Overexpression Of CK2 Research Articles

Abstract 6254: Casein kinase II (CK2) expression in pediatric solid tumors

Abstract Despite recent advances, high-risk neuroblastoma (NB) and sarcomas account for 15% and 5% of all pediatric cancer deaths, respectively. Many patients attain remission, but approximately half of patients relapse. Genomic alterations such as MYCN amplification and ALK mutations have been shown to be important in prognosis and treatment. Despite improvements, approximately 20% of patients have refractory or recurrent disease after therapy, underscoring the need for further genomic understanding and new approaches to treatment.CK2 is a serine/threonine kinase that regulates numerous cellular processes such as cell growth, proliferation, and survival. Overexpression of CK2 is associated with a poor prognosis in several adult solid tumors, making it a potential target of interest in pediatric solid tumors. This study aims to investigate CK2 expression among patients with NB, Ewing sarcoma (EWS), and rhabdomyosarcoma (RMS) and its correlation with patient survival. MethodsPatients were enrolled in NMTRC008/009: “Feasibility Trial Using Molecular Guided Therapy for Patients with Relapsed/Refractory Childhood Cancer” and BCC-001. WES and RNA-Seq were performed at the Translational Genomics Research Institute and Sema4 or Caris respectively. RNA expression levels were compared to a normal whole-body reference and a pediatric cancer reference. Differential expression data were interpreted in the context of systems biology.ResultsTumor WES was performed on 297 patients (212 NB, 50 EWS, 35 RMS) and RNA-Seq on 284 patients (210 NB, 39 EWS, 35 RMS). The most common mutations or copy number alterations were seen in MYCN, ATRX, ALK, CDKN2A, and chr17q+ in NB; EWSR1-FLI1 fusion in EWS; PAX3-FOXO1 fusion in RMS. RNA expression analysis was performed to understand MYCN-driver status by covariance and interactions and to identify subsets of patients with high/low expression of CK2 in different tumors. CK2 bimodal expression is seen across various cancers and normal tissues, implying distinct sub-groups exist in the data. In NB, overexpression of the CK2 gene with statistical significance was found in MYCN-amplified and chr17q+ tumors. A positive and negative correlation of the CK2 gene is seen with several target genes like MYCN, TRIM71, LIN28B, and HMGA2, JAK2, AR, STAT3, EGFR, LEF1, and NFKB2, respectively. In EWS and RMS, subgroups are identified with high and low expression of the CK2 gene.ConclusionGenomic sequencing of pediatric solid tumors showed a low mutation burden. RNA analysis resulted in the identification of potential novel therapeutic targets involving CK2 pathways. High expression of CK2 correlates with MYCN status. Further analysis of patient molecular subgroups is ongoing. Citation Format: Abhinav Beeravally Nagulapally, Chandrika Behura, Divya Gandra, Giselle Saulnier Sholler. Casein kinase II (CK2) expression in pediatric solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6254.

Emerging role of Protein Kinase CK2 in Tumor immunity.

Protein kinase CK2, a conserved serine/threonine-protein kinase, is ubiquitous in cells and regulates various intracellular processes, especially in tumor cells. As one of the earliest discovered protein kinases in humans, CK2 plays a crucial role in phosphorylating or associating with hundreds of substrates to modulate several signaling pathways. Excellent reviews have reported that the overexpression of CK2 could be observed in many cancers and was closely associated with tumor occurrence and development. The elevation of CK2 is also an indicator of a poor prognosis. Recently, increasing attention has been paid to the relationship between CK2 and tumor immunity. However, there is no comprehensive description of how CK2 regulates the immune cells in the tumor microenvironment (TME). Also, the underlying mechanisms are still not very clear. In this review, we systematically summarized the correlation between CK2 and tumor immunity, primarily the effects on various immune cells, both in innate and adaptive immunity in the TME. With the comprehensive development of immunotherapy and the mounting transformation research of CK2 inhibitors from the bench to the clinic, this review will provide vital information to find new treatment options for enhancing the efficacy of immunotherapy.

Protein kinase CK2 modulates the activity of Maf-family bZIP transcription factor NRL in rod photoreceptors of mammalian retina.

Maf-family basic motif leucine zipper protein NRL specifies rod photoreceptor cell fate during retinal development and, in concert with homeodomain protein CRX and other regulatory factors, controls the expression of most rod-expressed genes including the visual pigment gene Rhodopsin (Rho). Transcriptional regulatory activity of NRL is modulated by post-translational modifications, especially phosphorylation, and mutations at specific phosphosites can lead to retinal degeneration. During our studies to elucidate NRL-mediated transcriptional regulation, we identified protein kinase CK2 in NRL-enriched complexes bound to Rho promoter-enhancer regions and in NRL-enriched high molecular mass fractions from the bovine retina. The presence of CK2 in NRL complexes was confirmed by co-immunoprecipitation from developing and adult mouse retinal extracts. In vitro kinase assay and bioinformatic analysis indicated phosphorylation of NRL at Ser117 residue by CK2. Co-transfection of Csnk2a1 cDNA encoding murine CK2 with human NRL and CRX reduced the bovine Rho promoter-driven luciferase expression in HEK293 cells and mutagenesis of NRL-Ser117 residue to Ala restored the reporter gene activity. In concordance, overexpression of CK2 in the mouse retina in vivo by electroporation resulted in reduction of Rho promoter-driven DsRed reporter expression as well as the transcript level of many phototransduction genes. Thus, our studies demonstrate that CK2 can phosphorylate Ser117 of NRL. Modulation of NRL activity by CK2 suggests intricate interdependence of transcriptional and signaling pathways in maintaining rod homeostasis.

