Oliver Politz

Novel DNA methylation markers with potential prognostic relevance in advanced malignant melanoma identified using COBRA assays.

Aberrant methylation of promoter regions involved in silencing of tumor suppressor genes is a key feature of many human cancers including melanoma. These DNA methylation events occur early in cancer development, increase with progression, and may therefore serve as biomarkers for the detection and staging of cancer. In our study, we used an epigenomic reactivation screening approach including Combined Bisulfite Restriction Analyses (COBRA) assays to identify novel methylation markers in late-stage melanoma. Two human xenograft melanoma models have been used to identify genes methylated in cancer and reactivated upon treatment with a histone deacetylase inhibitor. Gene expression analysis and promoter scanning for DNA methylation by COBRA assays and bisulfite sequencing were used to identify candidate genes. The methylation status of the CpG island promoter region of genes related to melanoma pathophysiology in skin, lymph node, and visceral metastatic metastases in 28 patients (samples n=35) were assessed. These methylation markers have been evaluated in melanoma metastasis tissue and in control samples from normal skin. The screening in in-vitro and in-vivo systems for methylated genes in melanoma samples showed 10 candidate genes. Using COBRA assays, we detected a methylation pattern in the promoter region of 10 genes with two genes (BASP1, CDH11), together with the patient’s age and the log-S100B-level at biopsy, constructing a descriptor with a trend to correlate with shorter time to death.

Abstract 3685: BAY 1125976, is a selective allosteric AKT1/2 inhibitor with high efficacy in AKT1-mutated cancers

The PI3K-AKT-mTOR signaling cascade is activated in human cancers by elevated membrane receptor activity, mutation, amplification, and deletion of genes encoding components of the pathway. An oncogenic, single hotspot mutation in the AKT1 gene, G49A:E17K, in the pleckstrin homology domain of AKT1, was described in human cancers with highest relative incidence in breast cancer. The E17K mutation in AKT1 results in PI3K-independent membrane recruitment of AKT1. Recently we reported the development of a highly selective, potent allosteric AKT1/2 inhibitor BAY 1125976 with strong in vitro and in vivo activity in tumor models with activated AKT signaling and strong synergistic activity in combination. The efficacy of BAY 1125976 was evaluated in tumor models carrying activation of the PI3K-AKT pathway by either deletion of PTEN or activating mutation in PI3K. We investigated whether BAY 1125976 can also inhibit AKT signaling in cell lines carrying an activating mutation in AKT. To this extend, KU-19-19 (AKT1E17K; NRASQ61R) bladder cancer as well as LAPC4 (AKT1E17K) prostate cancer cell lines both bearing the AKT1E17K mutation were profiled. BAY 1125976 potently inhibited AKT activation as well as downstream signaling in KU-19-19 and LAPC4 cells. Furthermore, anti-tumor efficacy of BAY 1125976 was tested in the patient-derived anal cancer xenograft AXF 984 (AKT1E17K). BAY 1125976 shows dose-dependent potent pathway activity in AKT1mut patient derived tumor model with inhibition of pAKT473 as well as downstream targets p- PRAS40 and p-FOXO3a compared to selected PI3K, AKT and mTOR inhibitors. Continuous daily treatment of AXF 984 (AKT1E17K) mouse xenografts with 25 mg/kg and 50 mg/kg QD p.o. BAY 1125976 resulted in very potent statistically significant anti-tumor efficacy. All animals of these treatment groups exhibited tumor shrinkage or disease control with response rates of 88% and 83%, respectively. Furthermore, for animals treated with 50 mg/kg BAY 1125976 the delay of reaching a relative tumor volume of 600% was statically significant compared to the vehicle-treated control group. In summary, BAY 1125976 showed superior anti-tumor activity in AKT1E17K mutated models compared to PI3K and mTOR inhibitors. These results indicate that the clinical development of BAY 1125976 in patients with activating mutation in AKT could result in an innovative and more effective alternative to current treatments. Citation Format: Oliver Politz, Arne Scholz, Andrea Haegebarth, Ningshu Liu, Lars Baerfacker, Stuart Ince, Roland Neuhaus, Ulf Boemer, Martin Michels, Dominik Mumberg. BAY 1125976, is a selective allosteric AKT1/2 inhibitor with high efficacy in AKT1-mutated cancers. [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 3685. doi:10.1158/1538-7445.AM2014-3685

