Multiple Cytoproliferative Effects of Elevated

Review Journal of Pharmaceutical Sciences and Pharmacology

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Multiple Cytoproliferative Effects of Elevated Pyrimidines are DNA-Synthesis-Independent and Include p53 Repression Robert L. Gipson 4735 Doe Street, Shingle Springs, California 95682, USA

Several types of cytoproliferative diseases, including cancers and autoimmune diseases, are associated with elevated cellular levels of pyrimidine nucleotides. This review assembles literature evidence supporting a thesis that elevated pyrimidine levels drive cytoproliferation largely through DNA-synthesis-independent mechanisms, which circumstance, it is proposed, inherently limits clinical efficacy of DNA-synthesis-targeting cytostatic drugs. Five hypothetical mechanisms, with supporting literature evidence, are presented: (1) Pyrimidine nucleotides released from ion channels auto/paracrinally and persistently activate the EGFR, PKC and ERK signaling pathway, upregulating cytokines and Cdk2; (2) One effect of ERK activation is reversal of allosteric control of de novo pyrimidine biosynthesis; thus pyrimidines’ auto/paracrine ERK activation may reflect positive autoregulation; (3) Elevated intracellular pyrimidine nucleotides preferentially upregulate tumorigenic genes; (4) Elevated pyrimidines promote aberrant glycosylation of transcription factors; and (5) These diverse 67.187.171.106 On: Tue,to19repression Jun 2018of20:19:58 pyrimidine effects contribute, by IP: different paths yet collectively, p53 or of its function. Also reviewed are Copyright: American Scientific Publishers alternate pyrimidine biosynthesis pathways relevant to disease. Based on these hypotheses, a clinical strategy for cytoDelivered by Ingenta proliferative disease is proposed that co-targets three cellular processes seen as “linchpins” of pyrimidines’ auto/paracrine effects, including targeting alternate pyrimidine biosynthesis pathways.

KEYWORDS: Pyrimidines, Pyrimidine Biosynthesis, UTP, UDP, CTP, Cancer, Autoimmune Disease, Rheumatoid Arthritis, Lupus Erythematosus, Multiple Sclerosis, p53, DNA Synthesis, VRAC, P2Y, Cyclin Dependent Kinase, CDK, EGFR, Cytokines.

CONTENTS Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Past Clinical Antipyrimidine Strategies . . . . . . . . . . . . . . . Pyrimidine-Mediated Proliferation is Largely DNA-Synthesis-Independent . . . . . . . . . . . . . . . . . . . . Five Hypothetical Mechanisms, Unrelated to DNA Synthesis, via Which Elevated Pyrimidine Levels Mediate Cytoproliferation and Pathology . . . . . . . . . . . . . . . . . . Elevated Pyrimidines Auto/Paracrinally Activate the EGFR, PKC and ERK Signaling Pathway, Upregulating Cytokines and Cdk2 . . . . . . . . . . . . . Pericellular Pyrimidines May Autocrinally Reverse Allosteric Control of De Novo Pyrimidine Biosynthesis . . . . . . . . . . . . . . . . . . . . Elevated Pyrimidines Preferentially Upregulate Tumorigenic Genes . . . . . . . . . . . . . . . Elevated Pyrimidine Levels Promote Elevated Glycosylation of Transcription Factors . . . . . . . . . .

Email: [email protected] Received: 14 August 2013 Accepted: 30 September 2013

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Elevated Pyrimidine Nucleotide Levels Induce of P53 or Its Function by Multiple Paths . . Alternate Pyrimidine Biosynthesis Paths of Clinical Relevance . . . . . . . . . . . . . . . . . . . Possible Implications of Hypotheses for Clinical Strategies . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . References and Notes . . . . . . . . . . . . . . . . . . .

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INTRODUCTION In mammalian cells, diverse factors such as mitogens, carcinogens, estrogen and viruses can induce elevation of de novo pyrimidine biosynthesis, thereby elevating intracellular pyrimidine concentrations. Aberrant pyrimidine elevations have been associated with several types of cytoproliferative disease including cancers and autoimmune diseases (Bökkerink, 1986; de Korte, 1987; Huang, 2002; Jackson, 1980; Weber, 1983; Huang, 2004; Löffler 2333-3715/2014/1/004/022

doi:10.1166/jpsp.2014.1003

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Multiple Cytoproliferative Effects of Elevated Pyrimidines are DNA-Synthesis-Independent and Include p53 Repression

2005; Wahl 1979; Cysyk, 1995; Karle, 1984); uracil has been reported as a potent promoter of urinary bladder carcinogenesis in rats (Shirai, 1986). This and other evidence suggest the possibility of a causative role for elevated pyrimidines in pathological cytoproliferation. The exact mechanism(s) by which elevated pyrimidines mediate proliferation have not been conclusively defined, although elevation of DNA synthesis and/or RNA synthesis are commonly proposed mechanisms. Similarly, cytostatic effects of pyrimidine antagonist (antipyrimidine) drugs have been widely attributed to “pyrimidine starvation,” i.e., to depriving cells of pyrimidines necessary for biosynthetic roles, such as necessary nucleic acid or phospholipid synthesis. In contrast, the present review assembles evidence pointing to such drugs’ cytostatic effects being due to diminution of cellular events that are distinct from biosynthetic processes—specifically, to mitogenic and inflammatory events induced by aberrantly elevated pyrimidine levels. This insight suggests a reassessment of clinical strategies for cytoproliferative disease.

(5-FU), which has been used since the 1950s, and is still used for certain cancer types. Early work with N -(phosphonacetyl)-L-aspartic acid (PALA), acivicin, 6-azauracil and pyrazofurin, despite preclinical promise, showed poor clinical success, primarily due to cells’ acquired resistance or to dose-limiting toxicity. Acivicin, for example, exhibited central nervous system (CNS) toxicity (Earhart, 1983). Various modes of acquired resistance have included cells circumventing inhibition of a particular de novo synthesis pathway enzyme either by gene amplification of that enzyme (Wahl, 1979; Löffler, 2005), or by compensatorily increasing pyrimidine salvage (Weber, 1983; Karle, 1984; Natsumeda, 1984; Cysyk, 1995). Preclinical work with cyclopentyl uracil, a pyrimidine salvage inhibitor, found it to be ineffective as a solo agent, yet in combination with a de novo synthesis inhibitor (PALA) was lethal to rodents (Cyzyk, 1995). A combination of PALA and acivicin was shown to be synergistic in depleting pyrimidine levels (Ahmed, 1984), yet such combination has not been clinically developed (Löffler, 2005). Of solo agents, DHOD inhibitors have demonstrated considerable clinical promise, for a spectrum of cytoproliferative disease types ranging from various cancers PAST CLINICAL ANTIPYRIMIDINE (Loh, 1981; Löffler, 1998; Knecht, 2000a; Knecht, 2000b; STRATEGIES Löffler, 2005; Boyd, 2005; Baumann, 2009; O’Donnell, Antipyrimidine pharmacological strategies explored over 2012; Khutornenko, 2010; Zon, 2011; Martz, 2011; the last 60 years have included a variety of drugs that inhibit 2011), pyrimidine biosynthesis. Such IP: inhibitors have included 67.187.171.106 On: Tue,White, 19 Jun 2018autoimmune 20:19:58 diseases including rheumatoid and psoriatic arthritis, Copyright: American Scientific Publishers (Rückemann, 1998; Strand, 1999; pyrimidine analog antimetabolites and non-pyrimidineDelivered Ingenta 2000; Kremer, 2004; Maddison, 2005), sysanalogs, and have typically targeted specific enzymes byHerrmann, temic lupus erythematosus (Kulkarni, 2010) and multiple of one of the cell’s two main pyrimidine biosynthesis sclerosis (MS) (Linker, 2008; Borazanci, 2009; O’Conner, pathways: de novo pyrimidine biosynthesis, or the pyrim2010; Brinkmann, 2010). idine salvage pathway. As a partial review, Table I lists While the DHOD step is not the sole rate-limiting enzymajor enzymes of those pathways that have been preclinmatic step in de novo pyrimidine biosynthesis, it differs ically or clinically targeted, and the respective inhibitor from other steps in that both precursor (dihydroorotate) drugs used. The first three enzymes listed are components and product (orotate) must diffuse across the mitochondrial of the cytosolic trifunctional enzyme complex, carmembrane (Breedfeld, 2000; Löffler, 2005). That differbamoyl phosphate synthetase-aspartate transcarbamoylaseence may partly underlie it’s efficacy as clinical target. dihydroorotase (CAD). The fourth, dihydroorotate Inhibition of cytidine triphosphate (CTP) synthesis, such dehydrogenase (DHOD), is mitochondrially located. as by cyclopentyl cytosine (CPEC), has also shown conAmong the earliest antipyrimidine drugs explored were siderable clinical promise, either alone or in combination pyrimidine analog antimetabolites such as 5-fluorouracil

Robert L. Gipson graduated with a B.S. in Biological Sciences from Louisiana State University in Shreveport in 1978. In 1980 he published primary research in the field of environmental protozoology, and has worked in the environmental science field for 35 years, the last 25 years as an Environmental Scientist for the State of California. For the past 20 years, literature research in medicinal phytochemicals, naphthoquinone biochemistry and pyrimidine biology have been personal avocations. The present review, first in a series of articles, stems from those avocations. The author may be reached at [email protected].

