Establishment and Characterization of a Novel Inducible Apoc2 and Cd36 double Knockout Mouse Model

Apolipoprotein C-II (APOC2) and CD36 act together to promote acute myeloid leukemia (AML) growth via activating the ERK signaling pathway and present a therapeutic target in AML. APOC2 activates lipoprotein lipase, thereby facilitating lipid metabolism. CD36 is a fatty acid transporter involved in lipid metabolism. Targeting either APOC2 or CD36 exhibits limited impact on normal hematopoiesis. However, the impact of simultaneous targeting of APOC2 and CD36 on normal hematopoiesis remains unknown.

Here, we established and characterized an inducible APOC2 and CD36 double knockout mouse model. Gt(ROSA)26Sor^Cre/Cre, Apoc2^fl/fl mice recently generated by our group by breeding Apoc2^fl/fl mice (from the NIH) with Gt(ROSA)26Sor^Cre/Cre (Jackson laboratory) were crossed with Cd36^tm1Mfe/J CD36 knockout mice (Jackson laboratory). Gt(ROSA)26Sor^Cre/Cre, Apoc2^fl/fl, CD36 knockout homozygous mice were generated and confirmed by genotyping. Male (M) and female (F) mice were injected with tamoxifen (TAM: 20mg/mL dissolved in corn oil) intraperitoneally (75mg/kg per day for 5 days) to induce APOC2-KO, resulting in the generation of APOC2 and CD36 double knockout mice (APOC2/CD36-DKO). The CD45.2⁺ wild type (WT) mice were injected with TAM and served as control mice. The knockdown of APOC2 in the liver and blood was confirmed by qPCR, and the knockdown of CD36 in blood was confirmed by flow cytometry.

The APOC2/CD36-DKO appeared healthy with no obvious phenotypes compared with WT. Analysis of APOC2/CD36 DKO (n = 3F+2M) and WT (n=3F+2M) shows that the average total body weight did not differ significantly between APOC2/CD36-DKO and WT mice after injecting TAM (APOC2/CD36 DKO (n = 2M + 2F) vs WT (n = 2M + 2F) = 23.88g vs 25.05g, P = 0.047). APOC2/CD36 DKO exhibited a slight yet not significant increase in liver mass compared to WT mice (APOC2/CD36 DKO (n = 2M + 4F) vs WT (n = 2M + 4F) = 1414mg vs 1289mg, P = 0.465), and spleen weight was also increased relative to WT (APOC2/CD36 DKO (n = 2M + 4F) vs WT (n = 2M + 4F) = 195.7mg vs 103.8mg, P = 0.067) when the mice were of similar age.

Serum collected from the APOC2/CD36-DKO appeared white and viscous. The total cholesterol (TC) and triglyceride (TG) levels measured by TC and TG colorimetric assay were 9.5-fold and 193.9-fold respectively higher in APOC2/CD36-DKO than in WT (P < 0.0001). Also, the APOC2/CD36 DKO exhibited a 2-fold higher white blood cell (WBC) count than WT mice by hematology profile (P = 0.004). Yet, the number of WBCs remained within the normal range. T cells from APOC2/CD36-DKO show no significant difference in their in vitro cell proliferation in the presence of IL-2 and PHA. Similarly, hematopoietic stem and progenitor cells (HSPCs) cultured in vitro in the presence of thrombopoietin, and stem cell factor exhibited similar cell expansion compared with cells from WT. This is consistent with the colony forming assay showing no significant difference between the number of colonies generated from HSPCs of APOC2/CD36 DKO versus those from WT mice (P = 0.740).

In conclusion, we present the generation of the first homozygous APOC2 and CD36 double knockout mouse model, revealing that the absence of APOC2 and CD36 induces very severe hypertriglyceridemia. Ongoing studies will further characterize this model and evaluate the impact of dual deletion of APOC2 and CD36 on normal hematopoiesis in mice.

Alachkar:Servier: Consultancy.