Florian Puehler

Abstract 341: Preclinical mode of action and anti-tumor efficacy of the selective MKNK1 inhibitor BAY 1143269 in NSCLC models

MKNK1 (MAP kinase-interacting serine/threonine-protein kinase, also known as Mnk1) is activated by the mitogen-activated protein kinases ERK1/2 and p38. Thus, MKNK1 signaling is involved in the cellular response to environmental stress factors and cytokines. Of particular interest, MKNK1 kinase regulates mRNA translation by phosphorylating the translation initiation factor eIF4E (eukaryotic translation initiation factor 4E), known to be critical for malignant transformation but shown to be dispensable for translation in normal cells. Phosphorylated eIF4E levels were found to be elevated in several cancer tissues, including lung cancer. MKNK1 is also involved in resistance mechanisms to cancer therapeutics. Thus, the inhibition of MKNK1 activity may provide an innovative approach for anti-cancer therapy, and in particular for lung cancer, the main cancer-related cause of death worldwide. BAY 1143269 is a potent and selective MKNK1 inhibitor and inhibits eIF4E phosphorylation and reduces MKNK1-regulated translational downstream targets in non-small cell lung cancer (NSCLC) cell lines. In this study, BAY 1143269-mediated effects on molecular mechanisms in lung cancer models were analyzed. Epithelial-mesenchymal transition (EMT) is associated with the pathogenesis of numerous lung diseases including cancer progression, metastasis and resistance. BAY 1143269 reduced expression of EMT key regulators like Snail1 and cellular junction components, as well as reduced TGFβ1-induced EMT. Accumulating evidence suggests a role for proinflammatory cytokines in the development and progression of cancer; increased serum concentrations of cytokines like interleukin 6 (IL-6) are associated with diminished lung cancer survival rates. BAY 1143269 reduced the secretion of several proinflammatory cytokines, including TNFα and IL-6 in whole blood, and affected IFN-stimulated gene expression in cell lines. Consistent with the observed effects in vitro, BAY 1143269 showed significant anti-tumor effects in vivo in cell line as well as patient derived NSCLC xenograft models in monotherapy. In combination with chemotherapeutics approved for treatment of NSCLC, BAY 1143269 improved anti-tumor effects in comparison to chemotherapy alone. In conclusion, BAY 1143269 has the potential to provide therapeutic benefit in NSCLC. A phase I study of BAY 1143269 in combination with docetaxel for subjects with advance solid tumors is ongoing (NCT02439346). Citation Format: Susann Santag, Franziska Siegel, Antje M. Wengner, Claudia Lange, Ulf Boemer, Knut Eis, Florian Puehler, Martin Michels, Franz von Nussbaum, Karl Ziegelbauer, Dominik Mumberg, Kirstin Petersen. Preclinical mode of action and anti-tumor efficacy of the selective MKNK1 inhibitor BAY 1143269 in NSCLC models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 341.

Abstract 2604: Preclinical anti-tumor efficacy and mode of action of a novel, orally available, selective MKNK1 inhibitor [BAY 1143269]

MKNK1 (MAP kinase-interacting serine/threonine-protein kinase, also known as Mnk1) is activated by the mitogen-activated protein kinases ERK1/2 and p38. Thus, MKNK1 signaling is involved in the cellular response to environmental stress factors and cytokines. Of particular interest, MKNK1 kinase was shown to regulate mRNA translation by phosphorylating the translation initiation factor eIF4E (eukaryotic translation initiation factor 4E), known to be critical for malignant transformation but dispensable for translation in normal cells. In addition, MKNK is involved in resistance mechanisms to cancer therapeutics. Thus, the inhibition of MKNK1 activity may provide an innovative approach for anti-cancer therapy. BAY 1143269 was identified as a potent and selective inhibitor of MKNK1 activity with an unprecedented mode of action. It inhibits the phosphorylation of eIF4E in various cancer cell lines in vitro and leads to reduced expression of MKNK-regulated translational downstream targets, including survivin, Cdc25C and cyclin B1. In addition, BAY 1143269 potently inhibits cytokine release in LPS stimulated human blood. In vivo, BAY 1143269 shows a significant monotherapy efficacy in non-small cell lung cancer (NSCLC), colorectal cancer and melanoma xenograft models after an once daily, oral application of 200 mg/kg in mice and 70 mg/kg in rats. Furthermore, combination treatment with chemotherapy and BAY 1143269 gives additive efficacy in several NSCLC cell lines and patient-derived xenograft models leading to partial response (Lu7558, A549), stable disease (Lu7187, Lu7166) or significant delay in tumor re-growth versus chemotherapy alone after stop of treatment (Lu7558). In summary, BAY 1143269 is a selective, orally available MKNK1 inhibitor that demonstrates preclinical in vivo efficacy in mono- and combination therapy and may provide therapeutic benefit for patients with solid tumors. Citation Format: Susann Santag, Franziska Siegel, Antje Margret Wegner, Claudia Schneider, Ulf Boemer, Knut Eis, Florian Puehler, Martin Michels, Franz von Nussbaum, Karl Ziegelbauer, Dominik Mumberg, Kirstin Petersen. Preclinical anti-tumor efficacy and mode of action of a novel, orally available, selective MKNK1 inhibitor [BAY 1143269]. [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 2604. doi:10.1158/1538-7445.AM2015-2604