J. Pharm. Sci. Pharmacol. 1, 4–25, 2014

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Multiple Cytoproliferative Effects of Elevated Pyrimidines are DNA-Synthesis-Independent and Include p53 Repression Table I.

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Example enzyme targets of past antipyrimidine strategies.

Pathway De novo pyrimidine biosynthesis pathway

Enzyme Carbamoyl phosphate synthetase 2

Dihydroorotate dehydrogenase (DHOD)

Forms carbamoyl phosphate Forms N-carbamyl-L-aspartate Converts carbamoyl aspartic acid to 4,5-dihydroorotic acid Converts dihydroorotate (DHO) to orotate

Uridine monophosphate synthetase (UMPS) CTP synthetase

Converts uridine to uridine monophosphate (UMP) Converts UTP to CTP.

Thymidylate synthetase Uridine kinase

Forms thymidine. Converts uridine to UMP

Aspartate transcarbamylase Dihydroorotase (DHOase)

Pyrimidine salvage

Function

Inhibitor(s) Acivicin N-(phosphonacetyl)-L-aspartic acid (PALA) 5-aminoorotic acid

Dichloroallyl lawsone; Leflunomide; Brequinar; Redoxal. 6-azauracil; pyrazofurin. Acivicin; cyclopentyl cytosine (CPEC) 5-fluorouracil (5-FU) Cyclopentenyl uracil (CPEU)

with other agents. Huang and colleagues reported that drugs: Namely, the cellular events primarily driving upon depletion of UTP and CTP by A77 1726 (the active cytoproliferation are unrelated to DNA synthesis, although metabolite of leflunomide), addition of cytidine alone are nonetheless pyrimidine-driven. restored proliferation (Huang, 2002). Huang et al. also referenced past studies of a variety of malignancies wherein PYRIMIDINE-MEDIATED PROLIFERATION IS increased concentrations of ribonucleotides were found, LARGELY DNA-SYNTHESIS-INDEPENDENT with the largest increase being cytidine ribonucleotides Inhibition of DNA synthesis arrests proliferation. It does (Jackson, 1980; Weber, 1983; Huang, 2002) and noted follow, however, that elevation of DNA other studies in promyelocytic HL-60 cells where CPEC IP: 67.187.171.106 On: Tue,not 19 necessarily Jun 2018 20:19:58 synthesis is necessarily Copyright:accompaAmerican Scientific Publishers the driver of pathological proliferainduced growth inhibition and differentiation, Surely, it can be a driver in some cases, such as Grem Delivered Ingenta nied by decline in CTP levels but not in UTP, ATP, or GTP bytion. and coworkers’ finding that PALA-induced growth inhibi(Huang, 2004; Glazer, 1986). tion of L1210 cells was reversed by addition of 10 M Pyrimidine analog antimetabolites such as CPEC, 5dCyd and dThd (Grem, 1988). Consider, however, findings FU and ara-C ultimately target DNA synthesis, thus in by Bennet and coworkers that DHOD inhibitor dichloroalthat sense can be roughly grouped with other DNAlyl lawsone (DCL) arrested proliferation of L1210 cells synthesis-targeting cytostatic drugs such as antifolates without any effect on DNA or protein synthesis, the effects (e.g., methotrexate), DNA alkylators (e.g., nitrosurea), being reversed by uridine (Bennet, 1979). Huang and DNA crosslinkers (e.g., cisplatin, mitomycin) and topoisocolleagues, in separate studies in 2002 and 2004, found merase inhibitors. The pharmacologic basis for targeting that DHOD inhibitor A77 1726, arrested proliferation and DNA synthesis is that rapidly dividing cells depend more induced differentiation in myeloid leukemia K562 cells heavily upon DNA synthesis than normal cells. Clinical by depleting UTP and CTP, but that deoxyribonucleotide efficacies of DNA-targeting drugs are limited, however, (dCTP, dTTP) formation appeared to not be rate-limiting by non-target cells’ need for functional DNA. A fur(Huang, 2002; Huang, 2004). ther limitation exists for pyrimidine analogs such as 5-FU Huang’s 2004 study also reported that in certain and ara-C which require enzymatic bioactivation: their leukemic cell lines, under conditions of pyrimidine depleefficacy is dependent on level of genetic expression of tion by either leflunomide or CPEC, inhibition of the p38 the bioactivating enzymes, and of enzymes that confer kinase pathway—surprisingly—restored proliferation and resistance to the drugs (Maring, 2005). Similarly, certain de-differentiation; i.e., that differentiation and cell cycle mutations in CTP synthetase were shown to confer resisarrest caused by pyrimidine depletion was greatly suptance to CPEC in Chinese hamster ovary cells (Chang, pressed by inhibiting p38 (Huang, 2004). That finding 2008; Whelan 1993) and yeast (Ostrander, 1998; Chang, could be interpreted, I propose, as further evidence—albeit 2008), and the mutation sites of the encoding genes are indirect—that pyrimidine-dependent proliferation and dehighly conserved in human CTP synthetase (Weber, 1983; differentiation are not driven through fulfilling cells’ Yamauchi, 1990). normal metabolic needs for pyrimidines (e.g., for synthesis In addition to the above limitations, the present of DNA, RNA or phospholipids). I further suggest the review proposes existence of an even greater—inherent— limitation to clinical efficacy of DNA-synthesis-targeting possibility that the observed rescue of proliferation by 6


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p38 inhibition may have resulted from increased actiThe exit mechanism can be vesicular in exocrine cells; in a variety of other cell types it can occur via volvation of MEK 1/2 and ERK 1/2, given that other ume regulated anion channels (VRACs) (Davis, 2006; studies have shown that, due to phosphatase-mediated Scrivens, 2006; Wolk, 2008). Intracellular transporters of crosstalk between the ERK and p38 pathways, p38 inhibinucleotide sugars have been shown to facilitate their celtion increases MEK 1/2 and ERK 1/2 activity. This phelular release (Sesma, 2009). Davis, et al. observed that nomenon was reported in corneal epithelial cells (Wang, inhibition of de novo pyrimidine synthesis by leflunomide 2006) and cardiac myocytes (Liu, 2004). Relevant to that prevents VRAC release of UTP, and the authors comphenomenon, described further below is evidence that elemented that nucleotide concentrations at epithelial surfaces vated pyrimidines autocrinally activate the ERK pathway. may be 10-fold higher than extracellular measurements Therefore, it is possible that, in Huang’s 2004 study, p38 indicate, and further proposed that ectonucleotidases and inhibition—ironically—fulfilled the ERK-inducing role of ectonucleoside diphosphokinases may interconvert ATP the missing (depleted) pyrimidines. Moreover, pyrimidines and UTP to locally modulate cell-surface-ligand profiles nominally needed for proliferation may have come from (Davis, 2006); occurrence of this interconversion was also the pyrimidine salvage pathway, which in many cell types indicated by Scrivens et al. (Scrivens, 2006). Importantly, waxes in response to de novo synthesis inhibition. Huang Davis et al. also observed that volume of UTP release also found that p38 gene silencing did not completely is proportional to its intracellular concentration (Davis, suppress CPEC-induced erythroid differentiation, and pro2006), which is consistent with findings of Lazarowski and posed that this may have resulted from incomplete gene Boucher (Lazarowski, 2003). Evidence indicates that CTP silencing (Huang, 2004). In contrast, I suggest that the also exits VRACs (Hisadome, 2002). observed incompleteness of suppression may reflect the Uridine nucleotides and uridine nucleotide sugars are limit to which p38 silencing increases MEK 1/2 and ERK potent agonists of cell surface receptors of the P2Y fam1/2 activation. ily (Lazarowski, 2003; Scrivens, 2006). It is equally well The above-described studies are proposed as evidence of established that activation of P2Y receptors have a wide pyrimidine-dependent proliferation that is DNA-synthesisspectrum of downstream effects (Pavenstädt 1992; Boyer, independent. In such cases, presumably, any DNA syn2000; Teixeira, 2001; Lazarowski, 2001; Gounaris, 2002; thesis that does occur does so only to meet the demand Hou, 2002; Pham, 2003; Schäfer, 2003; Chen, 2004; (for DNA synthesis) created by some separate, unidenti2005;20:19:58 Wihlborg, 2006; Scrivens, 2006; Ando, IP:That 67.187.171.106 On: Tue,Communi, 19 Jun 2018 fied driving force of proliferation. model, it is here Copyright: American Publishers 2010), including activation of the extracellular signalargued, substantially defines the therapeutic limitations of Scientific Delivered byregulated Ingenta kinase (ERK) cascade. The ERK cascade sequenDNA-synthesis-targeting cytostatic drugs. For methotrextially activates Ras GTP-binding proteins, c-Raf, MEK, and ate, which actually elevates pyrimidine levels (BuesaERK1/2, and activated ERKs activate many transcription Perez, 1980; Kaminskas, 1982), that paradigm is even factors such as c-myc, Elk1, and c-fos (Marshall, 1995; more relevant. Lewis, 1998; Yoon, 2006; Nishimoto, 2006; Chan, 2010) Described below, with supporting literature evidence, and upregulate pro-inflammatory cytokines (Carter, 1999; are hypotheses for five DNA-synthesis-independent mechKurosawa, 2000; Maddahi, 2011; Singer, 2011). anisms (i.e., pathways) by which cytoproliferative and There is direct evidence of pyrimidine induction, via inflammatory effects of elevated pyrimidine levels are P2Y receptors, of pro-inflammatory cytokines (Warny, mediated. These hypotheses are based solely on literature 2001; Cox, 2005; Kukulski, 2007). Davis, Lazarowski and evidence and deduction. It is possible that all of the procoworkers showed that either DHOD inhibitor leflunoposed mechanisms exist. Their descriptions are followed mide or VRAC inhibitors such as Verapamil inhibited proby discussion of alternate pyrimidine biosynthesis pathinflammatory cytokines IFN-, IL-1, KC, and TNF- ways of clinical relevance. This review concludes with in alveolar cells infected with respiratory syncytial virus discussion of possible implications of these concepts for (Davis, 2006). Wolk and coworkers showed that either clinical strategy. leflunomide active metabolite A77-1726, VRAC inhibitor fluoxetine, or P2Y receptor antagonists were effective in FIVE HYPOTHETICAL MECHANISMS, blocking UTP’s induction of pro-inflammatory cytokines, UNRELATED TO DNA SYNTHESIS, VIA and confirmed that A77-1726’ effects were mediated via WHICH ELEVATED PYRIMIDINE LEVELS inhibition of de novo pyrimidine biosynthesis rather than MEDIATE CYTOPROLIFERATION inhibition of tyrosine kinases (Wolk, 2008). Nickcevich AND PATHOLOGY and coworkers showed leflunomide to directly inhibit both Elevated Pyrimidines Auto/Paracrinally Activate antigen and IL-2 stimulated tyrosine kinase activity in T the EGFR, PKC and ERK Signaling Pathway, cells, notably without effect on protein kinase C (PKC) Upregulating Cytokines and Cdk2 (Nickcevich, 1993). A77 1726 was also shown to sigPyrimidine nucleotides and pyrimidine sugars can exit the nificantly reverse estrogen-induced upregulation of proincell to exert potent paracrine or auto/paracrine effects. flammatory cytokine IL-6 (Montagna, 2010). Borazanci J. Pharm. Sci. Pharmacol. 1, 4–25, 2014

