Enzyme In Salvage Pathway Research Articles

Acyclic nucleoside phosphonates with unnatural nucleobases, favipiravir and allopurinol, designed as potential inhibitors of the human and Plasmodium falciparum 6-oxopurine phosphoribosyltransferases

Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) is a key enzyme of the purine salvage pathway and its activity is crucial for the survival of certain parasites e.g. Plasmodium falciparum (Pf). Acyclic nucleoside phosphonates (ANPs) containing either guanine or hypoxanthine as the purine base are inhibitors of this enzyme. In this part of a SAR study, these two naturally-occurring nucleobases attached to an acyclic moiety were replaced by allopurinol or favipiravir. Both allopurinol and favipiravir ANPs were prepared via Mitsunobu reaction. The alkylation of favipiravir was optimized to yield both N- and O- regioisomers but the N-regioisomers were unstable under deprotection conditions. Thus, only the ANPs containing the O-isomer of favipiravir and those containing allopurinol were evaluated as potential inhibitors of human HGPRT and PfHGXPRT. Two ANPs with allopurinol as the base have Ki values of 10 μM and 30 μM for PfHGXPRT but do not inhibit human HGPRT activity at concentrations of 100–150 μM.

Pyridoxal kinase: A vitamin B6 salvage pathway enzyme from Leishmania donovani

Pyridoxal kinase (PdxK, EC 2.7.1.35) is an important enzyme of vitamin B6 salvage pathway which is required for phosphorylation of B6 vitamers. In the present study, pyridoxal kinase (pdxK) gene from Leishmania donovani (LdPdxK) was cloned and a 33 kDa protein was expressed and kinetically characterized. Site-directed mutagenesis was performed to determine the functional significance of conserved GXGD motif. Mutation of Thr229 to Ala did not affect the catalytic function of LdPdxK however Gly228, Gly230 and Asp231 were found to be indispensible for enzyme activity. To determine the role of LdPdxK in Leishmania promastigotes, LdPdxK overexpressing parasites were generated by episomal expression of the enzyme. The overexpression studies revealed the role of this enzyme in growth and infection of the parasite. In silico analysis of the human and parasite PdxK structure revealed significant differences in the active site region thus highlighting its potential as an antileishmanial drug target. Homology model of LdPdxK was built and was subjected to molecular dynamics simulations. Based on the above information, a pharmacophore was developed and shape based virtual screening was performed to identify potential and selective inhibitors against this essential enzyme. The current data suggests that LdPdxK could be a promising antileishmanial drug target.

NAMPT: A pleiotropic modulator of monocytes and macrophages

Nicotinamide phosphoribosyltransferase (NAMPT) is the bottleneck enzyme of the NAD salvage pathway and thereby is a controller of intracellular NAD concentrations. It has been long known that the same enzyme can be secreted by a number of cell types and acts as a cytokine, although its receptor is at present unknown. Investigational compounds have been developed that target the enzymatic activity as well as the extracellular action (i.e. neutralizing antibodies). The present contribution reviews the evidence that links intracellular and extracellular NAMPT to myeloid biology, for example governing monocyte/macrophage differentiation, polarization and migration. Furthermore, it reviews the evidence that links this protein to some disorders in which myeloid cells have a prominent role (acute infarct, inflammatory bowel disease, acute lung injury and rheumatoid arthritis) and the data showing that inhibition of the enzymatic activity or the neutralization of the cytokine is beneficial in preclinical animal models.

Examination of Hypoxanthine Guanine Phosphoribosyltransferase as a biomarker for colorectal cancer patients

ABSTRACTThe aim of this study is to investigate these enzymes as possible biomarkers in two colorectal cancer cell lines: HT29, SW480, SW620, and Colo205. With 1,168,929 individuals currently diagnosed with colorectal cancer in the United States, there remains a need to find biomarkers to improve diagnosis and expand treatment options for patients. Due to their role in proliferation and cell cycle regulation, we hypothesized an increase in salvage pathway enzyme (APRT, DCK, and HPRT) expression and possible presentation within colon cancer cells. Enzyme surface localization was assessed utilizing confocal microscopy, flow cytometry, and scanning electron microscopy. General protein expression was evaluated utilizing immunohistochemistry and Western blot analysis. While we found no statistically significant presence of either APRT or DCK on the membranes of SW620, Colo205, and HT29 cells, but found significant expression of HPRT on the surface of HT29, SW480, and SW620 cells. The average population fluorescence increased by 28%, 58%, and 40% in HT29, SW620, and SW480 cells, respectively, when compared to isotype controls. Confocal microscopy images revealed direct overlap between SW620 cells stained with a membrane dye and anti-HPRT antibody, indicating co-localization on the plasma membrane. In addition, cells treated with gold labelled HPRT antibody experienced significant changes in gold weight percentage on both SW620 and HT29 cells when compared to isotype controls. When evaluating expression within normal tissue, there was insignificant levels of HPRT binding. These data collectively suggest that HPRT may be a possible biomarker target for the identification and treatment of colorectal cancer.

