R Keith Humphries

Publications (85)818.93 Total impact

• Article: CD34(+) Expansion With Delta-1 and HOXB4 Promotes Rapid Engraftment and Transfusion Independence in a Macaca nemestrina Cord Blood Transplant Model.

  Korashon L Watts, Colleen Delaney, Veronica Nelson, Grant D Trobridge, Brian C Beard, R Keith Humphries, Hans-Peter Kiem
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  ABSTRACT: Umbilical cord blood (CB) transplantation is a promising therapeutic approach but continues to be associated with delayed engraftment and infections. Here, we explored in our macaque CB transplant model expansion and engraftment kinetics of cells expanded with the combination of HOXB4 and Delta-1. CB cells were divided into two equal fractions; one fraction was transduced with HOXB4 yellow fluorescent protein (YFP) and expanded on control OP9 cells, and the other was transduced with HOXB4 green fluorescent protein (GFP) and expanded on Delta-expressing OP9 cells (OP9-DL1). Both fractions were transplanted into myeloablated subjects. Neutrophil and platelet recovery occurred within 7 and 19 days respectively, which was significantly earlier than in our previous study using cells expanded with HOXB4 alone, which resulted in neutrophil recovery within 12 days (P = 0.05) and platelet recovery within 37 days (P = 0.02). Furthermore, two of three animals in the current study remained fully transfusion-independent after transplantation. By day 30, reconstitution of lymphocytes was significantly greater with the HOXB4/OP9-DL1 expanded cells in all animals (P = 0.05). In conclusion, our data show that the combination of OP9-DL1 and HOXB4 can result in increased numbers of repopulating cells, thus leading to rapid engraftment and transfusion independence in macaques transplanted with autologous, expanded CB cells.Molecular Therapy (2013); doi:10.1038/mt.2013.40.
  Molecular Therapy 04/2013; · 6.87 Impact Factor
• Article: Enhanced normal short term human myelopoiesis in mice engineered to express human-specific myeloid growth factors.

  Paul H Miller, Alice M S Cheung, Philip A Beer, David J H F Knapp, Kiran Dhillon, Gabrielle Rabu, Shabnam Rostamirad, R Keith Humphries, Connie J Eaves
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  ABSTRACT: Better methods to characterize normal human hematopoietic cells with short term repopulating activity (STRCs) are needed to facilitate improving recovery rates in transplanted patients. We now show that 5-fold more human myeloid cells are produced in sublethally irradiated NOD/SCID-IL-2Receptor-γchain-null (NSG) mice engineered to constitutively produce human interleukin-3, granulocyte-macrophage colony-stimulating factor and Steel Factor (NSG-3GS mice) than in regular NSG mice 3 weeks after an intravenous injection of CD34(+) human cord blood cells. Importantly, the NSG-3GS mice also show a concomitant and matched increase in circulating mature human neutrophils. Imaging NSG-3GS recipients of lenti-luciferase-transduced cells showed the human cells being produced 3 weeks post-transplant are heterogeneously distributed, validating the blood as a more representative measure of transplanted STRC activity. Limiting dilution transplants further demonstrated that the early increase in human granulopoiesis in NSG-3GS mice reflects an expanded output of differentiated cells per STRC rather than an increase in STRC detection.
  Blood 12/2012; · 9.90 Impact Factor
• Dataset: Blood-EZH2-suppl-2011

  Damian B Yap, Justin Chu, Tobias Berg, Matthieu Schapira, S-W Grace Cheng, Annie Moradian, Ryan D Morin, Andrew J Mungall, Barbara Meissner, Merrill Boyle, Victor E Marquez, Marco A Marra, Randy D Gascoyne, R Keith Humphries, Cheryl H Arrowsmith, Gregg B Morin, Samuel A J R Aparicio
• Source

  Available from: Damian Yap

  Dataset: Blood-EZH2-suppl-2011

  Damian B Yap, Justin Chu, Tobias Berg, Matthieu Schapira, S-W Grace Cheng, Annie Moradian, Ryan D Morin, Andrew J Mungall, Barbara Meissner, Merrill Boyle, Victor E Marquez, Marco A Marra, Randy D Gascoyne, R Keith Humphries, Cheryl H Arrowsmith, Gregg B Morin, Samuel A J R Aparicio
• Article: Varying levels of aldehyde dehydrogenase activity in adult murine marrow hematopoietic stem cells are associated with engraftment and cell cycle status.