Abstract B42: RAS mutations detected by cell-free plasma DNA (BEAMing) assay may portend a favorable response to refametinib +/- sorafenib in hepatocellular carcinoma

Background: Per COSMIC database, RAS mutations are uncommon events in hepatocellular carcinoma (HCC). Refametinib (BAY 86-9766) is a potent (IC50=17-60 nM) allosteric dual MEK 1/2 inhibitor, which exhibits high selectivity for MEK as compared to other kinases as well as strong preclinical synergy in combination with sorafenib, suggesting it may have potential utility in treating HCC patients. As part of a phase II study that evaluated the efficacy and safety of refametinib plus sorafenib in Asian patients with HCC, a biomarker analysis was performed using plasma DNA to investigate a possible correlation between mutational status and clinical outcome. Methods: Refametinib was tested in a kinase panel of 205 enzymes at 10 μM and in a variety of cancer cell lines for antiproliferative effects. In vivo refametinib was tested for tumor growth inhibition in monotherapy or in combination with sorafenib mouse xenograft HCC models. Mutational analysis of clinical plasma specimens was performed by Inostics GmbH (Hamburg, Germany) using BEAMing (Beads, Emulsions, Amplification, and Magnetics) technology on DNA isolated from plasma samples collected at baseline. Mutational status was correlated with clinical outcome using descriptive analyses. Plasma from 69 patients was evaluated for the following mutations: KRAS (G12A, C, D, R, S, V; G13D; Q61H; A146T); NRAS (Q61H, K, L, R) and BRAF (V600E). Results: Based on it allosteric binding mode refametinib shows more than 100-fold selectivity for MEK. There was strong antiproliferative activity of BAY 86-9766 in HCC cell lines being most active in an NRAS amplified model (HepG2). In the orthotopic HBV-driven human Hep3B xenograft model, BAY 86-9766 (25 mg/kg) monotherapy was more effective than sorafenib at its maximally tolerated dose (30 mg/kg) in prolonging survival and exhibited strong synergism in improving this endpoint when combined with sorafenib. Based on the BEAMing data, the frequency of HCC patients with mutant RAS identified in our study (5.8%) was similar to the frequency of RAS mutations reported in HCC patients (5%) (COSMIC). A RAS mutation was identified in 4 patients, 3 of whom were still receiving study treatment at the cut-off date used for the final data analysis. These 3 patients had achieved confirmed PR, with duration of responses ranging from 128 to 382 days. The fourth patient with a RAS mutation discontinued study treatment after 41 days on therapy due to PD. No mutations in BRAF were identified in these 69 samples. Conclusions: In this exploratory retrospective study, HCC patients with mutant RAS exhibited a particularly robust clinical response to refametinib plus sorafenib compared to patients with wild-type RAS. Further investigation is required to assess the clinical activity of this drug combination in HCC patients with mutant RAS. Accordingly, we have initiated two single-arm phase 2 studies in patients with prospectively identified, BEAMing-confirmed RAS-mutated HCC; sorafenib and refametinib in first-line (NCT01915602), and refametinib as first- or second-line monotherapy (NCT01915589). Citation Format: Ho Yeong Lim, Heiko Krissel, Michael Teufel, Florian Puehler, Joachim Reischl, Frank Diehl, Barrett H. Childs, Josep M. Llovet. RAS mutations detected by cell-free plasma DNA (BEAMing) assay may portend a favorable response to refametinib +/- sorafenib in hepatocellular carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B42. doi: 10.1158/1557-3125.RASONC14-B42

Abstract 1052: Refametinib (BAY 86-9766): An allosteric inhibitor of MEK1/2, potently inhibits tumor growth in patient-derived xenograft models of gastric cancer.

