Medical Scientist Training Program (MSTP)

Individual Predoctoral Fellowship Awards

 
 

Current Students with Active Fellowships

Chris Nishimura NIH NRSA F30 Fellowship for a project entitled "Mechanistic Dissection and Therapeutic Targeting of B7x in Cancer" (Sponsor,  XingXing Zang, Microbiology & Immunology)

Ryan Graff NIH NRSA F30 Fellowship for a project entitled "Platelet PI3Kβ regulation of metastasis" (Sponsor,  Jonathan Backer and Anne Bresnick, Molecular Pharmacology)

Henrietta Bains NIH NRSA F31 Fellowship for a project entitled "How does mTOR sense lipid in vivo" (Sponsor,  Rajat Singh, Developmental & Molecular Biology)

Julio Flores NIH NRSA F31 Fellowship for a project entitled "Epigenetic regulation of stem cells and development by the DNA dioxygenase Te2" (Sponsor,  Meelad Dawlaty, Genetics)

Daniel Borger NIH NRSA F30 Fellowship for a project entitled "Developing a novel ex vivo platform to support hematopoietic cells and characterize the stem cell niche" (Sponsor,  Paul Frenette, Cell Biology)

Ryan Malonis NIH NRSA F30 Fellowship for a project entitled "Discovery & characterization of human monoclonal antibodies targeting multiple arthritogenic alphaviruses" (Sponsor, Jon Lai, Biochemistry)

Bianca Ulloa NIH NRSA F31 Fellowship for a project entitled "Deciphering the development of hematopoietic stem and progenitor cell self-renewal and differentiation" (Sponsor, Teresa Bowman, Developmental & Molecular Biology)

Taylor Thompson NIH NRSA F31 Fellowship for a project entitled "Transcriptional Regulatory and Cell Differentiation Influences of an Endocrine Disrupting Chemical" (Sponsor, John Greally, Genetics)

Michelle Gulfo NIH NRSA F31 Fellowship for a project entitled "Assessing dopaminergic modulation of an associative circuit within the dentate gyrus" (Sponsor, Pablo Castillo, Neuroscience)

Meera Trivedi NIH NRSA F31 Fellowship for a project entitled "Characterizing Novel Regulations of Dendritic Tiling in C. elegans" (Sponsor, Hannes Buelow, Neuroscience)

Jamie Moore NIH NRSA F31 Fellowship for a project entitled "Unraveling Mechanisms of Plasmacytoid Dendritic Cell Priming by CD169+ Macrophages in Severe Murine Malaria" (Sponsor, Gregoire Lauvau, Microbiology & Immunology)

Adam Spitz NIH NRSA F30 Fellowship for a project entitled "Direct Small Molecule Activation of Pro-apoptotic BAK" (Sponsor, Evris Gavathiotis, Biochemistry)

Hayden Hatch NIH NRSA F31 Fellowship for a project entitled "Transcriptional regulation, neuronal development, and function of the mushroom body in a Drosophila model of intellectual disability" (Co-Sponsors, Julie Secombe and Nicholas Baker, Neuroscience/Genetics)

Joshua Weinreb NIH NRSA F30 Fellowship for a project entitled "Uncovering the Role of the DEAD Box Helicase Ddx41 in Hematopoiesis" (Sponsor,  Teresa Bowman, Developmental & Molecular Biology)

Rosiris Leon-Rivera NIH NRSA F31 Fellowship for a project entitled "Molecular Mechanisms of Increased Risk of Racial and Ethnic Minorities for HIV Associated Neurocognitive Disorders" (Sponsor, Joan Berman, Pathology)

Kristin Palarz NIH NRSA F31 Fellowship for a project entitled "Serotoninergic modulation of cerebellar circuitry" (Sponsor, Kamran Khodakhah, Neuroscience)

Peter John NIH NRSA F30 Fellowship for a project entitled "B7x in Cancer: Mechanisms and Therapies" (Sponsor, XingXing Zang, Microbiology & Immunology)

Richard Piszczatowski NIH NRSA F30 Fellowship for a project entitled "Investigating the role of Nol3 in normal and malignant hematopoiesis" (Sponsor, Ulrich Steidl, Cell Biology)

     
     

    Current Students with Completed Fellowships

    Joshua Mayoral NIH NRSA F31 Fellowship for a project entitled "Secreted effectors of Toxoplasma gondii bradyzoites" (Sponsor, Louis Weiss, Pathology)

