highly-efficient-gene-disruption-murine-human-hematopoietic

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Bone marrow hematopoietic stem cells (HSCs) balance proliferation and differentiation by integrating complex transcriptional and post-translational mechanisms regulated by cell intrinsic and extrinsic factors. We found that transcripts of G(0)/G(1) switch gene 2 (G0S2) are enriched in lineage(-) Sca-1(+) c-kit(+) (LSK) CD150(+) CD48(-) CD41(-) cells, a population highly enriched for quiescent HSCs, whereas G0S2 expression is suppressed in dividing LSK CD150(+) CD48(-) cells. Gain-of-function analyses using retroviral expression vectors in bone marrow cells showed that G0S2 localizes to the mitochondria, endoplasmic reticulum, and early endosomes in hematopoietic cells. Co-transplantation of bone marrow cells transduced with the control or G0S2 retrovirus led to increased chimerism of G0S2-overexpressing cells in femurs, although their contribution to the blood was reduced. This finding was correlated with increased quiescence in G0S2-overexpressing HSCs (LSK CD150(+) CD48(-)) and progenitor cells (LS(-)K). Conversely, silencing of endogenous G0S2 expression in bone marrow cells increased blood chimerism upon transplantation and promoted HSC cell division, supporting an inhibitory role for G0S2 in HSC proliferation. A proteomic study revealed that the hydrophobic domain of G0S2 interacts with a domain of nucleolin that is rich in arginine-glycine-glycine repeats, which results in the retention of nucleolin in the cytosol. We showed that this cytosolic retention of nucleolin occurs in resting, but not proliferating, wild-type LSK CD150(+) CD48(-) cells. Collectively, we propose a novel model of HSC quiescence in which elevated G0S2 expression can sequester nucleolin in the cytosol, precluding its pro-proliferation functions in the nucleolus.

The cytosolic protein G0S2 maintains quiescence in hematopoietic stem cells.

Sexually dimorphic mammalian tissues, including sexual organs and the brain, contain stem cells that are directly or indirectly regulated by sex hormones. An important question is whether stem cells also exhibit sex differences in physiological function and hormonal regulation in tissues that do not show sex-specific morphological differences. The terminal differentiation and function of some haematopoietic cells are regulated by sex hormones, but haematopoietic stem-cell function is thought to be similar in both sexes. Here we show that mouse haematopoietic stem cells exhibit sex differences in cell-cycle regulation by oestrogen. Haematopoietic stem cells in female mice divide significantly more frequently than in male mice. This difference depends on the ovaries but not the testes. Administration of oestradiol, a hormone produced mainly in the ovaries, increased haematopoietic stem-cell division in males and females. Oestrogen levels increased during pregnancy, increasing haematopoietic stem-cell division, haematopoietic stem-cell frequency, cellularity, and erythropoiesis in the spleen. Haematopoietic stem cells expressed high levels of oestrogen receptor-α (ERα). Conditional deletion of ERα from haematopoietic stem cells reduced haematopoietic stem-cell division in female, but not male, mice and attenuated the increases in haematopoietic stem-cell division, haematopoietic stem-cell frequency, and erythropoiesis during pregnancy. Oestrogen/ERα signalling promotes haematopoietic stem-cell self-renewal, expanding splenic haematopoietic stem cells and erythropoiesis during pregnancy.

Oestrogen increases haematopoietic stem-cell self-renewal in females and during pregnancy.

Neurosphere formation is commonly used as a surrogate for neural stem cell (NSC) function but the relationship between neurosphere-initiating cells (NICs) and NSCs remains unclear. We prospectively identified, and isolated by flow cytometry, adult mouse lateral ventricle subventricular zone (SVZ) NICs as Glast(mid)EGFR(high)PlexinB2(high)CD24(-/low)O4/PSA-NCAM(-/low)Ter119/CD45(-) (GEPCOT) cells. They were highly mitotic and short-lived in vivo based on fate-mapping with Ascl1(CreERT2) and Dlx1(CreERT2). In contrast, pre-GEPCOT cells were quiescent, expressed higher Glast, and lower EGFR and PlexinB2. Pre-GEPCOT cells could not form neurospheres but expressed the stem cell markers Slc1a3-CreER(T), GFAP-CreER(T2), Sox2(CreERT2), and Gli1(CreERT2) and were long-lived in vivo. While GEPCOT NICs were ablated by temozolomide, pre-GEPCOT cells survived and repopulated the SVZ. Conditional deletion of the Bmi-1 polycomb protein depleted pre-GEPCOT and GEPCOT cells, though pre-GEPCOT cells were more dependent upon Bmi-1 for Cdkn2a (p16(Ink4a)) repression. Our data distinguish quiescent NSCs from NICs and make it possible to study their properties in vivo.DOI: http://dx.doi.org/10.7554/eLife.02669.001.