Abstract 562: HPRT surface localization on prostate cancer cells as a biomarker for immunotherapy

Abstract The purpose of this study is to evaluate the expression and possible upregulation of the salvage pathway enzyme hypoxanthine guanine phosphoribosyltransferase (HPRT) in prostate cancer cells to determine if it could serve as a biomarker for prostate cancer diagnosis and treatment. Prostate cancer is the second most lethal cancer in men, and an estimated 26,730 men will die from the disease in 2017. We chose to evaluate the HPRT enzyme due to its involvement in nucleotide synthesis and cell cycle progression. Two prostate cancer cell lines were used for this analysis (PC3 and DU145) along with malignant tissue from 35 patients with prostate carcinoma. The surface localization of HPRT was determined utilizing flow cytometry and scanning electron microscopy, while upregulation within tissue was assessed using immunohistochemistry. Additionally, RNA-seq data from TCGA was used to evaluate general HPRT upregulation in patients with prostate cancer (n = 502) when compared to healthy individuals (n = 52). Throughout our investigation, we found a significant association between HPRT and the plasma membrane of DU145 cells, but found no presence on PC3 cells. Flow cytometry showed insignificant (p = 0.14) changes in fluorescence when PC3 cells were exposed to HPRT antibodies, while there was a significant increase in fluorescence when DU145 cells were treated with fluorescent HPRT antibody (p = 0.0004). To determine the distribution of HPRT across the membrane and ensure the observed expression was not due to cytoplasmic HPRT, gold conjugated antibodies were used for analysis with an electron microscope. The distribution of the gold on the cell surface showed random HPRT binding across the membrane with no patterns of localization. This analysis aided in confirming HPRT surface presence as the gold weight % of DU145 cells increased significantly when exposed to HPRT antibodies (p < 0.0001). In addition to being presented on the surface of DU145 cells, tissue samples revealed variable HPRT expression as approximately 53% of prostate cancer patients evaluated had elevated levels of HPRT compared to normal controls, while 47% of patients had no upregulation. TCGA data revealed a significant (p = 1.53x10-4) increase in HPRT levels upon malignancy. While some patients had levels consistent with healthy controls, there was a significant number of patients with increased protein expression upon cancer development. The control of HPRT expression within these cancer cells has been linked preliminarily to p53 functionality. While PC3 cells are null for p53, DU145 cells have a gain of function (GOF) p53 mutation. GOF p53 mutations are known to influence salvage pathway enzyme expression and is an influential gene in HPRT expression in these cancer cells. These results strongly indicate a unique relationship between prostate cancer cells and HPRT and suggest HPRT as a possible biomarker for the detection and treatment of patients with prostate cancer. Citation Format: Michelle H. Townsend, Abigail M. Felsted, Taylor P. Cox, Zachary E. Ence, Stephen R. Piccolo, Richard A. Robison, Kim O'Neill. HPRT surface localization on prostate cancer cells as a biomarker for immunotherapy [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 562.

Abstract 3049: Dexamethasone pretreatment impairs the TS inhibition-mediated flare in thymidine salvage pathway activity

Abstract Introduction: Inhibition of thymidylate synthase (TS) by pemetrexed, an inhibitor of thymidylate synthase, results in an early transient burst or “flare” in thymidine salvage pathway activity at 2 hrs of therapy, which, we have demonstrated in prior publications, can be measurable with FLT ([18F]thymidine)-positron emission tomography (PET) in non-small cell lung cancer (NSCLC). Administration of dexamethasone with pemetrexed-based therapy, as typically practiced in the clinic, could potentially confound this imaging approach since dexamethasone is known to inhibit expression of thymidine kinase 1, a key enzyme in the thymidine salvage pathway. Here we examine the potential impact of dexamethasone on the TS inhibition-mediated thymidine salvage pathway “flare” in NSCLC. Materials and Methods: In order to determine NSCLC cell line sensitivity to dexamethasone and pemetrexed, IC50 studies were performed on NSCLC cell lines H23, H1975, H460, H1299. TS inhibition-mediated “flare” in thymidine salvage pathway activity was then measured at 2hrs of exposure to pemetrexed and cisplatin in NSCLC cells lines following using 3H-thymidine incorporation assays under the following conditions: control (no chemotherapy or dexamethasone), treated with pemetrexed and cisplatin without dexamethasone, with 24 pretreatment of dexamethasone, or with dexamethasone administered together with chemotherapy. These conditions were chosen to model the delivery of pemetrexed-based therapy in the clinic. Results: The IC50 of H23, H1975, H460, H1299 for dexamethasone and pemetrexed were 40, 5.9, 718, 362 uM and 0.22, 0.73, 0.14 and 0.66 uM, respectively. Significant blunting of the thymidine salvage pathway “flare” is observed at 2hrs of pemetrexed-based therapy when dexamethasone-sensitive cell lines H23 and H1975 were pretreated with dexamethasone but not when dexamethasone was given together with pemetrexed therapy or in the setting of dexamethasone resistance (H460 and H1299). Conclusion: Imaging of TS inhibition may be impacted by pretreatment with dexamethasone but not administration of dexamethasone on the same day of therapy delivery. Citation Format: Sharyn I. Katz, Xiao Chen, Yizeng Yang. Dexamethasone pretreatment impairs the TS inhibition-mediated flare in thymidine salvage pathway activity [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 3049.

