Publications by authors named "Ronan Chaligne"

18 Publications

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Integrating genetic and non-genetic determinants of cancer evolution by single-cell multi-omics.

Nat Rev Genet 2021 01 17;22(1):3-18. Epub 2020 Aug 17.

New York Genome Center, New York, NY, USA.

Cancer represents an evolutionary process through which growing malignant populations genetically diversify, leading to tumour progression, relapse and resistance to therapy. In addition to genetic diversity, the cell-to-cell variation that fuels evolutionary selection also manifests in cellular states, epigenetic profiles, spatial distributions and interactions with the microenvironment. Therefore, the study of cancer requires the integration of multiple heritable dimensions at the resolution of the single cell - the atomic unit of somatic evolution. In this Review, we discuss emerging analytic and experimental technologies for single-cell multi-omics that enable the capture and integration of multiple data modalities to inform the study of cancer evolution. These data show that cancer results from a complex interplay between genetic and non-genetic determinants of somatic evolution.
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http://dx.doi.org/10.1038/s41576-020-0265-5DOI Listing
January 2021

DNA methylation disruption reshapes the hematopoietic differentiation landscape.

Nat Genet 2020 04 23;52(4):378-387. Epub 2020 Mar 23.

New York Genome Center, New York, NY, USA.

Mutations in genes involved in DNA methylation (DNAme; for example, TET2 and DNMT3A) are frequently observed in hematological malignancies and clonal hematopoiesis. Applying single-cell sequencing to murine hematopoietic stem and progenitor cells, we observed that these mutations disrupt hematopoietic differentiation, causing opposite shifts in the frequencies of erythroid versus myelomonocytic progenitors following Tet2 or Dnmt3a loss. Notably, these shifts trace back to transcriptional priming skews in uncommitted hematopoietic stem cells. To reconcile genome-wide DNAme changes with specific erythroid versus myelomonocytic skews, we provide evidence in support of differential sensitivity of transcription factors due to biases in CpG enrichment in their binding motif. Single-cell transcriptomes with targeted genotyping showed similar skews in transcriptional priming of DNMT3A-mutated human clonal hematopoiesis bone marrow progenitors. These data show that DNAme shapes the topography of hematopoietic differentiation, and support a model in which genome-wide methylation changes are transduced to differentiation skews through biases in CpG enrichment of the transcription factor binding motif.
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http://dx.doi.org/10.1038/s41588-020-0595-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7216752PMC
April 2020

Somatic mutations and cell identity linked by Genotyping of Transcriptomes.

Nature 2019 07 3;571(7765):355-360. Epub 2019 Jul 3.

New York Genome Center, New York, NY, USA.

Defining the transcriptomic identity of malignant cells is challenging in the absence of surface markers that distinguish cancer clones from one another, or from admixed non-neoplastic cells. To address this challenge, here we developed Genotyping of Transcriptomes (GoT), a method to integrate genotyping with high-throughput droplet-based single-cell RNA sequencing. We apply GoT to profile 38,290 CD34 cells from patients with CALR-mutated myeloproliferative neoplasms to study how somatic mutations corrupt the complex process of human haematopoiesis. High-resolution mapping of malignant versus normal haematopoietic progenitors revealed an increasing fitness advantage with myeloid differentiation of cells with mutated CALR. We identified the unfolded protein response as a predominant outcome of CALR mutations, with a considerable dependency on cell identity, as well as upregulation of the NF-κB pathway specifically in uncommitted stem cells. We further extended the GoT toolkit to genotype multiple targets and loci that are distant from transcript ends. Together, these findings reveal that the transcriptional output of somatic mutations in myeloproliferative neoplasms is dependent on the native cell identity.
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http://dx.doi.org/10.1038/s41586-019-1367-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6782071PMC
July 2019

Epigenetic evolution and lineage histories of chronic lymphocytic leukaemia.

Nature 2019 05 15;569(7757):576-580. Epub 2019 May 15.