Background: Gastric cancer (GC) is a prevalent and deadly disease. Because the Ras/Raf/MEK/ERK signaling cascade plays a critical role in signal transduction pathways that regulate growth factor response, angiogenesis, tumor cell proliferation, and apoptosis, blocking this signaling pathway could thus have therapeutic efficacy. This study aims to target this pathway in preclinical models of GC with refametinib (BAY 86-9766, RDEA119), an orally available, allosteric inhibitor of MEK1/2. Methods: Eight patient-derived GC xenograft models with different histotypes were treated orally with refametinib or vehicle and tumor growth was monitored using caliper measurements. Tissue sections of vehicle- or refametinib-treated xenografts were analyzed by immunohistochemistry for cell proliferation, apoptosis and microvessel area, using antibodies against p-Histone H3Ser10, cleaved PARP and CD31, respectively. Tumor lysates were analyzed by Western Blotting with antibodies for effects on marker proteins for proliferation, apoptosis and angiogenesis.Results: Refametinib demonstrated significant tumor growth inhibition ranging from 66-87% in eight GC models at a dose of 15 mg/kg/d. Refametinib inhibited cell proliferation by 2-4 fold as observed by reduced staining for p-Histone H3Ser10. Increased staining for caspase-cleaved p85 PARP by 1.5 to 9 fold was detected, indicating that refametinib induced apoptosis. Furthermore the compound significantly inhibited angiogenesis in 2 of 8 models by 3-fold compared to the vehicle-treated group as indicated by reduced CD31 staining. Reduced cell proliferation and induction of apoptosis were associated with decreased levels of p-ERK1/2, p-cdk2Thr14/Tyr15, p-p90RSKSer359/363, p-p70S6KThr421/Ser424, p-4EBP1Thr70, p-S6RSer235/236 and p-eIF4ESer209 and elevation of p-AktSer473, Bim and cleaved PARP. Conclusion: We show that single-agent refametinib has significant anti-tumor activity in patient-derived gastric cancer preclinical models. Our findings support further investigation of refametinib either in monotherapy or in combination with inhibitors of the PI3K-AKT-mTOR pathway in the treatment of GC. Citation Format: Huynh T. Hung, Richard Ong, Khee Chee Soo, Florian Puehler, Arne Scholz, Dominik Mumberg, Dieter Zopf, Karl Ziegelbauer. Refametinib (BAY 86-9766): An allosteric inhibitor of MEK1/2, potently inhibits tumor growth in patient-derived xenograft models of gastric cancer. [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 1052. doi:10.1158/1538-7445.AM2013-1052

Abstract A44: 18F-FLT, a potential biomarker for predicting the synergistic combination of PI3K inhibitor BAY 80-6946* and MEK inhibitor BAY 86-9766.

Signaling pathways of particular interest in the cancer field are the RAS-RAF-MEK and PI3K-AKT pathways that act as parallel conduits to transduce signals involved in tumor cell proliferation, survival, and cell migration. Compared to targeting either pathway separately, simultaneous inhibition of these two key pathways has been considered to have greater therapeutic efficacy and broader applicability for the treatment of cancers. One of the challenges is the discovery of biomarkers that correlate with efficacy and synergy of PI3K and MEK combination therapy in early-stage clinical trials. To identify the best imaging tracer for monitoring the synergistic anti-tumor activity of BAY 80-6946* and BAY 86-9766, we first compared FDG and FLT uptake in vitro in 2 colorectal (CRC) and 2 non-small cell lung cancer (NSCLC) cell lines bearing either KRAS or KRAS plus PIK3CA mutations. Partial inhibition of FDG- and FLT-uptake ( 94%) was demonstrated in the combination group. Interestingly, the maximum inhibition of FDG-uptake was much weaker compared to FLT-uptake, indicating that FLT might be a better tracer for PI3K and MEK combination therapy. Further investigation iwas conducted in HCT116 CRC xenografts in mice and H460 NSCLC xenografts in rats. BAY 80-6946* was administered intraveneously (Q2D) at 14 mg/kg in mice and at 3 mg/kg in rats whereas BAY 86-9766 was dosed orally (QD) at 25 mg/kg in mice and at 15 mg/kg in rats. Tumor uptake of 18F-FLT was investigated by PET imaging as well as by quantitative biodistribution 1 day before and 1 day after treatment start. In both models, 18F-FLT tumor uptake was not significantly altered in animals treated with each single agent. However, one day after the treatment in the combination group, 18F-FLT tumor uptake was reduced from 8.50±0.99 %ID/g to 3.91±0.66 %ID/g (n=5, vs vehicle, p 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 A44.

Abstract B247: The MEK inhibitor BAY 869766 inhibits tumor growth and metastatic spread, prolongs the survival and acts synergistically with standard of care drugs in models of hepatocellular carcinoma and pancreatic cancer.

