Ulrike Rennefahrt

An Isoform-Selective, Small-Molecule Inhibitor Targets the Autoregulatory Mechanism of p21-Activated Kinase

Autoregulatory domains found within kinases may provide more unique targets for chemical inhibitors than the conserved ATP-binding pocket targeted by most inhibitors. The kinase Pak1 contains an autoinhibitory domain that suppresses the catalytic activity of its kinase domain. Pak1 activators relieve this autoinhibition and initiate conformational rearrangements and autophosphorylation events leading to kinase activation. We developed a screen for allosteric inhibitors targeting Pak1 activation and identified the inhibitor IPA-3. Remarkably, preactivated Pak1 is resistant to IPA-3. IPA-3 also inhibits activation of related Pak isoforms regulated by autoinhibition, but not more distantly related Paks, nor >200 other kinases tested. Pak1 inhibition by IPA-3 in live cells supports a critical role for Pak in PDGF-stimulated Erk activation. These studies illustrate an alternative strategy for kinase inhibition and introduce a highly selective, cell-permeable chemical inhibitor of Pak.

Specificity profiling of Pak kinases allows identification of novel phosphorylation sites

The p21-activated kinases (Paks) serve as effectors of the Rho family GTPases Rac and Cdc42. The six human Paks are divided into two groups based on sequence similarity. Group I Paks (Pak1 to -3) phosphorylate a number of substrates linking this group to regulation of the cytoskeleton and both proliferative and anti-apoptotic signaling. Group II Paks (Pak4 to -6) are thought to play distinct functional roles, yet their few known substrates are also targeted by Group I Paks. To determine if the two groups recognize distinct target sequences, we used a degenerate peptide library method to comprehensively characterize the consensus phosphorylation motifs of Group I and II Paks. We find that Pak1 and Pak2 exhibit virtually identical substrate specificity that is distinct from that of Pak4. Based on structural comparisons and mutagenesis, we identified two key amino acid residues that mediate the distinct specificities of Group I and II Paks and suggest a structural basis for these differences. These results implicate, for the first time, residues from the small lobe of a kinase in substrate selectivity. Finally, we utilized the Pak1 consensus motif to predict a novel Pak1 phosphorylation site in Pix (Pak-interactive exchange factor) and demonstrate that Pak1 phosphorylates this site both in vitro and in cultured cells. Collectively, these results elucidate the specificity of Pak kinases and illustrate a general method for the identification of novel sites phosphorylated by Paks.

Constitutive JNK Activation in NIH 3T3 Fibroblasts Induces a Partially Transformed Phenotype

The c-Jun N-terminal kinases (JNKs) (also known as stress-activated protein kinases or SAPKs), members of the mitogen-activated protein kinase (MAPK) family, regulate gene expression in response to a variety of physiological and unphysiological stimuli. Gene knockout experiments and the use of dominant interfering mutants have pointed to a role for JNKs in the processes of cell differentiation and survival as well as oncogenic transformation. Direct analysis of the transforming potential of JNKs has been hampered so far by the lack of constitutively active forms of these kinases. Recently, such mutants have become available by fusion of the MAPK with its direct upstream activator kinase. We have generated a constitutively active SAPKβ-MKK7 hybrid protein and, using this constitutively active kinase, we are able to demonstrate the transforming potential of activated JNK, which is weaker than that of classical oncogenes such as Ras or Raf. The inducible expression of SAPKβ-MKK7 caused morphological transformation of NIH 3T3 fibroblasts. Additionally, these cells formed small foci of transformed cells and grew anchorage-independent in soft agar. Furthermore, similar to oncogenic Ras and Raf, the expression of activated SAPKβ resulted in the disassembly of F-actin stress fibers. Our data suggest that constitutive JNK activation elicits major aspects of cellular transformation but is unable to induce the complete set of changes which are required to establish the fully transformed phenotype.

Identification of Novel in Vivo Phosphorylation Sites of the Human Proapoptotic Protein BAD PORE-FORMING ACTIVITY OF BAD IS REGULATED BY PHOSPHORYLATION

