Faculty Summaries
David L. Wiest, PhD
David L. Wiest, PhD
Professor
  • Deputy Chief Scientific Officer
  • Co-Leader, Blood Cell Development and Function
  • Adjunct Associate Professor, Thomas Jefferson University
David.Wiest@fccc.edu
Office Phone: 215-728-2966
Lab Phone: 215-728-2968
Fax: 215-728-2412
Office: 383A
Lab: R364
  • A. The Role of TCR Signal Strength in Lineage Commitment.
    Francis Coffey & Shawn Fahl, in collaboration with Dietmar Kappes, C Murre, Y. Zhuang & J.C. Zuniga-Pflucker
    Schematic of role of TCR signal strength in fate adoption
    Schematic of role of TCR signal strength in fate adoption

    αβ and γδ T lymphocytes comprise two distinct lineages that perform vital, non-overlapping roles in immune responses. These distinct lineages are thought to arise from a common thymic precursor, but despite much effort, very little is known about the developmental instructions that determine which of these lineages a developing thymocyte will adopt (PICT 1). Our laboratory seeks to gain insight into the molecular processes controlling alternate αβ/γδ lineage commitment. This is of critical importance not only because the divergence of T cells into functionally distinct αβ and γδ lineages is essential for normal immune responses, but also because differentiation of multipotential precursor cells into distinct cell types is a fundamental, yet poorly understood process, common to all multicellular organisms.
     

    Model by which prolonged ERK signals promote adoption of distinct fates
    Model by which prolonged ERK signals promote adoption of distinct fates

    Importantly, we have provided compelling evidence for a signal strength model of lineage commitment which posits that weak signals promote commitment to the αβ lineage while comparatively strong signals promote commitment to the γδ lineage, irrespective of the TCR complex from which they originate. We further proposed that the differences in signal strength that alter fate are dependent upon differential activation of the signaling pathway consisting of: 1) the proximal signaling molecule, extracellular-signal regulated kinase (ERK); 2) downstream transcription factors of the early growth response (Egr) family; and 3) the Egr target, inhibitor of DNA-binding 3 (ID3) (PICT 1). We propose that differences in the activation of this signaling axis specify fate through graded reductions in the activity of E box DNA-binding proteins.



     

    Schematic of the relationship between γδ lineage commitment and specification of effector fate
    Schematic of γδ lineage commitment and specification of effector fate relationship

    We are currently addressing a number of critical questions that remain regarding how lineage fate is specified. 1) Do the TCR signals that control adoption of the αβ and γδ fate differ in intensity, duration, or both? Our emerging evidence suggests that adoption of the γδ lineage fate is associated with prolonged ERK signals that specify fate through a novel target interaction interface. The prolonged signals appear to stabilize the protein products of immediate early genes, including those encoding transcription factors, thereby enabling them to transactivate addition gene targets not modulated by transient signals (PICT 2). 2) Graded repression of E protein function is likely to be responsible for specification of the αβ and γδ fates; however, the E protein targets that are critical for these processes remain to be identified. Genomic analysis is currently being employed to identify the collection of E protein targets required for adoption of the γδ fate and how they differ from those important for the αβ fate. 3) Unlike αβ lineage cells that acquire effector function in the periphery, γδ effector fate is largely specified in the thymus. Does γδ lineage commitment and specification of effector fate occur coincidently or sequentially? We have identified a TCR-ligand inducible marker, CD73, which has enabled us to determine that these processes are separable, and that TCR-ligand appears to play an important role in both events (PICT 3).

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  • B. Antagonistic control of embryonic development and hematopoiesis by Rpl22 and its paralog, Rpl22-Like1 (Like1).
    Bryan Harris, Suraj Peri & Yong Zhang, in collaboration with B. Kennedy, D. Liebermann , Stephen Sykes & A. Verma
    Extraribosomal functions of Rpl22 and Rpl22-like1 (Like1)
    Extraribosomal functions of Rpl22 and Rpl22-like1 (Like1).

