Engraftment of Human Primary Acute Myeloid Leukemia Defined by Integrated Genetic Profiling in NOD/SCID/IL2rγnull Mice for Preclinical Ceramide-Based Therapeutic Evaluation

Abstract

Acute Myeloid Leukemia (AML) is a highly heterogeneous and poor prognosis disease with few available therapeutic options. Novel advances are urgently needed, however effective models to test experimental therapeutics have been lacking. Recently, NOD/SCID/IL2rγnull (NSG) mice were shown to engraft primary human AML in a manner that recapitulated the natural disease and its progression. Additionally, integrated genomic profiling was used to refine risk stratification of AML. In this study, we demonstrated the engraftment of molecularly defined primary AML in NSG mice. We showed that AML that express DNMT3A mutations, which predict for adverse outcome, engrafted with exceptional efficacy. Lastly, we demonstrated that human AML-engrafted NSG mice can be effectively used to study novel ceramide-based therapeutics. Ceramide is a bioactive sphingolipid that has been implicated as an inducer of apoptosis. Elevation in cancer cell ceramide levels either via exogenous delivery or by provoking intracellular ceramide generation is the goal of ceramide-based therapeutics. In this study, we used the human AML-engrafted NSG mouse model to evaluate nanoliposomal short-chain C6-ceramide and a nanoliposomal formulation of the ceramide-inducer tamoxifen. Altogether, the NSG model is likely to prove invaluable in the study of novel agents, sushc as ceramide-based therapeutics, with the ability to define therapeutic activity against specific molecularly defined and risk stratified AML.

Keywords: Acute myeloid leukemia; Ceramide; DNMT3A; Integrated genetic profiling; NSG mouse; Nanoliposome; Tamoxifen.

Figure 1

Monitoring the therapeutic efficacy of C6-ceramide and tamoxifen nanoliposomes in NSG mice engrafted with human AML. NSG mice were given sublethal total body irradiation prior to engraftment with human AML via tail vein injection. Engraftment was evaluated by flow cytometry of the peripheral blood (A), and bone marrow (B), using anti-human CD13, CD33, HLA-DR, and CD45 antibodies. (C) Treatment was initiated following engraftment confirmation, and the anti-AML efficacy of C6-ceramide nanoliposomes (Lip-C6), or control nanoliposomes (Lip-Ghost), was evaluated using NSG mice engrafted with a poor prognosis human AML sample (inv3, -7), and leukemia burden was routinely monitored by analysis of blood collected from tail vein prick. *p<0.001, 2-way ANOVA, n=4 mice per group, error bars represent standard error of the mean. (D) Human AML engraftment was assessed by flow cytometry of spleen and bone marrow preparations following necropsy at day 98. Included is a comparison with tamoxifen nanoliposomes (Lip-Tam), as well as nanoliposomes loaded with both C6-ceramide and tamoxifen (Lip-C6/Tam) (*p=0.0317, unpaired t-test comparing Lip-Ghost and Lip-Tam).

Figure 2

DNMT3A mutation predicts for enhanced engraftment in NSG mice. The integrated genetic risk profile, or DNMT3A mutation, was used to rank cases for linear regression analysis (1=good, 2=intermediate, 3=poor, 4=DNMT3A mutation).