C J Der

University of North Carolina at Chapel Hill, North Carolina, United States

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Publications (321)2302.95 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Defining the full complement of substrates for each ubiquitin ligase remains an important challenge. Improvements in mass spectrometry instrumentation, computation and protein biochemistry methods have resulted in several new methods for ubiquitin ligase substrate identification. Here we used the parallel adapter capture (PAC) proteomics approach to study βTrCP2/FBXW11, a substrate adaptor for the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex. The processivity of the ubiquitylation reaction necessitates transient physical interactions between FBXW11 and its substrates, thus making biochemical purification of FBXW11-bound substrates difficult. Using the PAC-based approach, we inhibited the proteasome to 'trap' ubiquitylated substrates on the SCF(FBXW11) E3 complex. Comparative mass spectrometry analysis of immunopurified FBXW11 protein complexes before and after proteasome inhibition revealed 21 known and 23 putatively novel substrates. In focused studies, we found that SCF(FBXW11) bound, polyubiquitylated and destabilized RAPGEF2, a guanine nucleotide exchange factor that activates the small GTPase RAP1. High RAPGEF2 protein levels promoted cell-cell fusion and consequently multinucleation. Surprisingly, this occurred independently of the GEF catalytic activity and of RAP1. Our data establish new functions for RAPGEF2 that may contribute to aneuploidy in cancer. More broadly, this study supports the continued use of substrate trapping proteomics to comprehensively define targets for E3 ubiquitin ligases. All proteomic data are available via ProteomeXchange with identifier PXD001062.
    Molecular and cellular biology. 10/2014;
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    ABSTRACT: Despite more than three decades of intensive effort, no effective pharmacological inhibitors of the RAS oncoproteins have reached the clinic, prompting the widely held perception that RAS proteins are 'undruggable'. However, recent data from the laboratory and the clinic have renewed our hope for the development of RAS-inhibitory molecules. In this Review, we summarize the progress and the promise of five key approaches. Firstly, we focus on the prospects of using direct inhibitors of RAS. Secondly, we address the issue of whether blocking RAS membrane association is a viable approach. Thirdly, we assess the status of targeting RAS downstream effector signalling, which is arguably the most favourable current approach. Fourthly, we address whether the search for synthetic lethal interactors of mutant RAS still holds promise. Finally, RAS-mediated changes in cell metabolism have recently been described and we discuss whether these changes could be exploited for new therapeutic directions. We conclude with perspectives on how additional complexities, which are not yet fully understood, may affect each of these approaches.
    Nature reviews. Drug discovery. 10/2014;
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    ABSTRACT: Cancers driven by oncogenic Ras proteins encompass some of the most deadly human cancer types, and there is a pressing need to develop therapies for these diseases. Although recent studies suggest that mutant Ras proteins may yet be druggable, the most promising and advanced efforts involve inhibitors of Ras effector signaling. Most efforts to target Ras signaling have been aimed at the ERK mitogen-activated protein kinase and the phosphoinositide 3-kinase signaling networks. However, to date, no inhibitors of these Ras effector pathways have been effective against RAS-mutant cancers. This ineffectiveness is due, in part, to the involvement of additional effectors in Ras-dependent cancer growth, such as the Rac small GTPase and the p21-activated serine-threonine kinases (PAK). PAK proteins are involved in many survival, cell motility, and proliferative pathways in the cell and may present a viable new target in Ras-driven cancers. In this review, we address the role and therapeutic potential of Rac and group I PAK proteins in driving mutant Ras cancers. Clin Cancer Res; 20(18); 4740-6. ©2014 AACR.
    Clinical cancer research : an official journal of the American Association for Cancer Research. 09/2014; 20(18):4740-6.
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    ABSTRACT: Since their discovery in 1986, Ral (Ras-like) GTPases have emerged as critical regulators of diverse cellular functions. Ral-selective guanine nucleotide exchange factors (RalGEFs) function as downstream effectors of the Ras oncoprotein, and the RalGEF-Ral signaling network comprises the third best characterized effector of Ras-dependent human oncogenesis. Because of this, Ral GTPases as well as their effectors are being explored as possible therapeutic targets in the treatment of RAS mutant cancer. The two Ral isoforms, RalA and RalB, interact with a variety of downstream effectors and have been found to play key and distinct roles in both normal and neoplastic cell physiology including regulation of vesicular trafficking, migration and invasion, tumor formation, metastasis, and gene expression. In this review we provide an overview of Ral biochemistry and biology, and we highlight recent discoveries.
    Biochimica et biophysica acta. 09/2014;
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    ABSTRACT: G protein-coupled receptors (GPCRs) regulate organisation of the actin cytoskeleton by activating the Rac subfamily of small GTPases. The guanine nucleotide exchange factor (GEF) P-Rex1 is engaged downstream of GPCRs and PI3K in many cell types, and promotes tumorigenic signalling and metastasis in breast cancer and melanoma, respectively. Although P-Rex1-dependent functions have been attributed to its GEF activity towards Rac1, we show that P-Rex1 also acts as a GEF for the Rac-related GTPase RhoG, both in vitro and in GPCR-stimulated primary mouse neutrophils. Further, loss of either P-Rex1 or RhoG caused equivalent reductions in GPCR-driven Rac activation and Rac-dependent NADPH oxidase activity, suggesting they both function upstream of Rac in this system. Loss of RhoG also impaired GPCR-driven recruitment of the RacGEF DOCK2, and F-actin, to the leading edge of migrating neutrophils. Together, our results reveal a novel signalling hierarchy in which P-Rex1, acting as a GEF for RhoG, regulates Rac-dependent functions indirectly via RhoG-dependent recruitment of DOCK2. These findings thus have broad implications for our understanding of GPCR signalling to RhoGTPases and the actin cytoskeleton.
    Journal of Cell Science 03/2014; · 5.88 Impact Factor
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    ABSTRACT: Oncogenic KRAS mutation is the signature genetic event in the progression and growth of pancreatic ductal adenocarcinoma (PDAC), an almost universally fatal disease. Although it has been appreciated for some time that nearly 95% of PDAC harbor mutationally activated KRAS, to date no effective treatments that target this mutant protein have reached the clinic. A number of studies have shown that oncogenic KRAS plays a central role in controlling tumor metabolism by orchestrating multiple metabolic changes including stimulation of glucose uptake, differential channeling of glucose intermediates, reprogrammed glutamine metabolism, increased autophagy, and macropinocytosis. We review these recent findings and address how they may be applied to develop new PDAC treatments.
    Trends in Biochemical Sciences 01/2014; · 13.08 Impact Factor
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    ABSTRACT: Diverse environmental cues converge on and are integrated by the mTOR signaling network to control cellular growth and homeostasis. The mammalian Tsc1-Tsc2 GTPase activating protein (GAP) heterodimer is a critical negative regulator of Rheb and mTOR activation. The RalGAPα-RalGAPβ heterodimer shares sequence and structural similarity with Tsc1-Tsc2. Unexpectedly, we observed that C. elegans expresses orthologs for the Rheb and RalA/B GTPases and for RalGAPα/β, but not Tsc1/2. This prompted our investigation to determine whether RalGAPs additionally modulate mTOR signaling. We determined that C. elegans RalGAP loss decreased lifespan, consistent with a Tsc-like function. Additionally, RalGAP suppression in mammalian cells caused RalB-selective activation and Sec5- and exocyst-dependent engagement of mTORC1 and suppression of autophagy. Unexpectedly, we also found that Tsc1-Tsc2 loss activated RalA/B independently of Rheb-mTOR signaling. Finally, RalGAP suppression caused mTORC1-dependent pancreatic tumor cell invasion. Our findings identify an unexpected crosstalk and integration of the Ral and mTOR signaling networks.
    Molecular cell 12/2013; · 14.61 Impact Factor
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    ABSTRACT: The high prevalence of KRAS mutations and importance of the RalGEF-Ral pathway downstream of activated K-Ras in pancreatic ductal adenocarcinoma (PDAC) emphasize the importance of identifying novel methods by which to therapeutically target these pathways. It was recently demonstrated that phosphorylation of RalA S194 by Aurora A kinase is critical for PDAC tumorigenesis. We sought to evaluate the Aurora A kinase-selective inhibitor MLN8237 as a potential indirect anti-RalA targeted therapy for PDAC. We utilized a site-specific phospho-S194 RalA antibody and determined that RalA S194 phosphorylation levels were elevated in a subset of PDAC cell lines and human tumors relative to unmatched normal controls. Effects of MLN8237 on anchorage-independent growth in PDAC cell lines and growth of patient-derived xenografts (PDX) were variable, with a subset of cell lines and PDX showing sensitivity. Surprisingly, RalA S194 phosphorylation levels in PDAC cell lines or PDX tumors did not correlate with MLN8237 responsiveness. However, we identified Ki67 as a possible early predictive biomarker for response to MLN8237 in PDAC. These results indicate that MLN8237 treatment may be effective for a subset of PDAC patients independent of RalA S194 phosphorylation. Ki67 may be an effective pharmacodynamic biomarker to identify response early in the course of treatment.
    Molecular Cancer Therapeutics 11/2013; · 5.60 Impact Factor
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    ABSTRACT: Ect2, a Rho guanine nucleotide exchange factor (RhoGEF), is atypical among RhoGEFs in its predominantly nuclear localization in interphase cells. One current model suggests that Ect2 mislocalization drives cellular transformation by promoting aberrant activation of cytoplasmic Rho family GTPase substrates. However, in ovarian cancers, where Ect2 is both amplified and overexpressed at the mRNA level, we observed that the protein is highly expressed and predominantly nuclear and that nuclear but not cytoplasmic Ect2 increases with advanced disease. Knockdown of Ect2 in ovarian cancer cell lines impaired their anchorage-independent growth without affecting their growth on plastic. Restoration of Ect2 expression rescued the anchorage-independent growth defect, but not if either the DH catalytic domain or the nuclear localization sequences of Ect2 were mutated. These results suggested a novel mechanism whereby Ect2 could drive transformation in ovarian cancer cells by acting as a RhoGEF specifically within the nucleus. Interestingly, Ect2 had an intrinsically distinct GTPase specificity profile in the nucleus versus the cytoplasm. Nuclear Ect2 bound preferentially to Rac1, while cytoplasmic Ect2 bound to RhoA but not Rac. Consistent with nuclear activation of endogenous Rac, Ect2 overexpression was sufficient to recruit Rac effectors to the nucleus, a process that required a functional Ect2 catalytic domain. Furthermore, expression of active nuclearly targeted Rac1 rescued the defect in transformed growth caused by Ect2 knockdown. Our work suggests a novel mechanism of Ect2-driven transformation, identifies subcellular localization as a regulator of GEF specificity, and implicates activation of nuclear Rac1 in cellular transformation.
    Genes & cancer 11/2013; 4(11-12):460-75.
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    ABSTRACT: Histone deacetylase 6 (HDAC6) is well known for its ability to promote cell migration through deacetylation of its cytoplasmic substrates such as tubulin. However, how HDAC6 itself is regulated to control cell motility remains elusive. Previous studies have shown that one-third of extracellular signal-regulated kinase (ERK) is associated with the microtubule cytoskeleton in cells. Yet, no connection between HDAC6 and ERK has been discovered. Here, for the first time, we reveal that ERK binds to and phosphorylates HDAC6 to promote cell migration via deacetylation of tubulin. We have identified two novel ERK-mediated phosphorylation sites: Threonine 1031 (T1031) and Serine 1035 (S1035) in HDAC6. Both sites were phosphorylated by ERK1 in vitro, whereas S1035 was phosphorylated in response to the activation of EGFR-Ras-Raf-MEK-ERK signaling pathway in vivo. HDAC6-null mouse embryonic fibroblasts (MEFs) rescued by the nonphosphorylation mimicking mutant displayed significantly reduced cell migration as compared to those rescued by the wild type. Consistently, the nonphosphorylation mimicking mutant exerted lower tubulin deacetylase activity in vivo compared with the wild type. This data indicates that ERK/HDAC6-mediated cell motility is through deacetylation of tubulin. Overall, our results suggest that HDAC6-mediated cell migration could be governed by EGFR-Ras-Raf-MEK-ERK signaling.
    Journal of Biological Chemistry 10/2013; · 4.65 Impact Factor
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    ABSTRACT: DLC1 encodes a RhoA GTPase-activating protein and tumor suppressor lost in cancer by genomic deletion or epigenetic silencing and loss of DLC1 gene transcription. We unexpectedly identified non-small cell lung cancer (NSCLC) cell lines and tumor tissue that expressed DLC1 mRNA yet lacked DLC1 protein expression. We determined that DLC1 was ubiquitinated and degraded by cullin 4A-RING ubiquitin ligase (CRL4A) complex interaction with DDB1 and the FBXW5 substrate receptor. siRNA-mediated suppression of cullin 4A, DDB1, or FBXW5 expression restored DLC1 protein expression in NSCLC cell lines. FBXW5 suppression-induced DLC1 reexpression was associated with a reduction in the levels of activated RhoA-GTP and in RhoA effector signaling. Finally, FBXW5 suppression caused a DLC1-dependent decrease in NSCLC anchorage-dependent and -independent proliferation. In summary, we identify a posttranslational mechanism for loss of DLC1 and a linkage between CRL4A-FBXW5-associated oncogenesis and regulation of RhoA signaling.
    Proceedings of the National Academy of Sciences 09/2013; · 9.81 Impact Factor
  • D R Cook, K L Rossman, C J Der
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    ABSTRACT: The aberrant activity of Ras homologous (Rho) family small GTPases (20 human members) has been implicated in cancer and other human diseases. However, in contrast to the direct mutational activation of Ras found in cancer and developmental disorders, Rho GTPases are activated most commonly in disease by indirect mechanisms. One prevalent mechanism involves aberrant Rho activation via the deregulated expression and/or activity of Rho family guanine nucleotide exchange factors (RhoGEFs). RhoGEFs promote formation of the active GTP-bound state of Rho GTPases. The largest family of RhoGEFs is comprised of the Dbl family RhoGEFs with 70 human members. The multitude of RhoGEFs that activate a single Rho GTPase reflects the very specific role of each RhoGEF in controlling distinct signaling mechanisms involved in Rho activation. In this review, we summarize the role of Dbl RhoGEFs in development and disease, with a focus on Ect2 (epithelial cell transforming squence 2), Tiam1 (T-cell lymphoma invasion and metastasis 1), Vav and P-Rex1/2 (PtdIns(3,4,5)P3 (phosphatidylinositol (3,4,5)-triphosphate)-dependent Rac exchanger).Oncogene advance online publication, 16 September 2013; doi:10.1038/onc.2013.362.
    Oncogene 09/2013; · 8.56 Impact Factor
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    ABSTRACT: The effectiveness of cancer therapeutics targeting signal transduction pathways is comprised of a diversity of mechanisms that drive de novo or acquired resistance. Two recent studies identify mTOR activation as a point of convergence of mechanisms that cause resistance to inhibitors of the Raf-MEK-ERK and PI3K signaling.
    Cancer cell 09/2013; 24(3):284-6. · 25.29 Impact Factor
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    ABSTRACT: Signaling events mediated by Rho family GTPases orchestrate cytoskeletal dynamics and cell junction formation. The activation of Rho GTPases is tightly regulated by guanine nucleotide exchange-factors (GEFs). In this study, we identified a novel Rhospecific GEF called TEM4 (Tumor Endothelial Marker 4) that associates with multiple members of the cadherin-catenin complex and with several cytoskeleton-associated proteins. Depending on confluence, TEM4 localized to either actin stress fibers, or areas of cell-cell contact. The junctional localization of TEM4 was independent of actin binding. Depletion of endogenous TEM4 by shRNAs impaired Madin-Darby Canine Kidney (MDCK) and Human Umbilical Vein Endothelial Cell (HUVEC) cell junctions, disrupted MDCK acini formation in 3D culture, and negatively affected endothelial barrier function. Together, our findings implicate TEM4 as a novel and critical junctional RhoGEF that regulates cell junction integrity and epithelial and endothelial cell function.