  Maura Gasparetto, Sanja Sekulovic, Anush Zakaryan, Suzan Imren, David G Kent, R Keith Humphries, Vasilis Vasiliou, Clay Smith
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  ABSTRACT: Aldehyde dehydrogenase (ALDH) activity is a widely used marker for human hematopoietic stem cells (HSCs), yet its relevance and role in murine HSCs remain unclear. We found that murine marrow cells with a high level of ALDH activity as measured by Aldefluor staining (ALDH(br) cells) do not contain known HSCs or progenitors. In contrast, highly enriched murine HSCs defined by the CD48(-)EPCR(+) and other phenotypes contain two subpopulations, one that stains dimly with Aldefluor (ALDH(dim)) and one that stains at intermediate levels (ALDH(int)). The CD48(-)EPCR(+)ALDH(dim) cells are virtually all in G(0) and yield high levels of engraftment via both intravenous and intrabone routes. In contrast the CD48(-)EPCR(+)ALDH(int) cells are virtually all in G(1), have little intravenous engraftment potential, and yet can engraft long-term after intrabone transplantation. These data demonstrate that Aldefluor staining of unfractionated murine marrow does not identify known HSCs or progenitors. However, varying levels of Aldefluor staining when combined with CD48 and EPCR detection can identify novel populations in murine marrow including a highly enriched population of resting HSCs and a previously unknown HSC population in G(1) with an intravenous engraftment defect.
  Experimental hematology 06/2012; 40(10):857-866.e5. · 3.11 Impact Factor
• Article: Beyond Hox: the role of ParaHox genes in normal and malignant hematopoiesis.

  Vijay P S Rawat, R Keith Humphries, Christian Buske
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  ABSTRACT: During the past decade it was recognized that homeobox gene families such as the clustered Hox genes play pivotal roles both in normal and malignant hematopoiesis. More recently, similar roles have also become apparent for members of the ParaHox gene cluster, evolutionarily closely related to the Hox gene cluster. This is in particular found for the caudal-type homeobox genes (Cdx) genes, known to act as upstream regulators of Hox genes. The CDX gene family member CDX2 belongs to the most frequent aberrantly expressed proto-oncogenes in human acute leukemias and is highly leukemogenic in experimental models. Correlative studies indicate that CDX2 functions as master regulator of perturbed HOX gene expression in human acute myeloid leukemia, locating this ParaHox gene at a central position for initiating and maintaining HOX gene dysregulation as a driving leukemogenic force. There are still few data about potential upstream regulators initiating aberrant CDX2 expression in human leukemias or about critical downstream targets of CDX2 in leukemic cells. Characterizing this network will hopefully open the way to therapeutic approaches that target deregulated ParaHox genes in human leukemia.
  Blood 04/2012; 120(3):519-27. · 9.90 Impact Factor
• Article: Notch-mediated repression of miR-223 contributes to IGF1R regulation in T-ALL.

  Samuel Gusscott, Florian Kuchenbauer, R Keith Humphries, Andrew P Weng
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  ABSTRACT: To identify microRNAs regulated by oncogenic Notch signaling, we performed microarray-based miRNA profiling of T-cell acute lymphoblastic leukemia (T-ALL) cells before and after treatment with γ-secretase inhibitor (GSI) to block Notch signaling. We show miR-223 levels increase after GSI treatment suggesting that active Notch signaling represses miR-223 expression. We also demonstrate that insulin-like growth factor-1 receptor (IGF1R) is regulated by miR-223 in this context, but observe no apparent effects on cell growth by overexpression or knock-down of miR-223 alone. We conclude that miR-223 contributes to IGF1R regulation, but may act in concert with other genes and/or microRNAs to alter T-ALL biology.
  Leukemia research 03/2012; 36(7):905-11. · 2.36 Impact Factor
• Article: Functional regulation of pre-B-cell leukemia homeobox interacting protein 1 (PBXIP1/HPIP) in erythroid differentiation.

