Johannes Braegelmann

Abstract 1349: Systematic deconvolution of kinase inhibitor profiles identifies synthetic lethal targets in ERBB2-mutant and BRD4-NUT rearranged cancer

The development of targeted therapies that efficiently inhibit cancer signaling pathways is one of the main goals of modern precision cancer medicine. Consequently, genetic and biological phenotypic data of in vitro screens are increasingly utilized to develop compounds directed against distinct oncogenic alterations. However, current targeted therapies are often limited to small genetically defined patient cohorts due to the very finite number of proteins amenable to direct chemical inhibition. An alternative approach is the exploitation of synthetic lethality, i.e. inhibition of an unaltered protein required for cell viability in a certain genetic background. Systematic chemo-genomic analyses of cancer cell lines have been shown to be suitable tools for the identification of novel synthetic lethal dependencies in cancer (Chan et al. Sci Trans Med, 2011; Sos et al. PNAS, 2012; Kim et al. Cell 2013). To systematically extend this strategy to non-small cell lung cancer (NSCLC) we characterized the efficacy of 1505 chemical compounds based on a variety of kinase inhibitor motifs in a high-throughput screen against 80 NSCLC cell lines. We extracted patterns of biological activity based on chemical and genetic information and found that potency and selectivity of compounds are strongly related to their molecular scaffold, but independent of their overall chemical complexity. We thereby discovered a sunitinib derivative that exhibited exquisite activity against ERBB2-mutant cell lines but was devoid of ERBB2 kinase activity. Instead a kinome scan and an shRNA screen suggested a mechanism of synthetic lethality by activity against NTRK family members. Moreover a CDK9 inhibitor was identified as selective and potent against a midline carcinoma cell line - a tumor entity characterized by recurrent BRD4-NUT gene fusions. Using additional cell lines we validated the upregulation of c-Fos and selective induction of apoptosis in BRD4-NUT positive midline carcinoma compared to control cell lines following CDK9 inhibition. This can augment existing therapeutic approaches, which have primarily focused on directly targeting the fusion product with bromodomain inhibitors, and offers a novel target in this entity. In conclusion, by systematically screening a large number of compounds against a panel of genetically well characterized NSCLC cell lines and incorporating chemical information we were able to derive structure activity relationships and to identify potential synthetically lethal targets in two genetic entities in clinical need of advanced selective therapies. Citation Format: Johannes Braegelmann, Peter Habenberger, Felix Dietlein, Johannes M. Heuckmann, Sascha Menninger, Uwe Koch, Axel Choidas, Daniel Rauh, Bert Klebl, Martin L. Sos, Roman K. Thomas. Systematic deconvolution of kinase inhibitor profiles identifies synthetic lethal targets in ERBB2-mutant and BRD4-NUT rearranged cancer. [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 1349.

Abstract 5472: Role of Harvey Ras (HRAS) mutations in head and neck squamous cell carcinoma (HNSCC)

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Harvey Ras (HRAS) was recently reported being mutated in head and neck squamous cell carcinomas (HNSCC) and likely plays an important role as an oncogene. The precise role of HRAS mutations for signaling and carcinogenesis of HNSCC remains to be determined. Methods: We completed mutational screening (Sanger Sequencing) for tissues and cell lines focused on the known hotspot mutations G12X and Q61X. Furthermore we performed viability testing for various cell lines and visualized the signaling-effects by itself, in presence of PI3K-, EGFR inhibitors and likewise in combination, by immunoblotting. After suppression of HRAS using siRNA, we determined the cell-viability. Results: In our study we sequenced 100 HNSCC tumor tissues and HNSCC cell lines and identified several canonical HRAS mutations. One cell line contained a G12D HRAS mutation and was further examined. Additional two cell lines with atypical HRAS variants were identified and compared to the classic hotspot mutated cell line. The viability for the mentioned cell lines were indicative of resistance to EGFR inhibition to different degrees. The protein activation levels in important signaling pathways (PI3K/MAPK) confirmed our viability data. HRAS signaling was primarily via PI3K/AKT. Silencing HRAS showed significantly decreased viability. Conclusions: Previous studies have shown that EGFR-targeting agents remain insufficient as single targeted therapy. HRAS appears to contribute to the EGFR-resistance of HNSCC. The canonical mutation G12D appears to signal primarily via PI3K and PI3K inhibitors may be effective. The G12D cell line model indicates a central role of mutated HRAS for signaling and viability consistent with role as a driver mutation. 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 5472. doi:1538-7445.AM2012-5472

