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Urocortin-1 (UCN1) is a member of the corticotropin releasing hormone (CRH) family of peptides that acts through CRH-receptor 1 (CRHR1) and CRH-receptor 2 (CRHR2). UCN1 can induce the adrenocorticotropin hormone and downstream glucocorticoids through CRHR1 and promote beneficial metabolic effects through CRHR2. UCN1 has a short half-life and has been shown to improve experimental autoimmune disease. A pegylated UCN1 peptide (PEG-hUCN1) was generated to extend half-life and was tested in multiple experimental autoimmune disease models and in healthy mice to determine effects on corticosterone induction, autoimmune disease, and glucocorticoid induced adverse effects. Cardiovascular effects were also assessed by telemetry. PEG-hUCN1 demonstrated a dose dependent 4-6-fold elevation of serum corticosterone and significantly improved autoimmune disease comparable to prednisolone in several experimental models. In healthy mice, PEG-hUCN1 showed less adverse effects compared with corticosterone treatment. PEG-hUCN1 peptide induced an initial 30% reduction in blood pressure that was followed by a gradual and sustained 30% increase in blood pressure at the highest dose. Additionally, an adeno-associated viral 8 (AAV8) UCN1 was used to assess adverse effects of chronic elevation of UCN1 in wild type and CRHR2 knockout mice. Chronic UCN1 expression by an AAV8 approach in wild type and CRHR2 knockout mice demonstrated an important role of CRHR2 in countering the adverse metabolic effects of elevated corticosterone from UCN1. Our findings demonstrate that PEG-hUCN1 shows profound effects in treating autoimmune disease with an improved safety profile relative to corticosterone and that CRHR2 activity is important in metabolic regulation. SIGNIFICANCE STATEMENT: This study reports the generation and characterization of a pegylated UCN1 peptide and the role of CRHR2 in UCN1-induced metabolic effects. The potency/selectivity, pharmacokinetic properties, pharmacodynamic effects, and efficacy in four autoimmune models and safety profiles are presented. This pegylated UCN1 shows potential for treating autoimmune diseases with reduced adverse effects compared to corticosterone treatment. Continuous exposure to UCN1 through an AAV8 approach demonstrates some glucocorticoid mediated adverse metabolic effects that are exacerbated in the absence of the CRHR2 receptor.

Supplementary Figure Legends & Tables

Figure S1. Efficacy of pemetrexed in Lewis Lung carcinoma model and metabolomic analysis of MC38 tumor bearing mice treated with 50 mg/kg of pemetrexed Figure S2. T cell inflamed phenotype induced by pemetrexed is modestly reduced in tumors upon combination with cisplatin Figure S3. Combination Effects of pemetrexed -/+ anti-PD-L1 in MC38, LLC and Colon26 tumor models Figure S4 (A) Mice bearing Colon26 tumors were treated starting 10 days after tumor implantation with pemetrexed (50 mg/kg, 5 days on, 2 days off, IP) and/or anti-PD-L1 (αPD-L1) (500 ug/mouse, weekly IP), and tumors were isolated after 14 days of treatment and single cell suspensions were subjected to flow cytometry analysis. FACS plots show representative data of intra-tumor CD45+ tumor-infiltrating lymphocyte (TIL) T cell (CD3+) and Myeloid cell (CD11b+) subsets frequencies; and percentage of Ki67+ CD4+ T effector cells based on the gating scheme indicated. (B) Spleens, TDLNs and tumors were isolated and antigen specific T cell responses were measured by IFNγ ELISpot, gp70 Tetramer staining (Tet) and Intracellular cytokine staining. (Left) CD8 T cells isolated from pooled spleens and TDLNs from individual mice were cultured alone (control) with irrelevant BALB/c tumor EMT6 or cognate Colon26 tumor cells overnight and number of IFNγ producing T cells was measured by ELISpot. (Middle) Freshly isolated CD8+ T cells from spleens and TDLNs were assessed for % of gp70 Tet+ cells. (Right) Freshly isolated tumors single cell suspensions were stimulated with PMA/ phorbol myristate acetate (PMA) and ionomycin with monensin for 4 hrs and the % of TNF-alpha positive and gp70 Tet+ CD8 T cells were measured from the Live CD45+, CD3+ T cells by FACS. Figure S5. Concordance between QuantiGene and nCounter gene expression data for in vitro activated T cells treated in the presence or absence of pemetrexed (related to Figure 6F). Supplementary Table 1. Ingenuity pathway analysis (IPA) of MC38 tumors treated with pemetrexed or paclitaxel with or without carboplatin Supplementary Table 2: IPA results for Colon26 experiment in Figure 4 sorted by functional class. Pemetrexed monotherapy in this experiment at this timepoint did not have enough differentially expressed genes to generate IPA results.

