Abstract
Enhancer of Zeste 2 (EZH2) is the enzymatic subunit of Polycomb Repressive Complex 2 (PRC2) that is highly upregulated and related to poor progress in AML patients. We observed the synergistic effects of EZH2 inhibitor DZNeP with newly synthesized flavonoid compound GL-V9 on apoptosis and cell proliferation arrest in AML cell lines and AML primary cells in vitro. In this study, we further explore the anti-tumor effect of DZNeP with GL-V9 on AML in vivo.
The 5-6-week NSG mice (GemPharmatech) were inoculated via the tail vein with U937 (1×10^6 cells) for the human leukemia xenograft (CDX) mouse model, and the two AML patients' samples (3x10^7) for the PDX mouse model, respectively. Once leukemia engraftment was confirmed by flow cytometry of the mice's peripheral blood, the animals were randomly divided into 4 groups (16 mice/group) for treatment: vehicle (N.S.), DZNeP alone (8 mg/kg every other day, i.p.), GL-V9 alone (300mg/kg daily, oral gavage), and combo. Treatments were performed for 8 weeks in two PDXs and 2 weeks in U937 CDX. The 8 mice per group were euthanized by CO2 inhalation once the vehicle group reached the early removal criteria according to IACUC (Institutional Animal Care and Use Committee) protocols. Bone marrow (BM) and spleen were harvested. For tumor burden analysis, cells from BM and spleen were stained with anti-human CD45 and CD33 and analyzed with flow cytometry (Attune NxT Cytometer), and immunohistochemistry (IHC) was also applied to detect human CD45-positive cells in spleen tissue. The remaining 8 mice per group were used for survival analysis, and the dead mice were recorded daily and statistically analyzed using the Kaplan-Meier curve.
In the U937 CDX model, the combination of DZNeP with GL-V9 significantly extended the survival, and reduced the spleen size, spleen weight, and % hCD45+hCD33+ cells in spleen and BM compared to vehicle and either single control. IHC data showed the combo group significantly reduced the % hCD45+ cells in the spleen compared to the control.
For the PDX mouse models, the first patient was diagnosed as MDS-derived AML with complex karyotype and mutations in the genes TET2 (82.48%), TP53 (48.21%), U2AF1 (42.77%), WT1 (5.26%), and RUNX1 (49.16%). The second patient is a newly diagnosed AML-M5a, with MLL-AF6 fusion gene, KRAS missense mutations: p.G13D (27.19%) and p.G12D (7.62%). We successfully established PDX models via tail vein injection of the two AML patient samples, as evidenced by the flow analysis of the peripheral blood of the mice. Results showed that the combination of DZNeP with GL-V9 significantly extended the survival, reduced the spleen size, spleen weight, and % hCD45+hCD33+ cells in spleen and BM, and the % hCD45+ cells in the spleen slides (IHC) compared to vehicle and either single control. Overall, our findings suggest the combination of DZNeP with GL- V9 exhibits potential antileukemic activity in PDX mouse models with diverse genetic backgrounds.
Our results demonstrated the in vivo anti-leukemia efficacy of DZNeP in combination with GL-V9 in one CDX and two PDX mouse models. Our findings strongly indicate that the combined therapy holds potential as a clinical therapeutic strategy for AML patients.