    Justin Wheat NIH NRSA F30 Fellowship entitled "Uncovering Transcriptional Regulation of a Master Hematopoietic Transcription Factor at Single Molecule Resolution" (Sponsor, Ulrich Steidl & Robert Singer, Cell Biology and Anatomy & Structural Biology)

 
 

Abstracts of Recent Projects

Chris Nishimura - ABSTRACT: Immune checkpoints are proteins that regulate the body’s immune system via inhibition of immune cells. In diseases such as cancer, these same pathways may be commandeered in order to inappropriately inhibit immune responses. One approach to overcome this excessive inhibition and restore normal immune function is to physically block these inhibitory proteins using monoclonal antibodies (mAbs), a strategy known as immune checkpoint blockade. Our lab discovered the protein B7x (VTCN1/B7S1/B7-H4), an immune checkpoint whose regulation and mechanism of action are still being elucidated. B7x is expressed on a wide variety of cancers and is associated with poor clinical outcomes. It has been shown to inhibit effector T cell functions such as cytokine production and proliferation, and promotes T cell exhaustion; it has also been associated with tumor infiltration of immunosuppressive cell populations such as myeloid derived suppressor cells (MDSCs). Intriguingly, it is not typically co-expressed with the well-studied immune checkpoint PD-L1, and blocking B7x using anti-B7x mAbs improves anti-tumor responses and survival in mouse models. This suggests that B7x holds a non-overlapping but key role in cancer immune evasion. For these reasons I hypothesize that B7x mediates immune evasion through inhibiting effector cell functions while simultaneously promoting immunosuppressive cells and is therefore a promising target for cancer immunotherapy. Thus, we propose the following two aims: 1) Examine the mechanisms that regulate B7x expression and its role on MDSCs; and 2) Develop and characterize a new anti-B7x immunotherapy. In aim 1 I will examine the how hypoxia and other tumor microenvironment-associated cytokines regulate B7x expression. In addition, I will explore how B7x affects the immunosuppressive MDSC cell population generation and survival. In aim 2 I will test the anti-tumor efficacy of newly generated anti-B7x mAb-based immune checkpoint blockade in vivo using metastasis and spontaneous models of lung cancer. We will also characterize how this new immunotherapy alters T cell phenotypes using flow cytometry and single-cell RNA sequencing. Finally, we will explore if combining our anti-B7x mAb with anti-TIM3 and anti-PD1 mAbs confers superior anti-tumor efficacy than anti-B7x monotherapy in vivo. Together, these studies will allow us to broadly explore the role of B7x in tumorigenesis and therapeutic potential of anti-B7x immunotherapy, and combined with a personalized training plan and supportive research environment at Einstein, cultivate the skills required of a physician-scientist.

Ryan Graff - ABSTRACT: Metastasis is the primary cause of morbidity and mortality among patients with solid tumors. Platelets interact with tumor cells upon their entry into the vasculature and promote the metastatic spread of these cells by several mechanisms. Circulating tumor cells (CTCs) directly bind to and activate platelets resulting in the formation of platelet-fibrin complexes that envelope circulating tumor cells and protect them from immune clearance. Adhesion to tumor cells also induces the release of platelet cytokines and other soluble factors that promote epithelial-mesenchymal transition in the tumor cells. Finally, platelets promote the adhesion of CTCs to the endothelium, assisting in their extravasation at distant metastatic sites. Platelet activation, adhesion, and in vivo thrombus formation require the activity of the Class IA PI 3-kinase PI3K. This isoform of PI3K produces the majority of PI(3,4,5)P3 in platelets, suggesting that PI3K has a unique role in classical platelet activation. However, the role of PI3K in platelet-mediated cancer metastasis has yet to be defined. We propose that PI3K is required for platelet activation upon interaction with tumor cells. Aim 1 will test the role of platelet PI3K in platelet-stimulated tumor cell Matrigel invasion, transendothelial migration, epithelial-mesenchymal transition, cell stemness, NFB activation, and platelet TGF secretion. We will also use RNAseq and antibody arrays to more broadly examine the role of PI3K in platelet chemokine/cytokine release and in transcriptional responses by tumor cells. We will use both platelets from mice expressing mutant PI3K and human platelets pre-treated with the irreversible pan-PI3K inhibitor wortmannin. Aim 2 will directly evaluate the role of platelet PI3K in cancer metastasis in wild type and whole-animal or platelet-specific mutant PI3K mice. Murine mammary carcinoma cells will be injected intravenously to analyze in vivo tumor cell-platelet complex formation as well as tumor cell interactions with macrophages and other leukocytes. We will also test the role of PI3K in tumor cell-induced thrombocytopenia and experimental metastasis. Isoform selective PI3K inhibitors were originally developed to be used as anti-thrombotic agents, as they prevent thrombotic occlusion of injured arteries without increasing bleeding. More recently these inhibitors have also been explored as anti-cancer agents in the treatment of PTEN-deficient cancers. This proposal seeks to understand the potential for a broader application of these agents in cancer patients to inhibit platelet activity and prevent metastasis in a variety of cancer types.

