Olivier A. Bernard

Deregulated expression of cytokine receptor gene, CRLF2, is involved in lymphoid transformation in B-cell precursor acute lymphoblastic leukemia

We report 2 novel, cryptic chromosomal abnormalities in precursor B-cell acute lymphoblastic leukemia (BCP-ALL): a translocation, either t(X;14)(p22;q32) or t(Y;14)(p11;q32), in 33 patients and an interstitial deletion, either del(X)(p22.33p22.33) or del(Y)(p11.32p11.32), in 64 patients, involving the pseudoautosomal region (PAR1) of the sex chromosomes. The incidence of these abnormalities was 5% in childhood ALL (0.8% with the translocation, 4.2% with the deletion). Patients with the translocation were older (median age, 16 years), whereas the patients with the deletion were younger (median age, 4 years). The 2 abnormalities result in deregulated expression of the cytokine receptor, cytokine receptor-like factor 2, CRLF2 (also known as thymic stromal-derived lymphopoietin receptor, TSLPR). Overexpression of CRLF2 was associated with activation of the JAK-STAT pathway in cell lines and transduced primary B-cell progenitors, sustaining their proliferation and indicating a causal role of CRLF2 overexpression in lymphoid transformation. In Down syndrome (DS) ALL and 2 non-DS BCP-ALL cell lines, CRLF2 deregulation was associated with mutations of the JAK2 pseudokinase domain, suggesting oncogenic cooperation as well as highlighting a link between non-DS ALL and JAK2 mutations.

TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS.

TET proteins convert 5‐methylcytosine to 5‐hydroxymethylcytosine, an emerging dynamic epigenetic state of DNA that can influence transcription. Evidence has linked TET1 function to epigenetic repression complexes, yet mechanistic information, especially for the TET2 and TET3 proteins, remains limited. Here, we show a direct interaction of TET2 and TET3 with O‐GlcNAc transferase (OGT). OGT does not appear to influence hmC activity, rather TET2 and TET3 promote OGT activity. TET2/3–OGT co‐localize on chromatin at active promoters enriched for H3K4me3 and reduction of either TET2/3 or OGT activity results in a direct decrease in H3K4me3 and concomitant decreased transcription. Further, we show that Host Cell Factor 1 (HCF1), a component of the H3K4 methyltransferase SET1/COMPASS complex, is a specific GlcNAcylation target of TET2/3–OGT, and modification of HCF1 is important for the integrity of SET1/COMPASS. Additionally, we find both TET proteins and OGT activity promote binding of the SET1/COMPASS H3K4 methyltransferase, SETD1A, to chromatin. Finally, studies in Tet2 knockout mouse bone marrow tissue extend and support the data as decreases are observed of global GlcNAcylation and also of H3K4me3, notably at several key regulators of haematopoiesis. Together, our results unveil a step‐wise model, involving TET–OGT interactions, promotion of GlcNAcylation, and influence on H3K4me3 via SET1/COMPASS, highlighting a novel means by which TETs may induce transcriptional activation.

Recurrent mutations in epigenetic regulators, RHOA and FYN kinase in peripheral T cell lymphomas

Peripheral T cell lymphomas (PTCLs) are a heterogeneous and poorly understood group of non-Hodgkin lymphomas. Here we combined whole-exome sequencing of 12 tumor-normal DNA pairs, RNA sequencing analysis and targeted deep sequencing to identify new genetic alterations in PTCL transformation. These analyses identified highly recurrent epigenetic factor mutations in TET2, DNMT3A and IDH2 as well as a new highly prevalent RHOA mutation encoding a p.Gly17Val alteration present in 22 of 35 (67%) angioimmunoblastic T cell lymphoma (AITL) samples and in 8 of 44 (18%) PTCL, not otherwise specified (PTCL-NOS) samples. Mechanistically, the RHOA Gly17Val protein interferes with RHOA signaling in biochemical and cellular assays, an effect potentially mediated by the sequestration of activated guanine-exchange factor (GEF) proteins. In addition, we describe new and recurrent, albeit less frequent, genetic defects including mutations in FYN, ATM, B2M and CD58 implicating SRC signaling, impaired DNA damage response and escape from immune surveillance mechanisms in the pathogenesis of PTCL.

