Potent GCN2 Inhibitor Capable of Reversing MDSC-Driven T Cell Suppression Demonstrates In Vivo Efficacy as a Single Agent and in Combination with Anti-Angiogenesis Therapy.

General control nonderepressible 2 (GCN2) protein kinase is a cellular stress sensor within the tumor microenvironment (TME), whose signaling cascade has been proposed to contribute to immune escape in tumors. Herein, we report the discovery of cell-potent GCN2 inhibitors with excellent selectivity against its closely related Integrated Stress Response (ISR) family members heme-regulated inhibitor kinase (HRI), protein kinase R (PKR), and (PKR)-like endoplasmic reticulum kinase (PERK), as well as good kinome-wide selectivity and favorable PK. In mice, compound 39 engages GCN2 at levels ≥80% with an oral dose of 15 mg/kg BID. We also demonstrate the ability of compound 39 to alleviate MDSC-related T cell suppression and restore T cell proliferation, similar to the effect seen in MDSCs from GCN2 knockout mice. In the LL2 syngeneic mouse model, compound 39 demonstrates significant tumor growth inhibition (TGI) as a single agent. Furthermore, TGI mediated by anti-VEGFR was enhanced by treatment with compound 39 demonstrating the complementarity of these two mechanisms.

NRF2 Activation Promotes Aggressive Lung Cancer and Associates with Poor Clinical Outcomes.

PURPOSE: Stabilization of the transcription factor NRF2 through genomic alterations in KEAP1 and NFE2L2 occurs in a quarter of patients with lung adenocarcinoma and a third of patients with lung squamous cell carcinoma. In lung adenocarcinoma, KEAP1 loss often co-occurs with STK11 loss and KRAS-activating alterations. Despite its prevalence, the impact of NRF2 activation on tumor progression and patient outcomes is not fully defined. EXPERIMENTAL DESIGN: We model NRF2 activation, STK11 loss, and KRAS activation in vivo using novel genetically engineered mouse models. Furthermore, we derive a NRF2 activation signature from human non-small cell lung tumors that we use to dissect how these genomic events impact outcomes and immune contexture of participants in the OAK and IMpower131 immunotherapy trials. RESULTS: Our in vivo data reveal roles for NRF2 activation in (i) promoting rapid-onset, multifocal intrabronchiolar carcinomas, leading to lethal pulmonary dysfunction, and (ii) decreasing elevated redox stress in KRAS-mutant, STK11-null tumors. In patients with nonsquamous tumors, the NRF2 signature is negatively prognostic independently of STK11 loss. Patients with lung squamous cell carcinoma with low NRF2 signature survive longer when receiving anti-PD-L1 treatment. CONCLUSIONS: Our in vivo modeling establishes NRF2 activation as a critical oncogenic driver, cooperating with STK11 loss and KRAS activation to promote aggressive lung adenocarcinoma. In patients, oncogenic events alter the tumor immune contexture, possibly having an impact on treatment responses. Importantly, patients with NRF2-activated nonsquamous or squamous tumors have poor prognosis and show limited response to anti-PD-L1 treatment.

The Hippo pathway effector TAZ induces TEAD-dependent liver inflammation and tumors.

The Hippo signaling pathway regulates organ size and plays critical roles in maintaining tissue growth, homeostasis, and regeneration. Dysregulated in a wide spectrum of cancers, in mammals, this pathway is regulated by two key effectors, YAP and TAZ, that may functionally overlap. We found that TAZ promoted liver inflammation and tumor development. The expression of TAZ, but not YAP, in human liver tumors positively correlated with the expression of proinflammatory cytokines. Hyperactivated TAZ induced substantial myeloid cell infiltration into the liver and the secretion of proinflammatory cytokines through a TEAD-dependent mechanism. Furthermore, tumors with hyperactivated YAP and TAZ had distinct transcriptional signatures, which included the increased expression of inflammatory cytokines in TAZ-driven tumors. Our study elucidated a previously uncharacterized link between TAZ activity and inflammatory responses that influence tumor development in the liver.

Apoptotic cell-induced AhR activity is required for immunological tolerance and suppression of systemic lupus erythematosus in mice and humans.

