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4188 Dual Targeting Novel WDR5/ATAD2 Oncogenic Signaling through CK2/Ikaros Axis Demonstrates Synergistic Efficacy in T-ALL

Program: Oral and Poster Abstracts
Session: 605. Molecular Pharmacology and Drug Resistance: Lymphoid Neoplasms: Poster III
Hematology Disease Topics & Pathways:
Fundamental Science, Research, Translational Research, Combination therapy, Therapies
Monday, December 11, 2023, 6:00 PM-8:00 PM

Qi Han1*, Yan Gu1*, Chan Yang1*, Jun Li1*, Sinisa Dovat, MD, PhD2, Chunhua Song, MD, PhD3 and Zheng Ge, MD, PhD1

1Department of Hematology, Zhongda Hospital, School of Medicine, Southeast University, Institute of Hematology Southeast University, Nanjing, China
2Hershey Medical Center, Pennsylvania State University Medical College, Hershey, PA
3Division of Hematology, The Ohio State University Wexner Medical Center and The James Cancer Hospital, Columbus, OH

Background WD repeat-containing protein 5 (WDR5) is highly expressed in human cancers and its high expression is linked with adverse prognoses. However, the oncogenic roles of WDR5 and underlying mechanisms are undefined in T-cell acute lymphoblastic leukemia (T-ALL). Casein Kinase II (CK2) is a pro-oncogene, and CK2-mediated IKAROS dysfunction plays critical roles in high-risk ALL, and CK2 inhibitor CX-4945 has the anti-leukemic effect by restoring IKAROS function. This study aims to explore the synergistic anti-leukemic efficacy of dual targeting novel WDR5/ATAD2 oncogenic signaling via the CK2/IKAROS axis in T-ALL.

Methods Clinical samples from 38 T-ALL patients and 32 healthy controls were obtained from Zhongda Hospital Southeast University. The expression of WDR5 and ATAD2 were tested by q-PCR and Western blot. RNA-seq was performed after CEM cells were treated with WDR5 inhibitor (OICR-9429), CX-4945 and vehicle control for 72 hours. The human leukemia xenograft mouse model is established by intravenously injected with WDR5 knockdown CEM (CEM-shWDR5) cells and shRNA control (CEM-shNC) into NSG mice. Mice were treated for 25 days in 4 groups [CEM-shNC + vehicle, CEM-shWDR5 + vehicle; CEM-shNC + CX-4945 (100 mg/kg via gavage), CEM-shWDR5 + CX-4945]. Leukemia burden in mice spleen and bone marrow was analyzed by flow cytometry. The survival of mice was compared by the Kaplan-Meier method. The synergistic effect of CX-4945 and OICR-9429 was analyzed by CCK8 assay. This study was approved by the Ethics Committee of Zhongda Hospital Southeast University

Results WDR5 is highly expressed in T-ALL patients compared with normal controls (P<0.001) and is higher in the relapsed samples than paired newly-diagnosed controls (P<0.05) (Fig. 1A-B). WDR5 knockdown in T-ALL cells significantly suppressed cell proliferation and induced cell cycle arrest in vitro (P<0.01) (Fig. 1C), and in vivo leukemia development (Fig. 1D). Also, WDR5 inhibitor OICR-9429 induces cell proliferation and cell cycle arrest in the cells. RNA-seq revealed that ATAD2 is downregulated upon OICR-9429 treatment and indicated that ATAD2 is the downstream target of WDR5. WDR5 knockdown suppresses ATAD2 expression (P<0.01) (Fig. 1E). In addition, ATAD2 is highly expressed in T-ALL patients versus normal controls (P<0.05) (Fig. 1F), and ATAD2 silencing significantly suppresses cell proliferation and induces cell cycle arrest of T-ALL cells (P<0.01) (Fig. 1G). These data indicate WDR5/ATAD2 signaling act as a novel potential oncogenic pathway in T-ALL.

Furthermore, we found that IKAROS transcriptionally suppresses WDR5 expression, and CK2-mediated hyperphosphorylation of IKAROS is one of the major mechanisms of IKAROS dysfunction in ALL. CX-4945 strongly suppresses WDR5 expression in an IKAROS-dependent manner (Fig. 1H-J). The synergistic effect of CX-4945 with WDR5 disruption on cell proliferation suppression and cell cycle arrest was observed in T-ALL both in in vitro and in vivo xenograft mouse model. The combination of CX-4945 with WDR5 disruption significantly prolonged the survival of mice compared with single CX-4945 [55(52-58) days vs. 38.5(36-41) days,P<0.001] and single WDR5 disruption control [55(52-58) days vs. 41.5(39-44) days, P<0.001] (Fig. 1K). Consistently, the combination significantly reduces the size and weight of the spleen (Fig. 1L), % human CD45+ T-ALL cells in the spleen (Fig. 1M) and bone marrow (data not shown), and the expression of WDR5 and ATAD2 in the spleen versus single treatment controls (P<0.01) (Fig. 1N-P). Finally, a synergistic effect of CX-4945 with OICR-9429 on cell proliferation suppression and down-regulation of WDR5 and ATAD2 was observed in the primary leukemic cells from T-ALL patients (Fig. 2A-D). Together, these data indicated the synergistic efficacy of CX-4945 with OICR-9429 on T-ALL and the novel model was summarized in Fig.2E.

Conclusion In this study, we identified a novel WDR5/ATAD2 oncogenic signaling regulated by CK2/IKAROS axis in T-ALL. Our study reveals a model that dual targeting WDR5/ATAD2 signaling through direct inhibiting oncoproteins and via CK2/IKAROS axis to transcriptionally repress the oncoprotein to achieve synergistic efficacy. Our results further highlight the combination of CX-4945 with WDR5 inhibition is a potential option for the therapy of T-ALL patients.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH