Mechanism of homodimeric cytokine receptor activation and dysregulation by oncogenic mutations

Wilmes, Stephan; Hafer, Maximillian; Vuorio, Joni; Tucker, J.A.; Winkelmann, Hauke; Löchte, Sara; Stanly, Tess A.; Prieto, Katiuska Daniela Pulgar; Poojari, Chetan; Sharma, Vivek; Richter, Christian; Kurre, Rainer; Hubbard, Stevan R.; García, K. Christopher; Moraga, Ignacio; Vattulainen, Ilpo; Hitchcock, Ian S.; Piehler, Jacob

doi:10.1126/science.aaw3242

Cited by 135 publications

(216 citation statements)

References 90 publications

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“…Receptors were considered as dimerized if two individual particles in both spectral channels persistently co-localized for ³10 consecutive steps (~320 ms) in a proximity of 150 nm. These colocalization/co-tracking thresholds allowed reliable elimination of density-dependent random colocalizations (Wilmes et al, 2020). In the absence of IL-10, we did not observe heterodimerization of IL-10Rα and IL-10Rβ (Figure 2b and 2c).…”

Section: Enhanced Il-10rβ Binding Affinity Increases Receptor Heterodmentioning

confidence: 76%

“…The tags were designed to specifically recognise either one of two different anti-GFP nanobodies (Kirchhofer et al, 2010). These nanobodies (NBs) were conjugated to photostable organic fluorophores ATTO Rho11 and ATTO 643 suitable for simultaneous dualcolour single molecule tracking of IL-10Rα and IL-10Rβ in the plasma membrane of live cells as shown previously in other cytokine receptor systems (Martinez-Fabregas et al, 2019;Moraga et al, 2015a;Wilmes et al, 2020) (Figure 2a and Sup. Figure 3a).…”

Section: Enhanced Il-10rβ Binding Affinity Increases Receptor Heterodmentioning

confidence: 99%

“…Receptor homo-and heterodimerization was quantified by two-colour single-molecule co-tracking as described previously (Moraga et al, 2015c;Wilmes et al, 2015;Wilmes et al, 2020). Receptor dimerization experiments were performed in HeLa cells transienty expressing IL-10Rα and IL-10Rβ with N-terminally fused variants of monomeric ECFP and EGFP, respectively.…”

Section: Live-cell Dual Colour Single Molecule Imaging Studiesmentioning

confidence: 99%

“…Molecules were localized using the multiple-target tracing (MTT) algorithm (Serge et al, 2008). Receptor dimers were identified as molecules that co-localized within a distance threshold of 150 nm for at least 10 consecutive frames as described in detail previously (Moraga et al, 2015c;Wilmes et al, 2015;Wilmes et al, 2020). Supplementary Figure 1.…”

Section: Live-cell Dual Colour Single Molecule Imaging Studiesmentioning

confidence: 99%

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Engineered IL-10 variants elicit potent immuno-modulatory activities at therapeutic low ligand doses

Gorby

Wilmes

et al. 2020

Preprint

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Interleukin-10 is a dimeric cytokine with both immune-suppressive and immune-stimulatory activities. Despite its immuno-modulatory potential, IL-10-based therapies have shown only marginal benefits in the clinic. Here we have explored whether the stability of the IL-10-receptor complex contributes to IL-10 immuno-modulatory potency. For that, we have generated an IL-10 mutant with greatly enhanced affinity for its IL-10Rβ receptor via yeast surface display. The affinity enhanced IL-10 variants recruited IL-10Rβ more efficiently into active cell surface signaling complexes than the wild-type cytokine and triggered more potent STAT1 and STAT3 activation in human monocytes and CD8 T cells. This in turn led to more robust induction of IL-10-mediated gene expression programs at a wide range of ligand concentrations in both human cell subsets. IL-10 regulated genes are involved in monocyte energy homeostasis, migration and trafficking, and genes involved in CD8 T cell exhaustion. Interestingly, at non-saturating doses, IL-10 lost key components of its gene-expression program, which may explain its lack of efficacy in clinical settings. Remarkably, our engineered IL-10 variant exhibited a more robust bioactivity profile than IL-10 wt at all the doses tested in monocytes and CD8 T cells. Moreover, CAR-modified T cells expanded with the engineered IL-10 variant displayed superior cytolytic activity than those expanded with IL-10 wt. Our study provides unique insights into how IL-10-receptor complex stability fine-tunes IL-10 biology, and opens new opportunities to revitalize failed IL-10 therapies.

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Section: Enhanced Il-10rβ Binding Affinity Increases Receptor Heterodmentioning

confidence: 76%

Section: Enhanced Il-10rβ Binding Affinity Increases Receptor Heterodmentioning

confidence: 99%

Section: Live-cell Dual Colour Single Molecule Imaging Studiesmentioning

confidence: 99%

Section: Live-cell Dual Colour Single Molecule Imaging Studiesmentioning

confidence: 99%

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Engineered IL-10 variants elicit potent immuno-modulatory activities at therapeutic low ligand doses

Gorby

Wilmes

et al. 2020

Preprint

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“…If untreated, the chronic myeloproliferation driven by the BCR-ABL oncogene rapidly progresses to an accelerated phase and terminal blast crisis (BC). In PV, on the other hand, the gain-of-function mutation in the JAK2 gene (JAK2 V617F) constitutively activates type-1 myeloid cytokine receptor-mediated signaling [3][4][5][6]36], resulting in myeloproliferation and systemic inflammation with a protracted clinical course and near-normal life expectancy [37]. These differences in progression of CML and PV suggest distinctions in the nature of BCR-ABL and JAK2 V617F oncogene-induced intrinsic and extrinsic mechanisms that govern the Inactivation of TP53 or silencing of Atm signaling leads to fully compromised DDR, allowing acceleration of the disease course to full-blown blast crisis (BC) of CML.…”