Henrietta Bains - ABSTRACT: Alterations in lipid metabolism determine metabolic disease and mortality in the aging population. Despite our understanding of regulation of lipid metabolism, how organisms sense lipid remains unknown. It is conceivable that sensing of lipid will inform downstream decisions taken by the cell that modulate metabolism, proteostasis, stress response, and growth—each of which are dysregulated with age. The mechanistic target of rapamycin (mTOR), is a serine/threonine kinase and amino acid sensor, that drives growth and proliferation. More recently, mTOR in cultured cells has been shown to be activated by cholesterol and phosphatidic acid (PA) in absence of amino acids. Whether mTOR senses lipid in whole organisms is unclear. mTOR exists as two major complexes—mTORC1 and mTORC2. Activation of mTORC1 occurs at the lysosomal surface in presence of amino acids and requires key regulatory proteins that stimulate its activity. By contrast, mTORC2 responds to growth factors to regulate cytoskeletal organization. Hyperactivation of mTORC1 (hereafter, mTOR for simplicity) drives aging and age-related diseases in part by disrupting autophagy and promoting growth. Indeed, suppressing mTOR has been shown to increase lifespan in multiple organisms. However, how mTORC1 is hyperactivated with age remains unknown. It has been shown that there are quantitative and qualitative changes in membrane lipids with age including changes in lysosomal membrane lipids—the major site of mTOR activation. Whether changes in lysosomal membrane lipids are mechanistically linked to mTOR hyperactivation remain unknown. Our preliminary data show that subjecting mice to an oral gavage of corn oil causes activation of mTOR and its translocation to distinct cholesterol-rich microdomains (CRMs)/lipid rafts in lysosome membranes. Our preliminary data also show that immunoprecipitating mTOR from lysosome membranes from livers of oil-gavaged mice reveal its binding to diacylglycerol. These data suggest that mTOR is a sensor of diacylglycerol, a membrane lipid. Since mTOR senses nutrients at lysosome membranes, I hypothesize that mTOR senses lipid at lysosomal membranes, and that age-related changes in lysosomal membrane lipid composition lead to mTOR hyperactivation. To test our hypothesis, we present the following specific aims: Aim 1: To determine how mTOR senses lipid at the lysosome surface. In Aim 1, diverse approaches will be used to characterize lipid-driven mTOR activation at lysosome membranes. By pulling down mTOR from lysosome membranes for lipidomic and proteomic analyses, I will identify lipid species that bind to mTOR and its interacting partners. I will use an siRNA screen in vitro to silence each of the interacting partners, which will identify novel regulators of lipid-driven mTOR signaling. Aim 2: To determine the mechanism of mTOR hyperactivation with age. In Aim 2, I will characterize the changes in lipid composition of lysosome membrane CRMs and expansion of lysosome CRMs with age. I will determine whether alterations in membrane lipid composition with age correlate with increased mTOR activity. I will then determine whether inactivating the synthesis of specific membrane lipids, e.g., PA and DG, by shRNAs against relevant biosynthetic enzymes in liver will dampen age-related mTOR hyperactivation. I will also determine whether targeting key interacting partners of mTOR in liver will dampen age-related hyperactivation of mTOR signaling and reverse deleterious mTOR-dependent outcomes, i.e., blockage of autophagy and proteostasis failure.