Purpose:Combination strategies leveraging chemotherapeutic agents and immunotherapy have held the promise as a method to improve benefit for patients with cancer. However, most chemotherapies have detrimental effects on immune homeostasis and differ in their ability to induce immunogenic cell death (ICD). The approval of pemetrexed and carboplatin with anti-PD-1 (pembrolizumab) for treatment of non–small cell lung cancer represents the first approved chemotherapy and immunotherapy combination. Although the clinical data suggest a positive interaction between pemetrexed-based chemotherapy and immunotherapy, the underlying mechanism remains unknown.Experimental Design:Mouse tumor models (MC38, Colon26) and high-content biomarker studies (flow cytometry, Quantigene Plex, and nCounter gene expression analysis) were deployed to obtain insights into the mechanistic rationale behind the efficacy observed with pemetrexed/anti-PD-L1 combination. ICD in tumor cell lines was assessed by calreticulin and HMGB-1 immunoassays, and metabolic function of primary T cells was evaluated by Seahorse analysis.Results:Pemetrexed treatment alone increased T-cell activation in mouse tumors in vivo, robustly induced ICD in mouse tumor cells and exerted T-cell–intrinsic effects exemplified by augmented mitochondrial function and enhanced T-cell activation in vitro. Increased antitumor efficacy and pronounced inflamed/immune activation were observed when pemetrexed was combined with anti-PD-L1.Conclusions:Pemetrexed augments systemic intratumor immune responses through tumor intrinsic mechanisms including immunogenic cell death, T-cell–intrinsic mechanisms enhancing mitochondrial biogenesis leading to increased T-cell infiltration/activation along with modulation of innate immune pathways, which are significantly enhanced in combination with PD-1 pathway blockade.See related commentary by Buque et al., p. 6890

Context Tirzepatide substantially reduced hemoglobin A1c (HbA1c) and body weight in subjects with type 2 diabetes (T2D) compared with the glucagon-like peptide 1 receptor agonist dulaglutide. Improved glycemic control was associated with lower circulating triglycerides and lipoprotein markers and improved markers of beta-cell function and insulin resistance (IR), effects only partially attributable to weight loss. Objective Assess plasma metabolome changes mediated by tirzepatide. Design Phase 2b trial participants were randomly assigned to receive weekly subcutaneous tirzepatide, dulaglutide, or placebo for 26 weeks. Post hoc exploratory metabolomics and lipidomics analyses were performed. Setting Post hoc analysis. Participants 259 subjects with T2D. Intervention(s) Tirzepatide (1, 5, 10, 15 mg), dulaglutide (1.5 mg), or placebo. Main Outcome Measure(s) Changes in metabolite levels in response to tirzepatide were assessed against baseline levels, dulaglutide, and placebo using multiplicity correction. Results At 26 weeks, a higher dose tirzepatide modulated a cluster of metabolites and lipids associated with IR, obesity, and future T2D risk. Branched-chain amino acids, direct catabolic products glutamate, 3-hydroxyisobutyrate, branched-chain ketoacids, and indirect byproducts such as 2-hydroxybutyrate decreased compared to baseline and placebo. Changes were significantly larger with tirzepatide compared with dulaglutide and directly proportional to reductions of HbA1c, homeostatic model assessment 2-IR indices, and proinsulin levels. Proportional to metabolite changes, triglycerides and diglycerides were lowered significantly compared to baseline, dulaglutide, and placebo, with a bias toward shorter and highly saturated species. Conclusions Tirzepatide reduces body weight and improves glycemic control and uniquely modulates metabolites associated with T2D risk and metabolic dysregulation in a direction consistent with improved metabolic health.

OBJECTIVE Sex differences in insulin sensitivity are present throughout the life-span, with men having a higher prevalence of insulin resistance and diabetes compared with women. Differences in lean mass, fat mass, and fat distribution-particularly ectopic fat-have all been postulated to contribute to the sexual dimorphism in diabetes risk. Emerging data suggest ectopic lipid composition and subcellular localization are most relevant; however, it is not known whether they explain sex differences in obesity-induced insulin resistance. METHODS To address this gap, this study evaluated insulin sensitivity and subcellular localization of intramuscular triacylglycerol, diacylglycerol, and sphingolipids as well as muscle acylcarnitines and serum lipidomics in people with obesity. RESULTS Insulin sensitivity was significantly lower in men (P

© 2020 Tam et al. Obesity and elevation of circulating free fatty acids are associated with an accumulation and proinflammatory polarization of macrophages within metabolically active tissues, such as adipose tissue, muscle, liver, and pancreas. Beyond macrophages, neutrophils also accumulate in adipose and muscle tissues during high-fat diets and contribute to a state of local inflammation and insulin resistance. However, the mechanisms by which neutrophils are recruited to these tissues are largely unknown. Here we used a cell culture system as proof of concept to show that, upon exposure to a saturated fatty acid, palmitate, macrophages release nucleotides that attract neutrophils. Moreover, we found that palmitate up-regulates pannexin-1 channels in macrophages that mediate the attraction of neutrophils, shown previously to allow transfer of nucleotides across membranes. These findings suggest that proinflammatory macrophages release nucleotides through pannexin-1, a process that may facilitate neutrophil recruitment into metabolic tissues during obesity.

Background/Introduction In a Phase 2 trial, tirzepatide (TZP) dose-dependently reduced HbA1c, body weight and serum triglycerides in moderately obese type 2 diabetes (T2D) patients. Purpose To understand changes in fasting serum lipids with TZP, lipidomics profiling of multiple lipid classes was conducted. Methods Patients (n=314) were randomized to receive weekly subcutaneous TZP, dulaglutide, or placebo for 26 weeks. Shotgun lipidomic analysis was performed in positive electrospray using a Sciex Triple TOF 5600 mass spectrometer operating in MS/MSALL mode and sphingomyelin (SPM) data were acquired in positive electrospray TOF mode. Data were assessed using MMRM. Results At 26 weeks, total cholesterol esters (CE), phosphatidylcholines (PC) and phosphatidylinositols (PI) increased with placebo; dulaglutide treatment did not affect any lipid class (Table). With TZP 10 and 15 mg, total triacylglycerides (TAG), diacylglycerides (DAG), phosphatidylethanolamines (PE), PC and PI dose-dependently decreased at 4, 12 and 26 weeks, whereas total CE and phosphatidylserines (PS) levels did not change at any timepoint. At 26 weeks, individual, but not total, SPM and ceramide (Cer) changes were observed with TZP 15 mg. Most saturated, long chain SPM (14:0, 20:0, 21:0, 22:0, 23:0, 24:0) and Cer (22:0, 23:0, 24:0) were reduced by 8–16% and 14–20%, respectively. Conversely, unsaturated SPM (24:1,24:2) increased by 6.5% and 11.4%, and unsaturated glycosylated Cer 16:0 lactosyl and 24:1 hexosyl increased by 22.4% and 19.3%, respectively. TZP 10 and 15 mg decreased total SPM by 4–6% at 4 weeks, and total Cer between 8% and 13% at 4 and 12 weeks. Conclusions Tirzepatide dose-dependently reduced levels of total TAG, DAG, PE, PC, and PI at all timepoints, whereas levels of saturated long chain SPM and Cer were reduced and those of some unsaturated SPM and glycosylated Cer increased at 26 weeks. Net improvements in lipidomics profiling with tirzepatide warrant further evaluation of potential benefits on cardiovascular events and NASH. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Eli Lilly and Company

AICARFT is a folate dependent catalytic site within the ATIC gene, part of the purine biosynthetic pathway, a pathway frequently upregulated in cancers. LSN3213128 is a potent (16 nM) anti-folate inhibitor of AICARFT and selective relative to TS, SHMT1, MTHFD1, MTHFD2 and MTHFD2L. Increases in ZMP, accompanied by activation of AMPK and cell growth inhibition, were observed with treatment of LY3213128. These effects on ZMP and proliferation were dependent on folate levels. In human breast MDA-MB-231met2 and lung NCI-H460 cell lines, growth inhibition was rescued by hypoxanthine, but not in the A9 murine cell line which is deficient in purine salvage. In athymic nude mice, LSN3213128 robustly elevates ZMP in MDA-MB-231met2, NCI-H460 and A9 tumors in a time and dose dependent manner. Significant tumor growth inhibition in human breast MDA-MB231met2 and lung NCI-H460 xenografts and in the syngeneic A9 tumor model were observed with oral administration of LSN3213128. Strikingly, AMPK appeared activated within the tumors and did not change even at high levels of intratumoral ZMP after weeks of dosing. These results support the evaluation of LSN3213128 as an antineoplastic agent.

A hallmark of cancer is unbridled proliferation that can result in increased demand for de novo synthesis of purine and pyrimidine bases required for DNA and RNA biosynthesis. These synthetic pathways are frequently upregulated in cancer and involve various folate-dependent enzymes. Antifolates have a proven record as clinically used oncolytic agents. Our recent research efforts have produced LSN 3213128 (compound 28a), a novel, selective, nonclassical, orally bioavailable antifolate with potent and specific inhibitory activity for aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT), an enzyme in the purine biosynthetic pathway. Inhibition of AICARFT with compound 28a results in dramatic elevation of 5-aminoimidazole 4-carboxamide ribonucleotide (ZMP) and growth inhibition in NCI-H460 and MDA-MB-231met2 cancer cell lines. Treatment with this inhibitor in a murine based xenograft model of triple negative breast cancer (TNBC) resulted in tumor growth inhibition.