New York Genome Center, New York, NY, USA.

Genetic and epigenetic intra-tumoral heterogeneity cooperate to shape the evolutionary course of cancer. Chronic lymphocytic leukaemia (CLL) is a highly informative model for cancer evolution as it undergoes substantial genetic diversification and evolution after therapy. The CLL epigenome is also an important disease-defining feature, and growing populations of cells in CLL diversify by stochastic changes in DNA methylation known as epimutations. However, previous studies using bulk sequencing methods to analyse the patterns of DNA methylation were unable to determine whether epimutations affect CLL populations homogeneously. Here, to measure the epimutation rate at single-cell resolution, we applied multiplexed single-cell reduced-representation bisulfite sequencing to B cells from healthy donors and patients with CLL. We observed that the common clonal origin of CLL results in a consistently increased epimutation rate, with low variability in the cell-to-cell epimutation rate. By contrast, variable epimutation rates across healthy B cells reflect diverse evolutionary ages across the trajectory of B cell differentiation, consistent with epimutations serving as a molecular clock. Heritable epimutation information allowed us to reconstruct lineages at high-resolution with single-cell data, and to apply this directly to patient samples. The CLL lineage tree shape revealed earlier branching and longer branch lengths than in normal B cells, reflecting rapid drift after the initial malignant transformation and a greater proliferative history. Integration of single-cell bisulfite sequencing analysis with single-cell transcriptomes and genotyping confirmed that genetic subclones mapped to distinct clades, as inferred solely on the basis of epimutation information. Finally, to examine potential lineage biases during therapy, we profiled serial samples during ibrutinib-associated lymphocytosis, and identified clades of cells that were preferentially expelled from the lymph node after treatment, marked by distinct transcriptional profiles. The single-cell integration of genetic, epigenetic and transcriptional information thus charts the lineage history of CLL and its evolution with therapy.
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http://dx.doi.org/10.1038/s41586-019-1198-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533116PMC
May 2019

Corrupted coordination of epigenetic modifications leads to diverging chromatin states and transcriptional heterogeneity in CLL.

Nat Commun 2019 04 23;10(1):1874. Epub 2019 Apr 23.

New York Genome Center, New York, 10013, NY, USA.

Cancer evolution is fueled by epigenetic as well as genetic diversity. In chronic lymphocytic leukemia (CLL), intra-tumoral DNA methylation (DNAme) heterogeneity empowers evolution. Here, to comprehensively study the epigenetic dimension of cancer evolution, we integrate DNAme analysis with histone modification mapping and single cell analyses of RNA expression and DNAme in 22 primary CLL and 13 healthy donor B lymphocyte samples. Our data reveal corrupted coherence across different layers of the CLL epigenome. This manifests in decreased mutual information across epigenetic modifications and gene expression attributed to cell-to-cell heterogeneity. Disrupted epigenetic-transcriptional coordination in CLL is also reflected in the dysregulation of the transcriptional output as a function of the combinatorial chromatin states, including incomplete Polycomb-mediated gene silencing. Notably, we observe unexpected co-mapping of typically mutually exclusive activating and repressing histone modifications, suggestive of intra-tumoral epigenetic diversity. Thus, CLL epigenetic diversification leads to decreased coordination across layers of epigenetic information, likely reflecting an admixture of cells with diverging cellular identities.
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http://dx.doi.org/10.1038/s41467-019-09645-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478836PMC
April 2019

TARGET-seq Takes Aim at Cancer Evolution through Multi-omics Single-Cell Genotyping and Transcriptomics.

