Daniel C Link

Washington University in St. Louis, San Luis, Missouri, United States

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Publications (135)1708.02 Total impact


  • No preview · Conference Paper · Dec 2015
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    ABSTRACT: Small nucleolar RNAs (snoRNAs) are small (90-300nt) non-coding guide RNAs found in all multicellular organisms. H/ACA and CD box snoRNAs localize to the nucleolar region and are part of a catalytic multicomponent protein complex selecting target RNAs based on complementarity. They are responsible for the site-specific pseudouridylation and 2' O methylation of ribosomal RNAs, respectively. ScaRNAs localize to the Cajal body and are responsible for the methylation and pseudouridylation of splicesomal RNAs U1, U2, U4, U5, and U12. There are also orphan snoRNAs without any known RNA targets. Recent studies have suggested an expanded role for snoRNAs outside of ribosomal biogenesis, including the regulation of RNA splicing and chromatin remodeling. Moreover, emerging data suggest that aberrantly expressed snoRNAs may contribute to neoplastic transformation. Here, we characterize snoRNA expression in normal human hematopoiesis and in AML. We first developed a novel strategy to identify and quantify properly processed snoRNAs. This is important, since standard RNA sequencing or array-based analyses cannot distinguish between processed snoRNAs and primary mRNA transcripts of the host genes (most snoRNAs are contained in the introns of coding genes). In brief, we take advantage of the fact that, like miRNAs, snoRNAs contain a free 3'-hydroxyl group, which allows for efficient ligation to sequencing adaptors. Following size selection (20-200 nt) to enrich for small non-coding RNAs, the libraries are sequenced on the Illumina next generation sequencing platform. We also have developed a novel analysis pipeline that maps areas of contiguous alignment in the genome, forming ab initio "clusters" representing snoRNAs. Using this sequencing assay, we first interrogated snoRNA expression in hematopoietic cells from healthy individuals. Specifically, we sequenced small RNA libraries derived from CD34+ cells, promyelocytes, neutrophils, monocytes, T-cells, and B-cells. Of the 269 known snoRNAs, 132 (49%) were expressed in one or more hematopoietic cell population. Likewise, 80 of 112 (71%) of known H/ACA box snoRNAs, and all 21 scaRNAs were expressed. In addition, we identified (and computationally validated using SnoReport, snoGPS, and an in-house snoFinder script) 8 putative novel snoRNAs (1 H/ACA box and 7 CD box snoRNAs). Most snoRNAs were stably expressed across all hematopoietic lineages. However, there were numerous examples of snoRNAs that were specifically enriched in a specific hematopoietic cell population (e.g., CD34+ cells). We also identified several snoRNAs that were up- or down-regulated during granulocytic differentiation. For example, many of the orphan C/D box snoRNAs contained in the imprinted DLK/DIO3 locus on chromosome 14q32 are significantly down-regulated during granulocytic differentiation. To determine whether snoRNAs are frequently dysregulated in AML, we next sequenced small non-coding RNAs isolated from the bone marrow of 33 patients with de novo acute myeloid leukemia (all with a normal karyotype). Using a strict 5% false discovery rate, only 9.3% of CD box snoRNAs and 0.9% of H/ACA box snoRNAs were found to have significantly increased or decreased expression compared with normal CD34+ cells (including some of the putative novel snoRNAs). Of note, no differentially expressed snoRNAs were detected comparing AML with or without DNMT3A mutations or with or without IDH1/2 mutations. We next interrogated published whole genome sequencing data to determine whether there were any cytogenetically silent genetic alterations in snoRNAs. No recurring point mutations or small insertions/deletions were detected in snoRNA genes in 50 cases of AML. In summary, we developed a new next-generation sequencing approach and analysis pipeline to quantify snoRNAs. We show that, compared with coding genes, snoRNA expression is more stable across different hematopoietic lineages, consistent with a housekeeping function. However, examples of developmentally-regulated and lineage-restricted snoRNA expression were identified. Finally, we show that a small subset of snoRNAs appear to be dysregulated in AML, although genetic alterations the specifically target snoRNAs appear to be rare in AML.
    No preview · Conference Paper · Dec 2015
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    ABSTRACT: There is interest in using leukemia-gene panels and next-generation sequencing (NGS) to assess acute myelogenous leukemia (AML) response to induction chemotherapy. Studies have shown that patients with AML in morphologic remission may continue to have clonal hematopoiesis with populations closely related to the founding AML clone and that this confers an increased risk of relapse. However, it remains unknown how induction chemotherapy is influencing the clonal evolution of a patient's non-leukemic hematopoietic population. Here we report that 5 of 15 patients with genetic clearance of their founding AML clone after induction chemotherapy had a concomitant expansion of a hematopoietic population unrelated to the initial AML. These populations frequently harbored somatic mutations in genes recurrently mutated in AML or myelodysplastic syndromes (MDS) and were detectable at very low frequencies at the time of AML diagnosis. These results suggest that non-leukemic hematopoietic stem and progenitor cells (HSPCs), harboring specific aging-acquired mutations, may have a competitive fitness advantage after induction chemotherapy, expand, and persist long after the completion of chemotherapy. Although the clinical importance of these "rising" clones remains to be determined, it will be important to distinguish them from leukemia-related populations when assessing for molecular responses to induction chemotherapy.
    No preview · Article · Dec 2015 · Blood