Prospective identification of functionally distinct stem cells and neurosphere-initiating cells in adult mouse forebrain.

The liver kinase B1 (Lkb1)/AMP-activated protein kinase (AMPK) pathway maintains metabolic homeostasis of cells by promoting catabolism and inhibiting anabolism. Activation of this pathway induces tumor suppressors, including p53, and leads to inhibition of the mammalian target of rapamycin pathway, which often is overactivated in multiple cancers. Thus, the Lkb1/AMPK pathway suppresses cancer by negatively regulating cell proliferation. However, recent studies of mouse hematopoietic stem cells (HSCs) revealed that Lkb1 is critical for maintaining HSCs, rather than limiting their proliferation. Furthermore, the role of AMPK in cancer cells has remained elusive. Outstanding questions concern the role of the Lkb1/AMPK pathway in regulating the metabolism and proliferation of cancer cells within a physiological setting. This review focuses on the function of the Lkb1/AMPK pathway in HSCs and leukemia and provides an overview of the therapeutic strategies aimed at targeting this pathway in cancer.

The role of the Lkb1/AMPK pathway in hematopoietic stem cells and Leukemia.

Epigenetic dysregulation is involved in the initiation and progression of many epithelial cancers. BMI1, a component of the polycomb protein family, plays a key role in these processes by controlling the histone ubiquitination and long-term repression of multiple genomic loci. BMI1 has previously been implicated in pancreatic homeostasis and the function of pancreatic cancer stem cells. However, no work has yet addressed its role in the early stages of pancreatic cancer development. Here, we show that BMI1 is required for the initiation of murine pancreatic neoplasia using a novel conditional knockout of Bmi1 in combination with a Kras(G12D)-driven pancreatic cancer mouse model. We also demonstrate that the requirement for Bmi1 in pancreatic carcinogenesis is independent of the Ink4a/Arf locus and at least partially mediated by dysregulation of reactive oxygen species. Our data provide new evidence of the importance of this epigenetic regulator in the genesis of pancreatic cancer.

Bmi1 is required for the initiation of pancreatic cancer through an Ink4a-independent mechanism.

How cancer cells adapt to metabolically adverse conditions in patients and strive to proliferate is a fundamental question in cancer biology. Here we show that AMP-activated protein kinase (AMPK), a metabolic checkpoint kinase, confers metabolic stress resistance to leukemia-initiating cells (LICs) and promotes leukemogenesis. Upon dietary restriction, MLL-AF9-induced murine acute myeloid leukemia (AML) activated AMPK and maintained leukemogenic potential. AMPK deletion significantly delayed leukemogenesis and depleted LICs by reducing the expression of glucose transporter 1 (Glut1), compromising glucose flux, and increasing oxidative stress and DNA damage. LICs were particularly dependent on AMPK to suppress oxidative stress in the hypoglycemic bone marrow environment. Strikingly, AMPK inhibition synergized with physiological metabolic stress caused by dietary restriction and profoundly suppressed leukemogenesis. Our results indicate that AMPK protects LICs from metabolic stress and that combining AMPK inhibition with physiological metabolic stress potently suppresses AML by inducing oxidative stress and DNA damage.

AMPK Protects Leukemia-Initiating Cells in Myeloid Leukemias from Metabolic Stress in the Bone Marrow.

Metabolic homeostasis is a fundamental property of cells that becomes dysregulated in cancer to meet the altered, often heightened, demand for metabolism for increased growth and proliferation. Oncogenic mutations can directly change cellular metabolism in a cell-intrinsic manner, priming cells for malignancy. Additionally, cell-extrinsic cues from the microenvironment, such as hypoxia, nutrient availability, oxidative stress, and crosstalk from surrounding cells can also affect cancer cell metabolism, and produce metabolic heterogeneity within the tumor. Here, we highlight recent findings revealing the complexity and adaptability of leukemia cells to coordinate metabolism.

Cell intrinsic and extrinsic regulation of leukemia cell metabolism.

