Yali Ding

Targeting casein kinase II restores Ikaros tumor suppressor activity and demonstrates therapeutic efficacy in high-risk leukemia

Ikaros (IKZF1) is a tumor suppressor that binds DNA and regulates expression of its target genes. The mechanism of Ikaros activity as a tumor suppressor and the regulation of Ikaros function in leukemia are unknown. Here, we demonstrate that Ikaros controls cellular proliferation by repressing expression of genes that promote cell cycle progression and the phosphatidylinositol-3 kinase (PI3K) pathway. We show that Ikaros function is impaired by the pro-oncogenic casein kinase II (CK2), and that CK2 is overexpressed in leukemia. CK2 inhibition restores Ikaros function as transcriptional repressor of cell cycle and PI3K pathway genes, resulting in an antileukemia effect. In high-risk leukemia where one IKZF1 allele has been deleted, CK2 inhibition restores the transcriptional repressor function of the remaining wild-type IKZF1 allele. CK2 inhibition demonstrated a potent therapeutic effect in a panel of patient-derived primary high-risk B-cell acute lymphoblastic leukemia xenografts as indicated by prolonged survival and a reduction of leukemia burden. We demonstrate the efficacy of a novel therapeutic approach for high-risk leukemia: restoration of Ikaros tumor suppressor activity via inhibition of CK2. These results provide a rationale for the use of CK2 inhibitors in clinical trials for high-risk leukemia, including cases with deletion of one IKZF1 allele.

Transcriptional Regulation of JARID1B/KDM5B Histone Demethylase by Ikaros, Histone Deacetylase 1 (HDAC1), and Casein Kinase 2 (CK2) in B-cell Acute Lymphoblastic Leukemia

Impaired function of the Ikaros (IKZF1) protein is associated with the development of high-risk B-cell precursor acute lymphoblastic leukemia (B-ALL). The mechanisms of Ikaros tumor suppressor activity in leukemia are unknown. Ikaros binds to the upstream regulatory elements of its target genes and regulates their transcription via chromatin remodeling. Here, we report that Ikaros represses transcription of the histone H3K4 demethylase, JARID1B (KDM5B). Transcriptional repression of JARID1B is associated with increased global levels of H3K4 trimethylation. Ikaros-mediated repression of JARID1B is dependent on the activity of the histone deacetylase, HDAC1, which binds to the upstream regulatory element of JARID1B in complex with Ikaros. In leukemia, JARID1B is overexpressed, and its inhibition results in cellular growth arrest. Ikaros-mediated repression of JARID1B in leukemia is impaired by pro-oncogenic casein kinase 2 (CK2). Inhibition of CK2 results in increased binding of the Ikaros-HDAC1 complex to the promoter of JARID1B, with increased formation of trimethylated histone H3 lysine 27 and decreased histone H3 Lys-9 acetylation. In cases of high-risk B-ALL that carry deletion of one Ikaros (IKZF1) allele, targeted inhibition of CK2 restores Ikaros binding to the JARID1B promoter and repression of JARID1B. In summary, the presented data suggest a mechanism through which Ikaros and HDAC1 regulate the epigenetic signature in leukemia: via regulation of JARID1B transcription. The presented data identify JARID1B as a novel therapeutic target in B-ALL and provide a rationale for the use of CK2 inhibitors in the treatment of high-risk B-ALL.

Epigenetic regulation of gene expression by Ikaros, HDAC1 and Casein Kinase II in leukemia.

IKZF1 (Ikaros) encodes a DNA-binding protein that acts as a master regulatory of hematopoiesis and a tumor suppressor in acute lymphoblastic leukemia (ALL).1, 2, 3, 4 The deletion and/or mutation of Ikaros is associated with the development of B-cell acute lymphoblastic leukemia (B-ALL) with poor outcome.5, 6, 7, 8, 9, 10, 11 Ikaros directly associates with components of the histone deacetylase complex (NuRD), HDAC1, HDAC2 and Mi-2.12, 13, 14 Although Ikaros is hypothesized to regulate the transcription of target genes by recruiting the NuRD complex, the mechanism of Ikaros-mediated transcriptional regulation in leukemia is still unknown. Here we use a systems biology approach to determine the mechanism through which Ikaros and HDAC1 regulate gene expression in human B-ALL.

