Clinical characteristics and outcome of 318 families with familial monoclonal gammopathy: A multicenter Intergroupe Francophone du Myélome study.

Familial forms of monoclonal gammopathy, defined as multiple myeloma (MM) or Monoclonal Gammopathy of Undetermined Significance (MGUS), are relatively infrequent and most series reported in the literature describe a limited number of families. MM rarely occurs in a familial context. MGUS is observed much more commonly, which can in some cases evolve toward full-blown MM. Although recurrent cytogenetic abnormalities have been described in tumor cells of sporadic cases of MM, the pathogenesis of familial MM remains largely unexplained. In order to identify genetic factors predisposing to familial monoclonal gammopathy, the Intergroupe Francophone du Myélome identified 318 families with at least two confirmed cases of monoclonal gammopathy. There were 169 families with parent/child pairs and 164 families with cases in at least two siblings, compatible with an autosomal transmission. These familial cases were compared with sporadic cases who were matched for age at diagnosis, sex and immunoglobulin isotype, with 10 sporadic cases for each familial case. The gender distribution, age and immunoglobulin subtypes of familial cases were unremarkable in comparison to sporadic cases. With a median follow-up of 7.4 years after diagnosis, the percentage of MGUS cases having evolved to MM was 3%. The median overall survival of the 148 familial MM cases was longer than that of matched sporadic cases, with projected values of 7.6 and 16.1 years in patients older and younger than 65 years, respectively. These data suggest that familial cases of monoclonal gammopathy are similar to sporadic cases in terms of clinical presentation and carry a better prognosis.

A Proposed Methodology to Deal with the Impact of In Vitro Cellular Matrix on the Analytical Performances of a Targeted Metabolomic LC-HRMS Method.

Performances of metabolomic methods have been widely studied on biological matrices such as serum, plasma, and urine; but much less on in vitro cell extracts. While the impact of cell culture and sample preparation on results are well-described, the specific effect of the in vitro cellular matrix on the analytical performance remains uncertain. The aim of the present work was to study the impact of this matrix on the analytical performance of an LC-HRMS metabolomic method. For this purpose, experiments were performed on total extracts from two cell lines (MDA-MB-231 and HepaRG) using different cell numbers. Matrix effects, carryover, linearity, and variability of the method were studied. Results showed that the performances of the method depend on the nature of the endogenous metabolite, the cell number, and the nature of the cell line. These three parameters should, therefore, be considered for the processing of experiments and the interpretation of results depending on whether the study focuses on a limited number of metabolites or aims to establish a metabolic signature.

Ambulatory high-dose methotrexate administration as central nervous system prophylaxis in patients with aggressive lymphoma.

High-dose methotrexate (HD-MTX) at 3 g/m2 is one of the strategies for central nervous system (CNS) prophylaxis in the first-line treatment of aggressive lymphomas, especially in diffuse large B cell lymphoma patients with high-risk CNS-International Prognostic Index. The objective of our study was to retrospectively analyze the safety of 2 cycles of systemic HD-MTX administered as an ambulatory regimen. Between January 2013 and December 2016, 103 patients were carefully selected on 6 criteria, including age < 60, albumin > 34, performance status 0 or 1, normal renal and hepatic functions, good understanding of practical medical guidance, and no loss of weight. Strict procedures of HD-MTX infusion were observed including alkalinization, urine pH monitoring, and leucovorin rescue. Renal and hepatic functions were monitored at days 2 and 7. MTX clearance was not monitored. Toxicities and grades of toxicity were collected according to the NCI-CTCAE (version 4.0). Among the 103 selected patients, 92 (89%) patients successfully completed the planned 2 cycles of HD-MTX on an outpatient basis. Eleven patients completed only 1 cycle, 3 because of lymphoma progression and 8 because of toxicity including 3 grade II hepatotoxicity, 2 grade I/II renal toxicity, 1 grade III neutropenia, 1 active herpetic infection, and 1 grade III ileus reflex. Reported adverse events (AE) included 92 (84%) grade I/II and 18 (16%) grade III/IV. Grade III hepatotoxicity, mostly cytolysis, was the most frequent AE observed with 8 (8%) events. Grade III/IV hematologic toxicities concerned 9 patients with 8 grade III/IV neutropenia and 1 thrombocytopenia. Renal toxicity was rare, mild, and transient, observed with 4 (4%) grade I/II events. Ambulatory administration of HD-MTX at 3 g/m2 without MTX clearance monitoring is safe with strict medical guidance. It requires careful selection of patients before administration, and a renal and hepatic monitoring after the administration.