Abstract 2050: BAY 1125976, a highly selective and potent allosteric AKT1/2 inhibitor, for the treatment of cancers with aberrations in the PI3K-AKT-mTOR pathway.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The PI3K/AKT/mTOR pathway is frequently activated in human cancer. AKT, a central element in the pathway, is essential for tumor growth, proliferation, survival, invasion and metastasis. Activation of AKT is a key mechanism in resistance to chemo-, radio- and targeted therapies. Thus, AKT is considered an attractive drug target. Herein, we report on the preclinical profile and combinability of BAY 1125976, a potent, highly selective, allosteric AKT1/2 inhibitor, which is particularly effective in models with PI3K-AKT pathway aberrations. In biochemical assays, BAY 1125976 demonstrates equal potency against AKT1 and AKT2 in the low nanomolar range (IC50 ∼ 10 nM) while it displays weaker activity against AKT3 (IC50 ∼ 500 nM) and is inactive against ∼230 other protein/ lipid kinases (IC50 > 1 μM). Mechanistically, BAY 1125976 blocks AKT signalling by inhibiting the phosphorylation of AKT at both Thr308 and Ser473 (IC50 < 1 nM), as well as downstream phosphorylation of 4E-BP1 (IC50 < 50 nM). The strong inhibition of cellular p-AKT and downstream signalling translates to a broad inhibition of tumor cell proliferation in vitro. In particular, tumor cell lines carrying defects in the tumor suppressor PTEN, or oncogenic mutations in PIK3CA are most sensitive to BAY 1125976 treatment. Daily oral dosing of BAY 1125976 in human xenograft tumor models induces strong pharmacodynamic inhibition of AKT phosphorylation that correlates with drug exposure. In vivo, BAY 1125976 demonstrates dose-dependent anti-tumor efficacy in multiple xenograft tumor models of different histological types with PIK3CA mutations or PTEN deletions while being well tolerated. BAY 1125976 can be effectively combined with various anti-cancer therapies. In vitro combination profiling shows synergistic anti-proliferative effects with anti-hormonal therapeutics in breast and prostate cancer cell lines, which translates to enhanced anti-tumor efficacy with durable tumor regressions in vivo. Furthermore, in vivo combination of BAY 1125976 with external beam radiation results in strong additive to synergistic efficacy and significant tumor growth delay. Moreover, the combination of BAY 1125976 with the bone-targeting agent Radium 223 in a breast cancer bone metastasis model results in reduced tumor and metastases burden and increased necrotic and fibrotic bone area. In conclusion, BAY 1125976 is a highly selective, potent allosteric AKT1/2 inhibitor with strong in vitro and in vivo activity in tumor models with activated AKT signalling and strong synergistic activity in combination. Targeting AKT might also provide a promising strategy for overcoming chemo/radio-resistance and increasing radio-sensitization and radio-potentiation. Citation Format: Oliver Politz, Lars Baerfacker, Stuart Ince, William J. Scott, Roland Neuhaus, Ulf Boemer, Martin Michels, Dominik Mumberg, Franz von Nussbaum, Karl Ziegelbauer, Andrea Haegebarth. BAY 1125976, a highly selective and potent allosteric AKT1/2 inhibitor, for the treatment of cancers with aberrations in the PI3K-AKT-mTOR pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2050. doi:10.1158/1538-7445.AM2013-2050

Abstract B2: Regorafenib, an oral multi-kinase inhibitor of angiogenic, stromal and oncogenic receptor tyrosine kinases, potently inhibits tumor growth and angiogenesis in patient-derived xenograft models of gastric cancer.

Background: Gastric cancer (GC) is a prevalent and deadly disease. Because the VEGFR and PDGFR signaling cascade plays a critical role in the development and progression of gastric cancer and VEGF expression is correlated with angiogenesis, tumor progression and poor prognosis in patients with GC, blocking these signaling pathways could thus have therapeutic efficacy. This study aims to investigate the potential of a combined therapeutic approach to target these pathways in preclinical models of GC with regorafenib an oral multi-kinase inhibitor targeting VEGFR1–3, PDGFR, FGFR, TIE-2, RET and KIT. Methods: Eight patient-derived GC xenograft models with different histotypes were treated orally with regorafenib or vehicle and tumor growth was monitored using caliper measurements. Tissue sections of vehicle- or regorafenib-treated xenografts were analyzed by immunohistochemistry for apoptosis, microvessel area, and cell proliferation using antibodies against cleaved PARP, CD31 and phospho-Histone3 Ser10 , respectively. Tumor lysates were analyzed by Western Blotting with respective antibodies for effects on marker proteins for proliferation, apoptosis and angiogenesis. Results: Regorafenib demonstrated significant tumor growth inhibition of 81%, 85% and 88% in the GC09-0109 model after oral administration of 5, 10 and 15 mg/kg/d, respectively. In addition potent tumor growth inhibition ranging from 72–96% was observed in seven other GC models at a dose of 10 mg/kg/d. Regorafenib significantly inhibited angiogenesis in all models by 4–12 fold compared to the vehicle-treated group, independent of their degree of vascularization, as indicated by reduced CD31 staining. Furthermore inhibition of cell proliferation by 2–4 fold was observed by reduced staining for p-histone 3 Ser10 . Increased staining for caspase-cleaved p85 PARP by 2 to 20 fold was detected, indicating that regorafenib induced apoptosis. Finally, regorafenib also induced tumor necrosis as detected by histological staining. Inhibition of angiogenesis, reduced cell proliferation and induction of apoptosis and necrosis was associated with decreased levels of p-ERK1/2, p-cdk2 Thr14/Tyr15 , p-VEGFR2 Tyr951 , p-p70S6K Thr421/Ser424 , p-S6R Ser235/236 and p-eIF4E Ser209 and elevation of cleaved caspase-3 and cleaved PARP. Conclusion: We show that regorafenib is an efficacious anti-tumor targeted agent in patient-derived gastric cancer preclinical models. Our findings support further investigation of regorafenib in the treatment of GC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B2.

