Ronan Chaligne

Weill Cornell Medical College | Cornell

Metastasis is the principal cause of cancer death, yet we lack an understanding of metastatic cell states, their relationship to primary tumor states, and the mechanisms by which they transition. In a cohort of biospecimen trios from same-patient normal colon, primary and metastatic colorectal cancer, we show that while primary tumors largely adopt...

RNA splicing factors are recurrently mutated in clonal blood disorders, but the impact of dysregulated splicing in hematopoiesis remains unclear. To overcome technical limitations, we integrated genotyping of transcriptomes (GoT) with long-read single-cell transcriptomics and proteogenomics for single-cell profiling of transcriptomes, surface prote...

The response to tumor-initiating inflammatory and genetic insults can vary among morphologically indistinguishable cells, suggesting as yet uncharacterized roles for epigenetic plasticity during early neoplasia. To investigate the origins and impact of such plasticity, we performed single-cell analyses on normal, inflamed, premalignant, and maligna...

Interferon-alfa (IFN) as a therapeutic agent for myeloproliferative neoplasms (MPN) presents a unique opportunity to decipher type 1 IFN signaling in primary mutated vs wildtype (WT) human hematopoietic stem and progenitor cells. Thus, we applied our innovative Genotyping of Transcriptomics (GoT) that captures mutation status and single-cell RNA-se...

Somatic mutations in cancer genes have been detected in clonal expansions across healthy human tissue, including in clonal hematopoiesis. However, because mutated and wild-type cells are admixed, we have limited ability to link genotypes with phenotypes. To overcome this limitation, we leveraged multi-modality single-cell sequencing, capturing geno...

RNA splicing factors are recurrently affected by alteration-of-function mutations in clonal blood disorders, highlighting the importance of splicing regulation in hematopoiesis. However, our understanding of the impact of dysregulated RNA splicing has been hampered by the inability to distinguish mutant and wildtype cells in primary patient samples...

In normal somatic tissue differentiation, changes in chromatin accessibility govern priming and commitment of precursors towards cellular fates. In turn, somatic mutations can disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging du...

Somatic mutations in cancer genes have been ubiquitously detected in clonal expansions across healthy human tissue, including in clonal hematopoiesis. However, mutated and wildtype cells are morphologically and phenotypically similar, limiting the ability to link genotypes with cellular phenotypes. To overcome this limitation, we leveraged multi-mo...

Interferon-alpha (IFN), the first approved immunotherapy for cancer, remains an effective therapy for patients with myeloproliferative neoplasms (MPN). The mechanisms of action of IFN on MPN cells are poorly understood, particularly in patients with CALR mutated (MUT) MPNs, who often exhibit clinical but not molecular responses. Previously, by deve...

Splicing factor mutations are recurrent genetic alterations in blood disorders, highlighting the importance of alternative splicing regulation in hematopoiesis. Specifically, mutations in splicing factor 3B subunit 1 (SF3B1) are implicated in the pathogenesis of myelodysplastic syndromes (MDS) and linked to a high-risk of leukemic transformation in...

In hematopoiesis, changes in chromatin accessibility define priming and commitment of hematopoietic precursors towards cellular fates. In turn, somatic mutations in hematopoietic stem and progenitor cells (HSPCs) drive the onset and progression of myeloid disorders, such as myeloproliferative neoplasms (MPNs), and reshape differentiation topologies...

Single-cell RNA sequencing has revealed extensive transcriptional cell state diversity in cancer, often observed independently of genetic heterogeneity, raising the central question of how malignant cell states are encoded epigenetically. To address this, here we performed multiomics single-cell profiling—integrating DNA methylation, transcriptome...

The integration of DNA methylation and transcriptional state within single cells is of broad interest. Several single-cell dual- and multi-omics approaches have been reported that enable further investigation into cellular heterogeneity, including the discovery and in-depth study of rare cell populations. Such analyses will continue to provide impo...

Epigenetic alterations, such as promoter hypermethylation, may drive cancer through tumor suppressor gene inactivation. However, we have limited ability to differentiate driver DNA methylation (DNAme) changes from passenger events. We developed DNAme driver inference–MethSig–accounting for the varying stochastic hypermethylation rate across the gen...

Human diffuse gliomas are incurable malignancies, where cellular state diversity fuels tumor progression and resistance to therapy. Single-cell RNA-sequencing (scRNAseq) studies recently charted the cellular states of the two major categories of human gliomas, IDH-mutant gliomas (IDH-MUT) and IDH-wildtype glioblastoma (GBM), showing that malignant...

Leukemia driver mutations have been identified in clonal hematopoiesis (CH; Jaiswal et al, NEJM, 2014). This provides a window of opportunity to interrogate the downstream impact of driver mutations in the earliest stages of neoplasia, before the accumulation of additional drivers that lead to frank malignancy. However, CH mutated cells are morphol...

