3029 – 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.

Genome-wide analysis of transcriptional regulators in human HSPCs reveals a densely interconnected network of coding and noncoding genes

MLL leukemia induction by t(9;11) chromosomal translocation in human hematopoietic stem cells using genome editing

<div>Abstract<p>The conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is a key step in DNA demethylation that is mediated by ten–eleven translocation (TET) enzymes, which require ascorbate/vitamin C. Here, we report the 5hmC landscape of normal hematopoiesis and identify cell type–specific 5hmC profiles associated with active transcription and chromatin accessibility of key hematopoietic regulators. We utilized CRISPR/Cas9 to model <i>TET2</i> loss-of-function mutations in primary human hematopoietic stem and progenitor cells (HSPC). Disrupted cells exhibited increased colonies in serial replating, defective erythroid/megakaryocytic differentiation, and <i>in vivo</i> competitive advantage and myeloid skewing coupled with reduction of 5hmC at erythroid-associated gene loci. Azacitidine and ascorbate restored 5hmC abundance and slowed or reverted the expansion of <i>TET2</i>-mutant clones <i>in vivo</i>. These results demonstrate the key role of 5hmC in normal hematopoiesis and <i>TET2-</i>mutant phenotypes and raise the possibility of utilizing these agents to further our understanding of preleukemia and clonal hematopoiesis.</p>Significance:<p>We show that 5-hydroxymethylation profiles are cell type–specific and associated with transcriptional abundance and chromatin accessibility across human hematopoiesis. <i>TET2</i> loss caused aberrant growth and differentiation phenotypes and disrupted 5hmC and transcriptional landscapes. Treatment of <i>TET2</i> KO HSPCs with ascorbate or azacitidine reverted 5hmC profiles and restored aberrant phenotypes.</p><p><i><a href="https://aacrjournals.org/bloodcancerdiscov/article/doi/10.1158/2643-3230.BCD-3-4-ITI" target="_blank">This article is highlighted in the In This Issue feature, p. 265</a></i></p></div>

Recent evidence suggests that the environment-sensing transcription factor aryl hydrocarbon receptor (AHR) is an important regulator of hematopoiesis. Yet, the mechanisms and extent of AHR-mediated regulation within the most primitive hematopoietic cells, hematopoietic stem and progenitor cells (HSPCs), are poorly understood. Through a combination of transcriptomic and flow cytometric approaches, this study provides new insight into how the AHR influences hematopoietic stem and progenitor cells. Comparative analysis of intraphenotypic transcriptomes of hematopoietic stem cells (HSCs) and multipotent progenitor (MPP) cells from AHR knockout (AHR KO) and wild type mice revealed significant differences in gene expression patterns. Notable among these were differences in expression of cell cycle regulators, specifically an enrichment of G2/M checkpoint genes when Ahr was absent. This included the regulator Aurora A kinase (Aurka, AurA). Analysis of AurA protein levels in HSPC subsets using flow cytometry, in combination with inducible AHR KO or in vivo AHR antagonism, showed that attenuation of AHR increased levels of AurA in HSCs and lineage-biased MPP cells. Overall, these data highlight a potential novel mechanism by which AHR controls HSC homeostasis and HSPC differentiation. These findings advance the understanding of how AHR influences and regulates primitive hematopoiesis.

Whole-Exome Sequencing Identifies Loci Associated with Blood Cell Traits and Reveals a Role for Alternative GFI1B Splice Variants in Human Hematopoiesis

Ex vivo culture of hematopoietic stem and progenitor cells with platelet lysate: Investigating proliferation and erythroid-megakaryocytic lineage effects.

Regulation of in vitro human hematopoietic differentiation by dioxin-like compounds.

Dissecting ELANE neutropenia pathogenicity by human HSC gene editing.