Recurrent TET2 mutations in peripheral T-cell lymphomas correlate with TFH-like features and adverse clinical parameters.

Inactivating mutations of the Ten-Eleven Translocation 2 (TET2) gene were first identified in myeloid malignancies and more recently in peripheral T-cell lymphomas (PTCLs). In the present study, we investigated the presence of TET2 coding sequence mutations and their clinical relevance in a large cohort of 190 PTCL patients. TET2 mutations were identified in 40 of 86 (47%) cases of angioimmunoblastic T-cell lymphoma (AITL) and in 22 of 58 (38%) cases of peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), but were absent in all other PTCL entities, with the exception of 2 of 10 cases of enteropathy-associated T-cell lymphoma. Among PTCL-NOS, a heterogeneous group of lymphoma-comprising cases likely to derive from Th follicular (T(FH)) cells similarly to AITL, TET2 mutations were more frequent when PTCL-NOS expressed T(FH) markers and/or had features reminiscent of AITL (58% vs 24%, P = .01). In the AITL and PTCL-NOS subgroups, TET2 mutations were associated with advanced-stage disease, thrombocytopenia, high International Prognostic Index scores, and a shorter progression-free survival.

Clonal architecture of chronic myelomonocytic leukemias.

Genomic studies in chronic myeloid malignancies, including myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), and MPN/MDS, have identified common mutations in genes encoding signaling, epigenetic, transcription, and splicing factors. In the present study, we interrogated the clonal architecture by mutation-specific discrimination analysis of single-cell-derived colonies in 28 patients with chronic myelomonocytic leukemias (CMML), the most frequent MPN/MDS. This analysis reveals a linear acquisition of the studied mutations with limited branching through loss of heterozygosity. Serial analysis of untreated and treated samples demonstrates a dynamic architecture on which most current therapeutic approaches have limited effects. The main disease characteristics are early clonal dominance, arising at the CD34(+)/CD38(-) stage of hematopoiesis, and granulomonocytic differentiation skewing of multipotent and common myeloid progenitors. Comparison of clonal expansions of TET2 mutations in MDS, MPN, and CMML, together with functional invalidation of TET2 in sorted progenitors, suggests a causative link between early clonal dominance and skewed granulomonocytic differentiation. Altogether, early clonal dominance may distinguish CMML from other chronic myeloid neoplasms with similar gene mutations.

Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors

TET2 converts 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA and is frequently mutated in myeloid malignancies, including myeloproliferative neoplasms. Here we show that the level of 5-hmC is decreased in granulocyte DNA from myeloproliferative neoplasm patients with TET2 mutations compared with granulocyte DNA from healthy patients. Inhibition of TET2 by RNA interference decreases 5-hmC levels in both human leukemia cell lines and cord blood CD34(+) cells. These results confirm the enzymatic function of TET2 in human hematopoietic cells. Knockdown of TET2 in cord blood CD34(+) cells skews progenitor differentiation toward the granulomonocytic lineage at the expense of lymphoid and erythroid lineages. In addition, by monitoring in vitro granulomonocytic development we found a decreased granulocytic differentiation and an increase in monocytic cells. Our results indicate that TET2 disruption affects 5-hmC levels in human myeloid cells and participates in the pathogenesis of myeloid malignancies through the disturbance of myeloid differentiation.

