Das Hepatozellulare Karzinom (HCC) gilt als resistent gegen Chemotherapeutika, und erst durch die Entwicklung des Kinase-Inhibitors Sorafenib konnte eine systemische Behandlungsmethode gegen HCCs ermoglicht werden. Eine Monotherapie mit Sorafenib verbessert die durchschnittliche Uberlebensrate von HCC Patienten aber nur um wenige Monate. Daher ist es wichtig, die molekularen Mechanismen der Sorafenib Behandlung zu verstehen, um potenzielle Kombinationstherapien mit besserer Wirkung zu entwickeln.
Die Aktivierung des Ras/MAPK Signalwegs gilt als wichtig fur die Entstehung und Entwicklung von HCCs. Durch die Nutzung eines mosaiken HCC Mausmodels, konnten NrasG12V getriebene Lebertumore in p19Arf-/- Mausen erzeugt werden. Die Sorafenib Behandlung dieser Tumore zeigte einen geringen, aber signifikanten Behandlungseffekt, vergleichbar mit der Reaktion von humanen HCCs auf Sorafenib.
Fur die Identifizierung von Genen, die bei der Resistenzentwicklung der Sorafenib Behandlung eine Rolle spielen, wurde ein in vivo RNA-Interferenz Screening durchgefuhrt. Gepoolte shRNAs (gerichtet gegen amplifizierte Gene humaner HCCs) wurden zusammen mit NrasG12V in murinen, p19Arf-/- Tumoren exprimiert, welche mit Sorafenib oder einem Carrier behandelt wurden. Die Analyse der shRNA Verteilung in den Tumoren beider Gruppen zeigte mehrere shRNAs gegen Mapk14 (p38alpha) stark reduziert in Sorafenib behandelten HCCs, was auf eine Rolle von Mapk14 in der Sorafenib Resistenz hindeutet.
Bei Validierungsexperimenten konnte gezeigt werden, dass die shRNA vermittelte Reduktion von Mapk14 das Uberleben Sorafenib behandelte Mause signifikant verlangert und die Proliferation von HCC Zellen verringert. Mittels regulierbaren shRNAs konnte zudem illustriert werden, dass das Ausschalten von Mapk14 die Sorafenib Behandlung auch in fortgeschrittenen HCCs verbessert. Auch pharmakologische Inhibitoren gegen Mapk14 steigerten den Effekt der Sorafenib Behandlung.
Der Effekt dieser Kombinationstherapie konnte auch in HCC Zelllinien mit anderen genetischen Alterationen und in verschiedenen humanen HCC Zelllinien gezeigt werden. Der Transkriptionsfaktor Atf2 wurde zudem als wichtiger Mediator der Mapk14 vermittelten Resistenz gegen Sorafenib identifiziert.
Die hier prasentierte Studie beschreibt eine Sorafenib basierte Kombinationstherapie fur die Behandlung von HCCs und verdeutlich das Potenzial von in vivo RNA-Interferenz Screenings zur Identifizierung von Genen die Krebstherapien beeinflussen.
