Controlling stem cell fate via chemical inhibition of DYRK kinases

© 2016 Dr. Stephanie Frances BellmaineA high-throughput screen for small molecules that can enhance the self-renewal of embryonic stem (ES) cells led to the discovery of ID-8, an indole derivative that can maintain the pluripotency of ES cells in long term culture, under conditions that usually lead to spontaneous differentiation. A biotinylated derivative of ID-8 was found to bind to DYRK kinases, and DYRK1A miRNA knockdown elicited the same enhancement of ES self-renewal as ID-8 treatment. Of the five mammalian DYRK isoforms, DYRK1A is best characterised due to its overexpression in Down's Syndrome (DS), and is believed to promote neural differentiation processes, thus contributing to the neurodegenerative phenotype of DS. In this thesis we provide the first evidence that ID-8 is a potent DYRK1A inhibitor and through kinase profiling demonstrate that ID-8 is selective for DYRK1A and other closely related kinases. We investigate the effects of ID-8 on human ES cell processes, and show that it is a potent inhibitor of neural induction. A range of ID-8 analogues were synthesized and used to establish structure-activity-relationships for the ability of this class of small molecules to inhibit DYRK1A and related kinases, and neural induction of human ES cells, leading to the discovery of 28, an ID-8 analogue with more potent ability to inhibit neural differentiation. Finally, we show that structurally-unrelated DYRK1A inhibitors from the literature do not inhibit neural induction of human ES cells to the same extent as ID-8 and 28, suggesting that effective DYRK1A inhibition is not the sole criterion necessary for inhibition of neural differentiation

Inhibition of DYRK1A disrupts neural lineage specificationin human pluripotent stem cells

Genetic analysis has revealed that the dual specificity protein kinase DYRK1A has multiple roles in the development of the central nervous system. Increased DYRK1A gene dosage, such as occurs in Down syndrome, is known to affect neural progenitor cell differentiation, while haploinsufficiency of DYRK1A is associated with severe microcephaly. Using a set of known and newly synthesized DYRK1A inhibitors, along with CRISPR-mediated gene activation and shRNA knockdown of DYRK1A, we show here that chemical inhibition or genetic knockdown of DYRK1A interferes with neural specification of human pluripotent stem cells, a process equating to the earliest stage of human brain development. Specifically, DYRK1A inhibition insulates the self-renewing subpopulation of human pluripotent stem cells from powerful signals that drive neural induction. Our results suggest a novel mechanism for the disruptive effects of the absence or haploinsufficiency of DYRK1A on early mammalian development, and reveal a requirement for DYRK1A in the acquisition of competence for differentiation in human pluripotent stem cells