A humanized bone marrow ossicle xenotransplantation model enables improved engraftment of healthy and leukemic human hematopoietic cells
2016; 22 (7): 812-821
Leukemia-Associated Cohesin Mutants Dominantly Enforce Stem Cell Programs and Impair Human Hematopoietic Progenitor Differentiation.
Cell stem cell
2015; 17 (6): 675-688
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.
View details for DOI 10.1038/nm.4103
View details for Web of Science ID 000379366900021
View details for PubMedID 27213817
Tuning Cytokine Receptor Signaling by Re-orienting Dimer Geometry with Surrogate Ligands
2015; 160 (6): 1196-1208
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.
View details for DOI 10.1016/j.stem.2015.09.017
View details for PubMedID 26607380
View details for PubMedCentralID PMC4671831
Isocitrate dehydrogenase 1 and 2 mutations induce BCL-2 dependence in acute myeloid leukemia.
2015; 21 (2): 178-184
Most cell-surface receptors for cytokines and growth factors signal as dimers, but it is unclear whether remodeling receptor dimer topology is a viable strategy to "tune" signaling output. We utilized diabodies (DA) as surrogate ligands in a prototypical dimeric receptor-ligand system, the cytokine Erythropoietin (EPO) and its receptor (EpoR), to dimerize EpoR ectodomains in non-native architectures. Diabody-induced signaling amplitudes varied from full to minimal agonism, and structures of these DA/EpoR complexes differed in EpoR dimer orientation and proximity. Diabodies also elicited biased or differential activation of signaling pathways and gene expression profiles compared to EPO. Non-signaling diabodies inhibited proliferation of erythroid precursors from patients with a myeloproliferative neoplasm due to a constitutively active JAK2V617F mutation. Thus, intracellular oncogenic mutations causing ligand-independent receptor activation can be counteracted by extracellular ligands that re-orient receptors into inactive dimer topologies. This approach has broad applications for tuning signaling output for many dimeric receptor systems.
View details for DOI 10.1016/j.cell.2015.02.011
View details for Web of Science ID 000351951800018
View details for PubMedID 25728669
Hereditary erythrocytosis, thrombocytosis and neutrophilia
BEST PRACTICE & RESEARCH CLINICAL HAEMATOLOGY
2014; 27 (2): 95-106
Mutant isocitrate dehydrogenase (IDH) 1 and 2 proteins alter the epigenetic landscape in acute myeloid leukemia (AML) cells through production of the oncometabolite (R)-2-hydroxyglutarate (2-HG). Here we performed a large-scale RNA interference (RNAi) screen to identify genes that are synthetic lethal to the IDH1(R132H) mutation in AML and identified the anti-apoptotic gene BCL-2. IDH1- and IDH2-mutant primary human AML cells were more sensitive than IDH1/2 wild-type cells to ABT-199, a highly specific BCL-2 inhibitor that is currently in clinical trials for hematologic malignancies, both ex vivo and in xenotransplant models. This sensitization effect was induced by (R)-2-HG-mediated inhibition of the activity of cytochrome c oxidase (COX) in the mitochondrial electron transport chain (ETC); suppression of COX activity lowered the mitochondrial threshold to trigger apoptosis upon BCL-2 inhibition. Our findings indicate that IDH1/2 mutation status may identify patients that are likely to respond to pharmacologic BCL-2 inhibition and form the rational basis for combining agents that disrupt ETC activity with ABT-199 in future clinical studies.
View details for DOI 10.1038/nm.3788
View details for PubMedID 25599133
View details for PubMedCentralID PMC4406275
Preleukemic mutations in human acute myeloid leukemia affect epigenetic regulators and persist in remission.
