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.

TSLP as a Potential Therapy in the Treatment of CRLF2 B Cell Acute Lymphoblastic Leukemia.

Cytokine receptor-like factor 2 B-cell acute lymphoblastic leukemia (CRLF2 B-ALL) is a high-risk subtype characterized by CRLF2 overexpression with poor survival rates in children and adults. CRLF2 and interleukin-7 receptor alpha (IL-7Rα) form a receptor for the cytokine thymic stromal lymphopoietin (TSLP), which induces JAK/STAT and PI3K/AKT/mTOR pathway signals. Previous studies from our group showed that low TSLP doses increased STAT5, AKT, and S6 phosphorylation and contributed to CRLF2 B-ALL cell survival. Here we investigated the role of TSLP in the survival and proliferation of CRLF2 B-ALL cells in vitro and in vivo. We hypothesized that high doses of TSLP increase CRLF2 signals and contribute to increased proliferation of CRLF2 B-ALL cells in vitro and in vivo. Interestingly, we observed the opposite effect. Specifically, high doses of TSLP induced apoptosis in human CRLF2 B-ALL cell lines in vitro, prevented engraftment of CRLF2 B-ALL cells, and prolonged the survival of +TSLP patient-derived-xenograft mice. Mechanistically, we showed that high doses of TSLP induced loss of its receptor and loss of CRLF2 signals in vitro. These results suggest that high doses of TSLP could be further investigated as a potential therapy for the treatment of CRLF2 B-ALL.

The Current Genomic and Molecular Landscape of Philadelphia-like Acute Lymphoblastic Leukemia.

Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) is a high-risk B-cell Acute Lymphoblastic Leukemia (B-ALL) characterized by a gene expression profile similar to Ph-positive B-ALL but lacking the BCR-ABL1 translocation. The molecular pathogenesis of Ph-like B-ALL is heterogenous and involves aberrant genomics, receptor overexpression, kinase fusions, and mutations leading to kinase signaling activation, leukemogenic cellular proliferation, and differentiation blockade. Testing for the Ph-like signature, once only a research technique, is now available to the clinical oncologist. The plethora of data pointing to poor outcomes for this ALL subset has triggered investigations into the role of targeted therapies, predominantly involving tyrosine kinase inhibitors that are showing promising results.

Transcriptional Regulation of JARID1B/KDM5B Histone Demethylase by Ikaros, Histone Deacetylase 1 (HDAC1), and Casein Kinase 2 (CK2) in B-cell Acute Lymphoblastic Leukemia.

Impaired function of the Ikaros (IKZF1) protein is associated with the development of high-risk B-cell precursor acute lymphoblastic leukemia (B-ALL). The mechanisms of Ikaros tumor suppressor activity in leukemia are unknown. Ikaros binds to the upstream regulatory elements of its target genes and regulates their transcription via chromatin remodeling. Here, we report that Ikaros represses transcription of the histone H3K4 demethylase, JARID1B (KDM5B). Transcriptional repression of JARID1B is associated with increased global levels of H3K4 trimethylation. Ikaros-mediated repression of JARID1B is dependent on the activity of the histone deacetylase, HDAC1, which binds to the upstream regulatory element of JARID1B in complex with Ikaros. In leukemia, JARID1B is overexpressed, and its inhibition results in cellular growth arrest. Ikaros-mediated repression of JARID1B in leukemia is impaired by pro-oncogenic casein kinase 2 (CK2). Inhibition of CK2 results in increased binding of the Ikaros-HDAC1 complex to the promoter of JARID1B, with increased formation of trimethylated histone H3 lysine 27 and decreased histone H3 Lys-9 acetylation. In cases of high-risk B-ALL that carry deletion of one Ikaros (IKZF1) allele, targeted inhibition of CK2 restores Ikaros binding to the JARID1B promoter and repression of JARID1B. In summary, the presented data suggest a mechanism through which Ikaros and HDAC1 regulate the epigenetic signature in leukemia: via regulation of JARID1B transcription. The presented data identify JARID1B as a novel therapeutic target in B-ALL and provide a rationale for the use of CK2 inhibitors in the treatment of high-risk B-ALL.

TSLP or IL-7 provide an IL-7Rα signal that is critical for human B lymphopoiesis.

Thymic stromal lymphopoietin (TSLP) and IL-7 are cytokines that signal via the IL-7 receptor alpha (IL-7Rα) to exert both overlapping and unique functions during early stages of mouse B-cell development. In human B lymphopoiesis, the requirement for IL-7Rα signaling is controversial and the roles of IL-7 and TSLP are less clear. Here, we evaluated human B-cell production using novel in vitro and xenograft models of human B-cell development that provide selective IL-7 and human TSLP (hTSLP) stimulation. We show that in vitro human B-cell production is almost completely blocked in the absence of IL-7Rα stimulation, and that either TSLP or IL-7 can provide a signal critical for the production and proliferation of human CD19(+) PAX5(+) pro-B cells. Analysis of primary human bone marrow stromal cells shows that they express both IL-7 and TSLP, providing an in vivo source of these cytokines. We further show that the in vivo production of human pro-B cells under the influence of mouse IL-7 in a xenograft scenario is reduced by anti-IL-7 neutralizing antibodies, and that this loss can be restored by hTSLP at physiological levels. These data establish the importance of IL-7Rα mediated signals for normal human B-cell production.

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.

Cutting Edge: Hematopoietic Stem Cell Expansion and Common Lymphoid Progenitor Depletion Require Hematopoietic-Derived, Cell-Autonomous TLR4 in a Model of Chronic Endotoxin.

Hematopoietic stem and progenitors cells (HSPCs) are activated through TLR4 in vitro. However, it remains unclear whether in vivo TLR4 sensing by HSPCs occurs directly or via other cell intermediates. In this study, we examined the cellular mechanisms underlying murine hematopoietic stem cell (HSC) expansion and common lymphoid progenitor (CLP) depletion in a model of chronic low-dose LPS. Using adoptive-transfer approaches, we show that HSC and CLP sensitivity to chronic LPS depends on hematopoietic-derived, cell subset-autonomous TLR4. Like murine progenitors, human HSPCs are activated by TLR4 in vitro. Using humanized mice, a preclinical model relevant to human physiology, we show that persistent endotoxin increases the frequency of Ki-67(+) HSCs and severely depletes CLPs and B precursors. Together, our findings show that murine HSPCs directly respond to endotoxin in vivo and that persistent LPS, a feature of several diseases of global health significance, impairs human lymphopoiesis.

A novel xenograft model to study the role of TSLP-induced CRLF2 signals in normal and malignant human B lymphopoiesis.

Thymic stromal lymphopoietin (TSLP) stimulates in-vitro proliferation of human fetal B-cell precursors. However, its in-vivo role during normal human B lymphopoiesis is unknown. Genetic alterations that cause overexpression of its receptor component, cytokine receptor-like factor 2 (CRLF2), lead to high-risk B-cell acute lymphoblastic leukemia implicating this signaling pathway in leukemogenesis. We show that mouse thymic stromal lymphopoietin does not stimulate the downstream pathways (JAK/STAT5 and PI3K/AKT/mTOR) activated by the human cytokine in primary high-risk leukemia with overexpression of the receptor component. Thus, the utility of classic patient-derived xenografts for in-vivo studies of this pathway is limited. We engineered xenograft mice to produce human thymic stromal lymphopoietin (+T mice) by injection with stromal cells transduced to express the cytokine. Control (-T) mice were produced using stroma transduced with control vector. Normal levels of human thymic stromal lymphopoietin were achieved in sera of +T mice, but were undetectable in -T mice. Patient-derived xenografts generated from +T as compared to -T mice showed a 3-6-fold increase in normal human B-cell precursors that was maintained through later stages of B-cell development. Gene expression profiles in high-risk B-cell acute lymphoblastic leukemia expanded in +T mice indicate increased mTOR pathway activation and are more similar to the original patient sample than those from -T mice. +T/-T xenografts provide a novel pre-clinical model for understanding this pathway in B lymphopoiesis and identifying treatments for high-risk B-cell acute lymphoblastic leukemia with overexpression of cytokine-like factor receptor 2.

Differences in mouse and human nonmemory B cell pools.

