma a promising indication. Biomarker-positive GBM models are sensitive to BAY 2666605 both in vitro and in vivo. In a subset of orthotopic GBM models BAY 2666605 treatment has significant impact on survival. In BAY 2666605 treated models we have observed MCL1 downregulation and this biomarker will be evaluated in clinical settings. Our pre-clinical data indicate that BAY 2666605 is a potent anti-tumor agent with first-in-class potential and broad indication space. 1. de Waal et al. Identification of cancer-cytotoxic modulators of PDE3A by predictive chemogenomics, Nat. Chem. Biol. 12, 102-108 (2016) 2. Garvie et al. Structure of PDE3A-SLFN12 complex reveals requirements for activation of SLFN12 RNase, Nat. Commun. 12, 4375 (2021) Citation Format: Silvia Goldoni, Martin Lange, Charlotte Kopitz, Stefan Kaulfuss, Sven Golfier, Adrian Tersteegen, Stefanie Bunse, Melanie Berthold, Thibaud Jordan, Philip Lienau, Franziska Siegel, Annette Walter, Henrik Seidel, Elisa Aquilanti, Andrew Baker, Xiaoyun Wu, Sooncheol Lee, Stefan Gradl, Emmanuelle di Tomaso, Matthew Meyerson, Knut Eis, Ashley Eheim, Heidi Greulich. Preclinical profiling of BAY 2666605: The first PDE3A-SLFN12 complex inducer for cancer therapy [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 2663.

To identify novel therapeutic targets, we utilize the PRISM platform, a multiplexed cell line viability technology of 500 solid tumor cell lines and correlate responses to functional genomic and baseline genetic data. We describe ESD0140656, a small molecule with selective anti-proliferative effect on colorectal and PIK3CA-mutant cell lines. Response to ESD0140656 is correlated to sensitivity to CRISPR/Cas9 KO of components of the protein lipoylation pathway and OGDH complex members, which catalyze a step of the TCA cycle. Lipoylation is a rare post-translational modification attached to just four enzymes in humans, including the OGDH complex. Knockout of the protein that transfers lipoic acid to these four enzymes (LIPT1) sensitizes cells to ESD0140656, and ESD0140656 treatment leads to reduction of lipoic acid in cells. These results suggest ESD0140656 targets the lipoylation pathway and may represent a novel therapeutic angle for colorectal and PIK3CA-mutant tumors. Citation Format: Laura Doherty, Tenzin Sangpo, Peter Tsvetkov, John Davis, Navid Dianati, Wolfgang Schwede, Katja Zimmermann, Laura Evans, Aldo Amatucci, Henrik Seidel, Atanas Kamburov, Gizem Akcay, Todd Golub, Ashley Eheim, Nils Burkhardt, Knut Eis, Sven Christian, Matt Rees, Jennifer Roth. Small molecule targeting the lipoic acid post-translational modification impacts proliferation of colorectal and PIK3CA-mutant 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 2682.

89 Background: Combination treatment with regorafenib (80–120 mg/day, PO, 3 wks on/1 wk off) plus nivolumab (480 mg IV Q4W) showed manageable safety but modest efficacy in a phase 2 study of 70 pts from North America with pMMR/MSS chemotherapy-resistant metastatic CRC (mCRC). Five pts had a partial response (PR; objective response rate [ORR]: 7%); all did not have liver metastases at baseline (n = 5/23; ORR: 22%). One pt had a confirmed complete response (CR) after the primary completion analysis of the study (ASCO 2021). This retrospective exploratory analysis investigated the potential association between specific biomarkers and anti-tumor activity, and how those biomarkers are modulated by treatment with regorafenib plus nivolumab. Methods: In formalin-fixed paraffin-embedded tumor samples obtained at baseline and Cycle (C) 2 Day (D) 8, immune-related biomarkers were assessed via immunohistochemistry (IHC), and RNA sequencing was used for gene expression profiling/gene signatures. Pre-/on-treatment blood samples were collected to measure circulating tumor DNA (ctDNA) and soluble biomarkers. Results: A total of 40 and 27 baseline tumor samples and 14 and 5 paired tumor samples at baseline/C2D8 were available for IHC and RNA sequencing, respectively. Higher baseline protein and mRNA expression of biomarkers for pre-existing immune sensitivity (eg, effector T cells) trended with anti-tumor activity. These biomarkers were expressed at lower levels in pts with liver metastases vs those without liver metastases at baseline. Cytotoxic T cell density was elevated on C2D8 but did not correlate with anti-tumor activity. Increased mean variant allelic frequency in ctDNA at C2D1 predominated in pts with progressive disease (PD), while clearance of ctDNA at C2D1 was only noted for the one pt with a CR. High clonal tumor mutational burden in ctDNA showed a numerical trend with anti-tumor activity (PD vs. SD/PR; P=0.058) and PFS (P = 0.072). Baseline serum levels of select markers related to angiogenesis (eg, vascular endothelial growth factor [VEGF] D) were associated with inferior anti-tumor activity (P = 0.002). Serum levels of immune-related soluble biomarkers (eg, tumor necrosis factor alpha) increased on treatment (P < 0.005), while levels of soluble VEGF receptor 2 decreased (P < 0.001). Conclusions: This study of pts with MSS mCRC treated with regorafenib plus nivolumab suggests that baseline tumor biomarkers for pre-existing immune sensitivity trended with anti-tumor activity, whereas select baseline peripheral biomarkers related to angiogenesis trended with inferior anti-tumor activity. Pharmacodynamics effects were observed, yet no significant correlation with anti-tumor activity was found. Due to the small sample size and retrospective nature, these analyses are hypothesis-generating. Clinical trial information: NCT04126733.

Purpose Phosphatidylinositol 3-kinase (PI3K) signaling is critical for the proliferation and survival of malignant B cells. Copanlisib, a pan-class I PI3K inhibitor with predominant activity against PI3K-α and -δ isoforms, has demonstrated efficacy and a manageable safety profile in patients with indolent lymphoma. Patients and Methods In this phase II study, 142 patients with relapsed or refractory indolent lymphoma after two or more lines of therapy were enrolled to receive copanlisib 60 mg intravenously on days 1, 8, and 15 of a 28-day cycle. The primary end point was objective response rate; secondary end points included duration of response, progression-free survival, and overall survival. In addition, safety and gene expression were evaluated. Results Median age was 63 years (range, 25 to 82 years), and patients had received a median of three (range, two to nine) prior regimens. The objective response rate was 59% (84 of 142 patients); 12% of patients achieved a complete response. Median time to response was 53 days. Median duration of response was 22.6 months, median progression-free survival was 11.2 months, and median overall survival had not yet been reached. The most frequent treatment-emergent adverse events were transient hyperglycemia (all grades, 50%; grade 3 or 4, 41%) and transient hypertension (all grades, 30%; grade 3, 24%). Other grade ≥3 events included decreased neutrophil count (24%) and lung infection (15%). High response rates to copanlisib were associated with high expression of PI3K/B-cell receptor signaling pathway genes. Conclusion PI3K-α and -δ inhibition by copanlisib demonstrated significant efficacy and a manageable safety profile in heavily pretreated patients with relapsed or refractory indolent lymphoma.

