Jacqueline S. Coats

Posttraumatic reduction of edema with aquaporin-4 RNA interference improves acute and chronic functional recovery.

Traumatic brain injury (TBI) is common in young children and adolescents and is associated with long-term disability and mortality. The neuropathologic sequelae that result from juvenile TBI are a complex cascade of events that include edema formation and brain swelling. Brain aquaporin-4 (AQP4) has a key role in edema formation. Thus, development of novel treatments targeting AQP4 to reduce edema could lessen the neuropathologic sequelae. We hypothesized that inhibiting AQP4 expression by injection of small-interfering RNA (siRNA) targeting AQP4 (siAQP4) after juvenile TBI would decrease edema formation, neuroinflammation, neuronal cell death, and improve neurologic outcomes. The siAQP4 or a RNA-induced silencing complex (RISC)-free control siRNA (siGLO) was injected lateral to the trauma site after controlled cortical impact in postnatal day 17 rats. Magnetic resonance imaging, neurologic testing, and immunohistochemistry were performed to assess outcomes. Pups treated with siAQP4 showed acute (3 days after injury) improvements in motor function and in spatial memory at long term (60 days after injury) compared with siGLO-treated animals. These improvements were associated with decreased edema formation, increased microglial activation, decreased blood–brain barrier disruption, reduced astrogliosis and neuronal cell death. The effectiveness of our treatment paradigm was associated with a 30% decrease in AQP4 expression at the injection site.

Repeated mild traumatic brain injury results in long-term white-matter disruption

Mild traumatic brain injury (mTBI) is an increasing public health concern as repetitive injuries can exacerbate existing neuropathology and result in increased neurologic deficits. In contrast to other models of repeated mTBI (rmTBI), our study focused on long-term white-matter abnormalities after bilateral mTBIs induced 7 days apart. A controlled cortical impact (CCI) was used to induce an initial mTBI to the right cortex of Single and rmTBI Sprague Dawley rats, followed by a second injury to the left cortex of rmTBI animals. Shams received only a craniectomy. Ex vivo diffusion tensor imaging (DTI), transmission electron microscopy (TEM), and histology were performed on the anterior corpus callosum at 60 days after injury. The rmTBI animals showed a significant bilateral increase in radial diffusivity (myelin), while only modest changes in axial diffusivity (axonal) were seen between the groups. Further, the rmTBI group showed an increased g-ratio and axon caliber in addition to myelin sheath abnormalities using TEM. Our DTI results indicate ongoing myelin changes, while the TEM data show continuing axonal changes at 60 days after rmTBI. These data suggest that bilateral rmTBI induced 7 days apart leads to progressive alterations in white matter that are not observed after a single mTBI.

Tissue vulnerability is increased following repetitive mild traumatic brain injury in the rat

Repetitive mild traumatic brain injury (rmTBI) is an important medical concern for active sports and military personnel. Multiple mild injuries may exacerbate tissue damage resulting in cumulative brain injury and poor functional recovery. In the present study, we investigated the time course of brain vulnerability to rmTBI in a rat model of mild cortical controlled impact. An initial mild injury was followed by a second injury unilaterally at an interval of 1, 3, or 7 days. RmTBI animals were compared to single mTBI and sham treated animals. Neuropathology was assessed using multi-modal magnetic resonance imaging (MRI), followed by ex vivo tissue immunohistochemistry. Neurological and behavioral outcomes were evaluated in a subset of animals receiving rmTBI 3 days apart and shams. RmTBI 1 or 3 days apart but not 7 days apart revealed significantly exacerbated MRI-definable lesion volumes compared to single mTBI and shams. Increases in cortical tissue damage, extravascular iron and glial activation assessed by histology/immunohistochemistry correlated with in vivo MRI findings where shorter intervals (1 or 3 days apart) resulted in greater tissue pathology. There were no neurological deficits associated with rmTBI 3 day animals. At 1 mo post-injury, animals with rmTBI 3 days apart showed reduced exploratory behaviors and subtle spatial learning memory impairments were observed. Collectively, our findings suggest that the mildly-impacted brain is more vulnerable to repetitive injury when delivered within 3 days following initial mTBI.

Meta-Analysis of Apparent Diffusion Coefficients in the Newborn Brain

Diffusion-weighted imaging and its quantitative apparent diffusion coefficient can assess severity in newborn hypoxic-ischemic injuries. A meta-analysis established normative values in term newborns, in comparison to those values in hypoxic-ischemic newborns with good versus poor outcomes. Measurements from 14 reports were stratified into three levels of increasing specificity: tissue type (gray matter, white matter, or cerebellum), tissue distribution (e.g., cortex or white-matter tracts), and anatomic structures (e.g., frontal white matter or posterior limb of the internal capsule). Normative apparent diffusion coefficients constituted white matter > gray matter = cerebellum, with lowest values in the posterior limb of the internal capsule and thalamus, and the highest in frontal and occipital white matter. Differences between normative and hypoxic-ischemic injury good-outcome groups were not evident. Values in the poor outcome group were significantly lower than normative data in white matter, gray matter, cortical gray matter, white matter tracts, posterior limb of the internal capsule, and cortical, frontal, and occipital white matter. Comparisons between injury groups found that coefficients were only significantly lower in the occipital cortex among poor outcomes. Coefficient values were lower in deep brain compared with cortical structures, reflecting tissue maturation and myelination. Differences between normative and hypoxic-ischemic injury poor-outcome groups suggest pathologies associated with neurologic sequelae. This meta-analysis provides the basis for normative apparent diffusion coefficient values in the newborn brain.

