Kimberly J. Payne

IL-7 Dependence in human B lymphopoiesis increases during progression of ontogeny from cord blood to bone marrow.

IL-7 is critical for B cell production in adult mice; however, its role in human B lymphopoiesis is controversial. One challenge was the inability to differentiate human cord blood (CB) or adult bone marrow (BM) hematopoietic stem cells (HSCs) without murine stroma. Here, we examine the role of IL-7 in human B cell development using a novel, human-only model based on coculturing human HSCs on primary human BM stroma. In this model, IL-7 increases human B cell production by >60-fold from both CB and adult BM HSCs. IL-7-induced increases are dose-dependent and specific to CD19+ cells. STAT5 phosphorylation and expression of the Ki-67 proliferation Ag indicate that IL-7 acts directly on CD19+ cells to increase proliferation at the CD34+ and CD34− pro-B cell stages. Without IL-7, HSCs in CB, but not BM, give rise to a small but consistent population of CD19lo B lineage cells that express EBF (early B cell factor) and PAX-5 and respond to subsequent IL-7 stimulation. Flt3 ligand, but not thymic stromal-derived lymhopoietin (TSLP), was required for the IL-7-independent production of human B lineage cells. As compared with CB, adult BM shows a reduction of in vitro generative capacity that is progressively more profound in developmentally sequential populations, resulting in an ∼50-fold reduction in IL-7-dependent B lineage generative capacity. These data provide evidence that IL-7 is essential for human B cell production from adult BM and that IL-7-induced expansion of the pro-B compartment is increasingly critical for human B cell production during the progression of ontogeny.

Efficient Reprogramming of Human Cord Blood CD34+ Cells Into Induced Pluripotent Stem Cells With OCT4 and SOX2 Alone

The reprogramming of cord blood (CB) cells into induced pluripotent stem cells (iPSCs) has potential applications in regenerative medicine by converting CB banks into iPSC banks for allogeneic cell replacement therapy. Therefore, further investigation into novel approaches for efficient reprogramming is necessary. Here, we show that the lentiviral expression of OCT4 together with SOX2 (OS) driven by a strong spleen focus-forming virus (SFFV) promoter in a single vector can convert 2% of CB CD34+ cells into iPSCs without additional reprogramming factors. Reprogramming efficiency was found to be critically dependent upon expression levels of OS. To generate transgene-free iPSCs, we developed an improved episomal vector with a woodchuck post-transcriptional regulatory element (Wpre) that increases transgene expression by 50%. With this vector, we successfully generated transgene-free iPSCs using OS alone. In conclusion, high-level expression of OS alone is sufficient for efficient reprogramming of CB CD34+ cells into iPSCs. This report is the first to describe the generation of transgene-free iPSCs with the use of OCT4 and SOX2 alone. These findings have important implications for the clinical applications of iPSCs.The reprogramming of cord blood (CB) cells into induced pluripotent stem cells (iPSCs) has potential applications in regenerative medicine by converting CB banks into iPSC banks for allogeneic cell replacement therapy. Therefore, further investigation into novel approaches for efficient reprogramming is necessary. Here, we show that the lentiviral expression of OCT4 together with SOX2 (OS) driven by a strong spleen focus-forming virus (SFFV) promoter in a single vector can convert 2% of CB CD34(+) cells into iPSCs without additional reprogramming factors. Reprogramming efficiency was found to be critically dependent upon expression levels of OS. To generate transgene-free iPSCs, we developed an improved episomal vector with a woodchuck post-transcriptional regulatory element (Wpre) that increases transgene expression by 50%. With this vector, we successfully generated transgene-free iPSCs using OS alone. In conclusion, high-level expression of OS alone is sufficient for efficient reprogramming of CB CD34(+) cells into iPSCs. This report is the first to describe the generation of transgene-free iPSCs with the use of OCT4 and SOX2 alone. These findings have important implications for the clinical applications of iPSCs.

