Targeting WDR5/ATAD2 signaling by the CK2/IKAROS axis demonstrates therapeutic efficacy in T-ALL

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Targeting WDR5/ATAD2 signaling by the CK2/IKAROS axis demonstrates therapeutic efficacy in T-ALL

Mar 27, 2025

Key Points

Identify a novel oncogenic WDR5/ATAD2 signaling by affecting cell cycle progress in T-ALL.
Targeting WDR5/ATAD2 signaling through the CK2/IKAROS axis demonstrates the synergistic antileukemic efficacy in T-ALL.

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Abstract

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with a poor prognosis and limited options for targeted therapies. Identifying new molecular targets to develop novel therapeutic strategies is the pressing immediate issue in T-ALL. Here, we observed high expression of WD repeat-containing protein 5 (WDR5) in T-ALL. With in vitro and in vivo models, we demonstrated the oncogenic role of WDR5 in T-ALL by activating cell cycle signaling through its new downstream effector, ATPase family AAA domain-containing 2 (ATAD2). Moreover, the function of a zinc finger transcription factor of the Kruppel family (IKAROS) is often impaired by genetic alteration and casein kinase II (CK2) which is overexpressed in T-ALL. We found that IKAROS directly regulates WDR5 transcription; CK2 inhibitor, CX-4945, strongly suppresses WDR5 expression by restoring IKAROS function. Last, combining CX-4945 with WDR5 inhibitor demonstrates synergistic efficacy in the patient-derived xenograft mouse models. In conclusion, our results demonstrated that WDR5/ATAD2 is a new oncogenic signaling pathway in T-ALL, and simultaneous targeting of WRD5 and CK2/IKAROS has synergistic antileukemic efficacy and represents a promising potential strategy for T-ALL therapy.

Subjects:

Free Research Articles, Lymphoid Neoplasia

Introduction

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that originates from immature T lymphocytes or lymphoid progenitors.¹^,² T-ALL accounts for 10% to 15% of pediatric and up to 25% of adult ALL cases.³ An overall survival has been achieved at 80% in the pediatric setting by using a risk-based stratification toward intensive multiagent combination chemotherapeutic protocols.⁴ The overall survival for adult patients are <50%,³ particularly the outcome remains poor for those patients who do experience disease relapse or are refractory to induction therapy. The rapid progress in targeted therapies and immunotherapies such as the recently approved blinatumomab, inotuzumab, ozogamicin, and chimeric antigen receptor T-cell therapy has dramatically improved the outcome of B-cell acute lymphoblastic leukemia (B-ALL).⁵^,⁶ However, there have been no new agents specifically approved for relapsed/refractory T-ALL since nelarabine was approved in 2005.⁷ The targeted therapies benefiting T-ALL are still limited owing to the biological heterogeneity of T-ALL.⁸^,⁹ Therefore, identifying novel “druggable” molecular markers and illustrating the underlying mechanisms are immediate pressing issues in T-ALL.¹⁰

WD repeat-containing protein 5 (WDR5) is the core subunit of the histone 3 lysine 4(H3K4) methyltransferase complex.¹¹^,¹² WDR5 directly interacts with mixed lineage leukemia (MLL) and increases H3K4 trimethylation (H3K4me3) at the target genes’ promoters, leading to the target gene’s transcriptional activation and resulting in tumorigenesis.¹¹^,^13-15 We reported that WDR5 is highly expressed and associated with poor survival in B-ALL.¹⁶ Recently, it has been reported that ubiquitin-specific peptidase 44 enhanced the growth of T-ALL by interacting with WDR5 and repressing its ubiquitination.¹⁷ In addition, WDR5 inhibitors such as OICR-9429 have been reported to exhibit antitumor activities in many human cancers.¹⁵^,^18-21 However, little is known about the WDR5 oncogenic roles in T-ALL.

Casein kinase II (CK2) is a ubiquitous and highly conserved eukaryotic serine/threonine-protein kinase,²² which plays a vital role in cell cycle progression, cell differentiation, and transcription regulation.^23-25IKZF1 gene-encoding protein IKAROS is an important tumor suppressor in ALL and the direct target of CK2.²⁶ CK2 is an essential pro-oncogene that phosphorylates IKAROS, thus reducing its DNA-binding and leukemia-suppressor function in high-risk B-ALL.²⁵^,²⁷^,²⁸ CK2 is overexpressed in T-ALL.²⁷^,²⁹ CX-4945 is a small molecule inhibitor of CK2 that exhibits potent and highly selective anti-CK2 activity; we and others have demonstrated that CX-4945 has therapeutic efficacy in high-risk B-ALL.²⁶^,^29-33

Here, we reported the association of WDR5 high expression with activation of cell cycle signaling in T-ALL and revealed the antileukemia effect of targeting WDR5 by inducing cell cycle arrest. Interestingly, we identified ATPase family AAA domain-containing protein 2 (ATAD2) as a core mediator of cell cycle signaling and the downstream target of WDR5 in T-ALL. Targeting the CK2/IKAROS axis downregulates WDR5 expression by restoring IKAROS function and exhibits a strong synergistic antileukemic efficacy with WDR5 inhibition, either by short hairpin RNA (shRNA) ablation or pharmacological means in human leukemia cells and patient-derived xenograft (PDX) mouse models of T-ALL. Our results demonstrate the role of WDR5/ATAD2 signaling in the oncogenesis of T-ALL, revealing the potential clinical relevance of targeting this new oncogenic signaling pathway directly using WDR5 inhibitors or upstream through CK2/IKAROS axis and providing strong preclinical evidence for combining these 2 agents for T-ALL.

