• Ikaros controls cellular proliferation by repressing genes that regulate cell cycle progression and the PI3K pathway in leukemia.• CK2 inhibitor restores Ikaros tumor suppressor function in high-risk B-ALL with IKZF1 deletion and has a strong therapeutic effect in vivo.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. (Blood. 2015;126(15):1813-1822 Introduction Ikaros (IKZF1) activity is essential for normal hematopoiesis and immune development. [1][2][3][4] Ikaros knockout mice have severely impaired hematopoiesis, 5-7 whereas mice with the heterozygous loss of Ikaros develop T-cell leukemia. 8 In humans, impaired Ikaros activity due to the deletion or inactivating mutation of a single IKZF1 allele results in high-risk B-cell leukemia that is resistant to treatment.9-14 Ikaros regulates transcription of target genes via chromatin remodeling. [15][16][17] Ikaros activity is controlled through multiple mechanisms. Mouse studies suggest that the transcription of IKZF1 during normal hematopoiesis is regulated by a complex network. 18 However, Ikaros protein is expressed at high levels in most hematopoietic cells, and posttranslational modifications are hypothesized to play a critical role in regulating Ikaros activity. 19 Several groups have shown that phosphorylation, [19][20][21][22][23][24] sumoylation, 25 and ubiquitination 22 can regulate Ikaros function as a transcriptional repressor. However, the role of posttranslational modification in the regulation of Ikaros tumor suppressor activity in leukemia is unknown.Despite extensive global analyses of Ikaros DNA binding in normal murine hematopoietic cells, 26-28 the molecular mechanisms by which Ikaros exerts its tumor suppressor effects in human 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 tumorsuppressor 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.IKZF1 encodes the Ikaros DNA-binding zinc finger protein (1-4). Ikaros is essential for normal hematopoiesis and acts as a tumor suppressor (5, 6). In humans, deletion of a single Ikaros allele is associated with the development of high-risk B-cell precursor acute lymphoblastic leukemia (B-ALL) 3 that is characterized by resistance to chemotherapy and poor prognosis (7-9). Alterations in the Ikaros have also been associated with T cell ALL (10, 11) and myeloid leukemias (12-16). Ikaros regulates transcription of its target genes via chromatin remodeling (9). Ikaros has been shown to directly bind histone deacetylases HDAC1 and HDAC2 and to associate with the chromatin remodeling complex NuRD through interaction with the Mi-2 protein (9, 17).Ikaros is hypothesized to recruit chromatin remodeling complexes to the regulatory elements of its target genes, resulting in chromatin modifications (primarily histone deacetylation) and transcriptional repression or activation of its target genes (18 -20). Mechanisms of Ikaros-mediated repression that are independent of histone deacetylase have also been described (18, 3 The abbreviations used are: B-ALL, B-cell precursor acute lymphoblastic leukemia; CK2, casein kinase 2; TSS, transcriptional start site; ALL, acute lymphoblastic leukemia; TBB, 4,5,6,7-tetrabromobenzotriazole; IK haploid , Ikaros haploinsufficiency; Ikaros-CTS, C terminus o...

CK2 is an attractive target in treatment of various cancers. Currently only a few specific CK2 inhibitors are available. Preclinical studies using CK2 inhibitor, CX4945 in high risk pediatric leukemias have shown promising results and warrants further testing in other types of leukemia.

We report 3 cases of Visceral leishmaniasis, who presented with unusual clinical manifestations. One child was diagnosed as hemophagocytic syndrome; another masqueraded with features of leukemoid changes alongwith hemophagocytosis and trilineage myelodysplasia; the third case presented with pyothorax. All the three patients showed amastigote forms of Leishmania donovani and positive serology (rk39 antigen). They showed complete clinical, hematological and parasitological resolution with Amphotericin B therapy.

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We conducted this study to observe evidence of portal hypertension in children with visceral leishmaniasis (VL). Eighty-eight consecutive cases (50 male) of VL were subjected to ultrasonography. Those with evidence of portal hypertension also underwent upper gastrointestinal endoscopy and liver biopsy. Eight patients had portal hypertension as evidenced by dilated caliber of portal and splenic veins. Two patients had periportal, splenic and peripancreatic collaterals and one patient had cavernous transformation of portal vein. Out of eight patients, four patients had esophageal and gastric varices. Liver biopsy was done in four patients and revealed hepatic sinusoidal dilations without any evidence of fibrosis. Portal hypertension may be an independent manifestation of VL and remain undiagnosed unless a physician maintains a high index of suspicion.

IKZF1 (Ikaros) encodes a DNA-binding protein that acts as a tumor suppressor in acute lymphoblastic leukemia. Deletion of one Ikaros allele results in the development of high-risk B-cell acute lymphoblastic leukemia (B-ALL) with a high incidence of relapse and poor prognosis. The mechanisms through which Ikaros suppresses leukemogenesis and that regulate Ikaros tumor suppressor activity in leukemia are unknown. Using a systems biology approach, we determined that Ikaros regulates transcription of genes that control two pathways that are crucial in leukemia cell proliferation: 1) cell cycle progression and 2) the phosphatidylinositol 3-kinase (PI3K) pathway. Gain- and loss-of-function experiments demonstrate that Ikaros represses the transcription of genes that promote cell cycle progression and the PI3K pathway and activates transcription of a gene that suppresses the PI3K pathway. We show that in high-risk B-ALL with deletion of one Ikaros allele, the function of Ikaros as a transcriptional regulator is impaired due to reduced DNA-binding affinity for promoters of its target genes. It has been shown 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 high-risk B-ALL. Treatment of primary high-risk B-ALL (with deletion of one Ikaros allele) using the CK2 specific inhibitor, CX-4945, restored Ikaros function as a transcriptional regulator of the genes that regulate cell cycle progression and the PI3K pathway, and was associated with cell cycle arrest and loss of phosphorylation of the AKT kinase - a downstream target of the PI3K pathway. 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; 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. The therapeutic effect of CK2 inhibition by CX-4945 on high-risk B-ALL was demonstrated in vivo using 4 different xenografts: 3 different high-risk primary pre-B-ALL xenografts and Nalm6 xenografts. Treatment with CX-4945 showed a strong therapeutic effect in all 4 xenografts, as evidence by reduced leukemia cell number in bone marrow and in spleen, along with prolonged survival of all xenografts. Expression analysis of Ikaros target genes that regulate cell cycle progression and the PI3K pathway in leukemia cells treated in vivo with CX-4945 revealed an expression pattern that was highly similar to that observed with Ikaros overexpression. This suggests that CK2 inhibition in vivo exerts its therapeutic effect on high-risk B-ALL via restoration of Ikaros function as transcriptional regulator of genes that promote cell cycle progression and the PI3K pathway. In summary, our results reveal that: 1) Ikaros functions as a tumor suppressor by suppressing cell cycle progression and the PI3K pathway; 2) Ikaros regulates 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 in vivo, and has a strong therapeutic effect in primary xenografts of high-risk B-ALL. These results provide support for the use of CK2 inhibitors in clinical trials for high-risk B-ALL. Supported by the National Institutes of Health R01 HL095120, and the Four Diamonds Fund Endowment. Disclosures No relevant conflicts of interest to declare.