Acute myeloid leukemia (AML), the most common acute leukemia in adults, is an aggressive hematologic malignancy resulting in bone marrow failure with a poor outcome; overall survival is approximately 25% at five years. Treatment options, in particular for the elderly population, are limited. Induction chemotherapy of cytarabine and an anthracycline (7+3) remains unchanged standard of care since its introduction in the early 1970s and there is a high medical need for new therapies (Yates et al. Cancer Chemother Rep 1973). DHODH is a key enzyme in the de novo pyrimidine synthesis converting dihydroorotate to orotate. Using a HOXA9 driven phenotypic screen to overcome differentiation arrest in myeloid cells, we have recently identified DHODH as a surprising novel target to overcome differentiation blockade in AML (Sykes et al. Cell 2016). Differentiation therapy already showed its enormous clinical benefit potential in the small subset of patients diagnosed with acute promyelocytic leukemia (APL) following treatment with all-trans retinoic acid with five-year survival exceeding 85% and should be considered the ultimate therapeutic goal for all AML subsets (Lo-Coco et al. NEJM 2013). Here, we disclose for the first time the structure and functional characterization of the novel DHODH inhibitor BAY 2402234. BAY 2402234 is a selective low-nanomolar inhibitor of human DHODH enzymatic activity. In vitro, it potently inhibits proliferation of AML cell lines in the sub-nanomolar to low-nanomolar range. BAY 2402234 induces differentiation of AML cell lines also in a sub-nanomolar to low-nanomolar range, demonstrating the anticipated mode of action in cellular mechanistic assays. In vivo, BAY 2402234 exhibits strong in vivo anti-tumor efficacy in monotherapy in several subcutaneous and disseminated AML xenografts as well as AML patient-derived xenograft (PDX) models. Target engagement of the novel DHODH inhibitor BAY 2402234 can be observed by increase of tumoral and plasma dihydroorotate levels after treatment with the inhibitor. Consistent with the in vitro data BAY 2402234 induces AML differentiation in vivo as detected by upregulation of differentiation cell surface markers in xenograft and PDX models after treatment with the inhibitor. Furthermore, differentiation-associated transcriptomic changes were evident following a single administration of BAY 2402234 in vivo. The start of clinical investigations of BAY 2402234 is planned for early 2018. Citation Format: Andreas Janzer, Stefan Gradl, Sven Christian, Katja Zimmermann, Claudia Merz, Hanna Meyer, Timo Stellfeld, Judith Guenther, Detlef Stoeckigt, Henrik Seidel, Pascale Lejeune, Michael Bruening, Ashley Eheim, Thomas Mueller, Ralf Lesche, Martin Michels, Andrea Haegebarth, Marcus Bauser, Sherif El Sheikh, Steven Ferrara, David Sykes, David Scadden. BAY 2402234: A novel, selective dihydroorotate dehydrogenase (DHODH) inhibitor for the treatment of myeloid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr DDT02-04.

Acute myeloid leukemia (AML) is a devastating disease, with the majority of patients dying within a year of diagnosis. For patients with relapsed/refractory AML, the prognosis is particularly poor with currently available treatments. Although genetically heterogeneous, AML subtypes share a common differentiation arrest at hematopoietic progenitor stages. Overcoming this differentiation arrest has the potential to improve the long-term survival of patients, as is the case in acute promyelocytic leukemia (APL), which is characterized by a chromosomal translocation involving the retinoic acid receptor alpha gene. Treatment of APL with all-trans retinoic acid (ATRA) induces terminal differentiation and apoptosis of leukemic promyelocytes, resulting in cure rates of over 80%. Unfortunately, similarly efficacious differentiation therapies have, to date, been lacking outside of APL. Inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in the de novo pyrimidine synthesis pathway, was recently reported to induce differentiation of diverse AML subtypes. In this report we describe the discovery and characterization of BAY 2402234 - a novel, potent, selective and orally bioavailable DHODH inhibitor that shows monotherapy efficacy and differentiation induction across multiple AML subtypes. Herein, we present the preclinical data that led to initiation of a phase I evaluation of this inhibitor in myeloid malignancies.

Background: Activating KRAS mutations are reported in up to 90% of pancreatic cancers. Refametinib potently inhibits MEK1/2, part of the MAPK signaling pathway. This phase I/II study evaluated the safety and efficacy of refametinib plus gemcitabine in patients with advanced pancreatic cancer. Methods: Phase I comprised dose escalation, followed by phase II expansion. Refametinib and gemcitabine plasma levels were analyzed for pharmacokinetics. KRAS mutational status was determined from circulating tumor DNA. Results: Ninety patients overall received treatment. The maximum tolerated dose was refametinib 50mg twice daily plus standard gemcitabine (1000mg/m2 weekly). The combination was well tolerated, with no pharmacokinetic interaction. Treatment-emergent toxicities included thrombocytopenia, fatigue, anemia, and edema. The objective response rate was 23% and the disease control rate was 73%. Overall response rate, disease control rate, progression-free survival, and overall survival were higher in patients without detectable KRAS mutations (48% vs. 28%, 81% vs. 69%, 8.8 vs. 5.3months, and 18.2 vs. 6.6months, respectively). Conclusion: Refametinib plus gemcitabine was well tolerated, with a promising objective response rate, and had an acceptable safety profile and no pharmacokinetic interaction. There was a trend towards improved outcomes in patients without detectable KRAS mutations that deserves future investigation. [Figure not available: see fulltext.]

Colorectal carcinoma (CRC) is the third most common malignancy in the US, being responsible for approximately 50,000 deaths per year. The overall 5-year survival rates are high for localized disease (~90%), but low for metastatic disease (~14%), thereby underlining the high unmet medical need for novel therapeutics for CRC patients. DHODH is a key enzyme in the de novo pyrimidine synthesis, converting dihydroorotate to orotate. We recently discovered its role in AML differentiation (Sykes et al 2016, Cell) and we are investigating BAY 2402234 in an ongoing phase I study in myeloid malignancies (NCT03404726). Since then, several publications have also described a role of DHODH in solid tumor indications. Herein we disclose the functional preclinical characterization of the novel DHODH inhibitor BAY 2402234 in CRC. BAY 2402234 is a selective low-nanomolar inhibitor of human DHODH enzymatic activity. In vitro it potently inhibits proliferation of CRC cell lines in the sub-nanomolar to low-nanomolar range. The anti-proliferative effects can be rescued by uridine supplementation which is known to bypass DHODH via the salvage pathway and demonstrates the on-target specificity of the inhibitor. Interestingly, not all CRC cell lines are sensitive to the inhibitor. Similar to the in vitro observations, BAY 2402234 exhibits strong in vivo anti-tumor efficacy in monotherapy in some, but not all, subcutaneous CRC xenograft models. A large CRC PDX in vivo screen revealed tumor growth inhibition in over 60% of the models in response to BAY 2402234 treatment. To elucidate the mode of action of BAY 2402234 in CRC we analyzed transcriptomic changes in CRC PDX models in vivo after single treatment with BAY 2402234, and identified several hundred differentially regulated genes at early time points. These in vivo PDX models were also used to explore potential effects of BAY 2402234 treatment on a large panel of metabolites. These preclinical results support the clinical evaluation of BAY 2402234 in patients with CRC, and such a clinical study is planned for early in 2019. Citation Format: Claudia Merz, Sven Christian, Ashley Eheim, Henrik Seidel, Ralf Lesche, Merlin Luetke- Eversloh, Hanna Meyer, Steven Ferrara, Marcus Bauser, Andrea Haegebarth, Stefan Gradl, Andreas Janzer. BAY 2402234: Preclinical evaluation of a novel, selective dihydroorotate dehydrogenase (DHODH) inhibitor for the treatment of colorectal carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3599.