Our understanding of the mechanisms that regulate hematopoietic stem/progenitor cells (HSPCs) has been advanced by the ability to genetically manipulate mice; however, germline modification is time consuming and expensive. Here, we describe fast, efficient, and cost-effective methods to directly modify the genomes of mouse and human HSPCs using the CRISPR/Cas9 system. Using plasmid and virus-free delivery of guide RNAs alone into Cas9-expressing HSPCs or Cas9-guide RNA ribonucleoprotein (RNP) complexes into wild-type cells, we have achieved extremely efficient gene disruption in primary HSPCs from mouse (>60%) and human (∼75%). These techniques enabled rapid evaluation of the functional effects of gene loss of Eed, Suz12, and DNMT3A. We also achieved homology-directed repair in primary human HSPCs (>20%). These methods will significantly expand applications for CRISPR/Cas9 technologies for studying normal and malignant hematopoiesis.

Highly Efficient Genome Editing of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9.

Leukemia is characterized by the uncontrolled production of leukemic cells and impaired normal hematopoiesis. Although the combination of chemotherapies and hematopoietic stem cell transplantation has significantly improved the outcome of leukemia patients, a proportion of patients still suffer from relapse after treatment. Upon relapse, a phenomenon termed "lineage switch" is observed in a subset of leukemia patients, in which conversion of lymphoblastic leukemia to myeloid leukemia oris observed. A rare entity of leukemia called mixed-phenotype acute leukemia exhibits co-expression of markers representing two or three lineages. These two phenotypes regarding the lineage ambiguity suggest that the fate of some leukemia retain or acquire a certain degree of plasticity. Studies using animal models provide insight into how lineage specifying transcription factors can enforce or convert a fate in hematopoietic cells. Modeling lineage conversion in normal hematopoietic progenitor cells may improve our current understanding of how lineage switch occurs in leukemia. In this review, we will summarize the role of transcription factors and microenvironmental signals that confer fate plasticity to normal hematopoietic progenitor cells, and their potential to regulate lineage switching in leukemias. Future efforts to uncover the mechanisms contributing to lineage conversion in both normal hematopoiesis and leukemia may pave the way to improve current therapeutic strategies.

Transcriptional and Microenvironmental Regulation of Lineage Ambiguity in Leukemia.

ENLightening YEATS in antagonizing polycomb repression.

Activation of the unfolded protein response (UPR) sustains protein homeostasis (proteostasis) and plays a fundamental role in tissue maintenance and longevity of organisms. Long-range control of UPR activation has been demonstrated in invertebrates, but such mechanisms in mammals remain elusive. Here, we show that the female sex hormone estrogen regulates the UPR in hematopoietic stem cells (HSCs). Estrogen treatment increases the capacity of HSCs to regenerate the hematopoietic system upon transplantation and accelerates regeneration after irradiation. We found that estrogen signals through estrogen receptor α (ERα) expressed in hematopoietic cells to activate the protective Ire1α-Xbp1 branch of the UPR. Further, ERα-mediated activation of the Ire1α-Xbp1 pathway confers HSCs with resistance against proteotoxic stress and promotes regeneration. Our findings reveal a systemic mechanism through which HSC function is augmented for hematopoietic regeneration.

Article	Year
The cytosolic protein G0S2 maintains quiescence in hematopoietic stem cells. PloS one\| PubMed ID: 22693613	2012
Oestrogen increases haematopoietic stem-cell self-renewal in females and during pregnancy. Nature\| PubMed ID: 24451543	2014
Prospective identification of functionally distinct stem cells and neurosphere-initiating cells in adult mouse forebrain. eLife\| PubMed ID: 24843006	2014
The role of the Lkb1/AMPK pathway in hematopoietic stem cells and Leukemia. Critical reviews in oncogenesis\| PubMed ID: 25404152	2014
Bmi1 is required for the initiation of pancreatic cancer through an Ink4a-independent mechanism. Carcinogenesis\| PubMed ID: 25939753	2015
AMPK Protects Leukemia-Initiating Cells in Myeloid Leukemias from Metabolic Stress in the Bone Marrow. Cell stem cell\| PubMed ID: 26440282	2015
Cell intrinsic and extrinsic regulation of leukemia cell metabolism. International journal of hematology\| PubMed ID: 26897135	2016
Highly Efficient Genome Editing of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9. Cell reports\| PubMed ID: 27783956	2016
Transcriptional and Microenvironmental Regulation of Lineage Ambiguity in Leukemia. Frontiers in oncology\| PubMed ID: 29164065	2017
ENLightening YEATS in antagonizing polycomb repression. Blood\| PubMed ID: 29438969	2018
ERα promotes murine hematopoietic regeneration through the Ire1α-mediated unfolded protein response. eLife\| PubMed ID: 29451493	2018

Daisuke Nakada

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