    Journal of Cell Science 05/2013; · 5.88 Impact Factor
  • Nicole M Baker, Channing J Der
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    ABSTRACT: Scientists have long aimed to develop drugs against the cancer-associated protein KRAS, but without success. An approach that targets the oncoprotein's cellular localization reignites lost enthusiasm. See Letter p.638
    Nature 05/2013; · 38.60 Impact Factor
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    ABSTRACT: p21-activated kinases (Paks) are prominent mediators of Rac/Cdc42-dependent and -independent signaling and regulate signal transduction and cytoskeletal-based cell movements. We used the reproducible migrations of the Caenorhabditis elegans gonadal distal tip cells to show that two of the three nematode Pak proteins, MAX-2 and PAK-1, function redundantly in regulation of cell migration but are regulated by very different mechanisms. First, we suggest that MAX-2 requires CED-10/Rac function and thus functions canonically. Second, PIX-1 and GIT-1 function in the same role as PAK-1, and PAK-1 interaction with PIX-1 is required for PAK-1 activity; thus, PAK-1 functions noncanonically. The human Pak-Pix-Git complex is central to noncanonical Pak signaling and requires only modest Rac/CDC-42 input. Unlike the human complex, our results suggest that the C. elegans Pak-Pix-Git complex requires PAK-1 kinase domain activity. This study delineates signaling network relationships in this cell migration model, thus providing potential further mechanistic insights and an assessment of total Pak contribution to cell migration events.
    G3-Genes Genomes Genetics 02/2013; 3(2):181-95. · 1.79 Impact Factor
  • Leanna Gentry, Ahmed A Samatar, Channing J Der
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    ABSTRACT: Although recent success in identifying direct inhibitors of mutant Ras has begun to challenge the perception that Ras is "undruggable," the successful transition of these hits to the clinic remains uncertain. Therefore, current efforts to develop anti-Ras inhibitors are focused on indirect approaches, with inhibitors of the downstream effectors of Ras signaling having attracted the greatest current interest. Of the multitude of effectors, the Raf-MEK-ERK mitogen-activated protein kinase (MAPK) cascade is arguably the most attractive target for these efforts. In this chapter, we review the evidence for a key driver role for the ERK MAPK cascade in RAS mutant cancers and the status of efforts to develop inhibitors of MEK1/2 and ERK1/2 to block this pathway.
    The enzymes / edited by Paul D. Boyer. 01/2013; 34:67-106.
  • Tikvah K Hayes, Channing J Der
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    ABSTRACT: Although the functional interplay between mutant and wild-type Ras in driving tumor initiation and growth has been described, a clear picture of the precise ramifications and mechanisms of this association remains elusive, sometimes with conflicting conclusions. A report in this issue of Cancer Discovery tackles this question, which may have important implications for therapeutic strategies to block mutant Ras for cancer treatment. Cancer Discov; 3(1); 24-6. ©2012 AACR.
    Cancer Discovery 01/2013; 3(1):24-6. · 15.93 Impact Factor
  • Dominico Vigil, Channing J Der
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    ABSTRACT: Aberrant activation of the RhoA small GTPase has been implicated in cancer and other human diseases. Therefore, inhibitors of RhoA may have important therapeutic value. However, similar to the Ras small GTPases, RhoA itself is not considered a tractable target and is currently considered to be "undruggable." While recent efforts suggest that direct inhibitors of the Ras oncoprotein may yet be developed, the most promising directions for anti-Ras inhibitors involve inhibitors of protein kinases that are activated downstream of Ras. By analogy, protein kinases activated downstream of RhoA may provide more attractive directions for the development of anti-RhoA inhibitors. Among the multitude of RhoA effectors, the ROCK serine/threonine kinases have emerged as attractive targets for anti-RhoA drug discovery. In this review, we summarize the current status of the development of small molecule inhibitors of ROCK.