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and coworkers are currently investigating use of the antiERK 1/2, it is reasonable to hypothesize that leflunomide’s decrease of Cdk2 was mediated by inhibition of de novo inflammatory Teriflunomide, a leflunomide derivative, for pyrimidine biosynthesis. the treatment of MS (Borazanci, 2009).a The proposed auto/paracrine path of pyrimidine effects Mounting evidence identifies UTP as a physiologic actidescribed above are depicted schematically in Figure 1. vator of the epidermal growth factor receptor (EGFR). ERK 1/2 also elevates rRNA synthesis (discussed furRatchford and coworkers showed in human salivary gland ther below) and may factor in microtubule disruption, cells that extracellular UTP, via P2Y2 receptors, dosewhich is associated with several cancers and neuropatholodependently induced phosphorylation of ERK1/2 via both gies such as Huntingtons. 9-cis-retinoic acid was shown PKC and by EGFR, with the PKC pathway being rapid to inhibit microtubule disruption via compartmentalized and the EGFR pathway slower (Ratchford, 2010). The inactivation of ERK 1/2 in the perinuclear region (Asrih, reported PKC activation was likely via phospholipase C 2011). (PLC), which UTP has been shown to activate via P2Y All of the above-described ERK effects may be caused receptors in HL-60 cells (Kim, 2002) and frog semicircuautocrinally by elevated pyrimidine levels. This suggests lar canal ampulla (Teixeira, 2001). Boucher and coworkers the possibility that clinically reducing pericellular pyrimalso demonstrated UTP activation of EGFR in the conidine nucleotide concentrations may be as effective as text of wound healing, and commented on the role of ERKand/or EGFR-targeting drugs. crosstalk between P2Y receptors and EGFR in cell migration and wound closure (Boucher, 2011). Liu et al. showed Pericellular Pyrimidines May Autocrinally UTP to increase the colocalization of P2Y and EGFR in Reverse Allosteric Control of De Novo the plasma membrane, in an Src-dependent manner (Liu, Pyrimidine Biosynthesis 2004). One of many effects of ERK 1/2 is reversal of the normal In contrast, Ratchford showed UTP’s EGFR activation allosteric control of de novo pyrimidine biosynthesis. That to be Src independent yet dependent on metalloprotease control is normally mediated by UTP’s deactivation of caractivity and neuregulin 1 expression (Ratchford, 2010). bamoyl phosphate synthetase 2 (CPS 2) in the cytosolic This is supported by Baker, et al. who showed UTP inducCAD multienzyme complex. As first discovered by Carrey tion of vascular cell adhesion molecule-1 (VCAM-1) to and coworkers (Carrey, 1985) and further elucidated by be independent of Src tyrosine kinases yet dependent on IP: 67.187.171.106 On: Tue, 19 Jun 20181992; 20:19:58 others (Shaw, Irvine, 1997; Sigoillot, 2002), UTP’s ADAM17 of the adamalysin family of metalloproteases Copyright: American Scientific Publishers normal deactivation of CPS 2 is nullified when ERK 1/2 (ADAMs). Baker further proposed that UTP stimulates Delivered byphosphorylates Ingenta the Thr456 residue of CPS 2, with two TNF release via activation of metalloproteases (Baker, results: It reduces CPS 2’s sensitivity to allosteric deac2008). ADAMs have been implicated in G protein coupled tivator UTP, and increases CPS 2’s sensitivity to phosreceptor (GPCR)-mediated tumor cell migration (Schäfer, phoribosyl pyrophosphate (PRPP), an allosteric activator. 2004; Frolov, 2007). Ratchford also demonstrated that Moreover, depending upon relative concentrations of UTP pericellular UTP induces formation of EGFR/ErbB3 hetand PRPP, UTP can transform from being a deactivator to erodimers, which have elsewhere been associated with a weak activator of CPS 2, meaning that allostery is not pancreatic cancer (Ratchford, 2010, Frolov, 2007), breast merely nullified, but reversed. cancer (Yang, 2006; Ratchford, 2010) and melanoma All enzymes in the de novo pyrimidine biosynthesis (Ueno, 2008; Ratchford, 2010). The P2Y2 receptor has pathway are subject to allosteric control. For example, both separately been shown to interact with v3/5 integrins ATCase and CTP synthetase are subject to allosteric conthat regulate ATP- and UTP-induced cell chemokinesis and trol by CTP. Notwithstanding, allosteric control at CPS 2 chemotaxis (Kim, 2002; Wang, 2005). Pericellular UTP is particularly rate-limiting for de novo pyrimidine biosynalso potently induces hematopoietic stem cell migration, thesis; therefore ERK 1/2’s reversal of allostery at this parthe effect being blocked by pertussis toxin (Rossi, 2007). ticular step appears to be pivotal in deregulating de novo Evidence also indicates that UTP upregulates cyclinsynthesis. For example, ERK 1/2 phosphorylation of CPS dependent kinase 2 (Cdk2). Siemasko and coworkers 2 factors not only in pathologic cytoproliferation, but also showed DHOD inhibitor leflunomide to decrease Cdk2 in the growth phase of the normal cell cycle. In that levels in B cells, targeting two different stages in cell cycle cycle, the need for elevated de novo pyrimidine biosynthetransition: from G1 to S phase and from S phase to G2/M sis is transient; hence ERK 1/2 activity—and phosphoryphase; and that these effects were reversed by exogenous lation of CPS 2—is transient. Then, as cells emerge from uridine (Siemasko, 1996). Given preceding evidence of the growth phase, Thr456 is dephosphorylated by a phosVRAC-released pyrimidines activating the ERK pathway, phatase, and Ser1406 of CPS 2 is reciprocally phosphoryand the fact that Cdk2 induction is a downstream effect of lated by protein kinase A (PKA), which restores allosteric control by UTP. By contrast, if ERK 1/2 activation is pera UPD-sugars can promote inflammation via a second path: They are sistent (as in pathology), Thr456 is persistently phosphosubstrates for glycosylation of adhesion molecules, discussed in a later rylated, and evidence suggests that, due to proximity of section of this review. 8


