Andrea Hägebarth

Identification of KCa3.1 Channel as a Novel Regulator of Oxidative Phosphorylation in a Subset of Pancreatic Carcinoma Cell Lines.

Pancreatic ductal adenocarcinoma (PDAC) represents the most common form of pancreatic cancer with rising incidence in developing countries and overall 5-year survival rates of less than 5%. The most frequent mutations in PDAC are gain-of-function mutations in KRAS as well as loss-of-function mutations in p53. Both mutations have severe impacts on the metabolism of tumor cells. Many of these metabolic changes are mediated by transporters or channels that regulate the exchange of metabolites and ions between the intracellular compartment and the tumor microenvironment. In the study presented here, our goal was to identify novel transporters or channels that regulate oxidative phosphorylation (OxPhos) in PDAC in order to characterize novel potential drug targets for the treatment of these cancers. We set up a Seahorse Analyzer XF based siRNA screen and identified previously described as well as novel regulators of OxPhos. The siRNA that resulted in the greatest change in cellular oxygen consumption was targeting the KCNN4 gene, which encodes for the Ca2+-sensitive K+ channel KCa3.1. This channel has not previously been reported to regulate OxPhos. Knock-down experiments as well as the use of a small molecule inhibitor confirmed its role in regulating oxygen consumption, ATP production and cellular proliferation. Furthermore, PDAC cell lines sensitive to KCa3.1 inhibition were shown to express the channel protein in the plasma membrane as well as in the mitochondria. These differences in the localization of KCa3.1 channels as well as differences in the regulation of cellular metabolism might offer opportunities for targeted therapy in subsets of PDAC.

Abstract 772: Anti-tumor efficacy of the selective pan-FGFR Inhibitor BAY 1163877 in preclinical squamous cell carcinoma models of different origin

Squamous cell carcinoma (SCC) is a common subtype of lung cancer and is strongly associated with smoking. In contrast to adenocarcinomas of the lung, SCCs do not significantly harbor epidermal growth factor (EGFR) mutations or ALK, ROS1 or RET translocations, which are therapeutically tractable. SCC of the lung and SCC of the head and neck region and esophagus not only share risk factors, morphologic features and mechanisms of tumorigenesis but also frequent fibroblast growth factor receptor 1 (FGFR1) gene alterations. In addition, we found that FGFR2 and FGFR3 expression were also altered in patient derived models of SCC of various origins. FGFRs may thus represent viable therapeutic targets for the treatment of SCC. BAY 1163877, an orally available and potent inhibitor of FGFR1, FGFR2 and FGFR3, inhibited SCC cell proliferation as well as downstream FGFR signaling in vitro. When applied in vivo, BAY 1163877 was able to reduce growth of a FGFR1-overexpressing lung SCC xenograft model, a FGFR1-overexpressing esophageal SCC xenograft model, and a FGFR3-overexpressing head and neck SCC xenograft model, achieving reductions in tumor growth by 67 to 92% as compared to the vehicle control. BAY 1163877 was very well tolerated in all models performed. These data suggest that selective inhibition of FGFR1 to 3 with BAY 1163877 might have therapeutic potential for the treatment of SCC malignancies. A phase I clinical trial (NCT01976741) to determine the safety, tolerability and recommended Phase 2 dose in advanced cancer patients is ongoing. Citation Format: Melanie Heroult, Matthias Ocker, Charlotte Kopitz, Dieter Zopf, Andrea Hagebarth, Karl Ziegelbauer, Stuart Ince, Peter Ellinghaus. Anti-tumor efficacy of the selective pan-FGFR Inhibitor BAY 1163877 in preclinical squamous cell carcinoma models of different origin. [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 772. doi:10.1158/1538-7445.AM2015-772

Abstract 665: Inhibition of mitochondrial complex I enhances the therapeutic benefit of BRAF inhibition in mutant melanoma cell lines

