Tankyrase-mediated ADP-ribosylation is a regulator of TNF-induced death.

Tumor necrosis factor (TNF) is a key component of the innate immune response. Upon binding to its receptor, TNFR1, it promotes production of other cytokines via a membrane-bound complex 1 or induces cell death via a cytosolic complex 2. To understand how TNF-induced cell death is regulated, we performed mass spectrometry of complex 2 and identified tankyrase-1 as a native component that, upon a death stimulus, mediates complex 2 poly-ADP-ribosylation (PARylation). PARylation promotes recruitment of the E3 ligase RNF146, resulting in proteasomal degradation of complex 2, thereby limiting cell death. Expression of the ADP-ribose-binding/hydrolyzing severe acute respiratory syndrome coronavirus 2 macrodomain sensitizes cells to TNF-induced death via abolishing complex 2 PARylation. This suggests that disruption of ADP-ribosylation during an infection can prime a cell to retaliate with an inflammatory cell death.

Targeting triple-negative breast cancers with the Smac-mimetic birinapant.

Smac mimetics target inhibitor of apoptosis (IAP) proteins, thereby suppressing their function to facilitate tumor cell death. Here we have evaluated the efficacy of the preclinical Smac-mimetic compound A and the clinical lead birinapant on breast cancer cells. Both exhibited potent in vitro activity in triple-negative breast cancer (TNBC) cells, including those from patient-derived xenograft (PDX) models. Birinapant was further studied using in vivo PDX models of TNBC and estrogen receptor-positive (ER+) breast cancer. Birinapant exhibited single agent activity in all TNBC PDX models and augmented response to docetaxel, the latter through induction of TNF. Transcriptomic analysis of TCGA datasets revealed that genes encoding mediators of Smac-mimetic-induced cell death were expressed at higher levels in TNBC compared with ER+ breast cancer, resulting in a molecular signature associated with responsiveness to Smac mimetics. In addition, the cell death complex was preferentially formed in TNBCs versus ER+ cells in response to Smac mimetics. Taken together, our findings provide a rationale for prospectively selecting patients whose breast tumors contain a competent death receptor signaling pathway for the further evaluation of birinapant in the clinic.

NK cell-derived GM-CSF potentiates inflammatory arthritis and is negatively regulated by CIS.

Despite increasing recognition of the importance of GM-CSF in autoimmune disease, it remains unclear how GM-CSF is regulated at sites of tissue inflammation. Using GM-CSF fate reporter mice, we show that synovial NK cells produce GM-CSF in autoantibody-mediated inflammatory arthritis. Synovial NK cells promote a neutrophilic inflammatory cell infiltrate, and persistent arthritis, via GM-CSF production, as deletion of NK cells, or specific ablation of GM-CSF production in NK cells, abrogated disease. Synovial NK cell production of GM-CSF is IL-18-dependent. Furthermore, we show that cytokine-inducible SH2-containing protein (CIS) is crucial in limiting GM-CSF signaling not only during inflammatory arthritis but also in experimental allergic encephalomyelitis (EAE), a murine model of multiple sclerosis. Thus, a cellular cascade of synovial macrophages, NK cells, and neutrophils mediates persistent joint inflammation via production of IL-18 and GM-CSF. Endogenous CIS provides a key brake on signaling through the GM-CSF receptor. These findings shed new light on GM-CSF biology in sterile tissue inflammation and identify several potential therapeutic targets.

Generation of novel Id2 and E2-2, E2A and HEB antibodies reveals novel Id2 binding partners and species-specific expression of E-proteins in NK cells.

NK cells are cytotoxic lymphocytes with a key role in limiting tumour metastases. In mice, the NK cell lineage continually expresses high levels of the Inhibitor of DNA-binding 2 (Id2) protein and loss of Id2 is incongruous with their survival due to aberrant E-protein target gene activity. Using novel Id2 and E-protein antibodies that detect both mouse and human proteins, we have extensively characterised Id2 and E-protein expression in murine and human NK cells. We detected clear expression of E2 A and HEB, and to a lesser extent E2-2 in murine NK cells. In contrast HEB appears to be the major E-protein expressed in human NK cells, with minor E2-2 expression and surprisingly, no E2 A detected in primary NK cells nor human NK cell lines. These novel antibodies are also functional in immunofluorescence and immunoprecipitation. Mass spectrometry analysis of Id2 immuno-precipitated from murine NK cells revealed a number of novel associated proteins including several members of the SWI/SNF-related matrix-associated actin-dependent regulator chromatin (SMARC) and Mediator complex (MED) families. Taken together, these data highlight the utility of novel Id2 and E-protein antibodies and caution against mouse models for understanding Id2/E-protein biology in NK cells given their clearly disparate expression patternbetween species.

IL4 and IL21 cooperate to induce the high Bcl6 protein level required for germinal center formation.

Bcl6 (B-cell lymphoma 6) is a transcriptional repressor and critical mediator of the germinal center reaction during a T-cell-dependent antibody response, where it enables somatic hypermutation of immunoglobulin genes and inhibits terminal differentiation via repression of Blimp1. It can also contribute to the development of diffuse large B-cell lymphoma when expressed inappropriately. Bcl6 regulation is mediated both at the transcriptional and post-transcriptional levels, and in particular a strong signal through the B-cell receptor causes rapid proteasomal degradation of Bcl6. Despite the importance of Bcl6 in both immunity and cancer, little is known about how other extrinsic factors regulate Bcl6 in B cells. Here we show that Bcl6 is indeed highly unstable in B cells after a B-cell receptor (BCR) signal, but that the T-cell-derived cytokines interleukin 4 (IL4) and IL21 counteract BCR-mediated degradation, preserving Bcl6 protein levels. Stat6, downstream of IL4, can induce Bcl6 transcription directly. In vivo, B-cell intrinsic loss of IL4 or IL21 signaling reduces the magnitude or duration of the GC response, respectively, while their combined loss almost completely eliminates the GC response. This work provides key insights into the effect mediated by T-follicular helper cytokines on Bcl6 regulation.