DHODH is a key enzyme in the biosynthesis of pyrimidines and recent studies have renewed interest in this old anti-cancer target. Here, we disclose the discovery of 4-triazolosalicylamides as inhibitors of DHODH and their structure activity relationship (SAR). The hit cluster was discovered during a phenotypic high throughput screen (HTS) of 2.5 million compounds where proliferation of H460 lung cancer cells was used as read-out. DHODH was successfully identified as the molecular target by comparing the activity profile of the hits in a panel of cell lines to a set of inhibitors with known pharmacological activity. The hit compounds showed good cellular potency but had undesirable DMPK properties. Interestingly, the compounds are non-ionizable in contrast to many other DHODH inhibitors and show no potency shift from biochemical to cellular assays. Structural modifications lead to compounds with sub-nanomolar potency in cellular assays and increased metabolic stability enabling the proof of concept in vivo xenograft experiments. Further optimization guided by lipophilicity efficiency and identification of metabolic hot spots resulted in molecules with low clearance and improved solubility. BAY 2402234 was selected as the clinical candidate after side by side comparison of a number of promising compounds. It shows great oral bioavailability, target engagement in all preclinical species tested, induces differentiation in AML models, and has excellent activity in a variety of leukemia models. A clinical phase I study has been initiated in patients with myeloid malignancies. (NCT03404726) Citation Format: Stefan N. Gradl, Thomas Mueller, Steven Ferrara, Sherif El Sheikh, Andreas Janzer, Han-Jie Zhou, Anders Friberg, Judith Guenther, Martina Schaefer, Timo Stellfeld, Knut Eis, Michael Kroeber, Duy Nguyen, Claudia Merz, Michael Niehues, Detlef Stoeckigt, Sven Christian, Katja Zimmermann, Pascal Lejeune, Michael Bruening, Hanna Meyer, Vera Puetter, David T. Scadden, David B. Sykes, Henrik Seidel, Ashley Eheim, Martin Michels, Andrea Haegebarth, Marcus Bauser. Discovery of BAY 2402234 by phenotypic screening: A human Dihydroorotate Dehydrogenase (DHODH) inhibitor in clinical trials for the treatment of myeloid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2.

Androgen receptor (AR) signaling is essential at all prostate cancer stages and is clinically addressed by surgical or chemical castration, AR antagonists and androgen synthesis inhibitors. Second-generation AR antagonists include the approved drug enzalutamide and the late-stage clinical investigational oral compounds apalutamide and darolutamide. Comparing the gene expression patterns and the overarching epigenetic modifications elicited by AR antagonists will be essential for a detailed understanding of the molecular mode of action of these compounds. Methods: The transcriptome and AR binding patterns were analyzed by RNA-seq and qPCR, respectively. Genome-wide changes of the active transcription histone modification H3K27 acetylation were determined by ChIP-seq. Results: RNA-seq was performed in the androgen-dependent VCaP and LNCaP cell lines for a detailed analysis of the genome-wide transcriptional effects of the AR antagonists darolutamide, enzalutamide and apalutamide. Gene set enrichment analysis indicated that many genes involved in androgen response were up- or down-regulated by androgen treatment with R1881. Principal component analysis showed that treatment with pharmacologically relevant doses of AR antagonists had similar effects at the 8-hour time point, however some differences were visible at the 22-hour time point. A strong expression down-regulation of a number of genes from the Wnt and epithelial-mesenchymal transition (EMT) pathways was observed for darolutamide as compared to enzalutamide and apalutamide treatment in the VCaP cell line. In LNCaP cells the major differences between the AR antagonists were observed in the androgen-regulated genes, which is consistent with varying degrees of inhibition by the drugs in this cell line. Next we determined the global H3K27 acetylation profiles which characterize open chromatin and active transcription. ChIP-seq analysis performed in LNCaP cells revealed a strong impact of androgen treatment on the H3K27 acetylation profiles of numerous genes involved in hormonal regulation. Darolutamide significantly reversed genome-wide H3K27 acetylation patterns, almost to non-androgen stimulated levels. More specifically, a reduction of H3K27 peaks was observed at KLK3 and FKBP5 regulatory elements. In addition, we determined the impact of AR antagonists on AR binding at the KLK2 and KLK3 regulatory elements. Darolutamide and enzalutamide strongly reduced androgen-stimulated AR binding whereas the impact of apalutamide was slightly lower. Conclusions: AR antagonists strongly reversed the effects of the androgen R1881, both at the level of gene expression, and of H3K27 acetylation and AR binding. A detailed comparison between second-generation AR antagonists showed interesting differences in the regulation of genes in the Wnt and EMT pathways. Citation Format: Simon J. Baumgart, Ekaterina Nevedomskaya, Ralf Lesche, Henrik Seidel, Bernard Haendler. A detailed comparison between second-generation AR antagonists reveals differences in the overall impact on gene regulation patterns in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1798.

Regorafenib is a small molecule multi-kinase inhibitor approved for the treatment of patients with hepatocellular carcinoma (HCC) who have previously received sorafenib based on an improvement in overall survival versus placebo in the phase 3 randomized, double-blind RESORCE trial (NCT01774344; Bruix et al., Lancet, 2017). Expression of 776 plasma miRNAs was quantified by qPCR in baseline plasma samples collected from 343 of 573 patients (60%) from RESORCE (Teufel et al., ESMO2017, abs 705P). Predictive and prognostic effects of miRNAs were evaluated using a Cox proportional hazards model. Nine miRNAs showed significant predictive association with overall survival. A survival benefit in favor of regorafenib was associated with increased plasma levels of miR-30a, miR-122, miR-125b, miR-200a and miR-374b, and decreased plasma levels of miR-15b, miR-107, miR-320 and miR-645. Here, we focus on the biological interpretation of these statistical results. Expression data from The Cancer Genome Atlas was used to bridge from miRNAs via mRNAs to biological function. Gene set enrichment analysis of mRNAs based on correlation with expression of predictive miRNAs identified a list of the top 142 gene sets concordantly found for at least 6 of the 9 predictive miRNAs at a FDR of 0.01. Top gene sets showed consistent patterns related to HCC subtypes and several processes including liver cancer progression, metabolism of lipids, amino acids, bile acids and xenobiotics, glucuronidation, and doxorubicin resistance. Results suggested that response to regorafenib was improved in G4/G5/G6 versus G1/G2/G3 subtypes of the Boyault classification and in the CTNNB1 and Polysom7 subtypes of the Chiang classification which both coincide with the S3 subtype of the Hoshida classification (Hoshida et al., Seminars in liver disease, 30:35, 2010), a tumor subclass that is generally characterized by a retained hepatocyte-like phenotype, with well-differentiated and smaller tumors, and better survival. This is consistent with known roles of the predictive miRNAs in epithelial-mesenchymal transition, fibrogenesis, glycolysis, proliferation, and enhancement of apoptosis via several mechanisms. For example, miR-122 targets IGF1R, PDK4, LDHA and GALNT10, thereby regulating RAS/RAF/ERK signaling and glycolysis, which has been reported to overcome resistance to sorafenib. In summary, our results suggest that response to regorafenib is favorable in tumors with properties that are consistent with the S3 subtype of the Hoshida classification. Conclusions from this hypothesis-generating investigation require confirmation in follow-up studies with molecular characterization of tumor biopsies and regorafenib response data. Citation Format: Henrik Seidel, Karl Köchert, Michael Teufel. Biological interpretation of circulating miRNA biomarkers predicting regorafenib clinical benefit in patients with hepatocellular carcinoma (HCC) in the RESORCE trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2606.