Abstract 846: Transcriptional regulation of Aurora-B kinase expression in B-cell acute lymphoblastic leukemia (B-ALL)

Abstract The Aurora kinases are serine/threonine kinases that are essential for mitosis. Aurora-B kinase is a component of the chromosomal passenger complex (CPC), chromosome condensation, the spindle-assembly checkpoint, and for cytokinesis. The activity of Aurora-B is essential for cellular division and proliferation. Inhibition of Aurora-B is cytotoxic, and inhibitor of Aurora-B - Barasertib (AZD1152) has been tested in clinical trials against different types of malignancies, including leukemia. However, regulation of Aurora-B expression is still largely unknown. Here, we present evidence that expression of Aurora-B in B-cell acute lymphoblastic leukemia (B-ALL) is regulated at the transcriptional level by Ikaros, a transcription factor and tumor suppressor protein. Analysis of global chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) in several B-ALL primary human B-ALL cells and cell lines, showed a strong enrichment of Ikaros at the promoter of the Aurora-B gene. Ikaros functions as a tumor suppressor protein and deletion of Ikaros is associated with development of high-risk B-ALL. Ikaros binding to Aurora-B promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in primary B-ALL cells. The role of Ikaros in regulating Aurora-B transcription in B-ALL was tested using gain-of-function and loss-of-function experiments. Overexpression of Ikaros in human B-ALL was associated with strongly reduced transcription and overall expression of Aurora-B. Ikaros knock-down with shRNA results in increased expression of Aurora-B in B-ALL. Since Ikaros function in B-ALL is negatively regulated by pro-oncogenic Casein Kinase II (CK2), we tested whether CK2 can regulate expression of Aurora-B in B-ALL. Overexpression of CK2 via retroviral transduction resulted in increased expression of the Aurora-B gene. Increased expression of CK2 was associated with a loss of Ikaros binding to the Aurora-B gene promoter. Molecular inhibition of CK2 using shRNA resulted in reduced expression of Aurora-B in human B-ALL. Inhibition of CK2 was associated with increased Ikaros binding at the Aurora-B promoter. Ikaros knock-down abolished downregulation of Aurora-B expression in B-ALL cells following treatment with CK2 inhibitors. These data demonstrate that CK2 and Ikaros are critical regulators of Aurora-B expression in B-ALL. In conclusion, these results indicate that expression of the Aurora-B gene in B-ALL is regulated by the CK2-Ikaros signaling axis and provide a novel insight into mechanisms that regulate mitosis in leukemia. Citation Format: Yali Ding, Chunhua Song, Joseph Schramm, Soumya Maru, Daniel Bogus, Dhimant H. Desai, Arati K. Sharma, Chandrika Gowda, Sinisa Dovat. Transcriptional regulation of Aurora-B kinase expression in B-cell acute lymphoblastic leukemia (B-ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 846.

Abstract PD3-08: Novel cancer stem cell inhibitor 108600 modulates tumor immunomicroenvironment of triple negative breast cancer (TNBC)

Abstract 108600, a novel CK2/DYRK1/TNIK inhibitor, targets and inhibits cancer stem cells (CSC) in triple negative breast cancer (TNBC), inhibiting tumor growth and metastases in patient-derived xenograft models. CSC’s have been shown to promote immune evasion of several types of cancer. Specifically, over-expression of CK2 has been shown to promote intratumoral recruitment of myeloid derived suppressor cells. We investigated the effects of 108600 treatment on the immune microenvironment of triple negative breast cancer since targeting CSC’s could promote favorable anti-tumor immune responses and mechanistically contribute to tumor growth inhibition. Methods:C57BL/6 mice bearing murine triple negative E0771 tumors were generated by orthotopic injection and treated either with vehicle control or 108600 for 5 days. Tumor growth was assessed by daily caliper measurement All tumors were recovered at endpoint, and processed for RNA sequencing, flow cytometry or Western blot analysis. Results:108600 treatment significantly inhibited growth of E0771 tumors in vivo, demonstrating for the first time efficacy of 108600 against TNBC in immunocompetent models. 108600 treatment decreased intratumoral phosphorylation and expression of 108600 targets AKT1 (Ser 129) and Cyclin D1, which are substrates of CK2α and Dyrk1, respectively. To further explore nature of transcriptional and signaling pathways affected by 108600 treatment in vivo, RNA sequencing was performed. DGE and subsequent annotation analysis showed that various immune (GO:0006955, GO) and inflammatory (GO:0006954, GO) signaling pathways associated with Stat1 (mmu04062, KEGG) and IFNγ (mmu04060, KEGG) were down-regulated in 108600-treated E0771 tumors. GSEA (Gene set enrichment analysis) indicated that the regulatory T cell (Treg) population (GSE42021, GSE40685) was suppressed by 108600 treatment. To validate these findings, tumor infiltrating leukocytes (TIL) were isolated from vehicle or 108600-treated tumors and analyzed by flow cytometry. CD4, CD25, and FOXP3 expression was used to gate the Treg population. The Treg population was significantly suppressed by 108600 treatment, confirming the RNA sequencing results. 108600 also likely regulates expression of immunomodulatory molecules in TNBC tumor cells since 108600 treatment increased PD-L1 surface expression on E0771 cells in vitro. Conclusions:Our study supports 108600, an inhibitor that targets breast cancer stem cells, as a modulator of the immune microenvironment of TNBC. 108600 suppresses the Treg population among the TIL population and increases tumoral expression of PD-L1. Tregs have been associated with disease progression and metastases of TNBC. Further studies are ongoing to clarify the functional consequences of these changes in the setting of tumor growth inhibition. Synergies between 108600 and checkpoint inhibition are also under investigation and could lead to novel combination therapies for TNBC. Citation Format: Katsutoshi Sato, Stacey J. Baker, E. P. Reddy, Hanna Y. Irie. Novel cancer stem cell inhibitor 108600 modulates tumor immunomicroenvironment of triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-08.

Control of TRPM3 Ion Channels by Protein Kinase CK2-Mediated Phosphorylation in Pancreatic β-Cells of the Line INS-1.

In pancreatic β-cells of the line INS-1, glucose uptake and metabolism induce the openings of Ca2+-permeable TRPM3 channels that contribute to the elevation of the intracellular Ca2+ concentration and the fusion of insulin granules with the plasma membrane. Conversely, glucose-induced Ca2+ signals and insulin release are reduced by the activity of the serine/threonine kinase CK2. Therefore, we hypothesized that TRPM3 channels might be regulated by CK2 phosphorylation. We used recombinant TRPM3α2 proteins, native TRPM3 proteins from INS-1 β-cells, and TRPM3-derived oligopeptides to analyze and localize CK2-dependent phosphorylation of TRPM3 channels. The functional consequences of CK2 phosphorylation upon TRPM3-mediated Ca2+ entry were investigated in Fura-2 Ca2+-imaging experiments. Recombinant TRPM3α2 channels expressed in HEK293 cells displayed enhanced Ca2+ entry in the presence of the CK2 inhibitor CX-4945 and their activity was strongly reduced after CK2 overexpression. TRPM3α2 channels were phosphorylated by CK2 in vitro at serine residue 1172. Accordingly, a TRPM3α2 S1172A mutant displayed enhanced Ca2+ entry. The TRPM3-mediated Ca2+ entry in INS-1 β-cells was also strongly increased in the presence of CX-4945 and reduced after overexpression of CK2. Our study shows that CK2-mediated phosphorylation controls TRPM3 channel activity in INS-1 β-cells.

