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.

Signaling pathways and regulation of gene expression in hematopoietic cells.

Cellular functions are regulated by signal transduction pathway networks consisting of protein-modifying enzymes that control the activity of many downstream proteins. Protein kinases and phosphatases regulate gene expression by reversible phosphorylation of transcriptional factors, which are their direct substrates. Casein kinase II (CK2) is a serine/threonine kinase that phosphorylates a large number of proteins that have critical roles in cellular proliferation, metabolism and survival. Altered function of CK2 has been associated with malignant transformation, immunological disorders and other types of diseases. Protein phosphatase 1 (PP1) is a serine/threonine phosphatase, which regulates the phosphorylation status of many proteins that are essential for cellular functions. IKAROS is a DNA-binding protein, which functions as a regulator of gene transcription in hematopoietic cells. CK2 directly phosphorylates IKAROS at multiple phosphosites which determines IKAROS activity as a regulator of gene expression. PP1 binds to IKAROS via the PP1-consensus recognition site and dephosphorylates serine/threonine residues that are phosphorylated by CK2. Thus, the interplay between CK2 and PP1 signaling pathways have opposing effects on the phosphorylation status of their mutual substrate - IKAROS. This review summarizes the effects of CK2 and PP1 on IKAROS role in regulation of gene expression and its function as a tumor suppressor in leukemia.

IGF2BP family of RNA-binding proteins regulate innate and adaptive immune responses in cancer cells and tumor microenvironment.

Insulin-like growth factor 2 mRNA-binding proteins (IGF2BP1, IGF2BP2, and IGF2BP3) are a family of RNA-binding proteins that play an essential role in the development and disease by regulating mRNA stability and translation of critical regulators of cell division and metabolism. Genetic and chemical inhibition of these proteins slows down cancer cell proliferation, decreases invasiveness, and prolongs life span in a variety of animal models. The role of RNA-binding proteins in the induction of tissues' immunogenicity is increasingly recognized, but, the impact of the IGF2BPs family of proteins on the induction of innate and adaptive immune responses in cancer is not fully understood. Here we report that downregulation of IGF2BP1, 2, and 3 expression facilitates the expression of interferon beta-stimulated genes. IGF2BP1 has a greater effect on interferon beta and gamma signaling compared to IGF2BP2 and IGF2BP3 paralogs. We demonstrate that knockdown or knockout of IGF2BP1, 2, and 3 significantly potentiates inhibition of cell growth induced by IFNß and IFNγ. Mouse melanoma cells with Igf2bp knockouts demonstrate increased expression of MHC I (H-2) and induce intracellular Ifn-γ expression in syngeneic T-lymphocytes in vitro. Increased immunogenicity, associated with Igf2bp1 inhibition, "inflames" mouse melanoma tumors microenvironment in SM1/C57BL/6 and SW1/C3H mouse models measured by a two-fold increase of NK cells and tumor-associated myeloid cells. Finally, we demonstrate that the efficiency of anti-PD1 immunotherapy in the mouse melanoma model is significantly more efficient in tumors that lack Igf2bp1 expression. Our retrospective data analysis of immunotherapies in human melanoma patients indicates that high levels of IGF2BP1 and IGF2BP3 are associated with resistance to immunotherapies and poor prognosis. In summary, our study provides evidence of the role of IGF2BP proteins in regulating tumor immunogenicity and establishes those RBPs as immunotherapeutic targets in cancer.

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.

Establishment and optimization of a hemp (Cannabis sativa L.) agroinfiltration system for gene expression and silencing studies.

Industrial hemp (Cannabis sativa L.) is a high-yielding annual crop primarily grown for fiber, seeds, and oil. Due to the phytochemical composition of hemp, there has been an increased interest in the market for nutraceuticals and dietary supplements for human health. Recent omics analysis has led to the elucidation of hemp candidate genes involved in the syntheses of specialized metabolites. However, a detailed study of these genes has not been undertaken due to the lack of a stable transformation system. We report for the first time an agroinfiltration system in hemp utilizing vacuum infiltration, which is an alternative method to stable transformation. A combination of 0.015% Silwett L-77, 5 mM ascorbic acid, and thirty second sonication followed by a 10-minute vacuum treatment resulted in the highest ß-glucuronidase expression in the leaf, male and female flowers, stem, and root tissues. The phytoene desaturase gene was silenced with a transient hairpin RNA expression, resulting in an albino phenotype in the leaves and the male and female flowers. This agroinfiltration system would be useful for overexpression and silencing studies of target genes to regulate the yield of specialized metabolites in hemp.

Enhanced tolerance of industrial hemp (Cannabis sativa L.) plants on abandoned mine land soil leads to overexpression of cannabinoids.

Industrial activities have a detrimental impact on the environment and health when high concentrations of pollutants are released. Phytoremediation is a natural method of utilizing plants to remove contaminants from the soil. The goal of this study was to investigate the ability of Cannabis sativa L. to sustainably grow and remediate abandoned coal mine land soils in Pennsylvania. In this study, six different varieties of industrial hemp (Fedora 17, Felina 32, Ferimon, Futura 75, Santhica 27, and USO 31) were grown on two different contaminated soil types and two commercial soils (Miracle-Gro Potting Mix and PRO-MIX HP Mycorrhizae High Porosity Grower Mix). Plants growing in all soil types were exposed to two environmental conditions (outside and in the greenhouse). Seed germination response and plant height indicated no significant differences among all hemp varieties grown in different soils, however on an average, the height of the plants grown in the greenhouse exceeded that of the plants grown outdoors. In addition, heavy metal analysis of Arsenic, Lead, Nickel, Mercury, and Cadmium was performed. The concentration of Nickel was 2.54 times greater in the leaves of hemp grown in mine land soil outdoors when compared to greenhouse conditions. No differences were found between expression of heavy metal transporter genes. Secondary metabolite analysis of floral buds from hemp grown in mine land soil displayed a significant increase in the total Cannabidiol content (2.16%, 2.58%) when compared to Miracle-Gro control soil (1.08%, 1.6%) for outdoors and in the greenhouse, respectively. Molecular analysis using qRT-PCR indicated an 18-fold increase in the expression of the cannabidiolic acid synthase gene in plants grown on mine land soil. The data indicates a high tolerance to heavy metals as indicated from the physiological and metabolites analysis.