IKAROS and CK2 regulate expression of BCL-XL and chemosensitivity in high-risk B-cell acute lymphoblastic leukemia.

High-risk B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive disease, often characterized by resistance to chemotherapy. A frequent feature of high-risk B-ALL is loss of function of the IKAROS (encoded by the IKZF1 gene) tumor suppressor. Here, we report that IKAROS regulates expression of the BCL2L1 gene (encodes the BCL-XL protein) in human B-ALL. Gain-of-function and loss-of-function experiments demonstrate that IKAROS binds to the BCL2L1 promoter, recruits histone deacetylase HDAC1, and represses BCL2L1 expression via chromatin remodeling. In leukemia, IKAROS' function is impaired by oncogenic casein kinase II (CK2), which is overexpressed in B-ALL. Phosphorylation by CK2 reduces IKAROS binding and recruitment of HDAC1 to the BCL2L1 promoter. This results in a loss of IKAROS-mediated repression of BCL2L1 and increased expression of BCL-XL. Increased expression of BCL-XL and/or CK2, as well as reduced IKAROS expression, are associated with resistance to doxorubicin treatment. Molecular and pharmacological inhibition of CK2 with a specific inhibitor CX-4945, increases binding of IKAROS to the BCL2L1 promoter and enhances IKAROS-mediated repression of BCL2L1 in B-ALL. Treatment with CX-4945 increases sensitivity to doxorubicin in B-ALL, and reverses resistance to doxorubicin in multidrug-resistant B-ALL. Combination treatment with CX-4945 and doxorubicin show synergistic therapeutic effects in vitro and in preclinical models of high-risk B-ALL. Results reveal a novel signaling network that regulates chemoresistance in leukemia. These data lay the groundwork for clinical testing of a rationally designed, targeted therapy that combines the CK2 inhibitor, CX-4945, with doxorubicin for the treatment of hematopoietic malignancies.

Revisiting the melanomagenic pathways and current therapeutic approaches.

Melanoma is one of the most aggressive forms of skin cancer with a steady increase in global incidence and mortality rate over the past five decades. Paradoxically, both reduced and excessive sun exposure has been linked to increased risk of melanoma incidence and death. Although the histological classification of melanoma is useful in diagnosis, its molecular subtypes are often determined by somatic mutations, which could be UV-dependent or -independent. Multiple genes involved in cancer development are often mutated dysregulating molecular pathways with concomitant phenotypic heterogeneity. Hence, treating melanoma has been a challenge, with patients experiencing poor clinical outcomes to current therapeutic options. This presents an unmet need to understand the interaction of molecular networks underpinning melanogenesis. This review describes the crosstalk of signaling cascades in melanoma development and the putative druggable targets, with the view of elucidating newer and better therapeutic strategies for the disease.

Targeting Protein Translation in Melanoma by Inhibiting EEF-2 Kinase Regulates Cholesterol Metabolism though SREBP2 to Inhibit Tumour Development.

Decreasing the levels of certain proteins has been shown to be important for controlling cancer but it is currently unknown whether proteins could potentially be targeted by the inhibiting of protein synthesis. Under this circumstance, targeting protein translation could preferentially affect certain pathways, which could then be of therapeutic advantage when treating cancer. In this report, eukaryotic elongation factor-2 kinase (EEF2K), which is involved in protein translation, was shown to regulate cholesterol metabolism. Targeting EEF2K inhibited key parts of the cholesterol pathway in cancer cells, which could be rescued by the addition of exogenous cholesterol, suggesting that it is a potentially important pathway modulated by targeting this process. Specifically, targeting EEF2K significantly suppressed tumour cell growth by blocking mRNA translation of the cholesterol biosynthesis transcription factor, sterol regulatory element-binding protein (SREBP) 2, and the proteins it regulates. The process could be rescued by the addition of LDL cholesterol taken into the cells via non-receptor-mediated-uptake, which negated the need for SREBP2 protein. Thus, the levels of SREBP2 needed for cholesterol metabolism in cancer cells are therapeutically vulnerable by targeting protein translation. This is the first report to suggest that targeting EEF2K can be used to modulate cholesterol metabolism to treat cancer.

Cell-free fetal DNA testing and its correlation with prenatal indications.

