Discovery of the first small molecules targeting the mRNA binding protein IGF2BP2/IMP2 as potential target in cancer therapy

The mRNA binding protein IGF2BP2/IMP2 is overexpressed in several cancer types, promotes tumorigenesis and tumor progression, and has been suggested to worsen disease outcome. Therefore, inhibition of IMP2 represents a promising approach in cancer therapy. The hypothesis to be tested within this thesis was (I) validate the target, (II) set up a screening assay for small molecule inhibitors of IMP2, and (III) test the biological activity of the obtained hits in vitro and in vivo. In vitro target validation using IMP2 siRNA knockdown and CRISPR/Cas9-mediated IMP2 inactivation showed a reduction in cell viability and proliferation, compared to control cells as determined by MTT assay, and electric cell-substrate impedance sensing (ECIS). Different compound libraries were screened for IMP2 inhibitors using a fluorescence polarization assay, and results were confirmed by thermal shift assay, microscale thermophoresis and saturation transfer difference - NMR. The biological activity of hit compounds was tested in cells expressing different IMP2 levels and confirmed specificity for cells expressing high levels of the target. Hit compounds significantly reduced tumor growth in vivo. In conclusion, our findings support that IMP2 represents a druggable target to reduce tumor cell proliferation. The identified hits provide a basis for subsequent lead generation efforts.Das mRNA-bindende Protein IGF2BP2/IMP2 wird bei verschiedenen Krebserkrankungen überexprimiert. IMP2 fördert sowohl die Entstehung von Tumoren, als auch deren Progression, sodass es mit einer schlechten Krankheitsprognose in Verbindung gebracht wird. Daher stellt die Hemmung dieses Proteins einen vielversprechenden Ansatz in der Krebstherapie dar. Die im Rahmen dieser Arbeit zu testende Hypothese bestand darin, (I) das IMP2 als Ziel zu validieren, (II) robuste Testverfahren für niedermolekulare IMP2-Inhibitoren zu etablieren und (III) die biologische Aktivität der erhaltenen Trefferverbindungen in vitro und in vivo zu testen. Um IMP2 in vitro als Ziel validieren zu können, wurde dieses einerseits unter Verwendung von siRNA herunterreguliert und andererseits mittels CRISPR/Cas9 inaktiviert. Dabei konnte Sowohl im MTT-Test, als auch mit dem Verfahren der elektrischen Zellsubstrat-Impedanzmessung (ECIS) eine Verringerung der Lebensfähigkeit und Proliferation von Krebszellen im Vergleich zu Kontrollzellen gezeigt werden. Fluoreszenzpolarisationsassays wurden benutzt, um verschiedene Substanzbibliotheken auf IMP2-Inhibitoren zu durchsuchen. Positive Ergebnisse konnten unter Verwendung der folgenden Verfahren bestätigt werden: Thermische Verschiebung Assay, Mikroskalige Thermophorese und Sättigungs-Transfer-Differenz - NMR bestätigt. Die biologische Aktivität der Trefferverbindungen wurde in Zellen getestet, die unterschiedliche IMP2 – Spiegel exprimierten; in Zellen mit hoher IMP2-Expression konnte dadurch ebenfalls die Spezifität dieser Verbindungen untermauert werden. Des Weiteren reduzierten die Trefferverbindungen das Tumorwachstum in vivo signifikant. Zusammenfassend zeigen unsere Ergebnisse, dass IMP2 als therapeutisches Ziel die Tumorzellproliferation reduzieren kann. Dabei bilden die identifizierten Trefferverbindungen eine Grundlage bei nachfolgenden Bemühungen Leitstrukturen zu generieren

IGF2 mRNA Binding Protein 2 Transgenic Mice Are More Prone to Develop a Ductular Reaction and to Progress Toward Cirrhosis

The insulin-like growth factor 2 (IGF2) mRNA binding proteins (IMPs/IGF2BPs) IMP1 and 3 are regarded as oncofetal proteins, whereas the hepatic IMP2 expression in adults is controversially discussed. The splice variant IMP2-2/p62 promotes steatohepatitis and hepatocellular carcinoma. Aim of this study was to clarify whether IMP2 is expressed in the adult liver and influences progression toward cirrhosis. IMP2 was expressed at higher levels in embryonic compared to adult tissues as quantified in embryonic, newborn, and adult C57BL/6J mouse livers and suggested by analysis of publicly available human data. In an IMP2-2 transgenic mouse model microarray and qPCR analyses revealed increased expression of liver progenitor cell (LPC) markers Bex1, Prom1, Spp1, and Cdh1 indicating a de-differentiated liver cell phenotype. Induction of these LPC markers was confirmed in human cirrhotic tissue datasets. The LPC marker SPP1 has been described to play a major role in fibrogenesis. Thus, DNA methylation was investigated in order to decipher the regulatory mechanism of Spp1 induction. In IMP2-2 transgenic mouse livers single CpG sites were differentially methylated, as quantified by amplicon sequencing, whereas human HCC samples of a human publicly available dataset showed promoter hypomethylation. In order to study the impact of IMP2 on fibrogenesis in the context of steatohepatitis wild-type or IMP2-2 transgenic mice were fed either a methionine-choline deficient (MCD) or a control diet for 2-12 weeks. MCD-fed IMP2-2 transgenic mice showed a higher incidence of ductular reaction (DR), accompanied by hepatic stellate cell activation, extracellular matrix (ECM) deposition, and induction of the LPC markers Spp1, Cdh1, and Afp suggesting the occurrence of de-differentiated cells in transgenic livers. In human cirrhotic samples IMP2 overexpression correlated with LPC marker and ECM component expression. Progression of liver disease was induced by combined MCD and diethylnitrosamine (DEN) treatment. Combined MCD-DEN treatment resulted in shorter survival of IMP2-2 transgenic compared to wild-type mice. Only IMP2-2 transgenic livers progressed to cirrhosis, which was accompanied by strong DR. In conclusion, IMP2 is an oncofetal protein in the liver that promotes DR characterized by de-differentiated cells toward steatohepatitis-associated cirrhosis development with poor survival