Accumulation of Immune Responsive Gene 1(IRG1) in macrophage induced by lipopolysaccharide (LPS) and interferon gamma (IFN-γ) leads to production of itaconate by decarboxylation of cis-aconitate. The biology associated with IRG1 and itaconate is not fully understood. A rapid and sensitive method for measurement of itaconate will benefit the study of IRG1 biology. Multiple HPLC and derivatization methods were tested. An ion pairing LC-MS/MS method using tributylamine/formic acid as ion pairing agents and a HypercarbTM guard column we proposed demonstrated better peak shape and better sensitivity for itaconate. The current protocol allows baseline separation of itaconate, citraconate, and cis-aconitate without derivatization and direct analysis of analytes in 80% methanol/water solution to avoid the dry-down step. It provides the limit of quantitation (LOQ) of 30 pg itaconate on column with a 4.5-minute run time. This method is validated for measurement of itaconate and cis-aconitate in RAW264.7 cell extract and cell media in a 96-well plate format. We applied this method to successfully measure the increase of itaconate and the decrease of cis-aconitate in RAW cell extract and cell media after LPS/IFN-γ treatment.

Activation of GIP (glucose-dependent insulinotropic peptide) receptor (GIPR) in combination with GLP-1 receptor has demonstrated reduced adiposity and bodyweight in mice. Although the incretin action of GIP in pancreatic beta cells is well characterized, its regulation of adipose tissue metabolism are incompletely understood. Using differentiated human adipocytes we demonstrated GIP suppression of insulin stimulated [14C] glucose and [14C] acetate incorporation into the cellular lipid fraction suggesting GIP/insulin co-regulation of de novo lipogenesis. To further investigate these findings, we employed an NMR and mass spec strategy. Briefly, human adipocytes were differentiated then incubated with [13C] glucose or [13C] acetate overnight in the presence GIP, insulin, GIP + insulin, or control followed by lysate preparation for mass spec and NMR analysis. Isotope label incorporation in lysate fatty acids were determined by HPLC-MS detection of base-hydrolyzed lipids. GIP significantly reduced incorporation of labeled acetate and labeled glucose in fatty acids, both in the presence and absence of insulin. Sample analysis by NMR demonstrated a GIP regulated increase in glycerol production from labeled glucose both in the presence and absence of insulin with little to no acetate incorporation into glycerol under all conditions tested. These findings demonstrate GIP signaling in adipocytes reduces de novo fatty acid synthesis while increasing glycerol formation. This suggests a model whereby GIP suppresses generation of new fatty acids from substrate but enhances appropriate storage capacity of dietary fatty acids by increasing production of glycerol required for triglyceride generation. Further studies will be required to elucidate this potential benefit of GIP to improve nutrient partitioning. Disclosure P.L. Milligan: Employee; Self; Eli Lilly and Company. K.D. Roth: Employee; Self; Eli Lilly and Company. H. Hu: None. J. Moyers: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. X. Ruan: Employee; Self; Eli Lilly and Company. W.C. Roell: Employee; Self; Eli Lilly and Company. A. Regmi: Employee; Self; Eli Lilly and Company.

Serum lipids and sphingolipids circulate in plasma and are linked with insulin resistance and increased cardiovascular disease risk. We previously reported that dihydroceramides (DHCer) are elevated in people with insulin resistance compared to individuals with obesity and trained athletes. Despite associations with insulin resistance, little is known whether insulin sensitizing lifestyle interventions can improve sphingolipid profiles. Twenty-one individuals with obesity and prediabetes (18F; 46± 2y; 35.0± 0.9 kg/m2; fasting glucose: 113.8± 9.4 mg/dl, 2h OGTT: 143.0± 19.4 mg/dl) participated in a 3-month lifestyle intervention of combined weight loss and exercise. Insulin sensitivity (SI) was assessed by hyperinsulinemic-euglycemic clamps and both targeted and shotgun lipidomics were conducted before and after intervention. BMI and body fat percentage were reduced by 11+1% (mean± SEM) and 9± 1% after intervention (p These data show that serum sphingolipids species previously linked to insulin resistance in humans can be improved with insulin sensitizing lifestyle interventions. Decreases in serum sphingolipids may explain increased SI and decreased cardiovascular disease risk associated with weight loss and exercise training. Disclosure J.L. Broussard: Advisory Panel; Self; National Institutes of Health. Research Support; Self; National Institute of Diabetes and Digestive and Kidney Diseases, Sleep Research Society, Society in Science-The Branco Weiss Fellowship. D.E. Kahn: None. S.A. Newsom: None. J.T. Brozinick: Employee; Self; Eli Lilly and Company. H. Bui: Employee; Self; Eli Lilly and Company. K.D. Roth: Employee; Self; Eli Lilly and Company. L. Perreault: Advisory Panel; Self; Novo Nordisk A/S, Sanofi. Speaker's Bureau; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Janssen Pharmaceuticals, Inc., Merck & Co., Inc., Novo Nordisk A/S. B.C. Bergman: Advisory Panel; Spouse/Partner; AstraZeneca, Merck & Co., Inc., Novo Nordisk Inc. Funding American Diabetes Association (1-14-CE-05 to B.B.); National Institutes of Health (RR-00036, K01DK110138); Colorado Nutrition Obesity Research Center (P30DK048520)