Loss of TET2 in hematopoietic cells leads to DNA hypermethylation of active enhancers and induction of leukemogenesis

DNA methylation is tightly regulated throughout mammalian development, and altered DNA methylation patterns are a general hallmark of cancer. The methylcytosine dioxygenase TET2 is frequently mutated in hematological disorders, including acute myeloid leukemia (AML), and has been suggested to protect CG dinucleotide (CpG) islands and promoters from aberrant DNA methylation. In this study, we present a novel Tet2-dependent leukemia mouse model that closely recapitulates gene expression profiles and hallmarks of human AML1-ETO-induced AML. Using this model, we show that the primary effect of Tet2 loss in preleukemic hematopoietic cells is progressive and widespread DNA hypermethylation affecting up to 25% of active enhancer elements. In contrast, CpG island and promoter methylation does not change in a Tet2-dependent manner but increases relative to population doublings. We confirmed this specific enhancer hypermethylation phenotype in human AML patients with TET2 mutations. Analysis of immediate gene expression changes reveals rapid deregulation of a large number of genes implicated in tumorigenesis, including many down-regulated tumor suppressor genes. Hence, we propose that TET2 prevents leukemic transformation by protecting enhancers from aberrant DNA methylation and that it is the combined silencing of several tumor suppressor genes in TET2 mutated hematopoietic cells that contributes to increased stem cell proliferation and leukemogenesis.

Congenital Portosystemic Shunts in Children: Recognition, Evaluation, and Management

Congenital portosystemic shunts are present in one in 30,000 children. Among the associated risks of severe complications are neonatal cholestasis, benign and malignant liver tumors, hepatopulmonary syndrome, portopulmonary hypertension, and encephalopathy. They can be detected on prenatal ultrasonograms, during the investigation of a positive galactosemia screening test in neonates or of a complication, or be found fortuitously on an abdominal ultrasound. Small intrahepatic shunts may resolve spontaneously within one year of age, but other shunts such as extrahepatic, persistent ductus venosus or persisting intrahepatic shunts, must be closed in one or two steps, by interventional radiology techniques or surgically. The plasticity of the intrahepatic portal system allows revascularization of the liver after shunt closure, even when no intrahepatic portal structures can be detected on imaging studies. This leaves little or no place for liver transplantation in the management of these children.

Severe giant cell hepatitis with autoimmune hemolytic anemia in early childhood.

Four children, aged 6 1/2 months to 2 years, presented with liver disease and autoimmune hemolytic anemia. Clinical signs included fever, jaundice, firm or hard hepatomegaly, and splenomegaly. Direct Coombs test results were of the mixed (IgG + C) type. Liver function tests showed high direct bilirubin transaminase, and serum gamma globulin values, and a prolonged prothrombin time. The liver histology was characterized by marked lobular fibrosis and giant cell transformation. The course of the disease was severe, resulting in the death of three patients from liver failure. However, the liver disease seemed responsive to corticosteroid treatment, which in one patient was clearly beneficial.

Activating mutations in genes related to TCR signaling in angioimmunoblastic and other follicular helper T-cell-derived lymphomas

Angioimmunoblastic T-cell lymphoma (AITL) and other lymphomas derived from follicular T-helper cells (TFH) represent a large proportion of peripheral T-cell lymphomas (PTCLs) with poorly understood pathogenesis and unfavorable treatment results. We investigated a series of 85 patients with AITL (n = 72) or other TFH-derived PTCL (n = 13) by targeted deep sequencing of a gene panel enriched in T-cell receptor (TCR) signaling elements. RHOA mutations were identified in 51 of 85 cases (60%) consisting of the highly recurrent dominant negative G17V variant in most cases and a novel K18N in 3 cases, the latter showing activating properties in in vitro assays. Moreover, half of the patients carried virtually mutually exclusive mutations in other TCR-related genes, most frequently in PLCG1 (14.1%), CD28 (9.4%, exclusively in AITL), PI3K elements (7%), CTNNB1 (6%), and GTF2I (6%). Using in vitro assays in transfected cells, we demonstrated that 9 of 10 PLCG1 and 3 of 3 CARD11 variants induced MALT1 protease activity and increased transcription from NFAT or NF-κB response element reporters, respectively. Collectively, the vast majority of variants in TCR-related genes could be classified as gain-of-function. Accordingly, the samples with mutations in TCR-related genes other than RHOA had transcriptomic profiles enriched in signatures reflecting higher T-cell activation. Although no correlation with presenting clinical features nor significant impact on survival was observed, the presence of TCR-related mutations correlated with early disease progression. Thus, targeting of TCR-related events may hold promise for the treatment of TFH-derived lymphomas.