  Bramanandam Manavathi, Dennis Lo, Suresh Bugide, Oindrilla Dey, Suzan Imren, Mitchell J Weiss, R Keith Humphries
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  ABSTRACT: Pre-B-cell leukemia homeobox interacting protein 1 or human PBX1 interacting protein (PBXIP1/HPIP) is a co-repressor of pre-B-cell leukemia homeobox 1 (PBX1) and is also known to regulate estrogen receptor functions by associating with the microtubule network. Despite its initial discovery in the context of hematopoietic cells, little is yet known about the role of HPIP in hematopoiesis. Here, we show that lentivirus-mediated overexpression of HPIP in human CD34(+) cells enhances hematopoietic colony formation in vitro, whereas HPIP knockdown leads to a reduction in the number of such colonies. Interestingly, erythroid colony number was significantly higher in HPIP-overexpressing cells. In addition, forced expression of HPIP in K562 cells, a multipotent erythro-megakaryoblastic leukemia cell line, led to an induction of erythroid differentiation. HPIP overexpression in both CD34(+) and K562 cells was associated with increased activation of the PI3K/AKT pathway, and corresponding treatment with a PI3K-specific inhibitor, LY-294002, caused a reduction in clonogenic progenitor number in HPIP-expressing CD34(+) cells and decreased K562 cell differentiation. Combined, these findings point to an important role of the PI3K/AKT pathway in mediating HPIP-induced effects on the growth and differentiation of hematopoietic cells. Interestingly, HPIP gene expression was found to be induced in K562 cells in response to erythroid differentiation signals such as DMSO and erythropoietin. The erythroid lineage-specific transcription factor GATA1 binds to the HPIP promoter and activates HPIP gene transcription in a CCCTC-binding factor (CTCF)-dependent manner. Co-immunoprecipitation and co-localization experiments revealed the association of CTCF with GATA1 indicating the recruitment of CTCF/GATA1 transcription factor complex onto the HPIP promoter. Together, this study provides evidence that HPIP is a target of GATA1 and CTCF in erythroid cells and plays an important role in erythroid differentiation by modulating the PI3K/AKT pathway.
  Journal of Biological Chemistry 12/2011; 287(8):5600-14. · 4.77 Impact Factor
• Source

  Available from: Chad Brocker

  Article: Aldehyde dehydrogenases are regulators of hematopoietic stem cell numbers and B-cell development.

  Maura Gasparetto, Sanja Sekulovic, Chad Brocker, Patrick Tang, Anush Zakaryan, Ping Xiang, Florian Kuchenbauer, Maggie Wen, Katayoon Kasaian, Marie France Witty, Patty Rosten, Ying Chen, Suzan Imren, Gregg Duester, David C Thompson, Richard Keith Humphries, Vasilis Vasiliou, Clay Smith
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  ABSTRACT: High levels of the aldehyde dehydrogenase isoform ALDH1A1 are expressed in hematopoietic stem cells (HSCs); however, its importance in these cells remains unclear. Consistent with an earlier report, we find that loss of ALDH1A1 does not affect HSCs. Intriguingly, however, we find that ALDH1A1 deficiency is associated with increased expression of the ALDH3A1 isoform, suggesting its potential to compensate for ALDH1A1. Mice deficient in ALDH3A1 have a block in B-cell development as well as abnormalities in cell cycling, intracellular signaling, and gene expression. Early B cells from these mice exhibit excess reactive oxygen species and reduced metabolism of reactive aldehydes. Mice deficient in both ALDH3A1 and ALDH1A1 have reduced numbers of HSCs as well as aberrant cell cycle distribution, increased reactive oxygen species levels, p38 mitogen-activated protein kinase activity and sensitivity to DNA damage. These findings demonstrate that ALDH3A1 can compensate for ALDH1A1 in bone marrow and is important in B-cell development, both ALDH1A1 and 3A1 are important in HSC biology; and these effects may be due, in part, to changes in metabolism of reactive oxygen species and reactive aldehydes.
  Experimental hematology 12/2011; 40(4):318-29.e2. · 3.11 Impact Factor
• Article: Hematopoietic stem cell expansion facilitates multilineage engraftment in a nonhuman primate cord blood transplantation model.