Acute myeloid leukemia is associated with the abnormal proliferation of myeloid progenitor cells unable to differentiate. Somatic gain-of-function mutations in isocitrate dehydrogenase (IDH) 1 occur in 10% of newly diagnosed AML patients. IDH1 mutations cause intracellular accumulation of the oncometabolite, 2-hydroxyglutarate (2-HG), which results in a hyper-methylation phenotype and a block in differentiation. Inhibitors of IDH1 mutant enzyme reduce levels of 2-HG, which relieves the differentiation block allowing AML cells to achieve terminal maturation. Recently, Ivosidenib, an IDH1 inhibitor, has been approved for use in AML patients. However, based on clinical findings, a fraction of the IDH1 mutant AML patients treated with Ivosidenib are primary refractory or relapse while on therapy. This raises the need for development of more potent inhibitors targeting IDH1. Lilly Research Laboratories have developed a potent covalent inhibitor of mutant IDH1, LY3410738 that modifies a single cysteine (Cys269) in an allosteric binding pocket and rapidly inactivates the enzyme, selectively inhibiting 2-HG production without affecting alpha-ketoglutarate (a-KG) levels. Here, we have assessed the activity of LY3410738 in IDH1 mutated patient-derived AML models and AML cell lines engineered to express wild-type IDH1 or mutant IDH1R132H. In vitro, LY3410738 displayed greater potency for inhibition of 2-HG production and differentiation of the IDH1 mutant cells compared to AG-120. Similarly, in vivo, we observed sustained 2-HG inhibition leading to a more robust and durable efficacy for LY3410738 with respect to AG-120. We next evaluated the combination activity of LY3410738 with Cytarabine and Azacitidine or the FLT3 inhibitor Midostaurin, the latter in FLT3-mutated AML. Combining LY3410738 with the chemotherapeutics resulted in increased efficacy, exhibiting a potent anti-leukemic effect, reduction of 2-HG level, and enhanced differentiation of the leukemic blasts in the mice. In addition, since IDH1 mutant AML cells have been shown to strongly depend on the anti-apoptotic Bcl-2 for the survival, we also combined LY3410738 with FDA approved Bcl-2 inhibitor, venetoclax. In vitro, isogenic cells with IDH1R132H mutation were more sensitive to the combination than wild-type IDH1-expressing cells. Importantly, the combination of LY3410738 with Venetoclax was also efficacious in an AML xenograft model derived from a patient refractory to AG-120. In conclusion, LY3410738 exhibits enhanced efficacy in IDH1 mutant AML PDX models in combination with Cytarabine, Azacitidine, Midostaurin and Venetoclax and demonstrates improved potency and durability compared to Ivosidenib. Citation Format: Vivian Salama, Nathan Brooks, Anna Skwarska, Lisa Kays, Paul Milligan, Katherine Newell, Kenneth Roth, Sandaruwan Geeganage, Raymond Gilmour, Steven M. Chan, Jean-Emmanuel Sarry, Mary Sabatier, Courtney DiNardo, Marina Konopleva. LY3410738, a novel inhibitor of mutant IDH1 is more effective than Ivosidenib and potentiates antileukemic activity of standard chemotherapy in preclinical models of acute myeloid leukemia (AML) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6417.

