Monica Yabal

The Ubiquitin Ligase XIAP Recruits LUBAC for NOD2 Signaling in Inflammation and Innate Immunity

Nucleotide-binding and oligomerization domain (NOD)-like receptors constitute a first line of defense against invading bacteria. X-linked Inhibitor of Apoptosis (XIAP) is implicated in the control of bacterial infections, and mutations in XIAP are causally linked to immunodeficiency in X-linked lymphoproliferative syndrome type-2 (XLP-2). Here, we demonstrate that the RING domain of XIAP is essential for NOD2 signaling and that XIAP contributes to exacerbation of inflammation-induced hepatitis in experimental mice. We find that XIAP ubiquitylates RIPK2 and recruits the linear ubiquitin chain assembly complex (LUBAC) to NOD2. We further show that LUBAC activity is required for efficient NF-κB activation and secretion of proinflammatory cytokines after NOD2 stimulation. Remarkably, XLP-2-derived XIAP variants have impaired ubiquitin ligase activity, fail to ubiquitylate RIPK2, and cannot facilitate NOD2 signaling. We conclude that XIAP and LUBAC constitute essential ubiquitin ligases in NOD2-mediated inflammatory signaling and propose that deregulation of NOD2 signaling contributes to XLP-2 pathogenesis.

XIAP Restricts TNF- and RIP3-Dependent Cell Death and Inflammasome Activation

X-linked inhibitor of apoptosis protein (XIAP) has been identified as a potent regulator of innate immune responses, and loss-of-function mutations in XIAP cause the development of the X-linked lymphoproliferative syndrome type 2 (XLP-2) in humans. Using gene-targeted mice, we show that loss of XIAP or deletion of its RING domain lead to excessive cell death and IL-1β secretion from dendritic cells triggered by diverse Toll-like receptor stimuli. Aberrant IL-1β secretion is TNF dependent and requires RIP3 but is independent of cIAP1/cIAP2. The observed cell death also requires TNF and RIP3 but proceeds independently of caspase-1/caspase-11 or caspase-8 function. Loss of XIAP results in aberrantly elevated ubiquitylation of RIP1 outside of TNFR complex I. Virally infected Xiap(-/-) mice present with symptoms reminiscent of XLP-2. Our data show that XIAP controls RIP3-dependent cell death and IL-1β secretion in response to TNF, which might contribute to hyperinflammation in patients with XLP-2.

Disease-causing mutations in the XIAP BIR2 domain impair NOD2-dependent immune signalling.

X‐linked Inhibitor of Apoptosis (XIAP) is an essential ubiquitin ligase for pro‐inflammatory signalling downstream of the nucleotide‐binding oligomerization domain containing (NOD)‐1 and ‐2 pattern recognition receptors. Mutations in XIAP cause X‐linked lymphoproliferative syndrome type‐2 (XLP2), an immunodeficiency associated with a potentially fatal deregulation of the immune system, whose aetiology is not well understood. Here, we identify the XIAP baculovirus IAP repeat (BIR)2 domain as a hotspot for missense mutations in XLP2. We demonstrate that XLP2‐BIR2 mutations severely impair NOD1/2‐dependent immune signalling in primary cells from XLP2 patients and in reconstituted XIAP‐deficient cell lines. XLP2‐BIR2 mutations abolish the XIAP‐RIPK2 interaction resulting in impaired ubiquitylation of RIPK2 and recruitment of linear ubiquitin chain assembly complex (LUBAC) to the NOD2‐complex. We show that the RIPK2 binding site in XIAP overlaps with the BIR2 IBM‐binding pocket and find that a bivalent Smac mimetic compound (SMC) potently antagonises XIAP function downstream of NOD2 to limit signalling. These findings suggest that impaired immune signalling in response to NOD1/2 stimulation is a general defect in XLP2 and demonstrate that the XIAP BIR2‐RIPK2 interaction may be targeted pharmacologically to modulate inflammatory signalling.