  Korashon L Watts, Veronica Nelson, Brent L Wood, Grant D Trobridge, Brian C Beard, R Keith Humphries, Hans-Peter Kiem
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  ABSTRACT: The use of umbilical cord blood for allogeneic transplantation has increased dramatically over the past years. However, the limited number of cells available in a single cord blood unit remains a serious obstacle. Here, we wished to establish a nonhuman primate cord blood transplantation model that would allow us to test various hematopoietic stem cell expansion and gene therapy strategies. We implemented HOXB4-mediated expansion based on our previous experience with HOXB4 in autologous cells. Cord blood units were divided into two equal parts; half of the cells were transduced with a yellow fluorescent protein control vector and cryopreserved, and half were transduced with a HOXB4GFP vector, expanded, and cryopreserved. Both fractions of cells were transplanted into Macaca nemestrina subjects. We found that neutrophil recovery occurred within 19 days in all animals, and both neutrophil and platelet recovery were substantially accelerated compared to human single unit cord blood transplants. In addition, HOXB4-transduced and expanded cells resulted in superior engraftment of all hematopoietic lineages in all animals over nonexpanded controls. In conclusion, we have successfully established a nonhuman primate cord blood transplantation model and demonstrated that HOXB4 stimulates expansion and engraftment of repopulating cells. The availability of such a model has significant implications for developing and testing strategies to improve clinical cord blood transplantation, as it will allow comparison of different stem cell expansion methodologies within a single animal. Furthermore, it can be used in long-term follow-up studies to determine how specific expansion techniques affect engraftment of various hematopoietic lineages.
  Experimental hematology 12/2011; 40(3):187-96. · 3.11 Impact Factor
• Article: Concise review: Multidimensional regulation of the hematopoietic stem cell state.

  Il-Hoan Oh, R Keith Humphries
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  ABSTRACT: Hematopoietic stem cells (HSCs) are characterized by their unique function to produce all lineages of blood cells throughout life. Such tissue-specific function of HSC is attributed to their ability to execute self-renewal and multilineage differentiation. Accumulating evidence indicates that the undifferentiated state of HSC is characterized by dynamic maintenance of chromatin structures and epigenetic plasticity. Conversely, quiescence, self-renewal, and differentiation of HSCs are dictated by complex regulatory mechanisms involving specific transcription factors and microenvironmental crosstalk between stem cells and multiple compartments of niches in bone marrows. Thus, multidimensional regulatory inputs are integrated into two opposing characters of HSCs-maintenance of undifferentiated state analogous to pluripotent stem cells but execution of tissue-specific hematopoietic functions. Further studies on the interplay of such regulatory forces as "cell fate determinant" will likely shed the light on diverse spectrums of tissue-specific stem cells.
  Stem Cells 11/2011; 30(1):82-8. · 7.78 Impact Factor
• Article: Ontogeny stage-independent and high-level clonal expansion in vitro of mouse hematopoietic stem cells stimulated by an engineered NUP98-HOX fusion transcription factor.

  Sanja Sekulovic, Maura Gasparetto, Véronique Lecault, Corinne A Hoesli, David G Kent, Patty Rosten, Adrian Wan, Christy Brookes, Carl L Hansen, James M Piret, Clayton Smith, Connie J Eaves, R Keith Humphries
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  ABSTRACT: Achieving high-level expansion of hematopoietic stem cells (HSCs) in vitro will have an important clinical impact in addition to enabling elucidation of their regulation. Here, we couple the ability of engineered NUP98-HOXA10hd expression to stimulate > 1000-fold net expansions of murine HSCs in 10-day cultures initiated with bulk lin(-)Sca-1(+)c-kit(+) cells, with strategies to purify fetal and adult HSCs and analyze their expansion clonally. We find that NUP98-HOXA10hd stimulates comparable expansions of HSCs from both sources at ∼ 60% to 90% unit efficiency in cultures initiated with single cells. Clonally expanded HSCs consistently show balanced long-term contributions to the lymphoid and myeloid lineages without evidence of leukemogenic activity. Although effects on fetal and adult HSCs were indistinguishable, NUP98-HOXA10hd-transduced adult HSCs did not thereby gain a competitive advantage in vivo over freshly isolated fetal HSCs. Live-cell image tracking of single transduced HSCs cultured in a microfluidic device indicates that NUP98-HOXA10hd does not affect their proliferation kinetics, and flow cytometry confirmed the phenotype of normal proliferating HSCs and allowed reisolation of large numbers of expanded HSCs at a purity of 25%. These findings point to the effects of NUP98-HOXA10hd on HSCs in vitro being mediated by promoting self-renewal and set the stage for further dissection of this process.
  Blood 08/2011; 118(16):4366-76. · 9.90 Impact Factor
• Article: Prolonged self-renewal activity unmasks telomerase control of telomere homeostasis and function of mouse hematopoietic stem cells.