Abstract 569: AKT1 (E17K) mutation: coexistence with oncogenic alterations, prevalence, and correlation to clinical parameter in a large series of breast cancer patients

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%. It9s 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 Bodenmuller, 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

Abstract 3497: Pathway profiling for personalized medicine: Comparison of two immunoassays.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Cancer therapy is evolving to a detailed molecular analysis of the patients tumor followed by treatment with selective drug(s) targeting the individual properties of the tumor based upon identification of prognostic and predictive biomarkers. This requires comprehensive, highly sensitive test systems. We therefore sought to develop a sensitive diagnostic test for functionally profiling a spectrum of signaling pathway proteins in tumor samples. Here, we evaluated and compared two assay platforms: CEER (Collaborative Enzyme Enhanced Reactive-Immunoassay; Prometheus Labaratories) and MSD (Meso Scale Discovery). Breast, lung, and prostate cancers as well as glioblastoma and melanoma model cell lines (in all 10 cell lines) with various oncogenic pathway signatures were treated with different concentrations of PI3K inhibitor (BAY 806946) or an inhibitor targeting HER1 and HER2 (Lapatinib). Cells were lysed and the activation status as well as abundance of ten pathway proteins (HER1, HER2, cMET, PI3K, AKT, ERK, MEK, PRAS40, RPS6, and P70S6K) was measured by MSD at Bayer in Berlin and by CEER in a blinded fashion at Prometheus Laboratories. Target-specific inhibition (IC50) and downstream signal modulations were determined and compared. Overall we found a high concordance between the two assays. While target specific inhibitions were observed in relevant cell lines, varying mechanisms of treatment resistance due to redundant pathway activation, feedback loop or pathway cross talks were observed between the PI3K/AKT and RAS/ERK pathways. This study shows that sensitive immunoassays are a suitable tool for the detection and monitoring of biomarkers and provides new insights into the mode of action of targeted agents. Comprehensive profiling of signaling pathways holds promise as an approach to personalize selection of anticancer therapy. Citation Format: Antje Stresemann, Oliver von Ahsen, Oliver Politz, Phillip Kim, Sharat Singh, Khusru Asadullah, Karl Ziegelbauer, Thomas Krahn. Pathway profiling for personalized medicine: Comparison of two immunoassays. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3497. doi:10.1158/1538-7445.AM2013-3497

Abstract 252: Molecular characterization of cancer stem cells isolated from primary colon cancer cells.