Ikaros Stability and Pericentromeric Localization Are Regulated by Protein Phosphatase 1

Ikaros encodes a zinc finger protein that is involved in gene regulation and chromatin remodeling. The majority of Ikaros localizes at pericentromeric heterochromatin (PC-HC) where it regulates expression of target genes. Ikaros function is controlled by posttranslational modification. Phosphorylation of Ikaros by CK2 kinase determines its ability to bind DNA and exert cell cycle control as well as its subcellular localization. We report that Ikaros interacts with protein phosphatase 1 (PP1) via a conserved PP1 binding motif, RVXF, in the C-terminal end of the Ikaros protein. Point mutations of the RVXF motif abolish Ikaros-PP1 interaction and result in decreased DNA binding, an inability to localize to PC-HC, and rapid degradation of the Ikaros protein. The introduction of alanine mutations at CK2-phosphorylated residues increases the half-life of the PP1-nonbinding Ikaros mutant. This suggests that dephosphorylation of these sites by PP1 stabilizes the Ikaros protein and prevents its degradation. In the nucleus, Ikaros forms complexes with ubiquitin, providing evidence that Ikaros degradation involves the ubiquitin/proteasome pathway. In vivo, Ikaros can target PP1 to the nucleus, and a fraction of PP1 colocalizes with Ikaros at PC-HC. These data suggest a novel function for the Ikaros protein; that is, the targeting of PP1 to PC-HC and other chromatin structures. We propose a model whereby the function of Ikaros is controlled by the CK2 and PP1 pathways and that a balance between these two signal transduction pathways is essential for normal cellular function and for the prevention of malignant transformation.

Efficient Generation of Integration-Free iPS Cells from Human Adult Peripheral Blood Using BCL-XL Together with Yamanaka Factors

The ability to efficiently generate integration-free induced pluripotent stem cells (iPSCs) from the most readily available source—peripheral blood—has the potential to expedite the advances of iPSC-based therapies. We have successfully generated integration-free iPSCs from cord blood (CB) CD34+ cells with improved oriP/EBNA1-based episomal vectors (EV) using a strong spleen focus forming virus (SFFV) long terminal repeat (LTR) promoter. Here we show that Yamanaka factors (OCT4, SOX2, MYC, and KLF4)-expressing EV can also reprogram adult peripheral blood mononuclear cells (PBMNCs) into pluripotency, yet at a very low efficiency. We found that inclusion of BCL-XL increases the reprogramming efficiency by approximately 10-fold. Furthermore, culture of CD3−/CD19− cells or T/B cell-depleted MNCs for 4–6 days led to the generation of 20–30 iPSC colonies from 1 ml PB, an efficiency that is substantially higher than previously reported. PB iPSCs express pluripotency markers, form teratomas, and can be induced to differentiate in vitro into mesenchymal stem cells, cardiomyocytes, and hepatocytes. Used together, our optimized factor combination and reprogramming strategy lead to efficient generation of integration-free iPSCs from adult PB. This discovery has potential applications in iPSC banking, disease modeling and regenerative medicine.

Recruitment of Ikaros to Pericentromeric Heterochromatin Is Regulated by Phosphorylation

Ikaros encodes a zinc finger protein that is involved in heritable gene silencing. In hematopoietic cells, Ikaros localizes to pericentromeric heterochromatin (PC-HC) where it recruits its target genes, resulting in their activation or repression via chromatin remodeling. The function of Ikaros is controlled by post-translational modifications. CK2 kinase has been shown to phosphorylate Ikaros at its C terminus, affecting cell cycle progression. Using in vivo labeling of murine thymocytes followed by phosphopeptide mapping, we identified four novel Ikaros phosphorylation sites. Functional analysis of phosphomimetic mutants showed that the phosphorylation of individual amino acids determines the affinity of Ikaros toward probes derived from PC-HC. In vivo experiments demonstrated that targeting of Ikaros to PC-HC is regulated by phosphorylation. The ability of Ikaros to bind the upstream regulatory elements of its known target gene terminal deoxynucleotidyltransferase (TdT) was decreased by phosphorylation of two amino acids. In thymocytes, Ikaros acts as a repressor of the TdT gene. Induction of differentiation of thymocytes with phorbol 12-myristate 13-acetate plus ionomycin results in transcriptional repression of TdT expression. This process has been associated with increased binding of Ikaros to the upstream regulatory element of TdT. Phosphopeptide analysis of in vivo-labeled thymocytes revealed that Ikaros undergoes dephosphorylation during induction of thymocyte differentiation and that dephosphorylation is responsible for increased DNA binding affinity of Ikaros toward the TdT promoter. We propose a model whereby reversible phosphorylation of Ikaros at specific amino acids controls the subcellular localization of Ikaros as well as its ability to regulate TdT expression during thymocyte differentiation.

