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Supplemental Figure 2: STING agonists stimulate cytokines in tumor, plasma and peripheral tissues following IT dosing.

Figure S1A-C - Dot plots and histograms from flow cytometry analysis of SK-MEL-5-educated M-MDSCs and PBMCs cultured in medium alone.Figure S1D - FACS result of cell surface staining of cytokine-induced M-MDSCs and PBMCs cultured in medium alone.Figure S1E - Cell surface staining of monocytes cultured in the presence of M-CSF and IL4 and SK-MEL-5-educated M-MDSCs by staining with HLA-DR.Figure S2 A-B - ILT3 is highly expressed on CD45+CD33+CD14+ myeloid cells in SK-MEL-5 tumor-infiltrating lymphocytes.Figure S3 - Single-cell RNAseq (scRNAseq) of human immune cells in non-small cell lung cancer (NSCLC) public dataset.Figure S4 - Association of LILRB4 expression with deconvoluted cell fractions for human tumor types in the TCGA database.Figure S5 - Suppression of autologous CD8+ T cell proliferation by cytokine-induced M-MDSCs.Figure S6 - CD33+ cells from PBMCs cultured in medium alone are not suppressive.Figure S7 - Differentially expressed genes (DEG) between SK-MEL-5 educated M-MDSCs and monocytes.

Table S1 - Antibodies used for flow cytometry-based assessment of binding of anti-ILT3 and anti-ILT4 antibody to human primary cells and SK-MEL-5-educated monocytic myeloid-derived suppressor cells.Table S2 - Antibodies used for flow cytometry-based assessment of ILT3 expression on myeloid cells in SK-MEL-5 tumor-infiltrating lymphocytes.Table S3 - Differential gene expression in SK-MEL-5 educated MDSCs treated with anti-ILT3 vs. isotype control using RNAseq.Table S4 - Pantherdb.org analysis for GO term enrichment of the 623 signatures.

Supplementary Methods and Figures

Targeting the programmed death 1/programmed death ligand 1 (PD-1/PD-L1) pathway with immunotherapy has revolutionized the treatment of many cancers. Somatic tumor mutational burden (TMB) and T-cell–inflamed gene expression profile (GEP) are clinically validated pan-tumor genomic biomarkers that can predict responsiveness to anti-PD-1/PD-L1 monotherapy in many tumor types. We analyzed the association between these biomarkers and the efficacy of PD-1 inhibitor in 11 commonly used preclinical syngeneic tumor mouse models using murinized rat anti-mouse PD-1 DX400 antibody muDX400, a surrogate for pembrolizumab. Response to muDX400 treatment was broadly classified into three categories: highly responsive, partially responsive, and intrinsically resistant to therapy. Molecular and cellular profiling validated differences in immune cell infiltration and activation in the tumor microenvironment of muDX400-responsive tumors. Baseline and on-treatment genomic analysis showed an association between TMB, murine T-cell–inflamed gene expression profile (murine-GEP), and response to muDX400 treatment. We extended our analysis to investigate a canonical set of cancer and immune biology-related gene signatures, including signatures of angiogenesis, myeloid-derived suppressor cells, and stromal/epithelial-to-mesenchymal transition/TGFβ biology previously shown to be inversely associated with the clinical efficacy of immune checkpoint blockade. Finally, we evaluated the association between murine-GEP and preclinical efficacy with standard-of-care chemotherapy or antiangiogenic agents that previously demonstrated promising clinical activity, in combination with muDX400. Our profiling studies begin to elucidate the underlying biological mechanisms of response and resistance to PD-1/PD-L1 blockade represented by these models, thereby providing insight into which models are most appropriate for the evaluation of orthogonal combination strategies.

Figure S1A-C - Dot plots and histograms from flow cytometry analysis of SK-MEL-5-educated M-MDSCs and PBMCs cultured in medium alone.Figure S1D - FACS result of cell surface staining of cytokine-induced M-MDSCs and PBMCs cultured in medium alone.Figure S1E - Cell surface staining of monocytes cultured in the presence of M-CSF and IL4 and SK-MEL-5-educated M-MDSCs by staining with HLA-DR.Figure S2 A-B - ILT3 is highly expressed on CD45+CD33+CD14+ myeloid cells in SK-MEL-5 tumor-infiltrating lymphocytes.Figure S3 - Single-cell RNAseq (scRNAseq) of human immune cells in non-small cell lung cancer (NSCLC) public dataset.Figure S4 - Association of LILRB4 expression with deconvoluted cell fractions for human tumor types in the TCGA database.Figure S5 - Suppression of autologous CD8+ T cell proliferation by cytokine-induced M-MDSCs.Figure S6 - CD33+ cells from PBMCs cultured in medium alone are not suppressive.Figure S7 - Differentially expressed genes (DEG) between SK-MEL-5 educated M-MDSCs and monocytes.

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells that accumulate in the tumor microenvironment (TME). MDSCs have been shown to dampen antitumor immune responses and promote tumor growth; however, the mechanisms of MDSC induction and their role in promoting immune suppression in cancer remain poorly understood. Here, we characterized the phenotype and function of monocytic MDSCs (M-MDSC) generated by coculture of human peripheral blood mononuclear cells with SK-MEL-5 cancer cells in vitro. We selected the SK-MEL-5 human melanoma cell line to generate M-MDSCs because these cells form subcutaneous tumors rich in myeloid cells in humanized mice. M-MDSCs generated via SK-MEL-5 coculture expressed low levels of human leukocyte antigen (HLA)-DR, high levels of CD33 and CD11b, and suppressed both CD8+ T-cell proliferation and IFNγ secretion. M-MDSCs also expressed higher levels of immunoglobulin-like transcript 3 (ILT3, also known as LILRB4) and immunoglobulin-like transcript 4 (ILT4, also known as LILRB2) on the cell surface compared with monocytes. Therefore, we investigated how ILT3 targeting could modulate M-MDSC cell function. Treatment with an anti-ILT3 antibody impaired the acquisition of the M-MDSC suppressor phenotype and reduced the capacity of M-MDSCs to cause T-cell suppression. Finally, in combination with anti-programmed cell death protein 1 (PD1), ILT3 blockade enhanced T-cell activation as assessed by IFNγ secretion.Implications:These results suggest that ILT3 expressed on M-MDSCs has a role in inducing immunosuppression in cancer and that antagonism of ILT3 may be useful to reverse the immunosuppressive function of M-MDSCs and enhance the efficacy of immune checkpoint inhibitors.