Genomic translocation events frequently underlie cancer development through generation of gene fusions with oncogenic properties. Identification of such fusion transcripts by transcriptome sequencing might help to discover new potential therapeutic targets. We developed TRUP (Tumor-specimen suited RNA-seq Unified Pipeline) (https://github.com/ruping/TRUP), a computational approach that combines split-read and read-pair analysis with de novo assembly for the identification of chimeric transcripts in cancer specimens. We apply TRUP to RNA-seq data of different tumor types, and find it to be more sensitive than alternative tools in detecting chimeric transcripts, such as secondary rearrangements in EML4-ALK-positive lung tumors, or recurrent inactivating rearrangements affecting RASSF8. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0558-0) contains supplementary material, which is available to authorized users.

Effector functions and proliferation of T helper (Th) cells are influenced by cytokines in the environment. Th1 cells respond to a synergistic effect of interleukin-12 (IL-12) and interleukin-18 (IL-18) to secrete interferon-gamma (IFN-gamma). In contrast, Th2 cells respond to interleukin-4 (IL-4) to secrete IL-4, interleukin-13 (IL-13), interleukin-5 (IL-5), and interleukin-10 (IL-10). The authors were interested in identifying nonpeptide inhibitors of the Th1 response selective for the IL-12/IL-18-mediated secretion of IFN-gamma while leaving the IL-4-mediated Th2 cytokine secretion relatively intact. The authors established a screening protocol using human peripheral blood mononuclear cells (PBMCs) and identified the hydrazino anthranilate compound 1 as a potent inhibitor of IL-12/IL-18-mediated IFN-gamma secretion from CD3(+) cells with an IC(50) around 200 nM. The inhibitor was specific because it had virtually no effect on IL-4-mediated IL-13 release from the same population of cells. Further work established that compound 1 was a potent intracellular iron chelator that inhibited both IL-12/IL-18- and IL-4-mediated T cell proliferation. Iron chelation affects multiple cellular pathways in T cells. Thus, the IL-12/IL-18-mediated proliferation and IFN-gamma secretion are very sensitive to intracellular iron concentration. However, the IL-4-mediated IL-13 secretion does not correlate with proliferation and is partially resistant to potent iron chelation.

Effector functions and proliferation of T helper (Th) cells are influenced by cytokines in the environment. Th1 cells respond to a synergistic effect of interleukin-12 (IL-12) and interleukin-18 (IL-18) to secrete interferon-gamma (IFN-gamma). In contrast, Th2 cells respond to interleukin-4 (IL-4) to secrete IL-4, interleukin-13 (IL-13), interleukin-5 (IL-5), and interleukin-10 (IL-10). The authors were interested in identifying nonpeptide inhibitors of the Th1 response selective for the IL-12/IL-18-mediated secretion of IFN-gamma while leaving the IL-4-mediated Th2 cytokine secretion relatively intact. The authors established a screening protocol using human peripheral blood mononuclear cells (PBMCs) and identified the hydrazino anthranilate compound 1 as a potent inhibitor of IL-12/IL-18-mediated IFN-gamma secretion from CD3(+) cells with an IC(50) around 200 nM. The inhibitor was specific because it had virtually no effect on IL-4-mediated IL-13 release from the same population of cells. Further work established that compound 1 was a potent intracellular iron chelator that inhibited both IL-12/IL-18- and IL-4-mediated T cell proliferation. Iron chelation affects multiple cellular pathways in T cells. Thus, the IL-12/IL-18-mediated proliferation and IFN-gamma secretion are very sensitive to intracellular iron concentration. However, the IL-4-mediated IL-13 secretion does not correlate with proliferation and is partially resistant to potent iron chelation.

The reasons why human mammary tumors become resistant to tamoxifen therapy are mainly unknown. Changes in gene expression may occur as cells acquire resistance to antiestrogens. We therefore undertook a comparative gene expression analysis of tamoxifen-sensitive and tamoxifen-resistant human breast cancer in vivo models using Affymetrix oligonucleotide arrays to analyze differential gene expression. Total RNAs from the tamoxifen-sensitive patient-derived mammary carcinoma xenograft MaCa 3366 and the tamoxifen-resistant model MaCa 3366/TAM were hybridized to Affymetrix HuGeneFL and to Hu95Av2 arrays. Pairwise comparisons and clustering algorithms were applied to identify differentially expressed genes and patterns of gene expression. As revealed by cluster analysis, the tamoxifen-sensitive and the tamoxifen-resistant breast carcinomas differed regarding their gene expression pattern. More than 100 transcripts are changed in abundance in MaCa 3366/TAM as compared with MaCa 3366. Among the genes that are differentially expressed in the tamoxifen-resistant tumors, there are several IFN-inducible and estrogen-responsive genes, and genes known to be involved in breast carcinogenesis. The genes neuronatin (NNAT) and bone marrow stem cell antigen 2 (BST2) were sharply up-regulated in MaCa 3366/TAM. The differential expression of four genes (NNAT, BST2, IGFBP5, and BCAS1) was confirmed by Taqman PCR. Our results provide the starting point for deriving markers for tamoxifen resistance by differential gene expression profiling in a human breast cancer model of acquired tamoxifen resistance. Finally, genes whose expression profiles are distinctly changed between the two xenograft lines will be further evaluated as potential targets for diagnostic or therapeutic approaches of tamoxifen-resistant breast cancer.

Interleukin-10 (IL-10), originally identified as an inhibitor of pro-inflammatory cytokine production, exerts multiple immunomodulatory functions. Its ability to inhibit a Th1 response has been used in clinical trials for the treatment of inflammatory diseases including psoriasis. However, little is known about the molecular mechanisms of IL-10 functions. We aimed at identifying possible mediators of in vitro IL-10 treatment in monocytes by gene chip technology using Hu95a Affymetrix mRNA arrays with 12,000 genes. To prove relevance of the identified genes for the clinical situation we compared these in vitro results with genes being regulated by IL-10 in peripheral blood mononuclear cells from psoriatic patients undergoing IL-10 therapy. A high proportion of the 1,600 genes up-regulated and 1,300 genes down-regulated in vitro was found to be similarly regulated in vivo. Some genes, which were previously unknown to be regulated by IL-10, can be assigned to known IL-10 functions like e.g. the increase of pathogen clearance. Other new potentially immunomodulating genes have been identified to be regulated by IL-10, but their impact needs to be experimentally evaluated. We could confirm a recently reported up-regulation of heme oxygenase-1 (HO-1). However, we demonstrate that the anti-inflammatory mechanisms of IL-10 remain functional even when HO-1 is irreversibly inhibited.

Integrating a wide range of biomedical data such as that rapidly emerging from the use of next-generation sequencing is expected to have a key role in identifying and qualifying new biomarkers to support precision medicine. Here, we highlight some of the challenges for biomedical data integration and approaches to address them.

