Cathrine Hall

RIPK1 Regulates RIPK3-MLKL-Driven Systemic Inflammation and Emergency Hematopoiesis

Upon ligand binding, RIPK1 is recruited to tumor necrosis factor receptor superfamily (TNFRSF) and Toll-like receptor (TLR) complexes promoting prosurvival and inflammatory signaling. RIPK1 also directly regulates caspase-8-mediated apoptosis or, if caspase-8 activity is blocked, RIPK3-MLKL-dependent necroptosis. We show that C57BL/6 Ripk1(-/-) mice die at birth of systemic inflammation that was not transferable by the hematopoietic compartment. However, Ripk1(-/-) progenitors failed to engraft lethally irradiated hosts properly. Blocking TNF reversed this defect in emergency hematopoiesis but, surprisingly, Tnfr1 deficiency did not prevent inflammation in Ripk1(-/-) neonates. Deletion of Ripk3 or Mlkl, but not Casp8, prevented extracellular release of the necroptotic DAMP, IL-33, and reduced Myd88-dependent inflammation. Reduced inflammation in the Ripk1(-/-)Ripk3(-/-), Ripk1(-/-)Mlkl(-/-), and Ripk1(-/-)Myd88(-/-) mice prevented neonatal lethality, but only Ripk1(-/-)Ripk3(-/-)Casp8(-/-) mice survived past weaning. These results reveal a key function for RIPK1 in inhibiting necroptosis and, thereby, a role in limiting, not only promoting, inflammation.

RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL

RIPK3 and its substrate MLKL are essential for necroptosis, a lytic cell death proposed to cause inflammation via the release of intracellular molecules. Whether and how RIPK3 might drive inflammation in a manner independent of MLKL and cell lysis remains unclear. Here we show that following LPS treatment, or LPS-induced necroptosis, the TLR adaptor protein TRIF and inhibitor of apoptosis proteins (IAPs: X-linked IAP, cellular IAP1 and IAP2) regulate RIPK3 and MLKL ubiquitylation. Hence, when IAPs are absent, LPS triggers RIPK3 to activate caspase-8, promoting apoptosis and NLRP3–caspase-1 activation, independent of RIPK3 kinase activity and MLKL. In contrast, in the absence of both IAPs and caspase-8, RIPK3 kinase activity and MLKL are essential for TLR-induced NLRP3 activation. Consistent with in vitro experiments, interleukin-1 (IL-1)-dependent autoantibody-mediated arthritis is exacerbated in mice lacking IAPs, and is reduced by deletion of RIPK3, but not MLKL. Therefore RIPK3 can promote NLRP3 inflammasome and IL-1β inflammatory responses independent of MLKL and necroptotic cell death.

Novel monoclonal antibody specific for the de2-7 epidermal growth factor receptor (EGFR) that also recognizes the EGFR expressed in cells containing amplification of the EGFR gene

In some respects, the EGFR appears to be an attractive target for tumor‐targeted antibody therapy: it is overexpressed in many types of epithelial tumor and inhibition of signaling often induces an anti‐tumor effect. The use of EGFR specific antibodies, however, may be limited by uptake in organs that have high endogenous levels of the wild type EGFR such as the liver. The de2‐7 EGFR (or EGFRvIII) is a naturally occurring extracellular truncation of the EGFR found in a number of tumor types including glioma, breast, lung and prostate. Antibodies directed to this tumor specific variant of the EGFR provide an alternative targeting strategy, although the lower proportion of tumors that express the de2‐7 EGFR restricts this approach. We describe a novel monoclonal antibody (MAb 806) that potentially overcomes the difficulties associated with targeting the EGFR expressed on the surface of tumor cells. MAb 806 bound to de2‐7 EGFR transfected U87MG glioma cells (U87MG.Δ2‐7) with high affinity (∼1 × 109 M−1), but did not bind parental cells that express the wild type EGFR. Consistent with this observation, MAb 806 was unable to bind a soluble version of the wild type EGFR containing the extracellular domain. In contrast, immobilization of this extracellular domain to ELISA plates induced saturating and dose response binding of MAb 806, suggesting that MAb 806 can bind the wild type EGFR under certain conditions. MAb 806 also bound to the surface of A431 cells, which due to an amplification of the EGFR gene express large amounts of the EGFR. Interestingly, MAb 806 only recognized 10% of the total EGFR molecules expressed by A431 cells and the binding affinity was lower than that determined for the de2‐7 EGFR. MAb 806 specifically targeted U87MG.Δ2‐7 and A431 xenografts grown in nude mice with peak levels in U87MG.Δ2‐7 xenografts detected 8 h after injection. No specific targeting of parental U87MG xenografts was observed. Following binding to U87MG.Δ2‐7 cells, MAb 806 was rapidly internalized by macropinocytosis and subsequently transported to lysosomes, a process that probably contributes to the early targeting peak observed in the xenografts. Thus, MAb 806 can be used to target tumor cells containing amplification of the EGFR gene or de2‐7 EGFR but does not bind to the wild type EGFR when expressed on the cell surface.