Supplementary Data from Reverse Translating Molecular Determinants of Anti–Programmed Death 1 Immunotherapy Response in Mouse Syngeneic Tumor Models

Supplemental Table 1: Log2Ratio and p values for all 310 genes shown in Figure 6

Purpose: Adipocytes represent one of the most abundant constituents of the mammary gland. They are essential for mammary tumor growth and survival. Metabolically, one of the more important fat-derived factors (“adipokines”) is adiponectin (APN). Serum concentrations of APN negatively correlate with body mass index and insulin resistance. To explore the association of APN with breast cancer and tumor angiogenesis, we took an in vivo approach aiming to study its role in the mouse mammary tumor virus (MMTV)-polyoma middle T antigen (PyMT) mammary tumor model.Experimental Design: We compared the rates of tumor growth in MMTV-PyMT mice in wild-type and APN-null backgrounds.Results: Histology and micro-positron emission tomography imaging show that the rate of tumor growth is significantly reduced in the absence of APN at early stages. PyMT/APN knockout mice exhibit a reduction in their angiogenic profile resulting in nutrient deprivation of the tumors and tumor-associated cell death. Surprisingly, in more advanced malignant stages of the disease, tumor growth develops more aggressively in mice lacking APN, giving rise to a larger tumor burden, an increase in the mobilization of circulating endothelial progenitor cells, and a gene expression fingerprint indicative of more aggressive tumor cells.Conclusions: These observations highlight a novel important contribution of APN in mammary tumor development and angiogenesis, indicating that APN has potent angio-mimetic properties in tumor vascularization. However, in tumors deprived of APN, this antiangiogenic stress results in an adaptive response that fuels tumor growth through mobilization of circulating endothelial progenitor cells and the development of mechanisms enabling massive cell proliferation despite a chronically hypoxic microenvironment.

Supplementary Data from Proangiogenic Contribution of Adiponectin toward Mammary Tumor Growth In vivo

Targeting the programmed death 1/programmed death ligand 1 (PD-1/PD-L1) pathway with immunotherapy has revolutionized the treatment of many cancers. Somatic tumor mutational burden (TMB) and T-cell–inflamed gene expression profile (GEP) are clinically validated pan-tumor genomic biomarkers that can predict responsiveness to anti-PD-1/PD-L1 monotherapy in many tumor types. We analyzed the association between these biomarkers and the efficacy of PD-1 inhibitor in 11 commonly used preclinical syngeneic tumor mouse models using murinized rat anti-mouse PD-1 DX400 antibody muDX400, a surrogate for pembrolizumab. Response to muDX400 treatment was broadly classified into three categories: highly responsive, partially responsive, and intrinsically resistant to therapy. Molecular and cellular profiling validated differences in immune cell infiltration and activation in the tumor microenvironment of muDX400-responsive tumors. Baseline and on-treatment genomic analysis showed an association between TMB, murine T-cell–inflamed gene expression profile (murine-GEP), and response to muDX400 treatment. We extended our analysis to investigate a canonical set of cancer and immune biology-related gene signatures, including signatures of angiogenesis, myeloid-derived suppressor cells, and stromal/epithelial-to-mesenchymal transition/TGFβ biology previously shown to be inversely associated with the clinical efficacy of immune checkpoint blockade. Finally, we evaluated the association between murine-GEP and preclinical efficacy with standard-of-care chemotherapy or antiangiogenic agents that previously demonstrated promising clinical activity, in combination with muDX400. Our profiling studies begin to elucidate the underlying biological mechanisms of response and resistance to PD-1/PD-L1 blockade represented by these models, thereby providing insight into which models are most appropriate for the evaluation of orthogonal combination strategies.

Fibrosis or accumulation of extracellular matrix is an evolutionarily conserved mechanism adopted by an organism as a response to chronic injury. Excessive fibrosis, however, leads to disruption of organ homeostasis and is a common feature of many chronic diseases. G protein-coupled receptors (GPCRs) are important cell signaling mediators and represent molecular targets for many Food and Drug Administration-approved drugs. To identify new targets for fibrosis, we used a synthetic GPCR system named designed receptors exclusively activated by designer drugs (DREADDs) to probe signaling pathways essential for fibrotic response. We found that upon expression in human lung fibroblasts, activation of Gq- and Gs-DREADDs abrogated the induction of TGFβ-induced fibrosis marker genes. Genome-wide transcriptome analysis identified dysregulation of multiple GPCRs in lung fibroblasts treated with TGFβ To investigate endogenous GPCR modulating TGFβ signaling, we selected 13 GPCRs that signal through Gq or Gs and activated them by using specific agonists. We examined the impact of each agonist and how activation of endogenous GPCR affects TGFβ signaling. Among the agonists examined, prostaglandin receptor agonists demonstrated the strongest inhibitory effect on fibrosis. Together, we have demonstrated that the DREADDs system is a valuable tool to identify beneficial GPCR signaling for fibrosis. This study in fibroblasts has served as a proof of concept and allowed us to further develop in vivo models for fibrosis GPCR discovery. SIGNIFICANCE STATEMENT: Fibrosis is the hallmark of many end-stage cardiometabolic diseases, and there is an unmet medical need to discover new antifibrotic therapies, reduce disease progression, and bring clinically meaningful efficacy to patients. Our work utilizes designed receptors exclusively activated by designer drug chemogenetic tools to identify beneficial GPCR signaling for fibrosis, providing new insights into GPCR drug discovery.

Bromodomain and extraterminal domain (BET) proteins help direct the differentiation of helper T cell subsets, but their role in activated T cell function has not been described in detail. In this study, we investigate various consequences of epigenetic perturbation in human T lymphocytes using MK-8628, a potent and highly selective inhibitor of BET proteins. MK-8628 reduces the expression of canonical transcripts directing the proliferation, activation, and effector function of T lymphocytes. Treatment with MK-8628 abolishes the expression of key cyclins required for cell cycle progression and induces G1 cell cycle arrest in TCR-activated lymphocytes. This antiproliferative phenotype partially results from T lymphocyte apoptosis, which is exacerbated by MK-8628. In naive and memory T cell subsets, MK-8628 antagonizes T cell activation and suppresses polyfunctional cytokine production. Collectively, our results describe potent immunosuppressive effects of BET inhibition on human T cell biology. These results have important implications for immune modulatory targeting of BET proteins in the settings of T cell–driven autoimmune inflammation.

Prostaglandin E2 (PGE2), an arachidonic acid pathway metabolite produced by cyclooxygenase (COX)-1/2, has been shown to impair anti-tumor immunity through engagement with one or more E-type prostanoid receptors (EP1-4). Specific targeting of EP receptors, as opposed to COX-1/2 inhibition, has been proposed to achieve preferential antagonism of PGE2–mediated immune suppression. Here we describe the anti-tumor activity of MF-766, a potent and highly selective small-molecule inhibitor of the EP4 receptor. EP4 inhibition by MF-766 synergistically improved the efficacy of anti-programmed cell death protein 1 (PD-1) therapy in CT26 and EMT6 syngeneic tumor mouse models. Multiparameter flow cytometry analysis revealed that treatment with MF-766 promoted the infiltration of CD8+ T cells, natural killer (NK) cells and conventional dendritic cells (cDCs), induced M1-like macrophage reprogramming, and reduced granulocytic myeloid-derived suppressor cells (MDSC) in the tumor microenvironment (TME). In vitro experiments demonstrated that MF-766 restored PGE2-mediated inhibition of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production in THP-1 cells and human blood, and PGE2-mediated inhibition of interleukin (IL)-2-induced interferon (IFN)-γ production in human NK cells. MF-766 reversed the inhibition of IFN-γ in CD8+ T-cells by PGE2 and impaired suppression of CD8+ T-cells induced by myeloid-derived suppressor cells (MDSC)/PGE2. In translational studies using primary human tumors, MF-766 enhanced anti-CD3-stimulated IFN-γ, IL-2, and TNF-α production in primary histoculture and synergized with pembrolizumab in a PGE2 high TME. Our studies demonstrate that the combination of EP4 blockade with anti-PD-1 therapy enhances antitumor activity by differentially modulating myeloid cell, NK cell, cDC and T-cell infiltration profiles.