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Figure 1. Proposed auto/paracrine pathway by which pyrimidines activate mitogenic kinases. (1) Newly synthesized UTP, UDP, UDP-glucose, UTP glucose and CTP exit the cell via volume regulated ion channels (VRACs); (2) These pyrimidine specia except CTP potently activate various P2Y-family receptors, activating downstream mitogenic kinases. UTP in particular induces activation of ERK 1/2 via two paths: via PLC and PKC, and via EGFR and metalloproteases; UTP also induces EGFR/ErbB3 heterodimer formation; (3) In the nucleus, ERKIP: 1/267.187.171.106 activates several On: transcription and 20:19:58 upregulates proinflammatory cytokines and Tue, 19 factors Jun 2018 Cdk2. Copyright: American Scientific Publishers

Delivered by Ingenta Thr456 to Ser1406, phosphorylation of Thr456 interferes CAD activity (Lindsey-Boltz, 2004), each of which phenomena could similarly be viewed as pyrimidine positive with PKA’s ability to reciprocally phosphorylate Ser1406 autoregulation, given above-referenced evidence that UTP to reinstate allosteric control (Sigoillot, 2007). An example can autocrinally upregulate PKC and Rad9. of this allostery abrogation—with, I suggest, possible relevance to breast cancer—is that CPS 2 in lactating bovine mammary tissue has been shown to be inhibited less by Elevated Pyrimidines Preferentially UTP than in non-lactating tissue (Vekich, 1973; Robinson, Upregulate Tumorigenic Genes 1974), whereas uridine nucleotide concentrations increase White and coworkers, working with Zebrafish embryos, dramatically in lactating tissue (Denamur, 1959; Vekich, found that depletion of pyrimidine nucleotides, via DHOD 1973). inhibitors such as leflunomide, downregulates genes assoGiven forgoing evidence that VRAC-released pyrimidine ciated with melanoma tumorigenesis; and that the downnucleotides and sugars are capable of mediating persisregulation is mediated by transcription regulation at the tent, auto/paracrine ERK 1/2 activation, it is reasonable to level of elongation rather than initiation (White, 2011). hypothesize that reversal of pyrimidine synthesis’ allosteric Elongation factors have been separately associated with control is one result of that activation. If so, the resultboth neural crest development (Nguyen, 2010; White, ing further elevation of pyrimidine levels could conceivably 2011) and cancer growth (Mueller, 2009; White, 2011), initiate a self-sustaining ‘pyrimidine auto/paracrine loop’ and other workers showed that pyrimidine nucleotide which, by perpetuating VRAC-release of pyrimidines and depletion inhibits gene elongation generally (Wada, 1998; their persistent activation of the ERK pathway, would conWhite, 2011). Notably, however, White et al. showed that tinuously elevate pyrimidine levels, and may do so indepyrimidine depletion preferentially affected developmenpendent of whatever external stimulus (virus, estrogen or tal genes involved in tumor formation while having little carcinogen) may have initially elevated pyrimidine levels. effect on non-tumorigenic developmental genes such as Such a cycle—if it exists—could be considered pyrimidine genes for blood and notochord development. Genes most positive autoregulation (Fig. 2). In that regard, it is furaffected by pyrimidine depletion were neural crest markther notable that CAD is upregulated by PKC (Sigoillot, ers, especially BRAF (V600E), which encodes a mutant of BRAF (Graves, 2000; White, 2011), Myc target genes 2007) and that the Rad9 checkpoint protein stimulates J. Pharm. Sci. Pharmacol. 1, 4–25, 2014

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Figure 2. Hypothetical auto/paracrine loop cyclically elevating UTP concentrations: (1) Increasing intracellular UTP exits VRACs, IP: 67.187.171.106 On: Tue, 19 Jun 2018 20:19:58 causing increasing pericellular UTP concentrations; (2) Increasing, persistent UTP activation of P2Y receptors and thereby of Copyright: American Scientific Publishers ERK 1/2; (3) Persistent ERK 1/2 phosphorylation of CPS 2, whichby persistently Delivered Ingenta thwarts UTP’s allosteric control of CPS 2 activity.

and NOTCH pathway genes. White et al. further indicated that this paradigm applied to multiple species. These important findings of White et al. suggest the possibility that frequency of elongation of tumorigenic genes may be directly responsive to pyrimidine nucleotide concentrations. That possibility is the foundation of the following hypothesis: That aberrantly elevated pyrimidine nucleotide concentrations could commensurately increase elongation frequency of such genes, thereby elevating their transcription. Elevated Pyrimidine Levels Promote Elevated Glycosylation of Transcription Factors Protein glycosylation is underpinned by pyrimidines in two ways: UDP-sugars are its anabolic substrates, and CTP is the cofactor, activating dolichol kinase (the only CTPdependent enzyme). While glycosylation of cytosolic and nuclear proteins is a normal posttranslational modification, aberrant glycosylation is a well-established marker of carcinogenesis (Varki, 2009; Bachmaier, 2009) that follows initial oncogenic transformation and factors in invasion and metastasis. Metastatic human prostatic adenocarcinoma cells have higher levels of uridine diphosphosugars (Kurhanewicz, 1993). In O-GlcNAcylation, a form of glycosylation of nuclear and cytoplasmic proteins that is 10

catalyzed by O-GlcNAc transferase (OGT), the substrate sugar is UDP-acetylglucosamine (UDP-GlcNAc), which is O-linked to serine or threonine residues. While elevated O-GlcNAcylation can result from upregulation of OGT, it can also result solely from elevated levels of substrate UDP-GlcNAc (Yang, 2001; Gewinner, 2004; Love, 2005), and there is evidence that elevation of UTP or uridine levels concomitantly elevates UDP-sugar levels. Labeling studies in rat hepatocytes showed that UTP from the de novo biosynthesis pathway is preferentially channeled to UDP-sugar synthesis (Rijcken, 1993) and increased plasma uridine has been shown to increase UDP-glucose consumption via glycogenesis (Yamamoto, 2011). Similarly, exogenous uridine was shown to increase UDP-N acetylhexosamine levels in small cell lung cancer cells (Pederson, 1992). Significantly, Brequinar, an inhibitor of DHOD (i.e., of de novo pyrimidine biosynthesis), was shown to block glycosylation of adhesion molecules (Allison, 1993). UDP-glucose is precursor to hyaluronan (HA), which factors in normal signal transduction but also in proliferation, cancer growth and metastasis. HA synthesis activity has been shown to be directly responsive to nucleotide sugar levels (Yang, 2001; Love, 2005). Besides cancer, aberrant O-GlcNAcylation factors in pathologies such as neurodegeneration, diabetes and J. Pharm. Sci. Pharmacol. 1, 4–25, 2014