Julio Flores- ABSTRACT: The Ten-eleven translocation (Tet1/2/3) family of enzymes are epigenetic regulators of gene expression important for stem cell biology and embryonic development. Tet enzymes are dioxygenases that promote DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethycytosine (5hmC) and higher oxidized derivatives. In addition to this enzymatic activity, Tet enzymes can bind chromatin modifying complexes, to regulate genes in a presumably catalytic-independent manner. Tet2 is a key member of this family. It is highly expressed in embryonic stem cells (ESCs) and controls gene expression programs necessary for stem cell lineage specification. Tet2 is also frequently mutated in hematological malignancies and has been implicated in neurodegenerative diseases. While the catalytic functions of Tet2 have been well studied, its non-catalytic roles remain poorly defined. In this proposal, we seek to establish the significance of the catalytic dependent and independent functions of Tet2 in ESC gene regulation and lineage commitment. We hypothesize that Tet2, in addition to regulating genes through its DNA demethylase activity, can also modulate genes in a non-catalytic fashion by recruiting histone modifiers to the chromatin, and this dual mode of gene regulation is essential for proper ESC differentiation along the neural and hematopoietic lineages. To test this hypothesis, I have generated Tet2 catalytic mutant (Tet2m/m) and knock-out (Tet2–/–) ESCs, which I will use as a platform to: (1) identify the catalytic and non-catalytic direct target genes of Tet2 in ESCs by integrating changes in gene expression with Tet2 genomic occupancy, (2) establish Tet2-mediated activating and repressing mechanisms of gene regulation involving interactions with histone modifiers OGT and HDAC2, and finally (3) define the biological significance of Tet2 enzymatic and non-enzymatic functions in ESC differentiation and lineage commitment along the neural and hematopoietic lineages. Findings from these experiments will elucidate novel epigenetic mechanisms of gene regulation in ESCs involving Tet2 catalytic and non-catalytic functions. They will enhance our understanding of stem cell biology and development and can have implications in hematological malignancies where Tet2 is affected.

Daniel Borger- ABSTRACT: Current culture methods reduce the ability of hematopoietic stem cells (HSCs) to successfully engraft in a host. Emerging gene editing technologies such as CRISPR/Cas9 require time in culture to allow for the correction of disease-causing alleles. There is therefore a need to develop new methods of culturing HSCs. Coculture of HSCs with bone marrow niche cells such as mesenchymal stem cells (MSCs) is one possible solution to problems in HSC culture, as these cells provide factors that support HSCs in vivo. However, MSCs cannot be maintained in culture for extended periods of time and fairly rapidly lose expression of niche factors. Through a screen of candidate transcription factors, our lab identified 5 factors that when transduced together restore niche factor expression and allow for prolonged culture. These factors are Kruppel-like factor 7 (Klf7), Osteoclast stimulating factor (Ostf1), X-box binding protein (XBP1), Interferon regulatory factor 3 (Irf3), and Irf7, which we collectively dubbed the KOXII factors. KOXII-transduced MSCs were able to expand both murine and human funtional HSCs to a much greater extent than mock-transduced MSCs. These cells therefore may be useful in expanding HSCs ex vivo for gene correction. However, there are regulatory barriers to the application of murine cells in human therapeutics. The work proposed here will in part focus on the development and characterization of KOXII-transduced human MSCs. After generating these cells, I will determine if the KOXII factors affect expression of niche factors in human MSCs. I will also use flow cytometry and stem cell xenotransplantation to determine if KOXII-transduced MSCs are more effective at driving HSC expansion than unmodified MSCs. Finally, using CRISPR/Cas9-based gene editing of HSCs derived from patients with sickle cell disease as a model, I will determine if coculture of patient cells with KOXII-transduced MSCs can improve the efficiency of gene editing or increase the yield of properly edited cells over current standard HSC culture methods. In parallel, I will use murine KOXII-transduced MSCs to more closely examine niche signalling by MSCs. As these cells can be cultured in relatively large numbers, they are ideal for proteomic studies. In collaboration with the lab of Jeroen Krijgsveld, I will examine the secretome of these cells in order to identify proteins whose secretion is upregulated by the KOXII factors. Using both in vitro and in vivo assays, I will evaluate the effect of these factors on HSC maintenance and proliferation, with the aim of identifying secreted proteins with previously unappreciated roles in MSC-HSC niche interactions.