Purpose: Combination strategies leveraging chemotherapeutic agents and immunotherapy have held the promise as a method to improve benefit for patients with cancer. However, most chemotherapies have detrimental effects on immune homeostasis and differ in their ability to induce immunogenic cell death (ICD). The approval of pemetrexed and carboplatin with anti-PD-1 (pembrolizumab) for treatment of non–small cell lung cancer represents the first approved chemotherapy and immunotherapy combination. Although the clinical data suggest a positive interaction between pemetrexed-based chemotherapy and immunotherapy, the underlying mechanism remains unknown. Experimental Design: Mouse tumor models (MC38, Colon26) and high-content biomarker studies (flow cytometry, Quantigene Plex, and nCounter gene expression analysis) were deployed to obtain insights into the mechanistic rationale behind the efficacy observed with pemetrexed/anti-PD-L1 combination. ICD in tumor cell lines was assessed by calreticulin and HMGB-1 immunoassays, and metabolic function of primary T cells was evaluated by Seahorse analysis. Results: Pemetrexed treatment alone increased T-cell activation in mouse tumors in vivo, robustly induced ICD in mouse tumor cells and exerted T-cell–intrinsic effects exemplified by augmented mitochondrial function and enhanced T-cell activation in vitro. Increased antitumor efficacy and pronounced inflamed/immune activation were observed when pemetrexed was combined with anti-PD-L1. Conclusions: Pemetrexed augments systemic intratumor immune responses through tumor intrinsic mechanisms including immunogenic cell death, T-cell–intrinsic mechanisms enhancing mitochondrial biogenesis leading to increased T-cell infiltration/activation along with modulation of innate immune pathways, which are significantly enhanced in combination with PD-1 pathway blockade. See related commentary by Buque et al., p. 6890

2-Hydroxy-oleic acid (2OHOA) is a potent anticancer drug that induces cancer cell cycle arrest and apoptosis. Previous studies have suggested that 2OHOA's anticancer effect is mediated by SMS activation in cancer cells, including A549 and U118 cells. To confirm this phenomenon, in this study, we treated both A549 and U118 cells with 2OHOA and measured SMS activity. To our surprise, we found neither 2OHOA-mediated SMS activation nor sphingomyelin accumulation in the cells. However, we noted that 2OHOA significantly reduces phosphatidylcholine in these cells. We also did not observe 2OHOA-mediated SMS activation in mouse tissue homogenates. Importantly, 2OHOA inhibited rather than activated recombinant SMS1 (rSMS1) and rSMS2 in a dose-dependent fashion. Intra-gastric treatment of C57BL/6J mice with 2OHOA for 10 days had no effects on liver and small intestine SMS activities and plasma sphingomyelin levels. The treatment inhibited lysophosphatidylcholine acyltransferase (LPCAT) activity, consistent with the aforementioned reduction in plasma phosphatidylcholine. Because total cellular phosphatidylcholine is used as a predictive biomarker for monitoring tumor responses, the previously reported 2OHOA-mediated cancer suppression could be related to this phosphatidylcholine reduction, which may influence cell membrane structure and properties. We conclude that 2OHOA is not a SMS activator and that its anticancer property may be related to an effect on phosphatidylcholine metabolism.

Nonalcoholic fatty liver disease (NAFLD) affects a significant number of patients with T2DM and places them at an increased risk of steatohepatitis (NASH) and cirrhosis. However, it is widely underdiagnosed due to lack of a simple, non-invasive test for its diagnosis. The aim of this study was to establish a plasma biomarker profile to identify NAFLD and NASH in patients with T2DM. We recruited 220 patients with T2DM (59±8 years; BMI: 33.6±4.8 kg/m2; A1c: 7.1±1.2%) and screened them for NAFLD by liver magnetic resonance spectroscopy (No NAFLD: n=76; NAFLD: n=144). If positive, a liver biopsy was performed (Isolated steatosis: n=73; NASH: n=71). All patients underwent untargeted plasma metabolomics and lipidomics. To account for multiple comparisons, a Bonferroni’s adjusted p-value Conclusion: Metabolomics/lipidomics may hold promise to non-invasively diagnose NAFLD in patients with T2DM, although its ability to identify those with NASH remains to be established. Disclosure F. Bril: None. S. Kalavalapalli: None. K.L. Duffin: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Pfizer Inc. M.L. Hartman: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. Y. Chen: None. Q. Yang: None. J.V. Haas: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. P.L. Milligan: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. K.D. Roth: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. K. Cusi: Consultant; Self; Janssen Global Services, LLC., Eli Lilly and Company. Research Support; Self; Cirius Therapeutics. Other Relationship; Self; Nordic Bioscience. Research Support; Self; Novartis Pharmaceuticals Corporation, Novo Nordisk Inc.. Other Relationship; Self; Quest Diagnostics. Research Support; Self; Zydus Pharmaceuticals (USA) Inc.. Other Relationship; Self; OWL metabolomics, Echosens. Research Support; Self; Janssen Global Services, LLC.. Consultant; Self; Tobira Therapeutics, Pfizer Inc..