CK2 Inhibitor CX4945 Shows Strong In Vivo Synergistic Anti-Leukemia Activity with Rapamycin in B-ALL Due to Enhanced Ikaros Mediated Repression of mTOR Pathway

Casein Kinase II (CK2) is a pro-oncogenic protein that is overexpressed in various cancers including leukemia. Ikaros encodes a DNA-binding zinc finger protein that functions as a tumor suppressor. Loss of Ikaros activity (genetic alteration and functional inactivation) in high-risk B-ALL is associated with resistance to conventional chemotherapy and a poor prognosis. Ikaros binds to the promoters and/or upstream regulatory sequences of its target genes and regulates their transcription via chromatin remodeling. CK2 phosphorylates Ikaros and impairs its tumor suppressor functions and interferes with Ikaros' ability to repress cell cycle progression and the PI3K pathway. Targeted inhibition of CK2 using specific inhibitor CX4945 restores Ikaros tumor suppressor function in high-risk B-ALL even in cases with single allele Ikaros deletion. CX4945 showed strong in vivo anti-leukemia effect in high risk B-ALL patient derived xenograft models (Song et al, Blood 2015). CX4945 is currently in clinical trial for solid tumors. mTOR is the key component of the PI3K/AKT/mTOR pathway - a major signaling pathway that stimulates cellular proliferation in B-ALL. Rapamycin is a specific inhibitor of mTOR that is currently in clinical trial for the treatment of relapsed/refractory leukemia. Here we report in vivo efficacy of combination therapy of rapamycin with CX4945 using high risk B-ALL pre-clinical models and describe mechanism of synergistic action of CX4945 and rapamycin. Methods and results: The therapeutic effect of the CX-4945, in combination with rapamycin was tested using three different patient-derived xenografts (PDX) from patients with high-risk B-ALL and its efficacy will be compared to single-drug treatment. All these leukemia samples were obtained from high risk pediatric B-ALL patients with high CK2 expression and one had IKZF1 haplo - deficiency. After engraftment following tail vein inoculation of leukemia cells into immunocompromised (NRG) mice, mice were treated with vehicle, rapamycin or CX4945 alone or combination of both CX4945 and rapamycin. Anti-tumor effect was measured by quantifying leukemia burden in bone marrow, spleen and peripheral blood by cell count and immune-phenotyping using flow cytometry. Overall survival was measured using Kaplan -Myers analysis in another cohort after similar treatment regimen. The analysis of Ikaros' global genome-wide occupancy in the human B-ALL cell line, Nalm6, and in primary cells from patient with B-ALL, showed that Ikaros binds to the upstream regulatory element (URE) of MTOR gene. Further, Ikaros binding to MTOR promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in primary B-ALL cells. The role of Ikaros in regulating mTOR transcription in Nalm-6 B-ALL cell line was tested using gain-of-function and loss-of-function experiments. Ikaros knock-down with shRNA results in increased transcription of mTOR whereas overexpression of Ikaros by transduction of Nalm-6 cells with Ikaros-containing retrovirus resulted in sharp reduction of mTOR expression. Since Ikaros function in B-ALL is negatively regulated by pro-oncogenic Casein Kinase II (CK2), we tested whether CK2 can regulate expression of mTOR. Over-expression of CK2 via retroviral transduction resulted in increased transcription of the mTOR mRNA, as measured by qRT-PCR, as well as increased overall expression of mTOR protein, as measured by western blot. Increased expression of CK2 was associated with a loss of Ikaros binding to the MTOR gene promoter. CK2 inhibition was also associated with reduced histone H3K9ac and H3K4me3 marks at the mTOR promoter suggesting that CK2 and Ikaros regulate mTOR transcription via chromatin remodeling. In summary, these results establish in vivo anti-leukemia effect of combination therapy using rapamycin and CX4945 in high risk B-ALL PDX models. CK2 inhibition with CX4945 represses the mTOR pathway by enhancing Ikaros-mediated transcriptional repression of mTOR and PI3K pathway genes. Results provide the rationale for evaluating clinical use of CK2 inhibitors in combination with mTOR inhibitors in high risk leukemia with dysregulation of mTOR/PI3K pathway. The results of our study establish a new paradigm for targeting the transcriptional network that regulates expression of the gene in hyperactive oncogenic signaling pathways such as mTOR. Disclosures Payne: Elf Zone, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Abstract 871: Regulation of CDC25a expression by the ikaros and casein kinase II (CK2) in T-cell acute lymphoblastic leukemia (T-ALL)

Abstract CDC25A is a member of the CDC25 family of phosphatases that plays a major role in cell cycle progression. Here, we present evidence that expression of CDC25a in T-ALL is regulated at the transcriptional level by oncogenic Casein Kinase II (CK2) via direct phosphorylation of Ikaros, a transcription factor and tumor suppressor protein. Global chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) studies in both primary human acute lymphoblastic leukemia cells and cell lines, demonstrated that Ikaros binds to the promoter of the CDC25a gene. Ikaros functions as a tumor suppressor protein and deletion of which is associated with development of T-ALL. Ikaros binding to CDC25a promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in primary T-ALL cells. Ikaros knock-down with shRNA results in increased transcription of CDC25a in T-ALL. In mice, T-ALL cells that were derived from Ikaros-knockout mice express high levels of CDC25a. Transduction of these cells with Ikaros-containing retrovirus results in sharp reduction of CDC25a expression. Overexpression of CK2 via retroviral transduction resulted in increased transcription of the CDC25a gene, as measured by qRT-PCR, as well as increased overall expression of CDC25a, as measured by Western blot. Increased expression of CK2 was associated with a loss of Ikaros binding to the CDC25a gene promoter. Molecular inhibition of CK2 using shRNA, as well as pharmacological inhibition with a specific CK2 inhibitor resulted in reduced expression of CDC25a in primary human T-ALL. CK2 inhibition was also associated with strong reduction in AKT phosphorylation, emphasizing that CK2 inhibition downregulates CDC25a and other cell cycle progression genes. Inhibition of CK2 was associated with increased Ikaros binding at the promoter of CDC25a. Ikaros knock-down restored high expression of CDC25a in T-ALL cells that were treated with CK2 inhibitors. These data showed that CK2 and Ikaros are major transcriptional regulators of CDC25a transcription in T-ALL and that CK2 inhibition represses CDC25a transcription via Ikaros-mediated repression. In conclusion, these results indicate that expression of the CDC25a oncogene in T-ALL is regulated by the CK2 which modulates Ikaros activity. Presented data revealed a novel mechanism of therapeutic action of CK2 inhibitors - repression of CDC25a expression via Ikaros. Results provide a rationale for the use of novel CK2 inhibitors in T-ALL. Citation Format: Soumya C. Iyer, Shriya Kane, Chandrika Gowda, Chunhua Song, Yali Ding, Jon Payne, Pavan Kumar Dhanyam Raju, Bihua Tan, Mary McGrath, Yevgeniya Bamme, Mario Solimon, Nathalia Moreno Cury, Dhimant Desai, Arati Sharma, Kimberly J. Payne, Sinisa Dovat. Regulation of CDC25a expression by the ikaros and casein kinase II (CK2) in T-cell acute lymphoblastic leukemia (T-ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 871.