The field of cancer genomics has been empowered by increasingly sophisticated inference tools to distinguish driver mutations from the vastly greater number of passenger mutations. Epigenetic alterations such as promoter DNA hypermethylation have been shown to drive cancer through inactivation of tumor suppressor genes (TSGs), but growing malignant...

Mutations in the kinase binding domain of BTK at position C481 are associated with resistance to BTK inhibitor (BTKi) therapy in chronic lymphocytic leukemia (CLL). Nearly half of patients manifesting clinical progression with these alterations exhibit a subclonal burden of resistance. Intriguingly, measured BTKC481 variant allelic fractions (VAF)...

Human diffuse gliomas are incurable brain tumors, where cellular state diversity fuels tumor progression and resistance to therapy. Single-cell RNA-sequencing (scRNAseq) studies recently charted the cellular states of the two major categories of human gliomas, IDH-mutant gliomas (IDH-MUT) and IDH-wildtype glioblastoma (GBM), showing that malignant...

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 an...

Mutations in genes involved in DNA methylation (DNAme; for example, TET2 and DNMT3A) are frequently observed in hematological malignancies1–3 and clonal hematopoiesis4,5. Applying single-cell sequencing to murine hematopoietic stem and progenitor cells, we observed that these mutations disrupt hematopoietic differentiation, causing opposite shifts...

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-ce...

A single-cell approach is used to follow the heritable stochastic changes to DNA methylation that occur in primary chronic lymphocytic leukaemia and healthy B cells, allowing the tracing of cell lineage histories and evolution during treatment with ibrutinib.

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 R...

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.

Genetic, epigenetic and transcriptional heterogeneity cooperate to fuel cancer's ability to evolve and adapt to therapy. In chronic lymphocytic leukemia (CLL), we have shown through bulk DNA methylation (DNAme) sequencing that the growing CLL populations diversify through stochastic DNAme changes (epimutations), impacting transcriptional heterogene...

Somatic mutations in hematopoietic precursors underlie the development of myeloid disorders, such as myeloproliferative neoplasms (MPN). However, our ability to interrogate the transcriptional impact of these mutations on human hematopoiesis is limited by the frequent admixing of mutant (MUT) with wildtype (WT) cells or with other subclones. Recent...

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 GH secretion after oral glucose, resulting f...

Defining the transcriptomic identity of clonally related malignant cells is challenging in the absence of cell surface markers that distinguish cancer clones from one another or from admixed non-neoplastic cells. While single-cell methods have been devised to capture both the transcriptome and genotype, these methods are not compatible with droplet...

Background 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. Methods We performed molecular...

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 ad...

Le syndrome de Cushing dépendant de l’alimentation est dû à l’expression illégitime du glucose-dependent insulinotropic peptide receptor (GIPR) dans les adénomes ou dans les hyperplasies surrénaliennes. Les mécanismes moléculaires conduisant à l’expression ectopique du GIPR dans les lésions surrénaliennes, qui confèrent une sensibilité anormale au...

L’acromégalie est caractérisée par l’absence de freinage de l’hormone de croissance (growth hormone, GH) dans le test de l’hyperglycémie provoquée par voie orale. Un sous-groupe de patients acromégales présente non seulement un freinage insuffisant, mais aussi une augmentation paradoxale de la GH plasmatique suite à l’hyperglycémie, dont les mécani...

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...

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...

The transition of ductal carcinoma in situ (DCIS) to invasive breast carcinoma requires tumor cells to cross the basement membrane (BM). However, mechanisms underlying BM transmigration are poorly understood. Here, we report that expression of membrane-type 1 (MT1)-matrix metalloproteinase (MMP), a key component of the BM invasion program, increase...

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 or...

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 proteins, DNA methylation and nuclear organi...

TPO and MEK1SS/DD expression induces ERK phosphorylation without any changes in total ERK expression. (a) UT711oc1 cells were treated with GM-CSF or TPO ± PD98059 or UO126 for 2 d. ERK phosphorylation and total ERK expression were then evaluated by WB. (b) UT711oc1 cells transduced with either empty retroviral vector (pMigr) or the vector encoding...

Top coincident genes when TPO-induced gene expression profile was compared to the molecular signature of oncogenic ras-induced senescence established in fibroblasts by Mason et al. [29]. (a) Top coincident up-regulated genes and their involvement in “growth, proliferation, and apoptosis”; “inflammation”; or “DNA replication, recombination, and repa...

EGR1 shRNAs are functional and re-induce cell proliferation after TPO exposure. (a) EGR1 shRNAs inhibit EGR1 protein expression after TPO exposure. (b) BrdU incorporation at 5 d of culture shows a significant (but partial) increase in DNA replication with TPO when cells express EGR1 shRNAs. Error bar represents the standard deviation. We performed...