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.

Infratentorial strokes for posterior circulation folks: clinical correlations for current translational therapeutics.

Approximately 20% of all strokes will occur in the infratentorial brain. This is within the vascular territory of the posterior vascular circulation. Very few clinical specifics are known about the therapeutic needs of this patient sub-population. Most evidence-based practices are founded from research about the treatment of anterior circulatory stroke. As a consequence, little is known about how stroke in the infratentorial brain region would require a different approach. We characterized the neurovascular features of infratentorial stroke, pathophysiological responses, and experimental models for further translational study.

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.

Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying the mechanisms of normal and malignant hematopoiesis and are the gold standard for identifying effective chemotherapies for many malignancies. PDX models are possible because many of the mouse cytokines also act on human cells. However, this is not the case for all cytokines, including many that are critical for studying normal and malignant hematopoiesis in human cells. Techniques that engineer mice to produce human cytokines (transgenic and knock-in models) require significant expense before the usefulness of the model has been demonstrated. Other techniques are labor intensive (injection of recombinant cytokine or lentivirus) and in some cases require high levels of technical expertise (hydrodynamic injection of DNA). This report describes a simple method for generating PDX mice that have exogenous human cytokine (TSLP, thymic stromal lymphopoietin) via weekly intraperitoneal injection of stroma that have been transduced to overexpress this cytokine. Use of this method provides an in vivo source of continuous cytokine production that achieves physiological levels of circulating human cytokine in the mouse. Plasma levels of human cytokine can be varied based on the number of stromal cells injected, and cytokine production can be initiated at any point in the experiment. This method also includes cytokine-negative control mice that are similarly produced, but through intraperitoneal injection of stroma transduced with a control vector. We have previously demonstrated that leukemia cells harvested from TSLP-expressing PDX, as compared to control PDX, exhibit a gene expression pattern more like the original patient sample. Together the cytokine-producing and cytokine-negative PDX mice produced by this method provide a model system that we have used successfully to study the role of TSLP in normal and malignant hematopoiesis.

Abstract B46: A novel patient-derived xenograft model for evaluating therapies that target the CRLF2 signaling pathway to reduce health disparities for Hispanic children with leukemia

The purpose of the studies described here was to identify drug targets and develop a preclinical model for testing therapies that can reduce health disparities for Hispanic children with high-risk acute lymphoblastic leukemia (ALL). Hispanic children are 1.24 times more likely to develop ALL than non-Hispanic whites and that number rises to 2.09 by adolescence and early adulthood. A major contributor to this health disparity is a type high-risk B-cell ALL called CRLF2 B-ALL. CRLF2 B-ALL occurs 5 times more often in Hispanic children than others, is prevalent in adolescents and young adults, and is associated with a high relapse rate and poor prognosis. CRLF2 B-ALL is caused by genetic alterations that result in over expression of the cytokine receptor, CRLF2. The CRLF2 receptor is activated by the cytokine, TSLP, causing downstream activation of the JAK/STAT5 and PI3/AKT/MTOR pathways. A gene target of activated STAT5 in B cell precursors is Mcl-1, a Bcl2 family pro-survival molecule. In addition, Mcl-1 protein levels are known to be increased through post-transcriptional mechanisms by activation of the mTOR pathway. We hypothesized that the normal level of circulating TSLP cytokine could induce CRLF2 activation leading to increased Mcl-1 expression in CRLF2 B-ALL cells. Our data show that TSLP increases phosphorylation of STAT5, as well as AKT and S6 (downstream of mTOR) in primary CRLF2 B-ALL cells from Hispanic pediatric patients, even when activating JAK mutations are present. When CRLF2 B-ALL cells from Hispanic pediatric patients were cultured for 3 days with and without physiological levels of TSLP, flow cytometry showed that expression of the Mcl-1 protein was significantly increased in cultures with TSLP as compared to cultures without TSLP. CRLF2 B-ALL cells treated in vitro with Mcl-1 inhibitor showed dose-dependent increases in caspase 3 activation and apoptosis as indicated by flow cytometry. These data provide evidence that TSLP can contribute to leukemia cell survival and identify Mcl-1 inhibitor as a candidate therapy for CRLF2 B-ALL. Our next step was to develop a preclinical model for testing therapies that target genes, such as Mcl-1, that are regulated by TSLP-induced CRLF2 signals in this disease. Patient-derived xenograft (PDX) models produced by transplanting leukemia cells from patients into immune deficient mice provide an in vivo model of disease that includes contributions of the background genetic landscape that can influence disease progression or treatment outcome in health disparities diseases. PDX models are possible because most cytokines produced in the mouse are active on human cells, however mouse TSLP is species-specific. Thus classic PDX models do not provide TSLP that can activate the CRLF2 receptor that is overexpressed in CRLF2 B-ALL. To address this issue we engineered PDX mice to express physiological levels of human TSLP (+T PDX mice) and control -T mice that lacked human TSLP. In vivo TSLP activity was validated and +T PDX were successfully generated using leukemia cells from two Hispanic pediatric patients with CRLF2 B-ALL. To determine whether +T PDX mice provide a preclinical model of B-ALL that more closely mirrors patients than -T PDX mice, we compared RNAseq gene expression profiles of leukemia cells isolated from +T PDX and -T PDX mice to that from the original patient sample. The gene expression pattern in leukemia cells from +T mice was significantly closer to primary patient sample than that from -T mice. The +T PDX mice described here provide a novel in vivo preclinical model for evaluating efficacy of drugs, such as Mcl-1 inhibitor, in context of the background genetic landscape and physiological human TSLP present in patients. Citation Format: Kimberly J. Payne, Cornelia Stoian, Jacqueline S. Coats, Olivia Francis, Terry-Ann M. Milford, Ineavely Baez, Pierce J. McCarthy, George Mambo, Anna V.C. White, Mariah M.Z. Jackson, Juliette M. Personius, Veriah Vidales, Muhammad Omair Kamal, Shadi Farzin Gohar, Sinisa Dovat. A novel patient-derived xenograft model for evaluating therapies that target the CRLF2 signaling pathway to reduce health disparities for Hispanic children with leukemia. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B46.