Abstract 4400: Epigenetic regulation of CDC16 expression by the ikaros and casein kinase II (CK2) in T-cell acute lymphoblastic leukemia (T-ALL)

Abstract CDC16 is one of several subunits of the anaphase-promoting complex (APC) with crucial role in cell division. Here, we show evidence that expression of CDC16 in T-ALL is modulated at the transcriptional level by oncogenic Casein Kinase II (CK2) via direct phosphorylation of Ikaros, a tumor suppressor protein and transcription factor. Global chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) studies in both cell lines and primary human ALL, shows that Ikaros binds to the promoter of the CDC16 gene. Ikaros functions as a tumor suppressor protein and its deletion is associated with development of T-ALL. Ikaros binding to CDC16 promoter was confirmed via quantitative chromatin immunoprecipitation (qChIP) in primary T-ALL cells. The role of Ikaros in regulating CDC16 transcription in T-ALL was tested using gain-of-function and loss-of-function experiments. Ikaros knock-down with shRNA resulted in increased transcription of CDC16 in T-ALL. Overexpression of Ikaros in human T-ALL was associated with strong reduction in transcription of CDC16. In mice, T-ALL cells that are derived from Ikaros-knockout mice express high levels of CDC16. Transduction of these cells with Ikaros-containing retrovirus results in sharp reduction of CDC16 expression. Since Ikaros function in T-ALL is negatively regulated by pro-oncogenic Casein Kinase II (CK2), we tested whether CK2 can regulate expression of CDC16 in T-ALL. Overexpression of CK2 via retroviral transduction resulted in increased CDC16 gene transcription as measured by qRT-PCR, as well as increased overall expression of CDC16, as analyzed by Western blot. Increased expression of CK2 was associated with loss of Ikaros binding to the CDC16 gene promoter. Molecular inhibition of CK2 using shRNA, as well as pharmacological inhibition with a specific CK2 inhibitor led to reduced expression of CDC16 in primary human T-ALL. Inhibition of CK2 was associated with increased Ikaros binding at the promoter of CDC16. Ikaros knock-down restored high expression of CDC16 in T-ALL cells that were treated with CK2 inhibitors. These data demonstrate that CK2 and Ikaros are major transcriptional regulators of CDC16 transcription in T-ALL and that inhibition of CK2 represses CDC16 transcription via Ikaros-mediated repression. In summary, these results provide evidence that expression of the CDC16 gene in T-ALL is regulated by the CK2 signaling pathway which modulates Ikaros funtion. Targeting CK2 with specific inhibitors has been used as a therapeutic strategy in preclinical models of T-ALL. Presented data reveals a novel mechanism of therapeutic action of CK2 inhibitors – repression of CDC16 expression via Ikaros. These results provide a rationale for the use of novel CK2 inhibitors in T-ALL to target CDC16. Citation Format: Pavan Kumar Dhanyam Raju, Shriya Kane, Chandrika Gowda, Chunhua Song, Yali Ding, Jonathan Payne, Soumya Iyer, Nathalia Moreno Curry, Mary McGrath, Yevgeniya Bamme, Bihua Tan, Mario Soliman, Dhimant Desai, Arati Sharma, Kimberly J. Payne, Sinisa Dovat. Epigenetic regulation of CDC16 expression by the ikaros and casein kinase II (CK2) in T-cell acute lymphoblastic leukemia (T-ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4400.

Regulation of mTOR expression by Casein Kinase II (CK2) signaling pathway in leukemia

The mammalian target of rapamycin(mTOR) is a kinase encoded by the MTOR gene. mTOR is a critical component of PI3K/mTOR/AKT signaling pathway that positively regulates proliferation of leukemia cells. Targeting mTOR and PI3K/mTOR/AKT pathway has been used to treat T‐cell acute lymphoblastic leukemia (T‐ALL). However, little is known about mechanisms that regulate transcription and expression of mTOR gene in T‐ALL. Here, we present the evidence that expression of mTOR in T‐ALL is regulated at transcriptional level by oncogenic Casein Kinase II (CK2) via direct phosphorylation of the Ikaros, a transcription factor and a tumor suppressor protein. Global chromatin immunoprecipitation coupled with the next‐generation sequencing (ChIP‐seq) studies in primary human T‐ALL, demonstrated that Ikaros binds of the promoter of mTOR gene. Ikaros binding to mTOR promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in primary T‐ALL cells. The role of Ikaros in regulation of mTOR transcription in T‐ALL was tested using gain‐of‐function and loss‐of‐function experiments. Ikaros knock‐down with shRNA, results in increased transcription of mTOR in T‐ALL. Overexpression of Ikaros in human T‐ALL was associated with strongly reduced transcription of mTOR. In mice, T‐ALL cells that are derived from Ikaros‐knockout mice express high level of mTOR. Transduction of these cells with Ikaros‐containing retrovirus, results in sharp reduction of mTOR expression. Since Ikaros function in T‐ALL is negatively regulated by pro‐oncogenic Casein Kinase II (CK2), we tested whether CK2 can regulate expression of mTOR in T‐ALL. Overexpression of CK2 via retroviral transduction resulted in increased transcription of the MTOR gene, as well as increased overall expression of mTOR, as measured by Western blot. Increased expression of CK2 was associated with loss of Ikaros binding to the promoter of the MTOR gene. Molecular inhibition of CK2 using shRNA, as well as pharmacological inhibition with a specific CK2 inhibitor resulted in reduced expression of mTOR in primary T‐ALL. Inhibition of CK2 was associated with increased Ikaros binding at promoter of mTOR. Ikaros knock‐down restored high expression of mTOR in T‐ALL cells that are treated with CK2 inhibitors. These data showed that CK2 and Ikaros are major transcriptional regulators of mTOR transcription in T‐ALL and that CK2 inhibition represses mTOR transcription via Ikaros‐mediated repression. CK2 inhibition, along with an increased Ikaros binding was associated with reduced histone H3K9ac and H3K4me3 marks at mTOR promoter suggesting that CK2 and Ikaros regulate mTOR transcription via chromatin remodeling. In conclusion, results presented the evidence that expression of mTOR oncogene is regulated by CK2 signaling pathway that involves Ikaros activity in T‐ALL. Results provide the rationale for the use of CK2 inhibitors in T‐ALL to target PI3K/mTOR/AKT pathway.Support or Funding InformationR01CA209829‐02 (SD and KP) and R01CA213912‐01A1 (SD)St. Baldrick's foundation ( SD and CG) and Hyundai Hope on wheels (SD and CG)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract C44: Epigenetic regulation of CRLF2 oncogene expression by Casein Kinase II (CK2) signaling in B-cell acute lymphoblastic leukemia that occurs at high frequency in Hispanic children

