The disialoganglioside GD2 is overexpressed on several solid tumors, and monoclonal antibodies targeting GD2 have substantially improved outcomes for children with high-risk neuroblastoma. However, ...approximately 40% of patients with neuroblastoma still relapse, and anti-GD2 has not mediated significant clinical activity in any other GD2
malignancy. Macrophages are important mediators of anti-tumor immunity, but tumors resist macrophage phagocytosis through expression of the checkpoint molecule CD47, a so-called 'Don't eat me' signal. In this study, we establish potent synergy for the combination of anti-GD2 and anti-CD47 in syngeneic and xenograft mouse models of neuroblastoma, where the combination eradicates tumors, as well as osteosarcoma and small-cell lung cancer, where the combination significantly reduces tumor burden and extends survival. This synergy is driven by two GD2-specific factors that reorient the balance of macrophage activity. Ligation of GD2 on tumor cells (a) causes upregulation of surface calreticulin, a pro-phagocytic 'Eat me' signal that primes cells for removal and (b) interrupts the interaction of GD2 with its newly identified ligand, the inhibitory immunoreceptor Siglec-7. This work credentials the combination of anti-GD2 and anti-CD47 for clinical translation and suggests that CD47 blockade will be most efficacious in combination with monoclonal antibodies that alter additional pro- and anti-phagocytic signals within the tumor microenvironment.
Natural killer (NK) cells are effector cells of the innate immune system involved in defense against virus-infected and transformed cells. The effector function of NK cells is linked to their ability ...to migrate to sites of inflammation or damage. Therefore, understanding the factors regulating NK cell migration is of substantial interest. Here, we show that in the absence of aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, NK cells have reduced capacity to migrate and infiltrate tumors
. Analysis of differentially expressed genes revealed that ankyrin repeat and SOCS Box containing 2 (
) expression was dramatically decreased in
NK cells and that AhR ligands modulated its expression. Further, AhR directly regulated the promoter region of the
gene. Similar to what was observed with murine
NK cells,
knockdown inhibited the migration of human NK cells. Activation of AHR by its agonist FICZ induced ASB2-dependent filamin A degradation in NK cells; conversely, knockdown of endogenous
inhibited filamin A degradation. Reduction of filamin A increased the migration of primary NK cells and restored the invasion capacity of AHR-deficient NK cells. Our study introduces AHR as a new regulator of NK cell migration, through an AHR-ASB2-filamin A axis and provides insight into a potential therapeutic target for NK cell-based immunotherapies.
Ageing is characterized by the development of persistent pro-inflammatory responses that contribute to atherosclerosis, metabolic syndrome, cancer and frailty
. The ageing brain is also vulnerable to ...inflammation, as demonstrated by the high prevalence of age-associated cognitive decline and Alzheimer's disease
. Systemically, circulating pro-inflammatory factors can promote cognitive decline
, and in the brain, microglia lose the ability to clear misfolded proteins that are associated with neurodegeneration
. However, the underlying mechanisms that initiate and sustain maladaptive inflammation with ageing are not well defined. Here we show that in ageing mice myeloid cell bioenergetics are suppressed in response to increased signalling by the lipid messenger prostaglandin E
(PGE
), a major modulator of inflammation
. In ageing macrophages and microglia, PGE
signalling through its EP2 receptor promotes the sequestration of glucose into glycogen, reducing glucose flux and mitochondrial respiration. This energy-deficient state, which drives maladaptive pro-inflammatory responses, is further augmented by a dependence of aged myeloid cells on glucose as a principal fuel source. In aged mice, inhibition of myeloid EP2 signalling rejuvenates cellular bioenergetics, systemic and brain inflammatory states, hippocampal synaptic plasticity and spatial memory. Moreover, blockade of peripheral myeloid EP2 signalling is sufficient to restore cognition in aged mice. Our study suggests that cognitive ageing is not a static or irrevocable condition but can be reversed by reprogramming myeloid glucose metabolism to restore youthful immune functions.