The sources of hepatocellular carcinoma are liver progenitor cells or hepatocytes. Interestingly, we observed differences between progenitor cell- and hepatocyte- derived hepatocellular carcinomas in mice. Oncogenic Nras (NrasG12V) efficiently triggers hepatocellular carcinomas derived from p53-/- liver progenitor cells, however almost no tumor growth is observed, when NrasG12V is delivered into p53-/- hepatocytes. Since aggressive hepatocellular carcinomas develop after short latency upon delivery of oncogenic Nras into p19Arf-/- hepatocytes, we suggest that p19Arf mediates a p53-independent tumor suppressive function in hepatocytes. To identify mediators of this p53-independent tumor suppressive function of p19Arf in the mouse liver, we set up an in vivo RNAi screen. As a proof of principle we could show that co-delivery of NrasG12V and p19Arf-shRNAs into p53-/- hepatocytes via transposable elements was able to phenocopy NrasG12V-driven hepatocarcinogenesis in p19Arf-/- livers. A focused shRNAmir library was compiled consisting of shRNAs targeting genes differenzially expressed in NrasG12V; p53-/- mouse livers compared to NrasG12V; p19Arf-/- livers. This shRNA library was divided into several low complexity pools and subjected to a positive selection screen in a p53-/- background. Via deep sequencing of gDNA derived from NrasG12V; p53-/- tumors, we identified several shRNAs knocking down new candidate genes mediating p53-independent tumor suppressive functions of p19Arf in the mouse liver. Among them are genes involved in the mitotic spindle assembly and the spindle checkpoint. Functional validation experiments using single hairpins have already been pursued for several candidates. ShRNA-mediated knockdown of those candidate genes allows for Ras driven tumorigenesis in a p53-deficient background.
Abstract Increased glycolytic flux is a hallmark of cancer; however, an increasing body of evidence indicates that glycolytic ATP production may be dispensable in cancer, as metabolic plasticity allows cancer cells to readily adapt to disruption of glycolysis by increasing ATP production via oxidative phosphorylation. Using functional genomic screening, we show here that liver cancer cells show a unique sensitivity toward aldolase A (ALDOA) depletion. Targeting glycolysis by disrupting the catalytic activity of ALDOA led to severe energy stress and cell cycle arrest in murine and human hepatocellular carcinoma cell lines. With a combination of metabolic flux analysis, metabolomics, stable-isotope tracing and mathematical modelling, we demonstrate that inhibiting ALDOA induced a state of imbalanced glycolysis in which the investment phase outpaced the payoff phase. Targeting ALDOA effectively converted glycolysis from an energy producing into an energy-consuming process. Moreover, we found that depletion of ALDOA extended survival and reduced cancer cell proliferation in an animal model of hepatocellular carcinoma. Thus, our findings indicate that induction of imbalanced glycolysis by targeting ALDOA presents a unique opportunity to overcome the inherent metabolic plasticity of cancer cells.
Abstract Myc oncoproteins are causally involved in the genesis of a large fraction of human tumors. Three closely related Myc proteins, c-myc, N-myc and L-myc, have been implicated in cancer, whereas c-myc was identified as an important oncogenic driver in frequent solid tumors.Applying transposon-based mosaic liver cancer mouse models and direct in vivo shRNA screening technology, we found that bypassing a latent p19Arf- and Aurka mediated G2/M cell cycle arrest is required for development of p53 altered liver carcinomas with activated Ras/MAPK signalling. The resulting tumors depend on high MYC levels for survival. A direct interaction of Aurka and MYC was identified in p53 altered liver cancer cells, which could be efficiently disrupted by conformation changing Aurka inhibitors, resulting in MYC degradation and cell death specifically of p53 altered human and murine hepatoma cells. Treatment studies in mouse models of p53 deficient therapy resistant liver cancer revealed marked therapeutic efficacy of conformation changing Aurka inhibitors, thus suggesting a new therapeutic strategy against this major lethal cancer. Citation Format: Dauch Daniel, Ramona Rudalska, Giacomo Cossa, Jean Charles Nault, Sandrine Imbeaud, Nisar P. Malek, Thomas Longerich, Stefan Laufer, Antti Poso, Jessica Zucman-Rossi, Martin Eilers, Lars Zender. A MYC-Aurka protein complex represents an actionable target in p53 altered liver 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 1257.