Abstract B-cell acute lymphoblastic leukemia (B-ALL) occurs more frequently in Hispanic children as compared to non-Hispanic whites. The specific subtype of B-ALL that is caused by overexpression of CRLF2 (CRLF2 B-ALL), occurs 5 times more frequently in Hispanic children as compared to the others. Since this type of B-ALL is associated with poor prognosis, the death rate of B-ALL is 39% higher in Hispanic children than in non-Hispanic whites. Thus, understanding the molecular mechanisms that regulate CRLF2 expression in CRLF2 B-ALL is essential for the development of targeted therapy for this disease. Our previous work determined that transcription of CRLF2 is negatively regulated by a tumor-suppressor protein, Ikaros. Ikaros deregulation is a feature of over 80% of CRLF2 B-ALL. Here we present evidence that Ikaros-mediated repression of CRLF2 transcription in B-ALL in Hispanic children is regulated by Casein Kinase II (CK2). CK2 is an oncogenic kinase that is overexpressed in B-ALL. We have previously shown that CK2 can directly phosphorylate Ikaros and that phosphorylation by CK2 can impair Ikaros function as transcriptional regulator. We tested whether inhibition of CK2 affects the ability of Ikaros to regulate CRLF2 transcription. Molecular inhibition by shRNA that targets the catalytic subunit of CK2, as well as pharmacologic inhibition of CK2 by the specific inhibitor, CX-4945, resulted in reduced expression of CRLF2 as measured by qRT-PCR. This was associated with increased Ikaros binding to the CRLF2 promoter as measured by quantitative chromatin immunoprecipitation (qChIP). To determine whether Ikaros function is essential for CRLF2 repression following CK2 inhibition, we compared expression of CRLF2 in cells that have Ikaros knocked-down by shRNA vs. cells with control shRNA, following treatment with CX-4945. Results showed that Ikaros knockdown abolished the ability of CK2 inhibitors to repress transcription of CRLF2 in B-ALL. These results demonstrate that Ikaros is an essential component of CK2 signaling that regulates CRLF2 expression. Analysis of the epigenetic signature at the CRLF2 promoter performed by serial qChIP assays showed that increased Ikaros binding to the CRLF2 promoter, following CK2 inhibition, is associated with enrichment for the H3K9me3 histone modification, which is a marker of repressive chromatin. In conclusion, we demonstrate that expression of the CRLF2 oncogene in acute leukemia that disproportionally occurs in Hispanic children is epigenetically regulated by the CK2-Ikaros axis. In CRLF2 B-ALL, Ikaros-mediated repression of CRLF2 is impaired due to overexpression of CK2. Treatment of CRLF2 B-ALL with CK2 inhibitors restores Ikaros tumor suppressor function, resulting in CRLF2 repression. Results identified a signaling network that regulates CRLF2 expression and suggest that restoration of Ikaros activity with CK2 inhibitors can be a therapeutic approach for CRLF2 B-ALL to reduce the health disparity for Hispanic children with ALL. Supported by 1R01CA209829. Citation Format: Chunhua Song, Zheng Ge, Kimberly J. Payne, Sinisa Dovat. Epigenetic regulation of CRLF2 oncogene expression by Casein Kinase II (CK2) signaling in B-cell acute lymphoblastic leukemia that occurs at high frequency in Hispanic children [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr C44.

Abstract 2527: Regulation of mitotic progression in T-cell acute lymphoblastic leukemia by the Ikaros tumor suppressor

Abstract 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 high-risk T-ALL which is associated with poor prognosis harbor inactivating mutations or deletion of one allele of the IKZF1 tumor suppressor. The IKZF1 gene encodes the Ikaros protein that functions as a regulator of transcription and a tumor suppressor. However, the molecular mechanism of Ikaros' tumor suppressor function in T-ALL is unclear. The use of quantitative chromatin immunoprecipitation (qChIP) determined that Ikaros binds to the promoter regions of Anaphase Promoting Complex Subunit 1 (ANAPC1) and Anaphase Promoting Complex Subunit 7 (ANAPC7) cell cycle genes in T-ALL primary cells in vivo. ANAPC1 and ANAPC7 genes encode proteins whose function is essential for progression through mitosis, as well as through the G1 phase of the cell cycle. Ikaros overexpression in T-ALL via retroviral transduction, results in reduced expression of ANAPC1and ANAPC7, as evidenced by quantitative RT-PCR (qRT-PCR) and Western blot. The luciferase reporter assay further confirmed Ikaros' function as a transcriptional repressor of ANAPC1 and ANAPC7. The knock-down of Ikaros with shRNA in T-ALL resulted in increased transcription of ANAPC1 and ANAPC7, as evidenced by qRT-PCR. These data suggest that Ikaros can regulate mitotic progression in T-ALL by repressing transcription of ANAPC1 and ANAPC7 genes. Next, we studied the mechanisms that regulate Ikaros' ability to repress ANAPC1 and ANAPC7 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 ANAPC1 and ANAPC7 in human T-ALL. Molecular inhibition of CK2 with shRNA against the CK2 catalytic subunit resulted in reduced transcription of ANAPC1 and ANAPC7, as evidenced by qRT-PCR. This was associated with increased DNA-binding of Ikaros to the promoters of ANAPC1 and ANAPC7, as evidenced by qChIP. These data suggest that CK2 impairs Ikaros' ability to transcriptionally repress ANAPC1 and ANAPC7 and to regulate mitotic progression in T-ALL. Inhibition of CK2 enhances transcriptional repression of ANAPC1 and ANAPC7 by Ikaros, resulting in the cell cycle arrest of T-ALL. In conclusion, our results show that mitotic progression in T-ALL is controlled by transcriptional regulation of ANAPC1 and ANAPC7 by Ikaros. Overexpression of CK2 impairs Ikaros' ability to repress ANAPC1 and ANAPC7 expression, which contributes to deregulation of the control of mitotic progression in T-ALL. Results suggest the novel therapeutic mechanism of CK2 inhibitors for treatment of T-ALL. Citation Format: Jonathon L. Payne, Elanora Dovat, Mario Soliman, Chunhua Song, Sinisa Dovat. Regulation of mitotic progression in T-cell acute lymphoblastic leukemia by the Ikaros tumor suppressor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2527.