    The importance of ribosomal proteins (RP) in development and disease is clearly illustrated by a collection of diseases termed ribosomopathies, which share a constellation of features including defects in hematopoiesis, craniofacial and/or skeletal abnormalities, and often an increased risk for developing cancer. While the molecular basis for these developmental abnormalities remains unclear, the prevailing view is that they result from generalized defects in ribosome formation and/or function. However, because accumulating evidence suggests that many RP are RNA binding proteins, we hypothesize that the ribosome is a depot for RNA-binding proteins that can also function outside of the ribosome (i.e., extraribosomally) to regulate biological processes by binding to cellular RNAs. Thus, while it is possible that many RP play regulatory roles important in development and/or disease either from within specialized ribosomes or more likely from outside of the ribosome, the challenge in testing this possibility is that many RP are required for ribosome biogenesis and/or function; therefore, it is difficult to determine whether defects caused by RP loss result from impairment of ribosome core functions or a regulatory, extraribosomal function played by the RP itself.

    Antagonistic regulation of RNA splicing by Rpl22 and Like1.
    Antagonistic regulation of RNA splicing by Rpl22 and Like1.

    Importantly, we recently identified two RP that are not required for ribosome biogenesis or the core function of translation, yet play critical roles in hematopoiesis. These are Rpl22 and its paralog, Rpl22-Like1 (Like1), both of which are RNA-binding proteins. The recent crystal structure of the ribosome revealed that Rpl22 occupies a location in the ribosome (PICT 4) that is not near the site of mRNA entry, polypeptide exit, or the 40S/60S interface. While these proteins are dispensable for ribosome biogenesis and function, they nevertheless play important roles in regulating development. They do so by leaving the ribosome, binding cellular RNA molecules and controlling their translation and/or splicing. Despite their very high degree of homology (>70% identical protein sequence), Rpl22 and Like1 play antagonistic roles in regulating development. Rpl22 and Like1 antagonistically regulate the splicing of Smad2, which is required for gastrulation (PICT 5).

    When Like1 is knocked down in zebrafish embryos, gastrulation is blocked and exon 9 of Smad2 splicing is excised, causing the loss of Smad2 protein. Importantly, knockdown of Rpl22 rescues both the gastrulation and Smad2 splicing defect caused by Like1 knockdown. This indicates that Like1 is acting to promote accurate splicing of Smad2 while Rpl22 is acting to oppose it. These effects coincide with a stage of development when both Rpl22 and Like1 are translocated to the nucleus (PICT5).

    Control of hematopoietic stem cell emergence by Rpl22/Like1 antagonism
    Control of hematopoietic stem cell emergence by Rpl22/Like1 antagonism

    Using the zebrafish model, we also found that Like1 and Rpl22 function antagonistically in regulating the emergence of embryonic hematopoietic stem cells (HSC). Like1 knockdown arrests the development of CD41-marked embryonic HSC (PICT6) and arrest is rescued by knocking down Rpl22, indicating that Rpl22 antagonism is responsible for the arrest. The focus of this antagonism is Smad1, a critical signaling molecule required for fetal HSC emergence, with Rpl22 acting to repress Smad1 expression and Like1 acting to oppose that repression. These proteins are regulating Smad1 expression by controlling its translation. Therefore, HSC emergence is controlled by the antagonistic balance of Rpl22 and Like1 activity, with Rpl22 excess causing anemia and Like1 excess predisposing to transformation (PICT6). In Rpl22-deficient mice, the dominance of Like1 results in an expansion of the HSC compartment and a myeloid developmental bias that mimics MDS. Our current focus is on identifying: 1) the signaling processes responsible for translocation of Rpl22 and Like1 to the nucleus; 2) the spectrum of targets through which Rpl22 and Like1 regulate gastrulation and HSC emergence; and 3) the basis for Rpl22/Like1 antagonism.

  • C. Control of T Cell Development by the Ribosomal Protein Rpl22
    Shawn Fahl & Nehal Solanki-Patel, in collaboration with B. Kennedy
    P53-dependent impairment of T cell development caused by Rpl22-deficiency
    P53-dependent impairment of T cell development caused by Rpl22-deficiency

    We have found that mice lacking Rpl22 in the germline are grossly normal, but exhibit a strikingly specific defect in T cell development. Indeed, while Rpl22-deficiency had only a mild effect on development of γδ lineage T cells, it caused a profound and selective arrest of the development of αβ lineage T cells. The developmental arrest was accompanied by a significant increase in apoptosis among αβ lineage cells, which was caused by induction of p53 expression. Indeed, p53-deficiency blocked death and restored development of Rpl22-deficient thymocytes indicating that p53 is a critical target of Rpl22 regulation (PICT 7). Importantly, Rpl22-deficiency appears to induce p53 at least in part by increasing p53 synthesis. Taken together, these data indicate that Rpl22-deficiency activates a p53-dependent checkpoint that produces a remarkably selective block in αβ T cell development, but spares γδ lineage cells, suggesting that some ribosomal proteins may perform cell-type or stage-specific functions. Efforts are currently underway to elucidate the molecular basis for selective induction of p53 in Rpl22-deficient αβ lineage progenitors by addressing the following questions: 1) How is Rpl22 expression differentially regulated in αβ and γδ lineage cells?; 2) Is p53 regulation by Rpl22 direct and if so to what sites in p53 mRNA does Rpl22 bind?; 3) What is the basis for the tissue restriction of translational de-repression of p53? Recently, we have determined that Rpl22-deficiency perturbs endoplasmic reticulum stress (ER) pathways, which can induce p53 when exacerbated; and 4) How does conditional ablation of Like1 in T cell progenitors affect development? Preliminary data suggest that conditional Like1-deletion in T cell progenitors also arrests T cell development, but the basis for this arrest is unclear at present.

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  • D. Regulation of transformation by Rpl22/Like1 antagonism
    Suraj Peri & Shuyun Rao, in collaboration with Margie Clapper, B. Kennedy, D. Liebermann , Stephen Sykes & Joseph Testa
    Pathway by which loss of Rpl22 promotes transformation
    Pathway by which loss of Rpl22 promotes transformation

    Ribosomal proteins are increasingly implicated in regulating cellular transformation. We have recently uncovered a unique role for Rpl22 in transformation. Indeed, loss of one allele of Rpl22 appears to predispose T lineage precursors to transformation (PICT8). RPL22 is mutated in ~10% of T-ALL and is inversely correlated with survival. Moreover, monoallelic inactivation of Rpl22 in a mouse model of T-ALL accelerates the development of thymic lymphoma. Rpl22 inactivation does so through the activation of NFκB, which in turn induces the NFκB target Lin28B, a heterochronic gene that has been implicated in aggressive cancers in humans. What remains unclear is how Rpl22 inactivation induces NFκB. Recent evidence suggests that the exaggeration of ER stress may play a role. ER stress pathways have been found to activate NFκB and Rpl22 has been linked to growth control in yeast in an ER stress dependent manner. While pediatric T-ALL is successfully treated in most cases, the prognosis for the remaining cases in which relapse occurs continues to be dismal. Accordingly, additional therapeutic approaches are desperately needed. We are currently evaluating the efficacy of targeting ER stress pathways in T-ALL.

    Rpl22 loss appears to contribute to the etiology of other hematologic malignancies as well. Analysis of Rpl22 expression levels in AML has revealed that patients with reduced Rpl22 levels exhibit reduced survival. Moreover, Like1 protein levels are induced in MDS and AML. Because we have recently determined that ectopic expression of Like1 promotes transformation in vitro, it is possible that Like1 induction contributes to the pathogenesis of MDS and in AML.

    Our current focus is to elucidate the mechanistic basis by which Rpl22 loss regulates transformation by addressing the following questions: 1) How does Rpl22 regulate stress responses and does this influence the development of cancer?; 2) What are the cellular targets whose expression is regulated by Rpl22 and how are they involved in transformation? and 3) How does Like1 induction promote transformation?

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  • E. Forward Genetic Screens to Identify Genes Essential for T Cell Development
    Yong Zhang, in collaboration with Richard Hardy, Dietmar Kappes & Jennifer Rhodes

    Because of the conservation of essential elements of hematopoiesis between zebrafish and man, we are undertaking a forward genetic screen in zebrafish to identify genes essential for normal T cell development. Details may be found at the web site of Dr. Jennifer Rhodes. The motivation for this effort is our hypothesis that genes that are essential for normal T cell development will also be involved in transformation. Studies on the Rpl22 gene described above are supportive of this idea.