The prognosis for adults diagnosed with acute myeloid leukemia (AML) remains poor, with a five-year survival of only 25%. This prognosis is even more dismal in older patients who are not well enough to receive standard induction chemotherapy. Speaking to the need for new therapies is the fact that our therapeutic backbone - a combination of cytarabine and an anthracycline - remains unchanged since 1973. The promise of differentiation therapy was realized in the small subset of patients diagnosed with acute promyelocytic leukemia (APL). Here, treatment in the form of all-trans retinoic acid (ATRA) and arsenic trioxide inverted the survival curve; where APL was once the worst form of myeloid leukemia, it now carries the best prognosis, with a five-year survival exceeding 85%. The goal of this study was thus to develop differentiation therapy for patients with non-promyelocytic AML with the question: “Can we identify small molecules that overcome myeloid differentiation arrest?” A phenotypic differentiation screen in a HOXA9 driven leukemia model followed by target deconvolution, identified DHODH as an unexpected target for overcoming differentiation arrest in AML. We used 2 potent small molecule inhibitors of DHODH to validate this initial finding: Brequinar, a known DHODH inhibitor and an in house compound BAY DHODHi. In several in vitro experiments we demonstrated induction of AML differentiation in a dose dependent fashion. Interestingly, these effects could be completely rescued by addition of uridine, confirming target specificity. Treating mice in multiple genetically diverse AML in vivo models with a DHODH inhibitor led to tumor growth reduction and AML differentiation. Expression analysis of leukemia cells explanted from mice xenografts treated with a DHODH inhibitor demonstrate an early onset of differentiation markers indicating a direct role of DHODH with the onset of differentiation in vivo. The mechanism for selective vulnerability of leukemia cells to DHODH inhibition remains under investigation. Despite the observation that DHODH is expressed in all cells, normal and malignant, mice can tolerate DHODH inhibitor therapy for more than 100 days without weight-loss or other concerning side-effects. Thus, our pre-clinical studies point towards DHODH as a new metabolic target in the differentiation treatment of AML. Hopefully, small molecule DHODH inhibitors will provide a much-needed differentiation therapy for patients with acute myeloid leukemia. Citation Format: Andreas Janzer, David Sykes, Stefan Gradl, Steven Ferrara, Sven Christian, Claudia Merz, Henrik Seidel, Andreas Bernthaler, Ralf Lesche, Mathias Wawer, David T. Scadden. Inhibitors of the enzyme dihydroorotate dehydrogenase, overcome the differentiation blockade in acute myeloid leukemia [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 3086. doi:10.1158/1538-7445.AM2017-3086

Regorafenib is a small molecule inhibitor of multiple transmembrane and intracellular kinases involved in normal cellular functions and in pathologic processes such as oncogenesis, tumor angiogenesis, metastasis, and tumor immunity. Regorafenib is approved for the treatment of patients with metastatic colorectal cancer (CRC) who have been previously treated with fluoropyrimidine-, oxaliplatin- and irinotecan-based chemotherapy, an anti-VEGF therapy, and, if RAS wild-type, an anti-EGFR therapy or with locally advanced, unresectable or metastatic gastrointestinal stromal tumor (GIST) who have been previously treated with imatinib mesylate and sunitinib malate. Recently an overall survival benefit has been shown in patients with hepatocellular carcinoma (HCC) who had previously been treated with sorafenib (RESORCE). OncoTrack is an Innovative Medicines Initiative (IMI) sponsored project with the goal to improve the basis for identification of biomarkers based on the mechanisms of action of therapies approved for this indication. For this purpose, a panel of CRC-PDX xenografts was generated by the OncoTrack project. At the time of the analysis reported here fifty xenografts had been treated with regorafenib at a dose of 10 mg/kg/d or with vehicle for 24 days. The analysis of tumor growth rates (TGR) showed pronounced differences between different tumors and between vehicle and regorafenib treated models. The relative antitumor activity (relative TGR) of regorafenib varied between -0,13 (good response) and 0.0 (no response). Investigations of relative TGR in relationship to (non-) clinical parameters of the primary tumor such as age, gender, sidedness and tumor histology identified a marginally significant (p= 0.04) better response in tumors from younger patients. No other correlations were detected to this end, which may be due to the small sample number. To correlate antitumor activity of regorafenib with gene expression, RNA was isolated from sections of selected vehicle and regorafenib treated xenografts and hybrized on Affymetrix HuGene-2.1_st human transcriptome arrays. Expression profiles were subsequently analyzed using the Random Forests algorithm to identify gene expression signatures predicting response to regorafenib. The best signatures did not perform better than signatures derived after randomizing responses, i.e. no predictive signature could be identified. Further studies with larger samples sizes are necessary to improve the outcome of such an approach; however one should acknowledge that to date no predictive gene signatures could be identified for multikinase inhibitors, which may be intrinsic to their complex mechanism of action. The research reported here received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement 115234 (OncoTrack). Citation Format: Henrik Seidel, Jens Hoffmann, Ralf Lesche, Sylvia Grünewald, David Henderson, Dieter Zopf. Correlation of preclinical antitumor activity of regorafenib in CRC-PDX xenografts with gene expression and clinical parameters of the primary tumor [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 5036. doi:10.1158/1538-7445.AM2017-5036

Molecular mechanisms underlying the development of resistance to platinum-based treatment in patients with ovarian cancer remain poorly understood. This is mainly due to the lack of appropriate in vivo models allowing the identification of resistance-related factors. In this study, we used human whole-genome microarrays and linear model analysis to identify potential resistance-related genes by comparing the expression profiles of the parental human ovarian cancer model A2780 and its platinum-resistant variant A2780cis before and after carboplatin treatment in vivo. Growth differentiation factor 15 (GDF15) was identified as one of five potential resistance-related genes in the A2780cis tumor model. Although A2780-bearing mice showed a strong carboplatin-induced increase of GDF15 plasma levels, the basal higher GDF15 plasma levels of A2780cis-bearing mice showed no further increase after short-term or long-term carboplatin treatment. This correlated with a decreased DNA damage response, enhanced AKT survival signaling and abrogated cell cycle arrest in the carboplatin-treated A2780cis tumors. Furthermore, knockdown of GDF15 in A2780cis cells did not alter cell proliferation but enhanced cell migration and colony size in vitro. Interestingly, in vivo knockdown of GDF15 in the A2780cis model led to a basal-enhanced tumor growth, but increased sensitivity to carboplatin treatment as compared to the control-transduced A2780cis tumors. This was associated with larger necrotic areas, a lobular tumor structure and increased p53 and p16 expression of the carboplatin-treated shGDF15-A2780cis tumors. Furthermore, shRNA-mediated GDF15 knockdown abrogated p27 expression as compared to control-transduced A2780cis tumors. In conclusion, these data show that GDF15 may contribute to carboplatin resistance by suppressing tumor growth through p27. These data show that GDF15 might serve as a novel treatment target in women with platinum-resistant ovarian cancer.

Motivation: Image analysis is a major part of data evaluation for array hybridization experiments in molecular biology. The program presented here is designed to analyze automatically images from hybridization experiments with various arrangements: different kinds of probes (oligonucleotides or complex probes), different supports (nylon filters or glass slides), different labeling of probes (radioactively or fluorescently). The program is currently applied to oligonucleotide fingerprinting projects and complex hybridizations. The only precondition for the use of the program is that the targets are arrayed in a grid, which can be approximately transformed to an orthogonal equidistant grid by a projective mapping. Results: We demonstrate that our program can cope with the following problems: global distortion of the grid, missing of grid nodes, local deviation of the spot from its specified grid position. This is checked by different quality measures. The image analysis of oligonucleotide fingerprint experiments on an entire genetic library is used, in clustering procedures, to group related clones together. The results show that the program yields automatically generated high quality input data for follow up analysis such as clustering procedures. Availability: The executable files will be available upon request for academics. Contact: steinfat@molgen.mpg.de * To whom correspondence should be addressed.