The innate immune agonist STING (STimulator of INterferon Genes) binds its natural ligand 2′3′-cGAMP (cyclic guanosine-adenosine monophosphate) and initiates type I IFN production. This promotes systemic antigen-specific CD8+ T-cell priming that eventually provides potent antitumor activity. To exploit this mechanism, we synthesized a novel STING agonist, MSA-1, that activates both mouse and human STING with higher in vitro potency than cGAMP. Following intratumoral administration of MSA-1 to a panel of syngeneic mouse tumors on immune-competent mice, cytokine upregulation and its exposure were detected in plasma, other tissues, injected tumors, and noninjected tumors. This was accompanied by effective antitumor activity. Mechanistic studies in immune-deficient mice suggested that antitumor activity of intratumorally dosed STING agonists is in part due to necrosis and/or innate immune responses such as TNF-α activity, but development of a robust adaptive antitumor immunity is necessary for complete tumor elimination. Combination with PD-1 blockade in anti–PD-1–resistant murine models showed that MSA-1 may synergize with checkpoint inhibitors but can also provide superior tumor control as a single agent. We show for the first time that potent cyclic dinucleotides can promote a rapid and stronger induction of the same genes eventually regulated by PD-1 blockade. This may have contributed to the relatively early tumor control observed with MSA-1. Taken together, these data strongly support the development of STING agonists as therapy for patients with aggressive tumors that are partially responsive or nonresponsive to single-agent anti–PD-1 treatment by enhancing the anti–PD-1 immune profile.

Prostaglandin E2 (PGE2), an arachidonic acid pathway metabolite produced by cyclooxygenase (COX)-1/2, has been shown to impair anti-tumor immunity through engagement with one or more E-type prostanoid receptors (EP1-4). Specific targeting of EP receptors, as opposed to COX-1/2 inhibition, has been proposed to achieve preferential antagonism of PGE2–mediated immune suppression. Here we describe the anti-tumor activity of MF-766, a potent and highly selective small-molecule inhibitor of the EP4 receptor. EP4 inhibition by MF-766 synergistically improved the efficacy of anti-programmed cell death protein 1 (PD-1) therapy in CT26 and EMT6 syngeneic tumor mouse models. Multiparameter flow cytometry analysis revealed that treatment with MF-766 promoted the infiltration of CD8+ T cells, natural killer (NK) cells and conventional dendritic cells (cDCs), induced M1-like macrophage reprogramming, and reduced granulocytic myeloid-derived suppressor cells (MDSC) in the tumor microenvironment (TME). In vitro experiments demonstrated that MF-766 restored PGE2-mediated inhibition of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production in THP-1 cells and human blood, and PGE2-mediated inhibition of interleukin (IL)-2-induced interferon (IFN)-γ production in human NK cells. MF-766 reversed the inhibition of IFN-γ in CD8+ T-cells by PGE2 and impaired suppression of CD8+ T-cells induced by myeloid-derived suppressor cells (MDSC)/PGE2. In translational studies using primary human tumors, MF-766 enhanced anti-CD3-stimulated IFN-γ, IL-2, and TNF-α production in primary histoculture and synergized with pembrolizumab in a PGE2 high TME. Our studies demonstrate that the combination of EP4 blockade with anti-PD-1 therapy enhances antitumor activity by differentially modulating myeloid cell, NK cell, cDC and T-cell infiltration profiles.

Immunotherapy fails to cure most cancer patients. Preclinical studies indicate that radiotherapy synergizes with immunotherapy, promoting radiation-induced antitumor immunity. Most preclinical immunotherapy studies utilize transplant tumor models, which overestimate patient responses. Here, we show that transplant sarcomas are cured by PD-1 blockade and radiotherapy, but identical treatment fails in autochthonous sarcomas, which demonstrate immunoediting, decreased neoantigen expression, and tumor-specific immune tolerance. We characterize tumor-infiltrating immune cells from transplant and primary tumors, revealing striking differences in their immune landscapes. Although radiotherapy remodels myeloid cells in both models, only transplant tumors are enriched for activated CD8+ T cells. The immune microenvironment of primary murine sarcomas resembles most human sarcomas, while transplant sarcomas resemble the most inflamed human sarcomas. These results identify distinct microenvironments in murine sarcomas that coevolve with the immune system and suggest that patients with a sarcoma immune phenotype similar to transplant tumors may benefit most from PD-1 blockade and radiotherapy., Promising results of cancer therapies in transplant tumor models often fail to predict efficacy in clinical trials. Here the authors show that, while transplant tumors are cured by radiotherapy and PD-1 blockade, autochthonous sarcomas are resistant to the identical treatment, recapitulating the immune landscape and resistance to checkpoint blockade observed in most sarcoma patients.

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells that accumulate in the tumor microenvironment (TME). MDSCs have been shown to dampen antitumor immune responses and promote tumor growth; however, the mechanisms of MDSC induction and their role in promoting immune suppression in cancer remain poorly understood. Here, we characterized the phenotype and function of monocytic MDSCs (M-MDSC) generated by coculture of human peripheral blood mononuclear cells with SK-MEL-5 cancer cells in vitro. We selected the SK-MEL-5 human melanoma cell line to generate M-MDSCs because these cells form subcutaneous tumors rich in myeloid cells in humanized mice. M-MDSCs generated via SK-MEL-5 coculture expressed low levels of human leukocyte antigen (HLA)-DR, high levels of CD33 and CD11b, and suppressed both CD8+ T-cell proliferation and IFNγ secretion. M-MDSCs also expressed higher levels of immunoglobulin-like transcript 3 (ILT3, also known as LILRB4) and immunoglobulin-like transcript 4 (ILT4, also known as LILRB2) on the cell surface compared with monocytes. Therefore, we investigated how ILT3 targeting could modulate M-MDSC cell function. Treatment with an anti-ILT3 antibody impaired the acquisition of the M-MDSC suppressor phenotype and reduced the capacity of M-MDSCs to cause T-cell suppression. Finally, in combination with anti-programmed cell death protein 1 (PD1), ILT3 blockade enhanced T-cell activation as assessed by IFNγ secretion. Implications: These results suggest that ILT3 expressed on M-MDSCs has a role in inducing immunosuppression in cancer and that antagonism of ILT3 may be useful to reverse the immunosuppressive function of M-MDSCs and enhance the efficacy of immune checkpoint inhibitors.

