Targeting EZH2 Promotes Chemosensitivity of BCL-2 Inhibitor through PIK3IP1-PI3K/AKT Axis in Acute Myeloid Leukemia
Authors: Yang Chan , Zi Jie , Song Chunhua , Ge Zheng 
Published: November 2021

Abstract
Introduction
Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2), which plays critical roles in transcription repressions.[1] PRC2 is required for acute Myeloid Leukemia (AML) cell survival, and EZH2 is overexpressed and related to worse prognosis in AML, therefore, EZH2 may involve in leukemogenesis through inhibition of the tumor suppressor genes in AML. [2, 3] BCL-2, one of the key anti-apoptosis proteins is dysregulated in AML. Venetoclax (Ven, ABT-199), a BCL-2 selective inhibitor has anti-tumor activity in AML but its overall response rate of monotherapy was unsatisfied owing to the clinical resistance to the inhibitor. [4] In this study, we examined the effect of targeting EZH2 on chemosensitivity of BCL-2 inhibitor in AML and the underlying mechanisms.
Methods
Cell Counting Kit-8 assay was used for cell proliferation and cytotoxicity in U937 and MV-4-11 AML cells treated with vehicle control, EZH2 inhibitor (DZNeP), Ven and Combination (Combo, Ven+DZNeP) for 48 hours. Synergistic effect was analyzed with Calcusyn. RNA-seq was performed with total RNA isolated from U937 cells treated with 2μM DZNeP, 7.5μM Ven or vehicle for 48 hours. Apoptosis was measured by cell staining with Annexin V+propidium iodide (PI) following flow cytometry analysis. EZH2 mRNA level was examined by qPCR in 39 newly-diagnosed AML patients from February 1, 2016 to February 28, 2019 at our institute with an approval of the Ethics Committee. Level of the apoptotic effectors was detected by western blot. GEPIA (Gene Expression Profiling Interactive Analysis) and R2 genomics analysis and visualization application were utilized for survival analysis.
Results
Both Ven and DZNeP had a dose-dependent and time-dependent effect on cell proliferation arrest in U937 and MV4-11 cells. DZNeP significantly sensitized the effect of Ven on cell proliferation arrest compared to single drug only (P&lt;0.001) (Fig.1a). CalcuSyn analysis showed the synergistic effect of the Ven+DZNeP on cell proliferation arrest (Fig.1b). Total apoptosis rate increased significantly in the Ven +DZNeP group in U937(32.04%±2.83) and MV-4-11 (25.73%±0.34) cells compared to single drug controls (Fig.1c). Also, level of the apoptotic effectors, PARP, Cleaved caspase-3 and Cleaved caspase-9 were significantly increased in Ven+DZNeP group compared to single drug and vehicle control (Fig.1d). Moreover, 1727 significantly regulated genes were identified by RNA-seq in U937 cells upon Ven treatment compared to vehicle control, and 1376 upon DZNeP treatment, in which 333 were overlapped (104 genes changed in the same direction but 228 changed in the opposite) (Fig 2a). The overlapped regulated genes upon the two drugs treatment are mainly involved in PIK3/AKT/mTOR, G1/S transition of mitotic cell cycle, apoptosis, et al. PIK3AP1, PIK3C2B and PIK3R3, the activators of PI3K signaling are up-regulated upon Ven treatment; and PI3KIP1, the suppressor of PI3K/AKT/mTOR signaling pathway is down-regulated by Ven but up-regulated by DZNeP. qPCR data showed that Ven+DZNeP significantly upregulated PIK3IP1 and down-regulated the expression of PI3K activators in the cells (Fig 2b). It is reported that the PI3K/AKT/mTOR signaling pathway is related to the clinical resistance of Ven in AML patients. Thus, our data not only showed the synergistic effect of Ven+DZNeP but also revealed a model that targeting EZH2 by DZNeP might conquer the Ven-induced bypass-effect through upregulation of PIK3IP1 to repression of PI3K/AKT signaling pathway. In addition, EZH2 mRNA level was quantified in 39 newly diagnosis patients with AML and 20 health control, and data showed EZH2 is increased in AML (p&lt;0.05). Metadata analysis support our results, and EZH2 overexpression is associated with short overall survival (p&lt;0.05) (Fig 2c). Database analysis assured that the expression of PIK3IP1 in patients with AML is lower than normal control. High PIK3IP1 showed a trend to a better outcome, which further supports that PIK3IP is a tumor suppressor in AML (Fig 2d).
Conclusions
Our data demonstrate the DZNeP sensitizes the effect of Ven in AML. Our results also reveal a novel mechanism that accounts for the synergistic effect of the two drugs and the fact that DZNeP may increase the chemosensitivity of BCL-2 inhibitor through PIK3IP1-PI3K/AKT axis in AML. Our findings suggest the potential combined therapy of Ven+DZNeP for AML.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.