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Acute myeloid leukemia patients with FLT3-ITD mutations have a high risk of relapse and death. FLT3 tyrosine kinase inhibitors improve overall survival, but their efficacy is limited and most patients who relapse will ultimately die of the disease. Even with potent FLT3 inhibition, the disease persists within the bone marrow microenvironment, mainly due to bone marrow stroma activating parallel signaling pathways that maintain pro-survival factors. BET inhibitors suppress pro-survival factors such as MYC and BCL2, but these drugs thus far have shown only limited single-agent clinical potential. We demonstrate here, using pre-clinical and clinical correlative studies, that the novel 4-azaindole derivative, PLX51107, has BET-inhibitory activity in vitro and in vivo. The combination of BET and FLT3 inhibition induces a synergistic antileukemic effect in a murine xenograft model of FLT3-ITD AML, and against primary FLT3-ITD AML cells co-cultured with bone marrow stroma. Using suppression of MYC as a surrogate for BET inhibition, we demonstrate BET inhibition in human patients. The short plasma half-life of PLX51107 results in intermittent target inhibition to enable tolerability while overcoming the protective effect of the microenvironment. Mechanistically, the synergistic cytotoxicity is associated with suppression of key survival genes such as MYC. These data provide the scientific rationale for a clinical trial of a BET plus FLT3 inhibitor for the treatment of relapsed/refractory FLT3-ITD AML. A clinical trial of PLX51107 as monotherapy in patients with different malignancies is underway and will be reported separately.

Macrophages play a central role in the immune response, and their diverse functions are attributed to the spectrum of their functional states. To elucidate molecules involved in modulating the balance between the anti-inflammatory cytokine IL-10 and the pro-inflammatory cytokine TNF-α, we conducted genome-wide siRNA screening. First, we established an siRNA screening system using mouse bone marrow-derived macrophages, which are a suitable model for studying functional states of macrophages in vitro. In the primary screen and the subsequent reproducibility assay, 112 siRNA pools demonstrated enhancement of IL-10 production and 497 siRNA pools suppressed IL-10 production. After a deconvolution assay for IL-10-up-regulating siRNA pools, 8 genes were identified as IL-10 repressors, including Cnot1 and Rc3h1, components of the CCR4-NOT complex known to degrade cytokine mRNAs. On the other hand, siRNA pools targeting ribosomal proteins were frequently found among those that down-regulated IL-10 production and up-regulated TNF-α production. Four pools were assayed using deconvoluted siRNAs and identified as high-confidence hits. Thus, we found that the genome-wide knockdown of 19 ribosomal proteins resulted in decreased IL-10 and increased TNF-α production.

Background: Acute myeloid leukemia patients with FLT3-ITD mutations have a high risk of relapse and death. FLT3 tyrosine kinase inhibitors such as quizartinib and gilteritinib improve overall survival in relapsed patients, but their efficacy is limited and most such patients die of the disease. This is because even with potent FLT3 inhibition, the disease persists within the bone marrow microenvironment, mainly due to bone marrow stroma activating parallel signaling pathways that maintain pro-survival factors. BET inhibitors suppress pro-survival factors such as c-Myc and Bcl2, but these drugs thus far have shown only limited single-agent clinical potential. PLX51107 is a novel BET inhibitor designed to inhibit BET activity in intermittent daily fashion to allow for greater tolerability. We investigated whether the addition of PLX51107 to potent FLT3 inhibition with quizartinib could overcome the protective effect of the bone marrow stroma through inhibition of c-Myc expression. Methods: We developed a plasma inhibitory activity assay for c-Myc (c-Myc PIA) to assess in vivo efficacy of the PLX51107 in patients and to identify c-Myc-inhibitory doses of the drug. We tested PLX51107 alone and in combination with quizartinib in murine models of AML and against primary FLT3-ITD AML cells co-cultured with human bone marrow stroma. In addition, we analyzed gene expression patterns in the treatment models to explore the basis of any observed synergistic cytotoxic effect. Results: In a murine xenograft model of AML using MV4-11 cells, PLX51107 alone induced suppression of tumor growth in association with a 90% decrease in c-Myc gene expression. The combination of PLX51107 and quizartinib induced complete tumor regression in 5 out of 7 animals after 14 days of treatment (Figure 1). Animals treated with a 5-day course of quizartinib alone displayed tumor regression persisting until day 26 of treatment, while the addition of PLX51107 resulted in tumor regression until day 39. In patients treated with a dose of 120 mg/day of PLX51107, the c-Myc PIA demonstrated a robust suppression of c-Myc expression for roughly 6 hours, returning to baseline between 7 and 9 hours post-treatment (Figure 2). The mean plasma concentration to achieve this inhibition was 3.3 uM, which, accounting for the difference in protein drug binding between plasma and culture medium with 10% FBS, corresponded to a concentration of 250 nM PLX51107 in culture medium. With this same concentration and schedule (i.e., at concentrations and exposure times equivalent to what human patients would experience taking 120 mg daily of PLX51107 and 60 mg daily of quizartinib), the combination induced synergistic cytotoxicity in a series of 10 different FLT3-ITD AML patient blast samples co-cultured with bone marrow stroma (Figure 3). C-Myc RNA and protein were directly suppressed in these primary samples, and ingenuity pathway analysis of RNA expression confirmed that c-Myc associated genes displayed the highest level of down-regulation. Conclusions: These studies suggest that combination therapy with approximately 120 mg/day PLX51107 and 60 mg/day quizartinib will be a more effective therapy for relapsed FLT3-ITD AML than 60 mg/day of quizartinib alone. The combination of FLT3 inhibition and BET inhibition may represent an attractive therapeutic option for FLT3-ITD AML. Disclosures Hizukuri: Daiichi Sankyo Co, Ltd: Employment. Severson:Plexxikon Inc.: Employment. Powell:Plexxikon Inc.: Employment. Zhang:Plexxikon Inc.: Employment. Ma:Plexxikon Inc: Employment. Narahara:Daiichi Sankyo Co, Ltd: Employment. Sumi:Daiichi Sankyo, Inc.: Employment. Bollag:Plexxikon Inc.: Employment. Levis:FUJIFILM: Consultancy, Research Funding; Daiichi Sankyo Inc: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Astellas: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Amgen: Consultancy, Honoraria; Menarini: Consultancy, Honoraria.