  Sanja Sekulovic, Vala Gylfadottir, Irma Vulto, Maura Gasparetto, Yasmine Even, Christy Brookes, Clayton Smith, Connie J Eaves, Peter M Lansdorp, Fabio M Rossi, R Keith Humphries
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  ABSTRACT: Strategies for expanding hematopoietic stem cells (HSCs) could have significant utility for transplantation-based therapies. However, deleterious consequences of such manipulations remain unknown. Here we examined the impact of HSC self-renewal divisions in vitro and in vivo on their subsequent regenerative and continuing ability to sustain blood cell production in the absence of telomerase. HSC expansion in vitro was obtained using a NUP98-HOXA10hd transduction strategy and, in vivo, using a serial transplant protocol. We observed ~ 10kb telomere loss in leukocytes produced in secondary mice transplanted with HSCs regenerated in primary recipients of NUP98-HOXA10hd-transduced and in vitro-expanded Tert(-/-) HSCs 6 months before. The second generation leukocytes also showed elevated expression of γH2AX (relative to control) indicative of greater accumulating DNA damage. In contrast, significant telomere shortening was not detected in leukocytes produced from freshly isolated, serially transplanted wild-type (WT) or Tert(-/-) HSCs, suggesting that HSC replication posttransplant is not limited by telomere shortening in the mouse. These findings document a role of telomerase in telomere homeostasis, and in preserving HSC functional integrity on prolonged self-renewal stimulation.
  Blood 08/2011; 118(7):1766-73. · 9.90 Impact Factor
• Source

  Available from: Xiao-Bing Zhang

  Article: Differential effects of HOXB4 and NUP98-HOXA10hd on hematopoietic repopulating cells in a nonhuman primate model.

  Korashon L Watts, Xiaobing Zhang, Brian C Beard, Sum Ying Chiu, Grant D Trobridge, R Keith Humphries, Hans-Peter Kiem
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  ABSTRACT: Expansion of hematopoietic stem cells (HSCs) is beneficial in settings where HSC numbers are limited, such as cord blood transplantation. The human homeobox transcription factor HOXB4 has been shown to enhance stem cell expansion in several experimental models. We have shown previously that HOXB4 overexpression in monkey CD34(+) cells has a dramatic effect on expansion and engraftment of short-term repopulating cells. Here, we wished to compare the effects of HOXB4 and another candidate gene, NUP98-HOXA10hd (NA10hd). We used a competitive repopulation assay in pigtailed macaques to study engraftment of CD34(+) cells modified with gammaretroviral HOXB4YFP or NA10hdGFP. We found that HOXB4YFP contributed more to early hematopoiesis (<30 days), whereas NA10hdGFP contributed more to later hematopoiesis. In each case, we observed two distinct peaks in engraftment of NA10hd-transduced cells, one within 20 days post transplant and another after 5-6 months. Analysis of CD14(+), CD3(+), and CD20(+) subsets confirmed that higher percentages of cells of each lineage were derived from NA10hdGFP(+) progenitors than from HOXB4YFP(+) progenitors. In conclusion, we show that HOXB4 and NA10hd both have a significant impact on hematopoietic reconstitution; however, these effects are differential and therefore may offer complementary strategies for HSC expansion.
  Human gene therapy 07/2011; 22(12):1475-82. · 4.20 Impact Factor
• Article: Cell of origin in AML: susceptibility to MN1-induced transformation is regulated by the MEIS1/AbdB-like HOX protein complex.

