Jean Soulier

Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia

The β-haemoglobinopathies are the most prevalent inherited disorders worldwide. Gene therapy of β-thalassaemia is particularly challenging given the requirement for massive haemoglobin production in a lineage-specific manner and the lack of selective advantage for corrected haematopoietic stem cells. Compound βE/β0-thalassaemia is the most common form of severe thalassaemia in southeast Asian countries and their diasporas. The βE-globin allele bears a point mutation that causes alternative splicing. The abnormally spliced form is non-coding, whereas the correctly spliced messenger RNA expresses a mutated βE-globin with partial instability. When this is compounded with a non-functional β0 allele, a profound decrease in β-globin synthesis results, and approximately half of βE/β0-thalassaemia patients are transfusion-dependent. The only available curative therapy is allogeneic haematopoietic stem cell transplantation, although most patients do not have a human-leukocyte-antigen-matched, geno-identical donor, and those who do still risk rejection or graft-versus-host disease. Here we show that, 33 months after lentiviral β-globin gene transfer, an adult patient with severe βE/β0-thalassaemia dependent on monthly transfusions since early childhood has become transfusion independent for the past 21u2009months. Blood haemoglobin is maintained between 9 and 10u2009gu2009dl−1, of which one-third contains vector-encoded β-globin. Most of the therapeutic benefit results from a dominant, myeloid-biased cell clone, in which the integrated vector causes transcriptional activation of HMGA2 in erythroid cells with further increased expression of a truncated HMGA2 mRNA insensitive to degradation by let-7 microRNAs. The clonal dominance that accompanies therapeutic efficacy may be coincidental and stochastic or result from a hitherto benign cell expansion caused by dysregulation of the HMGA2 gene in stem/progenitor cells.

NOTCH1 directly regulates c-MYC and activates a feed-forward-loop transcriptional network promoting leukemic cell growth

The NOTCH1 signaling pathway directly links extracellular signals with transcriptional responses in the cell nucleus and plays a critical role during T cell development and in the pathogenesis over 50% of human T cell lymphoblastic leukemia (T-ALL) cases. However, little is known about the transcriptional programs activated by NOTCH1. Using an integrative systems biology approach we show that NOTCH1 controls a feed-forward-loop transcriptional network that promotes cell growth. Inhibition of NOTCH1 signaling in T-ALL cells led to a reduction in cell size and elicited a gene expression signature dominated by down-regulated biosynthetic pathway genes. By integrating gene expression array and ChIP-on-chip data, we show that NOTCH1 directly activates multiple biosynthetic routes and induces c-MYC gene expression. Reverse engineering of regulatory networks from expression profiles showed that NOTCH1 and c-MYC govern two directly interconnected transcriptional programs containing common target genes that together regulate the growth of primary T-ALL cells. These results identify c-MYC as an essential mediator of NOTCH1 signaling and integrate NOTCH1 activation with oncogenic signaling pathways upstream of c-MYC.

Exome sequencing identifies mutation in CNOT3 and ribosomal genes RPL5 and RPL10 in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is caused by the cooperation of multiple oncogenic lesions. We used exome sequencing on 67 T-ALLs to gain insight into the mutational spectrum in these leukemias. We detected protein-altering mutations in 508 genes, with an average of 8.2 mutations in pediatric and 21.0 mutations in adult T-ALL. Using stringent filtering, we predict seven new oncogenic driver genes in T-ALL. We identify CNOT3 as a tumor suppressor mutated in 7 of 89 (7.9%) adult T-ALLs, and its knockdown causes tumors in a sensitized Drosophila melanogaster model. In addition, we identify mutations affecting the ribosomal proteins RPL5 and RPL10 in 12 of 122 (9.8%) pediatric T-ALLs, with recurrent alterations of Arg98 in RPL10. Yeast and lymphoid cells expressing the RPL10 Arg98Ser mutant showed a ribosome biogenesis defect. Our data provide insights into the mutational landscape of pediatric versus adult T-ALL and identify the ribosome as a potential oncogenic factor.

