Hintergrund: Onkogene Kras Mutationen führen zur Entstehung von duktalen Pankreaskarzinomen. Welche Kras aktivierten Signalwege hierbei eine Rolle spielen, ist bisher allerdings weitgehend unbekannt. Der Phosphatidylinositol 3-kinase (PI3K) Signalweg ist eines der Haupttargets von onkogenem Kras. Ziel der Arbeit war die Analyse des Beitrags des PI3K Signalwegs zur Karzinogenese im Pankreas.
Einleitung: Effektive konservative Therapien zur Behandlung des Pankreaskarzinoms fehlen weiterhin und dies trägt zur schlechten Prognose der Erkrankung bei. Vor kurzem haben wir die Überexpression der Histon Deacetylase 2 (HDAC2) im Pankreaskarzinom demonstrieren können. Zusätzlich konnten wir zeigen, dass HDAC2 Resistenz gegen Topoisomerase II Inhibitoren vermittelt.
Abstract Colorectal cancer (CRC) can be classified in several molecular, morphological and histological subtypes. Modelling of these subtypes in genetically engineered mice contributed to a mechanistic understanding of CRC development. However, these models exhibit a late tumor onset and predominantly develop tumors in the small intestine, which is a rare location of human cancers. Furthermore, murine tumors rarely progress to advanced stages and poorly recapitulate the adenoma-carcinoma sequence observed in human CRC. Preliminary data suggest that the mutational make-up of the tumor may dictate its specific immune microenvironment, but may also drive microbial alterations in the gut, which in turn determine tumor development and survival. In order to study CRC in its native anatomical location (colon) and to elucidate the cross-talk between different molecular subtypes and the microenvironment in a spatio-temporal manner, we used an orthotopic transplantation model that is based on colonoscopy-guided injections of intestinal organoids into the submucosa of the rectum/distal colon. To investigate the role of genes altered in human CRC and their interactions, we performed CRISPR/Cas9-based gene editing in organoids followed by orthotopic allogenic transplantation into the colon of recipient syngeneic mice. Notably, organoids derived from normal and tumor tissues, which harbor CRC-relevant mutations, engraft in the colon. Tissues with activated oncogenes/inactivated tumor suppressors display the ability to form invasive tumors locally and seed to distant sites. Moreover, tumors generated by orthotopic injections of tumor-derived organoids not only recapitulate the histology of the primary tumor, but also show signs of tumor progression (e.g. towards undifferentiated sarcomatoid phenotypes). Whole exome sequencing data showed that organoids derived from tumors after implantation share common mutations with the parental organoids isolated from the endogenous tumor. Interestingly, after transplantation of the parental line specific mutations are selected for, as seen by their increased variant allelic frequency (VAF). Additionally, exclusive mutations were detected in both the parental- and the xenograft-derived organoid lines suggesting that in vivo tumor progression might be fuelled by a clonal selection event taking place after engraftment of the parental organoid line. In summary, these orthotopic transplantations allow us to efficiently model CRC subtypes in different syngeneic immuno-competent and immuno-deficient models, to functionally analyze tumour development from initiation to metastasis and to assess the combinatorial effect of mutations on tumor evolution, histomorphology and the cross talk of cancer cells with their microenvironment. Citation Format: Markus Tschurtschenthaler, Miguel G. Silva, Moritz Jesinghaus, Ankit Sinha, Jatin Roper, Roland Rad, Dieter Saur. Spatio-temporal analysis of the tumor microenvironment of colorectal cancer subtypes using an orthotopic organoid transplantation model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1116.
Einleitung: Der Beitrag von NFkB zur Karzinogenese ist für viele solide Tumoren gut belegt. Im Gegensatz zu NFkB ist der Transkriptionsfaktor p53 ein klassischer Tumorsuppressor. Als zentraler Regulator der geregelten Zellteilung und Apoptose induziert er die Expression von Genen, die die Apoptose, Seneszenz und das Anhalten des Zellzyklus fördern.