Casein Kinase II (CK2), Glycogen Synthase Kinase-3 (GSK-3) and Ikaros mediated regulation of leukemia

Signaling networks that regulate cellular proliferation often involve complex interactions between several signaling pathways. In this manuscript we review the crosstalk between the Casein Kinase II (CK2) and Glycogen Synthase Kinase-3 (GSK-3) pathways that plays a critical role in the regulation of cellular proliferation in leukemia. Both CK2 and GSK-3 are potential targets for anti-leukemia treatment. Previously published data suggest that CK2 and GSK-3 act synergistically to promote the phosphatidylinositol-3 kinase (PI3K) pathway via phosphorylation of PTEN. More recent data demonstrate another mechanism through which CK2 promotes the PI3K pathway - via transcriptional regulation of PI3K pathway genes by the newly-discovered CK2-Ikaros axis. Together, these data suggest that the CK2 and GSK-3 pathways regulate AKT/PI3K signaling in leukemia via two complementary mechanisms: a) direct phosphorylation of PTEN and b) transcriptional regulation of PI3K-promoting genes. Functional interactions between CK2, Ikaros and GSK3 define a novel signaling network that regulates proliferation of leukemia cells. This regulatory network involves both direct posttranslational modifications (by CK and GSK-3) and transcriptional regulation (via CK2-mediated phosphorylation of Ikaros). This information provides a basis for the development of targeted therapy for leukemia.

Protein signaling and regulation of gene transcription in leukemia: role of the Casein Kinase II-Ikaros axis

Protein signaling and regulation of gene expression are the two major mechanisms that regulate cellular proliferation in leukemia. Discerning the function of these processes is essential for understanding the pathogenesis of leukemia and for developing the targeted therapies. Here, we provide an overview of one of the mechanisms that regulates gene transcription in leukemia. This mechanism involves the direct interaction between Casein Kinase II (CK2) and the Ikaros transcription factor. Ikaros (IKZF1) functions as a master regulator of hematopoiesis and a tumor suppressor in acute lymphoblastic leukemia (ALL). Impaired Ikaros function results in the development of high-risk leukemia. Ikaros binds to the upstream regulatory elements of its target genes and regulates their transcription via chromatin remodeling. In vivo, Ikaros is a target for CK2, a pro-oncogenic kinase. CK2 directly phosphorylates Ikaros at multiple amino acids. Functional experiments showed that CK2-mediated phosphorylation of Ikaros, regulates Ikaros’ DNA binding affinity, subcellular localization and protein stability. Recent studies revealed that phosphorylation of Ikaros by CK2 regulates Ikaros binding and repression of the terminal deoxytransferase (TdT) gene in normal thymocytes and in T-cell ALL. Available data suggest that the oncogenic activity of CK2 in leukemia involves functional inactivation of Ikaros and provide a rationale for CK2 inhibitors as a potential treatment for ALL.

New universal primers for genotyping and resistance detection of low HBV DNA levels.

Abstract HBV (hepatitis B virus) genotyping is important in determining the clinical manifestation of disease and treatment response, particularly, in patients with low viral loads. Also, sensitive detection of HBV antiviral drug resistance mutations is essential for monitoring therapy response. Asensitive direct sequencing method for genotyping and the drug resistance mutation detection of low levels of HBV DNA in patients’ plasma is developed by PCR amplification of the DNA with novel universal primers. The novel, common, and universal primers were identified by alignment of RT region of all the HBV DNA sequences in databases. These primers could efficiently amplify the RT region of HBV virus at low DNA levels by directly sequencing the resulting PCR products, and mapping with the reference sequence made it possible to clearly obtain the HBV subtypes and identify the resistance mutations in the samples with HBV DNA level as low as 20 IU/mL. We examined the reliability of the method in clinical samples, and found it could detect the HBV subtypes and drug resistance mutations in 80 clinical HBV samples with low HBV DNA levels ranging from 20 to 200 IU/mL. This method is a sensitive and reliable direct sequencing method for HBV genotyping and antiviral drug resistance mutation detection, and is helpful for efficiently monitoring the response to therapy in HBV patients.

Amplexicaule A exerts anti-tumor effects by inducing apoptosis in human breast cancer

Chemotherapy is the main treatment for patients with breast cancer metastases, but natural alternatives have been receiving attention for their potential as novel anti-tumor reagents. Amplexicaule A (APA) is a flavonoid glucoside isolated from rhizomes of Polygonum amplexicaule D. Don var. sinense Forb (PADF). We found that APA has anti-tumor effects in a breast cancer xenograft mouse model and induces apoptosis in breast cancer cell lines. APA increased levels of cleaved caspase-3,-8,-9 and PARP, which resulted from suppression of MCL-1 and BCL-2 expression in the cells. APA also inactivated the Akt/mTOR pathway in breast cancer cells. Thus, APA exerts a strong anti-tumor effect on breast cancer cells, most likely through induction of apoptosis. Our study is the first to identify this novel anti-tumor compound and provides a new strategy for isolation and separation of single compounds from herbs.

