Rolf-Peter Ryseck

Multiorgan inflammation and hematopoietic abnormalities in mice with a targeted disruption of RelB, a member of the NF-κB/Rel family

RelB, a member of the NF-kappa B/Rel family of transcription factors, has been implicated in the constitutive expression of kappa B-regulated genes in lymphoid tissues. We have generated mice carrying a germline mutation of the relB gene, resulting in the absence of RelB protein and a dramatic reduction of constitutive kappa B-binding activity in thymus and spleen. Mice homozygous for the disrupted relB locus had phenotypic abnormalities including multifocal, mixed inflammatory cell infiltration in several organs, myeloid hyperplasia, splenomegaly due to extramedullary hematopoiesis, and a reduced population of thymic dendritic cells. RelB-deficient animals also had an impaired cellular immunity, as observed in contact sensitivity experiments. Thus, RelB plays a decisive role in the hematopoietic system, and its absence cannot be functionally compensated by any other member of the NF-kappa B/Rel family.

Synergistic Antitumor Activity of Ixabepilone (BMS-247550) Plus Bevacizumab in Multiple In vivo Tumor Models

Purpose: Angiogenesis is a critical step in the establishment, growth, and metastasis of solid tumors, and combination of antiangiogenic agents with chemotherapy is an attractive therapeutic option. We investigated the potential of ixabepilone, the first in a new class of antineoplastic agents known as epothilones, to synergize with antiangiogenic agents to inhibit tumor growth. Experimental Design:In vitro and in vivo cytotoxicity of ixabepilone as single agent and in combination with two targeted antiangiogenic agents, bevacizumab or sunitinib, were examined in preclinical tumor models. Direct effects of the agents against endothelial cells was also examined and compared with the effects of paclitaxel as single agent and in combination with bevacizumab. Results: Ixabepilone showed robust synergistic antitumor activity in combination with bevacizumab and sunitinib in preclinical in vivo models derived from breast, colon, lung, and kidney cancers. The synergistic antitumor effect was greater with ixabepilone compared with paclitaxel. Furthermore, ixabepilone was more effective than paclitaxel at killing endothelial cells expressing P-glycoprotein in vitro and inhibiting endothelial cell proliferation and tumor angiogenesis in vivo. Conclusions: Ixabepilone may enhance the antitumor effects of antiangiogenic therapy by direct cytotoxicity and also indirectly via the killing of tumor-associated endothelial cells. Given that ixabepilone has reduced susceptibility to drug efflux pumps compared with taxanes, these data may explain the increased antiangiogenic and antitumor activity of ixabepilone in combination with antiangiogenic agents. Phase II studies to assess the efficacy and safety of ixabepilone plus bevacizumab in locally recurrent or metastatic breast cancer are planned.

Exome Sequencing Reveals Comprehensive Genomic Alterations across Eight Cancer Cell Lines

It is well established that genomic alterations play an essential role in oncogenesis, disease progression, and response of tumors to therapeutic intervention. The advances of next-generation sequencing technologies (NGS) provide unprecedented capabilities to scan genomes for changes such as mutations, deletions, and alterations of chromosomal copy number. However, the cost of full-genome sequencing still prevents the routine application of NGS in many areas. Capturing and sequencing the coding exons of genes (the “exome”) can be a cost-effective approach for identifying changes that result in alteration of protein sequences. We applied an exome-sequencing technology (Roche Nimblegen capture paired with 454 sequencing) to identify sequence variation and mutations in eight commonly used cancer cell lines from a variety of tissue origins (A2780, A549, Colo205, GTL16, NCI-H661, MDA-MB468, PC3, and RD). We showed that this technology can accurately identify sequence variation, providing ∼95% concordance with Affymetrix SNP Array 6.0 performed on the same cell lines. Furthermore, we detected 19 of the 21 mutations reported in Sanger COSMIC database for these cell lines. We identified an average of 2,779 potential novel sequence variations/mutations per cell line, of which 1,904 were non-synonymous. Many non-synonymous changes were identified in kinases and known cancer-related genes. In addition we confirmed that the read-depth of exome sequence data can be used to estimate high-level gene amplifications and identify homologous deletions. In summary, we demonstrate that exome sequencing can be a reliable and cost-effective way for identifying alterations in cancer genomes, and we have generated a comprehensive catalogue of genomic alterations in coding regions of eight cancer cell lines. These findings could provide important insights into cancer pathways and mechanisms of resistance to anti-cancer therapies.