BACKGROUND: The prenatal test of cell-free fetal DNA (cffDNA) is also known as noninvasive prenatal testing (NIPT) with high sensitivity and specificity. This study is to evaluate the performance of NIPT and its clinical relevance with various clinical indications. METHODS: A retrospective analysis was conducted on 14,316 pregnant women with prenatal indications, including advanced maternal age (≥35 years), maternal serum screening abnormalities, the thickened nuchal translucency (≥2.5 mm) and other ultrasound abnormalities, twin pregnancy/IVF-ET pregnancy, etc. The whole-genome sequencing (WGS) of maternal plasma cffDNA was employed in this study. RESULTS: A total of 189 (1.32%) positive NIPT cases were identified, and 113/189 (59.79%)cases were confirmed by invasive prenatal testing. Abnormal serological screening (53.14%) was the most common indication, followed by elderly pregnancy (23.02%). The positive prediction value for T21, T18, T13, sex chromosome abnormalities, other autosomal aneuploidy abnormalities, and CNV abnormalities were 91.84, 68.75,37.50, 66.67, 14.29, and 6.45%, respectively. The positive rate and the true positive rate of nuchal translucency (NT) thickening were the highest (4.17 and 3.33%), followed by the voluntary requirement group (3.49 and 1.90%) in the various prenatal screening indications. The cffDNA concentration was linearly correlated with gestational age (≥10 weeks) and the positive NIPT group's Z-score values. CONCLUSIONS: whole-genome sequencing of cffDNA has extremely high sensitivity and specificity for T21, high sensitivity for T18, sex chromosome abnormalities, and T13. It also provides evidence for other abnormal chromosomal karyotypes (CNV and non-21/18/13 autosomal aneuploidy abnormalities). The cffDNA concentration is closely related to the gestational age and determines the specificity of NIPT. Our results highlight NIPT's clinical significance, which is an effective prenatal screening tool for high-quality care of pregnancy.

Transcriptional Regulation of PIK3CD and PIKFYVE in T-Cell Acute Lymphoblastic Leukemia by IKAROS and Protein Kinase CK2.

IKAROS, encoded by the IKZF1 gene, is a DNA-binding protein that functions as a tumor suppressor in T cell acute lymphoblastic leukemia (T-ALL). Recent studies have identified IKAROS's novel function in the epigenetic regulation of gene expression in T-ALL and uncovered many genes that are likely to be directly regulated by IKAROS. Here, we report the transcriptional regulation of two genes, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta (PIK3CD) and phosphoinositide kinase, FYVE-type zinc finger containing (PIKFYVE), by IKAROS in T-ALL. PIK3CD encodes the protein p110δ subunit of phosphoinositide 3-kinase (PI3K). The PI3K/AKT pathway is frequently dysregulated in cancers, including T-ALL. IKAROS binds to the promoter regions of PIK3CD and PIKFYVE and reduces their transcription in primary T-ALL. Functional analysis demonstrates that IKAROS functions as a transcriptional repressor of both PIK3CD and PIKFYVE. Protein kinase CK2 (CK2) is a pro-oncogenic kinase that is overexpressed in T-ALL. CK2 phosphorylates IKAROS, impairs IKAROS's DNA-binding ability, and functions as a repressor of PIK3CD and PIKFYVE. CK2 inhibition results in increased IKAROS binding to the promoters of PIK3CD and PIKFYVE and the transcriptional repression of both these genes. Overall, the presented data demonstrate for the first time that in T-ALL, CK2 hyperactivity contributes to PI3K signaling pathway upregulation, at least in part, through impaired IKAROS transcriptional regulation of PIK3CD and PIKFYVE. Targeting CK2 restores IKAROS's regulatory effects on the PI3K oncogenic signaling pathway.

Medulloblastoma: "Onset of the molecular era".

Among brain tumors, Medulloblastoma (MB) is one of the most common, malignant, pediatric tumors of the cerebellum. It accounts for ~20% of all childhood central nervous system (CNS) tumors. Despite, tremendous advances in drug development processes, as well as novel drugs for MB the morbidity and mortality rates, remain high. Craniospinal radiation, high-dose chemotherapy, and surgical resection are the primary therapeutic strategies. Tremendous progress in the field of "genomics" with vast amounts of data has led to the identification of four distinct molecular subgroups in medulloblastoma: WNT group, SHH group, group-III, and group-IV. The identification of these subgroups has led to individualized treatment strategies for each subgroup. Here, we discuss the various molecular subgroups of medulloblastoma as well as the differences between them. We also highlight the latest treatment strategies available for medulloblastoma.

Transcriptional Regulation of Genes by Ikaros Tumor Suppressor in Acute Lymphoblastic Leukemia.