Identifying cross-species similarities and differences in immune development and function is critical for maximizing the translational potential of animal models. Coexpression of CD21 and CD24 distinguishes transitional and mature B cell subsets in mice. In this study, we validate these markers for identifying analogous subsets in humans and use them to compare the nonmemory B cell pools in mice and humans, across tissues, and during fetal/neonatal and adult life. Among human CD19(+)IgM(+) B cells, the CD21/CD24 schema identifies distinct populations that correspond to transitional 1 (T1), transitional 2 (T2), follicular mature, and marginal zone subsets identified in mice. Markers specific to human B cell development validate the identity of marginal zone cells and the maturation status of human CD21/CD24 nonmemory B cell subsets. A comparison of the nonmemory B cell pools in bone marrow, blood, and spleen in mice and humans shows that transitional B cells comprise a much smaller fraction in adult humans than mice. T1 cells are a major contributor to the nonmemory B cell pool in mouse bone marrow, in which their frequency is more than twice that in humans. Conversely, in spleen, the T1:T2 ratio shows that T2 cells are proportionally ∼ 8-fold higher in humans than in mice. Despite the relatively small contribution of transitional B cells to the human nonmemory pool, the number of naive follicular mature cells produced per transitional B cell is 3- to 6-fold higher across tissues than in mice. These data suggest differing dynamics or mechanisms produce the nonmemory B cell compartments in mice and humans.

Human papillomavirus type 16 E6* induces oxidative stress and DNA damage.

UNLABELLED: 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.

Protein phosphatase 1 (PP1) and Casein Kinase II (CK2) regulate Ikaros-mediated repression of TdT in thymocytes and T-cell leukemia.

BACKGROUND: Ikaros is a DNA-binding protein that acts as master-regulator of hematopoiesis and a tumor suppressor. In thymocytes and T-cell leukemia, Ikaros negatively regulates transcription of terminal deoxynucleotide transferase (TdT), a key protein in lymphocyte differentiation. The signaling pathways that regulate Ikaros-mediated repression of TdT are unknown. Our previous work identified Casein Kinase II (CK2) and Protein Phosphatase 1 (PP1) as regulators of Ikaros DNA binding activity. Here, we investigated the role of PP1 and CK2 in regulating Ikaros-mediated control of TdT expression. PROCEDURES: Ikaros phosphomimetic and phosphoresistant mutants and specific CK2 and PP1 inhibitors were used in combination with quantitative chromatin immunoprecipitation (qChIP) and quantitative reverse transcriptase-PCR (q RT-PCR) assays to evaluate the role of CK2 and PP1 in regulating the ability of Ikaros to bind the TdT promoter and to regulate TdT expression. RESULTS: We demonstrate that phosphorylation of Ikaros by pro-oncogenic CK2 decreases Ikaros binding to the promoter of the TdT gene and reduces the ability of Ikaros to repress TdT expression during thymocyte differentiation. CK2 inhibition and PP1 activity restore Ikaros DNA-binding affinity toward the TdT promoter, as well as Ikaros-mediated transcriptional repression of TdT in primary thymocytes and in leukemia. CONCLUSION: These data establish that PP1 and CK2 signal transduction pathways regulate Ikaros-mediated repression of TdT in thymocytes and leukemia. These findings reveal that PP1 and CK2 have opposing effects on Ikaros-mediated repression of TdT and establish novel roles for PP1 and CK2 signaling in thymocyte differentiation and leukemia.

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.

Residency and activation of myeloid cells during remodeling of the prepartum murine cervix.

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.

Cell cycle-specific function of Ikaros in human leukemia.