TNFR1-dependent cell death drives inflammation in Sharpin-deficient mice

SHARPIN regulates immune signaling and contributes to full transcriptional activity and prevention of cell death in response to TNF in vitro. The inactivating mouse Sharpin cpdm mutation causes TNF-dependent multi-organ inflammation, characterized by dermatitis, liver inflammation, splenomegaly, and loss of Peyers patches. TNF-dependent cell death has been proposed to cause the inflammatory phenotype and consistent with this we show Tnfr1, but not Tnfr2, deficiency suppresses the phenotype (and it does so more efficiently than Il1r1 loss). TNFR1-induced apoptosis can proceed through caspase-8 and BID, but reduction in or loss of these players generally did not suppress inflammation, although Casp8 heterozygosity significantly delayed dermatitis. Ripk3 or Mlkl deficiency partially ameliorated the multi-organ phenotype, and combined Ripk3 deletion and Casp8 heterozygosity almost completely suppressed it, even restoring Peyers patches. Unexpectedly, Sharpin, Ripk3 and Casp8 triple deficiency caused perinatal lethality. These results provide unexpected insights into the developmental importance of SHARPIN. DOI: http://dx.doi.org/10.7554/eLife.03464.001

Overexpression of insulin‐like growth factor binding protein‐6 inhibits rhabdomyosarcoma growth in vivo

Rhabdomyosarcoma is the most common soft‐tissue sarcoma of childhood. Rhabdomyosarcoma cell lines overexpress insulin‐like growth factor‐II (IGF‐II), an autocrine growth factor that is inhibited by insulin‐like growth factor binding protein‐6 (IGFBP‐6). IGFBP‐6 is associated with myoblast quiescence, and expression in rhabdomyosarcoma cells is low. The effect of IGFBP‐6 on 2 rhabdomyosarcoma cell lines, RD and Rh30, was studied. IGFBP‐6 inhibited anchorage‐dependent growth of RD and Rh30 cells in a dose‐dependent manner (p < 0.0001). IGFBP‐6 also inhibited anchorage‐independent growth of RD cells in soft agar in a dose‐dependent manner (p < 0.01). Anchorage‐independent growth of RD cells on polyhydroxyethylmethacrylate‐coated plates was decreased to a minimum of 48% of control after treatment with IGFBP‐6 (p < 0.001). In this system, IGFBP‐6 increased apoptosis 4‐fold (p < 0.001). IGF‐II partially reversed the IGFBP‐6‐induced decrease in growth and increase in apoptosis. Rh30 cells were stably transfected with an IGFBP‐6 cDNA and subcutaneous xenografts established in BALB/c nude mice. After 18 days, sizes of 2 independent clones of IGFBP‐6‐overexpressing Rh30 cells were reduced to 12% and 26% of vector control‐transfected tumors (p = 0.0006 and 0.002, respectively). IGFBP‐6 therefore inhibits proliferation and promotes apoptosis of rhabdomyosarcoma in vitro and dramatically inhibits xenograft growth in vivo, at least in part by inhibiting IGF‐II. Low expression of IGFBP‐6 may therefore contribute to rhabdomyosarcoma growth and metastasis.

