cx-4945 and Precursor-Cell-Lymphoblastic-Leukemia-Lymphoma

The IKZF1 gene encodes the Ikaros protein, a zinc finger transcriptional factor that acts as a master regulator of hematopoiesis and a tumor suppressor in leukemia. Impaired activity of Ikaros is associated with the development of high-risk acute lymphoblastic leukemia (ALL) with a poor prognosis. The molecular mechanisms that regulate Ikaros' function as a tumor suppressor and regulator of cellular proliferation are not well understood. We demonstrated that Ikaros is a substrate for Casein Kinase II (CK2), an oncogenic kinase that is overexpressed in ALL. Phosphorylation of Ikaros by CK2 impairs Ikaros' DNA-binding ability, as well as Ikaros' ability to regulate gene expression and function as a tumor suppressor in leukemia. Targeting CK2 with specific inhibitors restores Ikaros' function as a transcriptional regulator and tumor suppressor resulting in a therapeutic, anti-leukemia effect in a preclinical model of ALL. Here, we review the genes and pathways that are regulated by Ikaros and the molecular mechanisms through which Ikaros and CK2 regulate cellular proliferation in leukemia.

Topics: Animals; Antineoplastic Agents; Casein Kinase II; Cell Cycle; Cell Proliferation; Chromatin; Chromatin Assembly and Disassembly; DNA Nucleotidylexotransferase; Gene Expression Regulation; Humans; Ikaros Transcription Factor; Jumonji Domain-Containing Histone Demethylases; Naphthyridines; Nuclear Proteins; Phenazines; Phosphorylation; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Repressor Proteins; Signal Transduction; T-Lymphocytes

Children of Hispanic/Latino ancestry have increased incidence of high-risk B-cell acute lymphoblastic leukemia (HR B-ALL) with poor prognosis. This leukemia is characterized by a single-copy deletion of the IKZF1 (IKAROS) tumor suppressor and increased activation of the PI3K/AKT/mTOR pathway. This identifies mTOR as an attractive therapeutic target in HR B-ALL. Here, we report that IKAROS represses MTOR transcription and IKAROS' ability to repress MTOR in leukemia is impaired by oncogenic CK2 kinase. Treatment with the CK2 inhibitor, CX-4945, enhances IKAROS activity as a repressor of MTOR, resulting in reduced expression of MTOR in HR B-ALL. Thus, we designed a novel therapeutic approach that implements dual targeting of mTOR: direct inhibition of the mTOR protein (with rapamycin), in combination with IKAROS-mediated transcriptional repression of the MTOR gene (using the CK2 inhibitor, CX-4945). Combination treatment with rapamycin and CX-4945 shows synergistic therapeutic effects in vitro and in patient-derived xenografts from Hispanic/Latino children with HR B-ALL. These data suggest that such therapy has the potential to reduce the health disparity in HR B-ALL among Hispanic/Latino children. The dual targeting of oncogene transcription, combined with inhibition of the corresponding oncoprotein provides a paradigm for a novel precision medicine approach for treating hematological malignancies.

Topics: Antineoplastic Agents; B-Lymphocytes; Casein Kinase II; Cell Line; Cell Line, Tumor; Child; Gene Expression Regulation, Leukemic; Genes, Tumor Suppressor; HEK293 Cells; Humans; Naphthyridines; Phenazines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Signal Transduction; TOR Serine-Threonine Kinases

The tumor suppressor protein phosphatase and tensin homolog (PTEN) is a key regulator of the PI3K/AKT pathway which is frequently altered in a variety of tumors including a subset of acute B-lymphoblastic leukemias (B-ALL). While PTEN mutations and deletions are rare in B-ALL, promoter hypermethylation and posttranslational modifications are the main pathways of PTEN inactivation. Casein Kinase II (CK2) is often upregulated in B-ALL and phosphorylates both PTEN and DNA methyltransferase 3A, resulting in increased PI3K/AKT signaling and offering a potential mechanism for further regulation of tumor-related pathways.. Here, we evaluated the effects of CK2 inhibitor CX-4945 alone and in combination with hypomethylating agent decitabine on B-ALL proliferation and PI3K/AKT pathway activation. We further investigated if CX-4945 intensified decitabine-induced hypomethylation and identified aberrantly methylated biological processes after CK2 inhibition. In vivo tumor cell proliferation in cell line and patient derived xenografts was assessed by longitudinal full body bioluminescence imaging and peripheral blood flow cytometry of NSG mice.. CX-4945 incubation resulted in CK2 inhibition and PI3K pathway downregulation thereby inducing apoptosis and anti-proliferative effects. CX-4945 further affected methylation patterns of tumor-related transcription factors and regulators of cellular metabolism. No overlap with decitabine-affected genes or processes was detected. Decitabine alone revealed only modest anti-proliferative effects on B-ALL cell lines, however, if combined with CX-4945 a synergistic inhibition was observed. In vivo assessment of CX-4945 in B-ALL cell line xenografts resulted in delayed proliferation of B-ALL cells. Combination with DEC further decelerated B-ALL expansion significantly and decreased infiltration in bone marrow and spleen. Effects in patient-derived xenografts all harboring a t(4;11) translocation were heterogeneous.. We herein demonstrate the anti-leukemic potential of CX-4945 in synergy with decitabine in vitro as well as in vivo identifying CK2 as a potentially targetable kinase in B-ALL.

