NOD2 activation enhances macrophage Fcγ receptor function and may increase the efficacy of antibody therapy.

Introduction: Therapeutic antibodies have become a major strategy to treat oncologic diseases. For chronic lymphocytic leukemia, antibodies against CD20 are used to target and elicit cytotoxic responses against malignant B cells. However, efficacy is often compromised due to a suppressive microenvironment that interferes with cellular immune responses. To overcome this suppression, agonists of pattern recognition receptors have been studied which promote direct cytotoxicity or elicit anti-tumoral immune responses. NOD2 is an intracellular pattern recognition receptor that participates in the detection of peptidoglycan, a key component of bacterial cell walls. This detection then mediates the activation of multiple signaling pathways in myeloid cells. Although several NOD2 agonists are being used worldwide, the potential benefit of these agents in the context of antibody therapy has not been explored. Methods: Primary cells from healthy-donor volunteers (PBMCs, monocytes) or CLL patients (monocytes) were treated with versus without the NOD2 agonist L18-MDP, then antibody-mediated responses were assessed. In vivo, the Eµ-TCL1 mouse model of CLL was used to test the effects of L18-MDP treatment alone and in combination with anti-CD20 antibody. Results: Treatment of peripheral blood mononuclear cells with L18-MDP led to activation of monocytes from both healthy donors and CLL patients. In addition, there was an upregulation of activating FcγR in monocytes and a subsequent increase in antibody-mediated phagocytosis. This effect required the NF-κB and p38 signaling pathways. Treatment with L18-MDP plus anti-CD20 antibody in the Eµ-TCL model of CLL led to a significant reduction of CLL load, as well as to phenotypic changes in splenic monocytes and macrophages. Conclusions: Taken together, these results suggest that NOD2 agonists help overturn the suppression of myeloid cells, and may improve the efficacy of antibody therapy for CLL.

Pyrimidine depletion enhances targeted and immune therapy combinations in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a fatal disease characterized by the accumulation of undifferentiated myeloblasts, and agents that promote differentiation have been effective in this disease but are not curative. Dihydroorotate dehydrogenase inhibitors (DHODHi) have the ability to promote AML differentiation and target aberrant malignant myelopoiesis. We introduce HOSU-53, a DHODHi with significant monotherapy activity, which is further enhanced when combined with other standard-of-care therapeutics. We further discovered that DHODHi modulated surface expression of CD38 and CD47, prompting the evaluation of HOSU-53 combined with anti-CD38 and anti-CD47 therapies, where we identified a compelling curative potential in an aggressive AML model with CD47 targeting. Finally, we explored using plasma dihydroorotate (DHO) levels to monitor HOSU-53 safety and found that the level of DHO accumulation could predict HOSU-53 intolerability, suggesting the clinical use of plasma DHO to determine safe DHODHi doses. Collectively, our data support the clinical translation of HOSU-53 in AML, particularly to augment immune therapies. Potent DHODHi to date have been limited by their therapeutic index; however, we introduce pharmacodynamic monitoring to predict tolerability while preserving antitumor activity. We additionally suggest that DHODHi is effective at lower doses with select immune therapies, widening the therapeutic index.

