Differential Engraftment of CEBPA Mutated Acute Myeloid Leukemia Cells in NSGS Mice

The transcription factor C/EBPα is a major regulator of granulopoiesis. Mutations in the gene that encodes C/EBPα are reported in around 10% of acute myeloid leukemia (AML) patients. The mutations reported in CEBPA are point mutations at C-terminus bZIP domain and/ or frame-shift mutations at N- terminus. CEBPA mutations can be mono-allelic or bi-allelic with one mutation located in N-terminus and other mutation in C-terminus. The type of the CEBPA mutation is critical determinants of AML prognosis-overall survival in patients whose AML carries a mono-allelic bZIP or bi-allelic CEBPA mutation is 60%, while the overall survival of those with mono-allelic N-terminal CEBPA mutations is 20%.

Patient-derived xenotransplantation (PDX) models represents a great tool for understanding disease biology and pre-clinical drug testing. PDX models utilizing human primary AML samples have provided novel insights on functional heterogeneity across patients, including the identification of phenotypes associated with leukemia-initiating cell populations. NSGS strain which express human stem cell factor, granulocyte-macrophage colony-stimulating factor and interleukin-3 has been reported to enhance engraftment of human primary AML samples. While engraftment characteristics of multiple AML subtypes have been studied in detail, whether CEBPA mutant AML cells engraft in PDX models, and whether there are any differences in engraftment between different subtypes of CEBPA mutations remain unexplored. Understanding the engraftment characteristics of CEBPA mutant AML samples provide us critical knowledge in disease biology, and will deliver a platform for future pre-clinical drug testing. In this study, we investigated engraftment characteristics of human CEBPA mutated primary AML samples in NSGS mice.

Male and female NSGS mice 6-8 weeks of age were sublethally irradiated 5 hours prior to cell injections. T-cell depleted human primary AML cells with 3 subclasses of CEBPA mutation - mono-allelic N-terminal mutant, mono-allelic C-terminal mutant and bi-allelic mutant (1 million per mouse, 5 independent AML samples/subclass) were transplanted via tail vein injection into NSGS mice. Human AML engraftment was assessed by flow cytometry for human CD45+CD33+ cells in bone marrow aspirates 5 days after transplantation. The mice were monitored for leukemia incidence, and peripheral blood was collected submandibularly at 4 weeks intervals after transplantation to study progression of AML.

Our data demonstrates that primary AML cells with mono-allelic N-terminal CEBPA mutations display superior engraftment than mono-allelic C-terminal and bi-allelic CEBPA mutations. NSGS mice transplanted with N-terminal CEBPA mutations succumbed to leukemia with a median latency of 8 weeks. Bone marrow and peripheral blood from leukemic mice showed presence of higher myeloid blast cells. Histological assessment by hematoxylin and eosin staining showed that AML cells infiltrated into multiple organs such as liver, spleen and lungs. Secondary transplantation of primary mouse leukemic cells in NSGS recipients developed AML with characteristics very similar to leukemia developed in primary recipients.

While mono-allelic C-terminal CEBPA mutated samples did not engraft in NSGS mice, bi-allelic CEBPA mutant samples with GATA2 zinc finger-1 mutation engrafted and developed acute erythroid leukemia (AEL). Bone marrow and peripheral blood from these leukemic mice showed presence of both myeloid and erythroid blast cells, consistent with recent studies showing development of AEL in a genetic model for bi-allelic CEBPA mutation and GATA2 zinc finger-1 mutation. We are currently investigating what are the factors contributing to engraftment and AEL development in AML samples with bi-allelic CEBPA mutation and GATA2 zinc finger-1 mutation.

Our studies suggests engraftment of CEBPA mutant AML samples depend on the subclass of CEBPA mutation and secondary mutation present in the AML sample. This knowledge of differential engraftment of CEBPA mutant sample in NSGS mice offers a valuable model for testing novel therapies for CEBPA mutant AML.

No relevant conflicts of interest to declare.