Mol Cell 2019 03;73(6):1092-1094

Division of Hematology and Medical Oncology, Department of Medicine and Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA; New York Genome Center, New York, NY, USA; Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY, USA. Electronic address:

In this issue of Molecular Cell, Rodriguez-Meira et al. (2019) present TARGET-seq, an elegant single-cell method that genotypes somatic mutations and captures whole transcriptomes in the same tumor cells, thus paving the way to directly link somatic mutations with resulting transcriptional phenotypes in clonally diverse cancer populations.
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http://dx.doi.org/10.1016/j.molcel.2019.03.009DOI Listing
March 2019

Hypermethylator Phenotype and Ectopic GIP Receptor in GNAS Mutation-Negative Somatotropinomas.

J Clin Endocrinol Metab 2019 05;104(5):1777-1787

Institut National de la Santé et de la Recherche Médicale, Le Kremlin Bicêtre, France.

Context: Besides GNAS gene mutations, the molecular pathogenesis of somatotroph adenomas responsible for gigantism and acromegaly remains elusive.

Objective: To investigate alternative driver events in somatotroph tumorigenesis, focusing on a subgroup of acromegalic patients with a paradoxical increase in growth hormone (GH) secretion after oral glucose, resulting from ectopic glucose-dependent insulinotropic polypeptide receptor (GIPR) expression in their somatotropinomas.

Design, Setting, And Patients: We performed combined molecular analyses, including array-comparative genomic hybridization, RNA/DNA fluorescence in situ hybridization, and RRBS DNA methylation analysis on 41 somatotropinoma samples from 38 patients with acromegaly and three sporadic giants. Ten patients displayed paradoxical GH responses to oral glucose.

Results: GIPR expression was detected in 13 samples (32%), including all 10 samples from patients with paradoxical GH responses. All GIPR-expressing somatotropinomas were negative for GNAS mutations. GIPR expression occurred through transcriptional activation of a single allele of the GIPR gene in all GIPR-expressing samples, except in two tetraploid samples, where expression occurred from two alleles per nucleus. In addition to extensive 19q duplications, we detected in four samples GIPR locus microamplifications in a certain proportion of nuclei. We identified an overall hypermethylator phenotype in GIPR-expressing samples compared with GNAS-mutated adenomas. In particular, we observed hypermethylation in the GIPR gene body, likely driving its ectopic expression.

Conclusions: We describe a distinct molecular subclass of somatotropinomas, clinically revealed by a paradoxical increase of GH to oral glucose related to pituitary GIPR expression. This ectopic GIPR expression occurred through hypomorphic transcriptional activation and is likely driven by GIPR gene microamplifications and DNA methylation abnormalities.
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http://dx.doi.org/10.1210/jc.2018-01504DOI Listing
May 2019

Adrenal GIPR expression and chromosome 19q13 microduplications in GIP-dependent Cushing's syndrome.

JCI Insight 2017 09 21;2(18). Epub 2017 Sep 21.

Inserm U1185, Le Kremlin Bicêtre, France.

GIP-dependent Cushing's syndrome is caused by ectopic expression of glucose-dependent insulinotropic polypeptide receptor (GIPR) in cortisol-producing adrenal adenomas or in bilateral macronodular adrenal hyperplasias. Molecular mechanisms leading to ectopic GIPR expression in adrenal tissue are not known. Here we performed molecular analyses on adrenocortical adenomas and bilateral macronodular adrenal hyperplasias obtained from 14 patients with GIP-dependent adrenal Cushing's syndrome and one patient with GIP-dependent aldosteronism. GIPR expression in all adenoma and hyperplasia samples occurred through transcriptional activation of a single allele of the GIPR gene. While no abnormality was detected in proximal GIPR promoter methylation, we identified somatic duplications in chromosome region 19q13.32 containing the GIPR locus in the adrenocortical lesions derived from 3 patients. In 2 adenoma samples, the duplicated 19q13.32 region was rearranged with other chromosome regions, whereas a single tissue sample with hyperplasia had a 19q duplication only. We demonstrated that juxtaposition with cis-acting regulatory sequences such as glucocorticoid response elements in the newly identified genomic environment drives abnormal expression of the translocated GIPR allele in adenoma cells. Altogether, our results provide insight into the molecular pathogenesis of GIP-dependent Cushing's syndrome, occurring through monoallelic transcriptional activation of GIPR driven in some adrenal lesions by structural variations.
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http://dx.doi.org/10.1172/jci.insight.92184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5621881PMC
September 2017

Efficient and versatile CRISPR engineering of human neurons in culture to model neurological disorders.