Integrated Transcript and Genome Analyses Reveal NKX2-1 and MEF2C as Potential Oncogenes in T Cell Acute Lymphoblastic Leukemia

To identify oncogenic pathways in T cell acute lymphoblastic leukemia (T-ALL), we combined expression profiling of 117 pediatric patient samples and detailed molecular-cytogenetic analyses including the Chromosome Conformation Capture on Chip (4C) method. Two T-ALL subtypes were identified that lacked rearrangements of known oncogenes. One subtype associated with cortical arrest, expression of cell cycle genes, and ectopic NKX2-1 or NKX2-2 expression for which rearrangements were identified. The second subtype associated with immature T cell development and high expression of the MEF2C transcription factor as consequence of rearrangements of MEF2C, transcription factors that target MEF2C, or MEF2C-associated cofactors. We propose NKX2-1, NKX2-2, and MEF2C as T-ALL oncogenes that are activated by various rearrangements.

NF1 microdeletions in neurofibromatosis type 1: from genotype to phenotype

In 5‐10% of patients, neurofibromatosis type 1 (NF1) results from microdeletions that encompass the entire NF1 gene and a variable number of flanking genes. Two recurrent microdeletion types are found in most cases, with microdeletion breakpoints located in paralogous regions flanking NF1 (proximal NF1‐REP‐a and distal NF1‐REP–c for the 1.4 Mb type‐1 microdeletion, and SUZ12 and SUZ12P for the 1.2 Mb type‐2 microdeletion). A more severe phenotype is usually associated with NF1 microdeletion patients than in those with intragenic mutations. We characterized NF1 microdeletions in 70 unrelated NF1 microdeleted patients using a high‐resolution NF1 custom array comparative genomic hybridization (CGH). Genotype‐phenotype correlations were studied in 58 of these microdeletion patients and compared to 389 patients with intragenic truncating NF1 mutations and phenotyped in the same standardized way. Our results confirmed in an unbiased manner the existence of a contiguous gene syndrome with a significantly higher incidence of learning disabilities and facial dysmorphism in microdeleted patients compared to patients with intragenic NF1 mutations. Microdeleted NF1 patients also showed a trend toward significance for childhood overgrowth. High‐resolution array‐CGH identified a new recurrent ∼1.0u2009Mb microdeletion type, designated as type‐3, with breakpoints in the paralogous regions middle NF1‐REP‐b and distal NF1‐REP–c.

Genome wide DNA-profiling of marginal zone lymphomas identifies subtype-specific lesions with an impact on the clinical outcome

Marginal zone B-cell lymphomas (MZLs) have been divided into 3 distinct subtypes (extranodal MZLs of mucosa-associated lymphoid tissue [MALT] type, nodal MZLs, and splenic MZLs). Nevertheless, the relationship between the subtypes is still unclear. We performed a comprehensive analysis of genomic DNA copy number changes in a very large series of MZL cases with the aim of addressing this question. Samples from 218 MZL patients (25 nodal, 57 MALT, 134 splenic, and 2 not better specified MZLs) were analyzed with the Affymetrix Human Mapping 250K SNP arrays, and the data combined with matched gene expression in 33 of 218 cases. MALT lymphoma presented significantly more frequently gains at 3p, 6p, 18p, and del(6q23) (TNFAIP3/A20), whereas splenic MZLs was associated with del(7q31), del(8p). Nodal MZLs did not show statistically significant differences compared with MALT lymphoma while lacking the splenic MZLs-related 7q losses. Gains of 3q and 18q were common to all 3 subtypes. del(8p) was often present together with del(17p) (TP53). Although del(17p) did not determine a worse outcome and del(8p) was only of borderline significance, the presence of both deletions had a highly significant negative impact on the outcome of splenic MZLs.

