Jonathon L. Payne

Regulator of myeloid differentiation and function:The secret life of Ikaros

Ikaros (also known as Lyf-1) was initially described as a lymphoid-specific transcription factor. Although Ikaros has been shown to regulate hematopoietic stem cell renewal, as well as the development and function of cells from multiple hematopoietic lineages, including the myeloid lineage, Ikaros has primarily been studied in context of lymphoid development and malignancy. This review focuses on the role of Ikaros in myeloid cells. We address the importance of post-transcriptional regulation of Ikaros function; the emerging role of Ikaros in myeloid malignancy; Ikaros as a regulator of myeloid differentiation and function; and the selective expression of Ikaros isoform-x in cells with myeloid potential. We highlight the challenges of dissecting Ikaros function in lineage commitment decisions among lymphoid-myeloid progenitors that have emerged as a major myeloid differentiation pathway in recent studies, which leads to reconstruction of the traditional map of murine and human hematopoiesis.

Ikaros regulation of the BCL6/BACH2 axis and its clinical relevance in acute lymphoblastic leukemia

B-Cell CLL/Lymphoma 6 (BCL6) is a proto-oncogene that is highly expressed in acute lymphoblastic leukemia (ALL). BTB and CNC Homology 1 Basic Leucine Zipper Transcription Factor 2 (BACH2) is a suppressor of transcription. The BACH2–BCL6 balance controls selection at the pre-B cell receptor checkpoint by regulating p53 expression. However, the underlying mechanism and the clinical relevance of the BCL6/BACH2 axis are unknown. Here, we found that Ikaros, a tumor suppressor encoded by IKZF1, directly binds to both the BCL6 and BACH2 promoters where it suppresses BCL6 and promotes BACH2 expression in B-cell ALL (B-ALL) cells. Casein kinase 2 (CK2) inhibitors increase Ikaros function thereby inhibiting BCL6 and promoting BACH2 expression in an Ikaros-dependent manner. We also found that the expression of BCL6 is higher while BACH2 expression is lower in patients with B-ALL than normal bone marrow control. High BCL6 and low BACH2 expression is associated with high leukemic cell proliferation, unfavorable clinical and laboratory features, and inferior outcomes. Moreover, IKZF1 deletion is associated with high BCL6 and low BACH2 expression in B-ALL patients. CK2 inhibitors increase Ikaros binding to the promoter of BCL6 and BACH2 and suppress BCL6 while promoting BACH2 expression in the primary B-ALL cells. Our data indicates that Ikaros regulates expression of the BCL6/BACH2 axis in B-ALL. High BCL6 and low BACH2 expression are associated with Ikaros dysregulation and have a potential effect on the development of B-ALL.

Abstract 5542: Regulation of cell cycle control in T-cell acute lymphoblastic leukemia by Ikaros and Casein Kinase II

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that represents a therapeutic challenge. Next-generation sequencing revealed that a subset of T-ALL harbors inactivating mutations or deletion of one allele of the IKZF1 tumor suppressor. These data suggest that IKZF1 acts as a tumor suppressor in T-ALL. The IKZF1 gene encodes the Ikaros protein that functions as a regulator of transcription and a tumor suppressor in B cell acute lymphoblastic leukemia. However, the molecular mechanism of Ikaros tumor suppressor function in T-ALL is unclear. Using quantitative chromatin immunoprecipitation (qChIP), we determined that Ikaros binds to the promoter regions of the CDC2 and CDC7 cell cycle genes in primary T-ALL cells in vivo. Gain-of function experiments showed that Ikaros overexpression in T-ALL results in reduced expression of CDC2 and CDC7, as evidenced by quantitative RT-PCR (qRT-PCR) and Western blot. The knock-down of Ikaros with shRNA in T-ALL cells resulted in increased transcription of CDC2 and CDC7 as indicated by qRT-PCR. These data suggest that Ikaros can regulate cell cycle progression in T-ALL by repressing transcription of the CDC2 and CDC7 genes. Next, we studied the mechanisms that regulate Ikaros’ ability to repress CDC2 and CDC7 in T-ALL. Ikaros function as a transcriptional repressor is regulated by Casein Kinase II (CK2). CK2 is overexpressed in hematopoietic malignancies and increased expression of CK2 results in T-ALL in murine models. We tested the effect of CK2 inhibition on Ikaros’ ability to regulate transcription of CDC2 and CDC7 in human T-ALL. Molecular inhibition of CK2 with shRNA against the CK2 catalytic subunit resulted in reduced transcription of CDC2 and CDC7, as evidenced by qRT-PCR. This was associated with increased DNA-binding of Ikaros to promoters of CDC2 and CDC7, as shown by qChIP. These data suggest that CK2 impairs Ikaros’ ability to transcriptionally repress CDC2 and CDC7 and to regulate cell cycle progression in T-ALL. Inhibition of CK2 enhances transcriptional repression of CDC2 and CDC7 by Ikaros, resulting in improved control of cell cycle progression in T-ALL. In conclusion, our results show that control of cell cycle progression in T-ALL occurs trough Ikaros-mediated transcriptional regulation of CDC2 and CDC7. Overexpession of CK2 impairs Ikaros ability to repress CDC2 and CDC7 expression, which contributes to deregulation of cell cycle control in T-ALL. Results suggest a potential mechanism of therapeutic action of CK2 inhibitors for the treatment of T-ALL. Note: This abstract was not presented at the meeting. Citation Format: Mario A. Soliman, Tommy Hu, Malika Kapadia, Elanora Dovat, Yali Ding, Chunhua Song, Jonathon L. Payne, Sinisa Dovat. Regulation of cell cycle control in T-cell acute lymphoblastic leukemia by Ikaros and Casein Kinase II [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5542. doi:10.1158/1538-7445.AM2017-5542