Active Hexose-Correlated Compound Shows Direct and Indirect Effects against Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a disease characterized by the accumulation of mature CD19+CD5+CD23+ B cells in the bloodstream and in lymphoid organs. It usually affects people over 70 years of age, which limits the options for treatments. The disease is typically well-managed, but to date is still incurable. Hence, the need for novel therapeutic strategies remains. Nurse-like cells (NLCs) are major components of the microenvironment for CLL, supporting tumor cell survival, proliferation, and even drug resistance. They are of myeloid lineage, guided toward differentiating into their tumor-supportive role by the CLL cells themselves. As such, they are analogous to tumor-associated macrophages and represent a major therapeutic target. Previously, it was found that a mushroom extract, Active Hexose-Correlated Compound (AHCC), promoted the death of acute myeloid leukemia cells while preserving normal monocytes. Given these findings, it was asked whether AHCC might have a similar effect on the abnormally differentiated myeloid-lineage NLCs in CLL. CLL-patient PBMCs were treated with AHCC, and it was found that AHCC treatment showed a direct toxic effect against isolated CLL cells. In addition, it significantly reduced the number of tumor-supportive NLCs and altered their phenotype. The effects of AHCC were then tested in the Eµ-TCL1 mouse model of CLL and the MllPTD/WT Flt3ITD/WT model of AML. Results showed that AHCC not only reduced tumor load and increased survival in the CLL and AML models, but it also enhanced antitumor antibody treatment in the CLL model. These results suggest that AHCC has direct and indirect effects against CLL and that it may be of benefit when combined with existing treatments.

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.

Disruption of Nurse-like Cell Differentiation as a Therapeutic Strategy for Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia, but, despite advances in treatment, many patients still experience relapse. CLL cells depend on interactions with supportive cells, and nurse-like cells (NLCs) are the major such cell type. However, little is known about how NLCs develop. Here, we performed DNA methylation analysis of CLL patient-derived NLCs using the 850K Illumina array, comparing CD14+ cells at day 1 (monocytes) versus day 14 (NLCs). We found a strong loss of methylation in AP-1 transcription factor binding sites, which may be driven by MAPK signaling. Testing of individual MAPK pathways (MEK, p38, and JNK) revealed a strong dependence on MEK/ERK for NLC development, because treatment of patient samples with the MEK inhibitor trametinib dramatically reduced NLC development in vitro. Using the adoptive transfer Eµ-TCL1 mouse model of CLL, we found that MEK inhibition slowed CLL progression, leading to lower WBC counts and to significantly longer survival time. There were also lower numbers of mouse macrophages, particularly within the M2-like population. In summary, NLC development depends on MEK signaling, and inhibition of MEK leads to increased survival time in vivo. Hence, targeting the MEK/ERK pathway may be an effective treatment strategy for CLL.

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.

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.

Active hexose-correlated compound enhances extrinsic-pathway-mediated apoptosis of Acute Myeloid Leukemic cells.

Active Hexose Correlated Compound (AHCC) has been shown to have many immunostimulatory and anti-cancer activities in mice and in humans. As a natural product, AHCC has potential to create safer adjuvant therapies in cancer patients. Acute Myeloid Leukemia (AML) is the least curable and second-most common leukemia in adults. AML is especially terminal to those over 60 years old, where median survival is only 5 to 10 months, due to inability to receive intensive chemotherapy. Hence, the purpose of this study was to investigate the effects of AHCC on AML cells both in vitro and in vivo. Results showed that AHCC induced Caspase-3-dependent apoptosis in AML cell lines as well as in primary AML leukopheresis samples. Additionally, AHCC induced Caspase-8 cleavage as well as Fas and TRAIL upregulation, suggesting involvement of the extrinsic apoptotic pathway. In contrast, monocytes from healthy donors showed suppressed Caspase-3 cleavage and lower cell death. When tested in a murine engraftment model of AML, AHCC led to significantly increased survival time and decreased blast counts. These results uncover a mechanism by which AHCC leads to AML-cell specific death, and also lend support for the further investigation of AHCC as a potential adjuvant for the treatment of AML.

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.

Targeting the RAS/MAPK pathway with miR-181a in acute myeloid leukemia.