Abstract Hepatocellular carcinoma (HCC), the third leading cause of cancer death worldwide, represents a highly chemoresistant tumor and only recently the multikinase inhibitor sorafenib was approved as the first active systemic treatment against HCC. This illustrates that targeted therapies can be effective against HCC and therefore heralds a new era in HCC treatment. However, sorafenib monotherapy increases survival of HCC patients by less than 3 month, thus emphasizing the strong need to understand the molecular mechanisms of sorafenib sensitivity and resistance in order to inform new combination therapies with higher therapeutic efficacy. Activation of the Ras/MAPK signaling pathway was reported to play a major role in liver tumor development and progression. Taking advantage of genetically defined mosaic liver cancer mouse models, we found that sorafenib treatment of NrasG12V driven murine liver carcinomas results in moderate but distinct treatment responses resembling the response rates of sorafenib treated human HCCs. To identify genes mediating resistance or sensitivity towards sorafenib, we conducted an in vivo RNAi screen. Pools of shRNAs targeting genes found amplified in human hepatocellular carcinomas were applied and mice harboring NrasG12V expressing liver carcinomas with stable expression of shRNA library pools were either treated with sorafenib or carrier control. After 5 weeks of treatment, shRNA distribution was quantified in tumors from both cohorts using deep sequencing, whereas enriched shRNAs pinpoint potential resistance genes and depleted shRNAs pinpoint targets that may be exploited as sorafenib sensitizers. Functional validation experiments were performed with multiple independent single shRNAs against each “sensitizing” target and confirmed a significant survival benefit over sorafenib treated tumors that express non-targeting control shRNAs. Importantly, our top-scoring candidate represents a kinase and, taking advantage of readily available pharmacological inhibitors, we show that combination therapy with sorafenib is well tolerated by the mice and results in a significant survival advantage. We also expanded our new combination treatment to murine HCCs of additional genetic backgrounds, overexpressing c-myc or AKT. As therapeutic efficacy was also shown in these models, it is likely that our new combination therapy will be active against a broad range of human HCCs. Furthermore, we found that the newly identified combination therapy significantly decreased proliferation and induced cell death in a panel of well established human hepatoma cell lines. In summary, our study establishes a new sorafenib based combination therapy for the treatment of hepatocellular carcinoma and highlights the potential of in vivo RNAi screens to identify genes that modulate the treatment response of targeted therapies. 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 4876. doi:1538-7445.AM2012-4876
Fibroblast Growth Factor Receptor 4 (FGFR4) is thought to be a driver in several cancer types, most notably in hepatocellular carcinoma. One way to achieve high potency and isoform-selectivity for FGFR4 is covalently targeting a rare cysteine (C552) in the hinge region of its kinase domain that is not present in other FGFR family members (FGFR1-3). Typically, this cysteine is addressed via classical acrylamide electrophiles. We demonstrate that non-canonical covalent “warheads” based on nucleophilic aromatic substitution (SNAr) chemistry can be employed in a rational manner to generate highly potent and (isoform )selective FGFR4 inhibitors with a low intrinsic reactivity. Key compounds showed low- to subnanomolar potency, efficient covalent inactivation, and excellent selectivity over other FGFRs, kinases with an equivalent cysteine and a representative subset of the kinome. Moreover, these compounds achieved nanomolar potencies in cellular assays and demonstrated good microsomal stability highlighting the potential of SNAr-based approaches in covalent inhibitor design.