Transient abnormal myelopoiesis (TAM) is a form of clonal hematopoiesis seen in infants with trisomy 21. TAM is caused by mutations in the transcription factor GATA1, leading to the expression of a shortened isoform (GATA1s). TAM clonally evolves at high percentage to myeloid leukemia in Down syndrome (ML-DS) upon acquisition of secondary mutations. Leveraging a virus-free CRISPR platform to introduce GATA1s and additional mutations in primary human fetal liver hematopoietic stem and progenitor cells (hFL-HSPCs) followed by in vitro testing and in vivo xenotransplantation assays, we revealed KANSL1 loss to be a potent oncogenic event driving progression from clonal hematopoiesis to frank leukemia. To define the role of KANSL1 mutations in normal and malignant hematopoiesis, we are performing loss-of-function assays to investigate KANSL1 essentiality. Furthermore, we are establishing dTAG degron knock-in cell lines for proteomic, transcriptomic and epigenomic assays in order to define the KANSL1 interactome in leukemic cells. Future research will explore potential new therapeutic vulnerabilities in ML-DS patients carrying mutated KANSL1.

Epithelial ovarian cancer (EOC) patients exhibit a poor 5-year overall survival rate of approximately 40%, underscoring the urgent need for innovative therapies. Allogeneic natural killer (NK) cell therapy presents a promising and safe therapeutic option, given its ability to discriminate between normal and malignant cells with potent cytotoxic effects against malignant cells. We present the first-in-human study exploiting the safety of the NK cell product designated RNK001, derived ex vivo from umbilical cord blood-derived hematopoietic stem and progenitor cells. This phase 1 INTRO-01 trial (NCT03539406) was initiated to assess the feasibility, safety, and toxicity of intraperitoneal (IP) infusion of RNK001 in EOC patients exhibiting elevated CA125 levels at the second recurrence. RNK001 infusion was supported by IP IL-2 in six patients, and was preceded in one patient by lymphodepleting chemotherapy with cyclophosphamide/fludarabine. RNK001 consisted of 1.2 to 3.0×109 highly activated CD56+CD3- NK cells and was well tolerated, with neither evidence of graft-versus-host disease nor cytokine release syndrome. One patient experienced a grade 3 transient elevation in liver enzymes, another patient exhibited grade 3 ileus caused by disease progression. Notably, five out seven patients demonstrated a reduction in CA125 serum levels of 20-53% at 14days post-infusion. Furthermore, one patient achieved a clinical and biochemical response with radiological stable disease and a progression-free survival of 9months. These findings suggest that intraperitoneal RNK001-based immunotherapy can be safely administered to recurrent EOC patients without inducing severe toxicity, while clinical and biochemical responses warrant further development.

Long-Term Engraftment and Self-Renewal of AML Stem Cells in the Newborn NOD-scid/IL2rgnull Immunodeficient Mouse Model.

Objective The interplay between intrinsic and extrinsic elements involved in the physiology of hematopoietic cells is not completely understood. In the present study, we analyzed the transcriptional profiles of human cord blood-derived hematopoietic stem cells (HSCs), as well as myeloid (MPCs) and erythroid (EPCs) progenitors, and assessed their proliferation and expansion kinetics in vitro. Methods All cell populations were obtained by cell-sorting, and were cultured in liquid cultures supplemented with different cytokine combinations. Their gene expression profiles were determined by RNA microarrays right after cell-sorting, before culture. Results HSCs showed the highest proliferation and expansion capacities in culture, and were found to be more closely related, in transcriptional terms, to MPCs than to EPCs. This correlated with the fact that after 30 days, only cultures initiated with HSCs and MPCs were sustained. Expression of cell cycle and cell division-related genes was enriched in EPCs. Such cells showed significantly higher proliferation than MPCs, however, their expansion potential was reduced, so that cultures initiated with EPCs declined after 15 days and became exhausted by day 30. Proliferation and expansion of HSCs and EPCs were higher in the presence of a cytokine combination that favors erythropoiesis, whereas the growth of MPCs was higher under a cytokine combination that favors myelopoiesis. Conclusion This study shows a correlation between the transcriptional profiles of HSCs, MPCs, and EPCs, and their respective in vitro growth under particular culture conditions. These results may be relevant in the development of ex vivo systems for the expansion of hematopoietic cells for clinical application.

Single Doses of Antibody Drug Conjugates (ADCs) Targeted to CD117 or CD45 Have Potent In Vivo Anti-Leukemia Activity and Survival Benefit in Patient Derived AML Models

Profound Signaling Abnormalities Due to Cytoplasmic Variants of CSF3R in Patients with Myelodysplastic/Myeloproliferative Syndromes Associated with CSF3R Mutations or Mutations of Splicing Factor U2AF1