Inhibition of ERK decreases p21 expression in human megakaryocytes. Human megakaryocytes were cultured for 10 d in presence of TPO and 10 µM of U0126 MAPK inhibitor. U0126 inhibited ERK phosphorylation (b) and p21 protein (b) and mRNA expression (a). We performed three independent experiments. (0.11 MB TIF)

Thrombopoietin induces cellular senescence of UT711oc1 and not apoptosis. (a) TPO and GM-CSF induce similar levels of apoptosis in UT711oc1 cells. Cells were treated with TPO for 3 and 6 d and compared to GM-CSF-cultured cells for Annexin V labeling. (b) No difference in PARP and Caspase 3 cleavage between TPO- and GM-CSF-treated cells. UT711oc1 we...

p21 mRNA expression is up-regulated by TPO via the RAS/MAPK pathway. (a) p21 mRNA expression in UT711oc1 cells. Cells were treated with GM-CSF or TPO in presence of PD98056 and U0126 inhibitors and assayed for gene expression by Taqman. (b) Cathepsin D mRNA expression in UT711oc1 cells. Cells were treated with GM-CSF or TPO in presence of PD98056 a...

Cathepsin D and megakaryocytic differentiation markers in normal human megakaryocytes. (a) Proportion of CD41 and CD42 expressing cells during the megakaryocytic culture. (b) Co-expression of vWF and CD41 markers in megakaryocytes after 12 d of cell culture. (c) Cathepsin D mRNA expression during megakaryocytic differentiation process of human cyta...

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 ind...

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 myeloprolif...

436 Polycythemia vera (PV), essential thrombocytemia (ET) and primary myelofibrosis (PMF) are myeloproliferative disorders (MPDs) without curative treatment, unless hematopoietic stem cell (HSC) transplantation is performed. However, for several years the use of interferon-alpha (IFNα) has provided an efficient therapeutic alternative for MPD patie...

Adenovirus (Ad)-mediated delivery of anti-angiogenic molecules into tumors constitutes an appealing approach for growth inhibition. However, lack of expression on tumors of Ad receptors leads to weak tumor transduction. Therefore, to provide Ad with a new entry pathway into tumors, an NGR peptide was inserted into either fiber (AdFNGR) or hexon (Ad...

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 MPLW515L mutant could signal intracellularly. Addition of...

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.

The molecular pathogenesis of classic Philadelphia-negative myeloproliferative disorders (MPDs) has been greatly elucidated since the discovery of the JAK2V617F mutation in 2005. This abnormality is present in almost all patients with polycythemia vera and half of patients with essential thrombocythemia and primitive myelofibrosis. The mutation rec...

Primitive myelofibrosis (PMF) is a lethal myeloproliferative disorder (MPD) biologically characterized by a spontaneous megakaryocytic growth and the development of bone marrow and spleen fibrosis. PMF and essential thrombocytemia (ET) differ by their clinical features despite similar oncogenic molecular events (i.e. JAK2V617F and MPLW515 mutations...

The activating mutation W515L in the thrombopoietin receptor Mpl has been identified in a minority of primitive myelofibrosis and essential thrombocythemia negative for the JAK2V617F mutation. Mpl and the erythropoietin receptor EpoR belong to a distinct subset of the cytokine receptor superfamily. Mpl and EpoR lack intrinsic kinase activity and re...

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 nono...

MPL (or thrombopoietin receptor, TPO-R) 515 mutations have recently been described in 5-10% of primitive myelofibrosis (PMF) cases as decisive oncogenic events capable of triggering the disease. Here we report additional mutations located in exon 10 of MPL in PMF patients. We investigated whether these new mutations also lead to cell transformation...

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 ma...

X-linked clonality studies showed that myeloproliferative disorders derive from an abnormal hematopoietic stem cell (HSC). This has been recently confirmed by studies showing that the JAK2 V617F mutation was present in multipotent cells from patients with Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primitive Myelofibrosis (PMF). How...

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...

The MPL 515L mutation has recently been described in 10% of Idiopathic Myelofibrosis (MMM) as an essential oncogenic event. Here we report another MPL mutation in the same codon of MPL (MPL515K). The MPL515L mutation has been detected in granulocytes but has not been yet detected in patient hematopietic stem cells. This raises the question whether...

Agnogenic myeloid metaplasia or idiopathic myelofibrosis become the more severe "classical" myeloproliferative disorder since Gleevec® have been used in clinical practice to treat chronic myeloid leukemia patients. Interestingly, this disorder have two mains characteristics: a myeloproliferation and a stromal secondary reaction that could be respon...