BACKGROUND: The loss of Ikaros is associated with the development of B and T cell leukemia. Data on Ikaros function, including its role as a tumor suppressor and a regulator of cell cycle progression, come almost exclusively from murine studies; little is known of the mechanisms that regulate human Ikaros function. Our studies are the first to examine the function and regulation of human Ikaros isoforms during the cell cycle in human ALL. PROCEDURES: Electromobility shift assay (EMSA), confocal microscopy, and phosphopeptide mapping were used to study Ikaros function during different stages of the cell cycle. RESULTS: The DNA-binding activity of human Ikaros complexes undergoes dynamic changes as the cell cycle progresses. In S phase, Ikaros DNA-binding affinity for regulatory regions of its target genes decreases, while its binding to pericentromeric heterochromatin is preserved and correlates with Ikaros pericentromeric localization. These S phase-specific changes in Ikaros function are controlled by phosphorylation via the CK2 kinase pathway. During cell cycle progression, the subcellular pericentromeric localization of the largest human Ikaros isoforms is different from that in mouse cells, suggesting unique functions for human Ikaros. CONCLUSIONS: Our results demonstrate that the function of Ikaros is cell cycle-specific and controlled by CK2-mediated phosphorylation during S phase of the cell cycle in human T-cell and B-cell ALL. The differences we observe in murine and human Ikaros function highlight the importance of using human cells in studies of ALL. These data identify the CK2 pathway as a target for therapies in ALL.

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.

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 CD19(lo) 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 approximately 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.

Platelet and neutrophil responses to Porphyromonas gingivalis in human whole blood.

Porphyromonas gingivalis is a causative agent for periodontal disease. Binding of platelets to this gram-negative anaerobe can regulate host hemostatic (thrombus forming) and immune (neutrophil interacting) responses during bacterial infection. Additionally, in response to bacterial pathogens neutrophils can release their DNA, forming highly prothrombotic neutrophil extracellular traps (NETs), which then further enhance platelet responses. This study evaluates the role of P. gingivalis on platelet expression of CD62P, platelet-neutrophil interactions, and labeled neutrophil-associated DNA. Human whole blood was preincubated with varying P. gingivalis concentrations, with or without subsequent addition of adenosine diphosphate (ADP). Flow cytometry was employed to measure platelet expression of CD62P using PerCP-anti-CD61 and PE-anti-CD62P, platelet-neutrophil interactions using PerCP-anti-CD61 and FITC-anti-CD16, and the release of neutrophil DNA using FITC-anti-CD16 and Sytox Blue labeling. Preincubation with a high (6.25 × 106  CFU/mL) level of P. gingivalis significantly increased platelet expression of CD62P in ADP treated and untreated whole blood. In addition, platelet-neutrophil interactions were significantly increased after ADP stimulation, following 5-22 min preincubation of blood with high P. gingivalis CFU. However, in the absence of added ADP, platelet-neutrophil interactions increased in a manner dependent on the preincubation time with P. gingivalis. Moreover, after ADP addition, 16 min preincubation of whole blood with P. gingivalis led to increased labeling of neutrophil-associated DNA. Taken together, the results suggest that the presence of P. gingivalis alters platelet and neutrophil responses to increase platelet activation, platelet interactions with neutrophils, and the level of neutrophil antimicrobial NETs.

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.

Platelet-Neutrophil Interactions Are Lower in Cord Blood of Premature Newborns.

OBJECTIVE: To quantify platelet-neutrophil interaction by flow cytometry, in newborn cord blood, as a function of gestational age. RATIONALE: Little is known about platelet function markers in the newborn, and developmental variations in these markers are not well described. METHODS: Cord blood samples were obtained from 64 newborns between 23 and 40 weeks' gestation. The neonates were grouped into three categories: preterm (< 34 weeks' gestation, n = 21), late preterm (34 to < 37 weeks' gestation, n = 22), and term (≥37 weeks' gestation, n = 21). We monitored the expression of P-selectin and the formation of platelet-neutrophil aggregates (PNAs) by flow cytometry while using adenosine 5'-diphosphate (ADP) or thrombin receptor-activating peptide (TRAP) as agonists. RESULTS: PNAs were significantly lower in preterm compared to term neonates after TRAP or ADP stimulations (11.5 ± 5.2% vs. 19.9 ± 9.1%, p < 0.001, or 24.0 ± 10.1% vs. 39.1 ± 18.2%, p = 0.008, respectively). The expression of P-selectin also tended to be lower in preterm neonates, with significant positive correlations between P-selectin expression and PNA formation. CONCLUSIONS: The potential formation of PNAs correlates with gestational age. This suggests that the development of functional competencies of platelets and neutrophils continues throughout gestation, progressively enabling interactions between them.