  • No preview · Article · Nov 2015 · Cancer Research
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    ABSTRACT: Familial clustering of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) can be caused by inherited factors. We screened 59 individuals from 17 families with 2 or more biological relatives with MDS/AML for variants in 12 genes with established roles in predisposition to MDS/AML, and identified a pathogenic germline variant in 5 families (29%). Extending the screen with a panel of 264 genes that are recurrently mutated in de novo AML, we identified rare, non-synonymous germline variants in 4 genes segregating with MDS/AML in an additional 2 families each. Somatic mutations are required for progression to MDS/AML in these familial cases. Using a combination of targeted and exome sequencing of tumor and matched normal samples from 26 familial MDS/AML cases and asymptomatic carriers, we identified recurrent frameshift mutations in the cohesin-associated factor PDS5B, co-occurrence of somatic ASXL1 mutations with germline GATA2 mutations, and recurrent mutations in other known MDS/AML drivers. Mutations in genes that are recurrently mutated in de novo AML were under-represented in the familial MDS/AML cases, although the total number of somatic mutations per exome was the same. Finally, clonal skewing of hematopoiesis was detected in 67% of young, asymptomatic RUNX1 carriers, providing a potential biomarker that could be used for surveillance in these high-risk families.
    No preview · Article · Oct 2015 · Blood
  • Laura M Calvi · Daniel C Link
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    ABSTRACT: The bone marrow microenvironment contains a heterogeneous population of stromal cells organized into niches that support hematopoietic stem cells (HSCs) and other lineage-committed hematopoietic progenitors. The stem cell niche generates signals that regulate HSC self-renewal, quiescence, and differentiation. Here, we review recent studies that highlight the heterogeneity of the stromal cells that comprise stem cell niches and the complexity of the signals that they generate. We highlight emerging data that stem cell niches in the bone marrow are not static but instead responsive to environmental stimuli. Finally, we review recent data showing that hematopoietic niches are altered in certain hematopoietic malignancies, and we discuss how these alterations might contribute to disease pathogenesis.
    No preview · Article · Oct 2015 · Blood
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    ABSTRACT: In acute lymphoblastic leukemia (ALL) the bone marrow microenvironment provides growth and survival signals that may confer resistance to chemotherapy. Granulocyte colony-stimulating factor (G-CSF) potently inhibits lymphopoiesis by targeting stromal cells that comprise the lymphoid niche in the bone marrow. To determine whether lymphoid niche disruption by G-CSF sensitizes ALL cells to chemotherapy, we conducted a pilot study of G-CSF in combination with chemotherapy in patients with relapsed or refractory ALL. Thirteen patients were treated on study; three patients achieved a complete remission (CR/CRi) for an overall response rate of 23%. In the healthy volunteers, G-CSF treatment disrupted the lymphoid niche, as evidenced by reduced expression of CXCL12, interleukin-7, and osteocalcin. However, in most patients with relapsed/refractory ALL expression of these genes was markedly suppressed at baseline. Thus, although G-CSF treatment was associated with ALL cell mobilization into the blood, and increased apoptosis of bone marrow resident ALL cells, alterations in the bone marrow microenvironment were modest and highly variable. These data suggest that disruption of lymphoid niches by G-CSF to sensitize ALL cells to chemotherapy may be best accomplished in the consolidation where the bone marrow microenvironment is more likely to be normal.
    No preview · Article · Oct 2015 · Leukemia research
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    ABSTRACT: Tests that predict outcomes for patients with acute myeloid leukemia (AML) are imprecise, especially for those with intermediate risk AML. To determine whether genomic approaches can provide novel prognostic information for adult patients with de novo AML. Whole-genome or exome sequencing was performed on samples obtained at disease presentation from 71 patients with AML (mean age, 50.8 years) treated with standard induction chemotherapy at a single site starting in March 2002, with follow-up through January 2015. In addition, deep digital sequencing was performed on paired diagnosis and remission samples from 50 patients (including 32 with intermediate-risk AML), approximately 30 days after successful induction therapy. Twenty-five of the 50 were from the cohort of 71 patients, and 25 were new, additional cases. Whole-genome or exome sequencing and targeted deep sequencing. Risk of identification based on genetic data. Mutation patterns (including clearance of leukemia-associated variants after chemotherapy) and their association with event-free survival and overall survival. Analysis of comprehensive genomic data from the 71 patients did not improve outcome assessment over current standard-of-care metrics. In an analysis of 50 patients with both presentation and documented remission samples, 24 (48%) had persistent leukemia-associated mutations in at least 5% of bone marrow cells at remission. The 24 with persistent mutations had significantly reduced event-free survival vs the 26 who cleared all mutations (median [95% CI]: 6.0 months [95% CI, 3.7-9.6] for persistent mutations vs 17.9 months [95% CI, 11.3-40.4] for cleared mutations, log-rank P < .001; hazard ratio [HR], 3.67 [95% CI, 1.93-7.11], P < .001) and reduced overall survival (median [95% CI]: 10.5 months [95% CI, 7.5-22.2] for persistent mutations vs 42.2 months [95% CI, 20.6-not estimable] for cleared mutations, log-rank P = .003; HR, 2.86 [95% CI, 1.39-5.88], P = .004). Among the 32 patients with intermediate cytogenetic risk, the 14 patients with persistent mutations had reduced event-free survival compared with the 18 patients who cleared all mutations (median [95% CI]: 8.8 months [95% CI, 3.7-14.6] for persistent mutations vs 25.6 months [95% CI, 11.4-not estimable] for cleared mutations, log-rank P = .003; HR, 3.32 [95% CI, 1.44-7.67], P = .005) and reduced overall survival (median [95% CI]: 19.3 months [95% CI, 7.5-42.3] for persistent mutations vs 46.8 months [95% CI, 22.6-not estimable] for cleared mutations, log-rank P = .02; HR, 2.88 [95% CI, 1.11-7.45], P = .03). The detection of persistent leukemia-associated mutations in at least 5% of bone marrow cells in day 30 remission samples was associated with a significantly increased risk of relapse, and reduced overall survival. These data suggest that this genomic approach may improve risk stratification for patients with AML.
    No preview · Article · Aug 2015 · JAMA The Journal of the American Medical Association
  • Irfan J Lodhi · Daniel C Link · Clay F Semenkovich