Combination strategies leveraging chemotherapy and immunotherapy have held the promise as a method to improve benefit to cancer patients. However, most chemotherapies have detrimental effects on immune homeostasis and do not induce immunogenic cell death. The positive phase III trial of pemetrexed/carboplatin with the PD-1 antibody pembrolizumab in NSCLC (Keynote-189) lead to the first chemotherapy/immunotherapy combination ever approved. While this suggests a positive interaction between pemetrexed-based chemotherapy and PD-1 therapy, the underlying mechanism is unknown. Therefore, it is important to understand the role of pemetrexed in modulating antitumor immune response to assure application of this therapy to the appropriate patients. To characterize the effects of pemetrexed on intra-tumor immune response, murine tumor models which were sensitive to pemetrexed and known to be sensitive to PD-L1, were treated with pemetrexed with or without carboplatin, or anti-mouse PD-L1. In MC38 tumors, pemetrexed monotherapy demonstrated a trend towards an increased frequency of intra-tumor leukocytes that was accompanied by immune-related gene expression changes indicative of enhanced T cell infiltration and/or activation. Gene expression induced by pemetrexed was largely unaffected by carboplatin. Treatment of both MC38 and Colon26 tumor cells in vitro with pemetrexed induced release of HMGB1, indicative of immunogenic cell death. Although proliferation of primary human T cells was slightly reduced by pemetrexed, at clinically relevant concentrations, treatment lead to an enhanced T cell activation phenotype, including upregulation of multiple interferon gamma-induced genes, and increased mitochondrial respiration. This correlated with improved antigen specific in vitro cytotoxic activity of OT-1 TCR transgenic CD8 T cells when treated with pemetrexed during priming with OVA peptide. Treatment with pemetrexed and PD-L1 demonstrated a combination benefit compared to either monotherapy in both tumor models. Pathway Analysis of gene expression data revealed that improved antigen presentation, enhanced T cell and cytokine signaling and an engagement of CD4+ T cell-mediated immunity during the combination. This correlated with upregulation of MHC-I & II on monocytes, macrophages and tumor cells, suggesting increased immune priming. Accordingly, treatment with S1P1R antagonist (FTY720, preventing T cell LN egress) after initiation of therapy resulted in a loss of combination efficacy. This data suggests that pemetrexed promotes intra-tumor T cell-mediated immune response through immunogenic tumor cell death and increased activation and metabolic fitness of T cells. The combination of these effects results in enhanced T cell function leading to an improved anti-tumor efficacy in combination with PD-L1 antibody Citation Format: David A. Schaer, Nelusha Amaladas, Zhao Hai Lu, Erik Rasmussen, Andreas Sonyi, Darin Chin, Andrew Capen, Yanxia Li, Catalina Meyer, Bonita Jones, Xiaodong Hong, Shuang Luo, Carmine Carpenito, Kenneth Roth, Alexander Nikolayev, Bo Tan, Manisha Brahmachary, Krishna Chodavarapu, Frank Charles Dorsey, Jason Manro, Thompson Doman, Greg Donoho, David Surguladze, Gerald Hall, Sandaruwan Geeganage, Michael Kalos, Ruslan Novosiadly. The folate pathway inhibitor pemetrexed pleiotropically enhances effects of cancer immunotherapy via immunogenic tumor cell death and T cell-intrinsic mechanisms [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 3945.

Somatic gain-of-function mutations in IDH1 have been identified in multiple tumor types including AML, glioma, cholangiocarcinoma and chondrosarcoma. The neo-enzymatic activity of mutant IDH1 results in accumulation of the oncometabolite 2-hydroxyglutarate (2-HG), leading to a hyper-methylation phenotype, a block in cell differentiation, and tumor growth. Using a structure-based drug design approach, we have developed a highly potent covalent inhibitor of mutant IDH1. LY3410738 modifies a single cysteine (Cys269) in the allosteric binding pocket and rapidly inactivates the enzyme with a KI/Kinact = 84,257 M-1sec-1. The compound selectively inhibits the 2-HG in IDH1 mutant tumor cells without depleting the levels of a-ketoglutarate. Using patient-derived primary AML cells, we demonstrated that LY3410738 was more potent than AG120 in reversing the block in differentiation associated with IDH1 mutant activity. In vivo, LY3410738 displayed prolonged pharmacodynamic activity, depleting 2-HG levels in tumors at low circulating exposures and for an extended time after clearance of compound. Importantly, LY3410738 has the ability to cross the blood-brain barrier and can achieve concentrations in the brain that exceed those needed to engage the target. Consistent with this, LY3410738 effectively inhibits 2HG in orthotopic glioma models. Using patient-derived IDH1 mutant orthotopic AML models, we demonstrated that LY3410738 effectively inhibited 2-HG, induced differentiation, and cleared AML from mice. Collectively, LY3410738 represents the first covalent brain-penetrant mutant IDH1 inhibitor with potential for Best-in-Class activity. Citation Format: Nathan Brooks, Robin DeWalt, Serge Boulet, ZhaoHai Lu, Lisa Kays, Rachel cavitt, Sandra Gomez, John Strelow, Paul Milligan, Kenneth Roth, Renato Bauer, Stephen Antonysamy, Patric Hahn, Zoran Rankovic, Denis McCann, Gary Mo, Ramon Tiu, Timothy Burkholder, Sandaruwan Geeganage, Raymond Gilmour. Identification and characterization of LY3410738, a novel covalent inhibitor of cancer-associated mutant Isocitrate Dehydrogenase 1 (IDH1) [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-274.