Motivation: The choice of probes is an important feature of hybridisation experiments. In this paper we present an algorithm that optimises probes with respect to a training set of sequences based on Shannon entropy as a quality criterion. The practical motivation for our algorithm is oligonucleotide fingerprinting, a method for the simultaneous identification of sequences (cDNA or genomic DNA) by their hybridisation tags according to a set of short probes such as octamers, although the algorithm is of course not restricted to that application. Results: We can show that our method is superior to the selection of probes according to their frequencies, which is a widely used strategy, and to randomly chosen probe sets. The quality of probe sets is assessed by a simulation pipeline that entails the set of probes as a simulation parameter. The performance of probe sets trained on sequences from different organisms shows additionally that probes should be chosen with regard to the organism under analysis. Case studies are presented on how constraints (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{G\ +\ C}\) \end{document}-content, complexity of the individual probes) influence the selection process. Availability: A description of the oligonucleotide fingerprinting pipeline is published on our web-page http://www.molgen.mpg.de/~ag_onf/met.htm. An executable of the algorithm and probe lists designed for human and rodents can be downloaded from the ftp-site ftp://ftp.molgen.mpg.de/pub/mpimg/probe_design/. Contact: herwig@molgen.mpg.de To whom correspondence should be addressed.

We studied the influence of respiratory and cardiac phase on responses of the cardiac pacemaker to brief (0.35-s) increases of carotid baroreceptor afferent traffic provoked by neck suction in seven healthy young adult subjects. Cardiac responses to neck suction were measured indirectly from electrocardiographic changes of heart period. Our results show that it is possible to separate the influences of respiratory and cardiac phases at the onset of a neck suction impulse by a product of two factors: one depending only on the respiratory phase and one depending only on the cardiac phase. This result is consistent with the hypothesis that efferent vagal activity is a function of afferent baroreceptor activity, whereas respiratory neurons modulate that medullary throughput independent of the cardiac phase. Furthermore, we have shown that stimulus broadening and stimulus cropping influence the outcome of neck suction experiments in a way that makes it virtually impossible to obtain information on the phase dependency of the cardiac pacemaker's sensitivity to vagal stimulation without accurate knowledge of the functional shape of stimulus broadening.

672 Background: In the randomized phase 3 CONCUR trial (NCT01584830), regorafenib significantly improved overall survival (OS) and progression-free survival (PFS) vs placebo (PBO) in Asian patients (n = 136 regorafenib; n = 68 PBO) with treatment-refractory mCRC (HR [95% CI]: OS 0.55 [0.40‒0.77]; PFS 0.31 (0.22‒0.44]). Protein biomarker data from the phase 3 CORRECT trial of mostly Western patients with mCRC identified TIE-1 as a potential predictor of clinical response to regorafenib; however, this association was not significant in multivariable analyses. We present an exploratory protein biomarker analysis of patients in CONCUR. Methods: Sixteen proteins of interest, many of which are involved in angiogenesis, were quantified by multiplex immunoassay or ELISA in plasma samples collected at study entry from 121/204 (59%) patients (n = 83 regorafenib; n = 38 PBO). Potential predictive and prognostic effects were evaluated. Results: The biomarker cohort was representative of the overall study population in major baseline demographic factors, OS (HR 0.61; 95% CI 0.40‒0.93), and PFS (HR 0.25; 95% CI 0.16‒0.39). Elevated levels of ANG-2 (HR 2.46; p = 0.0016) and VEGF-A (HR 1.38; p = 0.03) were associated with poor OS prognosis; however, no significant association with treatment OS benefit for regorafenib vs PBO was observed for either marker (ANG-2: HR 0.76; p = 0.3307; VEGF-A: HR 0.83; p = 0.20). Elevated levels of five plasma proteins were associated with poor PFS prognosis: ANG-2 (HR 1.73; p = 0.0085), VEGF-A (HR 1.30; p = 0.0308), IL-8 (HR 1.67; p = 0.0014), VWF (HR 2.39; p = 0.0029), and IGF-BP2 (HR 1.71; p = 0.0384). Elevated levels of IL-8 (HR 0.70; p = 0.019), VWF (HR 0.53; p = 0.0312), and IGF-BP2 (HR 0.60; p = 0.0336) showed a modest interaction with regorafenib PFS; however, these results did not reach statistical significance after adjustment for multiple testing (p = 0.0045). Conclusions: None of the plasma proteins analyzed were predictive of regorafenib clinical benefit as measured by both OS and PFS in Asian patients with mCRC. These results, as well as those in CORRECT, suggest that rationally selected protein markers are not suitable to predict treatment benefit of regorafenib. Clinical trial information: NCT01584830.

Tumorigenesis is a multi-step process involving several consecutive genetic alterations resulting in loss of genomic stability and deregulated signal transduction pathways. To study these deregulated processes in vitro, typically established cancer cell lines derived from primary tumors, ascites, or from metastases are used. However, these cancer cell lines reflect only late stages of the tumorigenic process. To better understand the consequences of the sequential genetic alterations in an in vitro model system, we applied consecutive immortalization and transformation of primary human mammary epithelial cells (HMECs) combining shRNA-mediated knockdown of tumor suppressor genes and overexpression of oncogenes. Thereby, we developed a panel of isogenic HMEC-derived cell lines reflecting the multi-step process of tumorigenesis. The immortalized cell lines have a normal epithelial morphology and proliferate indefinitely and anchorage-dependently. In contrast, the transformed cells exhibit mesenchymal-like morphological changes and strong colony-forming activity in soft agar. SNP array analysis showed that none of the cell lines displayed gross chromosomal aberrations in 80 % of the chromosomes. However, massive changes were observed in some chromosomes of the transformed cells indicating that the transformed phenotype is characterized by chromosomal alterations. The isogenic immortalized and transformed cells described here provide a powerful tool for the in vitro validation of target genes for cancer therapy.

The efficiency and effectiveness of target selection and validation could be improved with accessible, standardized and integrated biological reference data sets. Such resources should be established through precompetitive approaches.

Background Treatment of non-small cell lung cancer with novel targeted therapies is a major unmet clinical need. Alternative splicing is a mechanism which generates diverse protein products and is of functional relevance in cancer. Results In this study, a genome-wide analysis of the alteration of splicing patterns between lung cancer and normal lung tissue was performed. We generated an exon array data set derived from matched pairs of lung cancer and normal lung tissue including both the adenocarcinoma and the squamous cell carcinoma subtypes. An enhanced workflow was developed to reliably detect differential splicing in an exon array data set. In total, 330 genes were found to be differentially spliced in non-small cell lung cancer compared to normal lung tissue. Microarray findings were validated with independent laboratory methods for CLSTN1, FN1, KIAA1217, MYO18A, NCOR2, NUMB, SLK, SYNE2, TPM1, (in total, 10 events) and ADD3, which was analysed in depth. We achieved a high validation rate of 69%. Evidence was found that the activity of FOX2, the splicing factor shown to cause cancer-specific splicing patterns in breast and ovarian cancer, is not altered at the transcript level in several cancer types including lung cancer. Conclusions This study demonstrates how alternatively spliced genes can reliably be identified in a cancer data set. Our findings underline that key processes of cancer progression in NSCLC are affected by alternative splicing, which can be exploited in the search for novel targeted therapies.

The majority of the biological effects of estrogens in the reproductive tract are mediated by estrogen receptor (ER)alpha, which regulates transcription by several mechanisms. Because the tissue-specific effects of some ERalpha ligands may be caused by tissue-specific transcriptional mechanisms of ERalpha, we aimed to identify the contribution of DNA recognition to these mechanisms in two clinically important target organs, namely uterus and liver. We used a genetic mouse model that dissects DNA binding-dependent vs. independent transcriptional regulation elicited by ERalpha. The EAAE mutant harbors amino acid exchanges at four positions of the DNA-binding domain (DBD) of ERalpha. This construct was knocked in the ERalpha gene locus to produce ERalpha((EAAE/EAAE)) mice devoid of a functional ERalpha DBD. The phenotype of the ERalpha((EAAE/EAAE)) mice resembles the general loss-of-function phenotype of alphaER knockout mutant mice with hypoplastic uteri, hemorrhagic ovaries, and impaired mammary gland development. In agreement with this phenotype, the expression pattern of the ERalpha((EAAE/EAAE)) mutant mice in liver obtained by genome-wide gene expression profiling supports the observation of a near-complete loss of estrogen-dependent gene regulation in comparison with the wild type. Further gene expression analyses to validate the results of the microarray data were performed by quantitative RT-PCR. The analyses indicate that both gene activation and repression by estrogen-bound ERalpha rely on an intact DBD in vivo.