Deregulation of microRNAs' expression frequently occurs in acute myeloid leukemia (AML). Lower miR-181a expression is associated with worse outcomes, but the exact mechanisms by which miR-181a mediates this effect remain elusive. Aberrant activation of the RAS pathway contributes to myeloid leukemogenesis. Here, we report that miR-181a directly binds to 3'-untranslated regions (UTRs); downregulates KRAS, NRAS and MAPK1; and decreases AML growth. The delivery of miR-181a mimics to target AML cells using transferrin-targeting lipopolyplex nanoparticles (NP) increased mature miR-181a; downregulated KRAS, NRAS and MAPK1; and resulted in decreased phosphorylation of the downstream RAS effectors. NP-mediated upregulation of miR-181a led to reduced proliferation, impaired colony formation and increased sensitivity to chemotherapy. Ectopic expression of KRAS, NRAS and MAPK1 attenuated the anti-leukemic activity of miR-181a mimics, thereby validating the relevance of the deregulated miR-181a-RAS network in AML. Finally, treatment with miR-181a-NP in a murine AML model resulted in longer survival compared to mice treated with scramble-NP control. These data support that targeting the RAS-MAPK-pathway by miR-181a mimics represents a novel promising therapeutic approach for AML and possibly for other RAS-driven cancers.

Gradual Rarefaction of Hematopoietic Precursors and Atrophy in a Depleted microRNA 29a, b and c Environment.

BACKGROUND: The self-renewing ability of HSCs is fundamental for the maintenance of a pool of bone marrow precursors throughout the life of an individual. The genetic mechanisms underlying such a complex process are still poorly understood. RESULTS AND SIGNIFICANCE: Here, we show that constitutive in vivo deletion of miR29ab1 leads to reduced number of HSCs and that miR29ab1 deficient bone marrow cannot repopulate the bone marrow of irradiated mice. An Affymetrix analysis of the miR29ab1 knockout mice identifies key proteins that could be responsible for this phenotype, as DNMT3a and b. Moreover, our findings reveal that whereas miR29b2c knockout mice do not exhibit any spontaneous abnormality, the double knock out--miR29ab1b2c--has marked generalized atrophy, raising the possibility that the two bi-cistrons might cooperate in order to maintain the stem cell number in general, not only limited to the bone marrow.

Decitabine priming enhances the antileukemic effects of exportin 1 (XPO1) selective inhibitor selinexor in acute myeloid leukemia.

The prognosis of acute myeloid leukemia (AML) is poor, highlighting the need for novel treatments. Hypomethylating agents, including decitabine are used to treat elderly AML patients with relative success. Targeting nuclear export receptor (exportin 1 [XPO1]) is a novel approach to restore tumor suppressor (TS) function in AML. Here, we show that sequential treatment of AML blasts with decitabine followed by selinexor (XPO1 inhibitor) enhances the antileukemic effects of selinexor. These effects could be mediated by the re-expression of a subset of TSs (CDKN1A and FOXO3A) that are epigenetically silenced via DNA methylation, and cytoplasmic-nuclear trafficking is regulated by XPO1. We observed a significant upregulation of CDKN1A and FOXO3A in decitabine- versus control-treated cells. Sequential treatment of decitabine followed by selinexor in an MV4-11 xenograft model significantly improved survival compared with selinexor alone. On the basis of these preclinical results, a phase 1 clinical trial of decitabine followed by selinexor in elderly patients with AML has been initiated.

Intronic miR-3151 within BAALC drives leukemogenesis by deregulating the TP53 pathway.