The TLX1 oncogene drives aneuploidy in T cell transformation

The TLX1 oncogene (encoding the transcription factor T cell leukemia homeobox protein-1) has a major role in the pathogenesis of T cell acute lymphoblastic leukemia (T-ALL). However, the specific mechanisms of T cell transformation downstream of TLX1 remain to be elucidated. Here we show that transgenic expression of human TLX1 in mice induces T-ALL with frequent deletions and mutations in Bcl11b (encoding B cell leukemia/lymphoma-11B) and identify the presence of recurrent mutations and deletions in BCL11B in 16% of human T-ALLs. Most notably, mouse TLX1 tumors were typically aneuploid and showed a marked defect in the activation of the mitotic checkpoint. Mechanistically, TLX1 directly downregulates the expression of CHEK1 (encoding CHK1 checkpoint homolog) and additional mitotic control genes and induces loss of the mitotic checkpoint in nontransformed preleukemic thymocytes. These results identify a previously unrecognized mechanism contributing to chromosomal missegregation and aneuploidy active at the earliest stages of tumor development in the pathogenesis of cancer.

Human RTEL1 deficiency causes Hoyeraal–Hreidarsson syndrome with short telomeres and genome instability

Hoyeraal-Hreidarsson syndrome (HHS), a severe variant of dyskeratosis congenita (DC), is characterized by early onset bone marrow failure, immunodeficiency and developmental defects. Several factors involved in telomere length maintenance and/or protection are defective in HHS/DC, underlining the relationship between telomere dysfunction and these diseases. By combining whole-genome linkage analysis and exome sequencing, we identified compound heterozygous RTEL1 (regulator of telomere elongation helicase 1) mutations in three patients with HHS from two unrelated families. RTEL1 is a DNA helicase that participates in DNA replication, DNA repair and telomere integrity. We show that, in addition to short telomeres, RTEL1-deficient cells from patients exhibit hallmarks of genome instability, including spontaneous DNA damage, anaphase bridges and telomeric aberrations. Collectively, these results identify RTEL1 as a novel HHS-causing gene and highlight its role as a genomic caretaker in humans.

Hypomorphic Mutations in the Gene Encoding a Key Fanconi Anemia Protein, FANCD2, Sustain a Significant Group of FA-D2 Patients with Severe Phenotype

FANCD2 is an evolutionarily conserved Fanconi anemia (FA) gene that plays a key role in DNA double-strand-type damage responses. Using complementation assays and immunoblotting, a consortium of American and European groups assigned 29 patients with FA from 23 families and 4 additional unrelated patients to complementation group FA-D2. This amounts to 3%-6% of FA-affected patients registered in various data sets. Malformations are frequent in FA-D2 patients, and hematological manifestations appear earlier and progress more rapidly when compared with all other patients combined (FA-non-D2) in the International Fanconi Anemia Registry. FANCD2 is flanked by two pseudogenes. Mutation analysis revealed the expected total of 66 mutated alleles, 34 of which result in aberrant splicing patterns. Many mutations are recurrent and have ethnic associations and shared allelic haplotypes. There were no biallelic null mutations; residual FANCD2 protein of both isotypes was observed in all available patient cell lines. These analyses suggest that, unlike the knockout mouse model, total absence of FANCD2 does not exist in FA-D2 patients, because of constraints on viable combinations of FANCD2 mutations. Although hypomorphic mutations arie involved, clinically, these patients have a relatively severe form of FA.

An international study of intrachromosomal amplification of chromosome 21 (iAMP21): cytogenetic characterization and outcome

Intrachromosomal amplification of chromosome 21 (iAMP21) defines a distinct cytogenetic subgroup of childhood B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). To date, fluorescence in situ hybridisation (FISH), with probes specific for the RUNX1 gene, provides the only reliable detection method (five or more RUNX1 signals per cell). Patients with iAMP21 are older (median age 9 years) with a low white cell count. Previously, we demonstrated a high relapse risk when these patients were treated as standard risk. Recent studies have shown improved outcome on intensive therapy. In view of these treatment implications, accurate identification is essential. Here we have studied the cytogenetics and outcome of 530 iAMP21 patients that highlighted the association of specific secondary chromosomal and genetic changes with iAMP21 to assist in diagnosis, including the gain of chromosome X, loss or deletion of chromosome 7, ETV6 and RB1 deletions. These iAMP21 patients when treated as high risk showed the same improved outcome as those in trial-based studies regardless of the backbone chemotherapy regimen given. This study reinforces the importance of intensified treatment to reduce the risk of relapse in iAMP21 patients. This now well-defined patient subgroup should be recognised by World Health Organisation (WHO) as a distinct entity of BCP-ALL.