Abstract Solid tumors evolve significant changes in metabolic pathways during development by virtue of their specific biosynthetic demands, their particular microenvironment and the potential occurrence of toxic metabolites such as reactive oxygen species. However, the development of cancer treatment approaches that are based on the inhibition of biosynthetic routes is impaired due to the high plasticity of metabolic networks, e.g. resulting in activation of compensatory pathways or in an increased exchange of metabolites between cancer cells and the tumor environment. Therefore, such therapies could not be translated into efficient clinical applications so far. Here we show that an enhanced lipogenesis, triggered by a pharmacological activation of the Liver X receptor (LXR), represents a new therapeutic strategy for the treatment of liver carcinoma (HCC). A combination of LXR mediated fatty acid synthesis and concomitant Raf suppression results in oxidative stress, induction of a critical ER stress response and subsequently in apoptosis of different murine and human liver cancer cells. Our mechanistic studies identified Raf as an important regulator of lipid metabolism in liver cancer. We found that Raf-1 directly interacts with Stearoyl-CoA desaturase-1 (Scd1), the central enzyme for the conversion of saturated into mono-unsaturated fatty acids and thereby maintains Scd1 protein stability in HCC cells. Thus, inhibition of Raf by Sorafenib diminished Scd1 protein abundance leading to toxic accumulation of saturated fatty acids and metabolic stress in cancer cells under sustained lipogenesis. Treatment studies in autochthonous liver cancer mouse models and xenograft models of human HCC revealed that a combinatorial therapy, consisting of the LXR agonist T0901317 and Sorafenib is highly potent to suppress liver cancer development and to extend the survival of tumor bearing animals. Such a therapy was efficient against hepatic neoplasia with different metabolic phenotypes and well tolerated by mice, even by animals that already suffer from a fatty liver disease. Taken together, we here propose a pharmacologically induced accumulation of toxic metabolites in cancer cells as a new strategy for efficient metabolic targeting of therapy refractory solid tumors. Citation Format: Ramona Rudalska, Jule Harbig, Marteinn Snaebjoernsson, Lyudmyla Taranets, Florian Heinzmann, Stefan Zwirner, Wei Ciu Hu, Thales Kronenberger, Tae-Won Kang, Antti Poso, Stefan Laufer, Mathias Rosenfeldt, Nisar P. Malek, Bernd Pichler, Nikita Popov, Almut Schulze, Lars Zender, Daniel Dauch. Liver X receptor mediated lipotoxicity represents a treatment option for liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4377.
We established a unique system for performing direct in vivo RNAi screens to genetically dissect cellular signaling networks that regulate proliferation control of hepatocytes during liver damage and regeneration. By combining the well characterized FAH-/- mouse model with a transposon based vector system, we generated chimeric mouse livers. In these mouse livers each hepatocyte expresses FAH, a marker gene and a microRNA based shRNA. To such mouse livers any form of liver damaging protocol can be applied and the regenerative process can be studied. We conducted a first in vivo RNAi screen, using a focused shRNAmir library consisting of 631 constructs targeting 362 genes. Mouse livers were stably repopulated with the shRNAmir library and after repopulation mice were subjected to chronic CCl4 treatment to induce chronic liver damage. The representation of each shRNAmir in the pool, the chimeric mouse liver and after chronic liver damage was determined by deep sequencing analysis. ShRNAs targeting MKK4 showed strong enrichment during liver damage and regeneration. MKK4 belongs to the Map kinase family and is part of two stress kinase signaling pathways, the JNK as well as the p38 pathway. Functional in vivo validation experiments showed that stable knock down of MKK4 by different shRNAs can significantly increase the repopulation efficiency of mouse hepatocytes and also increases the regenerative capacity of chronically damaged mouse livers. In addition we could show that MKK4 suppression also confers an increased robustness of hepatocytes in a model of acute liver failure, resulting in better survival of these animals. Beyond this, primary hepatocytes with stable MKK4 knockdown showed prolonged survival in cell culture and these cells could even be passaged and used to repopulate FAH-/- mouse livers. Protein array and western blot analyses of MKK4 shRNAs versus control-shRNA expressing livers revealed a complex activation pattern of JNK- and p38-signaling downstream factors. Interestingly, the observed pattern does not merely represent an increased JNK over p38 signaling but our data rather suggest that complex cross signaling events between both pathways are involved. Also hepatocytes with stable MKK4 knock down already show higher levels of CyclinD1, suggesting a priming of the cells to leave the G0 state and entering the G1 state of the cell cycle. We developed a new screening platform for pinpointing new regulators of hepatocyte proliferation and liver regeneration. A first in vivo RNAi screen identified MKK4 as an interesting new therapeutic target structure for improving the regenerative capacity under acute and chronic liver damaging conditions.