Wellcome Open Res 2016 Nov 15;1:13. Epub 2016 Nov 15.

Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK.

The recent identification of multiple new genetic causes of neurological disorders highlights the need for model systems that give experimental access to the underlying biology. In particular, the ability to couple disease-causing mutations with human neuronal differentiation systems would be beneficial. Gene targeting is a well-known approach for dissecting gene function, but low rates of homologous recombination in somatic cells (including neuronal cells) have traditionally impeded the development of robust cellular models of neurological disorders. Recently, however, CRISPR/Cas9 gene editing technologies have expanded the number of systems within which gene targeting is possible. Here we adopt as a model system LUHMES cells, a commercially available diploid human female mesencephalic cell line that differentiates into homogeneous mature neurons in 1-2 weeks. We describe optimised methods for transfection and selection of neuronal progenitor cells carrying targeted genomic alterations using CRISPR/Cas9 technology. By targeting the endogenous X-linked locus, we introduced four independent missense mutations that cause the autism spectrum disorder Rett syndrome and observed the desired genetic structure in 3-26% of selected clones, including gene targeting of the inactive X chromosome. Similar efficiencies were achieved by introducing neurodevelopmental disorder-causing mutations at the autosomal locus on chromosome 20. Our results indicate that efficiency of genetic "knock-in" is determined by the location of the mutation within the donor DNA molecule. Furthermore, we successfully introduced an mCherry tag at the locus to yield a fusion protein, demonstrating that larger insertions are also straightforward in this system. We suggest that our optimised methods for altering the genome of LUHMES cells make them an attractive model for the study of neurogenetic disorders.
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http://dx.doi.org/10.12688/wellcomeopenres.10011.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5146642PMC
November 2016

Ordered chromatin changes and human X chromosome reactivation by cell fusion-mediated pluripotent reprogramming.

Nat Commun 2016 08 10;7:12354. Epub 2016 Aug 10.

Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK.

Erasure of epigenetic memory is required to convert somatic cells towards pluripotency. Reactivation of the inactive X chromosome (Xi) has been used to model epigenetic reprogramming in mouse, but human studies are hampered by Xi epigenetic instability and difficulties in tracking partially reprogrammed iPSCs. Here we use cell fusion to examine the earliest events in the reprogramming-induced Xi reactivation of human female fibroblasts. We show that a rapid and widespread loss of Xi-associated H3K27me3 and XIST occurs in fused cells and precedes the bi-allelic expression of selected Xi-genes by many heterokaryons (30-50%). After cell division, RNA-FISH and RNA-seq analyses confirm that Xi reactivation remains partial and that induction of human pluripotency-specific XACT transcripts is rare (1%). These data effectively separate pre- and post-mitotic events in reprogramming-induced Xi reactivation and reveal a complex hierarchy of epigenetic changes that are required to reactivate the genes on the human Xi chromosome.
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http://dx.doi.org/10.1038/ncomms12354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987517PMC
August 2016

The inactive X chromosome is epigenetically unstable and transcriptionally labile in breast cancer.

Genome Res 2015 Apr 4;25(4):488-503. Epub 2015 Feb 4.