Transcriptional Regulation of Cell Cycle Progression in T‐Cell Leukemia

The IKZF1 gene encodes the Ikaros protein that acts as a tumor suppressor in acute lymphoblastic leukemia (ALL). The role of Ikaros in T‐cell acute lymphoblastic leukemia (T‐ALL) has been documented by the presence of Ikaros deletions in a significant subset of human T‐ALL. Ikaros functions as a transcriptional regulator of a large set of its target genes and a tumor suppressor in B‐cell acute lymphoblastic leukemia. However, the molecular mechanisms of the tumor suppressor activity of Ikaros in T‐ALL is unclear. The use of quantitative chromatin immunoprecipitation (qChIP) showed that Ikaros binds to the promoter regions of the CDK2 and CDK6 cell cycle genes in primary T‐ALL cells in vivo. Ikaros gain‐of‐function experiments performed using retroviral transduction of T‐ALL cells showed that Ikaros overexpression in T‐ALL results in the reduced expression of CDK2 and CDK6, as evidenced by quantitative RT‐PCR (qRT‐PCR) and western blot. The knock‐down of Ikaros with shRNA in T‐ALL resulted in increased transcription of CDK2 and CDK6 as measured using qRT‐PCR. These data suggest that Ikaros can regulate cell cycle progression in T‐ALL by direct transcriptional repression of the CDK2 and CDK6 genes. Next, we studied the mechanisms that regulate Ikaros' ability to repress CDK2 and CDK6 in T‐ALL. Ikaros' function as a transcriptional repressor is regulated by its direct phosphorylation by Casein Kinase II (CK2). CK2 is an oncogenic kinase that is overexpressed in hematopoietic malignancies. We tested the effect of CK2 inhibition on Ikaros' ability to regulate transcription of CDK2 and CDK6 in human T‐ALL. Molecular inhibition with shRNA, as well as pharmacological inhibition of CK2 with a specific inhibitor, CX‐4945, resulted in reduced transcription of CDK2 and CDK6. This was associated with increased DNA‐binding of Ikaros to the promoters of CDK2 and CDK6 as indicated by qChIP. These data suggest that CK2 impairs Ikaros' ability to transcriptionally repress CDK2 and CDK6 and to regulate cell cycle progression in T‐ALL. Inhibition of CK2 restores Ikaros' function as a transcriptional repressor of CDK2 and CDK6 and as a regulator of cell cycle progression in T‐ALL. In conclusion, our results demonstrate that the Ikaros tumor suppressor can control cell cycle progression in T‐ALL via transcriptional regulation of CDK2 and CDK6 expression. Overexpression of CK2 impairs Ikaros' ability to repress CDK2 and CDK6 expression, which contributes to deregulation of cell cycle control in T‐ALL. Results reveal the therapeutic mechanism of targeted inhibition of CK2 as treatment for T‐ALL.

Expression of protein kinase CK2 in leukemia and its significance

Protein kinase CK2 is a heterodimer composed of two catalytic subunits (α or α′ subunit) and two regulatory β subunits, which plays a significant role in the process of cell growth, proliferation, and differentiation. The overexpression and hyperactivation of protein kinase CK2 is closely related to some blood malignancies, therefore the inhibition of CK2 may provide an innovative strategy for leukemia. This article summarizes literatures on mechanism of protein kinase CK2 in leukemia and its significance. Key words: Casein kinase Ⅱ; Leukemia; Molecular targeted therapy

Reduced phosphorylation of Stat3 at Ser-727 mediated by casein kinase 2 — Protein phosphatase 2A enhances Stat3 Tyr-705 induced tumorigenic potential of glioma cells

Signal transducer and activator of transcription 3 (Stat3) is a transcription factor that is involved in cell survival and proliferation and has been found to be persistently activated in most human cancers mainly through its phosphorylation at Tyr-705. However, the role and regulation of Stat3 Ser-727 phosphorylation in cancer cells have not been clearly evaluated. In our findings, correlation studies on the expression of CK2 and Stat3 Ser-727 phosphorylation levels in human glioma patient samples as well as rat orthotopic tumor model show a degree of negative correlation. Moreover, brain tumor cell lines were treated with various pharmacological inhibitors to inactivate the CK2 pathway. Here, increased Stat3 Ser-727 phosphorylation upon CK2 inhibition was observed. Overexpression of CK2 (α, α′ or β subunits) by transient transfection resulted in decreased Stat3 Ser-727 phosphorylation. Stat3 Tyr-705 residue was conversely phosphorylated in similar situations. Interestingly, we found PP2A, a protein phosphatase, to be a mediator in the negative regulation of Stat3 Ser-727 phosphorylation by CK2. In vitro assays prove that Ser-727 phosphorylation of Stat3 affects the transcriptional activity of its downstream targets like SOCS3, bcl-xl and Cyclin D1. Stable cell lines constitutively expressing Stat3 S727A mutant showed increased survival, proliferation and invasion which are characteristics of a cancer cell. Rat tumor models generated with the Stat3 S727A mutant cell line formed more aggressive tumors when compared to the Stat3 WT expressing stable cell line. Thus, in glioma, reduced Stat3 Ser-727 phosphorylation enhances tumorigenicity which may be regulated in part by CK2–PP2A pathway.

P03.08 - Inhibition of Casein Kinase II (CK2) Enhances Ikaros Tumor Suppressor Activity and Shows Therapeutic Efficacy in a Preclinical Leukemia Model

Casein kinase II (CK2) promotes cell cycle progression and cellular proliferation in leukemia. Overexpression of CK2 results in the development of high-risk leukemia, that is resistant to chemotherapy. The mechanism by which CK2 promotes cellular proliferation and malignant transformation is unknown. Our published data showed that CK2 phosphorylates the Ikaros tumor suppressor protein, which results in loss of its function as a transcriptional repressor. To determine the therapeutic efficacy of CK2 inhibitors, we tested their effect on Ikaros tumor suppressor function in vitro in human pre-B acute lymphoblastic leukemia (ALL) and in vivo using a preclinical human-mouse pre-B ALL xenograft model.