Abstract 2536: Unique HPRT1 upregulation in malignant tissue: Potential use as diagnostic biomarker

Abstract The aim of this study is to examine the gene expression of the purine salvage pathway enzyme Hypoxanthine Guanine Phosphribosyltransferase (HPRT) in malignant and normal tissue to determine potential upregulation. Due to the critical role HPRT plays in the cell cycle, it was hypothesized that in a rapidly proliferating malignant tissue there may be differential gene expression. In breast, colon, lung, and prostate cancer the 5-year survival rates decrease by nearly 72% when diagnosed in stage 3 or 4 as opposed to an early stage diagnosis. We focused our investigation on evaluating if HPRT could serve as an early stage diagnostic agent for the four most commonly diagnosed cancers (lung, colorectal, breast, and prostate cancer) which together make up 42.5% of all cancer diagnoses. Initially, we evaluated differences in HPRT expression levels via RNA-sequencing data in 3,147 tumor and 316 normal samples from The Cancer Genome Atlas (TCGA). Samples from 1119 breast invasive carcinoma (p-value= 1.66x10-42), 483 colon adenocarcinoma (p-value= 9x10-18), 541 lung adenocarcinoma (p-value=3.16x10-32), 502 lung squamous carcinoma (p-value= 1.49x10-59), and 502 prostate adenocarcinoma (p-value= 1.53x10-4) patients were compared to healthy individuals and showed significant HPRT over-expression shifts in malignant tumors. To continue this investigation we obtained histological tissue from 52 breast cancer patients, 54 lung cancer patients, 100 colorectal cancer patients, and 56 prostate cancer patients with a varying level of cancer stage and tumor type. Healthy tissue, margins of carcinoma, and pre-cancerous tissues were also stained to determine stage dependence of HPRT expression. Briefly, tissues were treated with a monoclonal anti-HPRT antibody along with a GAPDH positive control and an isotope control. Tissues were incubated with an HRP-polymer conjugated anti-HPRT antibody and followed by a diaminobenzidine (DAB) substrate which, when oxidized, results in antigen labelling. Tissues were imaged and analyzed using ImageJ software, which converted images to a grayscale. From there, we set a parameter for tissues with “HPRT high” expression, and “HPRT low” expression. Overall, we found 33-55% of malignant tissues to have a significant upregulation of HPRT (Lung-33%, Breast-55%, Colon-33%, Prostate-47%). These findings were consistent with our examination of HPRT expression in TCGA and our findings also indicated that the protein over-expression is not dependent on stage. These findings indicate that HPRT may potentially be a valuable biomarker in detecting early cases of malignancy in all of the four major cancers. Citation Format: Michelle H. Passey, Abigail M. Felsted, Zachary E. Ence, Stephen R. Piccolo, Kim L. O'Neill, Richard A. Robison. Unique HPRT1 upregulation in malignant tissue: Potential use as diagnostic biomarker [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 2536.

Abstract 771: HPRT: A biomarker and potential target for detection and treatment of colorectal cancer

