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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

Objectives: HOXA5 has been identified as a biomarker in pathogenesis of several cancers, such as non-small cell lung cancer (NSCLC) and breast cancer cells. The role has not been explored in cervical squamous cell carcinoma (CSCC). Methods: Tissues of 120 cases with CSCC and 30 controls with chronic cervicitis were constructed from our archived surgical pathology files and staining with HOXA5. Additional antibodies to E-cadherin and β-catenin were stained for comparison. For each marker, low expression was defined as staining score 0 to 3 points, whereas high expression referred to 4 points and above. Fifty-four patients in this research with cervical cancer were followed up for prognostic assessment. Result: HOXA5 had high expression in chronic cervicitis and low in CSCC (P=0.004). The positivity rates of HOXA5 in patients without muscular layer invasion (MLI) and lymphatic invasion (LI) was higher than that in metastasis (113 vs. 17; 117 vs. 3). Consistently, low expression of HOXA5 was more common in poorly differentiated carcinoma, CSCC subjects without MLI and LI. Expression of E-cadherin and β-catenin was parallel with the expression of HOXA5. Additionally, patients with higher expression of HOXA5 had much more favorable prognosis than those with lower expression among follow up of the 54 patients. Conclusion: In parallel with E-cadherin and β-catenin, low expression of HOXA5 was more common in CSCC patients with poor differentiation, without MLI and LI, among those which showed poor prognosis.

Rationale: Latent autoimmune diabetes in adults (LADA) refers to an autoimmune disorder characterized with detectable islets antibodies in the early diagnosis and increased autoimmune beta-cell failure progression. Notably, this kind of diabetes seems to be confused with other phenotypic diabetes. Patient concerns: A young woman suffered an emotional exhaustion-induced LADA, showing asthenia, polydipsia, polyuria, and visible weight loss. The patient emotionally ended a 14-year romantic relationship, leading to the emotional flooding. Diagnoses: The data from physical examination and laboratory tests exhibited as follows: glutamic acid decarboxylase antibody (GADA) = 63.83 U/mL, the fasting blood glucose (FBG) = 13.3 mmol/L, and glycated haemoglobin (HbA1c) = 10.9%. According to levels of GADA, the patient was diagnosed as LADA. Interventions: The patient was clinically treated with insulin for 3-month. Then, running, diet-control, and emotional treatment were combined, such as the patient started a new relationship. Outcomes: An emotional recovery initiated from a new romantic relationship and a baby, showing normal levels of GAD65 (27.007 IU/mL) and FBG (5.46) mmol/L. Lessons: The emotional exhaustion might play a significant role in induction of LADA. It is important that individuals should maintain optimism, cheer, and a positive attitude.

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.

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.

Background: Recently, pemetrexed and carboplatin in combination with PD–1 antibody (pembrolizumab) demonstrated markedly improved clinical outcome in NSCLC patients (KEYNOTE–021G trial) suggesting a positive interaction between pemetrexed–based chemotherapy and immunotherapy. However, the role of pemetrexed in modulating antitumor immune response is largely unknown. The objective of this study was to characterize the effects of pemetrexed on intra–tumor immune response in monotherapy and combination with carboplatin or PD–1 pathway blockade in preclinical models. Methods: Mice bearing syngeneic MC38 or Colon26 tumors were treated with pemetrexed with or without carboplatin or anti–mouse PD–L1 antibody (178G7). Immune cell subsets and immune–related changes in mouse tumor tissue and T cells were assessed by flow cytometry and gene expression analysis (Quantigene Plex and nanoString nCounter assays). Effects of pemetrexed on immunogenic cell death in tumor cells and mitochondrial respiration in T cells were evaluated by HMGB1 ELISA and Agilent Seahorse XF analysis, respectively. Results: In MC38 tumors, pemetrexed monotherapy demonstrated a trend towards an increased frequency of intratumoral leukocytes and total and cycling (Ki67+) T cells accompanied by immune–related gene expression changes indicative of enhanced antigen presentation, T cell infiltration and/or activation and down–modulation of the myeloid cell compartment. Immune gene expression signature induced by pemetrexed was largely unaffected by carboplatin. In both MC38 and Colon26 tumor cells, in vitro treatment with pemetrexed induced a robust release of HMGB1 indicative of immunogenic cell death. Proliferation of primary human T cells stimulated with CD3/CD28 was inhibited by pemetrexed in a dose–dependent manner. At clinically relevant concentrations pemetrexed also enhanced T cell activation phenotype exemplified by an increased frequency of CD137+, GITR+ and PD–L1+ T cells as well as upregulation of multiple interferon gamma–induced genes, and increased mitochondrial respiration. In both MC38 and Colon26 models, treatment with pemetrexed and 178G7 demonstrated a combination benefit compared to either monotherapy, and nCounter profiling of Colon26 tumors followed by Ingenuity Pathway Analysis revealed that improved antigen presentation, enhanced T cell and cytokine signaling and an engagement of CD4+ T cell–mediated immunity might underlie this combination effect. Conclusions: Pemetrexed promotes intra–tumor T cell–mediated immune response through immunogenic tumor cell death and increased activation and metabolic fitness of T cells leading to an enhanced anti–tumor efficacy in combination with PD–L1 antibody. Citation Format: Ruslan Novosiadly, David Schaer, Nelusha Amaladas, Erik Rasmussen, Zhao Hai Lu, Andreas Sonyi, Carmine Carpenito, Yanxia Li, Shuang Luo, Andrew Capen, Catalina Meyer, Xiaodong Huang, Jason Manro, Gregory Donoho, Thompson Doman, Gerald Hall, Sandaruwan Geeganage, Michael Kalos. Pemetrexed enhances anti-tumor efficacy of PD1 pathway blockade by promoting intra tumor immune response via immunogenic tumor cell death and T cell intrinsic mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4549.

