The association of Greig syndrome and mastocytosis reveals the involvement of the hedgehog pathway in advanced mastocytosis.

Mastocytosis is a heterogeneous disease characterized by an abnormal accumulation of mast cells (MCs) in 1 or several organs. Although a somatic KIT D816V mutation is detected in ∼85% of patients, attempts to demonstrate its oncogenic effect alone have repeatedly failed, suggesting that additional pathways are involved in MC transformation. From 3 children presenting with both Greig cephalopolysyndactyly syndrome (GCPS, Mendelian Inheritance in Man [175700]) and congenital mastocytosis, we demonstrated the involvement of the hedgehog (Hh) pathway in mastocytosis. GCPS is an extremely rare syndrome resulting from haploinsufficiency of GLI3, the major repressor of Hh family members. From these familial cases of mastocytosis, we demonstrate that the Hh pathway is barely active in normal primary MCs and is overactive in neoplastic MCs. GLI3 and KIT mutations had a synergistic, tumorigenic effect on the onset of mastocytosis in a GCPS mouse model. Finally, Hh inhibitors suppressed neoplastic MC proliferation in vitro and extend the survival time of mice with aggressive systemic mastocytosis (ASM). This work revealed, for the first time, the involvement of Hh signaling in the pathophysiology of mastocytosis and demonstrated the cooperative effects of the KIT and Hh oncogenic pathways in mice with ASM, leading to the identification of new promising therapeutic targets.

Identification of a novel cDNA, encoding a cytoskeletal associated protein, differentially expressed in diffuse large B cell lymphomas.

Diffuse large B-cell lymphomas (DLBL) constitute an heterogeneous clinico-pathological entity. To characterize molecular events related to histological subtypes, clinical presentation or outcome, we compared the mRNAs expressed in a limited series of DLBL by Differential display-reverse transcription (DDRT) and cloned a differential cDNA, that we called LB1. LB1 open reading frame encodes a 683 amino-acid polypeptide that does not show significant homology upon comparison to protein databases, nor any structural domain relating LB1 to an already known protein family. Immunofluorescence analysis of transfected COS cells showed a cytoplasmic filamentous staining, indicating that LB1 protein is tightly associated with cytoskeletal fibers. Two LB1 transcripts, a major 3.6-3.9 Kb and a minor 2.2 Kb transcripts, were detected among human haematopoietic and non-haematopoietic lines and tissues. LB1 transcripts were abundant in testis, thymus and in tumour derived cell lines, while barely detectable in liver, prostate and kidney. Concerning DLBL, LB1 expression was high in two cases of DLBL, and low or undetectable in four others, confirming the differential expression previously observed in the DDRT experiment. Furthermore, LB1 gene mapped to chromosome 13q14, a region that has been involved as a chromosomal breakpoint in DLBL. The cellular function of LB1 and its relationship with B cell maturation and/or oncogenesis remain to be established.

The MAL gene is expressed in primary mediastinal large B-cell lymphoma.

Primary mediastinal large B-cell lymphoma (PMBL) appears to be a distinct clinicopathologic entity among diffuse large B-cell lymphomas (DLBLs). To find molecular alterations associated with this disease, we compared the mRNAs expressed in 3 PMBLs and 3 peripheral DLBLs by differential display-reverse transcription (DDRT) and identified a mRNA specifically expressed in PMBLs. Sequence analysis showed that this mRNA is encoded by the MAL gene, the expression of which was shown to be restricted to the T-cell lineage during hematopoiesis. MAL gene expression was demonstrated by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) in 8 of 12 PMBLs. However, there was little or no MAL gene expression in 8 peripheral DLBLs. Immunohistochemical analysis evidenced expression of MAL protein in tumoral B cells restricted to the PMBL subtype. Finally, Southern blot studies did not demonstrate rearrangement of the MAL gene. Altogether, our results indicate that MAL expression is recurrent in PMBLs, providing further evidence that PMBL represents a distinct entity among DLBLs. Because MAL protein is located in detergent-insoluble glycolipid-enriched membrane (GEM) domains involved in lymphocyte signal transduction, abnormal expression of MAL protein in the B-lymphoid lineage may have significant implications in PMBL lymphomagenesis.

Chromatin immunoselection defines a TAL-1 target gene.

Despite the major functions of the basic helix-loop-helix transcription factor TAL-1 in hematopoiesis and T-cell leukemogenesis, no TAL-1 target gene has been identified. Using immunoprecipitation of genomic fragments bound to TAL-1 in the chromatin of murine erythro-leukemia (MEL) cells, we found that 10% of the immunoselected fragments contained a CAGATG or a CAGGTG E-box, followed by a GATA site. We studied one of these fragments containing two E-boxes, CAGATG and CAGGTC, followed by a GATA motif, and showed that TAL-1 binds to the CAGGTG E-box with an affinity modulated by the CAGATG or the GATA site, and that the CAGGTG-GATA motif exhibits positive transcriptional activity in MEL but not in HeLa cells. This immunoselected sequence is located within an intron of a new gene co-expressed with TAL-1 in endothelial and erythroid cells, but not expressed in fibroblasts or adult liver where no TAL-1 mRNA was detected. Finally, in vitro differentiation of embryonic stem cells towards the erythro/megakaryocytic pathways showed that the TAL-1 target gene expression followed TAL-1 and GATA-1 expression. These results establish that TAL-1 is likely to activate its target genes through a complex that binds an E-box-GATA motif and define the first gene regulated by TAL-1.