Abstract This study aims to evaluate the expression of the salvage pathway enzymes DCK, APRT, and HPRT in colorectal cancer cells to investigate their potential as biomarkers for diagnosis and treatment. As the third most common cancer diagnosed in the United States, there remains a need to identify biomarkers capable of detecting and characterizing colorectal cancers. Because of their involvement in nucleotide synthesis and their role in cell cycle regulation and proliferation, we hypothesized an increase in these enzymes within malignancy. In order to quantify expression, we utilized two colorectal cancer cell lines (HT29 and SW620) as well as healthy and malignant patient tissue samples. Expression in tissues was evaluated using immunohistochemistry (IHC), and surface presence of the enzymes was assessed via confocal microscopy, flow cytometry, and scanning electron microscopy. Staining of malignant tissue samples using IHC showed upregulation of HPRT in 59% of patients when compared to control tissues derived from healthy patients (p=0.001). Surface analysis was performed utilizing flow cytometry, confocal microscopy, and scanning electron microscopy. Upon flow cytometry evaluation no significant presence of either DCK or APRT was found on the membranes of SW620 and HT29 cells (p=0.234 and 0.93, respectively), while HPRT expression was significantly increased on the surface of both SW620 and HT29 cells (p=0.013). Cell population fluorescence increased 58% and 28% in SW620 and HT29 cells, respectively, compared to controls. Confocal microscopy images showed direct overlap between SW620 cells stained with membrane-specific dye and anti-HPRT antibody, suggesting localization on the membrane. To further confirm the surface presence of HPRT, cells were treated with gold-labelled HPRT antibody and visualized under a scanning electron microscope to determine if surface expression was randomly dispersed across the cell surface. When treated with anti-HPRT antibodies, there was an increase in gold weight percentage on both SW620 (p=8.14x10 -6) and HT29 (p=1.74x10 -4) cells compared to controls, confirming HPRT presence and showing random surface localization patterns. Surface analysis was also conducted on malignant samples of patients with colorectal cancer. Of three samples, one of them exhibited significant HPRT surface localization. This supports the variation found within the tissues, as HPRT overexpression is not found in every patient. One patient did exhibit significant levels of HPRT on the surface of their malignant cells; this indicates HPRT as a potential target in patients that exhibit upregulation of the enzyme. These results suggest a relationship between colorectal cancers and HPRT, and indicate HPRT as a potential target for the detection and treatment of colorectal cancers. Citation Format: Michelle H. Townsend, Eric C. Olsen, Evita G. Weagel, Edwin J. Velasquez, Abigail M. Felsted, Weston Burrup, K Scott Weber, Richard Robison, Kim L. O'Neill. HPRT: A biomarker and potential target for detection and treatment of colorectal cancer [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 771.

Druggability of the guanosine/adenosine/cytidine nucleoside hydrolase from Trichomonas vaginalis.

Trichomonas vaginalis infects approximately 300million people worldwide annually. Infected individuals have a higher susceptibility to more serious conditions such as cervical and prostate cancer. The parasite has developed increasing resistance to current drug therapies, with an estimated 5% of clinical cases resulting from resistant strains, creating the need for new therapeutic strategies with novel mechanisms of action. Nucleoside salvage pathway enzymes represent novel drug targets as these pathways are essential for the parasite's survival. The guanosine/adenosine/cytidine nucleoside hydrolase (GACNH) may be particularly important as its expression is upregulated under glucose-limiting conditions mimicking those that occur during infection establishment. GACNH was screened against the NIH Clinical Collection to explore its druggability. Seven compounds were identified with IC50 values <20μM. Extensive overlap was found between inhibitors of GACNH and the adenosine/guanosine nucleoside hydrolase (AGNH), but no overlap was found with inhibitors of the uridine nucleoside hydrolase. The guanosine analog ribavirin was the only compound found to be specific for GACNH. Compounds that inhibit both AGNH and GACNH purine salvage pathway enzymes may prove critical given the role that GACNH appears to play in the early stages of infection.

Promiscuity of methionine salvage pathway enzymes in Methanocaldococcus jannaschii.

The methionine salvage pathway (MSP) is critical for regeneration of S-adenosyl-l-methionine (SAM), a widely used cofactor involved in many essential metabolic reactions. The MSP has been completely elucidated in aerobic organisms, and found to rely on molecular oxygen. Since anaerobic organisms do not use O2, an alternative pathway(s) must be operating. We sought to evaluate whether the functions of two annotated MSP enzymes from Methanocaldococcus jannaschii, a methylthioinosine phosphorylase (MTIP) and a methylthioribose 1-phosphate isomerase (MTRI), are consistent with functioning in a modified anaerobic MSP (AnMSP). We show here that recombinant MTIP is active with six different purine nucleosides, consistent with its function as a general purine nucleoside phosphorylase for both AnMSP and purine salvage. Recombinant MTRI is active with both 5-methylthioribose 1-phosphate and 5-deoxyribose 1-phosphate as substrates, which are generated from phosphororolysis of 5'-methylthioinosine and 5'-deoxyinosine by MTIP, respectively. Together, these data suggest that MTIP and MTRI may function in a novel pathway for recycling the 5'-deoxyadenosine moiety of SAM in M. jannaschii. These enzymes may also enable biosynthesis of 6-deoxy-5-ketofructose 1-phosphate (DKFP), an essential intermediate in aromatic amino acid biosynthesis. Finally, we utilized a homocysteine auxotrophic strain of Methanosarcina acetivorans Δma1821-22Δoahs (HcyAux) to identify potential AnMSP intermediates in vivo. Growth recovery experiments of the M. acetivorans HcyAux were performed with known and proposed intermediates for the AnMSP. Only one metabolite, 2-keto-(4-methylthio)butyric acid, rescued growth of M. acetivorans HcyAux in the absence of homocysteine. This observation may indicate that AnMSP pathways substantially differ among methanogens from phylogenetically divergent genera.