Clinical, cytogenetic, and gene-based studies have been used to inform biology and improve prognostication for patients with acute myeloid leukemia (AML). Candidate gene and whole genome studies have ...identified recurrent somatic mutations in AML patients including TET2, DNMT3A, ASXL1, IDH1/2 and cohesin complex mutations. We have also investigated the role of epigenetic regulator mutations in AML pathogenesis, and shown these mutations cooperate with other disease alleles to induce leukemic transformation and to alter gene regulatory networks which impact self-renewal and differentiation. In addition, Recent studies of clonal hematopoiesis (CH) discovered a subset of AML disease alleles, most commonly in epigenetic modifiers, while other alleles are only observed in overt hematologic malignancies. These observations suggest an important pathogenetic role for the chronology of mutational acquisition. We will present novel data showing how sequential acquisition of somatic mutation dysregulates hematopoietic stem/progenitor cells in order to drive transformation and that these mutations have biologic, prognostic, and therapeutic relevance.
Acute myeloid leukemia (AML) is characterized by aberrant expansion of dysregulated myeloid progenitor cells. Genomic studies have identified somatic mutations with variable variant allele ...frequencies (VAF), suggestive of sequential clonal evolution with “newer”, low VAF, mutations altering the fitness of antecedent clones. Our single cell DNA-sequencing studies have shown that mutations in NPM1, NRAS and FLT3 are rarely found as mono-allelic clones and consistent with a late role in leukemic transformation and propagation. These studies revealed clonal evolutionary trajectories to AML, where NPM1c was present in the entire leukemic clone and FLT3-ITD mutations were subclonal. Despite being a late event, FLT3 mutations portend a poor prognosis particularly when co-mutant with DNMT3A and NPM1. Preclinical FLT3 mutant models have employed constitutive expression or retroviral overexpression precluding evaluation of FLT3 mutations in the context observed in AML patients. Here, we report the development of a Flpo-inducible Flt3-ITD allele which allows somatic mutation acquisition subsequent to antecedent disease alleles. Activation of Flt3-ITD induced marked myeloproliferation in vivo. In competitive transplantation studies Flt3-ITD-mutant cells initiated disease at the expense of wild-type HSPCs. Despite these findings, disease was incapable of transplanting into secondary recipients, consistent with the observed depletion of SLAM+ LSK cells. This suggests the FLlt3-ITD cannot propagate disease in self-renewing stem cells. By contrast, acquisition of the Flt3-ITD in NPM1 or DNMT3A mutant HPSCs induced fully penetrant, transplantable AML with immunophenotypic characteristics seen in human AML with these same genotypes. These studies provide mechanistic insights into FLT3-mutant leukemogenesis and offer a preclinical platform for testing novel AML therapies.
Approximately 200 BRAF mutant alleles have been identified in human tumours. Activating BRAF mutants cause feedback inhibition of GTP-bound RAS, are RAS-independent and signal either as active ...monomers (class 1) or constitutively active dimers (class 2). Here we characterize a third class of BRAF mutants-those that have impaired kinase activity or are kinase-dead. These mutants are sensitive to ERK-mediated feedback and their activation of signalling is RAS-dependent. The mutants bind more tightly than wild-type BRAF to RAS-GTP, and their binding to and activation of wild-type CRAF is enhanced, leading to increased ERK signalling. The model suggests that dysregulation of signalling by these mutants in tumours requires coexistent mechanisms for maintaining RAS activation despite ERK-dependent feedback. Consistent with this hypothesis, melanomas with these class 3 BRAF mutations also harbour RAS mutations or NF1 deletions. By contrast, in lung and colorectal cancers with class 3 BRAF mutants, RAS is typically activated by receptor tyrosine kinase signalling. These tumours are sensitive to the inhibition of RAS activation by inhibitors of receptor tyrosine kinases. We have thus defined three distinct functional classes of BRAF mutants in human tumours. The mutants activate ERK signalling by different mechanisms that dictate their sensitivity to therapeutic inhibitors of the pathway.