    The enzymes / edited by Paul D. Boyer. 01/2013; 33:193-212.
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    ABSTRACT: Persistent cellular migration requires efficient protrusion of the front of the cell, the leading edge where the actin cytoskeleton and cell-substrate adhesions undergo constant rearrangement. Rho family GTPases are essential regulators of the actin cytoskeleton and cell adhesion dynamics. Here, we examined the role of the RhoGEF TEM4, an activator of Rho family GTPases, in regulating cellular migration of endothelial cells. We found that TEM4 promotes the persistence of cellular migration by regulating the architecture of actin stress fibers and cell-substrate adhesions in protruding membranes. Furthermore, we determined that TEM4 regulates cellular migration by signaling to RhoC as suppression of RhoC expression recapitulated the loss-of-TEM4 phenotypes, and RhoC activation was impaired in TEM4-depleted cells. Finally, we showed that TEM4 and RhoC antagonize myosin II-dependent cellular contractility and the suppression of myosin II activity rescued the persistence of cellular migration of TEM4-depleted cells. Our data implicate TEM4 as an essential regulator of the actin cytoskeleton that ensures proper membrane protrusion at the leading edge of migrating cells and efficient cellular migration via suppression of actomyosin contractility.
    PLoS ONE 01/2013; 8(6):e66260. · 3.53 Impact Factor

Publication Stats

21k Citations
2,302.95 Total Impact Points

Institutions

  • 1993–2014
    • University of North Carolina at Chapel Hill
      • • Lineberger Comprehensive Cancer Center
      • • Department of Radiation Oncology
      • • Department of Pharmacology
      • • Department of Medicine
      North Carolina, United States
  • 2011
    • North Carolina Central University
      • Department of Biology
      Durham, North Carolina, United States
  • 2002–2009
    • Duke University Medical Center
      • • Department of Pharmacology and Cancer Biology
      • • Division of Neurology
      Durham, NC, United States
  • 2005
    • Temple University
      Philadelphia, Pennsylvania, United States
    • Cytoskeleton, Inc.
      Denver, Colorado, United States
  • 2004
    • Colorado College
      Colorado Springs, Colorado, United States
    • Moffitt Cancer Center
      Tampa, Florida, United States
  • 1999–2001
    • Rutgers New Jersey Medical School
      • Department of Microbiology and Molecular Genetics
      Newark, NJ, United States
    • Iowa State University
      Ames, Iowa, United States
  • 1979–2001
    • University of California, Irvine
      • Department of Microbiology & Molecular Genetics
      Irvine, CA, United States
  • 1998
    • University of Adelaide
      Tarndarnya, South Australia, Australia
    • University of Michigan
      • Department of Biological Chemistry
      Ann Arbor, MI, United States
  • 1992–1998
    • Massachusetts Institute of Technology
      • Department of Biology
      Cambridge, Massachusetts, United States
  • 1996
    • IT University of Copenhagen
      København, Capital Region, Denmark
    • Indiana University-Purdue University Indianapolis
      Indianapolis, Indiana, United States
  • 1995
    • Wistar Institute
      Philadelphia, Pennsylvania, United States
  • 1994
    • University of Zurich
      • Physiologisches Institut
      Zürich, ZH, Switzerland
  • 1990–1993
    • The Scripps Research Institute
      • Department of Cell and Molecular Biology
      La Jolla, CA, United States
  • 1989
    • Children's Hospital Los Angeles
      • Division of Hematology-Oncology
      Los Angeles, CA, United States
    • University of Texas Southwestern Medical Center
      • Department of Pharmacology
      Dallas, TX, United States