Disease modeling on tumor organoids implicates AURKA as a therapeutic target in liver metastatic colorectal cancer

Metastatic spread and resistance to chemotherapy still limit the treatment success of current colorectal cancer therapy, even though multimodal treatment approaches have improved and prolonged patient survival. Here, we used state-of-the-art patient-derived tumor organoids (PDTOs) of liver metastatic colorectal cancer to model the generation of tolerance towards chemotherapy. We achieved this by long-term ex vivo treatment of KRAS wild type PDTOs with a clinically employed first-line therapy consisting of the chemotherapeutic regimen FOLFIRI plus the EGFR-targeting antibody Cetuximab. After up to 9 months of treatment, the PDTOs generated a tolerance towards FOLFIRI/Cetuximab and failed to induce an efficient apoptotic response. This phenotype occurred without the gain of resistance-conferring mutations in clinically relevant genes. Instead, unbiased whole transcriptome sequencing (next generation RNA sequencing) revealed an enrichment in MYC target gene expression in two out of three tolerant PDTO lines. The third PDTO line developed the tolerance towards first-line therapy via a different mechanism, which included upregulation of interferon-α-related gene expression. Intriguingly, all three tolerant PDTO lines were derived from tumors with a genomic amplification of the chromosomal region 20q13.2, which contains the Aurora kinase A (AURKA) locus, and displayed elevated mRNA and protein levels of AURKA compared to normal colonic epithelium. Treatment with the AURKA inhibitor Alisertib, which also represents a strategy to target MYC indirectly in different cancer types, restored an apoptotic response in the three established chemotherapy tolerant PDTO lines. We then introduced a KRASG12D mutation into the FOLFIRI/Cetuximab tolerant PDTO lines via CRISPR/Cas9-mediated genomic engineering and confirmed that this clinically problematic mutation confers resistance towards therapeutic approaches of single or dual targeting of the EGFR-MAPK pathway. Notably, the combination of dual targeting of the EGFR-MAPK pathway with inhibition of AURKA reduced the cell viability of first-line chemotherapy tolerant KRAS mutant PDTOs to a higher extent than each treatment alone. More importantly, the treatment with the AURKA inhibitor restored the apoptotic response and largely diminished the tumor organoid reformation capacity in KRAS mutant PDTOs, sensitized by dual EGFR-MAPK pathway inhibition. This combination treatment strategy was especially effective in the two PDTO lines that had developed increased MYC levels after acquisition of tolerance to first-line therapy. To obtain a deeper insight into AURKA expression levels in metastatic disease, we performed immunohistochemical staining of AURKA of CRCs of a matched patient cohort. Here, we observed that AURKA expression was slightly increased in non-metastatic colorectal cancers compared to liver or lung metastatic colorectal cancers. Moreover, the AURKA expression was positively correlated with the abundance of nuclear beta-catenin, which is a marker of aggressive disease and poor overall patient survival. In conclusion, this Ph.D. thesis provides evidence for the potential of patient-derived tumor organoids for the ex vivo modeling of colorectal cancer therapy tolerance, mutational disease progression, and the evaluation of drug combinations to overcome treatment resistance in a preclinical setting.Metastasierung und Resistenzen gegenüber Chemotherapie limitieren die Behandlungsmöglichkeiten des kolorektalen Karzinoms, obwohl multimodale Behandlungsansätze das Überleben der Patienten verbessert und verlängert