Enhanced Efficacy of Radioimmunotherapy with 90Y-CHX-A″-DTPA-hu3S193 by Inhibition of Epidermal Growth Factor Receptor (EGFR) Signaling with EGFR Tyrosine Kinase Inhibitor AG1478

Purpose: Monoclonal antibodies and tyrosine kinase inhibitors specific for the epidermal growth factor receptor (EGFR) have been shown to enhance the effect of external beam radiation on EGFR-positive tumors. The effect of EGFR signaling abrogation by EGFR tyrosine kinase inhibitor on the efficacy of radioimmunotherapy has not been reported previously. This study investigated the effect of EGFR tyrosine kinase inhibition on the efficacy of radioimmunotherapy in a human cancer xenograft model. Experimental Design: The humanized anti–Lewis Y antibody hu3S193 and the EGFR tyrosine kinase inhibitor AG1478 were studied. BALB/c nude mice were engrafted with A431 squamous carcinoma cells. Initial biodistribution properties of the 90Y-CHX-A″-DTPA-hu3S193 were evaluated in this model. In therapy experiments, cohorts of four to five xenografted mice were treated with saline as placebo, 0.4 mg AG1478 i.p. (six doses over 2 weeks), single i.v. injections of unlabeled hu3S193, or 90Y-CHX-A″-DTPA-hu3S193 (12.5, 25, 50, or 100 μCi). The combination of 0.4 mg AG1478 i.p. and 25 μCi 90Y-CHX-A″-DTPA-hu3S193 i.v. was subsequently evaluated in the A431 model. Results:90Y-CHX-A″-DTPA-hu3S193 retained excellent immunoreactivity after radiolabeling. The biodistribution study showed excellent uptake in tumor (90.33 ± 38.84%ID/g) peaking at 24 to 72 hours after injection and with prolonged retention. 90Y-CHX-A″-DTPA-hu3S193 significantly inhibited A431 xenograft growth at 25, 50, and 100 μCi doses. The combination of 0.4 mg AG1478 with a single dose of 25 μCi 90Y-CHX-A″-DTPA-hu3S193 resulted in a significant enhancement of efficacy compared with either agent alone (P = 0.013). Conclusions: The efficacy of radioimmunotherapy with 90Y-CHX-A″-DTPA-hu3S193 is significantly enhanced by EGFR tyrosine kinase inhibitor AG1478. Further investigations of dosing regimens using EGFR tyrosine kinase inhibitors and radioimmunotherapy in the treatment of EGFR expressing tumors are warranted.

Targeting of Fn14 Prevents Cancer-Induced Cachexia and Prolongs Survival

The cytokine TWEAK and its cognate receptor Fn14 are members of the TNF/TNFR superfamily and are upregulated in tumors. We found that Fn14, when expressed in tumors, causes cachexia and that antibodies against Fn14 dramatically extended lifespan by inhibiting tumor-induced weight loss although having only moderate inhibitory effects on tumor growth. Anti-Fn14 antibodies prevented tumor-induced inflammation and loss of fat and muscle mass. Fn14 signaling in the tumor, rather than host, is responsible for inducing this cachexia because tumors in Fn14- and TWEAK-deficient hosts developed cachexia that was comparable to that of wild-type mice. These results extend the role of Fn14 in wound repair and muscle development to involvement in the etiology of cachexia and indicate that Fn14 antibodies may be a promising approach to treat cachexia, thereby extending lifespan and improving quality of life for cancer patients.

Necroptosis signalling is tuned by phosphorylation of MLKL residues outside the pseudokinase domain activation loop.