Human Ikaros Function in Activated T Cells Is Regulated by Coordinated Expression of Its Largest Isoforms

The Ikaros gene is alternately spliced to generate multiple zinc finger proteins involved in gene regulation and chromatin remodeling. Whereas murine studies have provided important information regarding the role of Ikaros in the mouse, little is known of Ikaros function in human. We report functional analyses of the two largest human Ikaros (hIK) isoforms, hIK-VI and hIK-H, in T cells. Abundant expression of hIK-H, the largest described isoform, is restricted to human hematopoietic cells. We find that the DNA binding affinity of hIK-H differs from that of hIK-VI. Co-expression of hIk-H with hIk-VI alters the ability of Ikaros complexes to bind DNA motifs found in pericentromeric heterochromatin (PC-HC). In the nucleus, hIK-VI is localized solely in PC-HC, whereas the hIK-H protein exhibits dual centromeric and non-centromeric localization. Mutational analysis defined the amino acids responsible for the distinct DNA binding ability of hIK-H, as well as the sequence required for the specific subcellular localization of this isoform. In proliferating cells, the binding of hIK-H to the upstream regulatory region of known Ikaros target genes correlates with their positive regulation by Ikaros. Results suggest that expression of hIK-H protein restricts affinity of Ikaros protein complexes toward specific PC-HC repeats. We propose a model, whereby the binding of hIK-H-deficient Ikaros complexes to the regulatory sequence of target genes would recruit these genes to the restrictive pericentromeric compartment, resulting in their repression. The presence of hIK-H in the Ikaros complex would alter its affinity for PC-HC, leading to chromatin remodeling and activation of target genes.

Human Papillomavirus Type 16 E6* Induces Oxidative Stress and DNA Damage

ABSTRACT High-risk types of human papillomavirus (HPV) are the causative agents of virtually all cases of cervical cancer and a significant proportion of other anogenital cancers, as well as both oral and pharyngeal cancers. The high-risk types encode two viral oncogenes, E6 and E7, which work together to initiate cell transformation. Multiple steps involving the activities and interactions of both viral and cellular proteins are involved in the progression from HPV infection to cell transformation to cancer. The E6 oncoprotein is expressed as several isoforms: a full-length variant referred to as E6 and a few shorter isoforms collectively referred to as E6*. In this study, we found that expression of E6* increased the level of reactive oxygen species (ROS) in both HPV-positive and HPV-negative cells. This increased oxidative stress led to higher levels of DNA damage, as assessed by the comet assay, quantification of 8-oxoguanine, and poly(ADP-ribose) polymerase 1. The observed increase in ROS may be due to a decrease in cellular antioxidant activity, as we found that E6* expression also led to decreased expression of superoxide dismutase isoform 2 and glutathione peroxidase. These studies indicate that E6* may play an important role in virus-induced mutagenesis by increasing oxidative stress and DNA damage. IMPORTANCE Our findings demonstrate for the first time that an HPV gene product, E6*, can increase ROS levels in host cells. This ability may play a significant role both in the viral life cycle and in cancer development, because an increase in oxidative DNA damage may both facilitate HPV genome amplification and increase the probability of HPV16 DNA integration. Integration, in turn, is thought to be an important step in HPV-mediated carcinogenesis.