Regulation of oncogenic gene expression by transcription factors that function as tumor suppressors is one of the major mechanisms that regulate leukemogenesis. Understanding this complex process is essential for explaining the pathogenesis of leukemia as well as developing targeted therapies. Here, we provide an overview of the role of Ikaros tumor suppressor and its role in regulation of gene transcription in acute leukemia. Ikaros (IKZF1) is a DNA-binding protein that functions as a master regulator of hematopoiesis and the immune system, as well as a tumor suppressor in acute lymphoblastic leukemia (ALL). Genetic alteration or functional inactivation of Ikaros results in the development of high-risk leukemia. Ikaros binds to the specific consensus binding motif at upstream regulatory elements of its target genes, recruits chromatin-remodeling complexes and activates or represses transcription via chromatin remodeling. Over the last twenty years, a large number of Ikaros target genes have been identified, and the role of Ikaros in the regulation of their expression provided insight into the mechanisms of Ikaros tumor suppressor function in leukemia. Here we summarize the role of Ikaros in the regulation of the expression of the genes whose function is critical for cellular proliferation, development, and progression of acute lymphoblastic leukemia.

Regulation of Small GTPase Rab20 by Ikaros in B-Cell Acute Lymphoblastic Leukemia.

Ikaros is a DNA-binding protein that regulates gene expression and functions as a tumor suppressor in B-cell acute lymphoblastic leukemia (B-ALL). The full cohort of Ikaros target genes have yet to be identified. Here, we demonstrate that Ikaros directly regulates expression of the small GTPase, Rab20. Using ChIP-seq and qChIP we assessed Ikaros binding and the epigenetic signature at the RAB20 promoter. Expression of Ikaros, CK2, and RAB20 was determined by qRT-PCR. Overexpression of Ikaros was achieved by retroviral transduction, whereas shRNA was used to knockdown Ikaros and CK2. Regulation of transcription from the RAB20 promoter was analyzed by luciferase reporter assay. The results showed that Ikaros binds the RAB20 promoter in B-ALL. Gain-of-function and loss-of-function experiments demonstrated that Ikaros represses RAB20 transcription via chromatin remodeling. Phosphorylation by CK2 kinase reduces Ikaros' affinity toward the RAB20 promoter and abolishes its ability to repress RAB20 transcription. Dephosphorylation by PP1 phosphatase enhances both Ikaros' DNA-binding affinity toward the RAB20 promoter and RAB20 repression. In conclusion, the results demonstrated opposing effects of CK2 and PP1 on expression of Rab20 via control of Ikaros' activity as a transcriptional regulator. A novel regulatory signaling network in B-cell leukemia that involves CK2, PP1, Ikaros, and Rab20 is identified.

Drug resistance mechanisms in cancers: Execution of pro-survival strategies.

One of the quintessential challenges in cancer treatment is drug resistance. Several mechanisms of drug resistance have been described to date, and new modes of drug resistance continue to be discovered. The phenomenon of cancer drug resistance is now widespread, with approximately 90% of cancer-related deaths associated with drug resistance. Despite significant advances in the drug discovery process, the emergence of innate and acquired mechanisms of drug resistance has impeded the progress in cancer therapy. Therefore, understanding the mechanisms of drug resistance and the various pathways involved is integral to treatment modalities. In the present review, I discuss the different mechanisms of drug resistance in cancer cells, including DNA damage repair, epithelial to mesenchymal transition, inhibition of cell death, alteration of drug targets, inactivation of drugs, deregulation of cellular energetics, immune evasion, tumor-promoting inflammation, genome instability, and other contributing epigenetic factors. Furthermore, I highlight available treatment options and conclude with future directions.

Nanomedicine Strategies for Targeting Tumor Stroma.

The tumor stroma, or the microenvironment surrounding solid tumors, can significantly impact the effectiveness of cancer therapies. The tumor microenvironment is characterized by high interstitial pressure, a consequence of leaky vasculature, and dense stroma created by excessive deposition of various macromolecules such as collagen, fibronectin, and hyaluronic acid (HA). In addition, non-cancerous cells such as cancer-associated fibroblasts (CAFs) and the extracellular matrix (ECM) itself can promote tumor growth. In recent years, there has been increased interest in combining standard cancer treatments with stromal-targeting strategies or stromal modulators to improve therapeutic outcomes. Furthermore, the use of nanomedicine, which can improve the delivery and retention of drugs in the tumor, has been proposed to target the stroma. This review focuses on how different stromal components contribute to tumor progression and impede chemotherapeutic delivery. Additionally, this review highlights recent advancements in nanomedicine-based stromal modulation and discusses potential future directions for developing more effective stroma-targeted cancer therapies.

