Activating Notch1 Mutations Are an Early Event in T-Cell Malignancy of Ikaros Point Mutant Mice.
Authors: Mantha Simon , Ward Maureen , McCafferty Jonathon , Palomero Teresa , Ferrando Adolfo , Bank Arthur , Richardson Christine 
Published: April 2020

The Ikaros and Notch1 genes are critical to T-cell formation and differentiation through transcriptional activation of target genes and interaction with chromatin remodeling complexes. Notch1 is a cell surface receptor that is cleaved upon activation to enter the nucleus, and mutated in over 50% of human T-ALL. Ikaros is a zinc finger transcription factor that acts as a homodimer or heterodimer paired with one of several Ikaros family related proteins. Dominant negative Ikaros isoforms generated by alternative splicing activities can induce T-cell tumors in mice and have been reported to participate in the pathogenesis of human T-cell leukemia. An ENU-induced point mutant allele of Ikaros named Plstc which inhibits activity of the normal isoform as well as of other Ikaros family members has been shown to cause a hereditary T-cell lymphoma with autosomal dominant transmission in C57B6 mice. We characterized this disease in heterozygote carriers (Plstc+/−). We now demonstrate for the first time that Notch1 activating mutations are present at a high frequency in the T-cell malignancies that arise in Plstc +/− mice. More than half of Plstc +/− mice developed cervical lymph node enlargement by 4 months of age, with disease-associated mortality close to 100% by 8 months. Pathologic examination revealed massive involvement of the thymus, spleen, liver and kidneys. Peripheral blood smears demonstrated a leukemic phase, but no significant differences in hemoglobin levels or platelet counts. FACS analysis of cervical lymph node tumors revealed a population of lymphocytes overwhelmingly CD3+/T-cell receptorβ+ and minimal CD34, CD117 or T-cell receptorγδ expression. 17% to 63% of T-cells in the samples were CD4+/CD8+, a characteristic of incompletely differentiated thymic lymphocytes, and 36% to 54% of T-cells in samples expressed CD25. Symptomatic lymphoma was preceded by expansion of a CD4+/CD8+ T cell population in the peripheral blood by 10 weeks of age and as early as 8 weeks. This is earlier than reported for other dominant negative forms of Ikaros which do not alter activity of Ikaros related family members. Mutation analysis of Notch1 in Plstc+/− T-cell lymphomas revealed the presence of activating mutations in Notch1 in 9 of 15 cases (60%), analogous to those reported in human T-ALL samples. In these cases, the isolated lymph node predominantly contained either an exon 26 point mutation within the activating cleavage domain, or an exon 34 insertion leading to a frameshift and early truncation of the C-terminal PEST domain. By contrast, DNA isolated from lymph nodes of wild type littermates and DNA isolated from tail tips of each mouse by 5 weeks of age did not contain Notch1 mutations consistent with a somatically acquired oncogenic event. The predominance of Notch1 mutations in the tumors of Plstc+/− mice shows that preferential interactions between Notch1 and Ikaros family members can be a significant early event in T-cell proliferation and tumorigenesis, and provides a mouse model for further defining interactions between the Ikaros and Notch1 oncoproteins in the pathogenesis of T-cell tumors.