The pseudokinase MLKL (mixed lineage kinase domain-like), has recently emerged as a critical component of the necroptosis cell death pathway. Although it is clear that phosphorylation of the activation loop in the MLKL pseudokinase domain by the upstream protein kinase RIPK3 (receptor-interacting protein kinase-3), is crucial to trigger MLKL activation, it has remained unclear whether other phosphorylation events modulate MLKL function. By reconstituting Mlkl(-/-), Ripk3(-/-) and Mlkl(-/-)Ripk3(-/-) cells with MLKL phospho-site mutants, we compared the function of known MLKL phosphorylation sites in regulating necroptosis with three phospho-sites that we identified by MS, Ser(158), Ser(228) and Ser(248). Expression of a phosphomimetic S345D MLKL activation loop mutant-induced stimulus-independent cell death in all knockout cells, demonstrating that RIPK3 phosphorylation of the activation loop of MLKL is sufficient to induce cell death. Cell death was also induced by S228A, S228E and S158A MLKL mutants in the absence of death stimuli, but was most profound in Mlkl(-/-)Ripk3(-/-) double knockout fibroblasts. These data reveal a potential role for RIPK3 as a suppressor of MLKL activation and indicate that phosphorylation can fine-tune the ability of MLKL to induce necroptosis.

TRAF2 regulates TNF and NF-κB signalling to suppress apoptosis and skin inflammation independently of Sphingosine kinase 1.

TRAF2 is a component of TNF superfamily signalling complexes and plays an essential role in the regulation and homeostasis of immune cells. TRAF2 deficient mice die around birth, therefore its role in adult tissues is not well-explored. Furthermore, the role of the TRAF2 RING is controversial. It has been claimed that the atypical TRAF2 RING cannot function as a ubiquitin E3 ligase but counterclaimed that TRAF2 RING requires a co-factor, sphingosine-1-phosphate, that is generated by the enzyme sphingosine kinase 1, to function as an E3 ligase. Keratinocyte-specific deletion of Traf2, but not Sphk1 deficiency, disrupted TNF mediated NF-κB and MAP kinase signalling and caused epidermal hyperplasia and psoriatic skin inflammation. This inflammation was driven by TNF, cell death, non-canonical NF-κB and the adaptive immune system, and might therefore represent a clinically relevant model of psoriasis. TRAF2 therefore has essential tissue specific functions that do not overlap with those of Sphk1. DOI: http://dx.doi.org/10.7554/eLife.10592.001

Targeting properties of an anti-CD16/anti-CD30 bispecific antibody in an in vivo system.

Abstract Bispecific antibodies are currently being used in clinical trials in increasing numbers in the areas of breast cancer, prostate cancer, non-Hodgkins lymphoma and Hodgkins lymphoma. We have previously performed two clinical trials in patients with Hodgkins disease with an anti-CD30/anti-CD16 bispecific antibody and demonstrated a 30% response rate in a cohort of patients otherwise resistant to standard therapeutic modalities. However, no surrogate marker could be defined in these trials indicative of optimal antibody dosing/scheduling or predictive for favorable response. In order to evaluate accurately the potential biodistribution properties of bispecific antibody in patients, we have performed a detailed analysis of the binding properties and animal model in vivo characteristics of these constructs. For this purpose, the parental antibodies (anti-CD30 and anti-CD16) and the bispecific antibody (anti-CD30/anti-CD16) were radiolabeled with either 125I or 111In. Antibody integrity and binding properties after labeling were confirmed by Scatchard plot and Lindmo analysis. 111In-labeled antibodies revealed superior targeting properties in a standard SCID mouse tumor model. Both the bivalent parental anti-CD30 monoclonal antibody and the monovalent anti-CD30/anti-CD16 bispecific antibody showed excellent uptake in CD30+ tumors which did not differ significantly between the two (maximum uptake 16.5% ± 4.2% vs. 18.4% ± 3.8% injected dose/gram tissue). The equivalent targeting properties of the bispecific antibody compared with the parental anti-CD30 antibody encourages the further clinical development of this bispecific antibody, and might help to explain the clinical responses seen with this antibody so far in patients suffering from Hodgkins disease.