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wherein ribonucleotide depletion is merely a signal for atherosclerosis via modification of transcription factors p53 activation. Other studies suggest the more active role: such as specificity protein 1 (SP1) and transforming Khutornenkoa et al. reported that exogenous uridine and growth factor-alpha (TGF-) (Jackson, 1988; Reason, orotate (but not precursor dihydroorotate) repressed p53 1992; Zachara, 1992; Kelly, 1993; Chou, 1995; Hanover, (Khutornenkoa, 2010).b Zhao and coworkers reported that 2001; Vigetti, 2006; Vigetti, 2008). Artificially elevating CTP administered intraperitoneally to rats at 30 mg/kg UDP-GlcNAc pools has been shown to increase TGF- reduced p53 expression in brain cortical cells in 3 hours, gene transcription in arterial smooth muscle cells, thereby the effect peaking at 24 hours (Zhao, 2003). mimicking the effect of hyperglycemia (Sayeski, 1996). Mechanisms by which elevated pyrimidine levels might Consistent with this finding, exogenous uridine has been actively negatively regulate p53 have not previously been shown to increase peripheral insulin resistance (Wang, identified. Described below are hypotheses for eight such 1998). mechanisms, all of which may exist. The first seven are, Based on literature reviewed thus far, it appears that in reality, established mechanisms by which the ERK pathglycosylation’s global cellular effects are predominantly way and/or pro-inflammatory cytokines mediate repression pro-growth, in that it activates many proteins involved in of p53 or its function. It is here recognized, however, proliferation. For example, glycosylation is dynamically that these mechanisms may in some cases tacitly be linked to phosphorylation of regulatory proteins such as ‘pyrimidine mechanisms,’ given pyrimidines’ roles, disc-Myc, estrogen receptors, Sp1, endothelial nitric oxide cussed above, in ERK activation and upregulation of prosynthase, and beta-catenin (Kamemura, 2003). O-linked inflammatory cytokines. Similarly, the eighth mechanism glycosylation protects Sp1 from proteolytic degradation listed below, p53 repression via glycosylation, is here (Comer, 2000), and glycosylation of hepatocyte growth posited as a ‘pyrimidine mechanism’ on the basis that elefactor increases its post-transcriptional synthesis (Fukuta, vated pyrimidine levels promote elevated glycosylation of 2005). Glycosylation was shown to activate signal transproteins. ducer and activator of transcription 5 (Stat5) (Bromberg, (a) The ERK pathway upregulates Cdk2. Evidence indi2001). The Stat family of transcription factors is involved cates that Cdk2 inhibits p53 (Price, 1995; Ferguson, 2004; in differentiation, proliferation, apoptosis, inflammation Payton, 2006). A3 adenosine receptor agonist IB-MECA, (Dumon, 1999; Bromberg, 2001) and malignant transforwhich was shown to suppress cyclin D1 and ERK1/2 in mation (Hirano, 2000, Bowman, 2000). 67.187.171.106 On: Tue,rat19prostate Jun 2018 20:19:58 cancer cells (Jajoo, 2009) was shown in the In contrast to glycosylation’s IP: activating effect on proCopyright: American Scientific Publishers same study to increase p53 levels after 48 hours expoteins such as c-myc, Sp1 and STAT5, glycosylation’s net Delivered by Ingenta sure. Notably, cyclin-dependent kinase inhibition has been effect on p53 is to impair its function, as discussed in the shown to be p53-dependent (El-Deiry, 1993; Dulić, 1994; following section. Waldman, 1995). (b) The ERK pathway upregulates Mdm2 (Ito, 2002; Elevated Pyrimidine Nucleotide Levels Ofir-Rosenfeld, 2008) which negatively regulates p53 in Induce Repression of P53 or Its two ways: by repressing its transcription (Ofir-Rosenfeld, Function by Multiple Paths 2008) and by mediating its proteosomal degradation (Ito, Several studies in the last two decades have shown that 2002; Brenkman, 2008). depletion of intracellular pyrimidine nucleotides induces (c) ERK upregulates c-Jun amino-terminal kinase (JNK) activation or upregulation of p53 (Lane, 1992; Huang, (Lopez-Bergami, 2007) which, like Mdm2, binds p53 to 1996; Wahl, 1997; Herrmann, 1997; Huang, 1999; Foxa, facilitate its proteosomal degradation (Fuchs, 1998). Also 1999; Abcouwer, 1999; Hail, 2012). While it is well estabof interest is that Qiao and coworkers reported that deoxylished that p53 is activated by DNA damage, possibly cholic acid (DCA)-induced proteasomal degradation of triggered by instructions from a sensory “checkpoint” prop53 was blocked by blocking the ERK, but not the PKC, tein which may be p19ARF (Khan, 2000), evidence also pathway (Qiao, 2000). shows that ribonucleotide depletion can activate p53 in (d) Pro-inflammatory cytokines upregulate CCAAT/ the absence of DNA damage (Linke, 1996) and without enhancer binding protein (C/EBP), a p53 repressor p19ARF involvement (Ries, 2000). Moreover, evidence protein. The p53-repressive mechanism of C/EBP is suggests that, in the case of ribonucleotide depletion, the unconfirmed but suspected to be either by C/EBP trigger for p53 activation may be repression of Mdm2. directly binding to p53’s proximal promoter or by C/EBP Significantly, Mdm2 was shown to be repressed by PALAinduced pyrimidine nucleotide depletion (Khan, 2000). VRAC-released pyrimidine nucleotides autocrinally activate ERK. This, coupled with the fact that Mdm2 is activated by ERK (Ito, 2002; Ofir-Rosenfeld, 2008), suggests the possibility that elevation of ribonucleotides could actively repress p53—as opposed to a more passive role J. Pharm. Sci. Pharmacol. 1, 4–25, 2014

b Khutornenkoa et al. also found that pyrimidine depletion caused relocalization of NQO1 and NQO2 from cytoplasm to nucleus where they colocalized with the induced p53 (Khutornenkoa, 2010). It is therefore tempting to speculate that elevated pyrimidine levels may inhibit localization of NQO1 and NQO2 to the nucleus. If so, this in itself would have important implications.

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it will be useful to review alternate pyrimidine biosyntheregulating genes involved in posttranslational modificasis paths of clinical relevance. tions of p53 (Appella, 2001; Ewing, 2003). (e) Pro-inflammatory cytokines upregulate Bcl-2 proteins, which repress p53’s apoptotic function (Lin, 2002). ALTERNATE PYRIMIDINE BIOSYNTHESIS (f) Pro-inflammatory cytokines induce p53-repressing PATHS OF CLINICAL RELEVANCE microRNAs (miRNAs) such as MiR-155, which has been As important as the proliferative effects of pyrimidine are shown to repress p53-induced nuclear protein 1; and MiRthe diversity and redundancy of paths by which pyrim21, which targets several tumor suppressor genes (Schetter, idines can be manufactured by cells. That redundancy, 2010). reflecting the criticality of pyrimidine supply to cytopro(g) ERK-mediated elevation of rRNA synthesis. Active liferation, can in some cases enable cells to adaptively rRNA synthesis induces Mdm2-mediated ubiquitination bypass one drug-blocked pyrimidine synthesis pathway to and degradation of p53 (Zhang, 2009); Sugimoto, 2003), use another. One such redundancy is the aforementioned and ERK 1/2 elevates rRNA synthesis by upregulating compensatory increase in pyrimidine salvage upon inhibiPol I by phosphorylating both its transcription initiation tion of de novo pyrimidine biosynthesis. Another possible factor (TIF-A) (Zhao, 2003) and upstream binding factor example, although controversial, is a 1973 report of lactat(UBF) (Stefanovsky, 2001; Brandenburger, 2003; Feltoning bovine mammary tissue utilizing ammonia in the place Edkins, 2003; Mariappan, 2011). Low-dose actinomycin of glutamine as nitrogen source for pyrimidine synthesis D (5–10 nM) restores p53 function by inhibiting Pol I and (Vekich, 1973). Also, as mentioned above, lactating bovine thereby rRNA synthesis (Perry, 1970; Ishiguro, 1978; Dai, mammary tissue showed decreased allosteric sensitivity to 2004a; Dai, 2004b; Dai, 2004c; Kalousek, 2007; Choong, UTP at CPS 2 (Denamur, 1959). 2009), whereas higher-dose actinomycin D, which inhibits Another pyrimidine synthesis path of clinical relevance all RNA polymerases, induces p53 but does not restore its is overproduction of carbamoyl phosphate (CP) in the function (Perry, 1970; Rajagopalan, 1996). mitochondria. This causes CP to diffuse to the cytosol, (h) Glycosylation. Elevated pyrimidine levels may proallowing it to enter the de novo pyrimidine biosynthesis mote aberrant glycosylation of proteins generally. Signifpathway in such way as to bypass the allosterically conicantly, glycosylation of p53 impairs its function. In the trolled CP synthesis step at cytosolic CPS 2 (in CAD). EB-1 human cell line, O-GlcNAcylation of p53 has been This bypass of allosteric control abnormally elevates orotic IP: 67.187.171.106 shown to impair its DNA binding (Shaw, 1996). InOn: rat Tue, 19 Jun 2018 20:19:58 acid levels. Vasudevan and coworkers reported this pheCopyright: American Scientific Publishers liver, O-GlcNAcylation of p53 inhibited its binding to the by Ingenta Delivered nomenon in rats and mice, with the condition being 2-macroglobulin gene promoter (Mihailović, 2008). reversed by co-administration of ornithine and glycine. Relevant to the latter mechanism, Patwardhan and They also found the mitochondrial CP overproduction to coworkers recently demonstrated that glycosylation of be arrested by actinomycin D (Vasudevan, 1998). ceramide actively represses wild-type (WT) p53, implicatMitochondrial overproduction of CP is caused by ing ceramide glycosylation in the prevalence of mutant decrease in mitochondrial ornithine levels, which condip53 associated with cancer (Patwardhan, 2010). Patwardtion can be caused by either of two events involving difhan’s colleagues also found that disrupting ceramide ferent mitochondrial enzymes: (a) inhibition of ornithine glycosylation resuscitated WT p53 expression and p53transcarbamoylase (OTC) or (b) overactivity of ornithine dependent apoptosis in mutant p53 tumors (Liu, 2011). decarboxylase (ODC). OTC inhibition can be induced by These findings are consistent with earlier findings that carcinogens and/or carcinogenesis (McLean, 1964; Jeffers, inhibition of the enzyme which glycosylates ceramide 1988; Riordan, 1997) although the mechanism is unknown. inhibits melanoma growth (Weiss, 1993). Of possible relevance to that mechanism is that OTC is For completeness, it should be noted that Oknown to be negatively regulated by acetylation (Weber, GlcNAcylation of p53 at Ser 149 has been shown to 1972), and deacetylated (activated) by Sirt3 (Xu, 2011). stabilize p53 by blocking ubiquitin-dependent proteolysis ODC overactivity can induce cell transformation (Yang, 2006). On balance, however, it appears that gycosy(Auvinen, 1992; Smith, 1997; Ahmad, 2001; Deng, 2008). lation is generally p53-deactivating, and, based on forgoing ODC inhibitors have shown both chemopreventive and evidence, it is reasonable to hypothesize that glycosylatherapeutic potential in cancer (Hung, 1981; Marton, tion of both p53 and ceramide would be elevated in cases 1981; Sondergaard, 1985; Taplaz, 1986; Levin, 1987; where glycosylation is globally elevated, as in the cases of Pegg, 1988a; Heby, 1990; Reddy, 1990; McCann, 1992; elevated glucose or uridine. Iishi, 1992; Tatsuda, 1995; Iishi, 1997; Myskens, 1999; Presented above are hypothetical models, with supportSalim, 2000). The mechanism by which ODC overactivity ing literature evidence, for five proliferative mechanisms promotes neoplastic transformation is widely deemed to of elevated pyrimidines that are unrelated to DNA syntheinvolve ODC’s role in polyamine synthesis (Pegg, 1986; sis. These models, if valid, suggest new paths for cliniPegg, 1988b; Porter, 1986). While that is clearly a sigcal antipyrimidine strategies. Before discussing such paths, nificant factor, I suggest the possibility that an equally 12