Abstract LB-398: Detection of copy number alterations in 124 head and neck squamous cell carcinomas using the Nanostring nCounter assay

Background: Head and Neck Squamous Cell Carcinoma (HNSCC) is the 6th most common cancer worldwide. Little is known about changes in copy number (CN) in many cancer associated genes in this tumor type, which may contribute to carcinogenesis and could become useful treatment targets in the future. Many analyses of CN are complicated by the need to use formalin-fixed paraffin-embedded (FFPE) tissues posing technical challenges. We determined copy number alterations (CNA) using a novel, medium-throughput technology (NanoString nCounter) in order to determine common cancer associated CNAs and assess its performance in FFPE tissues. Results were compared with more established technologies such as SNP array, aCGH, and qPCR. Methods: We investigated CNA in 124 tumor specimens and 22 cell lines for 100 literature curated cancer genes using the NanoString nCounter. Most samples were OCT frozen tissues, with a small subset having both OCT frozen, and FFPE tissues. Slides were assessed for tumor content by a HNC pathologist and samples with at least 60% tumor content selected. DNA was extracted using standard column-based methods (Qiagen). We performed CN analysis in 124 frozen (+4 matching FFPE) HNSCC specimens and cell lines (Nanostring nCounter assay) focusing on a selection of cancer associated genes. Furthermore we used aCGH and SNP-CHIP to analyse 20 and 4 cell lines respectively two of which were covered by all three methods. FGFR1 was assessed by qPCR. For FFPE samples a special Nanostring probeset was used with 3-5 probes per gene to provide redundancy with degraded DNA samples. HPV status of samples was assessed by a nested PCR for E6. Results Copy number changes detected by Nanostring and aCGH correlated well. The Nanostring nCounter assay appeared more accurate in calling deletions, which were detected in MST1R, PBRM1, PTPRD for instance. We found amplifications in multiple samples and genes, e.g. CCND1, EGFR, MDM4, MYC, VEGFA, PAX9, ITGB4, SSND1, CTTN, FADD, FGF19, ORAOV1, PPFIA1, some of which were frequently (n>50 samples) or highly amplified (>30 copies). Some of these amplifications such as ORAOV1 and PPFIA1 seemed higher/more frequent in HPV(-) compared to HPV(+) samples. Samples with FGFR1 amplification were validated using qPCR and correlated very closely. FFPE sample processing was uncomplicated using the FFPE probeset. While some probes failed, using degraded FFPE derived DNA, the redundancy of probes allowed accurate calling of CNA that closely correlated with frozen sample results. Conclusions Copy number alterations are frequent in HNSCC and involve many cancer associated genes, including potentially targetable genes such as EGFR, MDM4, and PIK3CA. Most of the CN changes are recurrent. Amplifications and deletions to some extent differed depending on HPV status. The role and implications of these CN aberrations in a clinical setting need to be further elucidated and validated. 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 LB-398. doi:1538-7445.AM2012-LB-398

Abstract 2832: Fibroblast growth factors in head and neck cancer: Genetic alterations and therapeutic targeting with ponatinib

Background: FGF signaling plays an important role in cancer. Little is known about its involvement in head and neck squamous cell carcinoma (HNSCC). FGFR1 focal amplification and high expression (Freier, Schwaenen et al.), expression of FG-BP (Li et al.) and FGF2 autocrine signalling loops (Marshall et al.) have been described in connection with HNSSC. We therefore examined copy number alterations for several FGFR and FGF-related Genes in 159 patient tissues and cell lines as well as expression levels in 116 patient tissues and 40 cell lines. Based upon our findings we tried a combined FGFR-inhibiton with ponatinib in 5 head and neck cancer-cell lines alone and in combination with gefitinib. Methods: Copy Number Data analysis for 144 patient tissues and cell lines (Nanostring) as well as for 20 cell lines (aCGH) of which 5 were covered by both techniques and showed comparable results. Expression data analysis for 116 tissues and 40 cell lines (Agilent). Viability for 5 cell lines treated with gefitinib and ponatinib alone and in combination. Immunoblotting was performed to deterimed PI3K-AKT and MAPK signaling. Results: Frequent copy number increase could be detected for FGF19 in tissues and cell lines. FGFR1 copy number increase could be seen in only 1 tissue sample, but appeared to be deleted in several samples. High relative expression could be detected for FGFBP1 in tissues and cell lines. Viability testing showed high efficacy in 5/5 cell lines tested for ponatinib but was not solely mediated by AKT or MAPK signaling. Combined treatment with ponatinib and gefitinib was more effective than treatment with either agent alone and synergy was present. Conclusions: FGF signaling is important in Head and Neck Cancer. FGF19 amplification is frequent. We were unable to replicate FGFR1 amplification with only one tissue showing FGFR1 copy number increase. High expression levels could be shown for FGFBP1, providing an alternative hypothesis for explaining the efficacy of FGF2 inhibition as previously shown (Marshall et. al.). Ponatinib is effective as a single agent on HNSSC cell line models and shows synergistic effect in combination with gefitinib. It is promising to evaluate FGF pathway inhibition (e.g. with ponatinib) in its ability to overcome EGFR-inhibitor resistance. 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 2832. doi:1538-7445.AM2012-2832

