MEN1-NULL MOUSE EMBRYONIC STEM CELL LINES ARE DEFICIENT IN THEIR ABILITY TO DIFFERENTIATE INTO HEMOPOIETIC LINEAGES

Background: Inherited mutations in the MENI gene, and the subsequent loss or inactivation of the remaining allele results in multiple endocrine neoplasia, type 1 (MEN1). However, homozygous loss of Men 1 in mouse results in embryonic lethality, indicating the importance of the ubiquitously expressed Men 1 encoded protein, menin, in early differentiation and development. Menin binds MLL (mixedlineage leukemia), and as components of a multiprotein complex, activate expression of target genes by methylating lysine 4 of histone H3 (H3K4). Though the precise biochemical function of menin is unclear, the loss of menin results in reduced H3K4 methylation, and the associated reduction in the expression of known MLL targets Hoxc8 and Hoxa9. MLL is essential for hematopoiesis, and is a key partner in translocations leading to leukemia via epigenetic regulation of Hox genes.

Aims: We are exploring the role of menin in early differentiation. Since menin is required for the MLL-mediated expression of Hoxc8 and Hoxa9, menin-null ES (embryonic stem) cell lines are likely to be deficient in their ability to differentiate into hematopoietic lineages.

Methods: We generated Men 1-/- ES cell lines, by targeting sequentially, both the Men 1 alleles. We induced these cell lines to differentiate in vitro to evaluate their ability to form embryoid bodies (EBs), express early hematopoietic markers, and subsequently to form hematopoietic colonies. Plasmids capable of expressing the Hoxa9 or Men 1 were transfected into the menin-null ES cell lines, and their ability to rescue the deficiency in hematopoiesis was evaluated.

Results: By XGal staining we showed that Cre was expressed in pancreatic a-cells and in gastro-intestinal glucagon gene-expressing cells. No extraneous Cre expression was detected in other endocrine or non-endocrine tissues. In Men IloxP/loxP; GluCre+ mice, the Men 1 gene was specifically disrupted in a-cells, as evidenced by menin/ glucagon double staining. Hyperplasia of a-cells was already apparent at 8 weeks of age. At 40 weeks glucagonomas started toappear, and by 54 weeks islet tumours have developed in all examined mutant mice (N=7). Serum glucose levels were slightly elevated in 6 month-old mutant mice, revealing a disturbed glucagon homeostasis. More detailed cellular, hormonal and molecular characterizations of these tumours are under the way, and the results will be presented at the meeting.

Conclusions: The new a-cell-specific Men 1 knockout model provides a powerful tool to study the biology of a-cell tumorigenesis.

Reference

1. Bertolino P, et al. Pancreatic beta-cell-specific ablation of the multiple endocrine neoplasia type 1 (MEN 1) gene causes full penetrance of insulinoma development in mice. Cancer Res. 2003, 15;63(16):4836-41.