haben. Wir haben Patienten-abgeleitete Tumor-Organoide (englisch: patient-derived tumor organoids, PDTOs) des lebermetastasierten kolorektalen Karzinoms angewandt, um die Entwicklung einer Toleranz gegenüber Chemotherapie nachzubilden. Das erreichten wir, indem wir die KRAS-wildtyp PDTOs ex vivo einer Langzeitbehandlung mit einer klinisch üblichen Erstlinientherapie unterzogen, die aus der Chemotherapie-Doublette FOLFIRI und dem EGFR-spezifischen Antikörper Cetuximab besteht. Nach einer bis zu neun Monate andauernden Behandlung entwickelten die PDTOs eine Toleranz gegenüber FOLFIRI/Cetuximab und reagierten nicht mehr mit Apoptose auf diese Behandlung. Dieser Phänotyp trat unabhängig von Resistenz-vermittelnden Mutationen in klinisch relevanten Genen auf. Stattdessen offenbarte die Sequenzierung des gesamten Transkriptoms (RNA-Sequenzierung der nächsten Generation) eine Induktion der Expression von MYC-Zielgenen in zwei von drei toleranten PDTO-Linien. Die dritte PDTO-Linie entwickelte eine Toleranz gegenüber der Erstlinientherapie durch andere Mechanismen, welche die Hochregulierung von Interferon-α-Zielgenen beinhalteten. Interessanterweise stammten alle drei PDTO-Linien von Tumoren ab, die genomische Amplifikation des chromosomalen Bereichs 20q13.2 zeigten, welcher den Aurora Kinase A (AURKA) Locus enthält. Weiterhin zeigten diese Ursprungstumore erhöhte AURKA mRNA- und Proteinmengen im Vergleich zu normalem Kolonepithel. Eine Behandlung mit dem AURKA-Inhibitor Alisertib stellt eine Strategie zur indirekten MYC-Inhibition in verschiedenen Tumorarten dar und induzierte Apoptose in den drei hier etablierten chemotoleranten PDTO-Linien. Anschließend führten wir mittels CRISPR/Cas9-vermittelter Genommanipulation eine KRASG12D-Mutation in die FOLFIRI/Cetuximab-toleranten PDTO-Linien ein. Wir bestätigten, dass diese klinisch problematische Mutation eine Resistenz gegenüber Ansätzen der einfachen oder dualen Inhibition des EGFR-MAPK-Signalwegs vermittelt. Interessanterweise reduzierte die Kombination aus dualer Inhibition des EGFR-MAPK-Signalweges und AURKA-Hemmer die Zellviabilität der Erstlinientherapie-toleranten KRAS-mutierten PDTOs in größerem Ausmaß als die jeweiligen Einzelbehandlungen. Zudem führte die Behandlung mit dem AURKA-Inhibitor von KRAS-mutierten PDTOs, die durch die Inhibierung des EGFR-MAPK-Signalweges sensibilisiert wurden, zu einer Wiederherstellung der Apoptosereaktion und reduzierte zum Großteil die Reetablierungskapazität der Organoide nach Behandlung. Diese Strategie der Kombinationsbehandlung war besonders erfolgreich in den beiden PDTO-Linien, die erhöhte MYC-Level nach der Ausbildung der Toleranz gegenüber der Erstlinientherapie gezeigt hatten. Um einen tieferen Einblick in das AURKA-Expressionslevel in metastasierten Fällen zu erhalten, führten wir einen immunhistochemischen Nachweis von AURKA in einer gepaarten Kohorte von Patienten mit kolorektalen Krebserkrankung durch. Dabei beobachteten wir, dass die AURKA-Expression in nicht-metastasierten kolorektalen Karzinomen im Vergleich zu exklusiv lebermetastasierten oder lungenmetastasierten kolorektalen Karzinomen leicht erhöht war. Weiterhin korrelierte die AURKA-Expression positiv mit der Abundanz von nukleärem Beta-Catenin, welches ein bekannter Marker für eine aggressive Erkrankung und eine schlechte Überlebensrate der Patienten ist. Letztendlich betont diese Ph.D.-Arbeit das Potential von PDTOs als ex vivo Modell für die Therapietoleranz des kolorektalen Karzinoms, für das mutationsbasierte Fortschreiten der Tumorkrankheit sowie für die Evaluierung solcher Wirkstoffkombinationen zu dienen, die in der Lage sind, Behandlungsresistenzen in einem präklinischen Setting zu überwinden