Inhibition of NF-κB in T cells blocks lymphoproliferation and partially rescues autoimmune disease ingld/gld mice

The Fas ligand (FasL)/Fas pathway is crucial for the maintenance of homeostasis of the peripheral immune system. Its importance is illustrated by the spontaneous mouse mutants gld andlpr which lack functional FasL and Fas receptor, respectively. These animals develop lymphadenopathy, splenomegaly, increased serum Ig and autoantibodies, leading to an autoimmune syndromeand premature death. The Rel/NF‐κB family of transcription factors plays an important role in peripheral lymphocyte proliferation and survival. In this report, we studied the consequences of T cell‐specific inhibition of NF‐κB on the development of the gld phenotype. Transgenic gld/gld mice expressing a non‐degradable form of IκBα  under the control of T cell‐specific regulatory elements show dramatically reduced lymphadenopathy, splenomegaly, and an almost complete elimination of Thy‐1+B220+CD4–CD8– abnormal T cells, correlating with reduced proliferative responses and increased apoptosis of peripheral T cells upon TCR triggering. Interestingly, the B cell abnormalities that are characteristic of gld/gld mice, such as the production of autoantibodies, high levels of serum Ig, and the development of glomerulonephritis, are partially corrected. These results suggest that the T cell‐specific inhibition of NF‐κB opens apoptotic pathways distinct from FasL/Fas which, along with a diminished proliferative response, blocks splenomegaly and lymphadenopathy and partially rescues autoimmune disease in gld/gld mice.

Peptides Corresponding to the N and C Termini of IκB-α, -β, and -ε as Probes of the Two Catalytic Subunits of IκB Kinase, IKK-1 and IKK-2

The signal-inducible phosphorylation of serines 32 and 36 of IκB-α is the key step in regulating the subsequent ubiquitination and proteolysis of IκB-α, which then releases NF-κB to promote gene transcription. The multisubunit IκB kinase (msIKK) responsible for this phosphorylation contains two catalytic subunits, termed IKK-1 and IKK-2. Using recombinant IKK-2, a kinetic pattern consistent with a random, sequential binding mechanism was observed with the use of a peptide corresponding to amino acids 26–42 of IκB-α. Values of 313 μm, 15.5 μm, and 1.7 min−1 were obtained forK peptide, K ATP, andk cat, respectively. The value of α, a factor by which binding of one substrate changes the dissociation constant for the other substrate, was determined to be 0.2. Interestingly, the recombinant IKK-1 subunit gave similar values for α andK ATP, but values of 1950 μm and 0.016 min−1 were calculated forK peptide and k cat, respectively. This suggests that the IKK-2 catalytic subunit provides nearly all of the catalytic activity of the msIKK complex with the IKK-1 subunit providing little contribution to catalysis. Using peptides corresponding to different regions of IκB-α within amino acids 21–47, it was shown that amino acids 31–37 provide most binding interactions (−4.7 kcal/mol of binding free energy) of the full-length IκB-α (−7.9 kcal/mol) with the IKK-2. This is consistent with the observation that IKK-2 is able to phosphorylate the IκB-β and IκB-ε proteins, which have consensus phosphorylation sites nearly identical to that of amino acids 31–37 of IκB-α. A peptide corresponding to amino acids 279–303 in the C-terminal domain of IκB-α was unable to activate IKK-2 to phosphorylate an N-terminal peptide, which is in contrast to the results observed with the msIKK. Moreover, the IKK-2 catalyzes the phosphorylation of the full-length IκB-α and the amino acid 26–42 peptide with nearly equal efficiency, while the msIKK catalyzes the phosphorylation of the full-length IκB-α 25,000 times more efficiently than the 26–42 peptide. Therefore, the C terminus of IκB-α is important in activating the msIKK through interactions with subunits other than the IKK-2.

IRS2 Copy Number Gain, KRAS and BRAF Mutation Status as Predictive Biomarkers for Response to the IGF-1R/IR Inhibitor BMS-754807 in Colorectal Cancer Cell Lines