Melanoma therapeutics: a literature review.

Melanoma is a relentless type of skin cancer which involves myriad signaling pathways which regulate many cellular processes. This makes melanoma difficult to treat, especially when identified late. At present, therapeutics include chemotherapy, surgical resection, biochemotherapy, immunotherapy, photodynamic and targeted approaches. These interventions are usually administered as either a single-drug or in combination, based on tumor location, stage, and patients' overall health condition. However, treatment efficacy generally decreases as patients develop treatment resistance. Genetic profiling of melanocytes and the discovery of novel molecular factors involved in the pathogenesis of melanoma have helped to identify new therapeutic targets. In this literature review, we examine several newly approved therapies, and briefly describe several therapies being assessed for melanoma. The goal is to provide a comprehensive overview of recent developments and to consider future directions in the field of melanoma.

Drug-Tolerant Persister Cells in Cancer Therapy Resistance.

One of the current stumbling blocks in our fight against cancer is the development of acquired resistance to therapy, which is attributable to approximately 90% of cancer-related deaths. Undercutting this process during treatment could significantly improve cancer management. In many cases, drug resistance is mediated by a drug-tolerant persister (DTP) cell subpopulation present in tumors, often referred to as persister cells. This review provides a summary of currently known persister cell subpopulations and approaches to target them. A specific DTP cell subpopulation with elevated levels of aldehyde dehydrogenase (ALDH) activity has stem cell-like characteristics and a high level of plasticity, enabling them to switch rapidly between high and low ALDH activity. Further studies are required to fully elucidate the functions of ALDH-high DTP cells, how they withstand drug concentrations that kill other cells, and how they rapidly adapt under levels of high cellular stress and eventually lead to more aggressive, recurrent, and drug-resistant cancer. Furthermore, this review addresses the processes used by the ALDH-high persister cell subpopulation to enable cancer progression, the ALDH isoforms important in these processes, interactions of ALDH-high DTPs with the tumor microenvironment, and approaches to therapeutically modulate this subpopulation in order to more effectively manage cancer.

Role of aberrant Sonic hedgehog signaling pathway in cancers and developmental anomalies.

Development is a sophisticated process maintained by various signal transduction pathways, including the Hedgehog (Hh) pathway. Several important functions are executed by the Hh signaling cascade such as organogenesis, tissue regeneration, and tissue homeostasis, among various others. Considering the multiple functions carried out by this pathway, any mutation causing aberrant Hh signaling may lead to myriad developmental abnormalities besides cancers. In the present review article, we explored a wide range of diseases caused by aberrant Hh signaling, including developmental defects and cancers. Finally, we concluded this mini-review with various treatment strategies for Hh-induced diseases.

Mechanistic Basis for In Vivo Therapeutic Efficacy of CK2 Inhibitor CX-4945 in Acute Myeloid Leukemia.

Protein Kinase CK2 (Casein Kinase 2 or CK2) is a constitutively active serine-threonine kinase overactive in human malignancies. Increased expression and activity of CK2 in Acute Myeloid Leukemia (AML) is associated with a poor outcome. CK2 promotes AML cell survival by impinging on multiple oncogenic signaling pathways. The selective small-molecule CK2 inhibitor CX-4945 has shown in vitro cytotoxicity in AML. Here, we report that CX-4945 has a strong in vivo therapeutic effect in preclinical models of AML. The analysis of genome-wide DNA-binding and gene expression in CX-4945 treated AML cells shows that one mechanism, by which CK2 inhibition exerts a therapeutic effect in AML, involves the revival of IKAROS tumor suppressor function. CK2 phosphorylates IKAROS and disrupts IKAROS' transcriptional activity by impairing DNA-binding and association with chromatin modifiers. Here, we demonstrate that CK2 inhibition decreases IKAROS phosphorylation and restores IKAROS binding to DNA. Further functional experiments show that IKAROS negatively regulates the transcription of anti-apoptotic genes, including BCL-XL (B cell Lymphoma like-2 like 1, BCL2L1). CX-4945 restitutes the IKAROS-mediated repression of BCL-XL in vivo and sensitizes AML cells to apoptosis. Using CX-4945, alongside the cytotoxic chemotherapeutic drug daunorubicin, augments BCL-XL suppression and AML cell apoptosis. Overall, these results establish the in vivo therapeutic efficacy of CX-4945 in AML preclinical models and determine the role of CK2 and IKAROS in regulating apoptosis in AML. Furthermore, our study provides functional and mechanistic bases for the addition of CK2 inhibitors to AML therapy.

