femoral-bone-marrow-aspiration-in-live-mice

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Memorial Sloan-Kettering Cancer Center

Recurrent somatic ASXL1 mutations occur in patients with myelodysplastic syndrome, myeloproliferative neoplasms, and acute myeloid leukemia, and are associated with adverse outcome. Despite the genetic and clinical data implicating ASXL1 mutations in myeloid malignancies, the mechanisms of transformation by ASXL1 mutations are not understood. Here, we identify that ASXL1 mutations result in loss of polycomb repressive complex 2 (PRC2)-mediated histone H3 lysine 27 (H3K27) tri-methylation. Through integration of microarray data with genome-wide histone modification ChIP-Seq data, we identify targets of ASXL1 repression, including the posterior HOXA cluster that is known to contribute to myeloid transformation. We demonstrate that ASXL1 associates with the PRC2, and that loss of ASXL1 in vivo collaborates with NRASG12D to promote myeloid leukemogenesis.

ASXL1 mutations promote myeloid transformation through loss of PRC2-mediated gene repression.

Gene discovery efforts in patients with hematopoietic malignancies have brought to the forefront a series of mutations in genes thought to be involved in the epigenetic regulation of gene transcription. These mutations occur in genes known, or suspected, to play a role in modifying cytosine nucleotides on DNA and/or altering the state of histone modifications. Genes such as ASXL1, DNMT3A, EZH2, IDH1/2, MLL1, and TET2 all have been shown to be mutated and/or translocated in patients with myeloid malignancies. Intriguingly, many of the alterations affecting DNA cytosine modifications in myeloid malignancies (mutations in DNMT3A, IDH1/2, and TET2) have also been found in patients with T-cell lymphomas, and EZH2 mutations appear to be critical in T-cell acute lymphoblastic leukemia development as well. In addition, the discovery of frequent mutations in CREBBP, EP300, EZH2, and MLL2 in B-cell lymphomas suggests that epigenetic alterations play a critical role in lymphomagenesis. The purpose of this review is to present functional evidence of how alterations in these epigenetic modifiers promote hematopoietic transformation. The conclusions drawn from these data are valuable in understanding biological mechanisms and potential therapeutic targets.

Epigenetic alterations in hematopoietic malignancies.

Vemurafenib, a selective RAF inhibitor, extends survival among patients with BRAF V600E-mutant melanoma. Vemurafenib inhibits ERK signaling in BRAF V600E-mutant cells but activates ERK signaling in BRAF wild-type cells. This paradoxical activation of ERK signaling is the mechanistic basis for the development of RAS-mutant squamous-cell skin cancers in patients treated with RAF inhibitors. We report the accelerated growth of a previously unsuspected RAS-mutant leukemia in a patient with melanoma who was receiving vemurafenib. Exposure to vemurafenib induced hyperactivation of ERK signaling and proliferation of the leukemic cell population, an effect that was reversed on drug withdrawal.

Progression of RAS-mutant leukemia during RAF inhibitor treatment.

The EZH2 histone methyltransferase is highly expressed in germinal center (GC) B cells and targeted by somatic mutations in B cell lymphomas. Here, we find that EZH2 deletion or pharmacologic inhibition suppresses GC formation and functions. EZH2 represses proliferation checkpoint genes and helps establish bivalent chromatin domains at key regulatory loci to transiently suppress GC B cell differentiation. Somatic mutations reinforce these physiological effects through enhanced silencing of EZH2 targets. Conditional expression of mutant EZH2 in mice induces GC hyperplasia and accelerated lymphomagenesis in cooperation with BCL2. GC B cell (GCB)-type diffuse large B cell lymphomas (DLBCLs) are mostly addicted to EZH2 but not the more differentiated activated B cell (ABC)-type DLBCLs, thus clarifying the therapeutic scope of EZH2 targeting.

EZH2 is required for germinal center formation and somatic EZH2 mutations promote lymphoid transformation.

During wound healing, stem cells provide functional mature cells to meet acute demands for tissue regeneration. Simultaneously, the tissue must maintain a pool of stem cells to sustain its future regeneration capability. However, how these requirements are balanced in response to injury is unknown. Here we demonstrate that after wounding or ultraviolet type B irradiation, melanocyte stem cells (McSCs) in the hair follicle exit the stem cell niche before their initial cell division, potentially depleting the pool of these cells. We also found that McSCs migrate to the epidermis in a melanocortin 1 receptor (Mc1r)-dependent manner and differentiate into functional epidermal melanocytes, providing a pigmented protective barrier against ultraviolet irradiation over the damaged skin. These findings provide an example in which stem cell differentiation due to injury takes precedence over stem cell maintenance and show the potential for developing therapies for skin pigmentation disorders by manipulating McSCs.