4-Substituted-1,2,3-triazolo nucleotide analogues as CD73 inhibitors, their synthesis, in vitro screening, kinetic and in silico studies.

Three series of nucleotide analogues were synthesized and evaluated as potential CD73 inhibitors. Nucleobase replacement consisted in connecting the appropriate aromatic or purine residues through a triazole moiety that is generated from 1,3-dipolar cycloaddition. The first series is related to 4-substituted-1,2,3-triazolo-ß-hydroxyphosphonate ribonucleosides. Additional analogues were also obtained, in which the phosphonate group was replaced by a bisphosphonate pattern (P-C-P-C, series 2) or the ribose moiety was removed leading to acyclic derivatives (series 3). The ß-hydroxyphosphonylphosphonate ribonucleosides (series 2) were found to be potent inhibitors of CD73 using both purified recombinant protein and cell-based assays. Two compounds (2a and 2b) that contained a bis(trifluoromethyl)phenyl or a naphthyl substituents proved to be the most potent inhibitors, with IC50 values of 4.8 ± 0.8 µM and 0.86 ± 0.2 µM, compared to the standard AOPCP (IC50 value of 3.8 ± 0.9 µM), and were able to reverse the adenosine-mediated immune suppression on human T cells. This series of compounds illustrates a new type of CD73 inhibitors.

Intracellular Cytidine Deaminase Regulates Gemcitabine Metabolism in Pancreatic Cancer Cell Lines.

Cytidine deaminase (CDA) is a determinant of in vivo gemcitabine elimination kinetics and cellular toxicity. The impact of CDA activity in pancreatic ductal adenocarcinoma (PDAC) cell lines has not been elucidated. We hypothesized that CDA regulates gemcitabine flux through its inactivation and activation pathways in PDAC cell lines. Three PDAC cell lines (BxPC-3, MIA PaCa-2, and PANC-1) were incubated with 10 or 100 µM gemcitabine for 60 minutes or 24 hours, with or without tetrahydrouridine, a CDA inhibitor. Extracellular inactive gemcitabine metabolite (dFdU) and intracellular active metabolite (dFdCTP) were quantified with liquid chromatography tandem mass spectrometry. Cellular expression of CDA was assessed with real-time PCR and Western blot. Gemcitabine conversion to dFdU was extensive in BxPC-3 and low in MIA PaCa-2 and PANC-1, in accordance with their respective CDA expression levels. CDA inhibition was associated with low or undetectable dFdU in all three cell lines. After 24 hours gemcitabine incubation, dFdCTP was highest in MIA PaCa-2 and lowest in BxPC-3. CDA inhibition resulted in a profound dFdCTP increase in BxPC-3 but not in MIA PaCa-2 or PANC-1. dFdCTP concentrations were not higher after exposure to 100 versus 10 µM gemcitabine when CDA activities were low (MIA PaCa-2 and PANC-1) or inhibited (BxPC-3). The results suggest a regulatory role of CDA for gemcitabine activation in PDAC cells but within limits related to the capacity in the activation pathway in the cell lines. SIGNIFICANCE STATEMENT: The importance of cytidine deaminase (CDA) for cellular gemcitabine toxicity, linking a lower activity to higher toxicity, is well described. An underlying assumption is that CDA, by inactivating gemcitabine, limits the amount available for the intracellular activation pathway. Our study is the first to illustrate this regulatory role of CDA in pancreatic ductal adenocarcinoma cell lines by quantifying intracellular and extracellular gemcitabine metabolite concentrations.

Regulation of tumor infiltrated innate immune cells by adenosine.

Cancer has the ability to escape the immune system using different molecular actors. Adenosine is known to be involved in mechanisms which control inflammatory reactions and prevent excessive immune response. This purine nucleoside can be translocated from the cell or produced in the extracellular space by 5'-ectonucleotidases. Once bound to its receptors on the surface of immune effector cells, adenosine activates various molecular pathways, which lead to functional inhibition of the cell or its death. Some tumors are infiltrated by the different cells of immune system but are able to use adenosine as an immunosuppressive molecule and thus inhibit immune anticancer response. This mechanism is well described on adaptive cells, but much less on innate cells. This review outlines major effects of adenosine on innate immune cells, its consequences on cancer progression, and possible ways to block the adenosine-dependent immunosuppressive effect.