CYLD Limits Lys63- and Met1-Linked Ubiquitin at Receptor Complexes to Regulate Innate Immune Signaling

Summary Innate immune signaling relies on the deposition of non-degradative polyubiquitin at receptor-signaling complexes, but how these ubiquitin modifications are regulated by deubiquitinases remains incompletely understood. Met1-linked ubiquitin (Met1-Ub) is assembled by the linear ubiquitin assembly complex (LUBAC), and this is counteracted by the Met1-Ub-specific deubiquitinase OTULIN, which binds to the catalytic LUBAC subunit HOIP. In this study, we report that HOIP also interacts with the deubiquitinase CYLD but that CYLD does not regulate ubiquitination of LUBAC components. Instead, CYLD limits extension of Lys63-Ub and Met1-Ub conjugated to RIPK2 to restrict signaling and cytokine production. Accordingly, Met1-Ub and Lys63-Ub were individually required for productive NOD2 signaling. Our study thus suggests that LUBAC, through its associated deubiquitinases, coordinates the deposition of not only Met1-Ub but also Lys63-Ub to ensure an appropriate response to innate immune receptor activation.

RIPK3 Restricts Myeloid Leukemogenesis by Promoting Cell Death and Differentiation of Leukemia Initiating Cells

Since acute myeloid leukemia (AML) is characterized by the blockade of hematopoietic differentiation and cell death, we interrogated RIPK3 signaling in AML development. Genetic loss of Ripk3 converted murine FLT3-ITD-driven myeloproliferation into an overt AML by enhancing the accumulation of leukemia-initiating cells (LIC). Failed inflammasome activation and cell death mediated by tumor necrosis factor receptor caused this accumulation of LIC exemplified by accelerated leukemia onset in Il1r1(-/-), Pycard(-/-), and Tnfr1/2(-/-) mice. RIPK3 signaling was partly mediated by mixed lineage kinase domain-like. This link between suppression of RIPK3, failed interleukin-1β release, and blocked cell death was supported by significantly reduced RIPK3 in primary AML patient cohorts. Our data identify RIPK3 and the inflammasome as key tumor suppressors in AML.

Re-activation of mitochondrial apoptosis inhibits T cell lymphoma survival and treatment resistance

T lymphocyte non-Hodgkins lymphoma (T-NHL) represents an aggressive and largely therapy-resistant subtype of lymphoid malignancies. As deregulated apoptosis is a frequent hallmark of lymphomagenesis, we analyzed gene expression profiles and protein levels of primary human T-NHL samples for various apoptotic regulators. We identified the apoptotic regulator MCL-1 as the only pro-survival BCL-2 family member to be highly expressed throughout all human T-NHL subtypes. Functional validation of pro-survival protein members of the BCL-2 family in two independent T-NHL mouse models identified that the partial loss of Mcl-1 significantly delayed T-NHL development in vivo. Moreover, the inducible reduction of MCL-1 protein levels in lymphoma-burdened mice severely impaired the continued survival of T-NHL cells, increased their susceptibility to chemotherapeutics and delayed lymphoma progression. Lymphoma viability remained unaffected by the genetic deletion or pharmacological inhibition of all alternative BCL-2 family members. Consistent with a therapeutic window for MCL-1 treatment within the context of the whole organism, we observed an only minimal toxicity after systemic heterozygous loss of Mcl-1 in vivo. We conclude that re-activation of mitochondrial apoptosis by blockade of MCL-1 represents a promising therapeutic strategy to treat T-cell lymphoma.

