P322: THERAPEUTIC EFFECT OF TARGETING CASEIN KINASE II / WDR5 AXIS IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

Abstract

Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignance with limit treatment choices and extremely poor prognosis. WD repeat domain 5 (WDR5) is essential for the methyltransferase activity of MLL1 complex and plays an important role in various biological functions. We reported that targeting Casein Kinase II (CK2)/Ikaros axis by CK2 inhibitor CX-4945 has therapeutic efficacy in B-ALL. However, the effect of WDR5 inhibitor and CX-4945 in T-ALL remains unknown. Aims: This study is to explore the synergistic effect of WDR5 inhibitor OICR-9429 and CK2 inhibitor CX-4945 and the potential underlying mechanisms of this novel drug combination in T-ALL. Methods: The expression profile and clinical significance of WDR5 and ATAD2 in T-ALL were explored by analyzing the transcriptome data of T-ALL patients from the GEO database. CCK-8 assay was performed to analyze the synergistic effect of OICR-9429 combined CX-4945 in CEM and MOLT4 cell lines. Cell cycle effect of inhibitors were detected by flow cytometry after 72h cultured. RNA-seq was performed after CEM cells were treated with OICR-9429, CX-4945, and vehicle control for 72 hours. Differential expression genes (DEGs) and pathways were analyzed by R-software. WDR5 and ATAD2 were knockdown by shRNA in CEM and MOLT4 cells. qPCR and Western Bolt were performed to test the DEGs detected by RNA-seq. Results:WDR5 is significantly over-expressed in T-ALL patients compared with healthy controls in two independent cohorts and is associated with shorter overall survival (P<0.01). Bioinformatic analysis revealed that the cell cycle was the most significantly altered pathway between WDR5 high and low expression patients (P<0.001) (Figure 1A). In vitro assay showed that OICR-9429 and CX-4945 alone exhibited an anti-proliferation effect on T-ALL cells after being treated for 72h. In addition, we found that OICR-9429 and CX-4945 had a significant synergistic effect on cell proliferation arrest in CEM (Figure 1B) and MOLT4 cells compared with a single drug (P<0.05). Cell cycle assay revealed that OICR-9429 (Figure 1C) and CX-4945 induced a G1-phase arrest in CEM and MOLT4 cells and this effect was significantly increased upon OICR-9429 and CX-4945 combination (P<0.01) (Figure 1D). The anti-proliferation and cell cycle arrest effects were also exhibited in WDR5 knockdown CEM and MOLT4 cells (P<0.05). Pathway analysis of the DEGs showed that the cell cycle pathway was obviously altered after OICR-9429 and CX-4945 treated (P<0.001) (Figure 1E). In addition, CX-4945 strongly down-regulated WDR5 (Figure 1F) expression in both transcriptome level and protein level, and knockdown of WDR5 increased the anti-leukemic effect of CX-4945 (P<0.01) (Figure 1G), suggesting that WDR5 was the downstream target of CK2 and CX-4945 in T-ALL. Moreover, RNA-seq data showed that ATAD2, an important oncogene was suppressed upon treatment of OICR-9429, CX-4945 (Figure 1F), and WDR5 knockdown (P<0.001). Moreover, the combination of OICR-9429 and CX-4945 strongly induced ATAD2 downregulation compared with either single drug (P<0.001) (Figure 1H). Likewise, we found that ATAD2 is up-regulated in T-ALL patients and associated with poor prognosis and cell cycle pathway abnormal activation (P<0.001). ATAD2 knockdown significantly inhibited cell proliferation and induced a G1-phase cell cycle arrest in T-ALL cells (P<0.01) (Fig 1I). Image:Summary/Conclusion: Our data showed that OICR-9429 and CX-4945 have synergistic efficacy in T-ALL. Dual targeting WDR5 and CK2 may be a potential therapeutic approach for T-ALL through the CK2-WDR5-ATAD2 axis.