Analysis of Wnt and Hedgehog Pathways Regulating Protein Kinases CK1 and CK2 in Acute Myeloid Leukemia Cells and Stem Cells: Correlation with the Expression of Wnt and Hedgehog Targets and Biological and Clinical Features.

AbstractAbstract 2501 Background.Leukemia stem cells (LSC) constitute the reservoir of acute and chronic leukemias, from which the relapse of the disease takes origin. The Wnt/βcatenin and the Hedgehog (Hh) signaling pathways regulate the balance between self-renewal and commitment of both normal hematopoietic stem cells (HSC) and LSC. Serine-Threonine protein kinases CK1 and CK2 phosphorylate members of Wnt and Hh and are therefore potential regulators of HSC and LSC biology; however, their function in HSC and LSC is unknown. Purpose.In the present work we have investigated the mRNA and protein levels of CK1 and CK2 in acute myeloid leukemia (AML) blasts and LSC isolated from patients. We have also integrated the expression data of CK1 and CK2 with the expression data of Wnt and Hh signaling pathways gene targets and with biological and clinical parameters of a group of AML patients and cell lines. We also tested the effects on the growth of AML blasts of CK1 and CK2 small chemical inhibitors. The aim of this study was to gain insights into the function of these pivotal protein kinases and on their expression levels relative to biological, clinical and prognostic variables that dictate the outcome of AML patients. Methods.mRNA was extracted from HSC and FACS-sorted LSC (as defined as Lineage- CD34+ CD38- CD123+ CD90+); proteins were obtained from the bulk of blasts from fifteen AML patients. Quantitative RT-PCR was employed to assess mRNA levels of: CK1α, CK1γ, CK2α (catalytic) and CK2β (regulatory subunit); Lef1, FoxO1, FoxO3, CyclinD1 (Wnt targets); Ptch, Gli1, Gli2, Gli3, Bmi1 (Hh targets); western blot analysis was performed to determine CK1α, CK2α, CK2β, p53, phospho-Ser529 NF-κB and phospho-Ser13 Cdc37 (two direct CK2 target sites) protein levels. Normal CD34+ bone marrow cells were used as controls. CK2 chemical inhibitors (CX4945 and tTBB) were assayed on LSC and AML cells. Results.CK1α, CK2α and CK2β mRNA and proteins were found overexpressed in LSC and in AML blasts. CK1γ mRNA levels were barely detectable. Notably, some AML cases displayed low levels of CK2β but high levels of CK2α, indicating a possible CK2β-independent function of CK2α. FoxO1, FoxO3 mRNAs were found upregulated while Lef1 and CyclinD1 ones were found slightly and Axin2 one strongly diminished, respectively. Ptch, Smo, Gli1, Gli2 and Gli3 mRNAs were unchanged or reduced, while we observed a strong upregulation of that of Bmi1. A correlation between the overexpression of CK1α and CK2 and high-risk cytogenetic groups was observed. Also, in most of CK2α high-expressing cases, TP53 was found downregulated, while phospho-Ser529 p65 upregulated. Importantly, inhibition of CK2 with selective compounds caused AML cell and LSC growth arrest, a restoration of TP53 and a downregulation of phospho-Ser529-NF-kB p65 and phospho-Ser13 Cdc37 protein levels. Conclusions.The present study is the first to report on the expression of CK1 and CK2 kinases in normal HSC and LSC. CK1 (α) and CK2 (α and β) mRNA and protein levels were higher in LSC (especially from high risk groups) than in HSC. CK1γ mRNA levels were low. Wnt and Hh pathways genes were differentially upregulated pointing to a gene specific LSC-associated function. Moreover, our data with CK2 inhibitors suggest that CK2 could be a suitable therapeutic target to eradicate residual AML LSCs. Future research will assess more in depth the in vivo functional role of CK1 and CK2 in HSC and LSC. Disclosures:No relevant conflicts of interest to declare.

Abstract 3765: Discovery and biological characterization of CX-8184, a potent inhibitor of protein kinase CK2

Abstract Protein kinase CK2 regulates multiple pathways that play key roles in carcinogenesis, including cell cycle progression, suppression of apoptosis, hypoxia, angiogenesis, inflammation and DNA damage repair. Overexpression of CK2 in various cancer tissues has been frequently linked to poor prognosis. All this makes targeting of CK2 highly attractive for both single agent and combination therapies. To date therapeutic targeting of CK2 proved to be challenging, with only one compound, CX-4945, entering clinical development. In phase I clinical trials CX-4945 was well tolerated, inhibited CK2 activity in PBMC, modulated the PI3K-AKT-mTOR oncogenic pathway, reduced tumor-related serum IL-6 and IL-8 biomarkers, resulted in stable disease for at least 16 weeks in 20% of treated patients, and caused single agent tumor shrinkage. Building on these successes with the first generation CK2 inhibitor, we sought to design a second generation CK2 inhibitor with distinct pharmacologic properties. The application of a focused screening cascade and iterative medicinal chemistry efforts generated CX-8184, a selective single digit nanomolar inhibitor of the CK2 enzyme that demonstrated desirable biologic properties. When evaluated against a panel of 32 cancer cell lines, CX-8184 demonstrated broad antiproliferative activity with median IC50 = 145 nM. CX-8184 suppressed NF-κB, Hif-1α and β-catenin driven transcription at concentrations of 100-300 nM, and inhibited IL-6 expression with IC50 = 30 nM. Since CK2 is known to potentiate AKT signaling, treatment of cancer cells with CX-8184 decreased phosphorylation of AKT, as well as its substrates. The Hsp90/Cdc37 machinery is regulated by CK2 and plays an important role in activation of various kinases, including members of the ErbB RTK family. CX-8184 rapidly suppressed phosphorylation of Cdc37 by CK2, leading to the deactivation of ErbB family members. In combination with erlotinib or lapatinib, CX-8184 produced synergistic killing of lung cancer and breast cancer cells, respectively. CX-8184 also inhibited the phosphorylation of XRCC1, a scaffold protein involved in DNA strand break repair that plays an important role in sensitivity of cancer cells to DNA damaging agents. Consequently, the treatment of ovarian cancer cells pre-challenged with gemcitabine and/or cisplatin with 300 nM CX-8184 led to synergistic induction of cell death. In vivo, CX-8184 demonstrated favorable PK properties with oral bioavailability and a half-life amenable to once daily dosing in mice. CX-8184 exhibited potent anti-tumor activity and was well tolerated in A431 xenograft model. The compound has also showed an acceptable pharmacological profile as evidenced by CYP, hERG and Ames testing. The pharmacokinetic and pharmacological properties and the biological activity of CX-8184, both as single agent or in combination, support its further development as a preclinical candidate.9 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3765. doi:1538-7445.AM2012-3765