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 A21: Epigenetic regulation of cell cycle progression at the G2/M transition and mitosis in high-risk leukemia

High-risk acute lymphoblastic leukemia (ALL) is a clinical challenge due to drug resistance and poor prognosis. A characteristic molecular defect of most high-risk ALL is the deletion or inactivating mutation of one allele of the IKZF1 (Ikaros) tumor suppressor. Ikaros encodes a DNA-binding protein that regulates transcription of its target genes via chromatin remodeling. The mechanisms through which Ikaros regulates cellular proliferation in high-risk leukemia, are unknown. Using a systems biology approach, we determined that Ikaros regulates transcription of genes that are critical in the control of G2/M transition (CDC2) and mitotic progression (ANAPC1 and ANAPC7) in leukemia. Gain- and loss-of-function experiments demonstrate that Ikaros represses the transcription of CDC2, ANAPC1 and ANAPC7. Overexpression of Ikaros in leukemia also results in cell cycle arrest. We studied the mechanism through which Ikaros represses CDC2, ANAPC1 ad ANAPC7. The use of serial quantitative chromatin immunoprecipitation (qChIP) analyses spanning the promoters of Ikaros target genes demonstrated that Ikaros can repress transcription of its target genes by two different mechanisms: 1) via recruitment of histone deacetylase 1 (HDAC1), which is associated with the formation of repressive chromatin characterized by H3K27me3 and loss of H3K9ac (for ANAPC1 and CDC2); and 2) via an HDAC1-independent mechanism which is associated with the formation of repressive chromatin characterized by H3K9me3, along with the loss of H3K9ac (for ANAPC7). In high-risk ALL that is characterized by deletion of one Ikaros allele, the function of Ikaros as a transcriptional regulator is impaired due to reduced binding to promoters of Ikaros target genes. We showed previously that Ikaros DNA-binding affinity is regulated via direct phosphorylation by pro-oncogenic Casein Kinase II (CK2). CK2 is overexpressed in high-risk B-ALL as compared to normal B-cell precursors, which further reduces Ikaros function in this disease. In vivo CK2 inhibition with the CK2 specific inhibitor, CX-4945, results in a strong therapeutic effect in primary high-risk ALL xenografts. Analysis of primary high-risk B-ALL (that have deletion of one Ikaros allele) showed that treatment with CX-4945, restored Ikaros function as a transcriptional regulator of CDC2, ANAPC1 and ANAPC7, and was associated with cell cycle arrest. Epigenetic analysis of promoters of CDC2, ANAPC1 and ANAPC7 genes revealed that restoration of Ikaros binding to the promoters of these genes is associated with epigenetic alterations that are consistent with Ikaros overexpression and formation of repressive heterochromatin. In conclusion, our results reveal that: 1) Ikaros functions as a tumor suppressor by repressing transcription of genes that are critical for G/M transition (CDC2) and mitotic progression (ANAPC1 and ANAPC7); 2) Ikaros represses transcription by inducing two distinct epigenetic alterations at promoters of its target genes and 3) CK2 inhibition with CX-4945 restores Ikaros function as a transcriptional regulator of CDC2, ANAPC1 and ANAPC7 in high-risk leukemia. These results provide novel insights into the control of cell cycle progression in high-risk leukemia and the mechanisms by which CK2 inhibitors exert their therapeutic effects. Supported by the National Institutes of Health R01 HL095120, and the Four Diamonds Fund Endowment. Citation Format: Chunhua Song, Chandrika Gowda, Yali Ding, Kimberly J. Payne, Sinisa Dovat. Epigenetic regulation of cell cycle progression at the G2/M transition and mitosis in high-risk leukemia. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A21.

Pediatric High Risk Leukemia — Molecular Insights

Acute leukemia comprises of 31% of all cancers in children making it the most com‐ mon childhood malignancy. Significant strides have been made in treatment, partly through risk stratification and intensified therapy. A number of subtypes remain at high risk for relapse and poor outcome, despite current therapies. Here we describe risk stratification and molecular diagnosis used to identify high risk leukemias and guide treatment. Specific cytogenetic alterations that contribute to high risk B and T cell acute lymphoblastic leukemia (ALL), as well as infant leukemia are discussed. Particular attention is given to genetic alterations in IKZF1, CRLF2, and JAK, that have been identified by whole genome sequencing and recently associated with Phlike ALL. Ongoing studies of disease mechanisms and challenges in developing pre-clinical patient-derived xenograft models to evaluate therapies are discussed.