BAD is a proapoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Although much attention has been devoted to the identification of phosphorylation sites in murine BAD, little data are available with respect to phosphorylation of human BAD protein. Using mass spectrometry, we identified here besides the established phosphorylation sites at serines 75, 99, and 118 several novel in vivo phosphorylation sites within human BAD (serines 25, 32/34, 97, and 124). Furthermore, we investigated the quantitative contribution of BAD targeting kinases in phosphorylating serine residues 75, 99, and 118. Our results indicate that RAF kinases represent, besides protein kinase A, PAK, and Akt/protein kinase B, in vivo BAD-phosphorylating kinases. RAF-induced phosphorylation of BAD was reduced to control levels using the RAF inhibitor BAY 43-9006. This phosphorylation was not prevented by MEK inhibitors. Consistently, expression of constitutively active RAF suppressed apoptosis induced by BAD and the inhibition of colony formation caused by BAD could be prevented by RAF. In addition, using the surface plasmon resonance technique, we analyzed the direct consequences of BAD phosphorylation by RAF with respect to association with 14-3-3 and Bcl-2/Bcl-XL proteins. Phosphorylation of BAD by active RAF promotes 14-3-3 protein association, in which the phosphoserine 99 represented the major binding site. Finally, we show here that BAD forms channels in planar bilayer membranes in vitro. This pore-forming capacity was dependent on phosphorylation status and interaction with 14-3-3 proteins. Collectively, our findings provide new insights into the regulation of BAD function by phosphorylation.

Validation of a metabolite panel for early diagnosis of type 2 diabetes.

BACKGROUND Accurate, early diagnosis of type 2 diabetes (T2D) would enable more effective clinical management and a reduction in T2D complications. Therefore, we sought to identify plasma metabolite and protein biomarkers that, in combination with glucose, can better predict future T2D compared with glucose alone. METHODS In this case-control study, we used plasma samples from the Bavarian Red Cross Blood Transfusion Center study (61 T2D cases and 78 non-diabetic controls) for discovering T2D-associated metabolites, and plasma samples from the Personalized Medicine Research Project in Wisconsin (56 T2D cases and 445 non-diabetic controls) for validation. All samples were obtained before or at T2D diagnosis. We tested whether the T2D-associated metabolites could distinguish incident T2D cases from controls, as measured by the area under the receiver operating characteristic curve (AUC). Additionally, we tested six metabolic/pro-inflammatory proteins for their potential to augment the ability of the metabolites to distinguish cases from controls. RESULTS A panel of 10 metabolites discriminated better between T2D cases and controls than glucose alone (AUCs: 0.90 vs 0.87; p=2.08×10(-5)) in Bavarian samples, and associations between these metabolites and T2D were confirmed in Wisconsin samples. With use of either a Bayesian network classifier or ridge logistic regression, the metabolites, with or without the proteins, discriminated incident T2D cases from controls marginally better than glucose in the Wisconsin samples, although the difference in AUCs was not statistically significant. However, when the metabolites and proteins were added to two previously reported T2D prediction models, the AUCs were higher than those of each prediction model alone (AUCs: 0.92 vs 0.87; p=3.96×10(-2) and AUCs: 0.91 vs 0.71; p=1.03×10(-5), for each model, respectively). CONCLUSIONS Compared with glucose alone or with previously described T2D prediction models, a panel of plasma biomarkers showed promise for improved discrimination of incident T2D, but more investigation is needed to develop an early diagnostic marker.

Metabolite Profiling Reveals the Glutathione Biosynthetic Pathway as a Therapeutic Target in Triple-Negative Breast Cancer

Cancer cells can exhibit altered dependency on specific metabolic pathways and targeting these dependencies is a promising therapeutic strategy. Triple-negative breast cancer (TNBC) is an aggressive and genomically heterogeneous subset of breast cancer that is resistant to existing targeted therapies. To identify metabolic pathway dependencies in TNBC, we first conducted mass spectrometry–based metabolomics of TNBC and control cells. Relative levels of intracellular metabolites distinguished TNBC from nontransformed breast epithelia and revealed two metabolic subtypes within TNBC that correlate with markers of basal-like versus non-basal–like status. Among the distinguishing metabolites, levels of the cellular redox buffer glutathione were lower in TNBC cell lines compared to controls and markedly lower in non-basal–like TNBC. Significantly, these cell lines showed enhanced sensitivity to pharmacologic inhibition of glutathione biosynthesis that was rescued by N-acetylcysteine, demonstrating a dependence on glutathione production to suppress ROS and support tumor cell survival. Consistent with this, patients whose tumors express elevated levels of γ-glutamylcysteine ligase, the rate-limiting enzyme in glutathione biosynthesis, had significantly poorer survival. We find, further, that agents that limit the availability of glutathione precursors enhance both glutathione depletion and TNBC cell killing by γ-glutamylcysteine ligase inhibitors in vitro. Importantly, we demonstrate the ability to this approach to suppress glutathione levels and TNBC xenograft growth in vivo. Overall, these findings support the potential of targeting the glutathione biosynthetic pathway as a therapeutic strategy in TNBC and identify the non-basal-like subset as most likely to respond. Mol Cancer Ther; 17(1); 264–75. ©2017 AACR.