Abstract 3512: Ikaros regulation of theBCL6/BACH2axis and its clinical relevance in acute lymphoblastic leukemia

B-Cell CLL/Lymphoma 6 (BCL6) is a proto-oncogene that is highly expressed in acute lymphoblastic leukemia (ALL). BTB and CNC Homology 1 Basic Leucine Zipper Transcription Factor 2 (BACH2) is a suppressor of transcription. The BACH2-BCL6 balance controls selection at the pre-B cell receptor checkpoint by regulating p53 expression. However, the underlying mechanism and the clinical relevance of the BCL6/BACH2 axis are unknown. Here, we found that Ikaros, a tumor suppressor encoded by IKZF1, directly binds to both the BCL6 and BACH2 promoters where it suppresses BCL6 and promotes BACH2 expression in B-cell ALL (B-ALL) cells. Casein kinase 2 (CK2) inhibitors increase Ikaros function thereby inhibiting BCL6 and promoting BACH2 expression in an Ikaros-dependent manner. We also found that the expression of BCL6 is higher while BACH2 expression is lower in patients with B-ALL than normal bone marrow control. High BCL6 and low BACH2 expression is associated with high leukemic cell proliferation, unfavorable clinical and laboratory features, and inferior outcomes. Moreover, IKZF1 deletion is associated with high BCL6 and low BACH2 expression in B-ALL patients. CK2 inhibitors increase Ikaros binding to the promoter of BCL6 and BACH2 and suppress BCL6 while promoting BACH2 expression in the primary B-ALL cells. Our data indicates that Ikaros regulates expression of the BCL6/BACH2 axis in B-ALL. High BCL6 and low BACH2 expression are associated with Ikaros dysregulation and have a potential effect on the development of B-ALL. Citation Format: Chunhua Song, Zheng Ge, Jianyong Li, Baoan Chen, Elanora Dovat, Katarina Dovat, Jonathon Payne, Sinisa Dovat. Ikaros regulation of the BCL6/BACH2 axis and its clinical relevance in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3512. doi:10.1158/1538-7445.AM2017-3512

Abstract 4463: Epigenetic regulation of gene expression in high-risk B-cell acute lymphoblastic leukemia by Casein Kinase II

Acute lymphoblastic leukemia (ALL) is the most common malignancy in childhood. Although advances in the treatment of ALL have resulted in a high cure rate for this disease, high-risk ALL is characterized by both resistance to conventional chemotherapy and a poor prognosis. The pathogenesis of high-risk ALL is still not understood. Casein Kinase II (CK2) is an oncogenic kinase that is overexpressed in both B-cell ALL (B-ALL) and T-cell ALL (T-ALL) and is associated with poor outcome. Inhibition of CK2 results in a strong therapeutic effect in a preclinical model of leukemia. However, the mechanism by which CK2 promotes oncogenesis in leukemia is unknown. Here, we studied how CK2 regulates expression of histone demethylase KDM5B in ALL. The KDM5B gene encodes a histone demethylase that regulates levels of histone modification H3K4me3 in leukemia. Molecular inhibition of CK2 using shRNA that targets the CK2 catalytic subunit resulted in transcriptional repression of KDM5B in ALL as evidenced by qRT-PCR. A similar effect was observed when leukemia cells were treated with the CK2 inhibitor CX-4945. Inhibition of CK2 resulted in reduced expression of KDM5B with an increase in the global cellular level of H3K4me3 as evidenced by Western blot. The use of quantitative chromatin immunoprecipitation (qChIP) showed that CK2 inhibition enhances DNA binding of the Ikaros tumor suppressor to the promoter of the KDM5B gene. Ikaros is a DNA-binding protein that regulates transcription of its target genes via chromatin remodeling. Loss of Ikaros function results in high-risk ALL. Serial qChIP analysis demonstrated that the increased Ikaros binding to the KDM5B promoter following CK2 inhibition is associated with an alteration of the epigenetic signature at the DNA region that surrounds the Ikaros binding site. Specifically, enhanced Ikaros binding results in increased occupancy of the H3K27me3 histone modification, along with a reduced occupancy of the H3K9ac histone modification at the KDM5B promoter. These results are consistent with the formation of heterochromatin and transcriptional repression. We tested the effect of CK2 inhibitors on Ikaros-mediated repression of KDM5B in primary, high-risk B-ALL cells that have a deletion of one Ikaros allele. Results showed that CK2 inhibition in high-risk B-ALL restores Ikaros binding to KDM5B promoter and represses KDM5B transcription. These data suggest that the inhibition of CK2 controls expression of KDM5B and the global H3K4me3 level in ALL by regulating the function of Ikaros as a transcriptional repressor of KDM5B. This presented data demonstrates the role of the CK2-Ikaros signaling axis in the regulation of both gene expression and the global epigenetic signature in ALL, and provide a mechanistic insight into the role of CK2 in the pathogenesis of ALL. Citation Format: Morgann Loaec, Jonathon Payne, Elanora Dovat, Chunhua Song, Kimberly J. Payne, Sinisa Dovat. Epigenetic regulation of gene expression in high-risk B-cell acute lymphoblastic leukemia by Casein Kinase II. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4463.