Dual targeting of MTOR as a novel therapeutic approach for high-risk B-cell acute lymphoblastic leukemia.

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.

The PI3K/AKT Pathway Inhibitor ISC-4 Induces Apoptosis and Inhibits Growth of Leukemia in Preclinical Models of Acute Myeloid Leukemia.

Acute myeloid leukemia is a heterogeneous disease with a 5-year survival rate of 28.3%, and current treatment options constrained by dose-limiting toxicities. One of the key signaling pathways known to be frequently activated and dysregulated in AML is PI3K/AKT. Its dysregulation is associated with aggressive cell growth and drug resistance. We investigated the activity of Phenybutyl isoselenocyanate (ISC-4) in primary cells obtained from newly diagnosed AML patients, diverse AML cell lines, and normal cord blood cells. ISC-4 significantly inhibited survival and clonogenicity of primary human AML cells without affecting normal cells. We demonstrated that ISC-4-mediated p-Akt inhibition caused apoptosis in primary AML (CD34+) stem cells and enhanced efficacy of cytarabine. ISC-4 impeded leukemia progression with improved overall survival in a syngeneic C1498 mouse model with no obvious toxic effects on normal myelopoiesis. In U937 xenograft model, bone marrow cells exhibited significant reduction in human CD45+ cells in ISC-4 (~87%) or AraC (~89%) monotherapy groups compared to control. Notably, combination treatment suppressed the leukemic infiltration significantly higher than the single-drug treatments (~94%). Together, the present findings suggest that ISC-4 might be a promising agent for AML treatment.

DYRK1B blocks canonical and promotes non-canonical Hedgehog signaling through activation of the mTOR/AKT pathway.

Hedgehog (Hh) signaling plays important roles in embryonic development and in tumor formation. Apart from the well-established stimulation of the GLI family of transcription factors, Hh ligands promote the phosphorylation and activation of mTOR and AKT kinases, yet the molecular mechanism underlying these processes are unknown. Here, we identify the DYRK1B kinase as a mediator between Hh signaling and mTOR/AKT activation. In fibroblasts, Hh signaling induces DYRK1B protein expression, resulting in activation of the mTOR/AKT kinase signaling arm. Furthermore, DYRK1B exerts positive and negative feedback regulation on the Hh pathway itself: It negatively interferes with SMO-elicited canonical Hh signaling, while at the same time it provides positive feed-forward functions by promoting AKT-mediated GLI stability. Due to the fact that the mTOR/AKT pathway is itself subject to strong negative feedback regulation, pharmacological inhibition of DYRK1B results in initial upregulation followed by downregulation of AKT phosphorylation and GLI stabilization. Addressing this issue therapeutically, we show that a pharmacological approach combining a DYRK1B antagonist with an mTOR/AKT inhibitor results in strong GLI1 targeting and in pronounced cytotoxicity in human pancreatic and ovarian cancer cells.

Histone deacetylase 6 represents a novel drug target in the oncogenic Hedgehog signaling pathway.

Uncontrolled Hedgehog (Hh) signaling is the cause of several malignancies, including the pediatric cancer medulloblastoma, a neuroectodermal tumor affecting the cerebellum. Despite the development of potent Hh pathway antagonists, medulloblastoma drug resistance is still an unresolved issue that requires the identification of novel drug targets. Following up on our observation that histone deacetylase 6 (HDAC6) expression was increased in Hh-driven medulloblastoma, we found that this enzyme is essential for full Hh pathway activation. Intriguingly, these stimulatory effects of HDAC6 are partly integrated downstream of primary cilia, a known HDAC6-regulated structure. In addition, HDAC6 is also required for the complete repression of basal Hh target gene expression. These contrasting effects are mediated by HDAC6's impact on Gli2 mRNA and GLI3 protein expression. As a result of this complex interaction with Hh signaling, global transcriptome analysis revealed that HDAC6 regulates only a subset of Smoothened- and Gli-driven genes, including all well-established Hh targets such as Ptch1 or Gli1. Importantly, medulloblastoma cell survival was severely compromised by HDAC6 inhibition in vitro and pharmacologic HDAC6 blockade strongly reduced tumor growth in an in vivo allograft model. In summary, our data describe an important role for HDAC6 in regulating the mammalian Hh pathway and encourage further studies focusing on HDAC6 as a novel drug target in medulloblastoma.