Insulin-like growth factor receptor 1 (IGF-1R)–targeting therapies are currently at an important crossroad given the low clinical response rates seen in unselected patients. Predictive biomarkers for patient selection are critical for improving clinical benefit. Coupling in vitro sensitivity testing of BMS-754807, a dual IGF-1R/IR inhibitor, with genomic interrogations in 60 human colorectal cancer cell lines, we identified biomarkers correlated with response to BMS-754807. The results showed that cell lines with BRAFV600E or KRASG13D mutation were resistant, whereas cell lines with wild-type of both KRAS and BRAF were particularly sensitive to BMS-754807 if they have either higher RNA expression levels of IR-A or lower levels of IGFBP6. In addition, the cell lines with KRAS mutations, those with either insulin receptor substrate 2 (IRS2) copy number gain (CNG) or higher IGF-1R expression levels, were more sensitive to the drug. Furthermore, cell lines with IRS2 CNG had higher levels of ligand-stimulated activation of IGF-1R and AKT, suggesting that these cell lines with IGF-IR signaling pathways more actively coupled to AKT signaling are more responsive to IGF-1R/IR inhibition. IRS2 siRNA knockdown reduced IRS2 protein expression levels and decreased sensitivity to BMS-754807, providing evidence for the functional involvement of IRS2 in mediating the drug response. The prevalence of IRS2 CNG in colorectal cancer tumors as measured by qPCR-CNV is approximately 35%. In summary, we identified IRS2 CNG, IGF-1R, IR-A, and IGFBP6 RNA expression levels, and KRAS and BRAF mutational status as candidate predictive biomarkers for response to BMS-754807. This work proposed clinical development opportunities for BMS-754807 in colorectal cancer with patient selection to improve clinical benefit. Mol Cancer Ther; 14(2); 620–30. ©2014 AACR.

Falk Herbert Weih (1959–2014)

We announce with deep sorrow the passing of our friend and colleague Prof. Dr. Falk Weih, who lost his two-year battle with colon cancer at the age of 54 on 28 October 2014. Falk was known to many investigators in the fields of immunology, signal transduction and gene expression as a dedicated researcher in pursuit of understanding the role of RelB, a member of the nuclear factor-κB (NF-κB) family of transcription factors, and associated biological pathways. In the late 80s, Falk started his career in the laboratory of Prof. Günther Schütz at the German Cancer Research Center (DKFZ, Heidelberg), where he studied the interaction of methylated DNA and transcription factors at the tyrosine aminotransferase promoter [1]. He published several papers in high impact journals during this period, already giving an indication of his highly focused and inquisitive mind [2–4]. After completing his Ph.D., Falk joined the laboratory of Dr. Rodrigo Bravo at the Bristol-Myers Squibb Pharmaceutical Research Institute in Princeton, USA, as a postdoc. Drawn to the various aspects of NF-κB pathways, he generated several mouse models to understand the function of these proteins. During this phase, guided by the analysis of the RelB knockout mouse, Falk also developed his interest in inflammation [5–7]. He also met his wife, Debbie, during this time and together they continued this line of research. Falk and Debbie returned to Germany in 1997, where Falk continued studies on the in vivo function of RelB at the Institute of Toxicology and Genetics in Karlsruhe. In 2004 he was appointed Professor of Immunology at the Friedrich Schiller University, Jena, and set up his laboratory at the Leibniz Institute for Age Research (Fritz Lipmann Institute) in Jena. Among Falk’s 70 publications, significant contributions to the understanding of the non-canonical NF-κB pathway and various aspects of RelB function can be counted. These include roles in lymphoid organ development, and the transcriptional regulation of chemokines and cell adhesion molecules involved in secondary lymphoid tissue organization and function [8, 9]. Moreover, his group demonstrated the specific roles of both RelA and RelB in the differentiation of natural killer T and IL-17+ γδ T cells [10–12], as well as a role for non-canonical NF-κB signaling in B-cell development and function [13, 14]. Falk’s research interests also extended beyond the immune system, as shown by his recent publication on the function of different NF-κB proteins during neuronal regeneration and adipocyte differentiation and function [15]. In spite of Falk’s struggle with cancer during the last year of his life, he continued to have meetings with students, postdocs and collaborators. He was also a critical reviewer, sensitive mentor and friend, open to many long and fruitful discussions. Falk enjoyed sharing social activities, motor cycling, horseback riding, photography and music, all of which helped maintain a balance between work and private life. Falk leaves a rich scientific heritage and many of his students and colleagues will follow on in his research path and benefit from his teachings and methodologies. We will miss him and keep him in our memories.