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important mechanism may be ODC’s elevation of cellular orotate levels. Notably, increased ODC activity is also seen in rapid tissue growth in mammals, particularly in the fetus (Manen, 1983). Also notable is that antizyme, a natural ODC inhibitor in mammals, has been shown to induce apoptosis through a cell cycle-independent pathway that includes inhibiting Bcl-2 (Liu, 2006). This is relevant in that Bcl-2 upregulation is one effect of proinflammatory cytokines, which can be auto/paracrinally induced by elevated pyrimidine levels.

POSSIBLE IMPLICATIONS OF HYPOTHESES FOR CLINICAL STRATEGIES

inhibition of pyrimidine-mediated ERK activation, with little impact on normal cell functions that rely on nominal pyrimidine availability, VRAC and/or P2Y function. Such concurrent partial inhibition of the three linchpins would obviate clinical inhibition of pyrimidine salvage. The clinical rationale for inhibiting salvage is the phenomenon, discussed above, that some cell types compensatorily increase pyrimidine salvage in response to de novo synthesis inhibition. However, past attempts to concurrently inhibit de novo pyrimidine biosynthesis and pyrimidine salvage resulted in cell death, underscoring the criticality of nominal pyrimidine levels to normal cell physiology. Indeed, varying degrees of partial inhibition of both salvage and de novo synthesis may be worth investigating; an optimal balance may exist that is selective in toxicity to proliferating cells. That possibility notwithstanding, and all evidence presented above considered, the more attractive course is concurrent, partial inhibition of the three proposed linchpins of pyrimidines’ auto/paracrine effects. On that presumption, described below are proposed pharmacologic approaches for inhibiting each of these three targets. The first approach, for inhibition of de novo pyrimidine biosynthesis, is novel and somewhat complex.

The above hypothetical models suggest that proliferative and pathological effects of elevated pyrimidines are substantially auto/paracrine in nature, with pyrimidines’ VRAC release and P2Y agonism being principal mediators, i.e., linchpins, of pyrimidines’ auto/paracrine effects, equally as de novo pyrimidine biosynthesis itself is a linchpin. Hence, the auto/paracrine effects are depedent upon functionality of all three linchpins. This suggests, pharmacologically, that inhibiting only one of the three linchpins would effectively “short-circuit” the proposed auto/paracrine path. Inhibition of De Novo Pyrimidine Biosynthesis: However, complete inhibition of any one given linchpin Combine Partial DHOD Inhibition with would have adverse side effects; normal cell physiology Acid Modulation IP: 67.187.171.106 On: Tue,Amino 19 Jun 2018 20:19:58 depends upon some degree of P2Y and VRAC functionCopyright: American Scientific Publishers For purposes of partial DHOD inhibition, well-established ality, as well as some degree of pyrimidine synthesis. For by Ingenta Delivered inhibitors such as leflunomide or Redoxal (Knecht, 2000) example, adverse effects of excessive inhibition of de novo would be adequate. It is further proposed that partial pyrimidine biosynthesis can include: DHOD inhibition be augmented by modulating certain • Immunosuppression. Some degree of active de novo metabolic amino acid profiles; specifically, by depletpyrimidine biosynthesis is necessary for normal immune ing glutamine and administering (or clinically elevating) response. glycine and ornithine, for reasons detailed below. • Impairment of mitochondrial electron transfer, to which Glutamine depletion. Glutamine is a metabolic nitrogen DHOD’s oxidation of DHO in the de novo pathway is source both for pyrimidine and purine synthesis. In the coupled at cytochrome II. Such impairment may underpyrimidine pathway, glutamine is required both for the first lie reported cardiotoxicity in rhesus monkeys of DHOD synthesis step and for the last step, CTP formation. Siginhibitor dichloroallyl lawsone (DCL) (Bennet, 1979; nificantly, glutamine’s uptake by malignant cells exceeds McKelvey, 1979). that of other amino acids, despite it not being an essential • Impairment of ontogeny. Neural crest formation is amino acid (Wasa, 1996). DHOD-dependent (White, 2011). This would be relevant In aforementioned studies, a combination of de novo not only to chemotherapy during pregnancy but also to the biosynthesis inhibitor PALA and glutamine analogue use of stem cells for tissue regeneration. acivicin had synergistic effects in reducing pyrimiSuch effects define the clinical limitations of DHOD dine levels. However, it is here proposed that using inhibitors such as leflunomide. phenylbutyrate—a glutamine depleting agent—would be To minimize such adverse effects, one alternative stratpreferable to glutamine analog acivicin, because phenylegy would be to concurrently partially inhibit all three butyrate is also a potent histone deacetylase (HDAC) “linchpins” (de novo pyrimidine synthesis, VRACs and inhibitor. For that reason, a clinical strategy of combining P2Ys). Intuitively, such combination should have mulDHOD inhibition with phenylbutyrate was proposed by tiplicative inhibitory effect, analogous to serial dilution. this author in 2004 (Gipson, 2004). Major genes silenced Theoretically, presuming the validity of the hypothesized by HDAC inhibitors are—ironically—several genes codauto/paracrine pathway—with a signature endpoint being ing for pyrimidine biosynthesis enzymes including DHOD, persistent ERK activation—inhibition of all three linchpins thymidylate synthetase, formyltetrahydrofolate dehydrogenase and CTP synthase 2 (Chittur, 2008). Phenylbutyrate by only 80% each could (ideally) result in nearly 99% J. Pharm. Sci. Pharmacol. 1, 4–25, 2014

