The stem cell-supporting small molecule UM171 triggers Cul3-KBTBD4-mediated degradation of ELM2 domain-harboring proteins

To chemically modulate the ubiquitin-proteasome system for degradation of specific target proteins is currently emerging as an alternative therapeutic modality. Earlier we discovered such properties of the stem cell-supporting small molecule UM171 and identified that members of the CoREST complex (RCOR1 and LSD1) are targeted for degradation. UM171 supports the in vitro propagation of hematopoietic stem cells by transiently perturbing the differentiation-promoting effects of CoREST. Here, we employed global proteomics to map the UM171-targeted proteome and identified the additional target proteins, namely RCOR3, RREB1, ZNF217, and MIER2. Further, we discovered that critical elements recognized by Cul3KBTBD4 ligase in the presence of UM171 are located within the EGL-27 and MTA1 homology 2 (ELM2) domain of the substrate proteins. Subsequent experiments identified conserved amino acid sites in the N-terminus of the ELM2 domain that are essential for UM171-mediated degradation. Overall, our findings provide a detailed account on the ELM2 degrome targeted by UM171 and identify critical sites required for UM171-mediated degradation of specific substrates. Given the target profile, our results are highly relevant in a clinical context and point towards new therapeutic applications for UM171

Transient inhibition of NF-κB signaling enhances ex vivo propagation of human hematopoietic stem cells

Despite extensive studies, defining culture conditions in which hematopoietic stem cells can be expanded ex vivo has been challenging. Here we show that chemical inhibition of the NF- κB signaling pathway leads to a significant improvement of hematopoietic stem cell function from ex vivo cultured human umbilical cord blood derived CD34+ cells. We found a distinct peak of activation of the NF-κB pathway shortly after cells were put in culture, and consequently inhibition of the pathway was both necessary and sufficient during the first 24 hours of culture where it reduced the levels of several pro-inflammatory cytokines. Taken together, NF-κB pathway inhibition facilitates propagation of hematopoietic stem cells in culture and may complement other strategies for hematopoietic stem cell expansion by relieving stress signals that are induced as an immediate response to culture initiation

Combined lentiviral- and RNA-mediated CRISPR/Cas9 delivery for efficient and traceable gene editing in human hematopoietic stem and progenitor cells

The CRISPR/Cas9 system is a versatile tool for functional genomics and forward genetic screens in mammalian cells. However, it has been challenging to deliver the CRISPR components to sensitive cell types, such as primary human hematopoietic stem and progenitor cells (HSPCs), partly due to lentiviral transduction of Cas9 being extremely inefficient in these cells. Here, to overcome these hurdles, we developed a combinatorial system using stable lentiviral delivery of single guide RNA (sgRNA) followed by transient transfection of Cas9 mRNA by electroporation in human cord blood-derived CD34+ HSPCs. We further applied an optimized sgRNA structure, that significantly improved editing efficiency in this context, and we obtained knockout levels reaching 90% for the cell surface proteins CD45 and CD44 in sgRNA transduced HSPCs. Our combinatorial CRISPR/Cas9 delivery approach had no negative influence on CD34 expression or colony forming capacity in vitro compared to non-treated HSPCs. Furthermore, gene edited HSPCs showed intact in vivo reconstitution capacity following transplantation to immunodeficient mice. Taken together, we developed a paradigm for combinatorial CRISPR/Cas9 delivery that enables efficient and traceable gene editing in primary human HSPCs, and is compatible with high functionality both in vitro and in vivo

Combinatorial gene targeting in primary human hematopoietic stem and progenitor cells

The CRISPR/Cas9 system offers enormous versatility for functional genomics but many applications have proven to be challenging in primary human cells compared to cell lines or mouse cells. Here, to establish a paradigm for multiplexed gene editing in primary human cord blood-derived hematopoietic stem and progenitor cells (HSPCs), we used co-delivery of lentiviral sgRNA vectors expressing either Enhanced Green Fluorescent Protein (EGFP) or Kusabira Orange (KuO), together with Cas9 mRNA, to simultaneously edit two genetic loci. The fluorescent markers allow for tracking of either single- or double-edited cells, and we could achieve robust double knockout of the cell surface molecules CD45 and CD44 with an efficiency of ~ 70%. As a functional proof of concept, we demonstrate that this system can be used to model gene dependencies for cell survival, by simultaneously targeting the cohesin genes STAG1 and STAG2. Moreover, we show combinatorial effects with potential synergy for HSPC expansion by targeting the Aryl Hydrocarbon Receptor (AHR) in conjunction with members of the CoREST complex. Taken together, our traceable multiplexed CRISPR/Cas9 system enables studies of genetic dependencies and cooperation in primary HSPCs, and has important implications for modelling polygenic diseases, as well as investigation of the underlying mechanisms of gene interactions

RNAi Screen Identifies MTA1 as an Epigenetic Modifier of Differentiation Commitment in Human HSPCs

The molecular mechanisms regulating key fate decisions of hematopoietic stem cells (HSCs) remain incompletely understood. Here, we targeted global shRNA libraries to primary human hematopoietic stem and progenitor cells (HSPCs) to screen for modifiers of self-renewal and differentiation, and identified metastasis-associated 1 (MTA1) as a negative regulator of human HSPC propagation in vitro. Knockdown of MTA1 by independent shRNAs in primary human cord blood (CB) HSPCs led to a cell expansion during culture and a relative accumulation of immature CD34 +CD90 + cells with perturbed in vitro differentiation potential. Transplantation experiments in immunodeficient mice revealed a significant reduction in human chimerism in both blood and bone marrow from HSPCs with knockdown of MTA1, possibly caused by reduced maturation of blood cells. We further found that MTA1 associates with the nucleosome remodeling deacetylase (NuRD) complex in human HSPCs, and on knockdown of MTA1, we observed an increase in H3K27Ac marks coupled with a downregulation of genes linked to differentiation toward the erythroid lineage. Together, our findings identify MTA1 as a novel regulator of human HSPCs in vitro and in vivo with critical functions for differentiation commitment