Metabolic reprogramming is considered to be one of the major hallmarks of cancer. Selective inhibitors of mutant BRAFV600E, are a widely used targeted therapy for melanoma. Unfortunately, acquired drug resistance, which commonly occurs, represents a major limitation to clinical efficacy. Recent studies have highlighted the role of BRAF inhibition in promoting oxidative phosphorylation (OXPHOS), over glycolysis, in BRAFV600E melanomas, a switch commonly seen in conjunction with development of drug resistance. This metabolic switch occurs at the transcriptional level, indicated by the up-regulation of PGC1α through the lineage-specific transcription factor MITF. Based on these data, we propose that increased OXPHOS is required for melanoma cells to acquire resistance against the BRAF inhibition, which supports our investigation of the therapeutic benefit of combining BRAF and OXPHOS inhibitors. For the latter, respiratory Complex I, an essential enzyme in the mitochondrial respiratory chain, is a logical target for OXPHOS inhibition. We investigated the molecular, metabolic and bioenergetic effects of a selective mutant BRAF inhibitor (Vemurafenib/PLX4032) and BAY 87-2243, a small molecule inhibiting respiratory Complex I, in vitro using various BRAF-mutant melanoma cell lines. First, our study revealed that BRAF inhibition increased the expression of mitochondrial genes and mitochondrial respiration, which is associated with elevated mitochondrial oxidative stress, independent of the expression of PGC1α. Furthermore, BAY 87-2243 inhibited OXPHOS, down-regulated the expression of mitochondrial genes and increased reactive oxygen species (ROS) production. We also demonstrated that inhibition of BRAF increased the OXPHOS phenotype and therefore enhanced the effect of BAY 87-2243 on mitochondrial metabolism; the combination of both inhibitors showed synergistic effects in vitro.Interestingly, long term BRAF inhibition resulted in BRAFV600E melanoma cells to become resistant to BAY 87-2243. Preliminary studies suggest that melanoma cells pretreated with a BRAF inhibitor, with higher rates of mitochondrial respiration and elevated ROS levels, are prone to cell death when targeted by pro-oxidants. Further studies are currently ongoing to understand this resistance phenotype as well as the underlying mechanism which drives the synergistic drug effect. Citation Format: Laura Schoeckel, Katharina Bitschar, Melanie Heroult, Charlotte Kopitz, Andrea Hagebarth. Inhibition of mitochondrial complex I enhances the therapeutic benefit of BRAF inhibition in mutant melanoma cell lines. [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 665. doi:10.1158/1538-7445.AM2015-665

Abstract 3383: Inhibition of Akt signaling: a strategy to overcome platinum resistance in ovarian cancer.

Targeted therapies in combination with cytotoxic agents are promising future treatment regimens for women with resistant ovarian cancer. Current standard treatment of serous ovarian cancer is a platinum- (carboplatin) based chemotherapy, usually combined with taxanes. A 90% cure rate is observed in early stage ovarian cancers, however, in advanced stages, about 20-30% of patients never have a remission or will suffer from recurrent disease within 5 years. One major reason is the development of a resistance against the platinum-based chemotherapy. In order to identify possible novel therapeutic targets for platinum-resistant ovarian cancer, we compared gene expression and oncogenic kinase phosphorylation patterns in the human ovarian cancer cell line A2780 and its platinum-resistant variant A2780cis in vitro and in vivo.In vivo, the untreated A2780cis-derived tumor model showed reduced proliferation activity and enhanced apoptosis, while carboplatin treatment failed to induce tumor reduction, as compared to the sensitive A2780-derived tumor model. Analysis of 47 kinase phosphorylation sites in carboplatin-treated A2780 and A2780cis derived tumors revealed that Akt was hyperphosphorylated at Ser273 but not altered during treatment in resistant tumors, whereas Akt phosphorylation was weak and further reduced during treatment in sensitive tumors. This was associated with an unchanged p53 phosphorylation status in resistant tumors during treatment. In line with this, the novel allosteric Akt inhibitor BAY 10001931 resensitized A2780 and A2780cis cells to carboplatin in vitro. To investigate if the Akt phosphorylation status correlated with an activation of downstream targets, RNA of A2780 and A2780cis vehicle- or carboplatin treated tumors was hybridized onto an Illumina Bead Chip array. Here, linear model analysis identified GDF15 as one of four p53-induced target genes possibly involved in the development of chemoresistance. GDF15 protein expression was highly induced during carboplatin treatment in both cell lines, whereas basal GDF15 expression was higher in resistant cells than in sensitive A2780 cells in vitro and in vivo. This was correlated with a higher secretion of GDF15 into the serum of A2780cis-tumor-bearing mice. Similar tendencies were observed in vitro. Interestingly, simultaneous Akt inhibition with BAY 10001931 markedly reduced the carboplatin-induced upregulation of GDF15 in A2780 and A2780cis cells. Additionally, shRNA-mediated knockdown of GDF15 in resistant A2780cis cells also abrogated the phosphorylation of Akt in vitro. These results indicate that a causal relationship exists between Akt activation and GDF15 expression in resistant ovarian cancer cells. Together, a combination treatment with an Akt inhibitor and carboplatin of GDF15-expressing ovarian cancer cells might represent a novel approach to treat resistant ovarian cancer. Citation Format: Julia C. Meier, Bernard Haendler, Anette Sommer, Andrea Hagebarth, Georg Beckmann, Bertolt Kreft, Karl Ziegelbauer, Charlotte C. Kopitz. Inhibition of Akt signaling: a strategy to overcome platinum resistance in ovarian 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 3383. doi:10.1158/1538-7445.AM2013-3383