Comprehensive Genomic Alterations in Common Cancer Cell Lines Revealed by Exome Sequencing

It is well established that genomic alterations play an essential role in oncogenesis, disease progression, and response of tumors to therapeutic intervention. The advances of next-generation sequencing technologies (NGS) provide unprecedented capabilities to scan genomes for changes such as mutations, deletions, and alterations of chromosomal copy number. However, the cost of full-genome sequencing still prevents the routine application of NGS in many areas. Capturing and sequencing the coding exons of genes (the “exome”) can be a cost-effective approach for identifying changes that result in alteration of protein sequences. We applied an exome sequencing technology (Roche NimbleGen capture paired with 454 sequencing) to identify sequence variation and mutations in eight commonly used cancer cell lines from a variety of tissue origins (A2780, A549, Colo205, GTL16, NCI-H661, MDA-MB468, PC3, and RD). We showed that this technology can accurately identify sequence variation, providing ~95 % concordance with Affymetrix SNP Array 6.0 performed on the same cell lines. Furthermore, we detected 19 of the 21 mutations reported in Sanger COSMIC database for these cell lines. We identified an average of 2,779 potential novel sequence variations/mutations per cell line, of which 1,904 were non-synonymous. Many non-synonymous changes were identified in kinases and known cancer-related genes. In addition we confirmed that the read depth of exome sequence data can be used to estimate high-level gene amplifications and identify homologous deletions. In summary, we demonstrate that exome sequencing can be a reliable and cost-effective way for identifying alterations in cancer genomes, and we have generated a comprehensive catalogue of genomic alterations in coding regions of eight cancer cell lines. These findings could provide important insights into cancer pathways and mechanisms of resistance to anticancer therapies.

Abstract 4115: Depletion of unsaturated fatty acids by stearoyl-coA desaturase inhibition results in endoplasmic reticulum stress-mediated tumor cell death

Over the past few years, a more detailed picture of the importance of metabolic pathways to the maintenance and progression of human cancers has begun to emerge. In particular, recent studies have implicated lipid biosynthesis and desaturation as a requirement for tumor cell survival. The endoplasmic reticulum-resident Stearoyl CoA Desaturase (SCD) is the rate limiting enzyme that catalyzes conversion of saturated fatty acids to monounsaturated fatty acids, a key step in the fatty acid metabolic network. In the studies reported here, we show that inhibition of SCD1 by siRNA or a small molecule antagonist results in strong induction of apoptosis and growth inhibition when tumor cells are cultured in reduced (2%) serum conditions but has little impact on cells cultured under normal (10%) serum conditions. Consistent with the observed dependence on SCD1, levels of SCD1 protein increased in response to decreasing serum levels. Both induction of SCD1 protein and sensitivity to growth inhibition by SCD1 inhibition could be reversed by supplementing growth media with unsaturated fatty acids. Transcription profiling of cells treated with an SCD inhibitor revealed strong induction of markers of endoplasmic reticulum stress. This finding was confirmed using siRNA specifically targeting SCD1. Underscoring its importance in cancer, SCD1 protein was found to be highly expressed in a large percentage of human cancer specimens. SCD inhibition resulted in tumor growth delay in a human gastric cancer xenograft model. Altogether, these results suggest that unsaturated fatty acids are required for tumor cell survival and that SCD may represent a viable target for the development of novel agents for cancer therapy. 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 4115. doi:1538-7445.AM2012-4115

Abstract C62: Cancer cell dependence on unsaturated fatty acids implicates stearoyl-coA desaturase as a target for cancer therapy

Emerging literature suggests that metabolic pathways play an important role in the maintenance and progression of human cancers. In particular, recent studies have implicated lipid biosynthesis and desaturation as a requirement for tumor cell survival. In the studies reported here, we aimed to understand whether tumor cells require the activity of either human isoform of stearoyl-CoA-desaturase (SCD1 or SCD5) for survival. Inhibition of SCD1 by siRNA or a small molecule antagonist results in strong induction of apoptosis and growth inhibition when tumor cells are cultured in reduced (2%) serum conditions but has little impact on cells cultured in 10% serum. Depletion of SCD5 had minimal effects on cell growth or apoptosis. Consistent with the observed dependence on SCD1 but not SCD5, levels of SCD1 protein increased in response to decreasing serum levels. Both induction of SCD1 protein and sensitivity to growth inhibition by SCD1 inhibition could be reversed by supplementing growth media with oleic acid, a product of the enzymatic reaction catalyzed by SCD1. Transcription profiling of cells treated with an SCD inhibitor revealed strong induction of markers of endoplasmic reticulum stress. Underscoring its importance in cancer, SCD1 protein was found to be highly expressed in a large percentage of human cancer specimens. SCD inhibition resulted in tumor growth delay in a human gastric cancer xenograft model. Altogether, these results suggest that desaturated fatty acids are required for tumor cell survival and that SCD may represent a viable target for the development of novel agents for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C62.