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A small fraction of all cells within individual tumours from colorectal cancer (CRC) patients drives long term tumor growth and metastases in immune-compromised mice. Targeting these tumor-initiating cells (TIC) may improve the long-term outcome in advanced CRC. To identify candidate genes which drive proliferation and survival of TIC, we have performed a large scale high throughput loss of function shRNA screen in three-dimensional TIC enriched patient spheroids. Spheroids were transduced with the barcoded Cellecta decipher library comprising 27,500 shRNAs targeting 5043 genes associated with cell signaling pathways (Module 1). Two weeks later, cells were harvested for DNA isolation, barcode amplification and high-throughput barcode sequencing. Amongst others, we found 5/6 shRNAs targeting Insulin growth factor 2 (IGF2) scoring within a 20% depletion threshold, presenting depletion levels very similar to positive control shRNAs. mRNA expression profiling and qPCR analyses demonstrated low to moderate expression of the IGF2 gene product in the majority of patient spheroid cultures analyzed (n=17). In contrast, two out of 15 patient derived spheroid cultures analyzed demonstrated very pronounced IGF2 overexpression (>250 fold). Integrative SCNA profiling, expression and TAD profiling using the CESAM algorithm, followed by 4C Seq, demonstrated a tandem duplication of the IGF2 locus in these two patients which interrupts a IGF2 adjacent TAD boundary and results in de novo contact domain formation between the IGF2 promoter and a normally hidden distant super-enhancer. A dual luciferase reporter assay revealed that the hijacked enhancer is functionally active in human CRC cells and thereby may drive unphysiological IGF2 expression following enhancer hijacking. To assess the functional relevance of IGF2 tandem duplications, spheroids were transduced with RFP expressing lentiviral vectors encoding for 3 shRNAs targeting IGF2 as well as control shRNAs targeting EIF3A or scrambled shRNA. Strikingly, IGF2 knockdown led to a marked reduction of RFP+ cells in competitive proliferation assays and markedly reduced viability assessed by ATPlight assay. Moreover, IGF2 knockdown strongly reduced tumor formation following xenotransplantation into immune deficient NSG mice. These data demonstrate that IGF2 is required for survival, proliferation and tumor-initiation of primary human TIC enriched spheroids. Notably, oncogenic miRNA-483 is encoded within intron 8 of IGF2, however, its function in IGF2 locus tandem duplicated cells remains elusive. Understanding the mechanisms of IGF2 dependency and the role of miRNA-483 in this context will be essential for the future development of therapeutic approaches targeting IGF2 expression in this patient subset harbouring tandem-duplications of the IGF2 locus. Citation Format: Taronish D. Dubash, Christine Siegl, Sebastian M. Dieter, Joachim Weischenfeldt, Alexandros P. Drainas, Laura Schwarzmueller, Malgorzata Oles, Balca Mardin, Mikolaj Slabicki, Huber Wolfgang, Martin Schneider, Jan Korbel, Hanno Glimm, Claudia R. Ball. IGF2 is essential for tumor initiating cell activity in human colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2893. doi:10.1158/1538-7445.AM2017-2893

Introduction: The International Cancer Genome Consortium (ICGC) is a worldwide network aiming to provide comprehensive molecular genetic profiles of 50 clinically and societally important tumors. A major focus of the ICGC PedBrain Tumor project is medulloblastoma (MB) - the most common malignant brain tumor in childhood. Importantly, recent work has shown that MB is not a single disease, but is in fact comprised of 4 distinct molecular subgroups (WNT, SHH, Group 3, Group 4). Using an integrative next-generation sequencing-based approach, we have investigated tumorigenic events underlying medulloblastoma, with the aim of identifying novel diagnostic or prognostic markers, and therapeutic targets. Methods: The whole genome of 50 tumor-normal DNA pairs, plus the whole exome or 2,500 target genes in a further 100 sample pairs from different histological and transcriptomic subgroups, was sequenced on an Illumina HiSeq platform. Sequencing-based high-resolution copy-number data was also generated for all cases. Results: Overall, a smaller number of non-synonymous somatic mutations was seen than in most adult malignancies. The number of somatic changes also clearly showed a positive correlation with patient age, suggesting either longer-term selection for more weakly transforming alterations, or the acquisition of additional passenger mutations in tumors which grew over a longer period. Whilst some point mutations and small InDels affected known MB or cancer-related genes (e.g. CTNNB1, PTCH1, MLL2), the vast majority were in genes which have not previously been implicated in medulloblastoma. Furthermore, even the most recurrently altered genes were mutated in 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 4872. doi:1538-7445.AM2012-4872

Prostate cancer is often heterogeneous with multiple tumor foci within the same prostate. MALDI (Matrix-Assisted Laser Desorption/Ionization) mass spectrometry imaging (MSI) allows for a comprehensive profiling of the molecular phenotype on the protein, peptide, lipid, and metabolite level across entire tissue slides without prior enrichment of tumor cells. This study aims in a MALDI-based discrimination of cancer areas that are heterogeneous with respect to morphology and molecular features. We established a method for cryopreservation of entire prostates from cancer patients, and build tissue microarrays from individual cancerous prostates representing >30 different areas of the primary tumor as well as multiple lymph node metastases. We analyzed these arrays for ERG fusion by FISH and for peptide signatures by MALDI MSI. In addition, 2 selected tumor areas and 4 different lymph node metastases from one prostate were subjected to paired end next generation sequencing. MALDI identified 5 masses that were specific for epithelial cells. One particular mass was strongly related to presence of ERG fusion. In one prostate, we identified another mass that was present only in 2/35 tissue spots from the primary tumor as well as in 4/10 lymph nodes. Deep sequencing of different tumor areas of this case identified distinct patterns of structural genetic alterations. A cluster analysis showed that all samples were clonally related, and identified the area inside the primary tumor that gave rise to the metastases. In conclusion, these data suggest that MALDI imaging and next generation sequencing allow for identification of genetic and proteome patterns that correspond to metastatic spread of prostate cancer cells. A combination of both techniques is suited to dissect the genetic background of distinct MALDI signatures. 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 1272. doi:1538-7445.AM2012-1272