Academic Appointments

Honors & Awards

  • K99/R00 Pathway to Independence Award, National Cancer Institute, National Institutes of Health (3/01/2017)
  • Honorary Award for Outstanding Presentation, Stanford Hematology-Oncology Retreat, Asilomar (2015)
  • Outstanding External Grant Reviewer for 2014, National Health and Medical Research Council of Australia (2015)
  • Institute's Best Publication for 2013, Centre for Cancer Biology (2014)
  • Leukemia Foundation New Investigator Award 2014, Leukemia Foundation: New Directions in Leukemia 2014 (2014)
  • Prize: Dean?s Commmendation for Doctoral Thesis Excellence, University of Adelaide (2012)
  • CJ Martin Fellowship, National Health and Medical Research Council of Australia (2011)
  • HOTT Haematology/Oncology Targetted Therapy Fellowship, Haematology Society of Australia & New Zealand/COSA/Roche/COGA (2010)
  • Nimmo Prize, best full-time research, Royal Adelaide Hospital (2010)
  • Outstanding Research Presentation Prize, New Directions in Leukaemia Conference (2010)
  • National FRACP Adult Medicine Research Award, Royal Australasian College of Physicians (2008)
  • Albert Baikie Memorial Medal, Haematology Society of Australia & New Zealand (2007)
  • John Chalmers Prize for Best Medical Research Presentation, Royal Australasian College of Physicians (2007)
  • Hugh Gilmore Prize, Royal Adelaide Hospital (2004)

Professional Education

  • PhD, University of Adelaide, Leukemia Stem Cells:- Signalling and Therapeutic Targets (2012)
  • FRACP, Fellow of the Royal Australasian College of Physicians, Hematology (2007)
  • FRCPA, Fellow of the Royal College of Pathologists of Australasia, Hematology (2007)

Community and International Work

  • Homes of Hope, Tijuana


    Community Development

    Partnering Organization(s)

    Youth with a Mission

    Populations Served

    Mexican families



    Ongoing Project


    Opportunities for Student Involvement


  • Medical Mission to Syrian Refugees, Lebanon

    Partnering Organization(s)

    Horizons International

    Populations Served

    Syrian refugee



    Ongoing Project


    Opportunities for Student Involvement


Research & Scholarship

Current Research and Scholarly Interests

Whilst cancer is first a disease of unregulated cell proliferation, a small proportion of cells within a tumour are paradoxically in a relative state of quiescence. These chemotherapy-resistant cells are the likely culprits responsible for relapse but the signalling events that control this state are unknown. My work involves the design of mutation-specific therapeutics to target acute myeloid leukemia cancer stem cells.


All Publications

  • Data mining for mutation-specific targets in acute myeloid leukemia. Leukemia Benard, B., Gentles, A. J., Kohnke, T., Majeti, R., Thomas, D. 2019


    Three mutation-specific targeted therapies have recently been approved by the FDA for the treatment of acute myeloid leukemia (AML): midostaurin for FLT3 mutations, enasidenib for relapsed or refractorycases with IDH2 mutations, and ivosidenib for cases with an IDH1 mutation. Together, these agents offer a mutation-directed treatment approach for up to 45% of de novo adult AML cases, a welcome deluge after a prolonged drought. At the same time, a number of computational tools have recently been developed that promise to further accelerate progress in mutation-specific therapy for AML and other cancers. Technical advances together with comprehensively annotated AML tissue banks have resulted in the availability of large and complex data sets for exploration by the end-user, including (i) microarray gene expression, (ii) exome sequencing, (iii) deep sequencing data of sub-clone heterogeneity, (iv) RNA sequencing of gene expression (bulk and single cell), (v) DNA methylation and chromatin, (vi) and germline quantitative trait loci. Yet few clinicians or experimental hematologists have the time or the training to access or analyze these repositories. This review summarizes the data sets and bioinformatic tools currently available to further the discovery of mutation-specific targets with an emphasis on web-based applications that are open, accessible, user-friendly, and do not require coding experience to navigate. We show examples of how available data can be mined to identify potential targets using synthetic lethality, drug repurposing, epigenetic sub-grouping, and proteomic networks while also highlighting strengths and limitations and the need for superior models for validation.

    View details for PubMedID 30728456

  • Predicting response to new drugs in AML from simulation modelling: Value of the BEAT AML project as a validation resource. Leukemia research Benard, B., Thomas, D. 2019

    View details for PubMedID 30827724

  • Accumulation of JAK activation loop phosphorylation is linked to type I JAK inhibitor withdrawal syndrome in myelofibrosis. Science advances Tvorogov, D., Thomas, D., Liau, N. P., Dottore, M., Barry, E. F., Lathi, M., Kan, W. L., Hercus, T. R., Stomski, F., Hughes, T. P., Tergaonkar, V., Parker, M. W., Ross, D. M., Majeti, R., Babon, J. J., Lopez, A. F. 2018; 4 (11): eaat3834


    Treatment of patients with myelofibrosis with the type I JAK (Janus kinase) inhibitor ruxolitinib paradoxically induces JAK2 activation loop phosphorylation and is associated with a life-threatening cytokine-rebound syndrome if rapidly withdrawn. We developed a time-dependent assay to mimic ruxolitinib withdrawal in primary JAK2V617F and CALR mutant myelofibrosis patient samples and observed notable activation of spontaneous STAT signaling in JAK2V617F samples after drug washout. Accumulation of ruxolitinib-induced JAK2 phosphorylation was dose dependent and correlated with rebound signaling and the presence of a JAK2V617F mutation. Ruxolitinib prevented dephosphorylation of a cryptic site involving Tyr1007/1008 in JAK2 blocking ubiquitination and degradation. In contrast, a type II JAK inhibitor, CHZ868, did not induce JAK2 phosphorylation, was not associated with withdrawal signaling, and was superior in the eradication of flow-purified JAK2V617F mutant CD34+ progenitors after drug washout. Type I inhibitor-induced loop phosphorylation may act as a pathogenic signaling node released upon drug withdrawal, especially in JAK2V617F patients.