Id2 and E Proteins Orchestrate the Initiation and Maintenance of MLL-Rearranged Acute Myeloid Leukemia.

E proteins and their antagonists, the Id proteins, are transcriptional regulators important for normal hematopoiesis. We found that Id2 acts as a key regulator of leukemia stem cell (LSC) potential in MLL-rearranged acute myeloid leukemia (AML). Low endogenous Id2 expression is associated with LSC enrichment while Id2 overexpression impairs MLL-AF9-leukemia initiation and growth. Importantly, MLL-AF9 itself controls the E-protein pathway by suppressing Id2 while directly activating E2-2 expression, and E2-2 depletion phenocopies Id2 overexpression in MLL-AF9-AML cells. Remarkably, Id2 tumor-suppressive function is conserved in t(8;21) AML. Low expression of Id2 and its associated gene signature are associated with poor prognosis in MLL-rearranged and t(8;21) AML patients, identifying the Id2/E-protein axis as a promising new therapeutic target in AML.

Germinal center-independent, IgM-mediated autoimmunity in sanroque mice lacking Obf1.

Mice homozygous for a point mutation in the Rc3h1 gene encoding Roquin1, designated sanroque mice, develop a severe antibody-mediated autoimmune condition. The disease is T-cell intrinsic, exacerbated by macrophage-intrinsic defects and driven by excessive T follicular helper cell generation and spontaneous germinal centre (GC) formation. This culminates in abnormally high numbers of plasma cells secreting high-affinity autoreactive immunoglobulin G (IgG). Obf1 is a transcriptional co-activator required for normal T-cell-dependent antibody responses, and it is essential for GC formation under all circumstances so far tested. We crossed sanroque mice with Obf1-null mice to determine whether the hyperactivity of sanroque T cells could drive Obf1(-/-) B cells to differentiate to GC B cells, or conversely, if Obf1 loss would prevent sanroque-mediated autoimmune disease. Surprisingly, while sanroque/Obf1(-/-) mice did not form GC, they still developed autoimmune disease and succumbed even more rapidly than did sanroque mice. The disease was mediated by autoreactive IgM, which may have been derived from a pre-existing population of autoreactive B cells in the Obf1(-/-) mice responding to the over-exuberant activity of sanroque CD4 cells.

Bak activation for apoptosis involves oligomerization of dimers via their alpha6 helices.

A pivotal step toward apoptosis is oligomerization of the Bcl-2 relative Bak. We recently reported that its oligomerization initiates by insertion of an exposed BH3 domain into the groove of another Bak monomer. We now report that the resulting BH3:groove dimers can be converted to the larger oligomers that permeabilize mitochondria by an interface between alpha6 helices. Cysteine residues placed in alpha6 could be crosslinked only after apoptotic signaling. Cysteines placed at both interfaces established that the BH3:groove dimer is symmetric and that the alpha6:alpha6 interface can link these dimers into homo-oligomers containing at least 18 Bak molecules. A putative zinc-binding site in alpha6 was not required to form the alpha6:alpha6 interface, and its mutation in full-length Bak did not affect Bak conformation, oligomerization, or function. We conclude that alpha6:alpha6 interaction occurs during Bak oligomerization and proapoptotic function, but we find no evidence that zinc binding to that interface regulates apoptosis.

To trigger apoptosis, Bak exposes its BH3 domain and homodimerizes via BH3:groove interactions.

The Bcl-2 relative Bak is thought to drive apoptosis by forming homo-oligomers that permeabilize mitochondria, but how it is activated and oligomerizes is unclear. To clarify these pivotal steps toward apoptosis, we have characterized multiple random loss-of-function Bak mutants and explored the mechanism of Bak conformation change during apoptosis. Single missense mutations located to the alpha helix 2-5 region of Bak, with most altering the BH3 domain or hydrophobic groove (BH1 domain). Loss of function invariably corresponded to impaired ability to oligomerize. An essential early step in Bak activation was shown to be exposure of the BH3 domain, which became reburied in dimers. We demonstrate that oligomerization involves insertion of the BH3 domain of one Bak molecule into the groove of another and may produce symmetric Bak dimers. We conclude that this BH3:groove interaction is essential to nucleate Bak oligomerization, which in turn is required for its proapoptotic function.

Determination of cell survival by RING-mediated regulation of inhibitor of apoptosis (IAP) protein abundance.

Inhibitor of apoptosis (IAP) proteins, which bind to caspases via their baculoviral IAP repeat domains, also bear RING domains that enable them to promote ubiquitylation of themselves and other interacting proteins. Here we show that the RING domain of cIAP1 allows it to bind directly to the RING of X-linked IAP, causing its ubiquitylation and degradation by the proteasome, thus revealing a mechanism by which IAPs can regulate their abundance. Expression of a construct containing the RING of cellular IAP1 was able to deplete melanoma cells of endogenous X-linked IAP, promoted apoptosis, and also markedly reduced their clonogenicity when treated with cisplatin. Cross control of protein levels by RING domains may therefore enable their levels to be manipulated therapeutically.