Plasmodium falciparum invasion and intraerythrocytic development are impaired by 2′, 3′-dialdehyde adenosine

Purine nucleotide synthesis in protozoa takes place exclusively via the purine salvage pathway and S-adenosyl-l-homocysteine hydrolase (SAHH) is an important enzyme in the Plasmodium salvage pathway which is not present in erythrocytes. Here, we describe the antimalarial effect of 2′3′-dialdehyde adenosine or oxidized adenosine (oADO), inhibitor of SAHH, on in vitro infection of human erythrocytes by P. falciparum. Treatment of infected erythrocytes with oADO inhibits parasite development and reinvasion of new cells. Erythrocytes pre-treated with oADO have a reduced susceptibility to invasion. Our results suggest that oADO interferes with one or more parasitic enzymes of the purine salvage pathway.

L. donovani XPRT: Molecular characterization and evaluation of inhibitors

Among all PRT enzymes of purine salvage pathway in Leishmania, XPRT (Xanthine phosphoribosyl transferase) is unique in its substrate specificity and their non-existence in human. It is an interesting protein not only for drug designing but also to understand the molecular determinants of its substrate specificity. Analysis of the 3D model of L. donovani XPRT (Ld-XPRT) revealed that Ile 209, Glu 215 and Tyr 208 may be responsible for the altered substrate specificity of Ld-XPRT. Comparisons with it's nearest homologue in humans, revealed significant differences between the two. A 28 residue long unique motif was identified in Ld-XPRT, which showed highest fluctuation upon substrate binding during MD simulations. In kinetic analysis, Ld-XPRT could phosphoribosylate xanthine, hypoxanthine and guanine with Km values of 7.27, 8.13, 8.48μM and kcat values of 2.24, 1.82, 1.19min−1 respectively. Out of 159 compounds from docking studies, six compounds were characterized further by fluorescence spectroscopy, CD spectroscopy and enzyme inhibition studies. Fluorescence quenching experiment was performed to study the binding of inhibitors with Ld-XPRT and dissociation constants were calculated. Four compounds are bi-substrate analogues and show competitive inhibition with both the substrates (Xanthine and PRPP) of Ld-XPRT. The CD spectral analysis revealed that the binding of inhibitors to Ld-XPRT induce change in its tertiary structure, where as its secondary structure pattern remains unchanged. Two Ld-XPRT inhibitors (dGDP and cGMP), which also have ability to inhibit Leishmanial HGPRT, are predicted as potential drug candidates as it can inhibit both the important enzymes of the purine salvage pathway.

Selective and membrane-permeable small molecule inhibitors of nicotinamide N-methyltransferase reverse high fat diet-induced obesity in mice

There is a critical need for new mechanism-of-action drugs that reduce the burden of obesity and associated chronic metabolic comorbidities. A potentially novel target to treat obesity and type 2 diabetes is nicotinamide-N-methyltransferase (NNMT), a cytosolic enzyme with newly identified roles in cellular metabolism and energy homeostasis. To validate NNMT as an anti-obesity drug target, we investigated the permeability, selectivity, mechanistic, and physiological properties of a series of small molecule NNMT inhibitors. Membrane permeability of NNMT inhibitors was characterized using parallel artificial membrane permeability and Caco-2 cell assays. Selectivity was tested against structurally-related methyltransferases and nicotinamide adenine dinucleotide (NAD+) salvage pathway enzymes. Effects of NNMT inhibitors on lipogenesis and intracellular levels of metabolites, including NNMT reaction product 1-methylnicotianamide (1-MNA) were evaluated in cultured adipocytes. Effects of a potent NNMT inhibitor on obesity measures and plasma lipid were assessed in diet-induced obese mice fed a high-fat diet. Methylquinolinium scaffolds with primary amine substitutions displayed high permeability from passive and active transport across membranes. Importantly, methylquinolinium analogues displayed high selectivity, not inhibiting related SAM-dependent methyltransferases or enzymes in the NAD+ salvage pathway. NNMT inhibitors reduced intracellular 1-MNA, increased intracellular NAD+ and S-(5′-adenosyl)-l-methionine (SAM), and suppressed lipogenesis in adipocytes. Treatment of diet-induced obese mice systemically with a potent NNMT inhibitor significantly reduced body weight and white adipose mass, decreased adipocyte size, and lowered plasma total cholesterol levels. Notably, administration of NNMT inhibitors did not impact total food intake nor produce any observable adverse effects. These results support development of small molecule NNMT inhibitors as therapeutics to reverse diet-induced obesity and validate NNMT as a viable target to treat obesity and related metabolic conditions. Increased flux of key cellular energy regulators, including NAD+ and SAM, may potentially define the therapeutic mechanism-of-action of NNMT inhibitors.