Abstract Pancreatic ductal adenocarcinoma (PDAC) represents the fourth leading cause of cancer death in the western world, with a 5‐year survival rate below 5%. Murine double minute 2 (Mdm2) is an important negative regulator of the tumor suppressor p53. Reactivation of wild‐type p53 is a promising treatment strategy, and inhibitors of Mdm2 have already entered clinical trials. To investigate the effects of Mdm2 inhibitors in PDAC, we used a murine cell line platform with a genetically defined status of p53 . Here, we describe that Mdm2 inhibitors can act on a subset of murine PDAC cell lines p53 independently. Furthermore, we observed that Mdm2 inhibitors increase the sensitivity of murine PDAC cell lines toward topoisomerase II inhibitors by inducing effector caspase‐independent cell death. The combination of Mdm2 inhibitors with topoisomerase II inhibitors acts independent of the survival factor NFκB/RelA. Mechanistically, Mdm2 inhibitors increase topoisomerase II inhibitor‐induced DNA double‐strand breaks. We show that Mdm2 binds to Nbs1 of the Mre11–Rad50–Nijmegen breakage syndrome (Nbs) 1 DNA repair complex. In addition, we provide evidence that Mdm2 inhibitors delay DNA repair. These findings may help to design novel therapeutic strategies to overcome therapeutic resistance of PDAC.
Abstract Pancreatic ductal adenocarcinoma (PDAC) remains a devastating disease with poor survival rates as almost all patients develop resistance towards chemotherapy and molecular-informed targeted therapies are reserved to a few. Here, we aim to establish a longitudinal precision oncology platform with a multi-dimensional characterization of PDAC biopsies including genomic, transcriptomic as well as functional analyses to identify and exploit treatment-induced vulnerabilities. In order to investigate adaptive processes of tumors under treatment we aimed to generate PDAC patient-derived organoids (PDOs) and 2D cell lines before and after chemotherapy. Therefore, we enrolled a patient with borderline resectable PDAC who received neoadjuvant FOLFIRINOX. Endoscopic fine needle (pre-FFX) and surgical biopsies (post-FFX) were used to generate PDOs and 2D cells. Whole exome sequencing (WES) and RNA sequencing data of the pre-FFX and post-FFX organoids were compared in order to evaluate the genetic landscape and PDAC subtypes. 2D cells were subjected to an unbiased automated drug screening of 415 compounds to investigate FFX-induced vulnerabilities. Top targets were validated manually in the 2D cells and organoids. Although transcriptional subtyping classified both PDOs as classical PDAC, gene set enrichment analysis (GSEA) revealed a reduced pathway activation linked to the basal-like phenotype such as KRAS signaling in the post-FFX organoids. WES did not show major differences in the genetic landscape of the tumor pre- and post-FFX induction suggesting a plasticity process rather than a clonal selection during chemotherapy. Importantly, post-FFX cells exhibited an increased sensitivity in the unbiased drug screening towards MEK and EGFR inhibition compared to pre-FFX cells. 2D cells and organoids were treated with different MEK inhibitors (MEKi) for validation and post-FFX cells showed a highly increased response compared to the treatment-naïve cells, as well. Interestingly, when placed into the context of a panel of 15 primary PDAC cell lines the pre-FFX cells cluster with highly MEKi resistant PDAC cells whereas post-FFX cells belong to the most sensitive cell lines. In sum, integrating functional layers into personalized medicine allowed us to identify chemotherapy-induced vulnerabilities as potent targeted therapy options in PDAC. Thus, this longitudinal precision oncology platform harbors a unique opportunity to understand adaptive processes in tumor evolution and/or treatment-imposed pressure in PDAC patients. Citation Format: Katja Peschke, Hannah Jakubowski, Arlett Schäfer, Carlo Maurer, Sebastian Lange, Felix Orben, Raquel Bernad, Felix Harder, Matthias Eiber, Rupert Öllinger, Melissa Schlitter, Wilko Weichert, Veit Phillip, Christoph Schlag, Roland Schmid, Rickmer Braren, Bo Kong, Ekin Demir, Helmut Friess, Roland Rad, Dieter Saur, Günter Schneider, Maximilian Reichert. Longitudinal precision oncology platform to identify chemotherapy-induced vulnerabilities in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-070.