The transcription factor AhR modulates immunity at multiple levels. Here we report that phagocytes exposed to apoptotic cells exhibited rapid activation of AhR, which drove production of the cytokine IL-10. Activation of AhR was dependent on interactions between apoptotic-cell DNA and the pattern-recognition receptor TLR9 that was required for the prevention of immune responses to DNA and histones in vivo. Moreover, disease progression in mouse systemic lupus erythematosus (SLE) correlated with strength of the AhR signal, and the disease course could be altered by modulation of AhR activity. Deletion of AhR in the myeloid lineage caused systemic autoimmunity in mice, and an enhanced AhR transcriptional signature correlated with disease in patients with SLE. Thus, AhR activity induced by apoptotic cell phagocytes maintains peripheral tolerance.

The amino acid sensor GCN2 inhibits inflammatory responses to apoptotic cells promoting tolerance and suppressing systemic autoimmunity.

Efficient apoptotic cell clearance and induction of immunologic tolerance is a critical mechanism preventing autoimmunity and associated pathology. Our laboratory has reported that apoptotic cells induce tolerance by a mechanism dependent on the tryptophan catabolizing enzyme indoleamine 2,3 dioxygenase 1 (IDO1) in splenic macrophages (MΦ). The metabolic-stress sensing protein kinase GCN2 is a primary downstream effector of IDO1; thus, we tested its role in apoptotic cell-driven immune suppression. In vitro, expression of IDO1 in MΦs significantly enhanced apoptotic cell-driven IL-10 and suppressed IL-12 production in a GCN2-dependent mechanism. Suppression of IL-12 protein production was due to attenuation of IL-12 mRNA association with polyribosomes inhibiting translation while IL-10 mRNA association with polyribosomes was not affected. In vivo, apoptotic cell challenge drove a rapid, GCN2-dependent stress response in splenic MΦs with increased IL-10 and TGF-ß production, whereas myeloid-specific deletion of GCN2 abrogated regulatory cytokine production with provocation of inflammatory T-cell responses to apoptotic cell antigens and failure of long-tolerance induction. Consistent with a role in prevention of apoptotic cell driven autoreactivity, myeloid deletion of GCN2 in lupus-prone mice resulted in increased immune cell activation, humoral autoimmunity, renal pathology, and mortality. In contrast, activation of GCN2 with an agonist significantly reduced anti-DNA autoantibodies and protected mice from disease. Thus, this study implicates a key role for GCN2 signals in regulating the tolerogenic response to apoptotic cells and limiting autoimmunity.

Amino acid metabolism inhibits antibody-driven kidney injury by inducing autophagy.

Inflammatory kidney disease is a major clinical problem that can result in end-stage renal failure. In this article, we show that Ab-mediated inflammatory kidney injury and renal disease in a mouse nephrotoxic serum nephritis model was inhibited by amino acid metabolism and a protective autophagic response. The metabolic signal was driven by IFN-γ-mediated induction of indoleamine 2,3-dioxygenase 1 (IDO1) enzyme activity with subsequent activation of a stress response dependent on the eIF2α kinase general control nonderepressible 2 (GCN2). Activation of GCN2 suppressed proinflammatory cytokine production in glomeruli and reduced macrophage recruitment to the kidney during the incipient stage of Ab-induced glomerular inflammation. Further, inhibition of autophagy or genetic ablation of Ido1 or Gcn2 converted Ab-induced, self-limiting nephritis to fatal end-stage renal disease. Conversely, increasing kidney IDO1 activity or treating mice with a GCN2 agonist induced autophagy and protected mice from nephritic kidney damage. Finally, kidney tissue from patients with Ab-driven nephropathy showed increased IDO1 abundance and stress gene expression. Thus, these findings support the hypothesis that the IDO-GCN2 pathway in glomerular stromal cells is a critical negative feedback mechanism that limits inflammatory renal pathologic changes by inducing autophagy.

Marginal zone CD169+ macrophages coordinate apoptotic cell-driven cellular recruitment and tolerance.