Elevated Expression of Hypoxanthine Guanine Phosphoribosyltransferase within Malignant Tissue

Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) is a housekeeping enzyme involved in the purine synthesis of guanine and inosine in the salvage pathway. While other salvage pathway enzymes, such as TK1, have been established as biomarkers for both cancer cell proliferation and cancer development, little research been done to evaluate whether HPRT has the same potential as a cancer biomarker. We designed this study to determine if HPRT has value as an identifier of malignancy within the most common types of cancer. We utilized histological samples from lung, colon, prostate, and breast cancer with additional normal tissue to evaluate whether there was any elevation of HPRT within malignant samples. In addition, we also assessed general HPRT expression within patient’s samples from The Cancer Genome Atlas (TCGA) to confirm clinical relevance. We found that within a subset of patients, there was significant elevation of HPRT when compared to normal tissue controls. This elevation was seen in 33-55% of the malignant samples and appears to have no dependence on cancer stage. There were slight differences in staining patterns among all the organ types, but overall each organ displayed the same pattern of ‘HPRT high’ and ‘HPRT low’ populations within malignant samples. We found that in our TCGA samples, there was a similar elevation of HPRT that was significant when compared to normal controls. Overall, as an upregulated enzyme that does not directly correlate with stage, HPRT could become a valuable marker in the early diagnosis of a variety of solid tumors.

Abstract 1949: Salvage pathway enzyme HPRT as a molecular marker for Burkitt’s Lymphoma

Abstract The aim of this study is to investigate the potential of Hypoxanthine phosphoribosyltransferase (HPRT) as a surface biomarker and target for future immunotherapies in Burkitt’s B-cell Lymphoma. B-cell malignancies are the most common type of childhood cancer. Development of immunotherapies could improve current treatment. HPRT is a cytosolic transferase involved in nucleotide production via the purine salvage pathway. Altered expression of TK1, a salvage pathway enzyme, is an indicator of prognosis and diagnosis in multiple cancer types due to active proliferation of cells and the resulting elevated nucleotide demand. It has previously been reported that some salvage pathway enzymes are found on the surface of Burkitt’s Lymphoma cells. HPRT presence on the surface of this B-cell lymphoma subtype could provide a target for adoptive cell transfer and other immunotherapies. The potential surface presentation of HPRT was assessed using flow cytometry, scanning electron microscopy (SEM), and cytoplasmic staining on both healthy lymphocytes and Burkitt’s Lymphoma cells (Raji). Flow cytometry experiments with HPRT antibodies and fluorescent-labeled secondary antibodies show that Raji cells exhibit an 81.4% (p-value .0001) positive fluorescence shift when compared to IgG controls (1.5%). Healthy lymphocytes had a fluorescence shift of 2.38% (p-value .9787). The presence of HPRT on the surface of both Raji cells and healthy lymphocytes was further confirmed using gold-labeled antibodies. Utilizing a scanning electron microscope, the presence of the protein on the surface was evaluated and quantified via increases in gold weight percentage of the sample. When treated with antibodies against HPRT, there was a significant increase in gold binding along with an increase in gold weight percentage. These results suggest a direct relationship between HPRT and the surface of Burkitt’s lymphoma cells, indicating HPRT as a potential target for future immunotherapeutic treatment in Burkitt’s B-cell lymphoma pediatric patients. Citation Format: Michelle H. Townsend, John Ellis Lattin, Michael D. Anderson, Abigail Felsted, Edwin Velazquez, Evita Weagel, Richard Robison, Kim L. O'Neill. Salvage pathway enzyme HPRT as a molecular marker for Burkitt’s Lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1949. doi:10.1158/1538-7445.AM2017-1949

Abstract 1239: The expression of salvage pathway enzymes in non-small cell lung cancer cells