Colon cancer is the 3 rd most common type of cancer and the 4 th leading cause of cancer-related deaths worldwide. OncoTrack is an international consortium that has launched one of Europe9s largest collaborative academic-industry research projects to develop and assess novel approaches for identification of new markers for colon cancer (1). To this end, colorectal tumors and metastases, as well as isolated circulating tumor cells and cancer stem cells are subjected to high-throughput sequencing of DNA and RNA. The laboratory data are combined with clinical data and used for in silico modeling of the patients9 tumors and prediction of biomarkers as well as a tailored therapy. These predictions are validated using patient-specific in vitro (primary cells) and in vivo (xenografts) models. Recent data show that a small subpopulation of cells within different tumors, including colon cancer, is responsible for tumor growth and maintenance. Due to their functional similarities to adult tissue stem cells and their likely origin, these cells have been termed cancer stem cells. We aim at characterizing patient-derived primary cell cultures and cancer stem cells isolated from these. Primary tumors and metastases from colon cancer patients are cultivated in vitro using a matrigel-based, serum-free culture system. This system allows for long-term expansion of primary colon cancer cells in vitro (2). Using this methodology, we were able to establish over 35 novel, patient-specific colon cancer cell lines, both from primary tumors and metastases. Using previously established markers of colon cancer stem cells (CD44+, CD133+, CD166+, EpCAM-high, Wnt-high), cells are sorted by flow cytometry and subsequently subjected to genome-wide analyses using next-generation sequencing. These experiments allow for a detailed characterization of colon cancer stem cells at the level of transcriptome and methylome. It has been shown that tumors are heterogeneous with regard to gene mutations and expression of cancer stem cell markers (3). Using molecular tools such as padlock probes (4), we are analyzing the distribution of gene mutations identified by next-generation sequencing within primary cells as well as primary tumors. Moreover, we are addressing the expression pattern of cancer stem cell markers in these cell types. These data will help in a better understanding of individual tumors and assist in design of a personalized therapy. OncoTrack is a project funded by the Innovative Medicines Initiative Joint Undertaking (IMI JU). 1 Elsner M, Nature Biotechnology 29, 378 (2011); www.oncotrack.eu 2 Sato T el al., Gastroenterology. 2011 Nov;141(5):1762-72. 3 Gerlinger M et al., N Engl J Med. 2012 Mar 8;366(10):883-92. 4 Larsson C, et al., Nat Methods. 2010 May;7(5):395-7. Citation Format: Martin Lange, Dirk Schumacher, Thomas Hauling, Christian Regenbrecht, Oliver Politz, Mats Nilsson, Jens Hoffmann, Reinhold Schaefer, David Henderson. Molecular characterization of cancer stem cells isolated from primary colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 252. doi:10.1158/1538-7445.AM2013-252

Abstract 3751: In vitro and in vivo pharmacological profile of BAY 1001931, a novel highly potent allosteric AKT1/2 inhibitor

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The PI3K/AKT/mTOR pathway is essential for tumor growth, proliferation, survival, invasion and metastasis. AKT, a central switch in this pathway, is deregulated in a broad range of refractory and primary tumors. Importantly, activation of AKT is one of the major mechanisms by which tumors escape from and become resistant to chemo-, radio- and targeted therapies. We report on preclinical studies of BAY 1001931, a highly selective and potent allosteric AKT1/2 inhibitor. In biochemical assays, BAY 1001931 inhibits AKT1 and AKT2 with similar potency (IC50 = 16 nM) while it displays weak activity against AKT3 (IC50 ∼ 1 µM) and is inactive against ∼230 other protein/ lipid kinases. Mechanistically, BAY 1001931 blocks AKT signalling by inhibiting the phosphorylation of AKT at both Thr308 and Ser473 (IC50 = 3.3 / 5.5 nM) as well as downstream phosphorylation of 4E-BP1 (IC50 = 70 nM). The strong inhibition of cellular p-AKT translates to a selective inhibition of tumor cell proliferation in vitro. Cell lines carrying defects in the tumor suppressor PTEN or oncogenic mutations in PIK3CA are most sensitive to BAY 1001931 treatment. Moreover, characterization of BAY 1001931 in a broader breast and prostate cancer cell line panel indicated strongest anti-proliferative efficacy in luminal and HER2 positive breast cancer cell lines and in androgen sensitive prostate cancer cell lines. In vitro combination profiling showed synergistic anti-proliferative effects with anti-hormonal therapeutics in breast and prostate cancer cell lines. When dosed orally in human xenograft tumor models, BAY 1001931 induced strong pharmacodynamic inhibition of AKT phosphorylation that correlated with drug exposure. BAY 1001931 was highly efficacious in multiple xenograft tumor models of different histological types with PIK3CA mutations or PTEN deletions. In tumor models predicted to be dependent on activated AKT signalling such as the KPL4 breast tumor model (PIK3CA H1047R and HER2 overexpression), daily oral treatment with BAY 1001931 induced tumor stasis or regression at well tolerated doses. Most importantly, when combined with anti-hormonal therapies such as tamoxifen in PIK3CA breast cancer xenograft models or bicalutamide or abiraterone acetate in PTEN deleted prostate cancer xenograft models, enhanced anti-tumor efficacy with durable tumor regressions were observed. In conclusion, BAY 1001931 is a highly selective, potent allosteric AKT1/2 inhibitor with strong in vitro and in vivo activity in tumor models with activated AKT signalling and strong synergistic activity in combination with anti-hormonals in breast and prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3751. doi:1538-7445.AM2012-3751