Tolerance to apoptotic cells is essential to prevent inflammatory pathology. Though innate responses are critical for immune suppression, our understanding of early innate immunity driven by apoptosis is lacking. Herein we report apoptotic cells induce expression of the chemokine CCL22 in splenic metallophillic macrophages, which is critical for tolerance. Systemic challenge with apoptotic cells induced rapid production of CCL22 in CD169(+) (metallophillic) macrophages, resulting in accumulation and activation of FoxP3(+) Tregs and CD11c(+) dendritic cells, an effect that could be inhibited by antagonizing CCL22-driven chemotaxis. This mechanism was essential for suppression after apoptotic cell challenge, because neutralizing CCL22 or its receptor, reducing Treg numbers, or blocking effector mechanisms abrogated splenic TGF-ß and IL-10 induction; this promoted a shift to proinflammatory cytokines associated with a failure to suppress T cells. Similarly, CCR4 inhibition blocked long-term, apoptotic cell-induced tolerance to allografts. Finally, CCR4 inhibition resulted in a systemic breakdown of tolerance to self after apoptotic cell injection with rapid increases in anti-dsDNA IgG and immune complex deposition. Thus, the data demonstrate CCL22-dependent chemotaxis is a key early innate response required for apoptotic cell-induced suppression, implicating a previously unknown mechanism of macrophage-dependent coordination of early events leading to stable tolerance.

Cutting edge: DNA sensing via the STING adaptor in myeloid dendritic cells induces potent tolerogenic responses.

Cytosolic DNA sensing via the stimulator of IFN genes (STING) adaptor incites autoimmunity by inducing type I IFN (IFN-αß). In this study, we show that DNA is also sensed via STING to suppress immunity by inducing IDO. STING gene ablation abolished IFN-αß and IDO induction by dendritic cells (DCs) after DNA nanoparticle (DNP) treatment. Marginal zone macrophages, some DCs, and myeloid cells ingested DNPs, but CD11b(+) DCs were the only cells to express IFN-ß, whereas CD11b(+) non-DCs were major IL-1ß producers. STING ablation also abolished DNP-induced regulatory responses by DCs and regulatory T cells, and hallmark regulatory responses to apoptotic cells were also abrogated. Moreover, systemic cyclic diguanylate monophosphate treatment to activate STING induced selective IFN-ß expression by CD11b(+) DCs and suppressed Th1 responses to immunization. Thus, previously unrecognized functional diversity among physiologic innate immune cells regarding DNA sensing via STING is pivotal in driving immune responses to DNA.

O death where is thy sting? Immunologic tolerance to apoptotic self.

In higher organisms, innate scavenging cells maintain physiologic homeostasis by removal of the billions of apoptotic cells generated on a daily basis. Apoptotic cell removal requires efficient recognition and uptake by professional and non-professional phagocytic cells, which are governed by an array of soluble and apoptotic cell-integral signals resulting in immunologically silent clearance. While apoptosis is associated with profound suppression of adaptive and innate inflammatory immunity, we have only begun to scratch the surface in understanding how immunologic tolerance to apoptotic self manifest at either the molecular or cellular level. In the last 10 years, data has emerged implicating professional phagocytes, most notably stromal macrophages and CD8α(+)CD103(+) dendritic cells, as critical in initiation of the regulatory cascade that will ultimately lead to long-term whole-animal immune tolerance. Importantly, recent work by our lab and others has shown that alterations in apoptotic cell perception by the innate immune system either by removal of critical phagocytic sentinels in secondary lymphoid organs or blockage of immunosuppressive pathways leads to pronounced inflammation with a breakdown of tolerance towards self. This challenges the paradigm that apoptotic cells are inherently immunosuppressive, suggesting that apoptotic cell tolerance is a "context-dependent" event.

Engineering DNA nanoparticles as immunomodulatory reagents that activate regulatory T cells.

Nanoparticles containing DNA complexed with the cationic polymer polyethylenimine are efficient vehicles to transduce DNA into cells and organisms. DNA/polyethylenimine nanoparticles (DNPs) also elicit rapid and systemic release of proinflammatory cytokines that promote antitumor immunity. In this study, we report that DNPs possess previously unrecognized immunomodulatory attributes due to rapid upregulation of IDO enzyme activity in lymphoid tissues of mice. IDO induction in response to DNP treatment caused dendritic cells and regulatory T cells (Tregs) to acquire potent regulatory phenotypes. As expected, DNP treatment stimulated rapid increase in serum levels of IFN type I (IFN-αß) and II (IFN-γ), which are both potent IDO inducers. IDO-mediated Treg activation was dependent on IFN type I receptor signaling, whereas IFN-γ receptor signaling was not essential for this response. Moreover, systemic IFN-γ release was caused by TLR9-dependent activation of NK cells, whereas TLR9 signaling was not required for IFN-αß release. Accordingly, DNPs lacking immunostimulatory TLR9 ligands in DNA stimulated IFN-αß production, induced IDO, and promoted regulatory outcomes, but did not stimulate potentially toxic, systemic release of IFN-γ. DNP treatment to induce IDO and activate Tregs blocked Ag-specific T cell responses elicited in vivo following immunization and suppressed joint pathology in a model of immune-mediated arthritis. Thus, DNPs lacking TLR9 ligands may be safe and effective reagents to protect healthy tissues from immune-mediated destruction in clinical hyperimmune syndromes.