Killing AML: RIPK3 leads the way

Acute myeloid leukemia (AML) is a heterogeneous group of haematopoietic neoplasms driven partly by the loss of differentiation and the blockade of cell death. AML is sustained by leukemia-initiating cells (LICs) that arise from pre-leukemic haematopoietic stem and progenitor cells (HSPCs) that carry genetic alterations being selected for during leukemogenesis. The resistance of LICs to standard chemotherapies presents a major clinical challenge as they eventually cause disease relapse and death. Understanding the mechanisms that protect LICs from cell death therefore provide an ideal target for future anti-leukemia therapy. The ability of LICs and pre-leukemic HSCs to evade cell death has been attributed to several pathways that are distinct from normal HSPCs. LICs for example have constitutive NF-kB activity while normal HSPCs do not. In addition, analysis of patient-derived cells showed that in some cases the apoptotic cell death pathway in LICs is skewed. However, work in mature myeloid cells has shown that these cells are exquisitely sensitive to undergoing cell death driven by receptor-interacting serine-threonine kinase 3 (RIPK3). In addition to inducing apoptosis, RIPK3 can also induce regulated necrosis, a form of necrotic cell death marked by cell and organelle swelling and plasma membrane rupture, which, in contrast to apoptosis, is accompanied by inflammation due to the release of cytokines and DAMPs from dying cells. We found that in a similar fashion LICs are also sensitive to RIPK3-dependent cell death and that loss of RIPK3 expression underlies the developmental block and oncogenic process in many subtypes of AML. In our study, we focused on one of the recurring genetic alterations found in AML that gives rise to internal tandem duplications of the tyrosine kinase receptor FLT3 (FLT3-ITD). FLT3-ITD mutatuions are found in 30–40% of cytogenetically normal AML patients and define a poor prognostic subgroup. Using a model of adoptive transfer of bone marrow (BM) cells transduced with FLT3-ITD we demonstrated that genetic loss of Ripk3 greatly accelerated FLT3-ITD-induced leukemia and mortality. The enhanced disease was marked by an expansion of common myeloid progenitors (CMPs) and short-term haematopoietic stem cells (ST-HSCs), being the cellular compartment to contain LICs. In addition, RIPK3 deletion rendered FLT3-ITD splenocytes serially transplantable, indicating an enhanced ability of transformed HSPCs to survive and propagate. RIPK3 functioned to induce cell death of ST-HSCs and CMPs and acted as a tumor suppressor. Further, consistent with their roles in RIPK3 activation, we showed that deletion of TNF receptors 1 and 2 (Tnfr1/2¡/¡) or inhibition of RIPK1 phenocopied the leukemic disease induced by Ripk3¡/¡ BM. However, Mlkl deficiency did not abrogate cell death in vitro, and disease progression in vivo was indistinguishable between WT and Mlkl¡/¡, suggesting that RIPK3 may also induce apoptosis of LICs, a function that is consistent with recently published results. Importantly, WT FLT3-ITD-transformed cells produced substantial amounts of the inflammatory cytokine IL-1b, while Ripk3 or Mlkl deficiency severely blunted expression of IL-1b. The role of IL-1 b in promoting LIC differentiation and thus restricting leukemogenesis was confirmed with Pycard¡/¡ and Il1r1¡/¡ HSPCs. Hence, Ripk3 signals through 2 complementary pathways, cell death and IL-1b production, to restrict LICs and to promote myeloid cell differentiation, respectively (Fig. 1). Using several human patient sample cohorts, we observed a reduced expression of both RIPK3 and MLKL in FLT3-mutated AML, and many other AML subtypes. For example, samples from patients harboring the AML-ETO fusion, which is characterized by t(8;21) translocation, also exhibited reduced RIPK3 and MLKL expression. Ripk3 deficiency similarly accelerated leukemic disease in mice transplanted with AML-ETO-transduced BM. By contrast, AML associated with mixed-lineage leukemia (MLL) translocations showed normal RIPK3 and MLKL expression, and Ripk3 deficiency did not alter the myeloproliferative neoplasm caused by MLL-ENL. This finding is consistent with independent studies showing that deletion of Ripk3 did not alter disease progression of MLL-ENL-, MLL-AF9-, NUP98HoxA9-, and HoxA9/Meis1-induced leukemia. Furthermore, the authors showed that these leukemia subsets were sensitive to killing by the SMAC mimetic birinapant in a RIPK1/TNFR1-dependent manner. Therefore, our work

RIPK3-dependent cell death and inflammasome activation in FLT3-ITD expressing LICs.