Section: Role Of Ddr In CML and Pvmentioning

confidence: 99%

Role of DNA Damage Response in Suppressing Malignant Progression of Chronic Myeloid Leukemia and Polycythemia Vera: Impact of Different Oncogenes

Stetka

Gurský

Velasquez

et al. 2020

Cancers

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Inflammatory and oncogenic signaling, both known to challenge genome stability, are key drivers of BCR-ABL-positive chronic myeloid leukemia (CML) and JAK2 V617F-positive chronic myeloproliferative neoplasms (MPNs). Despite similarities in chronic inflammation and oncogene signaling, major differences in disease course exist. Although BCR-ABL has robust transformation potential, JAK2 V617F-positive polycythemia vera (PV) is characterized by a long and stable latent phase. These differences reflect increased genomic instability of BCR-ABL-positive CML, compared to genome-stable PV with rare cytogenetic abnormalities. Recent studies have implicated BCR-ABL in the development of a "mutator" phenotype fueled by high oxidative damage, deficiencies of DNA repair, and defective ATR-Chk1-dependent genome surveillance, providing a fertile ground for variants compromising the ATM-Chk2-p53 axis protecting chronic phase CML from blast crisis. Conversely, PV cells possess multiple JAK2 V617F-dependent protective mechanisms, which ameliorate replication stress, inflammation-mediated oxidative stress and stress-activated protein kinase signaling, all through up-regulation of RECQL5 helicase, reactive oxygen species buffering system, and DUSP1 actions. These attenuators of genome instability then protect myeloproliferative progenitors from DNA damage and create a barrier preventing cellular stress-associated myelofibrosis. Therefore, a better understanding of BCR-ABL and JAK2 V617F roles in the DNA damage response and disease pathophysiology can help to identify potential dependencies exploitable for therapeutic interventions.CML is characterized by an indolent, chronic phase (CP) preceding an acute transformation to BC. Failure of DNA damage repair and loss or malfunction of DDR components accompanied by accumulation of DNA damage and genomic instability has been considered in CML evolution [38,39]. It was proposed that BCR-ABL-expressing cells feature reduced activation of the ATR-Chk1-mediated DDR signaling, with ensuing accumulation of substantial genomic instability due to replication stress and oxidative damage. Mechanistically, such disruption of ATR-dependent signaling was attributed to nuclear import of BCR-ABL after DNA damage and its binding to ATR [40]. However, contrasting data were also reported, showing that BCR-ABL does activate ATR-Chk1 signaling, reflecting responsiveness of BCR-ABL-positive myeloid cells to DNA damage following genotoxic treatment [41]. Some studies also addressed functionality of the ATM-Chk2 signaling axis in CML. Thus, c-Abl is a nuclear tyrosine kinase activated by DNA damage in an ATM-dependent manner [42,43]. Even though the BCR-ABL is predominantly localized to the cytoplasm [44], the aforementioned ability of BCR-ABL translocation to the nucleus after DNA damage led to a proposal that BCR-ABL and c-Abl share multiple protein interactions including that with ATM [40]. As a consequence, ATM-mediated activatory phosphorylation of Chk2 was detected in BCR-ABL-expressing cellular models and...

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Identification of a New Cholesterol‐Binding Site within the IFN‐γ Receptor that is Required for Signal Transduction

et al. 2022

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The cytokine interferon-gamma (IFN-𝜸) is a master regulator of innate and adaptive immunity involved in a broad array of human diseases that range from atherosclerosis to cancer. IFN-𝜸 exerts it signaling action by binding to a specific cell surface receptor, the IFN-𝜸 receptor (IFN-𝜸R), whose activation critically depends on its partition into lipid nanodomains. However, little is known about the impact of specific lipids on IFN-𝜸R signal transduction activity. Here, a new conserved cholesterol (chol) binding motif localized within its single transmembrane domain is identified. Through direct binding, chol drives the partition of IFN-𝜸R2 chains into plasma membrane lipid nanodomains, orchestrating IFN-𝜸R oligomerization and transmembrane signaling. Bioinformatics studies show that the signature sequence stands for a conserved chol-binding motif presented in many mammalian membrane proteins. The discovery of chol as the molecular switch governing IFN-𝜸R transmembrane signaling represents a significant advance for understanding the mechanism of lipid selectivity by membrane proteins, but also for figuring out the role of lipids in modulating cell surface receptor function. Finally, this study suggests that inhibition of the chol-IFN𝜸R2 interaction may represent a potential therapeutic strategy for various IFN-𝜸-dependent diseases.

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Mechanism of homodimeric cytokine receptor activation and dysregulation by oncogenic mutations

Cited by 135 publications

References 90 publications

Engineered IL-10 variants elicit potent immuno-modulatory activities at therapeutic low ligand doses

Engineered IL-10 variants elicit potent immuno-modulatory activities at therapeutic low ligand doses

Role of DNA Damage Response in Suppressing Malignant Progression of Chronic Myeloid Leukemia and Polycythemia Vera: Impact of Different Oncogenes

Identification of a New Cholesterol‐Binding Site within the IFN‐γ Receptor that is Required for Signal Transduction

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