Abstract The aim of this study is to evaluate the expression of the salvage pathway enzymes DCK, APRT, and HPRT in lung cancer cells to determine if they could serve as biomarkers for lung cancer diagnosis and potential treatment. In both men and women, lung cancer is the most lethal cancer in the world, and accounts for more than 30% of cancer-related deaths. We chose to evaluate the salvage pathway enzymes due to an established relationship between the serum biomarker Thymidine Kinase 1 (TK1) and lung cancer. Two non-small cell lung carcinoma cell lines were utilized for this analysis (NCI-H460 and A549) along with cancer tissue and healthy tissue from 27 lung squamous carcinoma patients. The surface localization of these enzymes was determined utilizing flow cytometry, confocal microscopy, and scanning electron microscopy, while upregulation within tissue was assessed using immunohistochemistry (IHC). Throughout our investigation, we found no significant expression of DCK or APRT on the surface of non-small cell lung cancer cells, but found a significant presence of HPRT on the plasma membrane of both NCI-H460 and A549 cells. The average population florescence of cells treated with HPRT antibodies increased by 24.3% and 12.9% in NCI-H460 and A549 cells, respectively, in comparison to controls. To ensure expression was not attributed to cytoplasmic HPRT, confocal microscopy was performed to visualize HPRT binding on the plasma membrane. After staining NCI-H460 cells treated with both fluorescent antibodies and a membrane-specific dye, we observed direct overlap between HPRT and the membrane of the cancer cells. Additionally, gold conjugated antibodies were used to label and quantify the amount of HPRT on the cell surface using scanning electron microscopy and EDAX. Further confirming HPRT presence, the gold weight percentage of the sample increased significantly when NCI-H460 cells were exposed to HPRT antibody (p-value 0.012) in comparison to isotype controls. Finally, the general upregulation of the protein was observed in patient tissue samples, with approximately 44% of lung cancer tissues showing significant HPRT upregulation when compared to healthy tissue samples. This differential upregulation shows an altered HPRT expression within some patients, which may help explain the presence of this presumed cytosolic enzyme on the surface of lung cancer cells. These results strongly indicate a unique relationship between cancer cells and HPRT and suggest HPRT as a possible biomarker for the detection and treatment of non-small cell lung cancers. Citation Format: Michelle H. Townsend, Evita G. Weagel, Michael D. Anderson, Edwin Velazquez, Richard A. Robison, Kim L. O'Neill. The expression of salvage pathway enzymes in non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1239. doi:10.1158/1538-7445.AM2017-1239

Abstract 2149: Unusual expression of HPRT on the surface of the colorectal cancer cell lines HT29 and SW620

Abstract Colorectal cancer is one of the most common cancers in the world, affecting nearly 1.2 million people in the United States alone. The aim of this study is to investigate the salvage pathway enzyme HPRT as a possible biomarker in two colorectal cancer cell lines: HT29 and SW620. HPRT is a transferase in the purine salvage pathway that functions primarily by catalyzing the conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate. Because of its role in proliferation and cell cycle regulation, we hypothesized an increase in HPRT expression within cancer cells, which could potentially lead to presentation on the surface of the cell. HPRT surface localization was assessed utilizing confocal microscopy, flow cytometry, and scanning electron microscopy. These techniques allowed us to visualize HPRT on the plasma membrane and quantify expression. There was statistically significant expression of HPRT on the surface of both HT29 and SW620 cells with a 28% and 58% fluorescent shift in the population, respectively. Confocal microscopy images revealed overlap between cells stained with a membrane dye and anti-HPRT FITC antibody, which indicates a direct relationship between HPRT and the plasma membrane of SW620 cells. To visualize the location of HPRT on the plasma membrane, anti-HPRT antibodies were labelled with gold and protein presence was quantified using an electron microscope, which measured the gold elemental weight percentage of each sample. When exposed to anti-HPRT antibody, the gold weight percentage of the samples significantly increased with 12.4% gold in SW620 cells and 11.2% gold in HT29 cells, indicating significant HPRT surface presence. The observed localization of HPRT to the plasma membrane was variable between the two different cancer cell lines. SW620 cells, which are faster growing and more aggressive, had significantly higher HPRT presentation that HT29 cells. These data collectively suggest that HPRT may be a possible biomarker target for the identification and possible treatment of colorectal cancer cells. Citation Format: Evita G. Weagel, Michelle H. Townsend, Michael D. Anderson, Edwin J. Velazquez, K Scott Weber, Richard A. Robison, Kim L. O'Neill. Unusual expression of HPRT on the surface of the colorectal cancer cell lines HT29 and SW620 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2149. doi:10.1158/1538-7445.AM2017-2149

Abstract 4660: HPRT as a biomarker for tumor classification and its variability of expression in breast cancer