Ryan Malonis - ABSTRACT: Alphaviruses are enveloped, positive sense single-stranded RNA viruses, which include several important human pathogens. Arthritogenic alphaviruses are globally distributed, mosquito-transmitted viruses that cause human rheumatic disease and include chikungunya virus (CHIKV) and Mayaro virus (MAYV). Symptomatic infection is characterized by fever, rash, myalgia, as well as both acute and chronic polyarthralgia that can persist for months to years after infection. More severe manifestations of alphaviral disease – including hemorrhage, encephalopathy and mortality – have been reported. These viruses cause endemic disease as well as large, sporadic epidemics worldwide. Currently, there are no approved vaccines or anti-viral therapies for the prevention or treatment of alphavirus infection; therefore, the development of new therapeutic strategies targeting one or multiple arthritogenic alphaviruses is of substantial interest. A number of potently neutralizing CHIKV monoclonal antibodies (mAbs) have been described, but currently the only broadly neutralizing alphavirus mAbs that have been reported are murine. Thus, the extent to which the human antibody response elicits broadly-neutralizing mAbs following alphavirus infection, and which epitope(s) such mAbs may target, remains unknown. To address this question, this proposal seeks to expand our knowledge of the neutralizing antibody response to alphaviruses by systematically investigating cross-reactive antibodies from CHIKV-infected patients. Towards this end, we have used single B cell sorting to isolate a large panel of MAYV-reactive mAbs from CHIKV patients in the convalescent phase. We will study the reactivity and neutralization profiles of these mAbs against related arthitogenic alphaviruses (Aim 1). We will then biochemically determine the requirements of neutralization (Aim 2) and elucidate the mechanism of mAb inhibition (Aim 3). These studies will contribute to our fundamental understanding of how the adaptive immune system combats infection by arthritogenic alphaviruses and may aid the development of novel mAb-based treatments and vaccines.

Bianca Ulloa - ABSTRACT: Hematopoietic stem and progenitor cells (HSPCs) are characterized by their self-renewal and multipotent differentiation capacities. As such, they give rise to all mature blood cell types (e.g., myeloid, lymphoid, and erythroid) to maintain life-long hematopoiesis. Their regenerative capacity makes HSPCs valuable for cell replacement therapies in patients with hematological diseases, including those that are secondary to chemotherapy and radiotherapy. Understanding HSPC properties of self-renewal and multipotency allows for the development of methods to improve and maximize their therapeutic potential. By studying the embryonic origins of HSPC self-renewal and differentiation capacities, we aim to advance what is known about these defining characteristics of stem cells. In addition to HSPCs, other multi-lineage progenitors are produced during embryogenesis. These are limited in their self-renewal and differentiation output and are mostly regarded as transient in nature. These progenitor populations share many features of stem cells, confounding studies of HSPC properties within their native embryonic environments. Several studies in murine and zebrafish models suggest that these embryonic progenitors, and not HSPCs, are the dominant population generating mature blood cells in the embryo. If HSPCs are not necessary to sustain the embryo, what is their function during development? To answer this question, we propose to use zebrafish to determine when and where during development HSPCs self-renew and contribute to mature blood cell output in myeloid, lymphoid, and erythroid lineages. We will use novel regeneration and transplantation assays (Aim 1) to study self- renewal and lineage-tracing experiments (Aim 2) to investigate HSPC differentiation. Understanding how these properties are established and maintained is critical to harnessing stem cells for regenerative medicine.

Taylor Thompson - ABSTRACT: One major group of environmental toxicant that affect humans negatively are endocrine disrupting chemicals (EDCs). These chemicals interfere with the body’s natural hormone regulation leading to a range of human diseases. Our research focuses on the EDC tributyltin (TBT), a chemical frequently used as a pesticide and plastic stabilizer. TBT has major adipogenic effects when exposed in utero or in adult multipotent stem cells. Previously published data have demonstrated that TBT exposure promotes differentiation of mesenchymal stem cells (MSCs) into adipogenesis, and also increases their lipid content, representing both numerical and qualitative effects on adipocytes. Mechanistically, TBT has been found to bind to the ligand-binding domain of the peroxisome proliferator-activated receptor gamma (PPARg) transcription factor (TF), which is known to form a heterodimer with the RXR TF when activated, promoting a transcriptional reprogramming of MSCs to commit them to adipogenesis. MSCs can differentiate into a number of lineages, including muscle, bone, cartilage and fibrocystic cells. When a cell undergoes transcriptional reprogramming, the sites at which the TFs bind change, reflected by alterations of the distributions of loci of open chromatin. In this project, we propose to differentiate MSCs to both adipocytes and myocytes, initially using the cell culture conditions known to promote specific differentiation of MSCs. We will map the loci of open chromatin and test gene expression in these samples, allowing us to identify TFs mediating these differentiation pathways by searching for motif enrichment corresponding to known TF binding sites. With this information available, we can then use the same approaches to test how TBT causes transcriptional reprogramming, which should reveal whether the process is identical or involves a different set of TFs. Finally, we will apply the new CellTagging approach to test cells at multiple stages of differentiation to myocytes to test whether TBT exposure affects only undifferentiated MSCs, or can also cause transdifferentiation of cells already developing in the myogenic lineage. These new insights into the mechanism of action of TBT will be valuable in understanding how EDCs have their disease- causing effects. We will also get insights from TBT into how we a small molecule can mediate ‘epigenetic therapy’, influencing transcriptional reprogramming but in a way that is targeted to specific genomic locations. Under the mentorship of Drs. John Greally and Paul Frenette, I will accomplish these goals while developing new skills in developmental biology and genetics. Additionally I will gain valuable experiences in presenting, networking, and manuscript writing, all of which are essential as I train to become and independent investigator and physician-scientist.

