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Small Molecule Approaches for Targeting Signaling Networks in Cancer and Cellular Reprogramming
Aberrations in signaling networks have long been studied for their contribution to tissue degeneration and cancer. Chemical modulation of such pathways provides an excellent opportunity to fine-tune their activity, owing to the rapid and dose-dependent effects of small molecules. The results of the presented thesis demonstrate the application of small molecules in: (i) studying one of the key pathways in cancer and development–TGFb/Smad signaling; and (ii)modulation of cellular fate towards stemness via targeting self-renewal- and pluripotency-associated signaling molecules. In the first chapter, I describe the use of newly-synthesized molecules derived from the indirubin family of natural compounds in the regulation of R-Smad signaling, with a focus on TGFb-related Smad2/3. Using a large screen encompassing cell lines of various cancerous- and non-cancerous tissue origins, I show that indirubin derivatives (IRDs) collectively, and the IRD E738 analogue specifically, interfere with TGFb/Smad signaling via a profound reduction in steady-state R-Smad levels. My analyses illustrate that this effect is not only due to reduced R-Smad gene transcription by E738, but also a result of protein degradation, demonstrating that the IRD modulates the TGFb/Smad pathway through different mechanisms. Further investigation by transient over-expression of wild-type-, truncated-, and phosphorylation-defective mutant forms of Smad2 and Smad3 reveals the significance of the "linker domain", in particular its phosphorylation status, in the small molecule's regulation of basal Smad stability. Given the role of Smad linker phosphorylation–referred to as "phospho-Smad signaling"–in the malignant switch of TGFb in some tumor types, the potential connection between E738's regulation of steady-state Smad2/3 stability and the phospho-Smad pathway is explored. Using patient-derived cholangiocarcinoma (CCA) cell lines, I show that IRD E738 inhibits oncogenic phospho-Smad isoforms (linker region sites), while maintaining those associated with a cytostatic phenotype (C-terminally phosphorylated sites). This effect reflects the molecule's inhibitory activity on kinases (e.g., GSK3 and CDKs) involved in the phosphorylation of Smad2/3 linker region. The results of the second chapter outline a combined chemical and genetic approach for enhancing the generation and maintenance of human iPSCs. Using a cell-based high-throughput screen, a series of OCT4-inducing compounds (O4Is) are identified, including imidazopyridine analogues, with the lead compound termed O4I3, as well as 4-(tert-Butyl)-N-(2,3-dimethylphenyl) thiazol-2-amine, herein O4I4. The small molecules either alone or in combination are shown to activate pluripotency-associated signaling, and to increase the reprogramming efficiency of human fibroblasts into iPSCs when combined with ectopic expression of the master pluripotency transcription factors: OCT4, SOX2, KLF4, and MYC (known as "OSKM"). Transcriptomic analyses (DNA microarray/RNA-sequencing) and ATAC-sequencing revealed previously unrecognized, targetable molecular events in the path towards pluripotency. Indeed, O4I3 lifts an epigenetic barrier of reprogramming through inhibiting the histone demethylase enzyme, KDM5A, thus allowing the enrichment of H3K4Me3 at the OCT4 promoter. Additionally, applying a combination of O4I3 and O4I4 to OSKM-based reprogramming reveals the involvement of bone morphogenetic protein (BMP)/Smad signaling upstream of high mobility group A1 (HMGA1) in O4I3/4-mediated induction of endogenous OCT4, which in turn initiates the reprogramming process. Altogether, the results of both chapters demonstrate the potential use of new classes of small molecules in targeting signaling networks associated with cancer and somatic cell reprogramming.
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500 Naturwissenschaften und Mathematik
500 Natural sciences and mathematics
570 Biowissenschaften, Biologie
570 Life sciences
610 Medizin
610 Medical sciences Medicine