Supplemental digital content is available in the text, Gastrointestinal (GI) disorders often manifest similar symptoms with overlapping clinical diagnosis and unmet medical needs. Traditional Chinese medicine (TCM) has history-proven benefits for GI diseases; albeit language barrier prevents Western readers from accessing the original reports in Chinese. The TCM formula Si-Ni-San (SNS) consists of 4 herbs targeting on homeostatic disturbances characterized by “reflux” and “irritable” problems. Here we used SNS as a therapeutic tool to explore the common mechanisms of pathogenesis in non-neoplastic GI diseases. Data sources from PUBMED, Chinese National Knowledge Infrastructure, and Wanfang databases were searched for clinical trials. Comparisons were SNS as intervention and Western conventional medicine as control, which treat patients with upper GI disorders (gastroesophageal reflux disease, peptic ulcer, chronic gastritis, duodenogastric reflux), lower GI diseases (irritable bowel syndrome, ulcerative colitis), and functional dyspepsia. Participants and studies in accordance with the Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Statement were eligible. We used the Jadad scale to assess methodological qualities, the fixed or random-effect model to evaluate therapeutic efficacy, and the funnel plots to explore publication bias. Outcome was clinical efficacy defined by symptom relief with normal GI endoscopy, radiology, and pathology. We included 83 studies involving 7762 participants: 1708 versus 1397 of the upper GI disorders in 34 studies, 901 versus 768 of the lower GI diseases in 19 studies, 1641 versus 1348 of functional dyspepsia in 30 studies, and 328 versus 287 of relapse rate in 8 studies. Six studies had a Jadad score >2 points and the rest were

The interaction of human immunodeficiency virus type 1 (HIV-1) with CD4 and one of a cadre of chemokine receptors triggers conformational changes in the HIV-1 envelope (Env) glycoprotein that lead to membrane fusion. The coreceptor activity of the second extracellular loop of CXCR4, which is restricted to dual tropic and T-tropic strains, was insensitive to the removal of charged residues either singly or in combinations by alanine scanning mutagenesis or to the conversion of acidic residues to lysine. Conversion of Asp-187 to a neutral residue exclusively unmasked activity with M-tropic Env in fusion and infection experiments. Insertion of the D187V mutation into chimeras containing extracellular loop 2 of CXCR4 in a CXCR2 framework also resulted in the acquisition of M-tropic coreceptor activity. The independence of CXCR4 coreceptor activity from charged residues and the extension of its repertoire by removing Asp-187 suggest that this interaction is not electrostatic and that coreceptors have the potential to be utilized by a spectrum of Env, which may be masked by charged amino acids in extracellular domains. These findings indicate that the primary structural determinants of coreceptors that program reactivity with M-, dual, and T-tropic Env are surprisingly subtle and that relatively insignificant changes in CXCR4 can dramatically alter utilization by Env of varying tropism.