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would therefore uniquely control pyrimidine synthesis pyrimidine synthesis as total glutamine depletion, but with both at the metabolic level and transcription level. lesser effect on purine synthesis. No clinical human GLDC Glutamine’s role in proliferation is more than simply inhibitors are known as of this writing. Of possible interthat of a nitrogen source. Glutamine induces transcription est in this regard, however, is that aminoacetonitrile is an of proteins characteristic of early embryogenesis, i.e., the inhibitor of plant GLDCs (LaCuesta, 1997; Scott-Taggart, “embryonic genome pattern,” including heat shock pro1997), plant GLDCs are significantly homologous to anitein 70 (HSP70) (Poueymirou, 1989; Cai, 1991; McCauley, mal and human GLDCs (Kopriva, 1995), and aminoace1998). Glutamine’s induction of HSP70 was recently tonitrile derivatives are used as anthelmintics in ruminant shown to be dependent on UDP-N -acetylglucosamine mammals, exhibiting negligible host toxicity (Kaminsky, (GlcNAc)-mediated glycosylation of transcription factors 2008; Ducraya, 2008). SP1 and HSF-1 (Singleton, 2008; Hamiel, 2009). DisAs previously discussed, elevating cellular ornithine cussed above is evidence that GlcNAc-mediated glycosylevels, either by administration of an ODC inhibitor lation can be directly increased by elevated pyrimidine or by dietary ornithine increase, would prevent bypass levels. Thus, it is possible that elevated pyrimidines, by of allosteric control at CPS 2. Notably, Vasudevan promoting glycosylation of SP1 and HSF-1, promote gluand coworkers found co-administration of ornithine and tamine’s induction of HSP70. Also, White et al. (White, glycine to effectively inhibit orotic acid production in 2011) noted that elevated pyrimidine nucleotide levels rats and mice, wherease glycine alone was ineffective upregulated HSPs. These facts suggest the possibility that (Vasudevan, 1998). elevated pyrimidine levels may act synergistically with The above-described amino acid modulation regiglutamine to induce HSP70, further underscoring the clinimen (glutamine depletion plus elevation of ornithine cal potential of combining glutamine depletion with pyrimand glycine), used in combination with partial DHOD idine reduction. inhibition, would further lower the degree of DHOD inhiOne clinical drawback to complete glutamine depletion, bition needed to effectively inhibit de novo synthesis, however, is that glutamine is also required for purine synreducing impact on normal physiologic processes depenthesis. Clinical inhibition of purines should be minimized, dent on DHOD activity. because—in this reviewer’s opinion—pathologically proliferating cells do not differ from normal cells in their VRAC Inhibition: Use Verapamil purine requirements nearly as much as in their pyrimidine IP: 67.187.171.106 On: Tue, 19 Jun 2018 20:19:58 Verapamil is an established VRAC inhibitor which has also American requirements. Likely for this reason, Copyright: chemotherapies that Scientific Publishers been shown to inhibit human prostate cancer cell proliferDelivered target purines are marked by low therapeutic index. There- by Ingenta ation (Rybalchenko, 2001), to reverse multidrug resistance fore, as clinical strategy, it is proposed that glutamine be of cancer (Mayur, 2006), to be synergistic in combinadepleted significantly—but only partially—with phenylbution with cancer drugs (Ho, 2007), and to inhibit EGFR tyrate, in combination with other agents described herein. expression in human lung cancer cells (Zhang, 2009). VerGlycine and Ornithine. It is proposed that partial glupamil’s anticancer effects have generally been attributed tamine depletion be augmented by administration of the to its effects on Ca2+ or Cl− transport. I propose, howamino acids glycine and ornithine or, alternatively, adminever, based on aforementioned evidence, an equally imporistration of inhibitors of glycine decarboxylase (GLDC) tant role to be Verapamil’s inhibition of cellular release of and ornithine decarboxylase (ODC), which would elepyrimidine nuceotides and pyrimidine sugars. vate glycine and ornithine levels, respectively. The benefit of glycine is that it acts as a ‘natural’ regulator of P2Y Antagonist: Use Pertussis Toxin or Suramin de novo pyrimidine biosynthesis, because it is a competiAntagonists specific for P2Y6 would be intuitive cantive inhibitor of the glutamine binding site on ATCase. Its didates, given that P2Y6 is specific for UDP or UTP. effect on ATCase is therefore similar to glutamine depleIntriguingly, however, one study found suramin, a nontion, with the exception that glycine’s effect—since it is specific P2Y receptor antagonist, more potent in inhibitspecific to ATCase—is specific for pyrimidine synthesis, ing UDP-induced IL-8 release (IC50: 5 mM) than reactive not impacting purine synthesis. This fact, I speculate, may blue, a P2Y6-specific antagonist (IC50: 35 nM) (Charlton, underlie glycine’s reported arrest of breast tumor angio1996). Suramin blocks receptor binding of growth facgenesis in rats (Rose, 1999; Amin, 2003), although the tors such as insulin-like growth factor I (IGF-I), epiderexact mechanism has not been conclusively established. mal growth factor (EGF), platelet-derived growth factor Consistent with those findings is that glycine decarboxy(PDGF), and tumor growth factor-beta (TGF-), has been lase (GLDC) has been shown to promote accumulation of found to inhibit P2Y and P2U stimulation of phospholipyrimidines and drive non-small cell lung cancer tumoripase C (Ahles, 2004), and has been investigated for use genesis, and aberrant GLDC expression is observed in in prostate cancer (Erguven, 2008). Moreover, specific to multiple cancer types (Zhang, 2012). Therefore, adding pyrimidine effects, suramin was found to inhibit, in a glycine—or a GLDC inhibitor—in combination with parconcentration-dependent manner, UTP-, UDP-, TTP-, and tial glutamine depletion would have the same net effect on 14


Gipson Table II.

Multiple Cytoproliferative Effects of Elevated Pyrimidines are DNA-Synthesis-Independent and Include p53 Repression Proposed multitarget therapy for cytoproliferative disease.

Target De novo pyrimidine biosynthesis 1. DHOD 2. Glutamine 3. Ornithine decarboxylase (ODC) 4. Glycine decarboxylase (GLDC) 5. VRAC function 6. P2Y function

Suggested% inhibition 85% 90% (depletion) 95% 95%

80% 80%

Suggested inhibitor(s) Leflunomide or Redoxal Phenylbutyrate 1,3-diaminopropane (DAP) Aminoacetonitrile derivative

Notes

A clinical alternative to ODC inhibition is supplemental dietary ornithine. Aminoacetonitrile derivatives are not yet approved for humans. A clinical alternative to GLDC inhibition is supplemental dietary glycine.

Verapamil PT or suramin

CTP-induced release of thromboxane A2 (TXA2) from inhibition of which may be as high as 95%, with only 85% cultured glia from newborn rat cerebral cortex (Langley, DHOD inhibition. This is a very rough estimate, especially 1998). Notably, TXA2 is also a vasoconstrictor causative given the possibility that the effects of co-targeting 1–4 in angina. Suramin is therefore an attractive candidate P2Y may be dynamic as well as additive. antagonist for antipyrimidine clinical strategies. Another candidate is P2Y6-specific antagonist MRS2578, which DISCUSSION was shown to significantly block both CD25 expression This review focuses on two somewhat obscure phenomena: and IL-2 production (Tsukimoto, 2009). (a) That pyrimidine elevation can be initiated by surprisPerhaps the most intriguing candidate, however, is peringly diverse factors. For example, in addition to abovetussis toxin (PT), a nonspecific P2Y antagonist which discussed elevation of pyrimidines by carcinogens and potently enhances immune response, including increasing viruses, estrogen upregulates the gene coding for CAD production of T cells and regulatory cytokines such as (Khan, 2003; Chen, 2005), which may explain estrogen’s Th1, Th2, IL-2, IL-4 and IL-5 (Ryan, 1998; Shive, 2000; attenuation of leflunomide’s inhibition of cytokine producDenkinger, 2007), yet has also been shown to inhibit pro2010). inflammatory cytokines IL-12 and (Bagley, 2002). IP:TNF- 67.187.171.106 On: Tue,tion 19 (Montagna, Jun 2018 20:19:58 (b) That pyrimidine American This is a unique dichotomy of effect.Copyright: PT has shown effi- Scientific Publishers elevation, in turn, exerts surprisingly proliferative effects. Delivered Ingenta cacy against autoimmune diseases (Weber, 2010) which bydiverse Thus, in cytoproliferative diseases of varied etiologic may, it is intriguing to speculate, be due partly or priorigin, deregulation of pyrimidine biosynthesis may be a marily to its P2Y antagonist role and differential effect common denominator and pivotal etiologic event. Particuon ‘good’ versus ‘bad’ cytokines. Notably, specific to larly pivotal may be abrogation of one or more allosteric pyrimidine effects, PT blocked UTP’s potent induction of controls on the de novo pathway. In addition to abrogation hematopoietic stem cell migration (Rossi, 2007), and abolof UTP’s allosteric control, mutations in CTP synthetase ished UTP’s inhibition of adrenaline-evoked cAMP accuthat abrogate CTP’s allosteric control on that enzyme have mulation in cultured equine epithelial cells (Wilson, 1999). been shown to elevate CTP levels up to 15-fold in yeast Those effects, it is reasonable to speculate, may be due to (Ostrander, 1998). PT’s antagonism of P2Y receptors, since those receptors’ Presented above is circumstantial evidence that elevated activation by pericellular UTP would be blocked. All the pyrimidines contribute, by different paths yet collectively, above considered, suramin or PT are proposed as primary to p53 repression. Further supporting that argument is that candidates for clinical P2Y receptor antagonist to be used several types of cancer and autoimmune diseases have in in clinical combination with partial de novo pyrimidine common both p53 repression and sensitivity to leflunosynthesis inhibition and VRAC inhibition. mide. One of several targets of gene therapy for cancer Described above are strategies for pharmacologically is restoration of functional p53. Also, one lab is currently co-targeting three proposed “linchpins” of pyrimidines’ resuscitating wild type p53 by inhibiting the enzyme that auto/paracrine effects: de novo pyrimidine biosynthesis, glycosylates ceramide. Given the several paths proposed VRAC function and P2Y function. Table II summarizes herein by which elevated pyrimidine nucleotide levels may this multitarget strategy and proposes, for each target, repress p53, it is intriguing to speculate that control of estimated optimal degree (%) of inhibition and candidate pyrimidine levels may be as effective as gene therapy in inhibitors. Such a combination is proposed to achieve sigrestoring p53 functionality, and as effective as inhibiting nificant diminution of pyrimidines’ auto/paracrine effects, ceramide glycosylation. and of pyrimidine-mediated aberrant glycosylation, with Persistent repression or inactivation of p53 has the same negligible impact on normal cell functions. Targets 1–4, pathologic end result as p53 mutation. Mammary tumors as decribed above, would additively contribute to inhibiin mice caused by somatic loss of both p53 and BRCA1 tion of de novo pyrimidine biosynthesis, the cumulative J. Pharm. Sci. Pharmacol. 1, 4–25, 2014