  Michael Heuser, Haiyang Yun, Tobias Berg, Eric Yung, Bob Argiropoulos, Florian Kuchenbauer, Gyeongsin Park, Iyas Hamwi, Lars Palmqvist, Courteney K Lai, [......], Gordon Robertson, Martin Hirst, David Kent, Nicola K Wilson, Bertie Göttgens, Connie Eaves, Michael L Cleary, Marco Marra, Arnold Ganser, R Keith Humphries
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  ABSTRACT: Pathways defining susceptibility of normal cells to oncogenic transformation may be valuable therapeutic targets. We characterized the cell of origin and its critical pathways in MN1-induced leukemias. Common myeloid (CMP) but not granulocyte-macrophage progenitors (GMP) could be transformed by MN1. Complementation studies of CMP-signature genes in GMPs demonstrated that MN1-leukemogenicity required the MEIS1/AbdB-like HOX-protein complex. ChIP-sequencing identified common target genes of MN1 and MEIS1 and demonstrated identical binding sites for a large proportion of their chromatin targets. Transcriptional repression of MEIS1 targets in established MN1 leukemias demonstrated antileukemic activity. As MN1 relies on but cannot activate expression of MEIS1/AbdB-like HOX proteins, transcriptional activity of these genes determines cellular susceptibility to MN1-induced transformation and may represent a promising therapeutic target.
  Cancer cell 07/2011; 20(1):39-52. · 25.29 Impact Factor
• Article: High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays.

  Véronique Lecault, Michael Vaninsberghe, Sanja Sekulovic, David J H F Knapp, Stefan Wohrer, William Bowden, Francis Viel, Thomas McLaughlin, Asefeh Jarandehei, Michelle Miller, Didier Falconnet, Adam K White, David G Kent, Michael R Copley, Fariborz Taghipour, Connie J Eaves, R Keith Humphries, James M Piret, Carl L Hansen
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  ABSTRACT: Heterogeneity in cell populations poses a major obstacle to understanding complex biological processes. Here we present a microfluidic platform containing thousands of nanoliter-scale chambers suitable for live-cell imaging studies of clonal cultures of nonadherent cells with precise control of the conditions, capabilities for in situ immunostaining and recovery of viable cells. We show that this platform mimics conventional cultures in reproducing the responses of various types of primitive mouse hematopoietic cells with retention of their functional properties, as demonstrated by subsequent in vitro and in vivo (transplantation) assays of recovered cells. The automated medium exchange of this system made it possible to define when Steel factor stimulation is first required by adult hematopoietic stem cells in vitro as the point of exit from quiescence. This technology will offer many new avenues to interrogate otherwise inaccessible mechanisms governing mammalian cell growth and fate decisions.
  Nature Methods 05/2011; 8(7):581-6. · 19.28 Impact Factor
• Article: Probing the complexity of miRNA expression across hematopoiesis.

  Oleh I Petriv, Carl L Hansen, R Keith Humphries, Florian Kuchenbauer
  Cell cycle (Georgetown, Tex.) 01/2011; 10(1):2-3. · 5.36 Impact Factor
• Article: Delineating domains and functions of NUP98 contributing to the leukemogenic activity of NUP98-HOX fusions.

  Eric Yung, Sanja Sekulovic, Bob Argiropoulos, Courteney K Lai, Malina Leung, Tobias Berg, Sarah Vollett, Vicky Chi-Dan Chang, Adrian Wan, Sandy Wong, R Keith Humphries
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  ABSTRACT: To determine the contribution of the common N-terminal truncation of NUP98 in NUP98-translocations resulting in acute myeloid leukemia, we have conducted a structure-function analysis of NUP98 in the context of NUP98-HOXA10HD, a novel, canonical NUP98-Hox fusion that significantly enhances the self-renewal capacity of hematopoietic stem cells and collaborates with Meis1 to induce AML in our mouse models. Our results identify that NUP98 functions by transcriptional activation likely by recruitment of CBP/p300 via its FG/GLFG repeats. In contrast, the functional interaction of NUP98 with Rae1 or the anaphase promoting complex appears non-essential for its role in NUP98-leukemogenic fusions.
  Leukemia research 12/2010; 35(4):545-50. · 2.36 Impact Factor
• Source

  Available from: Damian Yap

  Article: Somatic mutations at EZH2 Y641 act dominantly through a mechanism of selectively altered PRC2 catalytic activity, to increase H3K27 trimethylation.