Abstract The purpose of this study is to investigate the differential expression of HRPT between various breast cancer tissues in comparison to healthy breast tissue in order to evaluate its potential use as a diagnostic tool. Breast cancer is the most common malignancy diagnosed in women and remains incurable in the metastatic setting requiring accurate and reliable tools for early diagnosis. We analyzed HPRT, a purine salvage pathway enzyme responsible for synthesizing 90% of the available GMP and IMP in cellular maintenance. Due to its critical role in maintaining necessary nucleotides for cell proliferation, we hypothesized an increase in the expression of HPRT in cancer. Tissues from 48 patients with breast carcinoma were stained using standard immunohistochemistry (IHC) techniques. We examined infiltrating ductal carcinoma, margin of carcinoma, hyperplasia, atypical hyperplasia, adenosis, collagen fibers tissues, and normal breast tissue in order to determine expression between various breast cancers. Briefly, tissues were treated with a polyclonal anti-HPRT antibody along with a GADPH positive control and a universal negative control. Tissues were incubated with an HRP-polymer conjugated antibody, and DAP substrate which reacts to antibody-antigen binding sites and results in a color change. Adenosis and hyperplasia are benign breast conditions and are early signs of breast carcinoma development. Hyperplasia tissues, which are in a state of rapid proliferation, showed significant upregulation of HPRT and adenosis tissues showed approximately 42% upregulation. This HPRT upregulation in pre-stage 1 tissues indicates that it may be used as a biomarker to detect early stages of breast cancer. Expression of HPRT was also found to be upregulated in 65% of infiltrating duct carcinoma showing high expression in active malignant areas. In marginal tissue located on the perimeter of primary tumors only 31% of tissues show upregulation of the protein. Because the levels of HPRT in marginal tissue is significantly lower than those found in primary tumors, HPRT may serve as a useful diagnostic tool when evaluating successful tumor removal. These data suggests HPRT as a useful diagnostic tool when identifying tissue with a high proliferation rate indicative of potential cancer development. Additionally, HPRT may also be used as a tool for pathologists to evaluate the success of tumor removal. Citation Format: Michelle H. Townsend, Abigail M. Felsted, Jacob Clarke, Richard A. Robison, Kim L. O'Neill. HPRT as a biomarker for tumor classification and its variability of expression in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4660. doi:10.1158/1538-7445.AM2017-4660

Increase in furfural tolerance by combinatorial overexpression of NAD salvage pathway enzymes in engineered isobutanol-producing E. coli

To reduce the furfural toxicity for biochemical production in E. coli, a new strategy was successfully applied by supplying NAD(P)H through the nicotine amide salvage pathway. To alleviate the toxicity, nicotinamide salvage pathway genes were overexpressed in recombinant, isobutanol-producing E. coli. Gene expression of pncB and nadE respectively showed increased tolerance to furfural among these pathways. The combined expression of pncB and nadE was the most effective in increasing the tolerance of the cells to toxic aldehydes. By comparing noxE- and fdh-harbouring strains, the form of NADH, rather than NAD+, was the major effector of furfural tolerance. Overall, this study is the application of the salvage pathway to isobutanol production in the presence of furfural, and this system seems to be applicable to alleviate furfural toxicity in the production of other biochemical.

Anticystogenic activity of a small molecule PAK4 inhibitor may be a novel treatment for autosomal dominant polycystic kidney disease

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common hereditary renal disease with no currently available targeted therapies. Based on the established connection between β-catenin signaling and renal ciliopathies, and on data from our and other laboratories showing striking similarities of this disease and cancer, we evaluated the use of an orally bioavailable small molecule, KPT-9274 (a dual inhibitor of the protein kinase PAK4 and nicotinamide phosphoribosyl transferase), for treatment of ADPKD. Treatment of PKD-derived cells withthis compound not only reduces PAK4 steady-state protein levels and regulates β-catenin signaling, but also inhibits nicotinamide phosphoribosyl transferase, the rate-limiting enzyme in a key NAD salvage pathway. KPT-9274 can attenuate cellular proliferation and induceapoptosis associated with a decrease in active (phosphorylated) PAK4 and β-catenin in several Pkd1-null murine cell lines, with a less pronounced effect on the corresponding phenotypically normal cells. Additionally, KPT-9274 shows inhibition of cystogenesis in an exvivo model of cyclic AMP-induced cystogenesis as well as in the early stage Pkd1flox/flox:Pkhd1-Cre mouse model, the latter showing confirmation of specific anti-proliferative, apoptotic, and on-target effects. NAD biosynthetic attenuation by KPT-9274, while critical for highly proliferative cancer cells, does not appear to be important in the slower growing cystic epithelial cells during cystogenesis. KPT-9274 was not toxic in our ADPKD animal model or in other cancer models. Thus, this small molecule inhibitor could be evaluated in a clinical trial as a viable therapy of ADPKD.