Multiplex CRISPR/Cas9-Based Genome Editing in Human Hematopoietic Stem Cells Models Clonal Hematopoiesis and Myeloid Neoplasia.
ABSTRACT: Hematologic malignancies are driven by combinations of genetic lesions that have been difficult to model in human cells. We used CRISPR/Cas9 genome engineering of primary adult and umbilical cord blood CD34+ human hematopoietic stem and progenitor cells (HSPCs), the cells of origin for myeloid pre-malignant and malignant diseases, followed by transplantation into immunodeficient mice to generate genetic models of clonal hematopoiesis and neoplasia. Human hematopoietic cells bearing mutations in combinations of genes, including cohesin complex genes, observed in myeloid malignancies generated immunophenotypically defined neoplastic clones capable of long-term, multi-lineage reconstitution and serial transplantation. Employing these models to investigate therapeutic efficacy, we found that TET2 and cohesin-mutated hematopoietic cells were sensitive to azacitidine treatment. These findings demonstrate the potential for generating genetically defined models of human myeloid diseases, and they are suitable for examining the biological consequences of somatic mutations and the testing of therapeutic agents.
Project description:Cohesin complex members have recently been identified as putative tumor suppressors in hematologic and epithelial malignancies. The cohesin complex guides chromosome segregation, however cohesin-mutant leukemias do not show genomic instability. We hypothesized reduced cohesin function alters chromatin structure and disrupts cis-regulatory architecture of hematopoietic progenitors. We investigated the consequences of Smc3 deletion in normal and malignant hematopoiesis. Bi-allelic Smc3 loss induced bone marrow aplasia with premature sister chromatid separation, and revealed an absolute requirement for cohesin in hematopoietic stem cell function. In contrast, Smc3 haploinsufficiency increased self-renewal in vitro and in vivo including competitive transplantation. Smc3 haploinsufficiency reduced coordinated transcriptional output, including reduced expression of transcription factors and other genes associated with lineage commitment. Smc3 haploinsufficiency cooperated with Flt3-ITD to induce acute leukemia in vivo, with potentiated Stat5 signaling and altered nucleolar topology. These data establish a dose-dependency for cohesin in regulating chromatin structure and hematopoietic stem cell function. ATAC-seq in murine c-kit+ cells for the following genotypes: Smc3 fl/+, Smc3 del/+, Flt3-ITD, Smc3 fl/del Flt3-ITD
Project description:Recurrent mutations in cohesin complex proteins have been identified in pre-leukemic hematopoietic stem cells and during the early development of acute myeloid leukemia and other myeloid malignancies. Although cohesins are involved in chromosome separation and DNA damage repair, cohesin complex functions during hematopoiesis and leukemic development are unclear. Here, we show that mutant cohesin proteins block differentiation of human hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo and enforce stem cell programs. These effects are restricted to immature HSPC populations, where cohesin mutants show increased chromatin accessibility and likelihood of transcription factor binding site occupancy by HSPC regulators including ERG, GATA2, and RUNX1, as measured by ATAC-seq and ChIP-seq. Epistasis experiments show that silencing these transcription factors rescues the differentiation block caused by cohesin mutants. Together, these results show that mutant cohesins impair HSPC differentiation by controlling chromatin accessibility and transcription factor activity, possibly contributing to leukemic disease.
Project description:Cohesin complex members have recently been identified as putative tumor suppressors in hematologic and epithelial malignancies. The cohesin complex guides chromosome segregation; however, cohesin mutant leukemias do not show genomic instability. We hypothesized that reduced cohesin function alters chromatin structure and disrupts cis-regulatory architecture of hematopoietic progenitors. We investigated the consequences of Smc3 deletion in normal and malignant hematopoiesis. Biallelic Smc3 loss induced bone marrow aplasia with premature sister chromatid separation and revealed an absolute requirement for cohesin in hematopoietic stem cell (HSC) function. In contrast, Smc3 haploinsufficiency increased self-renewal in vitro and in vivo, including competitive transplantation. Smc3 haploinsufficiency reduced coordinated transcriptional output, including reduced expression of transcription factors and other genes associated with lineage commitment. Smc3 haploinsufficiency cooperated with Flt3-ITD to induce acute leukemia in vivo, with potentiated Stat5 signaling and altered nucleolar topology. These data establish a dose dependency for cohesin in regulating chromatin structure and HSC function.