Methods

Clinical samples

A total of 38 T-ALL patients’ bone marrow (BM) samples and 32 normal control BM mononuclear cells from healthy volunteers were obtained from Zhongda Hospital Southeast University. Measurable residual disease negative was defined as blasts <0.01% by multiparameter flow cytometry according to the 2024 European LeukemiaNet (ELN) recommendations.³⁴ High-risk T-ALL was defined as patients with either age >35 years old, white blood cell >100 × 10⁹/L, early T-cell precursor ALL (ETP-ALL), RAS/PTEN mutation, or NOTCH1/FBXW7 wild type according to the National Comprehensive Cancer Network (NCCN) guidelines version 2.2024. For drug treatment, primary cells were isolated with lymphocyte separation medium (MP Biomedicals LLC, Irvine, CA) in a sterile environment and cultured in RPMI-1640 (Gibco, Beijing, China) supplemented with 10% fetal bovine serum (HyClone, Shanghai, China) immediately to do further experiments as previously reported.³⁵^,³⁶ This study was approved by the Ethics Committee of Zhongda Hospital Southeast University, and all patients provided written informed consent.

Plasmid construction, lentiviral transduction, and target gene knockdown

Lentiviral shRNA plasmids for WDR5, ATAD2, and IKZF1 were constructed by subcloning the shRNA oligos into the lentiviral shRNA vector (pLV3ltr-ZsGreen-Puro-U6) (Corues Biotechnology, Nanjing, China) as per the manufacturer’s recommendations.²⁹^,³⁰

Global transcriptome analysis by RNA-seq

Briefly, CEM cells were treated with 5 μM CX-4945, 20 μM OICR-9429, or dimethyl sulfoxide (DMSO) control for 72 hours. Total RNA was extracted from the cells with a Qiagen RNA isolation kit (Qiagen, Shanghai, China). All samples were studied in triplicate. RNA sequencing (RNA-seq) was performed as previously reported.²⁶

T-ALL PDX mouse models

Three patient samples for the PDX mouse model were obtained from Zhongda Hospital Southeast University, and 5 × 10⁵ leukemia cells per mouse were transplanted intravenously (IV) into NOD/ShiLtJGpt-Prkdc^em26Cd52Il2rg^em26Cd22/Gpt (NCG) mice (GemPharmatech Co., Ltd, Nanjing, China).

Following engraftment, mice (n = 20 per group per patient sample × 3 patients) received the vehicle, CX-4945, OICR-9429, and combination (combo) for 25 days. Then, when the vehicle mice met the early removal criteria due to the excessive leukemia burden, 10 mice per group were euthanized to observe the drug efficacy on leukemia burden. The single-cell suspension of euthanized mice BM or spleen cells was collected, and the red blood cells were removed with red blood cell lysis buffer (Biosharp, Hefei, China). The resulting cells were used for living cell counts, quantitative chromatin immunoprecipitation–quantitative polymerase chain reaction assay, quantitative reverse transcription–polymerase chain reaction, and flow cytometry analysis of leukemia burden. The remaining 10 mice per group were followed until the mice died or met early removal criteria for survival analysis. The dead mice were counted daily, and the Kaplan-Meier method was used to generate the survival curves and analyze the survival difference.

All experimental operations were performed with the consent of the Animal Care Committee of Southeast University and complied with the Regulations for the Administration of Affairs Concerning Experimental Animals of China.

Details and additional experimental methods can be found in the supplemental Materials, available on the Blood website.

Ethical approval and informed consent

These written informed consents were provided by all the patients with the Declaration of Helsinki before enrollment in the study. The institutional review board of Zhongda Hospital Southeast University, Nanjing, China, approved the study.

Results

Association of WDR5 expression with cell cycle signaling in T-ALL

WDR5 is significantly overexpressed in our T-ALL cohorts vs normal controls (Figure 1A; supplemental Table 1). High WDR5 expression is associated with significantly higher white blood cell count (Figure 1B), higher BM blasts (Figure 1C), poorer survival (Figure 1D), higher relapse rate (Figure 1E), and more high-risk patients (Figure 1F) in our cohorts (supplemental Table 2). WDR5 messenger RNA (mRNA) level is significantly elevated in 5 pairs of the relapsed vs newly diagnosed T-ALL samples (Figure 1G). Also, WDR5 is significantly overexpressed in 3 public T-ALL cohorts and a B-ALL cohort (supplemental Figure 1A-D). In addition, WDR5 is highly expressed, and no statistical difference was observed in different immune phenotypes, ETP-ALL vs non-ETP-ALL, and the various molecular subtypes of the T-ALL cohorts (supplemental Figure 1E-G). Moreover, the patients were divided into WDR5-high and WDR5-low groups based on the WDR5 mRNA level in the 3 public T-ALL cohorts, and the enrichment of cell cycle genes and activation of cell cycle signaling was significantly associated with WDR5-high vs WDR5-low expression; the representative data can be found in supplemental Figure 1H-J. Taken together, these data revealed the oncogenic roles of WDR5 high expression which is possibly mediated by activating cell cycle signaling in T-ALL.

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