    View details for PubMedID 30498775

  • Systematic discovery of mutation-specific synthetic lethals by mining pan-cancer human primary tumor data. Nature communications Sinha, S., Thomas, D., Chan, S., Gao, Y., Brunen, D., Torabi, D., Reinisch, A., Hernandez, D., Chan, A., Rankin, E. B., Bernards, R., Majeti, R., Dill, D. L. 2017; 8: 15580-?


    Two genes are synthetically lethal (SL) when defects in both are lethal to a cell but a single defect is non-lethal. SL partners of cancer mutations are of great interest as pharmacological targets; however, identifying them by cell line-based methods is challenging. Here we develop MiSL (Mining Synthetic Lethals), an algorithm that mines pan-cancer human primary tumour data to identify mutation-specific SL partners for specific cancers. We apply MiSL to 12 different cancers and predict 145,891 SL partners for 3,120 mutations, including known mutation-specific SL partners. Comparisons with functional screens show that MiSL predictions are enriched for SLs in multiple cancers. We extensively validate a SL interaction identified by MiSL between the IDH1 mutation and ACACA in leukaemia using gene targeting and patient-derived xenografts. Furthermore, we apply MiSL to pinpoint genetic biomarkers for drug sensitivity. These results demonstrate that MiSL can accelerate precision oncology by identifying mutation-specific targets and biomarkers.

    View details for DOI 10.1038/ncomms15580

    View details for PubMedID 28561042

  • Biology and relevance of human acute myeloid leukemia stem cells. Blood Thomas, D., Majeti, R. 2017


    Evidence of human acute myeloid leukemia stem cells (AML LSCs) was first reported nearly 2 decades ago through the identification of rare subpopulations of engrafting cells in xenotransplantation assays. These AML LSCs were shown to reside at the apex of a cellular hierarchy that initiates and maintains the disease, exhibiting properties of self-renewal, cell cycle quiescence, and chemoresistance. This cancer stem cell model offers an explanation for chemotherapy resistance and disease relapse and implies that approaches to treatment must eradicate LSCs for cure. More recently, a number of studies have both refined and expanded our understanding of LSCs and intrapatient heterogeneity in AML using improved xenotransplant models, genome-scale analyses, and experimental manipulation of primary patient cells. Here, we review these studies with a focus on the immunophenotype, biological properties, epigenetics, genetics, and clinical associations of human AML LSCs and discuss critical questions that need to be addressed in future research.

    View details for DOI 10.1182/blood-2016-10-696054

    View details for PubMedID 28159741

  • Autophagy mediates proteolysis of NPM1 and HEXIM1 and sensitivity to BET inhibition in AML cells. Oncotarget Huang, M., Garcia, J. S., Thomas, D., Zhu, L., Nguyen, L. X., Chan, S. M., Majeti, R., Medeiros, B. C., Mitchell, B. S. 2016


    The mechanisms underlying activation of the BET pathway in AML cells remain poorly understood. We have discovered that autophagy is activated in acute leukemia cells expressing mutant nucleophosmin 1 (NPMc+) or MLL-fusion proteins. Autophagy activation results in the degradation of NPM1 and HEXIM1, two negative regulators of BET pathway activation. Inhibition of autophagy with pharmacologic inhibitors or through knocking down autophagy-related gene 5 (Atg5) expression increases the expression of both NPM1 and HEXIM1. The Brd4 inhibitors JQ1 and I-BET-151 also inhibit autophagy and increase NPM1 and HEXIM1 expression. We conclude that the degradation of NPM1 and HEXIM1 through autophagy in certain AML subsets contributes to the activation of the BET pathway in these cells.

    View details for DOI 10.18632/oncotarget.12493

    View details for PubMedID 27732946

  • Burning Fat Fuels Leukemic Stem Cell Heterogeneity. Cell stem cell Thomas, D., Majeti, R. 2016; 19 (1): 1-2


    Obese leukemia patients exhibit reduced survival after chemotherapy, suggesting an important role of adipose tissue in disease progression. In this issue of Cell Stem Cell, Ye et al. (2016) reveal metabolic heterogeneity in leukemic stem cell (LSC) subpopulations and show that chemotherapy-resistant CD36+ LSCs co-opt gonadal adipose tissue to support their metabolism and survival.

    View details for DOI 10.1016/j.stem.2016.06.014

    View details for PubMedID 27392217

  • A humanized bone marrow ossicle xenotransplantation model enables improved engraftment of healthy and leukemic human hematopoietic cells NATURE MEDICINE Reinisch, A., Thomas, D., Corces, M. R., Zhang, X., Gratzinger, D., Hong, W., Schallmoser, K., Strunk, D., Majeti, R. 2016; 22 (7): 812-821


    Xenotransplantation models represent powerful tools for the investigation of healthy and malignant human hematopoiesis. However, current models do not fully mimic the components of the human bone marrow (BM) microenvironment, and they enable only limited engraftment of samples from some human malignancies. Here we show that a xenotransplantation model bearing subcutaneous humanized ossicles with an accessible BM microenvironment, formed by in situ differentiation of human BM-derived mesenchymal stromal cells, enables the robust engraftment of healthy human hematopoietic stem and progenitor cells, as well as primary acute myeloid leukemia (AML) samples, at levels much greater than those in unmanipulated mice. Direct intraossicle transplantation accelerated engraftment and resulted in the detection of substantially higher leukemia-initiating cell (LIC) frequencies. We also observed robust engraftment of acute promyelocytic leukemia (APL) and myelofibrosis (MF) samples, and identified LICs in these malignancies. This humanized ossicle xenotransplantation approach provides a system for modeling a wide variety of human hematological diseases.