Project description:The cohesin complex (consisting of Rad21, Smc1a, Smc3, and Stag2 proteins) is critically important for proper sister chromatid separation during mitosis. Mutations in the cohesin complex were recently identified in a variety of human malignancies including acute myeloid leukemia (AML). To address the potential tumor-suppressive function of cohesin in vivo, we generated a series of shRNA mouse models in which endogenous cohesin can be silenced inducibly. Notably, silencing of cohesin complex members did not have a deleterious effect on cell viability. Furthermore, knockdown of cohesin led to gain of replating capacity of mouse hematopoietic progenitor cells. However, cohesin silencing in vivo rapidly altered stem cells homeostasis and myelopoiesis. Likewise, we found widespread changes in chromatin accessibility and expression of genes involved in myelomonocytic maturation and differentiation. Finally, aged cohesin knockdown mice developed a clinical picture closely resembling myeloproliferative disorders/neoplasms (MPNs), including varying degrees of extramedullary hematopoiesis (myeloid metaplasia) and splenomegaly. Our results represent the first successful demonstration of a tumor suppressor function for the cohesin complex, while also confirming that cohesin mutations occur as an early event in leukemogenesis, facilitating the potential development of a myeloid malignancy.
Project description:SMC3 encodes a subunit of the cohesin complex that has canonical roles in regulating sister chromatids segregation during mitosis and meiosis. Recurrent heterozygous mutations in SMC3 have been reported in acute myeloid leukemia (AML) and other myeloid malignancies. In this study, we investigated whether the missense mutations in SMC3 might have dominant-negative effects or phenocopy loss-of-function effects by comparing the consequences of Smc3-deficient and -haploinsufficient mouse models. We found that homozygous deletion of Smc3 during embryogenesis or in adult mice led to hematopoietic failure, suggesting that SMC3 missense mutations are unlikely to be associated with simple dominant-negative phenotypes. In contrast, haploinsufficiency was tolerated during embryonic and adult hematopoiesis. Under steady-state conditions, Smc3 haploinsufficiency did not alter colony forming in methylcellulose, only modestly decreased mature myeloid cell populations, and led to limited expression changes and chromatin alteration in Lin-cKit+ bone marrow cells. However, following transplantation, engraftment, and subsequent deletion, we observed a hematopoietic competitive disadvantage across myeloid and lymphoid lineages and within the stem/progenitor compartments. This disadvantage was not affected by hematopoietic stresses, but was partially abrogated by concurrent Dnmt3a haploinsufficiency, suggesting that antecedent mutations may be required to optimize the leukemogenic potential of Smc3 mutations.
Project description:Xenotransplantation is frequently used to study normal and malignant hematopoiesis of human cells. However, conventional mouse xenotransplantation models lack essential human-specific bone-marrow (BM)-microenvironment-derived survival, proliferation, and self-renewal signals for engraftment of normal and malignant blood cells. As a consequence, many human leukemias and other hematologic disorders do not robustly engraft in these conventional models. Here, we describe a complete workflow for the generation of humanized ossicles with an accessible BM microenvironment that faithfully recapitulates normal BM niche morphology and function. The ossicles, therefore, allow for accelerated and superior engraftment of primary patient-derived acute myeloid leukemia (AML) and other hematologic malignancies such as myelofibrosis (MF) in mice. The humanized ossicles are formed by in situ differentiation of BM-derived mesenchymal stromal cells (MSCs). Human hematopoietic cells can subsequently be transplanted directly into the ossicle marrow space or by intravenous injection. Using this method, a humanized engraftable BM microenvironment can be formed within 6-10 weeks. Engraftment of human hematopoietic cells can be evaluated by flow cytometry 8-16 weeks after transplantation. This protocol describes a robust and reproducible in vivo methodology for the study of normal and malignant human hematopoiesis in a more physiologic setting.
Project description:Xenotransplantation models represent powerful tools for the investigation of healthy and malignant human hematopoiesis. However, current models do not fully mimic the components of the human bone marrow (BM) microenvironment, and they enable only limited engraftment of samples from some human malignancies. Here we show that a xenotransplantation model bearing subcutaneous humanized ossicles with an accessible BM microenvironment, formed by in situ differentiation of human BM-derived mesenchymal stromal cells, enables the robust engraftment of healthy human hematopoietic stem and progenitor cells, as well as primary acute myeloid leukemia (AML) samples, at levels much greater than those in unmanipulated mice. Direct intraossicle transplantation accelerated engraftment and resulted in the detection of substantially higher leukemia-initiating cell (LIC) frequencies. We also observed robust engraftment of acute promyelocytic leukemia (APL) and myelofibrosis (MF) samples, and identified LICs in these malignancies. This humanized ossicle xenotransplantation approach provides a system for modeling a wide variety of human hematological diseases.
Project description:GABP is an ets transcription factor that regulates genes that are required for myeloid differentiation. The tetrameric GABP complex includes GABP?, which binds DNA via its ets domain, and GABP?, which contains the transcription activation domain. To examine the role of GABP in myeloid differentiation, we generated mice in which Gabpa can be conditionally deleted in hematopoietic tissues. Gabpa knockout mice rapidly lost myeloid cells, and residual myeloid cells were dysplastic and immunophenotypically abnormal. Bone marrow transplantation demonstrated that Gabp? null cells could not contribute to the myeloid compartment because of cell intrinsic defects. Disruption of Gabpa was associated with a marked reduction in myeloid progenitor cells, and Gabp? null myeloid cells express reduced levels of the transcriptional repressor, Gfi-1. Gabp bound and activated the Gfi1 promoter, and transduction of Gabpa knockout bone marrow with Gfi1 partially rescued defects in myeloid colony formation and myeloid differentiation. We conclude that Gabp is required for myeloid differentiation due, in part, to its regulation of the tran-scriptional repressor Gfi-1.
Project description:<i>ASXL1</i> is frequently mutated in a spectrum of myeloid malignancies with poor prognosis. Loss of <i>Asxl1</i> leads to myelodysplastic syndrome-like disease in mice; however, the underlying molecular mechanisms remain unclear. We report that ASXL1 interacts with the cohesin complex, which has been shown to guide sister chromatid segregation and regulate gene expression. Loss of <i>Asxl1</i> impairs the cohesin function, as reflected by an impaired telophase chromatid disjunction in hematopoietic cells. Chromatin immunoprecipitation followed by DNA sequencing data revealed that ASXL1, RAD21, and SMC1A share 93% of genomic binding sites at promoter regions in Lin<sup>-</sup>cKit<sup>+</sup> (LK) cells. We have shown that loss of <i>Asxl1</i> reduces the genome binding of RAD21 and SMC1A and alters the expression of ASXL1/cohesin target genes in LK cells. Our study underscores the ASXL1-cohesin interaction as a novel means to maintain normal sister chromatid separation and regulate gene expression in hematopoietic cells.
Project description:Insertional mutagenesis resulting from the integration of retroviral vectors has led to the discovery of many oncogenes associated with leukemia. We investigated the role of HOXC6, identified by proximal provirus integration in a large animal hematopoietic stem cell gene therapy study, for a potential involvement in hematopoietic stem cell activity and hematopoietic cell fate decision. HOXC6 was overexpressed in the murine bone marrow transplantation model and tested in a competitive repopulation assay in comparison to the known hematopoietic stem cell expansion factor, HOXB4. We have identified HOXC6 as a factor that enhances competitive repopulation capacity in vivo and colony formation in vitro. Ectopic HOXC6 expression also induced strong myeloid differentiation and expansion of granulocyte-macrophage progenitors/common myeloid progenitors (GMPs/CMPs) in vivo, resulting in myeloid malignancies with low penetrance (3 of 17 mice), likely in collaboration with Meis1 because of a provirus integration mapped to the 3' region in the malignant clone. We characterized the molecular basis of HOXC6-induced myeloid differentiation and malignant cell transformation with complementary DNA microarray analysis. Overexpression of HOXC6 induced a gene expression signature similar to several acute myeloid leukemia subtypes when compared with normal GMPs/CMPs. These results demonstrate that HOXC6 acts as a regulator in hematopoiesis and is involved in malignant transformation.