Inhibition of BET Proteins Regulates Fcγ Receptor Function and Reduces Inflammation in Rheumatoid Arthritis.

Overactivation of immune responses is a hallmark of autoimmune disease pathogenesis. This includes the heightened production of inflammatory cytokines such as Tumor Necrosis Factor α (TNFα), and the secretion of autoantibodies such as isotypes of rheumatoid factor (RF) and anticitrullinated protein antibody (ACPA). Fcγ receptors (FcγR) expressed on the surface of myeloid cells bind Immunoglobulin G (IgG) immune complexes. Recognition of autoantigen-antibody complexes by FcγR induces an inflammatory phenotype that results in tissue damage and further escalation of the inflammatory response. Bromodomain and extra-terminal protein (BET) inhibition is associated with reduced immune responses, making the BET family a potential therapeutic target for autoimmune diseases such as rheumatoid arthritis (RA). In this paper, we examined the BET inhibitor PLX51107 and its effect on regulating FcγR expression and function in RA. PLX51107 significantly downregulated expression of FcγRIIa, FcγRIIb, FcγRIIIa, and the common γ-chain, FcϵR1-γ, in both healthy donor and RA patient monocytes. Consistent with this, PLX51107 treatment attenuated signaling events downstream of FcγR activation. This was accompanied by a significant decrease in phagocytosis and TNFα production. Finally, in a collagen-induced arthritis model, PLX51107-treatment reduced FcγR expression in vivo accompanied by a significant reduction in footpad swelling. These results suggest that BET inhibition is a novel therapeutic approach that requires further exploration as a treatment for patients with RA.

Activation of the Intracellular Pattern Recognition Receptor NOD2 Promotes Acute Myeloid Leukemia (AML) Cell Apoptosis and Provides a Survival Advantage in an Animal Model of AML.

TLRs, a family of membrane-bound pattern recognition receptors found on innate immune cells, have been well studied in the context of cancer therapy. Activation of these receptors has been shown to induce inflammatory anticancer events, including differentiation and apoptosis, across a wide variety of malignancies. In contrast, intracellular pattern recognition receptors such as NOD-like receptors have been minimally studied. NOD2 is a member of the NOD-like receptor family that initiates inflammatory signaling in response to the bacterial motif muramyl dipeptide. In this study, we examined the influence of NOD2 in human acute myeloid leukemia (AML) cells, demonstrating that IFN-γ treatment upregulated the expression of NOD2 signaling pathway members SLC15A3 and SLC15A4, downstream signaling kinase RIPK2, and the NOD2 receptor itself. This priming allowed for effective induction of caspase-1-dependent cell death upon treatment with muramyl tripeptide phosphatidylethanolamine (MTP-PE), a synthetic ligand for NOD2. Furthermore, the combination of MTP-PE and IFN-γ on AML blasts generated an inflammatory cytokine profile and activated NK cells. In a murine model of AML, dual treatment with MTP-PE and IFN-γ led to a significant increase in mature CD27- CD11b+ NK cells as well as a significant reduction in disease burden and extended survival. These results suggest that NOD2 activation, primed by IFN-γ, may provide a novel therapeutic option for AML.

CD31 Acts as a Checkpoint Molecule and Is Modulated by FcγR-Mediated Signaling in Monocytes.

Monocytes and macrophages express FcγR that engage IgG immune complexes such as Ab-opsonized pathogens or cancer cells to destroy them by various mechanisms, including phagocytosis. FcγR-mediated phagocytosis is regulated by the concerted actions of activating FcγR and inhibitory receptors, such as FcγRIIb and SIRPα. In this study, we report that another ITIM-containing receptor, PECAM1/CD31, regulates FcγR function and is itself regulated by FcγR activation. First, quantitative RT-PCR and flow cytometry analyses revealed that human monocyte FcγR activation leads to a significant downregulation of CD31 expression, both at the message level and at surface expression, mainly mediated through FcγRIIa. Interestingly, the kinetics of downregulation between the two varied, with surface expression reducing earlier than the message. Experiments to analyze the mechanism behind this discrepancy revealed that the loss of surface expression was because of internalization, which depended predominantly on the PI3 kinase pathway and was independent of FcγR internalization. Finally, functional analyses showed that the downregulation of CD31 expression in monocytes by small interfering RNA enhanced FcγR-mediated phagocytic ability but have little effect on cytokine production. Together, these results suggest that CD31 acts as a checkpoint receptor that could be targeted to enhance FcγR functions in Ab-mediated therapies.

Anti-leukemic effects of all-trans retinoic acid in combination with Daratumumab in acute myeloid leukemia.

Acute myeloid leukemia (AML) remains a significant health problem, with poor outcomes despite chemotherapy and bone marrow transplants. Although one form of AML, acute promyelocytic leukemia (APL), is successfully treated with all-trans retinoic acid (ATRA), this drug is seemingly ineffective against all other forms of AML. Here, we show that ATRA up-regulates CD38 expression on AML blasts to sufficient levels that promote antibody-mediated fratricide following the addition of anti-CD38 daratumumab (DARA). The combination of ATRA plus DARA induced Fc-dependent conjugate formation and cytotoxicity among AML blasts in vitro. Combination treatment also led to reduction in tumor volume and resulted in increased overall survival in murine engraftment models of AML. These results suggest that, although ATRA does not induce differentiation of non-APL, it may be effective as a therapy in conjunction with DARA.