    No preview · Article · May 2015 · Cell metabolism
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    ABSTRACT: The mechanisms that mediate the shift from lymphopoiesis to myelopoiesis in response to infectious stress are largely unknown. We show that treatment with G-CSF, which is often induced during infection, results in marked suppression of B lymphopoiesis at multiple stages of B-cell development. Mesenchymal-lineage stromal cells in the bone marrow, including CXCL12-abundant reticular (CAR) cells and osteoblasts, constitutively support B lymphopoiesis through the production of multiple B trophic factors. G-CSF acting through a monocytic cell intermediate reprograms these stromal cells altering their capacity to support B lymphopoiesis. G-CSF treatment is associated with an expansion of CAR cells and a shift towards osteogenic lineage commitment. It markedly suppresses the production of multiple B trophic factors by CAR cells and osteoblasts, including CXCL12, kit ligand, interleukin-6, interleukin-7, and insulin-like growth factor-1. Targeting bone marrow stromal cells is one mechanism by which inflammatory cytokines, such as G-CSF, actively suppress lymphopoiesis. Copyright © 2015 American Society of Hematology.
    No preview · Article · Mar 2015 · Blood
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    ABSTRACT: MIR233 is genetically or epigenetically silenced in a subset of acute myeloid leukemia (AML). MIR223 is normally expressed throughout myeloid differentiation and highly expressed in hematopoietic stem cells (HSCs). However, the contribution of MIR223 loss to leukemic transformation and HSC function is largely unknown. Herein, we characterize HSC function and myeloid differentiation in Mir223 deficient mice.We show that Mir223 loss results in a modest expansion of myeloid progenitors, but is not sufficient to induce a myeloproliferative disorder. Loss of Mir223 had no discernible effect on HSC quiescence, long-term repopulating activity, or self-renewal capacity. These results suggest that MIR223 loss is likely not an initiating event in AML but may cooperate with other AML associated oncogenes to induce leukemogenesis.
    Preview · Article · Mar 2015 · PLoS ONE
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    ABSTRACT: Leukemia is one of the leading journals in hematology and oncology. It is published monthly and covers all aspects of the research and treatment of leukemia and allied diseases. Studies of normal hemopoiesis are covered because of their comparative relevance.
    Full-text · Article · Feb 2015 · Leukemia
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    ABSTRACT: Phosphatase and tensin homolog (PTEN) is a critical negative regulator of the phosphoinositide-3 kinase pathway, members of which play integral roles in natural killer (NK) cell development and function. However, the functions of PTEN in NK cell biology remain unknown. Here, we used an NK cell-specific PTEN-deletion mouse model to define the ramifications of intrinsic NK cell PTEN loss in vivo. In these mice, there was a significant defect in NK cell numbers in the bone marrow and peripheral organs despite increased proliferation and intact peripheral NK cell maturation. Unexpectedly, we observed a significant expansion of peripheral blood NK cells and the premature egress of NK cells from the bone marrow. The altered trafficking of NK cells from peripheral organs into the blood was due to selective hyperresponsiveness to the blood localizing chemokine S1P. To address the importance of this trafficking defect to NK cell immune responses, we investigated the ability of PTEN-deficient NK cells to traffic to a site of tumor challenge. PTEN-deficient NK cells were defective at migrating to distal tumor sites but were more effective at clearing tumors actively introduced into the peripheral blood. Collectively, these data identify PTEN as an essential regulator of NK cell localization in vivo during both homeostasis and malignancy.
    Full-text · Article · Feb 2015 · Proceedings of the National Academy of Sciences