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(a protein with which p53 intimately associates both in adenosine receptors including A3AR, and in some cases are reverse agonists (Baraldi, 1996; Baraldi, 1998; Chebib, its cell-cycle-control and DNA-repair roles) are phenotyp2000; Chang, 2004; Moro, 2006; Wei, 2009; Taliani, ically indistinguishable from tumors caused by germline 2010; Yaziji, 2011). The derivatives vary in potency, yet loss of p53 and BRCA1 (Liu, 2007). In familial breast the pyrimidine heteroaromatic core appears necessary for and ovarian cancer, p53 mutation often occurs coordiantagonistic activity (Chang, 2004). Similarly, a number nately with mutations of retinoblastoma protein gene (Rb) of derivatives of pyridine, which is chemically similar to and BRCA1; yet in non-familial (sporadic) forms of those pyrimidine, have been reported as potent antagonists of same cancers, p53, Rb and BRCA1 are repressed rather adenosine receptors including A3AR (van Rhee, 1996; than being mutated (Kumar, 2012). Even where BRCA1 Li, 1998; Li, 1999). Therefore, it might be of interis not mutated, p53 compromise (by either mutation or est to study the A3AR affinity of pyrimidines besides repression) causes BRCA1 dysfunction because nuclear UTP (e.g., UDP, CTP, UDP-glucose) to assess whether export of BRCA1 is p53 dependent (Jiang, 2011), as is they might be important as physiological A3AR antagoBRCA1-mediated upregulation of p21 (Buck, 2008). Only nists. If so, it would mean that they compete not only 4% of breast cancers have BRCA1 mutations (Xu, 2001), with adenosine for A3AR binding, but also with A3AR whereas 20–30% of breast cancers have mutated p53 agonist drugs. Such drugs have shown marked efficacy (Kumar, 2012) as do more than 50% of all solid tumors against several cancer types (Fishman, 2002a; Fishman, (Jiang, 2011). These statistics underscore a prospect that 2004; Nakamura, 2006; Bar Yehuda, 2008; Stemmer, deregulation of pyrimidine biosynthesis, whether sporadic 2010) and, evidence indicates, exert their antiproliferative or genetic, may be a more important etiologic determieffects via down-modulation of the Wnt/GSK-3b/b-catenin nant of such cancers than are mutations of genes such as signaling pathway, the downstream effects of which BRCA1. include downregulation of cyclin D1 and c-myc (Fishman, It is therefore intriguing to speculate that genetic 2002b; Fishman, 2003a; Fishman, 2003b; Kim, 2008; determinants for familial, tissue-specific cancers may Lee, 2009). include polymorphisms of genes coding for pyrimidine biosynthesis enzymes, e.g., CAD (Wahl, 1979), DHOD, thymidylate synthetase, or CTP synthetase, and/or mitoCONCLUSION chondrial enzymes OTC, ODC or GLDC. Even more This review concludes the following: important may be mutations that allosteric IP: compromise 67.187.171.106 On: Tue, 19 Jun 2018 20:19:58 (1) BothPublishers carcinogenesis and autoimmune diseases are Copyright: control at CPS 2 or at CTP synthetase. However, American notwith- Scientific obligately dependent upon aberrant elevation of pyrimiDelivered by Ingenta standing genotypic pressure by such mutations, whether of dine levels caused by deregulation of pyrimidine biosynBRCA1 or of pyrimidine synthesis enzymes, their effects thesis, which deregulation can have varied underlying may be substantially marginalized by pharmacologic revercauses. sal of pyrimidine deregulation and/or control of pyrim(2) Pathological effects of elevated pyrimidines are mediidines’ cellular mediators of pathology. ated substantially through DNA-synthesis-independent This review raises the possibility that pyrimidine elevamechanisms, which include: tion is a basal driver of elevated rRNA synthesis via pyrim• Persistent, auto/paracrine induction of mitogenic idines’ auto/paracrine activation of Pol I. A large body pathways; of evidence, only partially referenced here (Castle, 2010; • Pyrimidine positive autoregulation via abrogation or Drygin, 2010a; Drygin, 2010b), conclusively links elevated reversal of allosteric control of de novo pyrimidine biosynrRNA synthesis and resultant nucleolar hypertrophy to carthesis; cinogenesis. For that reason alone, most cancer chemother• Upregulation of tumorigenic genes; apy regimens could be enhanced by co-administration of a • Aberrant glycosylation of transcription factors; and Pol I inhibitor. This review suggests, however, that rRNA • p53 repression via multiple paths. synthesis could be substantially controlled solely by blocking pyrimidines’ auto/paracrine effects. This circumstance substantially defines the clinical limitaA question raised and unresolved by this review is the tions of DNA-synthesis-targeting cytostatic drugs for cytomechanism by which CTP induces p53 repression (as eviproliferative disease. denced by Zhao, 2003), given other evidence that CTP is (3) While inhibition of de novo pyrimidine biosynthenot a P2Y activator as are uridine nucleotides and uridine sis, by drugs such as leflunomide, offer pharmacologinucleotide sugars. cal advantages over DNA-synthesis-targeting, even these A final, arcane pyrimidine effect worth passing meninhibitors have dose limitations due to physiologic needs tion in this review is that UTP was found to have slight of non-proliferating cells for pyrimidines. That problem yet appreciable affinity for the A3 adenosine receptor can be averted, however, through the providence that (A3AR) (Zhou, 1992), on the order of 1/10th the affinpathologic effects of elevated pyrimidines are substanity of ADP and 1/20th the affinity of ATP. Intriguingly, tially auto/paracrine in nature, and thus controllable subseveral pyrimidine derivatives are potent antagonists of stantially through inhibition of auto/paracrine effectors 16


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(VRACs and P2Y receptors). That providence renders partial inhibition of de novo pyrimidine biosynthesis clinically adequate, if implemented in a clinical combination such as detailed above. (4) Overproduction of mitochondrial carbamoyl phosphate, resulting in bypass of the allosterically controlled CPS 2 step of de novo pyrimidine biosynthesis, and which may be induced by different factors, may be more prevalent in cytoproliferative disease than generally recognized. The above considerations appear to have been lacking in past antipyrimidine strategies. It is hoped that they will be considered for empirical evaluation by an interested research body.

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Acknowledgments: For their respective reviews of the Bachmaier, R., Aryee, D. N. T., Jug, G., Kauer, M., Kreppel, M., Lee, manuscript, I sincerely thank Dr. Ralph de Vere White, K. A., and Kovar, H. (2009). O-GlcNAcylation is involved in the tranDirector, University of California-Davis Cancer Center; scriptional activity of EWS-FLI1 in Ewing’s sarcoma. Oncogene 28, 1280–1284. Dr. Laurence M. Hardy, Professor Emeritus, Louisiana State University-Shreveport; Dr. Enoch Post Baldwin, Bagley, K. C., Abdelwahab, S. F., Tuskan, R. G., Fouts, T. R., and Lewis, G. K. (2002). Pertussis toxin and the adenylate cyclase toxin from bordeProfessor, Molecular and Cell Biology, University tella pertussis activate human monocyte-derived dendritic cells and domor California-Davis; Dr. Kermit Carraway, Professor, inantly inhibit cytokine production through a cAMP-dependent pathway. Biochemistry and Molecular Medicine, University J. Leukoc Biol. 72, 962–969. of California-Davis; and Dr. Subhash Padhye, Director Baker, O. J., Camden, J. M., Rome, D. E., Seye, C. I., and Weisman, of Research, University of Pune, Pune, India, Director of G. A. (2008). Nucleotide receptor activation up-regulates vascular cell the Interdisciplinary Science and Technology Research adhesion molecular-1 expression and enhances lymphocyte adherence to Academy, Pune, and visiting Professor at Karmanos Cana human submandibular gland cell line. Mol. Immunol. 45, 65–75. IP: 67.187.171.106 On: Tue,Bar19Yehuda, Jun 2018 20:19:58 cer Institute, Wayne State University, Detroit. I especially S., Stemmer, S. M., Madi, L., Castel, D., Ochaion, A., Copyright: Publishers thank Dr. Padhye for his helpful suggestion toAmerican expand Scientific Cohen, S., Barer, F., Perez-Liz, G., Del Valle, L., and Fishman, P. (2008). Delivered byEffect Ingenta of CF102 on growth suppression and apoptosis in an orthotopic on genetic aspects of antipyrimidine therapies, and Drs. model of hepatocellular carcinoma. J. Clin. Oncol. 2008 ASCO Annual Baldwin and Carraway for their numerous and helpful Meeting Proceedings 26, 22113. editorial suggestions and corrections. I also am indebted to Baraldi, P. G., Cacciari, B., Spalluto, G., Bergonzoni, M., Dionisotti, S., my colleague Dr. Dennis Guo for his gracious translation Ongini, E., Varani, K., and Borea, P. A. (1998). Design, synthesis, and from Chinese of an article (Zhao, 2003) referenced herein, biological evaluation of a second generation of pyrazolo[4,3-e]1,2,4and to colleague Linnah Marlow for her equally gracious triazolo[1,5-c]pyrimidines as potent and selective A2A adenosine receptor antagonists. J. Med. Chem. 41, 2126–2133. graphic representations for Figures 1 and 2.

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