Proimmunogenic impact of MEK inhibition synergizes with agonist anti-CD40 immunostimulatory antibodies in tumor therapy

Cancer types with lower mutational load and a non-permissive tumor microenvironment are intrinsically resistant to immune checkpoint blockade. While the combination of cytostatic drugs and immunostimulatory antibodies constitutes an attractive concept for overcoming this refractoriness, suppression of immune cell function by cytostatic drugs may limit therapeutic efficacy. Here we show that targeted inhibition of mitogen-activated protein kinase (MAPK) kinase (MEK) does not impair dendritic cell-mediated T cell priming and activation. Accordingly, combining MEK inhibitors (MEKi) with agonist antibodies (Abs) targeting the immunostimulatory CD40 receptor results in potent synergistic antitumor efficacy. Detailed analysis of the mechanism of action of MEKi shows that this drug exerts multiple pro-immunogenic effects, including the suppression of M2-type macrophages, myeloid derived suppressor cells and T-regulatory cells. The combination of MEK inhibition with agonist anti-CD40 Ab is therefore a promising therapeutic concept, especially for the treatment of mutant Kras-driven tumors such as pancreatic ductal adenocarcinoma. Immune checkpoint inhibitors have limited efficacy in tumors with lower mutational burden and non-permissive microenvironment. Here, the authors show that combining MEK inhibition with an agonist anti-CD40 immunostimulatory antibody improves antitumor treatment by inducing immunogenic changes in the tumor microenvironment

Targeting c-MYC through interference with NAMPT and SIRT1 and their association to oncogenic drivers in murine serrated intestinal tumorigenesis1

We recently described a positive feedback loop connecting c-MYC, NAMPT, DBC1 and SIRT1 that contributes to unrestricted cancer cell proliferation. Here we determine the relevance of the loop for serrated route intestinal tumorigenesis using genetically well-defined BrafV600E and K-rasG12D mouse models. In both models we show that c-MYC and SIRT1 protein expression increased through progression from hyperplasia to invasive carcinomas and metastases. It correlated with high NAMPT expression and was directly associated to activation of the oncogenic drivers. Assessing functional and molecular consequences of pharmacological interference with factors of the loop, we found that inhibition of NAMPT resulted in apoptosis and reduced clonogenic growth in human BRAF-mutant colorectal cancer cell lines and patient-derived tumoroids. Blocking SIRT1 activity was only effective when combined with a PI3K inhibitor, whereas the latter antagonized the effects of NAMPT inhibition. Interfering with the positive feedback loop was associated with down-regulation of c-MYC and temporary de-repression of TP53, explaining the anti-proliferative and pro-apoptotic effects. In conclusion we show that the c-MYC-NAMPT-DBC1-SIRT1 positive feedback loop contributes to murine serrated tumor progression. Targeting the feedback loop exerted a unique, dual therapeutic effect of oncoprotein inhibition and tumor suppressor activation. It may therefore represent a promissing target for serrated colorectal cancer, and presumably for other cancer types with deregulated c-MYC

Oncogenic KRas-induced Increase in Fluid-phase Endocytosis is Dependent on N-WASP and is Required for the Formation of Pancreatic Preneoplastic Lesions

Fluid-phase endocytosis is a homeostatic process with an unknown role in tumor initiation. The driver mutation in pancreatic ductal adenocarcinoma (PDAC) is constitutively active KRasG12D, which induces neoplastic transformation of acinar cells through acinar-to-ductal metaplasia (ADM). We have previously shown that KRasG12D-induced ADM is dependent on RAC1 and EGF receptor (EGFR) by a not fully clarified mechanism. Using three-dimensional mouse and human acinar tissue cultures and genetically engineered mouse models, we provide evidence that (i) KRasG12D leads to EGFR-dependent sustained fluid-phase endocytosis (FPE) during acinar metaplasia; (ii) variations in plasma membrane tension increase FPE and lead to ADM in vitro independently of EGFR; and (iii) that RAC1 regulates ADM formation partially through actin-dependent regulation of FPE. In addition, mice with a pancreas-specific deletion of the Neural-Wiskott–Aldrich syndrome protein (N-WASP), a regulator of F-actin, have reduced FPE and impaired ADM emphasizing the in vivo relevance of our findings. This work defines a new role of FPE as a tumor initiating mechanism