M2 macrophages have been subdivided into subtypes such as IL-4-induced M2a and IL-10-induced M2c in vitro. Although it was reported that IL-10 stimulation leads to an increase in IL-4Rα, the effect of IL-4 and IL-10 in combination with macrophage subtype differentiation remains unclear. Thus, we sought to clarify whether IL-10 enhanced the M2 phenotype induced by IL-4. In this study, we showed that IL-10 enhanced IL-4Rα expression in M-CSF-induced bone marrow-derived macrophages (BMDMs). Global gene expression analysis of M2 macrophages induced by IL-4, IL-10 or IL-4 + IL-10 showed that IL-10 enhanced gene expression of M2a markers induced by IL-4 in M-CSF-induced BMDMs. Moreover, IL-4 and IL-10 synergistically induced CCL24 (Eotaxin-2) production. Enhanced CCL24 expression was also observed in GM-CSF-induced BMDMs and zymosan-elicited, thioglycolate-elicited and naive peritoneal macrophages. CCL24 is a CCR3 agonist and an eosinophil chemoattractant. In vitro, IL-4 + IL-10-stimulated macrophages produced a large amount of CCL24 and increased eosinophil migration, which was inhibited by anti-CCL24 antibody. We also showed that IL-4 + IL-10-stimulated (but not IL-4 or IL-10 alone) macrophages transferred into the peritoneum of C57BL/6J mice increased eosinophil infiltration into the peritoneal cavity. These results demonstrate that IL-4 + IL-10-simulated macrophages have enhanced M2a macrophage-related gene expression, CCL24 production and eosinophil infiltration-inducing activity, thereby suggesting their contribution to eosinophil-related diseases.

A high-fat diet (HFD) is a well-known contributing factor in the development of obesity. Most rats fed HFDs become obese. Those that avoid obesity when fed HFDs are considered diet resistant (DR). We performed a microarray screen to identify genes specific to the mesenteric fat of DR rats and revealed high expression of guanylin and guanylyl cyclase C (GC-C) in some subjects. Our histologic studies revealed that the cellular source of guanylin and GC-C is macrophages. Therefore, we developed double-transgenic (Tg) rats overexpressing guanylin and GC-C in macrophages and found that they were resistant to the effects of HFDs. In the mesenteric fat of HFD-fed Tg rats, Fas and perilipin mRNAs were downregulated, and those of genes involved in fatty acid oxidation were upregulated, compared with the levels in HFD-fed wild-type rats. In vitro studies demonstrated that lipid accumulation was markedly inhibited in adipocytes cocultured with macrophages expressing guanylin and GC-C and that this inhibition was reduced after treatment with guanylin- and GC-C-specific siRNAs. Our results suggest that the macrophagic guanylin-GC-C system contributes to the altered expression of genes involved in lipid metabolism, leading to resistance to obesity.