Tryptophan metabolism plays a central role in immunosuppression through the local depletion of tryptophan with the concomitant production and accumulation of kynurenine, both of which are immunosuppressive. Indoleamine 2,3-dioxygenase 1 (IDO1), a heme-dependent enzyme, catalyzes the initial and rate-limiting step of the kynurenine pathway. Tumor cells selectively upregulate IDO1 as an immune-evasion mechanism either through intrinsic expression of IDO1, or in response to IFN-γ, a cytokine secreted by immune cells during an active immune response. In addition to the immuno-suppressive role of IDO1 and kynurenine, we investigated their additional roles in tumor cells. Here we describe an NMR-based readout assay for IDO1 activity, in which we are able to trace the extra- and intracellular destination of the single tryptophan carbon groups. This assay allowed us to detect contribution of the tryptophan catabolism to purine synthesis in tumor cells, and suggested further roles of tryptophan catabolism in tumors, apart from kynurenine production, that contribute to the modulation of tumor growth. We detected the incorporation of labeled tryptophan carbon units in intracellular purine pools. Our highly potent, orally available, and CNS penetrant small molecule inhibitor of IDO1 (LY3381916) inhibits this incorporation of tryptophan-derived one carbon units into purines in vitro and in vivo. This activity leads to pre-clinical efficacy and may be an added advantage of inhibiting IDO1 in cancer. Citation Format: Sandaruwan Geeganage, Lillian Sams, James Henry, Frank Dorsey, Kenneth Roth, Alexander Nikolayev, Karim Benhadji, Raymond Gilmour, Ana Cerezo, Sandra Peregrina, Gloria Martínez-del Hoyo, Ramón Campos-Olivas, Juan Manuel Funes, Laura Diezma, Susana Velasco-Miguel. Tryptophan Metabolism Plays a Central Role in Immunosuppression [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 2187.

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate-limiting step in converting nicotinamide to NAD+, essential for cellular metabolism, energy production, and DNA repair. NAMPT has been extensively studied because of its critical role in these cellular processes and the prospect of developing therapeutics against the target, yet how it regulates cellular metabolism is not fully understood. In this study we utilized liquid chromatography-mass spectrometry to examine the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and serine biosynthesis in cancer cells and tumor xenografts. We show for the first time that NAMPT inhibition leads to the attenuation of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step due to the reduced availability of NAD+ for the enzyme. The attenuation of glycolysis results in the accumulation of glycolytic intermediates before and at the glyceraldehyde 3-phosphate dehydrogenase step, promoting carbon overflow into the pentose phosphate pathway as evidenced by the increased intermediate levels. The attenuation of glycolysis also causes decreased glycolytic intermediates after the glyceraldehyde 3-phosphate dehydrogenase step, thereby reducing carbon flow into serine biosynthesis and the TCA cycle. Labeling studies establish that the carbon overflow into the pentose phosphate pathway is mainly through its non-oxidative branch. Together, these studies establish the blockade of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step as the central metabolic basis of NAMPT inhibition responsible for ATP depletion, metabolic perturbation, and subsequent tumor growth inhibition. These studies also suggest that altered metabolite levels in tumors can be used as robust pharmacodynamic markers for evaluating NAMPT inhibitors in the clinic. Background: NAMPT catalyzes the rate-limiting reaction in converting nicotinamide to NAD+ in cancers. Results: NAMPT inhibition attenuates glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step, resulting in perturbing metabolic pathways related to glycolysis. Conclusion: The metabolic basis of NAMPT inhibition is the attenuation of glycolysis by reducing NAD+ available to glyceraldehyde 3-phosphate dehydrogenase. Significance: This study sheds new light on how NAMPT regulates cancer metabolism.