Topics: Animals; Antineoplastic Agents; Apoptosis; Casein Kinase II; Cell Line, Tumor; Computational Biology; Disease Models, Animal; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3A; Epigenesis, Genetic; Gene Expression Profiling; Gene Expression Regulation, Leukemic; Humans; Mice; Naphthyridines; Phenazines; Phosphatidylinositol 3-Kinases; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Xenograft Model Antitumor Assays

Recently, we demonstrated the ability of inhibitors of protein kinase 2 (casein kinase II; CK2) to enhance the efficacy of 5-fluorouracil, a thymidylate synthase (TYMS)-directed drug for anticancer treatment. The present study aimed to investigate the antileukemic effect of simultaneous inhibition of dihydrofolate reductase (DHFR), another enzyme involved in the thymidylate biosynthesis cycle, and CK2 in CCRF-CEM acute lymphoblastic leukemia cells.. The influence of combined treatment on apoptosis and cell-cycle progression, as well as the endocellular level of DHFR protein and inhibition of CK2 were determined using flow cytometry and western blot analysis, respectively. Real-time quantitative polymerase chain reaction was used to examine the influence of silmitasertib (CX-4945), a selective inhibitor of CK2 on the expression of DHFR and TYMS genes.. The synergistic effect was correlated with the increase of annexin V-binding cell fraction, caspase 3/7 activation and a significant reduce in the activity of CK2. An increase of DHFR protein level was observed in CCRF-CEM cells after CX-4945 treatment, with the mRNA level remaining relatively constant.. The obtained results demonstrate a possibility to improve methotrexate-based anti-leukemia therapy by simultaneous inhibition of CK2. The effect of CK2 inhibition on DHFR expression suggests the important regulatory role of CK2-mediated phosphorylation of DHFR inside cells.

Topics: Antineoplastic Agents; Apoptosis; Casein Kinase II; Cell Line, Tumor; Drug Synergism; Folic Acid Antagonists; Humans; Methotrexate; Naphthyridines; Phenazines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protein Kinase Inhibitors; Tetrahydrofolate Dehydrogenase

The proteasome inhibitor bortezomib is a new targeted treatment option for refractory or relapsed acute lymphoblastic leukemia (ALL) patients. However, a limited efficacy of bortezomib alone has been reported. A terminal pro-apoptotic endoplasmic reticulum (ER) stress/unfolded protein response (UPR) is one of the several mechanisms of bortezomib-induced apoptosis. Recently, it has been documented that UPR disruption could be considered a selective anti-leukemia therapy. CX-4945, a potent casein kinase (CK) 2 inhibitor, has been found to induce apoptotic cell death in T-ALL preclinical models, via perturbation of ER/UPR pathway. In this study, we analyzed in T- and B-ALL preclinical settings, the molecular mechanisms of synergistic apoptotic effects observed after bortezomib/CX-4945 combined treatment. We demonstrated that, adding CX-4945 after bortezomib treatment, prevented leukemic cells from engaging a functional UPR in order to buffer the bortezomib-mediated proteotoxic stress in ER lumen. We documented that the combined treatment decreased pro-survival ER chaperon BIP/Grp78 expression, via reduction of chaperoning activity of Hsp90. Bortezomib/CX-4945 treatment inhibited NF-κB signaling in T-ALL cell lines and primary cells from T-ALL patients, but, intriguingly, in B-ALL cells the drug combination activated NF-κB p65 pro-apoptotic functions. In fact in B-cells, the combined treatment induced p65-HDAC1 association with consequent repression of the anti-apoptotic target genes, Bcl-xL and XIAP. Exposure to NEMO (IKKγ)-binding domain inhibitor peptide reduced the cytotoxic effects of bortezomib/CX-4945 treatment. Overall, our findings demonstrated that CK2 inhibition could be useful in combination with bortezomib as a novel therapeutic strategy in both T- and B-ALL.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Bortezomib; Casein Kinase II; Cell Line, Tumor; Cell Survival; Drug Synergism; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Heat-Shock Proteins; Humans; Jurkat Cells; Microscopy, Fluorescence; Naphthyridines; Neoplastic Stem Cells; Phenazines; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Transcription Factor RelA; Unfolded Protein Response