    View details for DOI 10.1038/nm.4103

    View details for PubMedID 27213817

  • Isocitrate dehydrogenase 1 and 2 mutations induce BCL-2 dependence in acute myeloid leukemia. Nature medicine Chan, S. M., Thomas, D., Corces-Zimmerman, M. R., Xavy, S., Rastogi, S., Hong, W., Zhao, F., Medeiros, B. C., Tyvoll, D. A., Majeti, R. 2015; 21 (2): 178-184


    Mutant isocitrate dehydrogenase (IDH) 1 and 2 proteins alter the epigenetic landscape in acute myeloid leukemia (AML) cells through production of the oncometabolite (R)-2-hydroxyglutarate (2-HG). Here we performed a large-scale RNA interference (RNAi) screen to identify genes that are synthetic lethal to the IDH1(R132H) mutation in AML and identified the anti-apoptotic gene BCL-2. IDH1- and IDH2-mutant primary human AML cells were more sensitive than IDH1/2 wild-type cells to ABT-199, a highly specific BCL-2 inhibitor that is currently in clinical trials for hematologic malignancies, both ex vivo and in xenotransplant models. This sensitization effect was induced by (R)-2-HG-mediated inhibition of the activity of cytochrome c oxidase (COX) in the mitochondrial electron transport chain (ETC); suppression of COX activity lowered the mitochondrial threshold to trigger apoptosis upon BCL-2 inhibition. Our findings indicate that IDH1/2 mutation status may identify patients that are likely to respond to pharmacologic BCL-2 inhibition and form the rational basis for combining agents that disrupt ETC activity with ABT-199 in future clinical studies.

    View details for DOI 10.1038/nm.3788

    View details for PubMedID 25599133

  • Epigenetic and in vivo comparison of diverse MSC sources reveals an endochondral signature for human hematopoietic niche formation. Blood Reinisch, A., Etchart, N., Thomas, D., Hofmann, N. A., Fruehwirth, M., Sinha, S., Chan, C. K., Senarath-Yapa, K., Seo, E., Wearda, T., Hartwig, U. F., Beham-Schmid, C., Trajanoski, S., Lin, Q., Wagner, W., Dullin, C., Alves, F., Andreeff, M., Weissman, I. L., Longaker, M. T., Schallmoser, K., Majeti, R., Strunk, D. 2015; 125 (2): 249-260


    In the last decade there has been a rapid expansion in clinical trials using mesenchymal stromal cells (MSCs) from a variety of tissues. However, despite similarities in morphology, immunophenotype and differentiation behavior in vitro, MSCs sourced from distinct tissues do not necessarily have equivalent biological properties. We performed a genome-wide methylation, transcription and in vivo evaluation of MSCs from human bone marrow (BM), white adipose tissue, umbilical cord and skin cultured in humanized media. Surprisingly, only BM-derived MSCs spontaneously formed a bone marrow cavity through a vascularized cartilage intermediate in vivo that was progressively replaced by hematopoietic tissue and bone. Only BM-derived MSCs exhibited a chondrogenic transcriptional program with hypomethylation and increased expression of RUNX3, RUNX2, BGLAP, MMP13 and ITGA10 consistent with a latent and primed skeletal developmental potential. The humanized MSC-derived microenvironment permitted homing and maintenance of long-term murine SLAM(+) hematopoietic stem cells (HSCs) as well as human CD34(+)/CD38(-)/CD90(+)/CD45RA(+) HSCs after cord blood transplantation. These studies underscore the profound differences in developmental potential between MSC sources independent of donor age with implications for their clinical use. We also demonstrate a tractable human niche model for studying homing and engraftment of human hematopoietic cells in normal and neoplastic states.

    View details for DOI 10.1182/blood-2014-04-572255

    View details for PubMedID 25406351

  • Mutant WT1 is associated with DNA hypermethylation of PRC2 targets in AML and responds to EZH2 inhibition. Blood Sinha, S., Thomas, D., Yu, L., Gentles, A. J., Jung, N., Corces-Zimmerman, M. R., Chan, S. M., Reinisch, A., Feinberg, A. P., Dill, D. L., Majeti, R. 2015; 125 (2): 316-326


    Acute myeloid leukemia (AML) is associated with deregulation of DNA methylation; however, many cases do not bear mutations in known regulators of CpG methylation. We found that mutations in WT1, IDH2, and CEBPA were strongly linked to DNA hypermethylation in AML using a novel integrative analysis of TCGA data based on Boolean implications, if-then rules that identify all individual CpG sites that are hypermethylated in the presence of a mutation. Introduction of mutant WT1 (WT1mut) into wildtype AML cells induced DNA hypermethylation, confirming mutant WT1 to be causally associated with DNA hypermethylation. Methylated genes in WT1mut primary patient samples were highly enriched for polycomb repressor complex 2 (PRC2) targets, implicating PRC2 dysregulation in WT1mut leukemogenesis. We found that PRC2 target genes were aberrantly repressed in WT1mut AML, and that expression of mutant WT1 in CD34+ cord blood cells induced myeloid differentiation block. Treatment of WT1mut AML cells with shRNA or pharmacologic PRC2/EZH2 inhibitors promoted myeloid differentiation, suggesting EZH2 inhibitors may be active in this AML subtype. Our results highlight a strong association between mutant WT1 and DNA hypermethylation in AML, and demonstrate that Boolean implications can be used to decipher mutation-specific methylation patterns that may lead to therapeutic insights.

    View details for DOI 10.1182/blood-2014-03-566018

    View details for PubMedID 25398938

  • Targeting of acute myeloid leukemia in vitro and in vivo with an anti-CD123 mAb engineered for optimal ADCC. Leukemia Busfield, S. J., Biondo, M., Wong, M., Ramshaw, H. S., Lee, E. M., Ghosh, S., Braley, H., Panousis, C., Roberts, A. W., He, S. Z., Thomas, D., Fabri, L., Vairo, G., Lock, R. B., Lopez, A. F., Nash, A. D. 2014


    Acute myeloid leukemia (AML) is a biologically heterogeneous group of related diseases in urgent need of better therapeutic options. Despite this heterogeneity, overexpression of the interleukin (IL)-3 receptor ?-chain (IL-3?R?/CD123) on both the blast and leukemic stem cell (LSC) populations is a common occurrence, a finding that has generated wide interest in devising new therapeutic approaches that target CD123 in AML patients. We report here the development of CSL362, a monoclonal antibody to CD123 that has been humanized, affinity-matured and Fc-engineered for increased affinity for human CD16 (Fc?RIIIa). In vitro studies demonstrated that CSL362 potently induces antibody-dependent cell-mediated cytotoxicity of both AML blasts and CD34(+)CD38(-)CD123(+) LSC by NK cells. Importantly, CSL362 was highly effective in vivo reducing leukemic cell growth in AML xenograft mouse models and potently depleting plasmacytoid dendritic cells and basophils in cynomolgus monkeys. Significantly, we demonstrated CSL362-dependent autologous depletion of AML blasts ex vivo, indicating that CSL362 enables the efficient killing of AML cells by the patient's own NK cells. These studies offer a new therapeutic option for AML patients with adequate NK-cell function and warrant the clinical development of CSL362 for the treatment of AML.Leukemia advance online publication, 9 May 2014; doi:10.1038/leu.2014.128.

    View details for DOI 10.1038/leu.2014.128

    View details for PubMedID 24705479

  • Role of cysteine 288 in nucleophosmin cytoplasmic mutations: sensitization to toxicity induced by arsenic trioxide and bortezomib LEUKEMIA Huang, M., Thomas, D., Li, M. X., Feng, W., Chan, S. M., Majeti, R., Mitchell, B. S. 2013; 27 (10): 1970-1980


    Mutations in exon 12 of the NPM1 gene (NPMc+) define a distinct subset of acute myelogenous leukemias (AML) in which the NPMc+ protein localizes aberrantly to the leukemic cell cytoplasm. We have found that introduction of the most common NPMc+ variant into K562 and 32D cells sensitizes these cells to apoptosis induced by drugs such as bortezomib and arsenic trioxide that induce reactive oxygen species (ROS) formation and that cytotoxicity is prevented in the presence of N-acetyl-1-cysteine, a ROS scavenger. The substitution of tryptophan288 by cysteine occurs in the great majority of NPM1c+ mutations. Mutagenesis of C288 to alanine re-localizes NPMc+ from the cytoplasm to the nucleolus and attenuates the sensitivity of cells expressing this mutation to bortezomib and arsenic trioxide. Primary AML leukemic cells expressing NPMc+ are also significantly more sensitive than other AML cells to apoptosis induced by both drugs at pharmacologically achievable doses. We conclude that the presence of a cysteine moiety at position 288 results in the cytoplasmic localization of NPM1c+ and the increased sensitivity to bortezomib and arsenic trioxide. These data suggest that bortezomib and arsenic trioxide may have increased therapeutic efficacy in NPM1c+ leukemias.Leukemia accepted article preview online, 23 July 2013. doi:10.1038/leu.2013.222.

    View details for DOI 10.1038/leu.2013.222

    View details for PubMedID 23877794

  • Phosphorylation of Serine 779 in Fibroblast Growth Factor Receptor 1 and 2 by Protein Kinase C{epsilon} Regulates Ras/Mitogen-activated Protein Kinase Signaling and Neuronal Differentiation. journal of biological chemistry Lonic, A., Powell, J. A., Kong, Y., Thomas, D., Holien, J. K., Truong, N., Parker, M. W., Guthridge, M. A. 2013; 288 (21): 14874-14885


    The FGF receptors (FGFRs) control a multitude of cellular processes both during development and in the adult through the initiation of signaling cascades that regulate proliferation, survival, and differentiation. Although FGFR tyrosine phosphorylation and the recruitment of Src homology 2 domain proteins have been widely described, we have previously shown that FGFR is also phosphorylated on Ser(779) in response to ligand and binds the 14-3-3 family of phosphoserine/threonine-binding adaptor/scaffold proteins. However, whether this receptor phosphoserine mode of signaling is able to regulate specific signaling pathways and biological responses is unclear. Using PC12 pheochromocytoma cells and primary mouse bone marrow stromal cells as models for growth factor-regulated neuronal differentiation, we show that Ser(779) in the cytoplasmic domains of FGFR1 and FGFR2 is required for the sustained activation of Ras and ERK but not for other FGFR phosphotyrosine pathways. The regulation of Ras and ERK signaling by Ser(779) was critical not only for neuronal differentiation but also for cell survival under limiting growth factor concentrations. PKC? can phosphorylate Ser(779) in vitro, whereas overexpression of PKC? results in constitutive Ser(779) phosphorylation and enhanced PC12 cell differentiation. Furthermore, siRNA knockdown of PKC? reduces both growth factor-induced Ser(779) phosphorylation and neuronal differentiation. Our findings show that in addition to FGFR tyrosine phosphorylation, the phosphorylation of a conserved serine residue, Ser(779), can quantitatively control Ras/MAPK signaling to promote specific cellular responses.

    View details for DOI 10.1074/jbc.M112.421669

    View details for PubMedID 23564461

  • Targeting acute myeloid leukemia by dual inhibition of PI3K signalling and Cdk9-mediated Mcl-1 transcription. Blood Thomas, D., Powell, J. A., Vergez, F., Segal, D. H., Nguyen, N. N., Baker, A., Teh, T. C., Barry, E. F., Sarry, J. E., Lee, E. M., Nero, T. L., Jabbour, A. M., Pomilio, G., Green, B. D., Manenti, S., Glaser, S. P., Parker, M. W., Lopez, A. F., Ekert, P. G., Lock, R. B., Huang, D. C., Nilsson, S. K., Récher, C., Wei, A. H., Guthridge, M. A. 2013


    Resistance to cell death is a hallmark of cancer and renders transformed cells resistant to multiple apoptotic triggers. The Bcl-2 family member, Mcl-1, is a key driver of cell survival in diverse cancers, including acute myeloid leukemia (AML). A screen for compounds down-regulating Mcl-1 identified the kinase inhibitor, PIK-75, which demonstrates marked pro-apoptotic activity against a panel of cytogenetically diverse primary human AML patient samples. We show that PIK-75 transiently blocks Cdk7/9, leading to transcriptional suppression of MCL-1, a rapid loss of Mcl-1 protein and alleviation of its inhibition of pro-apoptotic Bak. PIK-75 also targets the p110? isoform of PI3K which leads to a loss of association between Bcl-xL and Bak. The simultaneous loss of Mcl-1 and Bcl-xL association with Bak leads to rapid apoptosis of AML cells. Concordantly, low Bak expression in AML confers resistance to PIK-75-mediated killing. On the other hand, the induction of apoptosis by PIK-75 did not require the expression of the BH3 proteins Bim, Bid, Bad, Noxa or Puma. PIK-75 significantly reduced leukemia burden and increased the survival of mice engrafted with human AML without inducing overt toxicity. Future efforts to co-target PI3K and Cdk9 with drugs such as PIK-75 in AML are warranted.

    View details for DOI 10.1182/blood-2012-08-447441

    View details for PubMedID 23775716

  • Protein Kinase Activity of Phosphoinositide 3-Kinase Regulates Cytokine-Dependent Cell Survival PLOS Biology Daniel Thomas, Daniel Thomas equal contributor, Jason A. Powell equal contributor, Benjamin D. Green, Emma F. Barry, Yuefang Ma, Joanna Woodcock, Stephen Fitter, Andrew C. W. Zannettino, Stuart M. Pitson, Timothy P. Hughes, Angel F. Lopez, Peter R. Shepherd, Andrew H. Wei, Paul G. Ekert, Mark A. Guthridge 2013; 11 (3): e1001515
  • Molecular Basis of Cytokine Receptor Activation IUBMB LIFE Lopez, A. F., Hercus, T. R., Ekert, P., Littler, D. R., Guthridge, M., Thomas, D., Ramshaw, H. S., Stomski, F., Perugini, M., D'Andrea, R., Grimbaldeston, M., Parker, M. W. 2010; 62 (7): 509-518


    Cytokines are secreted soluble peptides that precisely regulate multiple cellular functions. Amongst these the GM-CSF/IL-3/IL-5 family of cytokines controls whether hematopoietic cells will survive or apoptose, proliferate, differentiate, migrate, or perform effector functions such as phagocytosis or reactive oxygen species release. Their potent and pleiotropic activities are mediated through binding to high affinity membrane receptors at surprisingly low numbers per cell. Receptor binding triggers a cascade of intracellular signaling events, including reversible phosphorylation of receptor subunits and associated signaling molecules, leading to multiple biological responses, with the prevention of apoptosis or "cell survival" being a key cellular function that underpins all others. Many chronic inflammatory diseases and a number of haematological malignancies are driven by deregulated GM-CSF, IL-3, or IL-5 cytokine receptor signaling, highlighting their importance in disease. A major step in understanding how these cytokine receptors function is to elucidate their three dimensional structure and to relate this to the many signaling pathways emanating from their receptors. We have recently solved the structure of the human GM-CSF receptor complexed to GM-CSF which revealed distinct forms of receptor assembly: a hexamer that comprises two molecules each of GM-CSF, GM-CSF receptor alpha chain and GM-CSF receptor beta chain; and an unexpected dodecamer in which two hexameric complexes associate through a novel site 4. This latter form is necessary to bring JAK2 molecules sufficiently close together to enable full receptor activation. In this review we focus on the most recent insights in cytokine receptor signaling, and in receptor assembly. The stage is now set to link distinct forms of cytokine receptor assembled structures to specific forms of cytokine receptor signaling and function. Armed with this knowledge it may be possible to map distinct cytokine receptor signaling pathways from the cell surface to the cell nucleus which may themselves become new therapeutic targets.

    View details for DOI 10.1002/iub.350

    View details for Web of Science ID 000279972200006

    View details for PubMedID 20540154

  • Essential Requirement for PP2A Inhibition by the Oncogenic Receptor c-KIT Suggests PP2A Reactivation as a Strategy to Treat c-KIT+ Cancers CANCER RESEARCH Roberts, K. G., Smith, A. M., McDougall, F., Carpenter, H., Horan, M., Neviani, P., Powell, J. A., Thomas, D., Guthridge, M. A., Perrotti, D., Sim, A. T., Ashman, L. K., Verrills, N. M. 2010; 70 (13): 5438-5447


    Oncogenic mutations of the receptor tyrosine kinase c-KIT play an important role in the pathogenesis of gastrointestinal stromal tumors, systemic mastocytosis, and some acute myeloid leukemias (AML). Although juxtamembrane mutations commonly detected in gastrointestinal stromal tumor are sensitive to tyrosine kinase inhibitors, the kinase domain mutations frequently encountered in systemic mastocytosis and AML confer resistance and are largely unresponsive to targeted inhibition by the existing agent imatinib. In this study, we show that myeloid cells expressing activated c-KIT mutants that are imatinib sensitive (V560G) or imatinib resistant (D816V) can inhibit the tumor suppressor activity of protein phosphatase 2A (PP2A). This effect was associated with the reduced expression of PP2A structural (A) and regulatory subunits (B55alpha, B56alpha, B56gamma, and B56delta). Overexpression of PP2A-Aalpha in D816V c-KIT cells induced apoptosis and inhibited proliferation. In addition, pharmacologic activation of PP2A by FTY720 reduced proliferation, inhibited clonogenic potential, and induced apoptosis of mutant c-KIT(+) cells, while having no effect on wild-type c-KIT cells or empty vector controls. FTY720 treatment caused the dephosphorylation of the D816V c-KIT receptor and its downstream signaling targets pAkt, pSTAT5, and pERK1/2. Additionally, in vivo administration of FTY720 delayed the growth of V560G and D816V c-KIT tumors, inhibited splenic and bone marrow infiltration, and prolonged survival. Our findings show that PP2A inhibition is essential for c-KIT-mediated tumorigenesis, and that reactivating PP2A may offer an attractive strategy to treat drug-resistant c-KIT(+) cancers.

    View details for DOI 10.1158/0008-5472.CAN-09-2544

    View details for Web of Science ID 000279396800026

    View details for PubMedID 20551067

  • Expression profiling of a hemopoietic cell survival transcriptome implicates osteopontin as a functional prognostic factor in AML BLOOD Powell, J. A., Thomas, D., Barry, E. F., Kok, C. H., McClure, B. J., Tsykin, A., To, L. B., Brown, A., Lewis, I. D., Herbert, K., Goodall, G. J., Speed, T. P., Asou, N., Jacob, B., Osato, M., Haylock, D. N., Nilsson, S. K., D'Andrea, R. J., Lopez, A. F., Guthridge, M. A. 2009; 114 (23): 4859-4870


    Deregulated cell survival programs are a classic hallmark of cancer. We have previously identified a serine residue (Ser585) in the betac subunit of the granulocyte-macrophage colony-stimulating factor receptor that selectively and independently promotes cell survival. We now show that Ser585 phosphorylation is constitutive in 20 (87%) of 23 acute myeloid leukemia (AML) patient samples, indicating that this survival-only pathway is frequently deregulated in leukemia. We performed a global expression screen to identify gene targets of this survival pathway and report a 138-gene betac Ser585-regulated transcriptome. Pathway analysis defines a gene network enriched for PI3-kinase target genes and a cluster of genes involved in cancer and cell survival. We show that one such gene, osteopontin (OPN), is a functionally relevant target of the Ser585-survival pathway as shown by siRNA-mediated knockdown of OPN expression that induces cell death in both AML blasts and CD34(+)CD38(-)CD123(+) leukemic progenitors. Increased expression of OPN at diagnosis is associated with poor prognosis with multivariate analysis indicating that it is an independent predictor of overall patient survival in normal karyotype AML (n = 60; HR = 2.2; P = .01). These results delineate a novel cytokine-regulated Ser585/PI3-kinase signaling network that is deregulated in AML and identify OPN as a potential prognostic and therapeutic target.

    View details for DOI 10.1182/blood-2009-02-204818

    View details for Web of Science ID 000272190700015

    View details for PubMedID 19805619

  • The granulocyte-macrophage colony-stimulating factor receptor: linking its structure to cell signaling and its role in disease BLOOD Hercus, T. R., Thomas, D., Guthridge, M. A., Ekert, P. G., King-Scott, J., Parker, M. W., Lopez, A. F. 2009; 114 (7): 1289-1298


    Already 20 years have passed since the cloning of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha-chain, the first member of the GM-CSF/interleukin (IL)-3/IL-5 family of hemopoietic cytokine receptors to be molecularly characterized. The intervening 2 decades have uncovered a plethora of biologic functions transduced by the GM-CSF receptor (pleiotropy) and revealed distinct signaling networks that couple the receptor to biologic outcomes. Unlike other hemopoietin receptors, the GM-CSF receptor has a significant nonredundant role in myeloid hematologic malignancies, macrophage-mediated acute and chronic inflammation, pulmonary homeostasis, and allergic disease. The molecular mechanisms underlying GM-CSF receptor activation have recently been revealed by the crystal structure of the GM-CSF receptor complexed to GM-CSF, which shows an unexpected higher order assembly. Emerging evidence also suggests the existence of intracellular signosomes that are recruited in a concentration-dependent fashion to selectively control cell survival, proliferation, and differentiation by GM-CSF. These findings begin to unravel the mystery of cytokine receptor pleiotropy and are likely to also apply to the related IL-3 and IL-5 receptors as well as other heterodimeric cytokine receptors. The new insights in GM-CSF receptor activation have clinical significance as the structural and signaling nuances can be harnessed for the development of new treatments for malignant and inflammatory diseases.