Background: The GRID study demonstrated that regorafenib provides a significant improvement in progression-free survival (HR 0.27; p Methods: 91 plasma samples from patients enrolled in the Ph 3 study (GRID) for which BEAMing data (Jeffers et al 2013 JCO 31:10503) were subjected to Safe-SeqS covering cKit Exon 8 to Exon 18. Results: In 6 of 32 samples reported to be cKIT wildtype by BEAMing, mutations were identified by SafeSeqS. The detection of primary KIT exon 9 mutations showed a high degree of concordance among the two mutation-detection methods evaluated. Secondary / resistance hotspot mutations were also readily detected by both methods, although a greater number of such mutations were detected by Safe-SeqS than by BEAMing. The localization of the additional mutations detected by Safe-SeqS in known mutational hotspots supports their legitimacy. Safe-SeqS also detected KIT mutations for which BEAMing assays had not been developed, whereas in 17 samples a mutation for which a BEAMing assay was available was not detectable by Safe SeqS. In 58% (10/17) of samples, the mutant allele frequency found by BEAMing was close to the detection limit of this platform ( Conclusion: Our data support the use of Safe-SeqS as a sensitive and specific “liquid biopsy” method for non-invasive tumor genotyping of patients with GIST, enabling the identification of known and novel tumor-associated mutations using circulating DNA. These results confirm and extend the genotypic heterogeneity that had previously been identified in GRID circulating DNA samples by BEAMing. The comprehensive tumor mutational profiles generated by Safe-SeqS will be used to evaluate potential correlations between tumor genotype and clinical outcome. Citation Format: Michael Jeffers, Henrik Seidel, Susanne Schwenke, Joachim Reischl, Mark Rutstein, Christian Kappeler, Iris Kuss, Michael Teufel. Tumor genotyping in the phase III GRID study of regorafenib vs placebo in tyrosine kinase inhibitor (TKI)-refractory GIST: Detection of KIT mutations in circulating tumor DNA comparing digital PCR and massive parallel sequencing. [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 929. doi:10.1158/1538-7445.AM2015-929

Background: Mutationally-activated KRAS is present in 90% of pancreatic ductal adenocarcinoma (PDAC) and may represent an early genetic driver, being commonly found in low-grade pancreatic lesions (Eser et al. 2014 BJC 111:817). Refametinib is a potent oral allosteric MEK 1/2 inhibitor with both single-agent activity and synergistic activity in combination with gemcitabine in preclinical models of pancreatic cancer (PC). A Phase 1B/2 study in patients with locally advanced, unresectable or metastatic PC and no prior systemic therapy was conducted and recently reported (Van Laethem et al., ASCO 2014; NCT01251640). We report here on the exploratory biomarker findings from this study. Methods: KRAS mutational analysis was conducted via liquid biopsy at baseline on circulating tumor DNA (ctDNA) by BEAMing (Sysmex-Inostics) as well as circulating micro RNA (miRNA) from plasma collected at baseline and post-dose and analyzed by qPCR using an Exiqon panel of 752 miRNAs and an innovative data preprocessing method for normalization and imputation of undetermined values. Tumor molecular characterization was performed on archival tumor tissue and included targeted tumor gene next-generation sequencing with FOUNDATION ONE and the analysis of Ki67 proliferation index. Results: Samples for biomarker analysis were obtained from 69 treated patients. Forty-six (67%) had detectable KRAS mutations by liquid biopsy. KRAS G12D, G12V and G12R were the most frequent mutations. Interestingly, KRAS wild-type patients had better efficacy outcomes compared to mutant KRAS patients (mut/WT, respectively): overall response rate 15%/30% (OR 2.4, p = 0.147), median progression-free survival (mPFS) 3.7/8.8 mo (HR 0.32, p = 0.001), and overall survival (OS) 7.1/18.2 mo (HR 0.28, p = 0.001). There was a trend correlating KRAS mutant allele frequency with response. The CA19.9 levels correlated with KRAS mutational status. Tumor exome sequencing was performed from 16 patients, 15 of which had a KRAS mutation (G12D or G12V). The discordancy rate compared to BEAMing KRAS data was 26% (4/15). Conclusions: The high prevalence of KRAS mutations in patients with PC has been confirmed using BEAMing technology. In this study, there was an association between improved mPFS and OS in KRAS WT patients. Together with lower baseline levels of CA19.9 in the KRAS WT cohort, we conclude that liquid biopsy may be an approach to identify prognostic or predictive markers in PDAC treated with refametinib and gemcitabine. This hypothesis is sustained by the finding that poor clinical response showed increasing allele frequency of mutant KRAS. These results require confirmation in a larger trial. Citation Format: Michael Teufel, Jean-Luc Van Laethem, Hanno Riess, Marius Giurescu, Vittorio L. Garosi, Anke Schulz, Richardus Vonk, Henrik Seidel, Joachim Reischl, Barrett H. Childs. KRAS wild-type status as detected by circulating tumor DNA analysis may be a prognostic or predictive factor for clinical benefit in patients with unresectable, locally advanced or metastatic pancreatic cancer (PC) treated with the MEK inhibitor refametin [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 5239. doi:10.1158/1538-7445.AM2015-5239

The AKT1 (E17K) mutation is rare and occurs in colon, ovarian, lung, and especially breast cancer where its frequency ranges between 1.4% and 8.2%. It's precise role in cancer development and progression in clinical context is still unknown. To increase our understanding of the AKT1 (E17K) mutation in breast cancer we analyzed more than 600 tumor samples from breast cancer patients (UICC I - IV, including untreated and neoadjuvantly treated patients) which were provided by the non-profit organization PATH (Patients’ Tumor Bank of Hope, Germany). Extensive clinical data with a median follow-up time of 4.8 years to record disease progression were available for 95% of the patients included in this study. The AKT1 (E17K) mutation was detected in ∼6% of samples in the analyzed cohort using the BEAMing technology. Correlation with clinical parameters showed that the prevalence of the AKT1 (E17K) mutation was statistically independent of age or post-/pre-menopausal stage and was comparable between HER-2 positive and negative patients. In addition, FOUNDATION ONE® targeted exome Next Generation Sequencing (NGS) analysis of some of the tumor samples was done to demonstrate the fingerprint of individual tumors in correlation with the AKT1 (E17K) mutation. NGS and BEAMing technology had a ∼98% concordance for AKT1 (E17K) mutated and non-mutated samples. In 12 out of 36 AKT1 (E17K) mutated samples no additional somatic mutations (SNVs, indels) described to drive cancer development were detected. Moreover, neither amplification nor deletion of tested genes known to be recurrently amplified or deleted in cancer were found in 10 out of these 12 samples. This supports the hypothesis that AKT1 (E17K) can be a driver mutation. However, in all of these samples mutations with yet unannotated function in additional oncogenes were detected. It remains open whether these aberrations impact the role of AKT1 (E17K) as a driver mutation in tumor growth. Analyses of patient cohort data from large databases, as demonstrated here, holds promise for discovering the role of rare somatic mutations in known oncogenes (such as AKT1 (E17K)) in the development of breast cancer. Citation Format: Marion Rudolph, Tobias Anzeneder, Matthias Ocker, Eleni Lagkadinou, Oliver Politz, Martin Michels, Anke Schulz, Georg Beckmann, Michael Teufel, Henrik Seidel, Richie Soong, Heinz Bodenmüller, Ulla Ohlms, Khusru Asadullah, Joachim Reischl. AKT1 (E17K) mutation: coexistence with oncogenic alterations, prevalence, and correlation to clinical parameter in a large series of breast cancer patients. [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 569. doi:10.1158/1538-7445.AM2014-569

4129 Background: Refametinib is a potent oral allosteric MEK 1/2 inhibitor with both single-agent and synergistic activity in combination with gemcitabine in preclinical models of pancreas cancer (...

