MTG16 Variants Provide Context to Notch Signaling to Direct Leukemic Phenotype

Notch signaling guides hematopoietic stem and progenitor cells (HS/PCs) toward distinct cell fates and when dysfunctional, contributes to development of hematopoietic malignancies. Normally, Notch signaling favors T-cell fate and causes attenuated expression of myeloid-specific genes. In contrast, Notch pathway inactivation causes HS/PCs to diverge disproportionately toward a myeloid fate and is accompanied by T-lymphopenia. Mutations that constitutively activate Notch signaling trigger T-cell leukemia, while Notch inactivation is associated with myeloid leukemias. This suggests that Notch-regulated cell fate decisions are context dependent and that determinants downstream of Notch can influence Notch-dependent phenotypes in development and disease.

Myeloid Translocation Gene (MTG) 16 binds the intracellular domain of Notch (NICD) and this interaction is necessary to establish CD4⁺/CD8⁺ (double positive-DP) T-cell fate. Mtg16 -/- progenitors give rise to GR1⁺/Mac1⁺ progeny in response to Notch activation. In ex vivo assays, normal DP T-cell development can be restored to Mtg16 -/- progenitors by enforced expression of MTG16, but not an MTG16 derivative which lacks NICD binding and mimics a naturally occurring Mtg16 splice variant (MTG16c). These findings suggest the status of expressed Mtg16 isoforms can determine lineage allocation in response to Notch signaling and intimates that Notch signaling could trigger alternate phenotypes depending upon the context in which it is activated.

To assess impact of the NICD-MTG16 interaction as a determinant of malignant phenotype in the context of constitutive Notch signaling, we created bicistronic retroviral constructs whose NICD and MTG16 expression cassettes were separated by the "self-cleaving" 2A peptide of Foot and Mouth disease virus (FMDV). In each transduced progenitor cell, this insures concurrent stoichiometric expression of NICD and the chosen MTG16 derivative (wild type or NICD binding-deficient). Transduced Mtg16 -/- HS/PCs were then transplanted into irradiated, syngeneic recipients and leukemic phenotype established by flow cytometry. Mice transplanted with Mtg16 -/- cells transduced with vector only fail to reconstitute hematopoiesis in the recipient bone marrow due to a previously described HS/PC defect. Instead, these mice succumb to marrow failure by day 18 post-transplant. However, enforced expression of NICD alone enables survival of Mtg16 -/- progenitors, reconstitution of hematopoiesis and development of T-cell leukemia from transplanted donor elements. This same phenotype was observed when NICD was co-expressed with MTG16, indicating MTG16 is required for normal T-lymphopoiesis but is dispensible for NICD-driven T-cell leukemia. Notably, when Mtg16 -/- progenitors are transduced with NICD and a NICD binding deficient derivative of MTG16, recipients develop T-myeloid bilineal leukemia. This finding suggests that integration of NICD into transcriptional regulatory complexes involving MTG16 can alter the features of leukemic blasts along a continuum from lymphoid to myeloid phenotypes. To gain additional insights, we characterized promoter occupancy and Notch transcription complex (NTC) composition upon expression of MTG16 and its NICD binding deficient derivative. We find both MTG16 forms can exist in the same transcriptional complexes via their NHR2 oligomerization domains and can co-occupy the Notch-responsive Hes1 promoter. Given that NICD-MTG16 binding disrupts interactions between MTG16, histone deacetylases (HDACs) and other co-repressors, these data suggest that leukemic phenotype arising from constitutive Notch signaling may be influenced by combinatorial diversity among MTG16 splice variants and differential integration of the NICD into the NTC.