Despite impressive responses in some patients, immunotherapy fails to cure most cancer patients. Preclinical studies indicate that radiotherapy synergizes with immunotherapy, promoting radiation-induced antitumor immunity. Nearly all preclinical immunotherapy studies utilize transplant tumor models, but cure rates of transplant tumor models treated with immunotherapy often overestimate patient responses. Here, we show that transplant tumors are cured by PD-1 blockade and radiotherapy, but identical treatment fails in autochthonous tumors. We generated a single-cell atlas of tumor-infiltrating immune cells from transplant and primary tumors treated with radiation and immunotherapy, which reveals striking differences in their immune landscapes. Although radiotherapy remodels myeloid cell phenotypes in primary and transplant tumors, only transplant tumors are enriched for CD8+ T cells that mediate tumor clearance while mice with primary sarcomas demonstrate tumor-specific tolerance. These results identify distinct microenvironments in tumors that coevolve with the immune system, which promote tolerance that must be overcome for immune-mediated cancer cures.

BackgroundImmunoglobulin-like transcript 3 (ILT3), an immune-inhibitory receptor expressed on myeloid cells, is highly expressed in acute myeloid leukemia (AML) with monocytic differentiation (M4 and M5 subtypes) and clinically is negatively correlated with overall survival. We examined the effects of a highly selective and potent chimeric anti-ILT3 mAb (c52B8) on tumor cell survival/growth and human immune cell modulation in AML PBMC and AML tumor cell lines in pre-clinical in vitro and in vivo models.MethodsILT3 mRNA and cell surface expression levels were measured by RNAseq and FACS, respectively. PBMC from AML patients with different levels of ILT3 cell surface expression were treated with c52B8 in vitro.A systemic AML model was generated by IV inoculation of NSG mice with luciferase-transfected MV-4-11 human AML cells, with or without human PBMC engraftment. Tumor bearing mice were treated weekly with c52B8, modified to contain the human Fc of either the IgG1 or IgG4 subclass. MV-4-11 growth in vivo was measured by bioluminescence imaging (BLI).CyTOF was performed to phenotype AML PBMC in in vitro culture and bone marrow samples from the systemic MV-4-11 model after the treatment.THP-1 cells/human T cell co-cultures stimulated with anti-CD3/CD28 antibodies were treated with c52B8. Human T cell activity was assessed by levels of IFNγ production in the culture using MSD ELISA.ResultsIn ILT3 high AML patient PBMC, c52B8 treatment in vitro decreased the frequency of tumor blasts, modulated Tregs, and increased monocytic myeloid cells. In the MV-4-11 model, weekly treatment with c52B8 significantly reduced tumor burden in vivo, regardless of the IgG subclass. Importantly, tumor cell growth inhibition by c52B8 occurred in the absence of human PBMC. Additional post-mortem CyTOF analysis of bone marrow cells from MV-4-11/human PBMC inoculated mice confirmed a reduction in MV-4-11 cells, identified by CD3-CD19- human myeloid cells following c52B8 treatment. In THP-1/human T cell co-cultures, c52B8 treatment reversed THP-1 induced T cell suppression, as measured by enhanced IFNγ production.ConclusionsAnti-ILT3 mAb (c52B8) is efficacious in blocking progression of AML with monocytic differentiation by directly influencing tumor cell growth and enhancing human T cell activity in pre-clinical models. These studies support a potential role for this compound in humans.Ethics ApprovalAll animal work was reviewed and approved by Merck IACUC before experiments were conducted.

GPR40 and GPR120 are fatty acid sensors that play important roles in glucose and energy homeostasis. GPR40 potentiates glucose-dependent insulin secretion and demonstrated in clinical studies robust glucose lowering in type 2 diabetes. GPR120 improves insulin sensitivity in rodents, albeit its mechanism of action is not fully understood. Here, we postulated that the antidiabetic efficacy of GPR40 could be enhanced by coactivating GPR120. A combination of GPR40 and GPR120 agonists in db/db mice, as well as a single molecule with dual agonist activities, achieved superior glycemic control compared with either monotherapy. Compared with a GPR40 selective agonist, the dual agonist improved insulin sensitivity in ob/ob mice measured by hyperinsulinemic-euglycemic clamp, preserved islet morphology, and increased expression of several key lipolytic genes in adipose tissue of Zucker diabetic fatty rats. Novel insights into the mechanism of action for GPR120 were obtained. Selective GPR120 activation suppressed lipolysis in primary white adipocytes, although this effect was attenuated in adipocytes from obese rats and obese rhesus, and sensitized the antilipolytic effect of insulin in rat and rhesus primary adipocytes. In conclusion, GPR120 agonism enhances insulin action in adipose tissue and yields a synergistic efficacy when combined with GPR40 agonism.

The MYC oncogene is upregulated in human cancers by translocation, amplification, and mutation of cellular pathways that regulate Myc. Myc/Max heterodimers bind to E box sequences in the promoter regions of genes and activate transcription., The MYC oncogene is upregulated in human cancers by translocation, amplification, and mutation of cellular pathways that regulate Myc. Myc/Max heterodimers bind to E box sequences in the promoter regions of genes and activate transcription. The MYC inhibitor Omomyc can reduce the ability of MYC to bind specific box sequences in promoters of MYC target genes by binding directly to E box sequences as demonstrated by chromatin immunoprecipitation (CHIP). Here, we demonstrate by both a proximity ligation assay (PLA) and double chromatin immunoprecipitation (ReCHIP) that Omomyc preferentially binds to Max, not Myc, to mediate inhibition of MYC-mediated transcription by replacing MYC/MAX heterodimers with Omomyc/MAX heterodimers. The formation of Myc/Max and Omomyc/Max heterodimers occurs cotranslationally; Myc, Max, and Omomyc can interact with ribosomes and Max RNA under conditions in which ribosomes are intact. Taken together, our data suggest that the mechanism of action of Omomyc is to bind DNA as either a homodimer or a heterodimer with Max that is formed cotranslationally, revealing a novel mechanism to inhibit the MYC oncogene. We find that in vivo, Omomyc distributes quickly to kidneys and liver and has a short effective half-life in plasma, which could limit its use in vivo.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathologic T cell–B cell interactions and autoantibody production. Defining the T cell populations that drive B cell responses in SLE may enable design of therapies that specifically target pathologic cell subsets. Here, we evaluated the phenotypes of CD4(+) T cells in the circulation of 52 SLE patients drawn from multiple cohorts and identified a highly expanded PD-1(hi)CXCR5(–)CD4(+) T cell population. Cytometric, transcriptomic, and functional assays demonstrated that PD-1(hi)CXCR5(–)CD4(+) T cells from SLE patients are T peripheral helper (Tph) cells, a CXCR5(–) T cell population that stimulates B cell responses via IL-21. The frequency of Tph cells, but not T follicular helper (Tfh) cells, correlated with both clinical disease activity and the frequency of CD11c(+) B cells in SLE patients. PD-1(hi)CD4(+) T cells were found within lupus nephritis kidneys and correlated with B cell numbers in the kidney. Both IL-21 neutralization and CRISPR-mediated deletion of MAF abrogated the ability of Tph cells to induce memory B cell differentiation into plasmablasts in vitro. These findings identify Tph cells as a highly expanded T cell population in SLE and suggest a key role for Tph cells in stimulating pathologic B cell responses.