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    ABSTRACT: Fatty acid synthase (FAS) is altered in metabolic disorders and cancer. Conventional FAS null mice die in utero, so effects of whole-body inhibition of lipogenesis following development are unknown. Inducible global knockout of FAS (iFASKO) in mice was lethal due to a disrupted intestinal barrier and leukopenia. Conditional loss of FAS was associated with the selective suppression of granulopoiesis without disrupting granulocytic differentiation. Transplantation of iFASKO bone marrow into wild-type mice followed by Cre induction resulted in selective neutrophil depletion, but not death. Impaired lipogenesis increased ER stress and apoptosis in neutrophils by preferentially decreasing peroxisome-derived membrane phospholipids containing ether bonds. Inducible global knockout of PexRAP, a peroxisomal enzyme required for ether lipid synthesis, also produced neutropenia. FAS knockdown in neutrophil-like HL-60 cells caused cell loss that was partially rescued by ether lipids. Inhibiting ether lipid synthesis selectively constrains neutrophil development, revealing an unrecognized pathway in immunometabolism. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Jan 2015 · Cell metabolism
  • Daniel C. Link
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    ABSTRACT: CXCL12 (stromal-derived factor 1, SDF-1) is a chemokine that is constitutively expressed at high levels in the bone marrow and is a key component of the stem cell niche. Its major receptor CXCR4 is broadly expressed on hematopoietic cells, including hematopoietic stem cells (HSCs). The CXCL12/CXCR4 axis plays a key role in regulating many aspects of hematopoiesis. It is essential for normal B-cell lymphopoiesis and is a key determinant of neutrophil release from the bone marrow. Activating mutations of CXCR4 are causative of the warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, which is characterized by abnormal neutrophil retention in the bone marrow and impaired B-cell development. The CXCL12/CXCR4 axis also plays an essential role in HSC maintenance and trafficking, and pharmaceuticals that inhibit CXCR4 signaling result in rapid mobilization.
    No preview · Article · Jan 2015
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    Full-text · Dataset · Dec 2014
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    ABSTRACT: Therapy-related acute myeloid leukaemia (t-AML) and therapy-related myelodysplastic syndrome (t-MDS) are well-recognized complications of cytotoxic chemotherapy and/or radiotherapy. There are several features that distinguish t-AML from de novo AML, including a higher incidence of TP53 mutations, abnormalities of chromosomes 5 or 7, complex cytogenetics and a reduced response to chemotherapy. However, it is not clear how prior exposure to cytotoxic therapy influences leukaemogenesis. In particular, the mechanism by which TP53 mutations are selectively enriched in t-AML/t-MDS is unknown. Here, by sequencing the genomes of 22 patients with t-AML, we show that the total number of somatic single-nucleotide variants and the percentage of chemotherapy-related transversions are similar in t-AML and de novo AML, indicating that previous chemotherapy does not induce genome-wide DNA damage. We identified four cases of t-AML/t-MDS in which the exact TP53 mutation found at diagnosis was also present at low frequencies (0.003-0.7%) in mobilized blood leukocytes or bone marrow 3-6 years before the development of t-AML/t-MDS, including two cases in which the relevant TP53 mutation was detected before any chemotherapy. Moreover, functional TP53 mutations were identified in small populations of peripheral blood cells of healthy chemotherapy-naive elderly individuals. Finally, in mouse bone marrow chimaeras containing both wild-type and Tp53(+/-) haematopoietic stem/progenitor cells (HSPCs), the Tp53(+/-) HSPCs preferentially expanded after exposure to chemotherapy. These data suggest that cytotoxic therapy does not directly induce TP53 mutations. Rather, they support a model in which rare HSPCs carrying age-related TP53 mutations are resistant to chemotherapy and expand preferentially after treatment. The early acquisition of TP53 mutations in the founding HSPC clone probably contributes to the frequent cytogenetic abnormalities and poor responses to chemotherapy that are typical of patients with t-AML/t-MDS.