Acute myeloid leukemia (AML) is sustained by leukemia-initiating cells (LICs) with blocked myeloid differentiation and increased self-renewal capacities. These cells arise from pre-leukemic hematopoietic stem and progenitor cells (HSPCs) that carry genetic alterations being selected for during leukemogenesis [1]. The resistance of LICs to standard chemotherapies presents a major clinical challenge as they eventually cause disease relapse and death [2]. Understanding the mechanisms of LIC resistance to undergoing cell death is therefore critical for a curative therapy of AML. The ability of LICs and pre-leukemic HSCs to evade cell death has been attributed to several pathways that are distinct from normal HSPCs. LICs for example have constitutive NF-κB activity while normal HSPCs do not. In addition, analysis of patient-derived cells showed that in some cases the apoptotic cell death pathway in LICs is skewed. However, work in mature myeloid cells has shown that these cells are exquisitely sensitive to undergoing cell death driven by receptor-interacting serine-threonine kinase 3 (RIPK3) [3]. In addition to inducing apoptosis, RIPK3 can also induce regulated necrosis. In contrast to apoptosis, regulated necrosis in myeloid cells is accompanied by inflammation due to the release of cytokines and DAMPs from dying cells. We found that in a similar fashion LICs are also sensitive to RIPK3dependent cell death and that loss of RIPK3 expression underlies the developmental block and oncogenic process in many subtypes of AML [4]. In our study, we focused on one of the recurring genetic alterations found in AML that gives rise to internal tandem duplications of the tyrosine kinase receptor FLT3 (FLT3-ITD). In human patients FLT3-ITD is associated with poor prognosis and lower overall survival. Using a model of adoptive transfer of bone marrow (BM) cells transduced with FLT3-ITD we demonstrated that in the absence of Ripk3, FLT3-ITD-induced leukemia was accelerated and significantly more aggressive. The enhanced disease was due to the expansion of common myeloid progenitors (CMPs) and short-term hematopoietic stem cells (ST-HSCs), being the cellular compartment to contain LICs. RIPK3 functioned to induce cell death of these cells and acted as a tumor suppressor. We further showed that RIPK1 and the TNF receptors 1 and 2 (TNFR1/2) functioned upstream of RIPK3 to regulate cell death. However, Mlkl deficiency did not abrogate cell death in vitro, and disease progression in vivo was also not significantly different, suggesting that RIPK3 may also induce apoptosis of LICs, a function that is consistent with recently published results [5]. Importantly, WT FLT3-ITD-transformed cells produced substantial amounts of the inflammatory cytokine IL-1β which was severely blunted by Ripk3 or Mlkl deficiency. The role of IL-1β in promoting LIC differentiation and thus restricting leukemogenesis was confirmed with Pycard-/and Il1r1-/HSPCs. Hence, RIPK3 signals through two complementary pathways, cell death and IL-1β production, to restrict LICs and to promote myeloid cell differentiation, respectively (Figure 1). Using several human patient sample cohorts, we observed a reduced expression of both RIPK3 and Commentary: Autophagy and Cell Death

XIAP as a regulator of inflammatory cell death: the TNF and RIP3 angle

There is currently immense interest in understanding the biological consequences of aberrant necroptosis. The recently uncovered role for X-linked inhibitor of apoptosis protein (XIAP) in blocking tumor necrosis factor-dependent necroptosis explains, at least in part, the systemic hyperinflammatory syndrome XLP-2. However, it also points to rather unexpected differences between XIAP and the related proteins baculoviral IAP repeat containing 2 and 3 (cIAP1/2).