Joshua Weinreb - ABSTRACT: Myelodysplastic syndromes (MDS), a group of pre-malignant bone marrow failure syndromes arising from defects in hematopoietic stem cells (HSCs), are amongst the most common hematological malignancies of the elderly with ~30% progressing to acute myeloid leukemia (AML). Recently, loss-of-function germline and somatic mutations in the DEAD-box Helicase 41 gene (DDX41) have been identified in patients with MDS and AML, and are thought to contribute to disease pathogenesis. Both germline and somatic DDX41 mutations are thought to promote hematopoietic deregulation and leukemogenesis. Although a strong clinical correlation is found between mutations in DDX41 and MDS, the in vivo role of DDX41 in hematopoiesis has not been elucidated. To address this question, we will characterize hematopoiesis in a zebrafish ddx41 loss-of-function mutant. Our preliminary studies indicate that ddx41 mutants develop neutropenia and anemia. This animal model will allow us to elucidate the in vivo role of Ddx41 in normal hematopoiesis and the underlying mechanism for any defects. DDX41 has been implicated in splicing and shown to associate with components of the spliceosome including Splicing Factor 3B, subunit 1 (SF3B1), the most commonly mutated splicing factor in MDS. Additionally, MDS/AML patient samples with DDX41 mutations displayed errors in mRNA splicing that typically occur when components of the spliceosome are defective. Although these data point towards a role for DDX41 in splicing, it is still unknown if splicing aberrations contribute to the observed abnormalities in DDX41-mutated hematologic diseases. Using ddx41-deficient zebrafish will permit us to delineate the functionally relevant early molecular events leading to blood cell defects when ddx41 is mutated, which is anticipated to provide novel insight into the origins of MDS. In this proposal, we will use our novel in vivo model of Ddx41-deficiency to test the hypothesis that Ddx41 is critical for normal hematopoiesis via regulation of splicing, and that malfunctioning of this process contributes to the hematological defects seen in DDX41-mutated MDS/AML. In Aim 1, we will determine which blood populations require Ddx41 and which functional domains of Ddx41 are required for healthy hematopoiesis. In Aim 2, we will elucidate connections between DDX41 and SF3B1 using protein and genetic interaction studies. Combined these studies will reveal insights into the pathophysiology of DDX41-mutated MDS/AML.