Abstract 2444: TSLP regulates expression of Bcl2 family proteins in Ph-like ALL with CRLF2 alterations

B cell precursor acute lymphoblastic leukemia (B-ALL) is the most common childhood malignancy. A subset of children with B-ALL are at high risk for relapse and death. Gene expression profiles in these high-risk B-ALLs is similar to that of Philadelphia chromosome positive ALL. Approximately half of these Ph-like B-ALL are characterized by genetic alterations that result in overexpression of CRLF2. CRLF2, together with the IL-7 receptor α chain, forms a receptor complex for the cytokine, TSLP. When TSLP binds, the receptor initiates downstream JAK2/STAT5 and PI3/AKT/mTOR pathway activation. The activating JAK mutations found in some CRLF2 B-ALL led to speculation that TSLP stimulation is not a factor in this disease. However, we find that TSLP increases phosphorylation of STAT5, AKT and S6 (downstream of mTOR) in CRLF2 B-ALL cells, including those with JAK defects. Activation of these pathways has been associated with oncogenesis and chemoresistance and their downstream targets include members of the Bcl2 family. The Bcl2 family pro-survival molecule Bcl-XL is a down stream target of STAT5 in Ph+ B-ALL. Mcl-1, another BCL2 family pro-survival molecule is known to be upregulated by mTOR activation via post-translational mechanisms in B cell lymphoma. We hypothesized that TSLP-induced JAK2/STAT5 and PI3/AKT/mTOR pathway activation contribute to chemoresistance in high risk CRLF2 B-ALL by upregulating the expression of Bcl-XL and Mcl-1. To test this hypothesis we cultured human CRLF2 B-ALL cell lines (MUTZ5 and CALL4) with and without TSLP and evaluated expression of the Bcl2 family pro-survival proteins, Bcl-XL, Mcl-1, and Bcl2. We found that TSLP induced significant increases in Bcl-XL and Mcl-1 proteins, but not Bcl2 in CRLF2 B-ALL cells. These cell lines have activating Jak mutations and thus reflect the ability of TSLP to increase expression of the Bcl2 family proteins in cases where activating JAK mutations are present. Next we evaluated the effect of Mcl-1 inhibitor on MUTZ5 and CALL4 cells. Preliminary data from these experiments show that cell counts in cultures treated with Mcl-1 inhibitor are reduce by >90% and this reduction is maintained in the presence of TSLP. These data provide evidence that TSLP-induced CRLF2 signals increase expression of Bcl2 pro-survival proteins, even in CRLF2 B-ALL cells with activating JAK mutations. These data also suggest that Mcl-1 inhibitors could be an effective treatment for this disease. Ongoing studies will evaluate the effect of TSLP and Mcl-1 inhibitors in primary CRLF2 B-ALL samples. Citation Format: Cornelia Stoian, Muhammad Omair Kamal, Olivia Francis, Rhaya Johnson, Simone Montgomery, Jacqueline Coats, Hannah Choi, Shania Aponte-Paris, Micheal Reed, Shanalee Martinez, Karina Mayagoitia, Evgeny Chirshev, Chunhua Song, Sinisa Dovat, Kimberly J. Payne. TSLP regulates expression of Bcl2 family proteins in Ph-like ALL with CRLF2 alterations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2444.