    View details for DOI 10.1182/blood-2008-12-164004

    View details for Web of Science ID 000268924700006

    View details for PubMedID 19436055

    View details for PubMedCentralID PMC2727416

  • Monoclonal Antibody-Mediated Targeting of CD123, IL-3 Receptor alpha Chain, Eliminates Human Acute Myeloid Leukemic Stem Cells CELL STEM CELL Jin, L., Lee, E. M., Ramshaw, H. S., Busfield, S. J., Peoppl, A. G., Wilkinson, L., Guthridge, M. A., Thomas, D., Barry, E. F., Boyd, A., Gearing, D. P., Vairo, G., Lopez, A. F., Dick, J. E., Lock, R. B. 2009; 5 (1): 31-42


    Leukemia stem cells (LSCs) initiate and sustain the acute myeloid leukemia (AML) clonal hierarchy and possess biological properties rendering them resistant to conventional chemotherapy. The poor survival of AML patients raises expectations that LSC-targeted therapies might achieve durable remissions. We report that an anti-interleukin-3 (IL-3) receptor alpha chain (CD123)-neutralizing antibody (7G3) targeted AML-LSCs, impairing homing to bone marrow (BM) and activating innate immunity of nonobese diabetic/severe-combined immunodeficient (NOD/SCID) mice. 7G3 treatment profoundly reduced AML-LSC engraftment and improved mouse survival. Mice with pre-established disease showed reduced AML burden in the BM and periphery and impaired secondary transplantation upon treatment, establishing that AML-LSCs were directly targeted. 7G3 inhibited IL-3-mediated intracellular signaling of isolated AML CD34(+)CD38(-) cells in vitro and reduced their survival. These results provide clear validation for therapeutic monoclonal antibody (mAb) targeting of AML-LSCs and for translation of in vivo preclinical research findings toward a clinical application.

    View details for DOI 10.1016/j.stem.2009.04.018

    View details for Web of Science ID 000267879200008

    View details for PubMedID 19570512

  • Haematopoietic Growth Factors In: Encyclopedia of Life Sciences (ELS) John Wiley and Sons, Ltd: Chichester. Daniel Thomas, Angel Lopez 2009: March
  • Growth factor pleiotropy is controlled by a receptor Tyr/Ser motif that acts as a binary switch EMBO JOURNAL Guthridge, M. A., Powell, J. A., Barry, E. F., Stomski, F. C., McClure, B. J., Ramshaw, H., Felquer, F. A., Dottore, M., Thomas, D. T., To, B., BEGLEY, C. G., Lopez, A. F. 2006; 25 (3): 479-489


    Pleiotropism is a hallmark of cytokines and growth factors; yet, the underlying mechanisms are not clearly understood. We have identified a motif in the granulocyte macrophage-colony-stimulating factor receptor composed of a tyrosine and a serine residue that functions as a binary switch for the independent regulation of multiple biological activities. Signalling occurs either through Ser585 at lower cytokine concentrations, leading to cell survival only, or through Tyr577 at higher cytokine concentrations, leading to cell survival as well as proliferation, differentiation or functional activation. The phosphorylation of Ser585 and Tyr577 is mutually exclusive and occurs via a unidirectional mechanism that involves protein kinase A and tyrosine kinases, respectively, and is deregulated in at least some leukemias. We have identified similar Tyr/Ser motifs in other cell surface receptors, suggesting that such signalling switches may play important roles in generating specificity and pleiotropy in other biological systems.

    View details for DOI 10.1038/sj.emboj.7600948

    View details for Web of Science ID 000235803100004

    View details for PubMedID 16437163

  • 14-3-3 protein signaling in development and growth factor responses CURRENT TOPICS IN DEVELOPMENTAL BIOLOGY, VOL 67 Thomas, D., Guthridge, M., Woodcock, J., Lopez, A. 2005; 67: 285-303


    Tyrosine and serine phosphorylation are central to cellular signaling in growth and development. 14-3-3 proteins function as dimeric phosphoserine-binding proteins with documented interactions throughout the eukaryotic proteome and are highly conserved in both the animal and plant kingdoms. Binding of 14-3-3 to a client protein can have a range of context-dependent effects, including conformational change, enzyme inhibition, a shielding effect, re-localization, and bridging between two molecules. Proteome-based strategies utilizing mass spectrometry have revealed an unprecedented central stage for 14-3-3 in signal transduction with interacting partners composing at least 0.6% of the cellular proteome. 14-3-3 has been shown to bind to the human GM-CSF, IL-3, and IL-5 receptors and is required for the transmission of cell survival. 14-3-3 is involved in survival-specific signals, acting not only at the receptor level but also at critical steps downstream of the receptor. This phosphoserine-mediated pathway works independently of tyrosine kinases, highlighting an alternative mechanism of signaling for this receptor family. Other growth factor receptors and their adaptors are also being shown to associate with 14-3-3 and/or have putative 14-3-3 interaction sequences, such as the prolactin receptor, IGF-1 receptor, and some G-protein coupled receptors. 14-3-3 proteins are remarkably conserved through eukaryotic organisms and in Drosophila are required for photoreceptor development, learning, timing of cell cycles, and maintenance of cellular polarity. These findings are elevating our initial description of biochemical interactions to a better understanding of 14-3-3 function at the level of the whole organism. Further study should explore the integration of phosphoserine and phosphotyrosine signaling by 14-3-3 proteins and the role of isoform-specific functions in higher organisms. The prevalence of functional 14-3-3 binding sites throughout the proteome, and especially among growth factor receptors and signaling molecules, reflects a global role for 14-3-3 in multiple cellular decision making.