  Damian B Yap, Justin Chu, Tobias Berg, Matthieu Schapira, S-W Grace Cheng, Annie Moradian, Ryan D Morin, Andrew J Mungall, Barbara Meissner, Merrill Boyle, Victor E Marquez, Marco A Marra, Randy D Gascoyne, R Keith Humphries, Cheryl H Arrowsmith, Gregg B Morin, Samuel A J R Aparicio
  [show abstract] [hide abstract]
  ABSTRACT: Next-generation sequencing of follicular lymphoma and diffuse-large B-cell lymphoma has revealed frequent somatic, heterozygous Y641 mutations in the histone methyltransferase EZH2. Heterozygosity and the presence of equal quantities of both mutant and wild-type mRNA and expressed protein suggest a dominant mode of action. Surprisingly, B-cell lymphoma cell lines and lymphoma samples harboring heterozygous EZH2(Y641) mutations have increased levels of histone H3 Lys-27-specific trimethylation (H3K27me3). Expression of EZH2(Y641F/N) mutants in cells with EZH2(WT) resulted in an increase of H3K27me3 levels in vivo. Structural modeling of EZH2(Y641) mutants suggests a "Tyr/Phe switch" model whereby structurally neutral, nontyrosine residues at position 641 would decrease affinity for unmethylated and monomethylated H3K27 substrates and potentially favor trimethylation. We demonstrate, using in vitro enzyme assays of reconstituted PRC2 complexes, that Y641 mutations result in a decrease in monomethylation and an increase in trimethylation activity of the enzyme relative to the wild-type enzyme. This represents the first example of a disease-associated gain-of-function mutation in a histone methyltransferase, whereby somatic EZH2 Y641 mutations in lymphoma act dominantly to increase, rather than decrease, histone methylation. The dominant mode of action suggests that allele-specific EZH2 inhibitors should be a future therapeutic strategy for this disease.
  Blood 12/2010; 117(8):2451-9. · 9.90 Impact Factor
• Article: MicroRNA-146a disrupts hematopoietic differentiation and survival.

  Daniel T Starczynowski, Florian Kuchenbauer, Joanna Wegrzyn, Arefeh Rouhi, Oleh Petriv, Carl L Hansen, R Keith Humphries, Aly Karsan
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  ABSTRACT: MicroRNAs (miRNAs) are short noncoding RNAs capable of exerting dramatic effects by postranscriptionally regulating numerous messenger RNA targets. Toll-like receptor-4 (TLR-4) activation by lipopolysaccharide (LPS) induces the expression of three miRNAs in myeloid cells. The aim of this study was to investigate the in vivo consequences of expressing one of the LPS-induced miRNA, miR-146a, in bone marrow cells. The role of miR-146a in hematopoiesis was investigated by using retroviral infection and overexpression of miR-146a in mouse hematopoietic stem/progenitor cells, followed by bone marrow transplantations. miR-146a is mainly expressed in primitive hematopoietic stem cells and T lymphocytes. Overexpression of miR-146a in hematopoietic stem cells, followed by bone marrow transplantation, resulted in a transient myeloid expansion, decreased erythropoiesis, and impaired lymphopoiesis in select anatomical locations. Enforced expression of miR-146a also impaired bone marrow reconstitution in recipient mice and reduced survival of hematopoietic stem cells. Our results indicate that miR-146a, an LPS-induced miRNA, regulates multiple aspects of hematopoietic differentiation and survival. Furthermore, the consequences of miR-146a expression in hematopoietic cells mimics some of the reported effects with acute LPS exposure.
  Experimental hematology 10/2010; 39(2):167-178.e4. · 3.11 Impact Factor