Metabolic dysregulation and mitochondrial dysfunction are important features of acute and chronic tissue injury across species, and human genetics and preclinical data suggest that the master metabolic regulator 5'-adenosine monophosphate-activated protein kinase (AMPK) may be an effective therapeutic target for chronic kidney disease (CKD). We have recently disclosed a pan-AMPK activator, MK-8722, that was shown to have beneficial effects in preclinical models. In this study we investigated the effects of MK-8722 in a progressive rat model of diabetic nephropathy to determine whether activation of AMPK would be of therapeutic benefit. We found that MK-8722 administration in a therapeutic paradigm is profoundly renoprotective, as demonstrated by a reduction in proteinuria (63% decrease in MK-8722 10 mg/kg per day compared with vehicle group) and a significant improvement in glomerular filtration rate (779 and 430 μl/min per gram kidney weight in MK-8722 10 mg/kg per day and vehicle group, respectively), as well as improvements in kidney fibrosis. We provide evidence that the therapeutic effects of MK-8722 may be mediated by modulation of renal mitochondrial quality control as well by attenuating fibrotic and lipotoxic mechanisms in kidney cells. MK-8722 (10 mg/kg per day compared with vehicle group) achieved modest blood pressure reduction (10 mmHg lower for mean blood pressure) and significant metabolic improvements (decreased plasma glucose, triglyceride, and body weight) that could contribute to renoprotection. These data further validate the concept that targeting metabolic dysregulation in CKD could be a potential therapeutic approach. SIGNIFICANCE STATEMENT: We demonstrate in the present study that the pharmacological activation of AMPK using a small-molecule agent provided renoprotection and improved systemic and cellular metabolism. We further indicate that modulation of renal mitochondrial quality control probably contributed to renoprotection and was distinct from the effects of enalapril. Our findings suggest that improving renal mitochondrial biogenesis and function and attenuating fibrosis and lipotoxicity by targeting key metabolic nodes could be a potential therapeutic approach in management of CKD that could complement the current standard of care.

This study reports the establishment of a bone marrow mononuclear cell (BMMC) 3D culture model and the application of this model to define sensitivity and resistance biomarkers of acute myeloid leukaemia (AML) patient bone marrow samples in response to Cytarabine (Ara‐C) treatment. By mimicking physiological bone marrow microenvironment, the growth conditions were optimized by using frozen BMMCs derived from healthy donors. Healthy BMMCs are capable of differentiating into major hematopoietic lineages and various types of stromal cells in this platform. Cryopreserved BMMC samples from 49 AML patients were characterized for ex vivo growth and sensitivity to Ara‐C. RNA sequencing was performed for 3D and 2D cultures to determine differential gene expression patterns. Specific genetic mutations and/or gene expression signatures associated with the ability of the ex vivo expansion and response to Ara‐C were elucidated by whole‐exome and RNA sequencing. Data analysis identified unique gene expression signatures and novel genetic mutations associated with sensitivity to Ara‐C treatment of proliferating AML specimens and can be used as predictive therapeutic biomarkers to determine the optimal treatment regimens. Furthermore, these data demonstrate the translational value of this ex vivo platform which should be widely applicable to evaluate other therapies in AML.

Physical activity promotes metabolic and cardiovascular health benefits that derive in part from the transcriptional responses to exercise that occur within skeletal muscle and other organs. There is interest in discovering a pharmacologic exercise mimetic that could imbue wellness and alleviate disease burden. However, the molecular physiology by which exercise signals the transcriptional response is highly complex, making it challenging to identify a single target for pharmacological mimicry. The current studies evaluated the transcriptome responses in skeletal muscle, heart, liver, and white and brown adipose to novel small molecule activators of AMPK (pan-activators for all AMPK isoforms) compared to that of exercise. A striking level of congruence between exercise and pharmacological AMPK activation was observed across the induced transcriptome of these five tissues. However, differences in acute metabolic response between exercise and pharmacologic AMPK activation were observed, notably for acute glycogen balances and related to the energy expenditure induced by exercise but not pharmacologic AMPK activation. Nevertheless, intervention with repeated daily administration of short-acting activation of AMPK was found to mitigate hyperglycemia and hyperinsulinemia in four rodent models of metabolic disease and without the cardiac glycogen accretion noted with sustained pharmacologic AMPK activation. These findings affirm that activation of AMPK is a key node governing exercise mediated transcription and is an attractive target as an exercise mimetic.

Similar efficacy of the cathepsin K inhibitor odanacatib (ODN) and the bisphosphonate alendronate (ALN) in reducing bone turnover markers and increasing bone mineral density in spine and hip were previously demonstrated in ovariectomized (OVX)-monkeys treated for 20 months in prevention mode. Here, we profiled RNA from tibial metaphysis and diaphysis of the same study using Affymetrix microarrays, and selected 204 probe sets (p < 0.001, three-group ANOVA) that were differentially regulated by ODN or ALN versus vehicle. Both drugs produced strikingly different effects on known bone-related genes and pathways at the transcriptional level. Although ALN either reduced or had neutral effects on bone resorption-related genes, ODN significantly increased the expression of osteoclast genes (eg, APC5, TNFRSF11A, CTSK, ITGB3, and CALCR), consistent with previous findings on the effects of this agent in enhancing the number of nonresorbing osteoclasts. Conversely, ALN reduced the expression of known bone formation-related genes (eg, TGFBR1, SPP1, RUNX2, and PTH1R), whereas ODN either increased or had neutral effects on their expression. These differential effects of ODN versus ALN on bone resorption and formation were highly correlative to the changes in bone turnover markers, cathepsin K (Catk) target engagement marker serum C-terminal cross-linked telopeptide (1-CTP) and osteoclast marker tartrate resistant acid phosphatase isoform 5b (TRAP5b) in the same monkeys. Overall, the molecular profiling results are consistent with the known pharmacological actions of these agents on bone remodeling and clearly differentiate the molecular mechanisms of ODN from the bisphosphonates.

Fibrosis, or the accumulation of extracellular matrix, causes loss of organ function and is a common feature of many chronic diseases. To interrogate the core molecular pathways of fibrosis, we cross-examined human primary cells from various tissues treated with TGFβ. Transcriptome analyses revealed that in addition to the known TGFβ signature, top-regulated cluster of genes are involved in fatty acid metabolism. To further evaluate this observation in vivo, we characterized a renal fibrosis model through unilateral ureteral obstruction (UUO) in mice. TGFβ signaling was greatly augmented in UUO kidneys and preventive treatment with an anti-TGFβ antibody resulted in significant reduction of fibrosis. Transcriptome analysis also identified fatty acid metabolism as one of the top dysregulated pathways in UUO kidneys, which was further supported by a substantial accumulation of acylcarnitines. Additional rodent models of liver fibrosis revealed a similar metabolic signature upon fibrosis induction. Lastly, a compound library phenotypic screen for suppressers of fibrosis identified AMPK and PPAR activators. As a proof of concept, we demonstrated that pharmacological treatment of telmisartan, an angiotensin receptor and PPARγ dual-agent, significantly reduced fibrosis in UUO kidneys, suggesting that metabolic defect is integral to TGFβ signaling and fibrosis. Altogether, our work has revealed a common node of metabolic signature underlying fibrosis that could represent a promising therapeutic target for multiple fibrotic diseases.