    Full-text · Article · Dec 2014 · Nature
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    ABSTRACT: Mutations in the gene for neutrophil elastase, ELANE, cause cyclic neutropenia (CyN) and severe congenital neutropenia (SCN). This study summarized data from the Severe Chronic Neutropenia International Registry (SCNIR) on genotype-phenotype relationships of ELANE mutations to important clinical outcomes. We also summarize findings for ELANE mutations not observed in SCNIR patients. There were 307 SCNIR patients with 104 distinctive ELANE mutations who were followed longitudinally for up to 27 years. The ELANE mutations were diverse; there were 65 single amino acid substitutions; 61 of these mutations (94%) were 'probably' or 'possibly damaging' by PolyPhen-2 analysis, and one of the 'benign' mutations was associated with two cases of acute myeloid leukemia (AML). All frame-shift mutations (19/19) were associated with the SCN. The pattern of mutations in the SCN versus CyN was significantly different (P < 10), but some mutations were observed in both groups (overlapping mutations). The cumulative incidence of severe adverse events, that is, myelodysplasia, AML, stem cell transplantation, or deaths was significantly greater for patients with SCN versus those with CyN or overlapping mutations. Specific mutations (i.e. G214R or C151Y) had a high risk for evolution to AML. Sequencing is useful for predicting outcomes of ELANE-associated neutropenia.
    Full-text · Article · Nov 2014 · Current Opinion in Hematology
  • Ryan B Day · Daniel C Link
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    ABSTRACT: Mesenchymal stromal cells are key components of hematopoietic stem cell (HSC) niches in the bone marrow. Two studies now show that hematopoietic-derived megakaryocytes also contribute to the HSC niche, regulating HSC quiescence and function.
    No preview · Article · Nov 2014 · Nature Medicine
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    ABSTRACT: The contribution of osteoclasts to hematopoietic stem/progenitor cell (HSPC) retention in the bone marrow is controversial. Studies of HSPC trafficking in osteoclast-deficient mice are limited by osteopetrosis. Here, we employed two non-osteopetrotic mouse models to assess the contribution of osteoclasts to basal and granulocyte colony-stimulating factor (G-CSF) induced HSPC mobilization. We generated Rank-/- fetal liver chimeras using Csf3r-/- recipients to produce mice lacking G-CSF receptor expression in osteoclasts. Basal and G-CSF-induced HSPC mobilization was normal in these chimeras. We next acutely depleted osteoclasts in wild-type mice using the RANK ligand inhibitor osteoprotegerin (OPG). Marked suppression of osteoclasts was observed after a single injection of OPG-Fc. Basal and G-CSF-induced HSPC mobilization in OPG-Fc treated mice were comparable to control mice. Together, these data show that osteoclasts are not required for the efficient retention of HSPCs in the bone marrow and are dispensable for HSPC mobilization by G-CSF.
    No preview · Article · Nov 2014 · Experimental Hematology

Publication Stats

11k Citations
1,708.02 Total Impact Points

Institutions

  • 1994-2015
    • Washington University in St. Louis
      • • Department of Medicine
      • • Division of Oncology
      San Luis, Missouri, United States
  • 2007
    • Indiana University-Purdue University Indianapolis
      Indianapolis, Indiana, United States
  • 2005
    • American University Washington D.C.
      Washington, Washington, D.C., United States
  • 1998
    • Beth Israel Deaconess Medical Center
      • Division of Hematology/Oncology
      Boston, Massachusetts, United States
  • 1991-1995
    • Barnes Jewish Hospital
      San Luis, Missouri, United States