Abstract 2223: Screening for oncoviruses using next-generation sequencing

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: During the last decades numerous viruses have been found to play a causative role in cancer. Besides well established connections like HPV in cancers of the uterine cervix or EBV in nasopharyngeal carcinoma recent advances in next-generation sequencing technologies have led to the discovery of new tumor viruses e.g. a previously unknown Polyoma-virus subtype linked to Merkel-cell carcinoma of the skin. Method: To gain insight into the mechanisms leading to tumor formation we sequenced the transcriptome from an HPV16 positive cell line using short-read Illumina RNA-Seq and established a computational pipeline to screen the short reads for viral sequences based on freely available alignment tools. Results: Due to the large amount of data and in order to narrow down the search space and reduce the chance for false positives the first screening step consists in a subtraction of sequences that align to the human reference genome reducing the amount of reads by ∼70%. To find both known viruses and yet unknown strains/subtypes we apply a two-fold approach: 1. A rapid search for known viruses is done using a short-read alignment tool with a set of stringent parameters to map non-human reads to an index of the viral genbank and subsequent quantification of the hits. 2. Since slight differences between read and index sequence will cause the rapid search to not call a match we implemented a more lenient approach using standalone BLAST. Applying approach 1) to the control cell line we clearly identified HPV16 as being present. To test the possibility of finding a potentially unknown virus we deleted HPV16 from both the rapid approach and the index which caused the first algorithm to no longer identify the virus. Running the BLAST basec approach 2) resulted in multiple hits the most prominent being alignments to HPV subtypes 35 and 58, thus indicating the successful identification of related strains. Conclusion: We demonstrated the feasibility of our dual viral screening approach in an in-silico experiment using an HPV16 positive control. Our approach appears to be able to identify novel related viruses. In the future this will allow screening of patient samples and viral screening was added to our data pipeline. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2223.

Abstract A207: Whole transcriptome profiling in upper aerodigestive tract cancers

Background: RNA‐seq is a powerful technology that allows to obtain sequence and expression information simultaneously. We applied this technology to 4 upper aerodigestive tract cancers. Methods: RNA from four upper aerodigestive tract tumor specimens was extracted and sequencing libraries constructed. Samples were analyzed using an Illumina Genome Analyzer with a paired end module (36 or 54 base read length). Raw data was processed with a proprietary data pipeline. Potential mutations were filtered using both in house and external bioinformatic solutions, involving previously established databases of mutations (COSMIC) and SNPs (dbSNP), and removal of ancestral alleles identified from multiple sequence alignments. These SNPs were then parsed via in house scripts to determine whether the SNPs were present in coding regions, 3′UTR, 5′UTR, or in splice acceptor/donor sites. The coding SNPs were further parsed to determine which SNPs result in non‐synonymous changes. RNA‐Seq expression data was analyzed using Partek Genomics Suite and compared to microarray based expression data. Pathway analysis was performed using GeneGO Metacore and DAVID. Results: 500Mb to 2GB of data were obtained per sample. Between 700 and 2000 nsSNPs were identified, as well as a large number of alteration in the 3′ and 5′ untranslated regions. Genetic alterations in several commonly mutated genes were identified including TP53, ErbB2, H‐RAS, and Cyclin D1. SNPs were enriched in pathways commonly involved in upper aerodigestive tract tumors including cell cycle control, cytoskeleton, and receptor tyrosine kinases. Furthermore we screened for viral sequences identifying one sample that was HPV16 positive. RNA‐seq based and microarray based expression data generally correlated, but significant differences were also observed, that were at least partly due to the limited number of RNA‐seq reads. Conclusion: Cancer transcriptome sequencing is a promising and cost effective approach for identifying mutations and obtaining expression analysis simultaneously. Transcriptome sequencing holds promise as a readily available platform for assessing potential treatment targets in a specific tumor. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A207.