Synergistic Effect of CK2 Inhibitor CX-4945 with HDACi Chidamide in B-Cell Acute Lymphoblastic Leukemia By Downregulating TCL1A Expression
Authors: Xiang Huimin , Han Qi , Song Chunhua , Ge Zheng 
Published: December 2024

Background B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive hematological malignancy and high-risk B-ALL still has poor outcomes although many advanced treatment options have been developed. Ikaros dysfunction is the hallmark of high-risk B-ALL, we found Casein kinase II (CK2)-induced Ikaros dysfunction by inducing Ikaros hyperphosphorylation and targeting the CK2/IKAROS axis by CK2 inhibitor CX-4945 restores Ikaros function and demonstrated therapeutic efficacy in high-risk B-ALL (Song C, Blood, 2020; Zheng G, Leukemia 2021). Chidamide (CHI), a histone deacetylase inhibitor (HDACi), has been found to exert anticancer activity in hematologic malignancies by inhibiting cell proliferation, inducing cell cycle arrest, and promoting apoptosis (Kai C, Cell Death Dis 2020). However, whether and how the combination of the two drugs exerts synergistic anti-leukemic effects remains undetermined. This study aims to investigate the synergy of CX-4945 in combination with CHI in B-ALL.
Methods Cell Counting Kit-8 (CCK-8) cell proliferation assay, Annexin V apoptosis assay, and Propidium Iodide cell cycle assay were performed in B-ALL cells in the presence of CX-4945 only, CHI only, CX-4945 + CHI, and vehicle control for 48h. Differential expression genes (DEGs) and pathways analysis were conducted using R-software. TCL1A was knocked down by lentiviral shRNA in Nalm6 and BALL1 cells. q-PCR was performed to test TCL1A expression. T-test statistical analysis was employed to determine the significant difference between groups. A synergistic effect was determined by the combination indices (CIs) with CalcuSyn software.
Results Treatment of CX-4945 or CHI significantly led to cell proliferation arrest of Nalm6 and BALL1 cells in a dose-dependent manner. The combination of various doses of CHI with IC50 of CX-4945 significantly decreases the cell viability of the cells versus single-drug controls; CalcuSyn analysis showed the significant synergistic effects of CHI with CX-4945. The combination also significantly increased the apoptotic cell rates compared to the single-drug controls (p&lt;0.001). Cell cycle assay revealed that CX-4945 and CHI induced a G1-phase arrest in Nalm6 and BALL1 cells and this effect was significantly increased in the combination group (p&lt;0.01). Moreover, the combination significantly enhanced the cell growth arrest and increased the % of apoptotic cells in the primary cells from the B-ALL patients versus single drug controls (p&lt;0.001). To further understand the underlying mechanism of the synergy, we overlapped significantly binding genes of our Ikaros ChIP-seq (GSE141572) with Differentially Expressed genes (DEGs) of the B-ALL transcriptome (GSE13159 ). Results showed that a total of 264 genes were identified and TCL1A is one of the top targets, CX4945 treatment significantly enhanced the binding of IKAROS at the promoter region of TCL1A in Nalm6 cells, and Ikaros overexpression suppresses but its knockdown increases the expression of TCL1A in the cells. Moreover, we found that TCL1A is highly expressed (p&lt;0.001) and associated with poor prognosis in B-ALL patients (p&lt;0.05). q-PCR assay revealed that CX-4945 and CHI decreased TCL1A expression and the repression is more remarkable upon the CX-4945 combined with CHI compared with either single drug in B-ALL cell lines and primary sample (p&lt;0.001). Moreover, TCL1A knockdown significantly suppresses the proliferation of B-ALL cell lines (p&lt;0.05).
Conclusion Our results indicate that the combination of CX-4945 and CHI exerts an anti-leukemic effect by suppressing TCL1A expression in B-ALL. Our study reveals a new potential combination therapy for high-risk B-ALL.