Indian rhesus macaques (Macaca mulatta) are routinely used in preclinical studies to evaluate therapeutic Abs and candidate vaccines. The efficacy of these interventions in many cases is known to ...rely heavily on the ability of Abs to interact with a set of Ab FcγR expressed on innate immune cells. Yet, despite their presumed functional importance, M. mulatta Ab receptors are largely uncharacterized, posing a fundamental limit to ensuring accurate interpretation and translation of results from studies in this model. In this article, we describe the binding characteristics of the most prevalent allotypic variants of M. mulatta FcγR for binding to both human and M. mulatta IgG of varying subclasses. The resulting determination of the affinity, specificity, and glycan sensitivity of these receptors promises to be useful in designing and evaluating studies of candidate vaccines and therapeutic Abs in this key animal model and exposes significant evolutionary divergence between humans and macaques.
The citrus pathogen Phyllosticta citricarpa was first described 117 years ago in Australia; subsequently, from the summer rainfall citrus-growing regions in China, Africa, and South America; and, ...recently, the United States. Limited information is available on the pathogen's population structure, mode of reproduction, and introduction pathways, which were investigated by genotyping 383 isolates representing 12 populations from South Africa, the United States, Australia, China, and Brazil. Populations were genotyped using seven published and eight newly developed polymorphic simple-sequence repeat markers. The Chinese and Australian populations had the highest genetic diversities, whereas populations from Brazil, the United States, and South Africa exhibited characteristics of founder populations. The U.S. population was clonal. Based on principal coordinate and minimum spanning network analyses, the Chinese populations were distinct from the other populations. Population differentiation and clustering analyses revealed high connectivity and possibly linked introduction pathways between South Africa, Australia, and Brazil. With the exception of the clonal U.S. populations that only contained one mating type, all the other populations contained both mating types in a ratio that did not deviate significantly from 1:1. Although most populations exhibited sexual reproduction, linkage disequilibrium analyses indicated that asexual reproduction is important in the pathogen's life cycle.
ETS family transcription factors regulate diverse genes through binding at cognate DNA sites that overlap substantially in sequence. The DNA-binding domains of ETS proteins (ETS domains) are highly ...conserved structurally yet share limited amino acid homology. To define the mechanistic implications of sequence diversity within the ETS family, we characterized the thermodynamics and kinetics of DNA site recognition by the ETS domains of Ets-1 and PU.1, which represent the extremes in amino acid divergence among ETS proteins. Even though the two ETS domains bind their optimal sites with similar affinities under physiologic conditions, their nature of site recognition differs strikingly in terms of the role of hydration and counter ion release. The data suggest two distinct mechanisms wherein Ets-1 follows a “dry” mechanism that rapidly parses sites through electrostatic interactions and direct protein-DNA contacts, whereas PU.1 utilizes hydration to interrogate sequence-specific sites and form a long-lived complex relative to the Ets-1 counterpart. The kinetic persistence of the high affinity PU.1·DNA complex may be relevant to an emerging role of PU.1, but not Ets-1, as a pioneer transcription factor in vivo. In addition, PU.1 activity is critical to the development and function of macrophages and lymphocytes, which present osmotically variable environments, and hydration-dependent specificity may represent an important regulatory mechanism in vivo, a hypothesis that finds support in gene expression profiles of primary murine macrophages.
Background: ETS family transcription factors recognize DNA via structurally conserved DNA-binding domains that share limited amino acid homology.
Results: DNA recognition by the ETS domains of Ets-1 and PU.1, two extreme sequence-divergent paralogs, was compared.
Conclusion: Preferential hydration differentiates DNA recognition by Ets-1 and PU.1.
Significance: Preferential hydration represents a potential mechanism for PU.1 regulation and its activity as a pioneer transcription factor in vivo.
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