DISSECTION OF THE MOLECULAR PATHWAYS INVOLVED IN PANCREATIC CANCER INITIATION AND PROGRESSION WITH A NOVEL IN VIVO APPROACH
Creators: name: CARUGO, ALESSANDRO
Publisher: name: Universit� degli Studi di Milano
Year: 2016

description: We have seen great advances in our knowledge of the genetic regulation of various cancers in recent years, thanks in large part to large-scale genome sequencing efforts. As we catalogue and characterize the genomic aberrations associated with cancers with increasing detail and accuracy, we are faced with the challenge of having to cull bystanders from biologically active drivers and establish relevant disease context in which these drivers are rate-limiting. To address this challenge, we have adapted a loss-of-function screening approach to function in the context of an intact tumor microenvironment using patient-derived xenografts that more faithfully recapitulate the human disease compared to established cell lines. Due to the relevant genetic heterogeneity between human tumors with the same clinico-pathological indications, we have integrated independent screening approaches in a flexible platform for the interrogation of patient-derived samples as well as genetically-defined mouse models in exactly the same experimental conditions. The goal of this platform is to identify context-specific genetic vulnerabilities and translate these findings into drug discovery opportunities. As proof of concept for this approach, we describe the development of an in vivo loss-of-function screen to systematically interrogate epigenetic dependencies in pancreatic ductal adenocarcinoma (PDAC). In addition to the well-known genetic alterations (Kras, TP53, CDKN2A/p16, SMAD4), some epigenetic mechanisms demonstrated to play a central role in PDAC evolution and progression. The screening system utilizes tumor cells isolated from low-passage PDAC xenografted tissue and a lentiviral library of pooled shRNAs targeting 236 potentially ?druggable? epigenetic regulators. The custom-designed shRNA library (10 shRNAs per gene) was engineered with unique molecular barcodes that allow quantitation of each clone by massively parallel sequencing. Hairpins are clustered according to their depletion or enrichment in comparison to a control population before transplantation. To date, we have completed a total of 5 in vivo screens using diverse PDAC target cell models that have informed on novel epigenetic dependencies. So far, the main limitation for the systematic exploitation of in vivo loss-of-function screens to identify specific patient vulnerabilities come from the limited number of human cells contributing to tumor establishment in a transplantation setting. The frequency of these tumors initiating cells (TICs) is commonly estimated by time-consuming limiting dilution assays and may consistently vary between different tumor origins. With this in mind, we have integrated in our platform a system based on scrambled barcoded libraries that allows to directly assess the required coverage of screening libraries in each model and adjust the shRNA screens for this factor. Our coverage study demonstrated to be a powerful tool to identify the minimal number of cells/barcode required to sustain a complex library in transplantation assay and at the same time a step forward to personalize the in vivo screening patient by patient. We optimized a comprehensive data analytics pipeline and developed a high-throughput validation scheme to triage "hits" that emerge from each screen. The most potent "hits" have been enrolled in both functional and clinico-pathological validation studies to determine the highest priority targets for this devastating disease. Significantly, different components of the COMPASS histone H3 Lys4 (H3K4) methyltransferase complexes were identified as candidates in our screens. COMPASS and COMPASS-like complexes are characterized by unique subunits composition, whose identities provide insight into the different biological functions of these complexes. The methyltransferase unit of the COMPASS complexes is directly involved into the methylation of Lys4 on histone H3, a commonly accepted sign of open-chromatin and active transcription. Chromosomal translocations involving MLL gene are frequent events characterizing the Mixed Lineage Leukemia. In this disease, it has been shown that fusion events with a variety of different partners compromise the MLL methyltransferase activity. However, multiple members of the MLL family could be deregulated via different oncogenic mechanisms in PDAC, as the genetic alteration in MLL2 (amplification) and MLL3 (mutation) suggested. Our platform represents an ideal starting point to understand the COMPASS functionalities. So, a deeper understanding of genes and pathways regulated by each MLL subunit in the context of PDAC is critical to better elucidate the molecular dynamics of this disease and identify additional key points of vulnerability. Our study identified the core different subunits of the COMPASS complexes (WDR5-ASH2L-RBBP5) as broad relevant players in sustain PDAC progression, while the dependency on the MLL subunits appears to be more context-dependent and potentially consequent to specific genetic alterations. Mechanistically, WDR5 functions to sustain proper execution of DNA replication in PDAC cells, as previously suggested by replication stress studies involving MLL1, a critical ATR substrate, and c-Myc, also found to interact with WDR5. By showing that ATR inhibition mimicked the effects of WDR5 suppression, we open up the possibility of testing inhibitors currently in development for activity in this disease. These findings are proposing a new layer of complexities in trapping the COMPASS complexes during tumor development and unmasking unexplored directions for new therapeutical opportunities.