Centre de Recherche, Institut Curie, 75248 Paris Cedex 05, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche 3215, Institut Curie, 75248 Paris Cedex 05, France; Institut National de la Santé et de la Recherche Médicale U934, Institut Curie, 75248 Paris Cedex 05, France; Equipe Labellisée Ligue Contre le Cancer, UMR3215, 75248 Paris Cedex 05, France;

Disappearance of the Barr body is considered a hallmark of cancer, although whether this corresponds to genetic loss or to epigenetic instability and transcriptional reactivation is unclear. Here we show that breast tumors and cell lines frequently display major epigenetic instability of the inactive X chromosome, with highly abnormal 3D nuclear organization and global perturbations of heterochromatin, including gain of euchromatic marks and aberrant distributions of repressive marks such as H3K27me3 and promoter DNA methylation. Genome-wide profiling of chromatin and transcription reveal modified epigenomic landscapes in cancer cells and a significant degree of aberrant gene activity from the inactive X chromosome, including several genes involved in cancer promotion. We demonstrate that many of these genes are aberrantly reactivated in primary breast tumors, and we further demonstrate that epigenetic instability of the inactive X can lead to perturbed dosage of X-linked factors. Taken together, our study provides the first integrated analysis of the inactive X chromosome in the context of breast cancer and establishes that epigenetic erosion of the inactive X can lead to the disappearance of the Barr body in breast cancer cells. This work offers new insights and opens up the possibility of exploiting the inactive X chromosome as an epigenetic biomarker at the molecular and cytological levels in cancer.
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http://dx.doi.org/10.1101/gr.185926.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4381521PMC
April 2015

X-chromosome inactivation in development and cancer.

FEBS Lett 2014 Aug 14;588(15):2514-22. Epub 2014 Jun 14.

Mammalian Developmental Epigenetics Group, Genetics and Developmental Biology Unit, Institut Curie, CNRS UMR3215, INSERM U934, 75248 Paris, France. Electronic address:

X-chromosome inactivation represents an epigenetics paradigm and a powerful model system of facultative heterochromatin formation triggered by a non-coding RNA, Xist, during development. Once established, the inactive state of the Xi is highly stable in somatic cells, thanks to a combination of chromatin associated proteins, DNA methylation and nuclear organization. However, sporadic reactivation of X-linked genes has been reported during ageing and in transformed cells and disappearance of the Barr body is frequently observed in cancer cells. In this review we summarise current knowledge on the epigenetic changes that accompany X inactivation and discuss the extent to which the inactive X chromosome may be epigenetically or genetically perturbed in breast cancer.
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http://dx.doi.org/10.1016/j.febslet.2014.06.023DOI Listing
August 2014

A senescence-like cell-cycle arrest occurs during megakaryocytic maturation: implications for physiological and pathological megakaryocytic proliferation.

PLoS Biol 2010 Sep 7;8(9). Epub 2010 Sep 7.

INSERM, U790, Institut Gustave Roussy, Villejuif, France.

Thrombopoietin (TPO) via signaling through its cognate receptor MPL is a key cytokine involved in the regulation of megakaryocyte differentiation leading to platelet production. Mature megakaryocytes are polyploid cells that have arrested DNA replication and cellular proliferation but continue sustained protein synthesis. Here, we show that TPO induces cell-cycle arrest in the megakaryocytic UT7-MPL cell line by the activation of the ERK/MAPK pathway, induction of p21CIP transcription, and senescence markers through EGR1 activation. A similar senescence-like process was also detected in normal primary postmitotic megakaryocytes. In contrast, senescence was not observed in malignant megakaryocytes derived from primary myelofibrosis patients (a form of chronic myeloid hemopathy). Our data indicate that polyploid mature megakaryocytes receive signals from TPO to arrest cell proliferation and enter a senescent-like state. An escape from this physiological process may be associated with certain myeloproliferative neoplasms leading to abnormal megakaryocytic proliferation.
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http://dx.doi.org/10.1371/journal.pbio.1000476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2935456PMC
September 2010

Induction of myeloproliferative disorder and myelofibrosis by thrombopoietin receptor W515 mutants is mediated by cytosolic tyrosine 112 of the receptor.

Blood 2010 Feb 8;115(5):1037-48. Epub 2009 Dec 8.