Clinicians and experimentalists routinely estimate vagal-cardiac nerve traffic from respiratory sinus arrhythmia. However, evidence suggests that sympathetic mechanisms may also modulate respiratory sinus arrhythmia. Our study examined modulation of respiratory sinus arrhythmia by sympathetic outflow. We measured R-R interval spectral power in 10 volunteers that breathed sequentially at 13 frequencies, from 15 to 3 breaths/min, before and after β-adrenergic blockade. We fitted changes of respiratory frequency R-R interval spectral power with a damped oscillator model: frequency-dependent oscillations with a resonant frequency, generated by driving forces and modified by damping influences. β-Adrenergic blockade enhanced respiratory sinus arrhythmia at all frequencies (at some, fourfold). The damped oscillator model fit experimental data well (39 of 40 ramps; r = 0.86 ± 0.02). β-Adrenergic blockade increased respiratory sinus arrhythmia by amplifying respiration-related driving forces ( P < 0.05), without altering resonant frequency or damping influences. Both spectral power data and the damped oscillator model indicate that cardiac sympathetic outflow markedly reduces heart period oscillations at all frequencies. This challenges the notion that respiratory sinus arrhythmia is mediated simply by vagal-cardiac nerve activity. These results have important implications for clinical and experimental estimation of human vagal cardiac tone.

The approaches presented in this article to analyzing nonlinear dynamics in multivariate data (interacting quantities) may help to uncover new insights into the physiological functional organization by considering mechanisms of particular nonlinear coordinated behavior. The systems-analytical approaches are demonstrated with regard to different aspects of the cardiorespiratory and cardiovascular interactions, which are: couplings between respiratory movements and heart-rate fluctuations; couplings between heartbeats and respiratory rhythms; and couplings between cardiorespiratorily related generators in the brain stem. A representative overview of experimental-mathematical techniques applied to nonlinear coordinations includes the following approaches: isofunctional mathematical modeling and simulation; and analysis of measured experimental bivariate data including multivariate phase-space sets, surrogate data, mutual information, phase synchronizations and bicoherence.

Entrainment between two rhythms is a very well-known phenomenon in the theory of nonlinear dynamics. Although heart and respiration influence each other by several mechanisms, and though modulation of heart rate by respiration is a very well-known and investigated phenomenon, there are surprisingly few indications of true entrainment between the two rhythms. This absence might be due to an insufficient coupling strength or to disturbances by other physiological rhythms. Nonetheless, we sometimes observe intermittent phases where cardiac and respiratory rhythms run in parallel; i.e., where both rhythms seem to be entrained. However, it is not obvious how to decide whether this effect is true entrainment or whether it is just quasi-entrainment that occurs when two rhythms have an approximate frequency ratio of for example, 4:1. Therefore, we use a surrogate-data technique to determine the probability of quasi-entrainment as a function of its duration. This probability can be used to obtain a significance level for true entrainment.

We focus on certain types of cardiac arrhythmias that are caused by atrioventricular block (AV block). We investigate how the dynamics of such arrhythmias are altered by the influence of the feedback introduced by the baroreceptor-cardiac reflex. The baroreceptor-cardiac reflex (baroreflex) is the predominant mechanism for the short-term control of blood pressure. Baroreceptors are sensitive to blood pressure and to changes of blood pressure. They convert these variables into neural signals that are sent to the brain. Other time-varying neural activities such as respiratory neurons, affect the cerebral information processing of baroreceptor signals. Autonomic nerves convey the neural impulses to effector organs such as the heart and peripheral resistance vessels, which in turn affect blood pressure to counteract the changes detected by the baroreceptors. This closed-loop control system contains several nonlinearities and time delays that can induce complicated rhythms. In this article we present two different mathematical descriptions of the atrioventricular conduction delay, one with and one without the baroreflex feedback, and compare the dynamics of atrioventricular conduction generated by these models.

The purpose of this study was to determine how breathing protocols requiring varying degrees of control affect cardiovascular dynamics. We measured inspiratory volume, end-tidal CO2, R-R interval, and arterial pressure spectral power in 10 volunteers who followed the following 5 breathing protocols: 1) uncontrolled breathing for 5 min; 2) stepwise frequency breathing (at 0.3, 0.25, 0.2, 0.15, 0.1, and 0.05 Hz for 2 min each); 3) stepwise frequency breathing as above, but with prescribed tidal volumes; 4) random-frequency breathing (∼0.5–0.05 Hz) for 6 min; and 5) fixed-frequency breathing (0.25 Hz) for 5 min. During stepwise breathing, R-R interval and arterial pressure spectral power increased as breathing frequency decreased. Control of inspired volume reduced R-R interval spectral power during 0.1 Hz breathing ( P < 0.05). Stepwise and random-breathing protocols yielded comparable coherence and transfer functions between respiration and R-R intervals and systolic pressure and R-R intervals. Random- and fixed-frequency breathing reduced end-tidal CO2modestly ( P < 0.05). Our data suggest that stringent tidal volume control attenuates low-frequency R-R interval oscillations and that fixed- and random-rate breathing may decrease CO2 chemoreceptor stimulation. We conclude that autonomic rhythms measured during different breathing protocols have much in common but that a stepwise protocol without stringent control of inspired volume may allow for the most efficient assessment of short-term respiratory-mediated autonomic oscillations.

Psoriasis is a common skin disease involving keratinocyte proliferation and altered differentiation, as well as T-cell activation. Here, we show that altered gene transcription in psoriatic skin lesions is highly reproducible between independent data sets. Analysis of gene expression confirmed dysregulation in all expected functional categories, such as IFN signaling and keratinocyte differentiation, and allowed molecular fingerprinting of a previously characterized dendritic cell subset associated with psoriasis tumor necrosis factor alpha (TNF-alpha)- and inducible nitric oxide synthase (iNOS)-producing CD11b(INT) DC (Tip-DC). Unexpectedly, a large group of dysregulated transcripts was related to fatty acid signaling and adipocyte differentiation, exhibiting a pattern consistent with the activation of peroxisome proliferator-activated receptor delta (PPARdelta). PPARdelta itself was strongly induced in psoriasis in vivo. In primary keratinocytes, PPARdelta was induced by the transcription factor activator protein 1, in particular by junB, but not by canonical WNT signaling, in contrast to its regulation in colon carcinoma cells. Activation of PPARdelta enhanced proliferation of keratinocytes, while this was inhibited by knockdown of PPARdelta. Finally, heparin-binding EGF-like growth factor (HB-EGF), known to induce epidermal hyperplasia and itself overexpressed in psoriasis, was identified as a direct target gene of PPARdelta. The present data suggest that activation of PPARdelta is a major event in psoriasis, contributing to the hyperproliferative phenotype by induction of HB-EGF.