Abstract 1415: Prostate cancer: An integrated evaluation of metabolomics, transcriptomics, and proteomics expression data

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background Metabolite profiling research offers a deeper insight into biochemical changes in cancer metabolism. Moreover the integrated analysis of transcription, metabolomics and proteomics data can improve the understanding of the underlying biological processes.Material and Methods A set of 254 metabolites was determined by gas chromatography/liquid chromatography-mass spectrometry in matched malignant and non-malignant prostatectomy samples from 95 prostate cancer (PCa) patients. Transcription profiling data obtained from 15 PCa patients by means of Affymetrix U133 arrays was analysed together with public GEO expression data. Expression levels of selected proteins were determined by means of immunohistochemistry and tissue micro array technology in 41 matched frozen tissue samples. The association with clinicopathological variables and clinical outcome was tested. Transcription and metabolomics data were statistically analysed (ANOVA, Mann-Whitney U test) and significant differentially regulated metabolites/genes/proteins were selected.Results Differentially regulated metabolites/genes discrimination between malignant and non-malignant tissues was used for network analysis. Enriched pathways which are involved in PCa progression or recurrence such as carbohydrate and fatty acid metabolism were identified. The role of fatty acid metabolism in PCa was analysed in more detail. Several fatty acids such as cerebronic acid, 2-hydroxybehenic acid, tricosanoic acid showed higher concentrations in malignant than in non-malignant tissues. This finding is in concordance to the observed higher mRNA and protein expression level of fatty acid synthase (FASN) in PCa. In contrast to normal prostate tissue, where protein expression level of FASN was correlated to the level of measured metabolites we found in malignant tissues a deregulation of the corresponding pathway. Conclusion Our integrated analysis of transcription, metabolite and proteomics data confirms and extends the role of several biological pathways which are involved in PCa progression Citation Format: Ulrike Rennefahrt, Hellmuth-A. Meyer, Beate Kamlage, Regina Reszka, Philipp Schatz, Carsten Stephan, Klaus Jung, Dimo Dietrich, Glen Kristiansen. Prostate cancer: An integrated evaluation of metabolomics, transcriptomics, and proteomics expression data. [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 1415. doi:10.1158/1538-7445.AM2014-1415

Abstract A73: Metabolite profiling reveals the glutathione biosynthetic pathway as a therapeutic target in triple negative breast cancers

Identifying metabolic pathway alterations that are critical to support cancer growth is a key hurdle for developing therapeutic strategies that exploit these pathways. We have applied metabolomic and pharmacological approaches to identify targetable pathways in triple negative breast cancer (TNBC). TNBC is an aggressive and genomically heterogeneous subset of breast cancer that is resistant to existing targeted therapies. To identify dysregulated metabolic pathways in TNBC, we conducted mass spectrometry-based metabolomics of TNBC and control cells. The relative steady-state levels of 155 intracellular metabolites distinguished TNBC from non-transformed breast epithelia, and revealed two metabolic subtypes within TNBC that, unexpectedly, correlate with markers of basal-like versus non-basal-like status. Distinguishing metabolites included amino acids, lipids and the cellular redox buffer glutathione. Levels of glutathione were generally lower in TNBC cell lines compared to controls, and markedly lower in the metabolic subtype containing non-basal-like TNBC. Further, these cell lines showed enhanced sensitivity to inhibition of glutathione biosynthesis, demonstrating a dependence on glutathione production for survival. These findings demonstrate the potential of targeting the glutathione biosynthetic pathway as a therapeutic strategy in TNBC, and suggest that existing clinical biomarkers may provide a means for stratifying TNBC tumors to identify likely responders to anti-glutathione therapy. Note: This abstract was not presented at the conference. Citation Format: Alexander Beatty, Lauren Fink, Alexander Strigun, Ulrike Rennefahrt, Erik Peter, Regina Reszka, Hajo Schiewe, Jeffrey R. Peterson. Metabolite profiling reveals the glutathione biosynthetic pathway as a therapeutic target in triple negative breast cancers. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A73.