Objective Identify a gene expression signature in white adipose tissue (WAT) that reports on WAT browning and is associated with a healthy phenotype. Methods RNA from several different adipose depots across three species were analyzed by whole transcriptome profiling, including 1) mouse subcutaneous white fat, brown fat, and white fat after in vivo treatment with FGF21; 2) human subcutaneous and omental fat from insulin-sensitive and insulin-resistant patients; and 3) rhesus monkey subcutaneous fat from healthy and dysmetabolic individuals. Results A “browning” signature in mice was identified by cross-referencing the FGF21-induced signature in WAT with the brown adipose tissue (BAT) vs. WAT comparison. In addition, gene expression levels in WAT from insulin-sensitive/healthy vs. insulin-resistant/dysmetabolic humans and rhesus monkeys, respectively, correlated with the gene expression levels in mouse BAT vs. WAT. A subset of 49 genes were identified that were consistently regulated or differentially expressed in the mouse and human data sets that could be used to monitor browning of WAT across species. Conclusions Gene expression profiles of WATs from healthy insulin-sensitive individuals correlate with those of BAT and FGF21-induced browning of WAT.

Acute myeloid leukemia (AML) is a malignancy with complex genetics, poor survival rates and high rates of chemotherapy resistance. This study reports the establishment of a novel bone marrow mononuclear cell 3D-culture model and the application of this model to define sensitivity and resistance biomarkers as evident by DNA and RNA expression signatures in response to standard Cytarabine (Ara-C) chemotherapy. By simulating the bone marrow microenvironment, we optimized the growth conditions using frozen bone marrow mononuclear cells (BMMC) derived from healthy donors. We validated our platform by demonstrating that healthy BMMCs are capable of differentiating into all hematopoietic lineages and various types of stromal cells. Using this platform, we then characterized BMMC samples from 47 AML patients. Sixty percent of AML samples were capable of proliferating and 23 samples demonstrated robust growth. RNA sequencing was performed for 3D and 2D cultures to determine which conditions are superior at maintaining original gene expression signatures as compared to the uncultured bone marrow, To elucidate specific genetic mutations and/or gene expression signatures associated with the ability of ex vivo growth of these AML donors, whole exosome sequencing and RNA Sequencing were performed. The model identified unique gene expression signatures that correlated with sensitive as well as resistant donors in response to Ara-C treatment. These data demonstrate the translational value of our platform which should be widely applicable to evaluate other therapies in AML. Citation Format: Haiyan Xu, Eric S. Muise, Sarah Javaid, Lan Chen, Razvan Cristescu, My Sam Mansueto, Nicole Follmer, Jennifer Cho, Kimberley Kerr, Rachel Altura, Michelle Machacek, Benjamin Nicholson, George Addona, Ilona Kariv, Hongmin Chen. Identification of predictive genetic signatures of Cytarabine responsiveness in acute myeloid leukemia using a novel 3D translational culture model of primary bone marrow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-029.

Selective peroxisome proliferator-activated receptor γ (PPARγ) modulators (SPPARγMs) have been actively pursued as the next generation of insulin-sensitizing antidiabetic drugs, because the currently marketed PPARγ full agonists, pioglitazone and rosiglitazone, have been reported to produce serious adverse effects among patients with type 2 diabetes mellitus. We conducted extensive transcriptome profiling studies to characterize and to contrast the activities of 70 SPPARγMs and seven PPARγ full agonists. In both 3T3-L1 adipocytes and adipose tissue from db/db mice, the SPPARγMs generated attenuated and selective gene-regulatory responses, in comparison with full agonists. More importantly, SPPARγMs regulated the expression of antidiabetic efficacy-associated genes to a greater extent than that of adverse effect-associated genes, whereas PPARγ full agonists regulated both gene sets proportionally. Such SPPARγM selectivity demonstrates that PPARγ ligand regulation of gene expression can be fine-tuned, and not just turned on and off, to achieve precise control of complex cellular and physiological functions. It also provides a potential molecular basis for the superior therapeutic window previously observed with SPPARγMs versus full agonists. On the basis of our profiling results, we introduce two novel, gene expression-based scores, the γ activation index and the selectivity index, to aid in the detection and characterization of novel SPPARγMs. These studies provide new insights into the gene-regulatory activity of SPPARγMs as well as novel quantitative indices to facilitate the identification of PPARγ ligands with robust insulin-sensitizing activity and improved tolerance among patients with type 2 diabetes, compared with presently available PPARγ agonist drugs.

Inhibition of cannabinoid receptor 1 (CB1) has shown efficacy in reducing body weight and improving metabolic parameters, with the effects correlating with target engagement in the brain. The peripheral effects of inhibiting the CB1 receptor has been appreciated through studies in diet-induced obese and liver-specific CB1 knockout mice. In this article, we systematically investigated gene expression changes in peripheral tissues of diet-induced obese mice treated with the CB1 inverse agonist AM251 [1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide]. CB1 receptor inhibition led to down-regulation of genes within the de novo fatty acid and cholesterol synthetic pathways, including sterol regulatory element binding proteins 1 and 2 and their downstream targets in both liver and adipose tissue. In addition, genes involved in fatty acid beta-oxidation were up-regulated with AM251 treatment, probably through the activation of peroxisome proliferator-activated receptor alpha (PPARalpha). In adipose tissue, CB1 receptor inhibition led to the down-regulation of genes in the tumor necrosis factor alpha signal transduction pathway and possibly to the activation of PPARgamma, both of which would result in improved insulin sensitivity.

Purpose: Adipocytes represent one of the most abundant constituents of the mammary gland. They are essential for mammary tumor growth and survival. Metabolically, one of the more important fat-derived factors (“adipokines”) is adiponectin (APN). Serum concentrations of APN negatively correlate with body mass index and insulin resistance. To explore the association of APN with breast cancer and tumor angiogenesis, we took an in vivo approach aiming to study its role in the mouse mammary tumor virus (MMTV)-polyoma middle T antigen (PyMT) mammary tumor model. Experimental Design: We compared the rates of tumor growth in MMTV-PyMT mice in wild-type and APN-null backgrounds. Results: Histology and micro-positron emission tomography imaging show that the rate of tumor growth is significantly reduced in the absence of APN at early stages. PyMT/APN knockout mice exhibit a reduction in their angiogenic profile resulting in nutrient deprivation of the tumors and tumor-associated cell death. Surprisingly, in more advanced malignant stages of the disease, tumor growth develops more aggressively in mice lacking APN, giving rise to a larger tumor burden, an increase in the mobilization of circulating endothelial progenitor cells, and a gene expression fingerprint indicative of more aggressive tumor cells. Conclusions: These observations highlight a novel important contribution of APN in mammary tumor development and angiogenesis, indicating that APN has potent angio-mimetic properties in tumor vascularization. However, in tumors deprived of APN, this antiangiogenic stress results in an adaptive response that fuels tumor growth through mobilization of circulating endothelial progenitor cells and the development of mechanisms enabling massive cell proliferation despite a chronically hypoxic microenvironment.