Introduction: BAY 869766 (RDEA119) is an orally available, allosteric MEK1/2 inhibitor, targeting the central switch in the MAP kinase pathway. BAY 869766 demonstrates potent in vitro and in vivo activity in different human cancer cell lines and preclinical models. BAY 869766 is currently in clinical phase I/II studies in the indications HCC (hepatocellular carcinoma) and pancreatic cancer (PaCa). In the described preclinical studies BAY 869766 was studied in two orthotopic, syngeneic HCC and PaCa models and five subcutaneous PaCa xenografts as a single agent and combination therapy. In contrast to subcutaneous xenografts, orthotopic syngeneic models provide additional important information about microenvironment-dependent tumor growth, metastatic spread, immune response and survival. Results: To establish a syngeneic, orthotopic HCC model, the rat HCC cell line MH3924a was first evaluated in vitro. BAY 869766 induced antiproliferative effects in MH3924a HCC cells in monotherapy and showed synergistic effects in combination with sorafenib. In the corresponding rat allograft model efficacy of BAY 869766 in mono- and combination therapy with sorafenib was confirmed. Primary tumor growth and metastatic spread were strongly reduced. These effects were accompanied by a reduction of pERK levels, proliferation index and microvessel density. Finally, the median overall survival in combination treatment was increased by 100% in comparison with vehicle treatment. To characterize BAY 869766 in an orthotopic syngenic PaCa model, the Panc02 mouse allograft model was established. BAY 869766 was tested in mono- and combination therapy with Gemcitabine. Especially, combination treatment led to significant lower tumor weight, reduction of metastatic spread and increase of median survival by 135%. To test BAY 869766 in further clinical relevant pancreatic cancer models, five patient-derived pancreatic cancer tumor samples were subcutaneously implanted in mice and subsequently treated with BAY 869766. Significant tumor growth inhibition in these patient-derived pancreatic cancer xenograft models was observed (Treatment/Control ratios from 23,5% − 44%). Conclusion: These preclinical results support the clinical development of the MEK inhibitor BAY 869766 in HCC and pancreatic cancer. Inhibition of Ras-Raf-MEK-Erk-signaling, direct antiproliferative effects on tumor cells, reduction of metastatic spread and anti-angiogenic properties were demonstrated in these studies. In combination with the respective SoC drugs a significant benefit in survival could be achieved in the orthotopic models. 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 B247.

Abstract B3: Allosteric MEK inhibitor BAY 86-9766 acts synergistically with sorafenib to inhibit tumor growth, angiogenesis, and tumor cell survival in ectopic patient-derived xenograft models of human hepatocellular carcinoma.

Background: Hepatocellular carcinoma (HCC) is a particularly vascularized solid tumor and the Raf/MEK/ERK pathway is activated in HCC. Sorafenib has been shown to prolong survival of patients with advanced HCC. BAY 86-9766 (RDEA119) is an orally available, potent, allosteric MEK inhibitor currently in Phase I/II trials. A combination therapy of MEK inhibitor BAY 86-9766 with sorafenib might enhance the therapeutic efficacy of sorafenib in HCC. This study aims to investigate the potential of such a combined therapeutic approach in preclinical models of HCC. Methods: We treated 15 patient-derived HCC xenograft models (Asian origin) with i) sorafenib, ii) BAY 86-9766, and iii) sorafenib plus BAY 86-9766. Western blotting was employed to determine pharmacodynamic changes in biomarkers relevant to both angiogenesis and MEK signaling. Apoptosis, microvessel density, and cell proliferation were analyzed by immunohistochemistry. Results: We report here that sorafenib significantly suppressed tumor growth with the T/C ratios ranging from 0.25 to 0.6, angiogenesis and cell proliferation with modest elevation of pERK Thr202/Tyr204. BAY 86-9766 treatment caused a modest tumor growth inhibition (ranging from 30 to 60%) and dephosphorylation of ERK, Bim and p90RSK. The addition of BAY 86-9766 to sorafenib significantly augmented the antitumor activity of sorafenib in 15 models of HCC. The T/C ratios for the sorafenib/BAY 86-9766 ranged from 0.03 to 0.2. Such inhibition led to further increase in Bad, Bim and apoptosis. Profound inhibition of cell proliferation, angiogenesis and inactivation of p90RSK and mTOR targets were also observed in the combined therapy. Conclusion: We show that sorafenib/BAY 86-9766 treatment is considerably more effective than either single agents in suppressing tumor growth, angiogenesis, cell proliferation and cell survival. Our findings underscore the potential of sorafenib/BAY 86-9766 combination in the treatment of HCC. 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 B3.