Tolerance to apoptotic cells is regulated by indoleamine 2,3-dioxygenase.

Tolerance to self-antigens present in apoptotic cells is critical to maintain immune-homeostasis and prevent systemic autoimmunity. However, mechanisms that sustain self-tolerance are poorly understood. Here we show that systemic administration of apoptotic cells to mice induced splenic expression of the tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). IDO expression was confined to the splenic marginal zone and was abrogated by depletion of CD169(+) cells. Pharmacologic inhibition of IDO skewed the immune response to apoptotic cells, resulting in increased proinflammatory cytokine production and increased effector T-cell responses toward apoptotic cell-associated antigens. Presymptomatic lupus-prone MRL(lpr/lpr) mice exhibited abnormal elevated IDO expression in the marginal zone and red pulp and inhibition of IDO markedly accelerated disease progression. Moreover, chronic exposure of IDO-deficient mice to apoptotic cells induced a lupus-like disease with serum autoreactivity to double-stranded DNA associated with renal pathology and increased mortality. Thus, IDO limits innate and adaptive immunity to apoptotic self-antigens and IDO-mediated regulation inhibits inflammatory pathology caused by systemic autoimmune disease.

Heterologous protein incites abnormal plasma cell accumulation and autoimmunity in MRL-MpJ mice.

Although it is evident that there is complex interplay among genetic and environmental factors contributing to systemic autoimmunity, the events inciting autoreactivity are incompletely understood. Previously we demonstrated that MRL-MpJ mice posses a genetic background susceptible to autoimmunity development under conditions of altered inhibitory signaling. To gain better understanding of the influence of exogenous factors on autoreactivity in susceptible individuals, young MRL-MpJ mice were challenged with a single injection of heterologous protein and evaluated for evidence of autoimmunity. We found that MRL-MpJ mice developed high titer serum reactivity to DNA within 1 week of protein administration reaching maximal levels within 1 month. Importantly, the level of autoimmunity was sustained for an extended period of time (6 months). This was accompanied by a substantial increase in germinal center B cell and plasma cell numbers. In contrast, control mice showed no change in autoreactivity or lymphocyte homeostasis. Autoimmunity was dependent on marginal zone B cells as their depletion reduced serum auto-reactivity after challenge, thus suggesting immune stimulation with heterologous proteins can precipitate loss of B cell tolerance and autoimmunity in genetically prone individuals. This model may provide an important tool to further investigate the mechanisms whereby environmental stimuli trigger autoimmune reactivity in susceptible hosts.

Marginal zone macrophages suppress innate and adaptive immunity to apoptotic cells in the spleen.

Marginal zone macrophages (MZMs) are a small subset of specialized splenic macrophages known to interact with apoptotic material entering the spleen from circulation. To evaluate whether MZMs regulate immunity to apoptotic material we depleted MZMs and assessed innate and adaptive immune responses to apoptotic cells administered systemically. MZM depletion altered the spatial localization of apoptotic cells, which accumulated in T-cell areas of the lymphoid follicles. MZM depletion also enhanced phagocytosis of apoptotic cells by red pulp (CD68(+)F4/80(+)) macrophages, which expressed increased CD86, MHCII, and CCR7. MZM depletion led to increased production of proinflammatory cytokines and enhanced lymphocyte responsiveness to apoptotic cell antigens. Furthermore, we found that MZM depletion accelerated autoimmune disease progression in mice genetically prone to systemic lupus erythematosus and caused significant mortality in wild-type mice repeatedly exposed to exogenous apoptotic thymocytes. These findings support the hypothesis that MZMs are central in the clearance of apoptotic cells to minimize the immunogenicity of autoantigens.