Abstract 1209: Dissecting by 13C tracers the utilization of glucose and glutamine for the synthesis of fatty acids in triple negative breast cancer cells

Triple negative breast cancer (TNBC) is an aggressive cancer, which is resistant to the majority of available chemotherapies. We have recently identified significant differences in the levels of membrane lipids and fatty acids between two distinct TNCB metabolic subtypes (MST2 and MST3) (Beatty et al., 2014, manuscript submitted). Fatty acid synthesis has recently been discussed as potential pharmaceutical target. In order to evaluate whether glucose or glutamine is the preferred carbon source for palmitic acid synthesis, TNBC cell lines (BT549 and HCC1806) were fed with fully labelled glucose and fully labelled glutamine ([U-13C6]glucose and [U-13C5]glutamine) in independent experiments. Fully labelled glutamine allows conclusions on total usage of glutamine via the reductive and the oxidative part of the TCA-cycle. In order to assess the contribution of glutamine to palmitic acid synthesis via the reductive pathway we fed the subtypes with single labelled glutamine [5-13C1]glutamine. Enrichment of 13C in palmitic acid from all labelling experiments was measured via GC-MS (gas chromatography coupled mass spectrometry). Feeding the TNBC subtypes with [U-13C6]glucose and [U-13C5]glutamine resulted in a higher enrichment of 13C in palmitic acid derived from glucose as compared to glutamine in both subtypes, indicating that glucose is the main carbon source at the tested conditions. Feeding the cells with [5-13C1]glutamine also resulted in an enrichment of 13C in palmitic acid in both subtypes, which indicates that reductive glutamine metabolism contributes to palmitic acid synthesis in both subtypes. Based on the 13C-palmitic acid data from both glutamine isotopes we found that palmitic acid synthesis preferably occurs via the reductive path in MST3 (61% of total glutamine use) while the oxidative path seems the preferred route (34% of total glutamine use) in MST2. The presented data indicate that glucose is the main carbon source for fatty acid synthesis in both MSTs, suggesting a higher sensitivity towards pharmacological targeting of glucose metabolism. Despite of the lower dependency on glutamine, TNCBs of the MST3 subtype seem more sensitive towards targeting the reductive glutamine metabolism. Citation Format: Alexander Strigun, Regina Reszka, Hans-Joerg Schiewe, Jean-Philippe Laine, Ulrike Rennefahrt. Dissecting by 13C tracers the utilization of glucose and glutamine for the synthesis of fatty acids in triple negative breast cancer cells. [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 1209. doi:10.1158/1538-7445.AM2015-1209

Abstract 4333: Metabolite profiling reveals druggable metabolic distinctions between basal-like and non-basal-like triple-negative breast cancers

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer that represents about 15-20% of all breast cancers. Because TNBC tumors do not express the estrogen or progesterone receptor and lack HER2 amplification, the disease is not responsive to current targeted therapies. The development of therapeutic approaches specific for TNBC is hindered by genetic heterogeneity, and significant efforts are being made to subtype the disease. To this end, we performed metabolite profiling (metabolomics) to characterize metabolic fingerprints within TNBC in order to define metabolic subtypes, and identify molecular drivers for the development of targeted therapies. We profiled twelve well-characterized TNBC-derived cell lines as well as a non-transformed, immortalized breast cell line and two primary human mammary epithelial cell lines. Those cancer cell lines recapitulate all 7 genetic subtypes of TNBC which were proposed recently based on mRNA gene expression profiles (1). Our approaches used and data generated have implications for drug target discovery. Hierarchical clustering based on high quality intracellular metabolites clearly and reproducibly segregated the TNBC cell lines from the non-transformed lines. Alterations in energy utilization, lipid metabolism, and other pathways of importance to highly proliferative cells differed significantly from the control cell line MCF-10A. In addition, TNBC cell lines segregated into two discrete groups, suggesting the existence of two major metabolic subtypes of TNBC, which correlated with basal-like vs. non-basal-like gene expression. Metabolites like glutamate and glutamine, serine, glycine, trans-4-hydroxyproline, 5-oxoproline, several complex lipids (phosphatidylcholines and sphingomyelins), myo-inositol, polyamines spermidine and putrescine represented metabolites differing significantly between TNBC metabolic subtypes. Ongoing studies are evaluating whether these differences represent dependencies with therapeutic relevance. Metabolite profiling was also used to identify potential metabolic liabilities generated by treatment with clinical kinase inhibitors. Response of the metabolome to treatment with rapamycin, sorafenib, imatinib, and lapatinib in four genetically diverse TNBC cell lines and MCF-10A control cell lines revealed specific drug-induced metabolic alterations. Co-targeting kinases and metabolic targets may offer an approach to synthetic lethality with a reduced likelihood for the development of drug resistance. (1) (Lehmann BD, Bauer JA, Chen X, Sanders ME, Chakravarthy AB, Shyr Y, et al. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest. 2011;121) Citation Format: Alexander Beatty, Lauren Fink, Ulrike Rennefahrt, Alexander Strigun, Erik Peter, Hajo Schiewe, Regina Reszka, Jeffrey R. Peterson. Metabolite profiling reveals druggable metabolic distinctions between basal-like and non-basal-like triple-negative breast 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 4333. doi:10.1158/1538-7445.AM2014-4333