The BAALC/miR-3151 locus on chromosome 8q22 contains both the BAALC gene (for brain and acute leukemia, cytoplasmic) and miR-3151, which is located in intron 1 of BAALC. Older acute myeloid leukemia (AML) patients with high expression of both miR-3151 and the BAALC mRNA transcript have a low survival prognosis, and miR-3151 and BAALC expression is associated with poor survival independently of each other. We found that miR-3151 functioned as the oncogenic driver of the BAALC/miR-3151 locus. Increased production of miR-3151 reduced the apoptosis and chemosensitivity of AML cell lines and increased leukemogenesis in mice. Disruption of the TP53-mediated apoptosis pathway occurred in leukemia cells overexpressing miR-3151 and the miR-3151 bound to the 3' untranslated region of TP53. In contrast, BAALC alone had only limited oncogenic activity. We found that miR-3151 contains its own regulatory element, thus partly uncoupling miR-3151 expression from that of the BAALC transcript. Both genes were bound and stimulated by a complex of the transcription factors SP1 and nuclear factor κB (SP1/NF-κB). Disruption of SP1/NF-κB binding reduced both miR-3151 and BAALC expression. However, expression of only BAALC, but not miR-3151, was stimulated by the transcription factor RUNX1, suggesting a mechanism for the partly discordant expression of miR-3151 and BAALC observed in AML patients. Similar to the AML cells, in melanoma cell lines, overexpression of miR-3151 reduced the abundance of TP53, and knockdown of miR-3151 increased caspase activity, whereas miR-3151 overexpression reduced caspase activity. Thus, this oncogenic miR-3151 may also have a role in solid tumors.

SPARC promotes leukemic cell growth and predicts acute myeloid leukemia outcome.

Aberrant expression of the secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) gene, which encodes a matricellular protein that participates in normal tissue remodeling, is associated with a variety of diseases including cancer, but the contribution of SPARC to malignant growth remains controversial. We previously reported that SPARC was among the most upregulated genes in cytogenetically normal acute myeloid leukemia (CN-AML) patients with gene-expression profiles predictive of unfavorable outcome, such as mutations in isocitrate dehydrogenase 2 (IDH2-R172) and overexpression of the oncogenes brain and acute leukemia, cytoplasmic (BAALC) and v-ets erythroblastosis virus E26 oncogene homolog (ERG). In contrast, SPARC was downregulated in CN-AML patients harboring mutations in nucleophosmin (NPM1) that are associated with favorable prognosis. Based on these observations, we hypothesized that SPARC expression is clinically relevant in AML. Here, we found that SPARC overexpression is associated with adverse outcome in CN-AML patients and promotes aggressive leukemia growth in murine models of AML. In leukemia cells, SPARC expression was mediated by the SP1/NF-κB transactivation complex. Furthermore, secreted SPARC activated the integrin-linked kinase/AKT (ILK/AKT) pathway, likely via integrin interaction, and subsequent ß-catenin signaling, which is involved in leukemia cell self-renewal. Pharmacologic inhibition of the SP1/NF-κB complex resulted in SPARC downregulation and leukemia growth inhibition. Together, our data indicate that evaluation of SPARC expression has prognosticative value and SPARC is a potential therapeutic target for AML.

Eradicating acute myeloid leukemia in a Mll(PTD/wt):Flt3(ITD/wt) murine model: a path to novel therapeutic approaches for human disease.

The coexpression of the MLL partial tandem duplication (PTD) and the FLT3 internal tandem duplication (ITD) mutations associate with a poor outcome in cytogenetically normal acute myeloid leukemia (AML). In mice, a double knock-in (dKI) of Mll(PTD/wt) and Flt3(ITD/wt) mutations induces spontaneous AML with an increase in DNA methyltransferases (Dnmt1, 3a, and 3b) and global DNA methylation index, thereby recapitulating its human AML counterpart. We determined that a regulator of Dnmts, miR-29b, is downregulated in bone marrow of dKI AML mice. Bortezomib exerted a dose-dependent increase in miR-29b expression in AML blasts ex vivo, followed by decreased Dnmts, reduced proliferation, and increased apoptosis. In vivo, bortezomib was not active against dKI AML, yet liposomal-encapsulated bortezomib, as a single agent, reversed downregulation of miR-29b in vivo and induced a long-term (90-day) disease-free remission in 80% of dKI AML mice that exhibited high leukemic burden at the start of therapy, yet showed no signs of relapse at autopsy. Taken together, these data support that liposomal bortezomib, as a single agent, eradicates Mll(PTD/wt):Flt3(ITD/wt) AML in mouse and may represent a powerful and potentially curative approach to high-risk human disease.