Unexpected Growth-Promoting Effect of Oxaliplatin in Excision Repair Cross-Complementation Group 1 Transfected Human Colon Cancer Cells.

The nucleotide excision repair protein excision repair cross-complementation group 1 (ERCC1) has been repeatedly shown to be involved in the sensitivity of cancer cells to platinum derivatives. In order to better understand this process, we transfected HCT-116 cells with a plasmid encoding ERCC1 and studied their in vitro and in vivo behaviour. No main differences were observed for sensitivity to platinum drugs, DNA repair capacity and clonogenicity in vitro. However, -ERCC1-transfected HCT-116 cells showed paradoxical behaviour in vivo with increased growth in mice treated with oxaliplatin as compared to untreated mice. The Trop2 protein was identified as being potentially involved in the underlying mechanism for these observations, as it was overexpressed in transfected cells. Our results suggest complex regulation of signalling in cancer cells exposed to cancer drugs.

Determination of the enzymatic activity of cytosolic 5'-nucleotidase cN-II in cancer cells: development of a simple analytical method and related cell line models.

The cytosolic 5'-nucleotidase (cN-II) has been shown to be involved in the response of cancer cells to cytotoxic agents, and the quantification of its activity in biological samples is of great interest. In this context, we developed and validated an analytical method for determination of cN-II activity in cultured cancer cells. This non-radioactive method, using a Hypercarb column as stationary phase, was validated with a lower limit of quantification of 0.1 µM inosine. We used it to characterize cell line models with modified cN-II expression obtained with stable transfections. We show that the short hairpin RNA (shRNA)-mediated inhibition of cN-II expression in various malignant blood cells is associated with decreased protein expression and enzymatic activity (1.7-6.2-fold) as well as an increased sensitivity to cytotoxic agents (up to 14-fold). On the other hand, expression of green fluorescent protein (GFP)-fused wild type or hyperactive mutant (R367Q) cN-II increased the activity and also decreased the sensitivity to nucleoside analogues. Our results confirm the biological relevance of modulating cN-II in cancer cells, and we present a straightforward validated method for the determination of cN-II activity in cellular samples.

Expression of domains for protein-protein interaction of nucleotide excision repair proteins modifies cancer cell sensitivity to platinum derivatives and genomic stability.

Nucleotide excision repair (NER) is involved in the repair of DNA damage caused by platinum derivatives and has been shown to decrease the cytotoxic activity of these drugs. Because protein-protein interactions are essential for NER activity, we transfected human cancer cell lines (A549 and HCT116) with plasmids coding the amino acid sequences corresponding to the interacting domains between excision repair cross-complementation group 1 (ERCC1) and xeroderma pigmentosum, complementation group A (XPA), as well as ERCC1 and xeroderma pigmentosum, complementation group F (XPF), all NER proteins. Using the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and annexin V staining, we showed that transfected A549 cells were sensitized 1.2-2.2-fold to carboplatin and that transfected HCT116 cells were sensitized 1.4-5.4-fold to oxaliplatin in vitro. In addition, transfected cells exhibited modified in vivo sensitivity to the same drugs. Finally, in particular cell models of the interaction between ERCC1 and XPF, DNA repair was decreased, as evidenced by increased phosphorylation of the histone 2AX after exposure to mitomycin C, and genomic instability was increased, as determined by comparative genomic hybridization studies. The results indicate that the interacting peptides act as dominant negatives and decrease NER activity through inhibition of protein-protein interactions.

Small molecule inhibitors of ERCC1-XPF protein-protein interaction synergize alkylating agents in cancer cells.

The benefit of cancer chemotherapy based on alkylating agents is limited because of the action of DNA repair enzymes, which mitigate the damage induced by these agents. The interaction between the proteins ERCC1 and XPF involves two major components of the nucleotide excision repair pathway. Here, novel inhibitors of this interaction were identified by virtual screening based on available structures with use of the National Cancer Institute diversity set and a panel of DrugBank small molecules. Subsequently, experimental validation of the in silico screening was undertaken. Top hits were evaluated on A549 and HCT116 cancer cells. In particular, the compound labeled NSC 130813 [4-[(6-chloro-2-methoxy-9-acridinyl)amino]-2-[(4-methyl-1-piperazinyl)methyl]] was shown to act synergistically with cisplatin and mitomycin C; to increase UVC-mediated cytotoxicity; to modify DNA repair as indicated by the staining of phosphorylated H2AX; and to disrupt interaction between ERCC1 and XPF in cells. In addition, using the Biacore technique, we showed that this compound interacts with the domain of XPF responsible for interaction with ERCC1. This study shows that small molecules targeting the protein-protein interaction of ERCC1 and XPF can be developed to enhance the effects of alkylating agents on cancer cells.