Proceedings of the National Academy of Sciences of the United States of America
2014; 111 (7): 2548-2553
Hereditary erythrocytosis, thrombocytosis, and neutrophilia are rare inherited syndromes which exhibit Mendelian inheritance. Some patients with primary hereditary erythrocytosis exhibit a mutation in the erythropoietin receptor (EPOR) which is associated with low serum erythropoietin (EPO) levels. Secondary congenital erythrocytosis may be characterized by normal or high serum EPO levels, and is related to high oxygen affinity haemoglobin variants, mutation of the enzyme biphosphoglycerate mutase (BPGM), or defects in components of the oxygen-sensing pathway. Hereditary thrombocytosis was first linked to mutations in genes encoding thrombopoietin (THPO) or the thrombopoietin receptor, MPL. More recently, germline mutations in JAK2, distinct from JAK2 V617F, and mutation of the gelsolin gene, were uncovered in several pedigrees of hereditary thrombocytosis. Hereditary neutrophilia has been described in one family with an activating germline mutation in CSF3R. The mutational basis for most hereditary myeloproliferative disorders has yet to be identified.
View details for DOI 10.1016/j.beha.2014.07.002
View details for Web of Science ID 000342534800003
View details for PubMedID 25189721
Centrosome-kinase fusions promote oncogenic signaling and disrupt centrosome function in myeloproliferative neoplasms.
2014; 9 (3)
Cancer is widely characterized by the sequential acquisition of genetic lesions in a single lineage of cells. Our previous studies have shown that, in acute myeloid leukemia (AML), mutation acquisition occurs in functionally normal hematopoietic stem cells (HSCs). These preleukemic HSCs harbor some, but not all, of the mutations found in the leukemic cells. We report here the identification of patterns of mutation acquisition in human AML. Our findings support a model in which mutations in "landscaping" genes, involved in global chromatin changes such as DNA methylation, histone modification, and chromatin looping, occur early in the evolution of AML, whereas mutations in "proliferative" genes occur late. Additionally, we analyze the persistence of preleukemic mutations in patients in remission and find CD34+ progenitor cells and various mature cells that harbor preleukemic mutations. These findings indicate that preleukemic HSCs can survive induction chemotherapy, identifying these cells as a reservoir for the reevolution of relapsed disease. Finally, through the study of several cases of relapsed AML, we demonstrate various evolutionary patterns for the generation of relapsed disease and show that some of these patterns are consistent with involvement of preleukemic HSCs. These findings provide key insights into the monitoring of minimal residual disease and the identification of therapeutic targets in human AML.
View details for DOI 10.1073/pnas.1324297111
View details for PubMedID 24550281
How we treat: risk mitigation for ABO-incompatible plasma in plateletpheresis products
2012; 52 (10): 2081-2085
Unfavorable-risk cytogenetics in acute myeloid leukemia
EXPERT REVIEW OF HEMATOLOGY
2011; 4 (2): 173-184
Chromosomal translocations observed in myeloproliferative neoplasms (MPNs) frequently fuse genes that encode centrosome proteins and tyrosine kinases. This causes constitutive activation of the kinase resulting in aberrant, proliferative signaling. The function of centrosome proteins in these fusions is not well understood. Among others, kinase centrosome localization and constitutive kinase dimerization are possible consequences of centrosome protein-kinase fusions. To test the relative contributions of localization and dimerization on kinase signaling, we targeted inducibly dimerizable FGFR1 to the centrosome and other subcellular locations and generated a mutant of the FOP-FGFR1 MPN fusion defective in centrosome localization. Expression in mammalian cells followed by western blot analysis revealed a significant decrease in kinase signaling upon loss of FOP-FGFR1 centrosome localization. Kinase dimerization alone resulted in phosphorylation of the FGFR1 signaling target PLC?, however levels comparable to FOP-FGFR1 required subcellular targeting in addition to kinase dimerization. Expression of MPN fusion proteins also resulted in centrosome disruption in epithelial cells and transformed patient cells. Primary human MPN cells showed masses of modified tubulin that colocalized with centrin, Smoothened (Smo), IFT88, and Arl13b. This is distinct from acute myeloid leukemia (AML) cells, which are not associated with centrosome-kinase fusions and had normal centrosomes. Our results suggest that effective proliferative MPN signaling requires both subcellular localization and dimerization of MPN kinases, both of which may be provided by centrosome protein fusion partners. Furthermore, centrosome disruption may contribute to the MPN transformation phenotype.
