Development of Small Molecule MEIS Inhibitors that modulate HSC activity

Abstract

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 (LSKCD34 l°w cells) and human (CD34 + , CD133 + , and ALDH hi 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. Meis1 is a member of TALE class of transcription factors 1. Through interaction domains in the N terminus, MEIS1 cooperates in transcription factors PBX1 and HOXA9 to transactivate target genes 2,3. MEIS2 and MEIS3 protein sequences demonstrate a high degree of similarity with MEIS1 4. Meis1 was first described in leukemia mouse model and identified as a viral integration site (reviewed in 5). MEIS proteins are characterized by PBX interaction domains and a highly conserved homeodomain (HD). MEIS1 HD shares identical MEIS2 HD amino acid sequence. Studies to understand how MEIS1 HD interacts with DNA led to crystallization of MEIS1 HD with target DNA and identification of DNA sequence preferentially bound by MEIS proteins as "TGACAG" 6-8. Meis1 is highly expressed in the bone marrow 2,9. Lethality occurs in Meis1 knockout mice at mid gestation (E14.5-15.5) with a number of hematopoietic, vascular and cardiac abnormalities 10-12. Conditional and tissue specific deletion of Meis1 in bone marrow led to loss of HSC quiescence associated with expansion of HSC pool in vivo 13. Meis1 has been shown to regulate HSC metabolism through transcriptional regulation of hypoxia factors including Hif1a and Hif2a 13-16. Deletion of Meis1 or Hif-1α in HSCs leads to reduction in the cytoplasmic glycolysis and induction of mitochondrial phosphorylation. Intriguingly, studies showed a fundamental role of Meis1 in neonatal cardiac regeneration. Increased Me...

Mapping the native interaction surfaces of PREP1 with PBX1 by cross-linking mass-spectrometry and mutagenesis

“…It must be noticed that the use of the HXR9 in clinics has not been reported. Moreover, targeting the MEIS homeodomain by using small molecule derived from compound collections screenings seems effective 73 , but does not impair dimerization with PBX nor nuclear localization of MEIS.…”

Pluripotin facilitates the expansion of hematopoietic stem cells, but restricts the growth of fibroblasts and the proliferation of mesenchymal stem cells from the bone marrow

“…These studies include the use of StemRegenin 1 (AhR antagonist), Garcinol (nonspeci c HAT inhibitor) and Nicotinamide (SIRT1 inhibitor) to reproduce human and mouse HSCs (Boitano et al, 2010;Peled et al, 2012;Singh et al, 2011). We have recently shown that the regulation of hematopoietic factors such as Meis1 and Hif-1a in HSCs and that these factors are needed for HSC quiescence (Kocabas et al, 2012)and targeting with MEIS inhibitors could allow ex vivo HSC expansion (Turan et al, 2020).…”

“…After 7 days of the treatment with the small molecules which are CHIR-99021, Pluripotin and DMSO as a control, lineage negative cells were labelled with Fc Blocker into 1X PBS then surface marker staining for APC-lineage cocktail, PE-C-kit, PE-Cy7-Sca-1 and FITC-CD34 (BD StemFlow Cat No 560492) according to the manufacturer's manual (Stemcell Technologies). The expressions of CD34 Sca-1 and c-Kit, surface markers in the labelled cells were analysed by ow cytometry (BD FACSARIA III) (Turan et al, 2020).…”

MEIS1 and its potential as a cancer therapeutic target (Review)

“…In a recent study, two small molecule MEIS1 inhibitors (MEISi-1 and MEISi-2) were identified using high-throughput in silico screening, and induced hematopoietic stem cell expansion ( 82 ).…”