Ikaros, CK2 kinase, and the road to leukemia

Ikaros encodes a zinc finger protein that is essential for hematopoiesis and that acts as a tumor suppressor in leukemia. Ikaros function depends on its ability to localize to pericentromeric-heterochromatin (PC-HC). Ikaros protein binds to the upstream regulatory elements of target genes, aids in their recruitment to PC-HC, and regulates their transcription via chromatin remodeling. We identified four novel Ikaros phosphorylation sites that are phosphorylated by CK2 kinase. Using Ikaros phosphomimetic and phosphoresistant mutants of the CK2 phosphorylation sites, we demonstrate that (1) CK2-mediated phosphorylation inhibits Ikaros’ localization to PC-HC; (2) dephosphorylation of Ikaros at CK2 sites increases its binding to the promoter of the terminal deoxynucleotidetransferase (TdT) gene, leading to TdT repression during thymocyte differentiation; and (3) hyperphosphorylation of Ikaros promotes its degradation by the ubiquitin/proteasome pathway. We show that Ikaros is dephosphorylated by Protein Phosphatase 1 (PP1) via interaction at a consensus PP1-binding motif, RVXF. Point mutations that abolish Ikaros-PP1 interaction result in functional changes in DNA-binding affinity and subcellular localization, similar to those observed in hyperphosphorylated Ikaros and/or Ikaros phosphomimetic mutants. Phosphoresistant Ikaros mutations at CK2 sites restored Ikaros’ DNA-binding activity and localization to PC-HC and prevented accelerated Ikaros degradation. These results demonstrate the role of CK2 kinase in lymphocyte differentiation and in regulation of Ikaros’ function, and suggest that CK2 promotes leukemogenesis by inhibiting the tumor suppressor activity of Ikaros. We propose a model whereby a balance between CK2 kinase and PP1 phosphatase is essential for normal lymphocyte differentiation and for the prevention of malignant transformation.

Residency and Activation of Myeloid Cells During Remodeling of the Prepartum Murine Cervix

ABSTRACT Remodeling of the cervix is a critical early component of parturition and resembles an inflammatory process. Infiltration and activation of myeloid immune cells along with production of proinflammatory mediators and proteolytic enzymes are hypothesized to regulate cervical remodeling as pregnancy nears term. The present study standardized an approach to assess resident populations of immune cells and phenotypic markers of functional activities related to the mechanism of extracellular matrix degradation in the cervix in preparation for birth. Analysis of cells from the dispersed cervix of mice that were nonpregnant or pregnant (Days 15 and 18 postbreeding) by multicolor flow cytometry indicated increased total cell numbers with pregnancy as well as increased numbers of macrophages, the predominant myeloid cell, by Day 18, the day before birth. The number of activated macrophages involved in matrix metalloproteinase induction (CD147) and signaling for matrix adhesion (CD169) significantly increased by the day before birth. Expression of the adhesion markers CD54 and CD11b by macrophages decreased in the cervix by Day 18 versus that on Day 15 or in nonpregnant mice. The census of cells that expressed the migration marker CD62L was unaffected by pregnancy. The data suggest that remodeling of the cervix at term in mice is associated with recruitment and selective activation of macrophages that promote extracellular matrix degradation. Indices of immigration and activities by macrophages may thus serve as markers for local immune cell activity that is critical for ripening of the cervix in the final common mechanism for parturition at term.

Targeting casein kinase II restores Ikaros tumor suppressor activity and demonstrates therapeutic efficacy in high-risk leukemia

Ikaros (IKZF1) is a tumor suppressor that binds DNA and regulates expression of its target genes. The mechanism of Ikaros activity as a tumor suppressor and the regulation of Ikaros function in leukemia are unknown. Here, we demonstrate that Ikaros controls cellular proliferation by repressing expression of genes that promote cell cycle progression and the phosphatidylinositol-3 kinase (PI3K) pathway. We show that Ikaros function is impaired by the pro-oncogenic casein kinase II (CK2), and that CK2 is overexpressed in leukemia. CK2 inhibition restores Ikaros function as transcriptional repressor of cell cycle and PI3K pathway genes, resulting in an antileukemia effect. In high-risk leukemia where one IKZF1 allele has been deleted, CK2 inhibition restores the transcriptional repressor function of the remaining wild-type IKZF1 allele. CK2 inhibition demonstrated a potent therapeutic effect in a panel of patient-derived primary high-risk B-cell acute lymphoblastic leukemia xenografts as indicated by prolonged survival and a reduction of leukemia burden. We demonstrate the efficacy of a novel therapeutic approach for high-risk leukemia: restoration of Ikaros tumor suppressor activity via inhibition of CK2. These results provide a rationale for the use of CK2 inhibitors in clinical trials for high-risk leukemia, including cases with deletion of one IKZF1 allele.