Background Phenotype-based high-throughput screening is a useful technique for identifying drug candidate compounds that have a desired phenotype. However, the molecular mechanisms of the hit compounds remain unknown, and substantial effort is required to identify the target proteins associated with the phenotype. Methods In this study, we propose a new method to predict target proteins of drug candidate compounds based on drug-induced gene expression data in Connectivity Map and a machine learning classification technique, which we call the “transcriptomic approach.” Results Unlike existing methods such as the chemogenomic approach, the transcriptomic approach enabled the prediction of target proteins without dependence on prior knowledge of compound chemical structures. The prediction accuracy of the chemogenomic approach was highly depended on compounds structure similarities in data sets. In contrast, the prediction accuracy of the transcriptomic approach was maintained at a sufficient level, even for benchmark data consisting of structurally diverse compounds. Conclusions The transcriptomic approach reported here is expected to be a useful tool for structure-independent prediction of target proteins for drug candidate compounds.

Macrophage activation is the main immunological process occurring during the development of several diseases, and the heterogeneity of macrophage activation or differentiation has been suggested to be involved in disease progression. In the present study, we attempted to identify molecules specifically expressed on human classically activated macrophages (M1) to investigate the significance of the M1-like phenotype in human diseases. Human monocyte-derived macrophages were differentiated into M1, M2a, M2b and M2c phenotypes, and also M1(-) (the M1 phenotype differentiated with interferon-γ) to eliminate the strong effects of lipopolysaccharides (LPS) on the gene expression profile. The gene expression profiles of those macrophage phenotypes were analyzed by a cDNA microarray analysis and were used for a bioinformatics examination to identify the markers of the M1 phenotype that are expressed in both M1 and M1(-). The gene expression profiles of murine macrophages were also evaluated. We identified guanylate-binding protein 5 (GBP5), which is associated nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 (NLRP3)-mediated inflammasome assembly in the M1 macrophages of both humans and mice. Notably, the expression of GBP5 protein was detected in cultured M1(-) as well as in M1 macrophages by western blotting, which means that GBP5 is a more generalized marker of the M1 phenotype compared with the M1 markers that can be induced by LPS stimulation. GBP5 is a useful candidate marker of the M1 phenotype.

The metabolic network is an important biological network which consists of enzymes and chemical compounds. However, a large number of metabolic pathways remains unknown, and most organism-specific metabolic pathways contain many missing enzymes. We present a novel method to identify the genes coding for missing enzymes using available genomic and chemical information from bacterial genomes. The proposed method consists of two steps: (a) estimation of the functional association between the genes with respect to chromosomal proximity and evolutionary association, using supervised network inference; and (b) selection of gene candidates for missing enzymes based on the original candidate score and the chemical reaction information encoded in the EC number. We applied the proposed methods to infer the metabolic network for the bacteria Pseudomonas aeruginosa from two genomic datasets: gene position and phylogenetic profiles. Next, we predicted several missing enzyme genes to reconstruct the lysine-degradation pathway in P. aeruginosa using EC number information. As a result, we identified PA0266 as a putative 5-aminovalerate aminotransferase (EC 2.6.1.48) and PA0265 as a putative glutarate semialdehyde dehydrogenase (EC 1.2.1.20). To verify our prediction, we conducted biochemical assays and examined the activity of the products of the predicted genes, PA0265 and PA0266, in a coupled reaction. We observed that the predicted gene products catalyzed the expected reactions; no activity was seen when both gene products were omitted from the reaction.

Glycans, which are carbohydrate sugar chains attached to some lipids or proteins, have a huge variety of structures and play a key role in cell communication, protein interaction and immunity. The availability of a number of glycan structures stored in the KEGG/GLYCAN database makes it possible for us to conduct a large-scale comparative research of glycans. In this paper, we present a novel approach to compare glycan structures and extract characteristic glycan substructures of certain organisms. In the algorithm we developed a new similarity measure of glycan structures taking into account of several biological aspects of glycan synthesis and glycosyltransferases, and we confirmed the validity of our similarity measure by conducting experiments on its ability to classify glycans between organisms in the framework of a support vector machine. Finally, our method successfully extracted a set of candidates of substructrues which are characteristic to human, rat, mouse, bovine, pig, chicken, yeast, wheat and sycamore, respectively. We confirmed that the characteristic substructures extracted by our method correspond to the substructures which are known as the species-specific sugar chain of gamma-glutamyltranspeptidases in the kidney.