Mutant BCL2 Co-Operates with CBFβ/PEBP2β-MYH11 to Promote Expansion of Leukemia Initiating Cells with a Predominantly Pro-Apoptotic Mechanism Via Recruitment of Ras-GTP In a Mouse Model of Progressive Acute Myeloid Leukemia.

Abstract 1050

Mutations and translocation of the core binding factor complexes frequently co-operate with other lesions in Acute Myeloid Leukemia (AML). In order to study the molecular mechanisms and develop therapeutic platforms we have developed highly penetrant and tractable mouse models.

We crossed single transgenic mice that express the human (h)BCL2 or NRASD12 under the control of the myeloid promoter MRP8 with single transgenic mice that express the protein fusion CBFβ/PEBP2β-MYH11 (Inv16) under the same promoter. Whereas single transgenic mice have a normal lifespan, the double transgenic mice develop rapid and fatal hematologic disease. The NRASD12/Inv16 mice are already known to develop strong features of dysmyelopoiesis, developing severe thrombopenia and anemia associated with leucocytosis (Kogan SC, et al. PNAS USA 95:11863-11868). Here within marrow and spleen cells we find abnormal eosinophils with Charcot-Leydan crystals associated with an increase of myeloid blasts, features that are found in human AML4 with eosinophilia. The hBCL2/Inv16 mice develop anemia and leucopenia, with greater than 30% bone marrow blast cells associated with enlarged infiltrated liver and spleens. Consequently hBCL2/Inv16 animals demonstrated significantly reduced survival as compared to litter mate controls (median 65 days; p<0.001). Immunophenotyping of circulating myeloid cells revealed lack of differentiation; namely a raised Mac-1^hi/Gr-1^lo profile. We further demonstrate that there is an expansion of the primitive Lin-/Sca-1+/c-Kit+ compartment for both NRASD12/Inv16 and hBCL2/Inv16 models in comparison with the singles. The colony assays performed show an increase of all myeloid progenitors for the hBCL2/Inv16 mice whereas NRASD12/Inv16 assays revealed a strong increase of CFU-M. The diseases are transplantable into irradiated syngenic recipients, supporting the expansion of the leukemic initiating cell compartment.

To further elucidate signaling mechanisms we employed phospho-interrogation of spleen samples using the capillary isoelectric focusing methodology via NanoPro CB1000 (Cell Biosciences). Novel data reveals the balanced role of ERK/BCL2 phosphorylation states illustrating pro versus anti-apoptotic signaling. Surprisingly during disease progression, hBCL2/Inv16 recruits endogenous Ras, with a pro-apoptotic phenotype, providing tangible targets for the treatment of progressive AML.