Rosiris Leon-Rivera - ABSTRACT: Approximately 37 million people worldwide are living with HIV. HIV enters the CNS within 10 days of peripheral infection, with development of HIV Associated Neurocognitive Disorders (HAND) in ~50% of infected people despite ART. This is due to host/viral inflammatory and toxic factors within the CNS promoting neuronal injury. Studies have shown increased risk and incidence of HIV infected Hispanics for HAND compared to Whites. Molecular mechanisms underlying this increased risk have not been extensively studied. One mechanism by which HIV enters the CNS is by transmigration of infected monocytes across the blood brain barrier (BBB), establishing CNS HIV reservoirs, and inducing inflammation and neurotoxicity. A mature monocyte subset expressing the LPS receptor, CD14, and FcγIII receptor, CD16, is key to HIV neuropathogenesis, increased in the peripheral blood of HIV-infected people, and preferentially infected with HIV. We showed that CD14+CD16+ monocytes from HIV-infected people transmigrate preferentially across our human BBB model to CCL2 and CXCL12, chemokines elevated in the CNS of HIV-infected people. We also showed that CD14+CD16+ monocytes from HIV-infected people with HAND transmigrate in greater numbers to CCL2 than those from people with normal cognition. We showed that an antagonist to CXCR7, a recently identified CXCL12 receptor on monocytes, blocks CXCL12-mediated transmigration of specifically CD14+CD16+ monocytes from HIVinfected people, and we propose this as a therapeutic target to reduce their entry into the CNS. HIV DNA copies/106 PBMC from HIV-infected people, specifically within CD14+CD16+ monocytes, correlate with HAND. Using cultured CD14+CD16+ monocytes, we showed that monocytes harboring HIV (HIV+) preferentially transmigrate across the BBB to CCL2 as compared to uninfected but HIV-exposed monocytes, and that this selective advantage is due, in part, to increased junctional proteins JAM-A and ALCAM. However, there are no studies addressing whether CXCL12-mediated transmigration of mature monocytes, nor whether CXCL12-mediated transmigration specifically of HIV+CD14+CD16+ monocytes, correlate with HAND in Hispanics. Monocytes contribute to peripheral immune activation in HIV-infected people. Once CD14+CD16+ monocytes enter the CNS, they produce host/viral toxic factors that promote neuronal damage and HAND. We hypothesize that: 1. In a select cohort of HIV-infected Hispanics on ART, Hispanics with HAND, compared to those with normal cognition, have a higher percent of peripheral CD14+CD16+ monocytes expressing CCL2, CXCL12, TNF-a, and/or IL-6, and have higher levels of these mediators; 2. CD14+CD16+ monocytes from this cohort, particularly those harboring HIV, will preferentially transmigrate to CXCL12, and this will correlate with HAND; 3. Preferential transmigration of HIV+ monocytes to CXCL12 will be blocked with antibodies to JAM-A and ALCAM, and an inhibitor to CXCR7; 4. Hispanics with HAND will have higher HIV DNA copies/106 PBMC compared to Whites with HAND, contributing to their increased risk for HAND.

Richard Piszczatowski - ABSTRACT: Hematopoiesis relies on the proper function of hematopoietic stem and progenitor cells (HSPCs), in their capacity for both self-renewal and differentiation. Genetic lesions in these cell populations can give rise to myeloid malignancies including myeloproliferative neoplasms (MPN) and acute myeloid leukemia (AML). Myeloproliferative neoplasms, which result in the expansion of mature myeloid compartments, commonly harbor aberrations or mutations involving the JAK/STAT signaling pathway. Mutations in JAK2, CALR, and MPL are found in the majority of MPN cases, however cooperating co-driver mutations and disease modifiers are relatively poorly understood. Current treatment options for Philadelphia-chromosome-negative (Ph-) MPNs provide symptomatic relief and do not significantly alter overall survival. Moreover, leukemic transformation to AML is a common occurrence in Ph- MPN patients, and is thought to be due to acquisition of additional mutations which ultimately lead to a dramatic reduction in patient survival. The process of leukemic transformation of MPN to AML still remains unclear, and gaining insight into the molecular mechanisms of this phenomenon may provide openings for new interventions for preventing disease progression. We have discovered that loss of the Nol3 gene in mice leads to an MPN-like disease closely resembling primary myelofibrosis (PMF). Paradoxical to its canonical functions in repressing apoptosis, deletion of Nol3 results in an increased expansion and cycling of HSPCs in the bone marrow and spleen, suggesting a novel role for Nol3 within the hematopoietic system. Our analyses show that Nol3 is frequently downregulated or deleted in patients with PMF and other myeloid malignancies, and our data provides a link between Nol3 expression and JAK/STAT activation in MPN and AML cells, implicating Nol3 in the JAK/STAT signaling pathway. Our proposal seeks to (1) characterize the molecular role of Nol3 in hematopoiesis by defining molecular functions and subcellular localization, as well as identifying functional binding partners, (2) relate Nol3 loss to human disease by dissecting the role of Nol3 within the JAK/STAT pathway and identifying critical functional domains, and (3) gain insight into cooperativity between Nol3 loss and mutational drivers in MPN and transformation to AML. Our study will not only provide a novel and robust model for studying myeloid malignancies, but will also help define for the first time a role for Nol3 as a myeloid tumor suppressor and key component of the JAK/STAT signaling pathway. More importantly, our studies will also link Nol3 to human myeloid malignancy, which will provide a deeper understanding of the molecular pathogenesis of disease.

Awards

 
 
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