The Duffy antigen receptor for chemokines (DARC) is expressed in human erythrocytes and on endothelial cells lining postcapillary venules in kidney and spleen. DARC is a promiscuous chemokine receptor and a binding protein for the malarial parasite Plasmodium vivax. The expression of DARC by subsets of endothelial cells and neurons in discrete anatomic sites in the brain suggests that this enigmatic receptor may have multiple roles in normal and pathological physiology. Conservation of this promiscuous chemokine binding function is evident from the similarity in nucleotide sequence of DARC homologues from multiple species, as well as the high-affinity binding of human chemokines to murine and avian erythrocytes. Analysis of the functional domains of DARC using chimeric receptors and monoclonal antibodies to multiple extracellular domains localized chemokine binding to structures in the amino terminal extracellular domain (E1). Scatchard analysis demonstrated that a chimeric DARC receptor, composed of the E1 domain of DARC and the predicted hydrophobic helices and loops of interleukin-8RB (IL-8RB), bound IL-8, and MGSA with K D values almost identical to the wild type receptors and bound a repertoire of C-X-C and C-C chemokines characteristic of DARC. Although numerous reports have demonstrated that chemokines such as IL-8 are expressed in the brain, presumably by glial cells, little insight into the nature of their role in normal or pathological physiology in the nervous system has developed because the target cells that express the corresponding receptors have not yet been identified. Northern blotting experiments suggest that mRNA encoding DARC are expressed in the central nervous system, however, interpretation of this is unclear because of the ubiquitous expression of DARC lining postcapillary venules. This study provides direct evidence to localize expression of DARC in the cental nervous system. Immunohistochemical examination of human archival sections of the brain with monoclonal antibodies specific for DARC localize expression of DARC to cell bodies and processes of Purkinjie cells in the cerebellum. The immunohistochemical findings were supported by analysis of chemokine binding and radioligand crosslinking with membranes made from various brain fractions. The hierarchical expression of DARC in neurons in the cerebellum suggests that chemokines may play an important role in the modulation of neuronal activity by glial cells.

The Duffy antigen/receptor for chemokines (DARC), first identified on erythrocytes, functions not only as a promiscuous chemokine receptor but also as a receptor for the malarial parasite, Plasmodium vivax. The recent finding that DARC is ubiquitously expressed by endothelial cells lining postcapillary venules provides a possible insight into the function of this receptor because this anatomic site is an active interface for leukocyte trafficking. However, the biological significance of DARC is questionable since it has not yet been determined whether individuals lacking the expression of this protein on their erythrocytes (Duffy negative individuals), who are apparently immunologically normal, express the receptor on endothelial cells. However, we report here that DARC is indeed expressed in endothelial cells lining postcapillary venules and splenic sinusoids in individuals who lack the erythrocyte receptor. These findings are based on immunohistochemical, biochemical, and molecular biological analysis of tissues from Duffy negative individuals. We also present data showing that, in contrast to erythrocyte DARC, cells transfected with DARC internalize radiolabeled ligand. We conclude that the DARC may play a critical role in mediating the effects of proinflammatory chemokines on the interactions between leukocyte and endothelial cells since the molecular pathology of the Duffy negative genotype maintains expression on the latter cell type.

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.

The human erythrocyte chemokine receptor has recently been shown to be identical to the Duffy blood group antigen and is expressed in multiple organs, including kidney. Here we have examined the molecular properties of the renal isoform. Immunoblot analysis of erythrocyte and kidney detergent lysates, with a monoclonal antibody (Fy6) to the Duffy antigen, revealed that the renal isoform had a molecular mass of 43-45 kD, which could be distinguished from that observed in erythroid cells (38-47 kD). Chemical cross-linking of kidney membranes to 125I-melanoma growth stimulatory activity (MGSA) indicated that the renal chemokine receptor had a molecular mass of 38-45 kD. Binding of 125I-labeled MGSA to kidney membranes was competitively inhibited by the addition of unlabeled MGSA, IL-8, regulated on activation, normal T expressed and secrted, and monocyte chemotactic protein-1. Scatchard analysis of MGSA binding showed that the chemokine receptor from renal tissues had a binding affinity of 3.5 nM similar to that observed for the erythroid isoform (5-10 nM). The primary structure of the renal chemokine receptor predicted from the nucleotide sequence of cDNA from renal tissues is identical to that reported for the erythroid isoform. Immunocytochemical staining of kidney with Fy6 localized expression to endothelial cells present in postcapillary venules. These studies implicate the Duffy antigen/chemokine receptor in the complex interactions between postcapillary endothelial cells and granulocytes, which are modulated by pro-inflammatory chemokines.