Ludwig Institute for Cancer Research, Brussels, Belgium.

Constitutively active JAK2V617F and thrombopoietin receptor (TpoR) W515L/K mutants are major determinants of human myeloproliferative neoplasms (MPNs). We show that a TpoRW515 mutation (W515A), which we detected in 2 myelofibrosis patients, and the Delta5TpoR active mutant, where the juxtamembrane R/KW(515)QFP motif is deleted, induce a myeloproliferative phenotype in mouse bone marrow reconstitution experiments. This phenotype required cytosolic Y112 of the TpoR. Phosphotyrosine immunoprofiling detected phosphorylated cytosolic TpoR Y78 and Y112 in cells expressing TpoRW515A. Mutation of cytosolic Y112 to phenylalanine prevented establishment of the in vivo phenotype and decreased constitutive active signaling by Delta5TpoR and TpoRW515A, especially via the mitogen-activated protein (MAP)-kinase pathway, without decreasing Janus kinase 2 (JAK2) activation. In contrast, mutation of cytosolic Y78 to phenylalanine enhanced the myeloproliferative syndrome induced by the TpoRW515 mutants, by enhancing receptor-induced JAK2 activation. We propose that TpoR cytosolic phosphorylated Y112 and flanking sequences could become targets for pharmacologic inhibition in MPNs.
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http://dx.doi.org/10.1182/blood-2008-10-183558DOI Listing
February 2010

Ligand-independent thrombopoietin mutant receptor requires cell surface localization for endogenous activity.

J Biol Chem 2009 May 4;284(18):11781-91. Epub 2009 Mar 4.

INSERM, U790, Villejuif 94805, France.

The activating W515L mutation in the thrombopoietin receptor (MPL) has been identified in primary myelofibrosis and essential thrombocythemia. MPL belongs to a subset of the cytokine receptor superfamily that requires the JAK2 kinase for signaling. We examined whether the ligand-independent MPL(W515L) mutant could signal intracellularly. Addition of the endoplasmic reticulum (ER) retention KDEL sequence to the receptor C terminus efficiently locked MPL(W515L) within its natural ER/Golgi maturation pathway. In contrast to cells expressing the parental MPL(W515L), MPL(W515L)-KDEL-expressing FDC-P1 cells were unable to grow autonomously and to produce tumors in nude mice. When observed, tumor nodules resulted from in vivo selection of cells leaking the receptor at their surface. JAK2 co-immunoprecipitated with MPL(W515L)-KDEL but was not phosphorylated. We generated disulfide-bonded MPL(W515L) homodimers by the S402C substitution, both in the normal and KDEL context. Unlike MPL(W515L)-KDEL, MPL(W515L-S402C)-KDEL signaled constitutively and exhibited cell surface localization. These data establish that MPL(W515L) with appended JAK2 matures through the ER/Golgi system in an inactive conformation and suggest that the MPL(W515L)/JAK2 complex requires membrane localization for JAK2 phosphorylation, resulting in autonomous receptor signaling.
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http://dx.doi.org/10.1074/jbc.M808703200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2673247PMC
May 2009

The hematopoietic stem cell compartment of JAK2V617F-positive myeloproliferative disorders is a reflection of disease heterogeneity.

Blood 2008 Sep 8;112(6):2429-38. Epub 2008 Jul 8.

Inserm, U876, Bordeaux, France.