Adipocytes are embedded in a unique extracellular matrix whose main function is to provide mechanical support, in addition to participating in a variety of signaling events. During adipose tissue expansion, the extracellular matrix requires remodeling to accommodate adipocyte growth. Here, we demonstrate a general upregulation of several extracellular matrix components in adipose tissue in the diabetic state, therefore implicating "adipose tissue fibrosis" as a hallmark of metabolically challenged adipocytes. Collagen VI is a highly enriched extracellular matrix component of adipose tissue. The absence of collagen VI results in the uninhibited expansion of individual adipocytes and is paradoxically associated with substantial improvements in whole-body energy homeostasis, both with high-fat diet exposure and in the ob/ob background. Collectively, our data suggest that weakening the extracellular scaffold of adipocytes enables their stress-free expansion during states of positive energy balance, which is consequently associated with an improved inflammatory profile. Therefore, the disproportionate accumulation of extracellular matrix components in adipose tissue may not be merely an epiphenomenon of metabolically challenging conditions but may also directly contribute to a failure to expand adipose tissue mass during states of excess caloric intake.

Adipose tissue is a metabolically responsive endocrine organ that secretes a myriad of adipokines. Antidiabetic drugs such as peroxisome proliferator-activated receptor (PPAR) gamma agonists target adipose tissue gene expression and correct hyperglycemia via whole-body insulin sensitization. The mechanism by which altered gene expression in adipose tissue affects liver and muscle insulin sensitivity (and thus glucose homeostasis) is not fully understood. One possible mechanism involves the alteration in adipokine secretion, in particular the up-regulation of secreted factors that increase whole-body insulin sensitivity. Here, we report the use of transcriptional profiling to identify genes encoding for secreted proteins the expression of which is regulated by PPARgamma agonists. Of the 379 genes robustly regulated by two structurally distinct PPARgamma agonists in the epididymal white adipose tissue (EWAT) of db/db mice, 33 encoded for known secreted proteins, one of which was FGF21. Although FGF21 was recently reported to be up-regulated in cultured adipocytes by PPARgamma agonists and in liver by PPARalpha agonists and induction of ketotic states, we demonstrate that the protein is transcriptionally up-regulated in adipose tissue in vivo by PPARgamma agonist treatment and under a variety of physiological conditions, including fasting and high fat diet feeding. In addition, we found that circulating levels of FGF21 protein were increased upon treatment with PPARgamma agonists and under ketogenic states. These results suggest a role for FGF21 in mediating the antidiabetic activities of PPARgamma agonists.

Similar efficacy of the cathepsin K inhibitor odanacatib (ODN) and the bisphosphonate alendronate (ALN) in reducing bone turnover markers and increasing bone mineral density in spine and hip were previously demonstrated in ovariectomized (OVX)-monkeys treated for 20 months in prevention mode. Here, we profiled RNA from tibial metaphysis and diaphysis of the same study using Affymetrix microarrays, and selected 204 probe sets (p0.001, three-group ANOVA) that were differentially regulated by ODN or ALN versus vehicle. Both drugs produced strikingly different effects on known bone-related genes and pathways at the transcriptional level. Although ALN either reduced or had neutral effects on bone resorption-related genes, ODN significantly increased the expression of osteoclast genes (eg, APC5, TNFRSF11A, CTSK, ITGB3, and CALCR), consistent with previous findings on the effects of this agent in enhancing the number of nonresorbing osteoclasts. Conversely, ALN reduced the expression of known bone formation-related genes (eg, TGFBR1, SPP1, RUNX2, and PTH1R), whereas ODN either increased or had neutral effects on their expression. These differential effects of ODN versus ALN on bone resorption and formation were highly correlative to the changes in bone turnover markers, cathepsin K (Catk) target engagement marker serum C-terminal cross-linked telopeptide (1-CTP) and osteoclast marker tartrate resistant acid phosphatase isoform 5b (TRAP5b) in the same monkeys. Overall, the molecular profiling results are consistent with the known pharmacological actions of these agents on bone remodeling and clearly differentiate the molecular mechanisms of ODN from the bisphosphonates.

Identify a gene expression signature in white adipose tissue (WAT) that reports on WAT browning and is associated with a healthy phenotype.RNA from several different adipose depots across three species were analyzed by whole transcriptome profiling, including 1) mouse subcutaneous white fat, brown fat, and white fat after in vivo treatment with FGF21; 2) human subcutaneous and omental fat from insulin-sensitive and insulin-resistant patients; and 3) rhesus monkey subcutaneous fat from healthy and dysmetabolic individuals.A "browning" signature in mice was identified by cross-referencing the FGF21-induced signature in WAT with the brown adipose tissue (BAT) vs. WAT comparison. In addition, gene expression levels in WAT from insulin-sensitive/healthy vs. insulin-resistant/dysmetabolic humans and rhesus monkeys, respectively, correlated with the gene expression levels in mouse BAT vs. WAT. A subset of 49 genes were identified that were consistently regulated or differentially expressed in the mouse and human data sets that could be used to monitor browning of WAT across species.Gene expression profiles of WATs from healthy insulin-sensitive individuals correlate with those of BAT and FGF21-induced browning of WAT.

Phosphodiesterase 4 (PDE4) inhibitors elevate cyclic adenosine 5'-monophosphate (cAMP), and this elevation has been shown to inhibit inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). Using TNF-alpha as a biomarker, we have developed transcription-based assays to examine inhibition of PDE4 activity in human and guinea pig whole blood. In vitro inhibition by PDE4 inhibitors was measured using quantitative PCR (qPCR) analysis of TNF-alpha mRNA levels in whole blood stimulated with lipopolysaccharide (LPS). The kinetics of human TNF-alpha mRNA production were analyzed and shown to be highest 4 hr following LPS stimulation. The guinea pig displayed kinetics of TNF-alpha transcription similar to those of the human. Analysis of inhibition of human TNF-alpha protein production was performed by immunoassay and shown to correlate with inhibition of transcription for three of the four compounds tested. Roflumilast was found to be 9-fold more potent for TNF-alpha inhibition in the qPCR assay than in the protein assay. The potencies of L-826,141 and roflumilast were determined in human and guinea pig whole blood by qPCR, with IC(50) values of 270 and 20 nM, respectively, in humans and 100 and 10 nM, respectively, in guinea pigs. These results show that the potency of PDE4 inhibitors can be monitored in whole blood using a transcription-based assay, and that this type of assay can be adapted to various species provided the TNF-alpha nucleotide sequence is known. The in vitro whole blood IC(50) for TNF-alpha inhibition was compared to inhibition in the ovalbumin-challenged guinea pig model of bronchoconstriction. Obtaining plasma levels at the IC(50) determined in vitro for L-826,141 and roflumilast provides significant inhibition of bronchoconstriction. This suggests that TNF-alpha can be used as a whole blood biomarker in the guinea pig for PDE4 inhibition in this inflammatory model.

