In two independent ongoing next-generation sequencing projects for individuals with holoprosencephaly and individuals with disorders of sex development, and through international research ...collaboration, we identified twelve individuals with de novo loss-of-function (LoF) variants in protein phosphatase 1, regulatory subunit 12a (PPP1R12A), an important developmental gene involved in cell migration, adhesion, and morphogenesis. This gene has not been previously reported in association with human disease, and it has intolerance to LoF as illustrated by a very low observed-to-expected ratio of LoF variants in gnomAD. Of the twelve individuals, midline brain malformations were found in five, urogenital anomalies in nine, and a combination of both phenotypes in two. Other congenital anomalies identified included omphalocele, jejunal, and ileal atresia with aberrant mesenteric blood supply, and syndactyly. Six individuals had stop gain variants, five had a deletion or duplication resulting in a frameshift, and one had a canonical splice acceptor site loss. Murine and human in situ hybridization and immunostaining revealed PPP1R12A expression in the prosencephalic neural folds and protein localization in the lower urinary tract at critical periods for forebrain division and urogenital development. Based on these clinical and molecular findings, we propose the association of PPP1R12A pathogenic variants with a congenital malformations syndrome affecting the embryogenesis of the brain and genitourinary systems and including disorders of sex development.
IKAROS, encoded by the
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 (
) and phosphoinositide kinase, FYVE-type zinc finger containing (
), by IKAROS in T-ALL.
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.
The developing lens is a powerful system for investigating the molecular basis of inductive tissue interactions and for studying cataract, the leading cause of blindness. The formation of tightly ...controlled cell-cell adhesions and cell-matrix junctions between lens epithelial (LE) cells, between lens fiber (LF) cells, and between these two cell populations enables the vertebrate lens to adopt a highly ordered structure and acquire optical transparency. Adhesion molecules are thought to maintain this ordered structure, but little is known about their identity or interactions. Cysteine-rich motor neuron 1 (Crim1), a type I transmembrane protein, is strongly expressed in the developing lens and its mutation causes ocular disease in both mice and humans. How Crim1 regulates lens morphogenesis is not understood. We identified a novel ENU-induced hypomorphic allele of Crim1, Crim1(glcr11), which in the homozygous state causes cataract and microphthalmia. Using this and two other mutant alleles, Crim1(null) and Crim1(cko), we show that the lens defects in Crim1 mouse mutants originate from defective LE cell polarity, proliferation and cell adhesion. Crim1 adhesive function is likely to be required for interactions both between LE cells and between LE and LF cells. We show that Crim1 acts in LE cells, where it colocalizes with and regulates the levels of active β1 integrin and of phosphorylated FAK and ERK. The RGD and transmembrane motifs of Crim1 are required for regulating FAK phosphorylation. These results identify an important function for Crim1 in the regulation of integrin- and FAK-mediated LE cell adhesion during lens development.
Abstract
The utilization of next generation sequencing has been shown to accelerate gene discovery in human disease. However, our confidence in the correct disease-associations of rare variants ...continues to depend on functional analysis. Here, we employ a sensitive assay of human FGF8 variants in zebrafish to demonstrate that the spectrum of isoforms of FGF8 produced by alternative splicing can provide key insights into the genetic susceptibility to human malformations. In addition, we describe novel mutations in the FGF core structure that have both subtle and profound effects on ligand posttranslational processing and biological activity. Finally, we solve a case of apparent digenic inheritance of novel variants in SHH and FGF8, two genes known to functionally coregulate each other in the developing forebrain, as a simpler case of FGF8 diminished function.
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.
Abstract
Cellular proliferation in T-cell acute lymphoblastic leukemia is regulated by multiple signaling pathways. The Phosphoinositide 3-kinase (PI3K)/AKT pathway is frequently dysregulated in ...T-ALL. Targeting the PI3K pathway has shown promise as a novel therapeutic approach for T-ALL. However, regulation of the PI3K pathway is still not well understood. Here, we report that PI3K activity in T-ALL can be controlled by transcriptional regulation of key members of this pathway, PIK3CD and PIKFYVE. DNA binding analysis of primary T-ALL using qChIP revealed that the tumor suppressor protein, Ikaros, binds the promoter regions of PIK3CD and PIKFYVE. Since Ikaros acts as a regulator of transcription, we tested whether Ikaros binding to PIK3CD and PIKFYVE affects their expression. Overexpression of Ikaros results in reduced transcription of PIK3CD and PIKFYVE in T-ALL. Targeting Ikaros with a specific shRNA, resulted in increased transcription of PIK3CD and PIKFYVE in T-ALL. Together, these results demonstrate that Ikaros functions as a transcriptional repressor of both PIK3CD and PIKFYVE, and suggest that Ikaros can regulate the PI3K pathway in T-ALL. It has been previously shown that Ikaros function in B-cell acute lymphoblastic leukemia is regulated by oncogenic Casein Kinase II (CK2). We tested whether Ikaros ability to repress transcription of PIK3CD and PIKFYVE is regulated by CK2. Inhibition of CK2 by a specific pharmacological inhibitor, CX-4945, resulted in increased Ikaros binding to the promoters of PIK3CD and PIKFYVE, as well as in transcriptional repression of both of these genes. These results suggest that Ikaros function as a repressor of PIK3CD and PIKFYVE transcription is impaired by CK2 in T-ALL. CK2 inhibition restores Ikaros-mediated transcriptional repression of PIK3CD and PIKFYVE, which results in downregulation of the PI3K pathway. In conclusion, the presented data demonstrate that the PI3K signaling pathway is regulated by transcriptional repression of PIK3CD and PIKFYVE by Ikaros in T-ALL. Results reveal interplay between two signaling pathways in T-ALL, CK2 and PI3K, where CK2 positively regulates the PI3K pathway by inhibiting Ikaros function. These data reveal novel mechanisms that regulate cellular proliferation in T-ALL.