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  • F. Exome resequencing to identify genetic causes of human Severe Combined Immunodeficiency Disease (SCID).
    Yong Zhang, in collaboration with J. Puck & Jennifer Rhodes

    Causes of SCID in humans have all been mapped to mutations that affect coding sequences. Efforts are ongoing to identify causes of SCID in patients by performing massively parallel sequencing of the exomes of SCID kindreds using the Illumina platform and then screening candidate genes for function in zebrafish. This represents yet another strategy with which to identify genes that are essential for normal immune cell development. Following identification, their role in the etiology or diagnosis of hematologic malignancies will be explored.

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  • G. Lab Member Research Interests
  • Identifying intracellular signaling pathways that influence αβ/γδ lineage commitment during thymocyte development

    Francis Coffey

    The role of TCR-mediated signals in the adoption of the αβ or γδ lineage by developing thymocytes remains under investigation. Data from our lab and others suggest that quantitative differences in the strength of TCR-mediated signals promote development to either the αβ or γδ lineage. Accordingly, we have shown that stronger signals promote the development of γδ T cells, while weaker signals result in αβ lineage commitment. Our current studies aim to identify molecular pathways that have a role in the differentiation of T cell precursors to the αβ or γδ lineage, including the impact of TCR:ligand interactions on this signaling. We aim to identify genes and signaling pathways that play a role in the lineage fate of developing αβ or γδ T cells, in part by manipulating the duration and intensity of TCR-mediated signals.

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  • Identify new genes essential for normal T cell development and diseases

    Yong Zhang

    There is now more and more evidence indicates that many genes and signaling pathways which control the ontogeny and progress of diseases are also essential for normal embryonic development. The zebrafish (Danio rerio) is a powerful developmental and genetic system for the dissection of events in the thymic organogenesis and T-cell development which will provide important information for unraveling the molecular pathogenesis of human T cell development and disease. 1) To further our understanding the function of rpl22 and rpl22 like1, we are now using the zebrafish model system to address how rpl22 and rpl22 like1 may contribute to T cell development and thymopoiesis in vivo. 2) Based on the "phenotype-driven" forward genetic screening, we are undertaking the gene trapper screen contains GFP/RFP reporters in zebrafish to identify new genes essential for normal T cell development and diseases.

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  • Identifying the molecular mechanisms that control αβ/γδ lineage commitment during thymocyte development

    Shawn Fahl

    αβ and γδ T cell arise from a common precursor in the thymus. The molecular mechanisms that control αβ versus γδ lineage commitment, however, remain poorly understood. We have previously demonstrated that differences in TCR-mediated signal strength play a crucial role during lineage commitment. Stronger signals promote the adoption of the γδ T cell fate while weaker signals result in αβ T cell commitment and these lineage fate decisions are dependent on the amplitude of the activation of the ERK-Egr-Id3 pathway downstream of the TCR. Id3 suppresses the activity of the E protein family of transcription factors, which have been described to play crucial roles during T cell development. Our current studies aim to identify the downstream targets of E proteins that influence αβ versus γδ lineage commitment. In addition, we aim to identify novel transcription regulators that are modulated downstream of TCR signaling to influence of the development of αβ or γδ T cells.

  • Role of Rpl22 in maintaining genomic integrity of hematopoietic stem cells

    Bryan Harris

    The goal of my thesis research in the Wiest lab to date has been to identify the role of RpL22 in maintaining the hematopoietic stem cell population (HSC), as well as the mechanisms by which its loss confers resistance to leukemia treatments. HSCs in the bone marrow represent a unique cell population that is capable of long-term multi-lineage reconstitution. An essential feature of cells with self-renewal capacity is proficiency in maintaining genomic integrity in response to frequent insults. Alternatively, irreparable DNA damage causes HSCs to undergo senescence or differentiation in order to prevent leukemogenesis. We have found that Rpl22 is indispensable for the ability of HSCs to appropriately respond to genotoxic stress. In moving forward, I will elucidate the mechanisms by which RpL22 accomplishes this task and the relevance this has on treating leukemia patients with decreased expression of Rpl22.