Interferon-γ Promotes Antibody-mediated Fratricide of Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is characterized by the proliferation of immature myeloid lineage blasts. Due to its heterogeneity and to the high rate of acquired drug resistance and relapse, new treatment strategies are needed. Here, we demonstrate that IFNγ promotes AML blasts to act as effector cells within the context of antibody therapy. Treatment with IFNγ drove AML blasts toward a more differentiated state, wherein they showed increased expression of the M1-related markers HLA-DR and CD86, as well as of FcγRI, which mediates effector responses to therapeutic antibodies. Importantly, IFNγ was able to up-regulate CD38, the target of the therapeutic antibody daratumumab. Because the antigen (CD38) and effector receptor (FcγRI) were both simultaneously up-regulated on the AML blasts, we tested whether IFNγ treatment of the AML cell lines THP-1 and MV4-11 could stimulate them to target one another after the addition of daratumumab. Results showed that IFNγ significantly increased daratumumab-mediated cytotoxicity, as measured both by 51Cr release and lactate dehydrogenase release assays. We also found that the combination of IFNγ and activation of FcγR led to the release of granzyme B by AML cells. Finally, using a murine NSG model of subcutaneous AML, we found that treatment with IFNγ plus daratumumab significantly attenuated tumor growth. Taken together, these studies show a novel mechanism of daratumumab-mediated killing and a possible new therapeutic strategy for AML.

EZH2-driven immune evasion defines high-risk pediatric AML with t(16;21) FUS::ERG gene fusion.

Despite decades of research, acute myeloid leukemia (AML) remains a remarkably lethal malignancy. While pediatric AML (pAML) carries a more favorable prognosis than adult AML, the past 25 years of large clinical trials have produced few improvements in pAML survival. Nowhere is this more evident than in patients carrying a t(16;21)(p11;q22) translocation, which yields the FUS::ERG fusion transcript. Patients with FUS::ERG-positive AML are often primary refractory, and most responders quickly relapse. In COG clinical trials, allogeneic stem cell transplantation was of no benefit to FUS::ERG pAML patients; 100% of transplanted patients succumbed to their disease. Expression of major histocompatibility complex (MHC) class I & II and costimulatory molecules is absent at diagnosis in FUS::ERG AML, mirroring the epigenetic mechanism of post-transplant relapse seen in adult AML and its associated dismal outcomes. Here we show that this class-defining immune-repressive phenotype is driven by overexpression of the EZH2 histone lysine methyltransferase in vitro and in multiple clinical cohorts. We show that treatment with the FDA-approved EZH2 inhibitor tazemetostat along with IFN-γ reverses this phenotype, re-establishes MHC presentation, and severely impairs the viability of FUS::ERG AML cells. EZH2 inhibitors may thus provide the first targeted therapeutic option for patients with this high-risk subtype of pAML, with particular benefit as a bridge to successful allogeneic stem cell transplantation.

Activation of plasmacytoid dendritic cells promotes AML-cell fratricide.

Acute myeloid leukemia (AML) is characterized by the proliferation of immature myeloid blasts and a suppressed immune state. Interferons have been previously shown to aid in the clearance of AML cells. Type I interferons are produced primarily by plasmacytoid dendritic cells (pDCs). However, these cells exist in a quiescent state in AML. Because pDCs express TLR 7-9, we hypothesized that the TLR7/8 agonist R848 would be able to reprogram them toward a more active, IFN-producing phenotype. Consistent with this notion, we found that R848-treated pDCs from patients produced significantly elevated levels of IFNß. In addition, they showed increased expression of the immune-stimulatory receptor CD40. We next tested whether IFNß would influence antibody-mediated fratricide among AML cells, as our recent work showed that AML cells could undergo cell-to cell killing in the presence of the CD38 antibody daratumumab. We found that IFNß treatment led to a significant, IRF9-dependent increase in CD38 expression and a subsequent increase in daratumumab-mediated cytotoxicity and decreased colony formation. These findings suggest that the tolerogenic phenotype of pDCs in AML can be reversed, and also demonstrate a possible means of enhancing endogenous Type I IFN production that would promote daratumumab-mediated clearance of AML cells.

Antibody Conjugation of Fluorescent Nanodiamonds for Targeted Innate Immune Cell Activation.

BACKGROUND: fluorescent nanodiamonds (FND) are nontoxic, infinitely photostable nanoparticles that emit near-infrared fluorescence and have a modifiable surface allowing for the generation of protein-FND conjugates. FND-mediated immune cell targeting may serve as a strategy to visualize immune cells and promote immune cell activation. METHODS: uncoated-FND (uFND) were fabricated, coated with glycidol (gFND), and conjugated with immunoglobulin G (IgG-gFND). In vitro studies were performed using a breast cancer/natural killer/monocyte co-culture system, and in vivo studies were performed using a breast cancer mouse model. RESULTS: in vitro studies demonstrated the targeted immune cell uptake of IgG-gFND, resulting in significant immune cell activation and no compromise in immune cell viability. IgG-gFND remained at the tumor site following intratumoral injection compared to uFND which migrated to the liver and kidneys. CONCLUSION: antibody-conjugated FND may serve as immune drug delivery vehicles with "track and trace capabilities" to promote directed antitumor activity and minimize systemic toxicities.