Direct migration of follicular melanocyte stem cells to the epidermis after wounding or UVB irradiation is dependent on Mc1r signaling.

Somatic Addition of Sex Combs Like 1 (ASXL1) mutations occur in 10-30% of patients with myeloid malignancies, most commonly in myelodysplastic syndromes (MDSs), and are associated with adverse outcome. Germline ASXL1 mutations occur in patients with Bohring-Opitz syndrome. Here, we show that constitutive loss of Asxl1 results in developmental abnormalities, including anophthalmia, microcephaly, cleft palates, and mandibular malformations. In contrast, hematopoietic-specific deletion of Asxl1 results in progressive, multilineage cytopenias and dysplasia in the context of increased numbers of hematopoietic stem/progenitor cells, characteristic features of human MDS. Serial transplantation of Asxl1-null hematopoietic cells results in a lethal myeloid disorder at a shorter latency than primary Asxl1 knockout (KO) mice. Asxl1 deletion reduces hematopoietic stem cell self-renewal, which is restored by concomitant deletion of Tet2, a gene commonly co-mutated with ASXL1 in MDS patients. Moreover, compound Asxl1/Tet2 deletion results in an MDS phenotype with hastened death compared with single-gene KO mice. Asxl1 loss results in a global reduction of H3K27 trimethylation and dysregulated expression of known regulators of hematopoiesis. RNA-Seq/ChIP-Seq analyses of Asxl1 in hematopoietic cells identify a subset of differentially expressed genes as direct targets of Asxl1. These findings underscore the importance of Asxl1 in Polycomb group function, development, and hematopoiesis.

Deletion of Asxl1 results in myelodysplasia and severe developmental defects in vivo.

Hairy cell leukemia (HCL) is a chronic lymphoproliferative disorder characterized by somatic BRAFV600E mutations. The malignant cell in HCL has immunophenotypic features of a mature B cell, but no normal counterpart along the continuum of developing B lymphocytes has been delineated as the cell of origin. We find that the BRAFV600E mutation is present in hematopoietic stem cells (HSCs) in HCL patients, and that these patients exhibit marked alterations in hematopoietic stem/progenitor cell (HSPC) frequencies. Quantitative sequencing analysis revealed a mean BRAFV600E-mutant allele frequency of 4.97% in HSCs from HCL patients. Moreover, transplantation of BRAFV600E-mutant HSCs from an HCL patient into immunodeficient mice resulted in stable engraftment of BRAFV600E-mutant human hematopoietic cells, revealing the functional self-renewal capacity of HCL HSCs. Consistent with the human genetic data, expression of BRafV600E in murine HSPCs resulted in a lethal hematopoietic disorder characterized by splenomegaly, anemia, thrombocytopenia, increased circulating soluble CD25, and increased clonogenic capacity of B lineage cells-all classic features of human HCL. In contrast, restricting expression of BRafV600E to the mature B cell compartment did not result in disease. Treatment of HCL patients with vemurafenib, an inhibitor of mutated BRAF, resulted in normalization of HSPC frequencies and increased myeloid and erythroid output from HSPCs. These findings link the pathogenesis of HCL to somatic mutations that arise in HSPCs and further suggest that chronic lymphoid malignancies may be initiated by aberrant HSCs.

Article	Year
ASXL1 mutations promote myeloid transformation through loss of PRC2-mediated gene repression. Cancer cell\| PubMed ID: 22897849	2012
Epigenetic alterations in hematopoietic malignancies. International journal of hematology\| PubMed ID: 23015417	2012
Progression of RAS-mutant leukemia during RAF inhibitor treatment. The New England journal of medicine\| PubMed ID: 23134356	2012
EZH2 is required for germinal center formation and somatic EZH2 mutations promote lymphoid transformation. Cancer cell\| PubMed ID: 23680150	2013
Direct migration of follicular melanocyte stem cells to the epidermis after wounding or UVB irradiation is dependent on Mc1r signaling. Nature medicine\| PubMed ID: 23749232	2013
Deletion of Asxl1 results in myelodysplasia and severe developmental defects in vivo. The Journal of experimental medicine\| PubMed ID: 24218140	2013
Hematopoietic Stem Cell Origin of BRAFV600E Mutations in Hairy Cell Leukemia. Science translational medicine\| PubMed ID: 24871132	2014

Young Rock Chung

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