Phosphorylation by CK2 Enhances the Rapid Light-induced Degradation of Phytochrome Interacting Factor 1 in Arabidopsis

The phytochrome family of sensory photoreceptors interacts with phytochrome interacting factors (PIFs), repressors of photomorphogenesis, in response to environmental light signals and induces rapid phosphorylation and degradation of PIFs to promote photomorphogenesis. However, the kinase that phosphorylates PIFs is still unknown. Here we show that CK2 directly phosphorylates PIF1 at multiple sites. α1 and α2 subunits individually phosphorylated PIF1 weakly in vitro. However, each of four β subunits strongly stimulated phosphorylation of PIF1 by α1 or α2. Mapping of the phosphorylation sites identified seven Ser/Thr residues scattered throughout PIF1. Ser/Thr to Ala scanning mutations at all seven sites eliminated CK2-mediated phosphorylation of PIF1 in vitro. Moreover, the rate of degradation of the Ser/Thr to Ala mutant PIF1 was significantly reduced compared with wild-type PIF1 in transgenic plants. In addition, hypocotyl lengths of the mutant PIF1 transgenic plants were much longer than the wild-type PIF1 transgenic plants under light, suggesting that the mutant PIF1 is suppressing photomorphogenesis. Taken together, these data suggest that CK2-mediated phosphorylation enhances the light-induced degradation of PIF1 to promote photomorphogenesis.

CX-4945, a Selective and Orally Bioavailable Inhibitor of Protein Kinase CK2 Inhibits PI3K/Akt, JAK-STAT and NF-κB Signaling and Induces Apoptosis In Multiple Myeloma Cells

AbstractAbstract 787Multiple myeloma (MM) is an incurable malignancy characterized by infiltration of malignant plasma cells in the bone marrow. There are many subtypes of MM each of which relies on distinct oncogenic pathways. Oncogene addiction has been regarded as an Achilles' heel of cancer and has consequently been the focus of extensive efforts to design molecularly targeted anticancer therapies based on the premise that silencing an oncogene's expression will prove lethal in certain cancers. However, recent evidence suggests that MM may have a fatal vulnerability that is better described as non-oncogene addiction. Non-oncogenes do not directly transform cells but rather encode for normal cellular proteins that become overexpressed or otherwise deregulated in order to maintain oncogenic signaling. Protein kinase CK2 represents the prototypical non-oncogene. CK2 activity and expression levels are elevated in many cancers and it has been reported that MM cells rely on elevated CK2 for survival. CK2 is a highly conserved, constitutively active serine/threonine protein kinase that regulates multiple pathways important in MM including PI3K/AKT, JAK-STAT and NF-κB signaling cascades. Further, CK2 is elevated under hypoxic conditions regulating hypoxia-inducible transcription factor 1 alpha (HIF-1α) activity and other mediators of angiogenesis that are known to play an important role in MM pathogenesis. Given the generalized overexpression of CK2 in cancers, the central role of CK2 in multiple pro-survival pathways and the evidence that CK2 is an essential protein for MM cell survival, the development of selective CK2 inhibitors has emerged as an attractive targeted approach for the treatment of MM. Herein we describe CX-4945, a selective, orally bioavailable, small molecule inhibitor of CK2. CX-4945 is a potent, ATP-competitive inhibitor of both isoforms of the CK2 catalytic subunits CK2α and CK2α' with IC50 values of 1 nM. In a panel of 13 MM cell lines, CX-4945 exhibited a range of antiproliferative activity in the low micromolar range as measured by CellTiter-Glo® Luminescent Cell Viability Assay. Molecular characterization of the MM cell line panel revealed that on average CK2α mRNA levels and CK2α protein levels were elevated 9.5-fold and 14-fold, respectively, compared to normal CD138+ plasma cells. CX-4945 effectively reduced CK2 enzymatic activity in the MM cell lines. In immunoblots, the attenuation of PI3K/Akt signaling by CX-4945 was evidenced by dephosphorylation of Akt on the CK2-specific Ser129 site as well as the Ser473 and Thr308 sites. Further evidence of diminished Akt activity was demonstrated by decreased phosphorylation of the downstream Akt target protein p21 at Thr145. An additional biochemical consequence of CK2 inhibition was noted as CX-4945 selectively reduced phosphorylation of the p65 subunit of NF-κB at the CK2-specific Ser529 phosphorylation site but not at the S536 site. Moreover, CX-4945 inhibited the JAK/STAT pathway as demonstrated by the reduction in phosphorylation of regulatory sites on STAT3 (Tyr705) and JAK2 (Tyr1007/1008). PCR Arrays were used to analyze the global effect of CK2 inhibition on the NFκB and JAK/STAT mediated signal transduction pathways. Several key mediators, e.g. IL-6, CyclinD1, MYC, STAT1, STAT3, IFNγ, and IL-1R, were significantly downregulated upon treatment with CX-4945. Furthermore, CX-4945 reduced IL-6 secretion in a concentration dependent manner. In culture, MM cell lines have adapted to maintain HIF1α protein expression. CX-4945 effectively inhibited HIF1α expression and a well-known HIF1α transcriptional target, VEGF. Inhibition of CK2 by CX-4945 induced apoptosis in MM cells as shown by an increase in Caspase 3/7 activity and PARP cleavage. Interestingly, CX-4945 failed to induce apoptosis and had no effect on viability in the normal human-derived stromal cell line HS-5. These data lend further support to the CK2 non-oncogene addiction hypothesis in MM. A Phase 1 clinical trial evaluating CX-4945 as a single agent in solid tumors has already established a favorable pharmacokinetic, pharmacodynamic and safety profile. These findings, coupled with our preclinical data, suggest CX-4945 is a promising therapeutic agent for targeting aberrant CK2 activity in MM and a Phase 1 clinical trial in MM is underway. Disclosures:No relevant conflicts of interest to declare.