    View details for DOI 10.1016/S0070-2153(05)67009-3

    View details for Web of Science ID 000229969000009

    View details for PubMedID 15949538

  • Chromatin remodelling in the biology and treatment of acute leukaemia Cancer Reviews: Asia-Pacific Daniel Thomas, Bik To 2004; 2 (2): 109-119
  • Regulation of Haematopoiesis by Growth Factors - Emerging Insights and Therapies Expert Opinion in Biological Therapy Daniel Thomas, Angel Lopez 2004; 4 (6): 869-79
  • Granulocyte Macrophage Colony Stimulating Factor Encyclopedia of Hormones; Academic Press Daniel Thomas, Angel Lopez 2003: April 25
  • Identification of a 14-3-3 binding sequence in the common beta chain of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors that is serine-phosphorylated by GM-CSF BLOOD Stomski, F. C., Dottore, M., Winnall, W., Guthridge, M. A., Woodcock, J., BAGLEY, C. J., Thomas, D. T., Andrews, R. K., Berndt, M. C., Lopez, A. F. 1999; 94 (6): 1933-1942


    The common beta chain (beta(c)) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors is the major signaling subunit of these receptors coupling ligand binding to multiple biological activities. It is thought that these multiple functions arise as a consequence of the recruitment of specific signaling molecules to tyrosine-phosphorylated residues in the cytoplasmic domain of beta(c). However, the contribution of serine phosphorylation in beta(c) to the recruitment of signaling molecules is not known. We show here the identification of a phosphoserine motif in the cytoplasmic domain of beta(c) that interacts with the adaptor protein 14-3-3zeta. Coimmunoprecipitation and pull-down experiments with a glutathione S-transferase (GST):14-3-3zeta fusion protein showed that 14-3-3 directly associates with beta(c) but not the GM-CSF receptor alpha chain. C-terminal truncation mutants of beta(c) further showed that a region between amino acids 544 and 626 in beta(c) was required for its association with 14-3-3zeta. This region contains the sequence (582)HSRSLP(587), which closely resembles the RSXSXP (where S is phosphorylated) consensus 14-3-3 binding site identified in a number of signaling molecules, including Raf-1. Significantly, substitution of (582)HSRSLP(587) for EFAAAA completely abolished interaction of beta(c) with GST-14-3-3zeta. Furthermore, the interaction of beta(c) with GST-14-3-3 was greatly reduced in the presence of a peptide containing the 14-3-3 binding site, but only when (585)Ser was phosphorylated. Direct binding experiments showed that the peptide containing phosphorylated (585)Ser bound 14-3-3zeta with an affinity of 150 nmol/L. To study the regulation of (585)S phosphorylation in vivo, we raised antibodies that specifically recognized (585)Ser-phosphorylated beta(c). Using these antibodies, we showed that GM-CSF stimulation strongly upregulated (585)Ser phosphorylation in M1 myeloid leukemic cells. The proximity of the SHC-binding site ((577)Tyr) to the 14-3-3-binding site ((582)HSRSLP(587)) and their conservation between mouse, rat, and human beta(c) but not in other cytokine receptors suggest that they form a distinct motif that may subserve specialized functions associated with the GM-CSF, IL-3, and IL-5 receptors.

    View details for Web of Science ID 000082484500012

    View details for PubMedID 10477722

  • Mechanism of activation of the GM-CSF IL-3, and IL-5 family of receptors STEM CELLS Guthridge, M. A., Stomski, F. C., Thomas, D., Woodcock, J. M., BAGLEY, C. J., Berndt, M. C., Lopez, A. F. 1998; 16 (5): 301-313


    The process of ligand binding leading to receptor activation is an ordered and sequential one. High-affinity binding of GM-CSF, interleukin 3 (IL-3), and IL-5 to their receptors induces a number of key events at the cell surface and within the cytoplasm that are necessary for receptor activation. These include receptor oligomerization, activation of tyrosine kinase activity, phosphorylation of the receptor, and the recruitment of SH2 (src-homology) and PTB (phosphotyrosine binding) domain proteins to the receptor. Such a sequence of events represents a recurrent theme among cytokine, growth factor, and hormone receptors; however, a number of very recent and interesting findings have identified unique features in this receptor system in terms of: A) how GM-CSF/IL-3/IL-5 bind, oligomerize, and activate their cognate receptors; B) how multiple biological responses such as proliferation, survival, and differentiation can be transduced from activated GM-CSF, IL-3, or IL-5 receptors, and C) how the presence of novel phosphotyrosine-independent signaling motifs within a specific cytoplasmic domain of betaC may be important for mediating survival and differentiation by these cytokines. This review does not attempt to be all-encompassing but rather to focus on the most recent and significant discoveries that distinguish the GM-CSF/IL-3/IL-5 receptor subfamily from other cytokine receptors.

    View details for Web of Science ID 000076174100001

    View details for PubMedID 9766809

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