Citation Format: Tommy Hu, Mario Soliman, Malika Kapadia, Elanora Dovat, Jonathan Payne, Chunhua Song, Sinisa Dovat. Transcriptional control of signaling pathways in T-cell lymphoblastic leukemia by Ikaros tumor suppressor abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5540. doi:10.1158/1538-7445.AM2017-5540
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 T-ALL ...harbors inactivating mutations or deletion of one allele of the IKZF1 tumor suppressor. These data suggest that IKZF1 acts as a tumor suppressor in T-ALL. The IKZF1 gene encodes the Ikaros protein that functions as a regulator of transcription and a tumor suppressor in B cell acute lymphoblastic leukemia. However, the molecular mechanism of Ikaros tumor suppressor function in T-ALL is unclear. Using quantitative chromatin immunoprecipitation (qChIP), we determined that Ikaros binds to the promoter regions of the CDC2 and CDC7 cell cycle genes in primary T-ALL cells in vivo. Gain-of function experiments showed that Ikaros overexpression in T-ALL results in reduced expression of CDC2 and CDC7, as evidenced by quantitative RT-PCR (qRT-PCR) and Western blot. The knock-down of Ikaros with shRNA in T-ALL cells resulted in increased transcription of CDC2 and CDC7 as indicated by qRT-PCR. These data suggest that Ikaros can regulate cell cycle progression in T-ALL by repressing transcription of the CDC2 and CDC7 genes. Next, we studied the mechanisms that regulate Ikaros’ ability to repress CDC2 and CDC7 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 CDC2 and CDC7 in human T-ALL. Molecular inhibition of CK2 with shRNA against the CK2 catalytic subunit resulted in reduced transcription of CDC2 and CDC7, as evidenced by qRT-PCR. This was associated with increased DNA-binding of Ikaros to promoters of CDC2 and CDC7, as shown by qChIP. These data suggest that CK2 impairs Ikaros’ ability to transcriptionally repress CDC2 and CDC7 and to regulate cell cycle progression in T-ALL. Inhibition of CK2 enhances transcriptional repression of CDC2 and CDC7 by Ikaros, resulting in improved control of cell cycle progression in T-ALL. In conclusion, our results show that control of cell cycle progression in T-ALL occurs trough Ikaros-mediated transcriptional regulation of CDC2 and CDC7. Overexpession of CK2 impairs Ikaros ability to repress CDC2 and CDC7 expression, which contributes to deregulation of cell cycle control in T-ALL. Results suggest a potential mechanism of therapeutic action of CK2 inhibitors for the treatment of T-ALL.
Note: This abstract was not presented at the meeting.
Citation Format: Mario A. Soliman, Tommy Hu, Malika Kapadia, Elanora Dovat, Yali Ding, Chunhua Song, Jonathon L. Payne, Sinisa Dovat. Regulation of cell cycle control in T-cell acute lymphoblastic leukemia by Ikaros and Casein Kinase II abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5542. doi:10.1158/1538-7445.AM2017-5542
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.
Cornelia de Lange syndrome in diverse populations Dowsett, Leah; Porras, Antonio R.; Kruszka, Paul ...
American journal of medical genetics. Part A,
February 2019, Letnik:
179, Številka:
2
Journal Article
Recenzirano
Odprti dostop
Cornelia de Lange syndrome (CdLS) is a dominant multisystemic malformation syndrome due to mutations in five genes—NIPBL, SMC1A, HDAC8, SMC3, and RAD21. The characteristic facial dysmorphisms include ...microcephaly, arched eyebrows, synophrys, short nose with depressed bridge and anteverted nares, long philtrum, thin lips, micrognathia, and hypertrichosis. Most affected individuals have intellectual disability, growth deficiency, and upper limb anomalies. This study looked at individuals from diverse populations with both clinical and molecularly confirmed diagnoses of CdLS by facial analysis technology. Clinical data and images from 246 individuals with CdLS were obtained from 15 countries. This cohort included 49% female patients and ages ranged from infancy to 37 years. Individuals were grouped into ancestry categories of African descent, Asian, Latin American, Middle Eastern, and Caucasian. Across these populations, 14 features showed a statistically significant difference. The most common facial features found in all ancestry groups included synophrys, short nose with anteverted nares, and a long philtrum with thin vermillion of the upper lip. Using facial analysis technology we compared 246 individuals with CdLS to 246 gender/age matched controls and found that sensitivity was equal or greater than 95% for all groups. Specificity was equal or greater than 91%. In conclusion, we present consistent clinical findings from global populations with CdLS while demonstrating how facial analysis technology can be a tool to support accurate diagnoses in the clinical setting. This work, along with prior studies in this arena, will assist in earlier detection, recognition, and treatment of CdLS worldwide.
PDF