A CHO-K1 cell line stably expressing a recombinant full-length human PDE-IVa (HSPDE4A4B) enzyme was established under hygromycin B selection. Full-length expression of the protein was determined by Western blot analysis, which revealed the presence of a 125-kDa immunoreactive band using rabbit anti-PDE-IVa antibodies. The potency of inhibitor compounds was examined by their ability to increase cAMP in the whole-cell, and by their ability to inhibit cAMP hydrolysis in a 100,000 g supernatant (soluble enzyme preparation) obtained from the same cell line. Inhibition of the expressed PDE-IVa activity by selective PDE-IV inhibitors--(R) and (S)-rolipram, RS 14203, and CDP 840--at 100 nM substrate demonstrated that RS 14203 and CDP 840 were the most potent with IC50 = 9 nM, followed by (R)-rolipram (IC50 = 110 nM) and (S)-rolipram (IC50 = 420 nM). The rank order of potencies of the inhibitors in elevating cAMP in the whole-cell assay was quite different from that on the soluble enzyme. RS 14203 was still the most potent compound in elevating cAMP. Moreover, the relative rank order of potencies between CDP 840 and (R)-rolipram changed dramatically, such that (R)-rolipram was more potent than CDP 840 = (S)-rolipram. An apparent 30-fold stereoselectivity between (R)- and (S)-rolipram was also noted. The whole-cell rank order of potencies was also maintained when PKA activity ratios were measured in place of cAMP levels. The ability of the compounds to elevate cAMP in the stable CHO-K1 cells appeared to track better with the potency of the compounds against the high-affinity (Sr) conformer of the enzyme rather than the low-affinity catalytic state.

The T cell protein tyrosine phosphatase (TC-PTP) is one of the most abundant mammalian tyrosine phosphatases in hematopoietic cells; however, its role in hematopoietic cell function remains unknown. In this report, we investigated the physiological function(s) of TC-PTP by generating TC-PTP–deficient mutant mice. The three genotypes (+/+, +/−, −/−) showed mendelian segregation at birth (1:2:1) demonstrating that the absence of TC-PTP was not lethal in utero, but all homozygous mutant mice died by 3–5 wk of age, displaying runting, splenomegaly, and lymphadenopathy. Homozygous mice exhibited specific defects in bone marrow (BM), B cell lymphopoiesis, and erythropoiesis, as well as impaired T and B cell functions. However, myeloid and macrophage development in the BM and T cell development in the thymus were not significantly affected. BM transplantation experiments showed that hematopoietic failure in TC-PTP −/− animals was not due to a stem cell defect, but rather to a stromal cell deficiency. This study demonstrates that TC-PTP plays a significant role in both hematopoiesis and immune function.

The mouse chromosome 6 locus Cmv1 controls replication of mouse Cytomegalovirus (MCMV) in the spleen of the infected host. In our effort to clone Cmv1, we have constructed a high-resolution genetic linkage map in the proximity of the gene. For this, a total of 45 DNA markers corresponding to either cloned genes or microsatellites were mapped within a 7.9-cM interval overlapping the Cmv1 region. We have followed the cosegregation of these markers with respect to Cmv1 in a total of 2248 backcross mice from a preexisting interspecific backcross panel of 281 (Mus spretus x C57BL/6J)F1 x C57BL/6J and 2 novel panels of 989 (A/ J x C57BL6)F1 x A/J and 978 (BALB/c x C57BL/6J)F1 x BALB/c segregating Cmv1. Combined pedigree analysis allowed us to determine the following gene order and intergene distances (in cM) on the distal region of mouse chromosome 6: D6Mit216-(1.9)-D6Mit336-(2.2)- D6Mit218-(1.0)-D6Mit52-(0.5)-D6Mit194-(0.2)-Nkrp 1/ D6Mit61/135/257/289/338-(0.4)-Cmv1/Ly49A/D6Mit370++ +- (0.3)-Prp/Kap/D6Mit13/111/219-(0.3)-Tel/D6Mit374/290/ 220/196/195/110-(1.1)-D6Mit25. Therefore, the minimal genetic interval for Cmv1 of 0.7 cM is defined by 13 tightly linked markers including 2 markers, Ly49A and D6Mit370, that did not show recombination with Cmv1 in 1967 meioses analyzed; the proximal limit of the Cmv1 domain was defined by 8 crossovers between Nkrp1/ D6Mit61/135/257/289/338 and Cmv1/Ly49A/D6Mit370, and the distal limit was defined by 5 crossovers between Cmv1/Ly49A/D6Mit370 and Prp/Kap/D6Mit13/111/219. This work demonstrates tight linkage between Cmv1 and genes from the natural killer complex (NKC), such as Nkrp1 and Ly49A, suggesting that Cmv1 may represent an NK cell recognition structure encoded in the NKC region.

Inhibition of Diacylglycerol O-acyltransferase 1 (DGAT1) has been a mechanism of interest for metabolic disorders. DGAT1 inhibition has been shown to be a key regulator in an array of metabolic pathways; however, based on the DGAT1 KO mouse phenotype the anticipation is that pharmacological inhibition of DGAT1 could potentially lead to skin related adverse effects. One of the aims in developing small molecule DGAT1 inhibitors that target key metabolic tissues is to avoid activity on skin-localized DGAT1 enzyme. In this report we describe a modeling-based approach to identify molecules with physical properties leading to differential exposure distribution. In addition, we demonstrate histological and RNA based biomarker approaches that can detect sebaceous gland atrophy pre-clinically that could be used as potential biomarkers in a clinical setting.

FGF21 is a novel secreted protein with robust anti-diabetic, anti-obesity, and anti-atherogenic activities in preclinical species. In the current study, we investigated the signal transduction pathways downstream of FGF21 following acute administration of the growth factor to mice. Focusing on adipose tissues, we identified FGF21-mediated downstream signaling events and target engagement biomarkers. Specifically, RNA profiling of adipose tissues and phosphoproteomic profiling of adipocytes, following FGF21 treatment revealed several specific changes in gene expression and post-translational modifications, specifically phosphorylation, in several relevant proteins. Affymetrix microarray analysis of white adipose tissues isolated from both C57BL/6 (fed either regular chow or HFD) and db/db mice identified over 150 robust potential RNA transcripts and over 50 potential secreted proteins that were changed greater than 1.5 fold by FGF21 acutely. Phosphoprofiling analysis identified over 130 phosphoproteins that were modulated greater than 1.5 fold by FGF21 in 3T3-L1 adipocytes. Bioinformatic analysis of the combined gene and phosphoprotein profiling data identified a number of known metabolic pathways such as glucose uptake, insulin receptor signaling, Erk/Mapk signaling cascades, and lipid metabolism. Moreover, a number of novel events with hitherto unknown links to FGF21 signaling were observed at both the transcription and protein phosphorylation levels following treatment. We conclude that such a combined "omics" approach can be used not only to identify robust biomarkers for novel therapeutics but can also enhance our understanding of downstream signaling pathways; in the example presented here, novel FGF21-mediated signaling events in adipose tissue have been revealed that warrant further investigation.

A PCR-based random mutagenesis procedure was employed to identify several thermosensitive mutants of the MPTP enzyme, the murine homologue of the human T-cell PTPase and rat PTP-S enzymes. Four mutants with varying degrees of thermosensitivity were characterized for their thermostability and refolding properties following incubation at the nonpermissive temperature. Structure analysis of these mutations based on the hPTP1B co-ordinate structure demonstrates a clear relationship between the position of each mutated amino acid relative to the catalytic cysteine residue and their thermostability. Introduction of two of these mutations in the related enzyme hPTP1B suggests that the structural defects and the resulting thermosensitivity of these mutations may represent an intrinsic property of all PTPase catalytic domains.