Development of Small Molecule MEIS Inhibitors that modulate HSC activity.

Meis1, which belongs to TALE-type class of homeobox gene family, appeared as one of the key regulators of hematopoietic stem cell (HSC) self-renewal and a potential therapeutical target. However, small molecule inhibitors of MEIS1 remained unknown. This led us to develop inhibitors of MEIS1 that could modulate HSC activity. To this end, we have established a library of relevant homeobox family inhibitors and developed a high-throughput in silico screening strategy against homeodomain of MEIS proteins using the AutoDock Vina and PaDEL-ADV platform. We have screened over a million druggable small molecules in silico and selected putative MEIS inhibitors (MEISi) with no predicted cytotoxicity or cardiotoxicity. This was followed by in vitro validation of putative MEIS inhibitors using MEIS dependent luciferase reporter assays and analysis in the ex vivo HSC assays. We have shown that small molecules named MEISi-1 and MEISi-2 significantly inhibit MEIS-luciferase reporters in vitro and induce murine (LSKCD34l°w cells) and human (CD34+, CD133+, and ALDHhi cells) HSC self-renewal ex vivo. In addition, inhibition of MEIS proteins results in downregulation of Meis1 and MEIS1 target gene expression including Hif-1α, Hif-2α and HSC quiescence modulators. MEIS inhibitors are effective in vivo as evident by induced HSC content in the murine bone marrow and downregulation of expression of MEIS target genes. These studies warrant identification of first-in-class MEIS inhibitors as potential pharmaceuticals to be utilized in modulation of HSC activity and bone marrow transplantation studies.

c-Myc Inhibitor 10074-G5 Induces Murine and Human Hematopoietic Stem and Progenitor Cell Expansion and HDR Modulator Rad51 Expression.

BACKGROUND: c-Myc plays a major role in the maintenance of glycolytic metabolism and hematopoietic stem cell (HSC) quiescence. OBJECTIVE: Targeting modulators of HSC quiescence and metabolism could lead to HSC cell cycle entry with concomitant expansion. METHODS AND RESULTS: Here we show that c-Myc inhibitor 10074-G5 treatment leads to 2-fold increase in murine LSKCD34low HSC compartment post 7 days. In addition, c-Myc inhibition increases CD34+ and CD133+ human HSC number. c-Myc inhibition leads to downregulation of glycolytic and cyclindependent kinase inhibitor (CDKI) gene expression ex vivo and in vivo. In addition, c-Myc inhibition upregulates major HDR modulator Rad51 expression in hematopoietic cells. Besides, c-Myc inhibition does not alter proliferation kinetics of endothelial cells, fibroblasts or adipose-derived mesenchymal stem cells, however, it limits bone marrow derived mesenchymal stem cell proliferation. We further demonstrate that a cocktail of c-Myc inhibitor 10074-G5 along with tauroursodeoxycholic acid (TUDCA) and i-NOS inhibitor L-NIL provides a robust HSC maintenance and expansion ex vivo as evident by induction of all stem cell antigens analyzed. Intriguingly, the cocktail of c-Myc inhibitor 10074-G5, TUDCA and L-NIL improves HDR related gene expression. CONCLUSION: These findings provide tools to improve ex vivo HSC maintenance and expansion, autologous HSC transplantation and gene editing through modulation of HSC glycolytic and HDR pathways.

Emerging Roles of Meis1 in Cardiac Regeneration, Stem Cells and Cancer.

BACKGROUND: Meis1 is a member of three-amino-acid loop extension (TALE) homeodomain transcription factors. Studies in the last decade have shown that Meis1 has crucial roles in cardiac regeneration, stem cell function, and tumorigenesis. OBJECTIVE: We have recently demonstrated that knocking out of Meis1 in adult cardiomyocytes resulted in the induction of cardiomyocyte proliferation. This suggests that targeting of Meis1 might be utilized in the manipulation of cardiomyocyte cell cycle post cardiac injuries. In addition, hematopoietic stem cell (HSC) specific deletion of Meis1 leads to in vivo expansion of HSCs pool. Thus, targeting Meis1 may lead to not only cell cycle entry but also ex vivo and in vivo expansion of HSCs. On the other hand, Meis1 transcriptionally regulates the expression of hypoxic tumor markers, namely Hif-1α and Hif-2α. Hif-1α and Hif-2α are involved in the induction of cytoplasmic glycolysis and scavenging of reactive oxygen species (ROS), respectively. CONCLUSION: Studies highlight emerging roles of Meis1 towards development of new therapeutic approaches in the treatment of myocardial injuries, bone failure, and cancer.