  • Regulation of cellular transformation by Rpl22

    Shuyun Rao

    My primary research interest is in cancer biology. I seek to understand the molecular mechanism by which ribosomal proteins exert either anti-tumor or pro-tumor effects. Lymphoma/leukemia is a group of cancer types involving abnormal blood cells. I have discovered a ribosomal protein called Rpl22 that functions as a tumor suppressor in thymic lymphoma. Recent data revealed two other critical oncogenic proteins are upregulated in tumorigenesis caused by Rpl22 loss, (Lin28b and ribosomal protein Rpl22-Like1). I am studying the molecular mechanism of these two proteins in oncogenesis and how Rpl22 regulates their expression levels.

  • Investigate the role of Ribosomal protein L22 in lymphocyte development and transformation

    Nehal Solanki-Patel

    My graduate research project seeks to understand how Rpl22 controls both the normal development of lymphocytes and how they become transformed. We have shown that Rpl22-deficiency causes a selective block in the development of αβ T cells at the pre-T cell receptor controlled β-selection checkpoint in a p53-dependent manner. My first goal is to investigate the basis of selective induction on p53 upon Rpl22 loss. We hypothesize that Rpl22 regulates αβ T cell development by functioning as a translational repressor that can both directly repress p53 synthesis and control the activity of signaling processes that induce p53, such as endoplasmic reticulum (ER) stress. Our lab has also identified a role for Rpl22 as a tumor suppressor. The RPL22 gene is inactivated in a variety of cancers and serves as a negative prognostic indicator in some leukemias. In spite of the association between Rpl22 and transformation, the mechanism of transformation and its link to L22 remains unclear. We hypothesize that ribosomal protein Rpl22 talks to ER stress and cellular survival pathways to regulate lymphocyte development and transformation.

  • Ribosomal proteins (RPLs) are ubiquitously expressed proteins involved in the cellular process of translation

    Suraj Peri

    Ribosomal proteins (RPLs) are ubiquitously expressed proteins involved in the cellular process of translation. While the extra-ribosomal functions of many RPLs have been described, if not extensively, the sweeping effects of functional loss of a ribosomal protein are currently unknown. My colleagues in the lab have recently described Rpl22 as a tumor suppressor by demonstrating that its loss leading to Lin28B mediated transformation in T-ALL. Moreover, the results from TCGA and other comprehensive genome analyses revealed that Rpl22 function is frequently lost in many cancers including lung, endometrial and colon cancers. Since Rpl22 is located on p36 locus of chromosome 1, and that this locus is frequently deleted in colon cancer, we reasoned loss of Rpl22 play role in transformation of colon. A bioinformatics analyses of publicly available aCGH datasets indicated loss of Rpl22 in adenoma and dysplastic lesions of colon suggesting Rpl22 loss is an early event in colon cancer. My research interests in the lab in this context are:

    1. Assess expression of RPL22 in human colon pathogenesis using comprehensive bioinformatics evaluation of genomic datasets.
    2. To examine the role of Rpl22 in development of colonic neoplasia and to identify the molecular targets of Rpl22 using murine colon cancer models.
    3. Determine how loss of Rpl22 affects cellular response.
  • RPL22 Role in Childhood Acute Lymphoblasric Leukemia

    Noa Greenberg-Kushnir

    Recent work from our lab identified RPL22’s role as a crucial mediator of both T and B cells development. RPL22 locus is inactivated in a large proportion of pediatric patients with high-risk acute lymphoblastic leukemia (ALL) and is correlated with poor clinical outcome; nevertheless the way that RPL22 loss accelerates leukemic transformation remains poorly understood. Our current studies focus on interrogating genomic data from large cohorts of pediatric ALL patients in order to assess RPL22’s role in leukemogenesis, as well as its clinical significance. We hope to identify dysregulated novel pathways that are associated with RPL22 loss, which will serve as targets for therapeutic intervention. By targeting those dysregulated pathways we wish to expand and improve standard of care treatment for high-risk pediatric ALL patients.