The JAK2V617F somatic point mutation has been described in patients with myeloproliferative disorders (MPDs). Despite this progress, it remains unknown how a single JAK2 mutation causes 3 different MPD phenotypes, polycythemia vera (PV), essential thrombocythemia, and primitive myelofibrosis (PMF). Using an in vivo xenotransplantation assay in nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice, we tested whether disease heterogeneity was associated with quantitative or qualitative differences in the hematopoietic stem cell (HSC) compartment. We show that the HSC compartment of PV and PMF patients contains JAK2V617F-positive long-term, multipotent, and self-renewing cells. However, the proportion of JAK2V617F and JAK2 wild-type SCID repopulating cells was dramatically different in these diseases, without major modifications of the self-renewal and proliferation capacities for JAK2V617F SCID repopulating cells. These experiments provide new insights into the pathogenesis of JAK2V617F MPD and demonstrate that a JAK2 inhibitor needs to target the HSC compartment for optimal disease control in classical MPD.
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http://dx.doi.org/10.1182/blood-2008-02-137877DOI Listing
September 2008

Evidence for MPL W515L/K mutations in hematopoietic stem cells in primitive myelofibrosis.

Blood 2007 Nov 20;110(10):3735-43. Epub 2007 Aug 20.

Institut National de la Santé et de la Recherche Médicale (INSERM), U790, Université Paris XI, Institut Gustave Roussy, Villejuif, France.

The MPL (W515L and W515K) mutations have been detected in granulocytes of patients suffering from certain types of primitive myelofibrosis (PMF). It is still unknown whether this molecular event is also present in lymphoid cells and therefore potentially at the hematopoietic stem cell (HSC) level. Toward this goal, we conducted MPL genotyping of mature myeloid and lymphoid cells and of lymphoid/myeloid progenitors isolated from PMF patients carrying the W515 mutations. We detected both MPL mutations in granulocytes, monocytes, and platelets as well as natural killer (NK) cells but not in T cells. B/NK/myeloid and/or NK/myeloid CD34(+)CD38(-)-derived clones were found to carry the mutations. Long-term reconstitution of MPL W515 CD34(+) cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice was successful for as long as 12 weeks after transplantation, indicating that MPL W515 mutations were present in HSCs. Moreover, the 2 MPL mutations induced a spontaneous megakaryocytic growth in culture with an overall normal response to thrombopoietin (TPO). In contrast, erythroid progenitors remained EPO dependent. These results demonstrate that in PMF, the MPL W515L or K mutation induces a spontaneous megakaryocyte (MK) differentiation and occurs in a multipotent HSCs.
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http://dx.doi.org/10.1182/blood-2007-05-089003DOI Listing
November 2007

Proteasome inhibitor bortezomib impairs both myelofibrosis and osteosclerosis induced by high thrombopoietin levels in mice.

Blood 2007 Jul 20;110(1):345-53. Epub 2007 Mar 20.

Institut National de la Santé et de la Recherche Médicale, U790, Université Paris XI, Villejuif, France.

Primary myelofibrosis (PMF) is the most serious myeloproliferative disorder, characterized by clonal myeloproliferation associated with cytokine-mediated bone marrow stromal reaction including fibrosis and osteosclerosis. Current drug therapy remains mainly palliative. Because the NF-kappaB pathway is implicated in the abnormal release of cytokines in PMF, the proteasome inhibitor bortezomib might be a potential therapy. To test its effect, we used the lethal murine model of myelofibrosis induced by thrombopoietin (TPO) overexpression. In this TPO(high) model, the development of the disease is related to a deregulated MPL signaling, as recently described in PMF patients. We first demonstrated that bortezomib was able to inhibit TPO-induced NF-kappaB activation in vitro in murine megakaryocytes. It also inhibited NF-kappaB activation in vivo in TPO(high) mice leading to decreased IL-1alpha plasma levels. After 4 weeks of treatment, bortezomib decreased TGF-beta1 levels in marrow fluids and impaired marrow and spleen fibrosis development. After 12 weeks of treatment, bortezomib also impaired osteosclerosis development through osteoprotegerin inhibition. Moreover, this drug reduced myeloproliferation induced by high TPO level. Finally, bortezomib dramatically improved TPO(high) mouse survival (89% vs 8% at week 52). We conclude that bortezomib appears as a promising therapy for future treatment of PMF patients.
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http://dx.doi.org/10.1182/blood-2006-10-054502DOI Listing
July 2007