View details for DOI 10.1371/journal.pone.0092641
View details for PubMedID 24658090
Local false discovery rate facilitates comparison of different microarray experiments
NUCLEIC ACIDS RESEARCH
2009; 37 (22): 7483-7497
Cytogenetic analysis at diagnosis is one of the most significant prognostic factors in acute myeloid leukemia (AML). AML patients with unfavorable-risk cytogenetic abnormalities account for 16-30% of younger adult patients and have poor response to standard treatment, with only 32-55% achieving a complete response. Overall survival is also extremely poor with only 5-12% patients alive at 5-10 years after diagnosis. Owing to the poor response in this subset of patients, risk-adapted treatment has been investigated. Allogeneic stem cell transplant has been shown to provide a survival benefit in patients with unfavorable-risk cytogenetic abnormalities in complement receptor 1. Other risk-adapted treatment strategies, such as reduced-intensity conditioning regimens prior to allogeneic stem cell transplant for older patients with AML, have also shown some survival benefit, without increasing treatment-related toxicities. Risk-stratification models that include cytogenetic abnormalities, as well as other molecular markers, are being developed to allow for individualized risk-adapted treatment for patients with AML. Prospective multicenter trials will be needed to validate these prognostic models.
View details for DOI 10.1586/EHM.11.10
View details for Web of Science ID 000290371000014
View details for PubMedID 21495927
Immune signatures in follicular lymphoma
NEW ENGLAND JOURNAL OF MEDICINE
2005; 352 (14): 1496-1496
Toxicity from radiation therapy associated with abnormal transcriptional responses to DNA damage
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2004; 101 (17): 6635-6640
The local false discovery rate (LFDR) estimates the probability of falsely identifying specific genes with changes in expression. In computer simulations, LFDR <10% successfully identified genes with changes in expression, while LFDR >90% identified genes without changes. We used LFDR to compare different microarray experiments quantitatively: (i) Venn diagrams of genes with and without changes in expression, (ii) scatter plots of the genes, (iii) correlation coefficients in the scatter plots and (iv) distributions of gene function. To illustrate, we compared three methods for pre-processing microarray data. Correlations between methods were high (r = 0.84-0.92). However, responses were often different in magnitude, and sometimes discordant, even though the methods used the same raw data. LFDR complements functional assessments like gene set enrichment analysis. To illustrate, we compared responses to ultraviolet radiation (UV), ionizing radiation (IR) and tobacco smoke. Compared to unresponsive genes, genes responsive to both UV and IR were enriched for cell cycle, mitosis, and DNA repair functions. Genes responsive to UV but not IR were depleted for cell adhesion functions. Genes responsive to tobacco smoke were enriched for detoxification functions. Thus, LFDR reveals differences and similarities among experiments.
View details for DOI 10.1093/nar/gkp813
View details for Web of Science ID 000272935000021
View details for PubMedID 19825981
Toxicity from radiation therapy is a grave problem for cancer patients. We hypothesized that some cases of toxicity are associated with abnormal transcriptional responses to radiation. We used microarrays to measure responses to ionizing and UV radiation in lymphoblastoid cells derived from 14 patients with acute radiation toxicity. The analysis used heterogeneity-associated transformation of the data to account for a clinical outcome arising from more than one underlying cause. To compute the risk of toxicity for each patient, we applied nearest shrunken centroids, a method that identifies and cross-validates predictive genes. Transcriptional responses in 24 genes predicted radiation toxicity in 9 of 14 patients with no false positives among 43 controls (P = 2.2 x 10(-7)). The responses of these nine patients displayed significant heterogeneity. Of the five patients with toxicity and normal responses, two were treated with protocols that proved to be highly toxic. These results may enable physicians to predict toxicity and tailor treatment for individual patients.
View details for DOI 10.1073/pnas.0307761101
View details for Web of Science ID 000221107900056
View details for PubMedID 15096622