A systemically administered detoxified TLR4 agonist displays potent antitumor activity and an acceptable tolerance profile in preclinical models.

Bacterial lipopolysaccharides (LPS) are potent innate immunostimulants targeting the Toll-like receptor 4 (TLR4), an attractive and validated target for immunostimulation in cancer therapy. Although LPS possess anti-tumor activity, toxicity issues prevent their systemic administration at effective doses in humans. We first demonstrated that LPS formulated in liposomes preserved a potent antitumor activity per se upon systemic administration in syngeneic models, and significantly enhance the antitumor activity of the anti-CD20 antibody rituximab in mice xenografted with the human RL lymphoma model. Liposomal encapsulation also allowed a 2-fold reduction in the induction of pro-inflammatory cytokines by LPS. Mice receiving an intravenous administration demonstrated a significant increase of neutrophils, monocytes and macrophages at the tumor site as well as an increase of macrophages in spleen. Further, we chemically detoxified LPS to obtain MP-LPS that was associated with a 200-fold decrease in the induction of proinflammatory cytokines. When encapsulated in a clinically approved liposomal formulation, toxicity, notably pyrogenicity (10-fold), was limited while the antitumor activity and immunoadjuvant effect were maintained. This improved tolerance profile of liposomal MP-LPS was associated with the preferential activation of the TLR4-TRIF pathway. Finally, in vitro studies demonstrated that stimulation with encapsulated MP-LPS reversed the polarization of M2 macrophages towards an M1 phenotype, and a phase 1 trial in healthy dogs validated its tolerance upon systemic administration up to very high doses (10µg/kg). Altogether, our results demonstrate the strong therapeutic potential of MPLPS formulated in liposomes as a systemically active anticancer agent, supporting its evaluation in patients with cancer.

Second-Generation CD73 Inhibitors Based on a 4,6-Biaryl-2-thiopyridine Scaffold.

Various series of 4,6-biaryl-2-thiopyridine derivatives were synthesized and evaluated as potential ecto-5'-nucleotidase (CD73) inhibitors. Two synthetic routes were explored and the coupling of 4,6-disubstituted 3-cyano-2-chloro-pyridines with selected thiols allowed us to explore the structural diversity. Somehow divergent results were obtained in biological assays on CD73 inhibition using either the purified recombinant protein or cell-based assays, highlighting the difficulty to target protein-protein interface on proteins existing as soluble and membrane-bound forms. Among the 18 new derivatives obtained, three derivatives incorporating morpholino substituents on the 4,6-biaryl-2-thiopyridine core were shown to be able to reverse the adenosine-mediated immune suppression on human T cells. The higher blockade efficiency was observed for 2-((3-cyano-4,6-bis(4-morpholinophenyl)pyridin-2-yl)thio)-N-(isoxazol-3-yl)acetamide (with total reversion at 100 µM) and methyl 2-((3-cyano-4,6-bis(4-morpholinophenyl)pyridin-2-yl)thio)acetate (with partial reversion at 10 µM). Thus, this series of compounds illustrates a new chemotype of CD73 allosteric inhibitors.

Netrin-1 expression and targeting in multiple myeloma.

Deleted in colorectal cancer (DCC) and uncoordinated-5 (UNC5) receptors, play a key role in tumor progression of several solid tumors by inducing apoptosis when unbound to their ligand netrin-1. Netrin 1 is currently being evaluated as a therapeutic target. These receptors, known as dependence receptors, and their ligands, have not yet been extensively explored in hematological malignancies. Here, we performed a screening of various human myeloma cell lines and bone marrow samples from multiple myeloma patients for netrin-1 and its receptors to determine the expression of netrin 1 and its receptors in multiple myeloma as well as to assess the potential anti-myeloma activity of a novel anti-netrin-1 treatment (NP137). Our results showed heterogeneous expression of netrin-1 and its receptors DCC and UNC5H2(B) in six human myeloma lines. Additionally, immunohistochemistry and flow cytometry showed expression of these molecules in a majority of myeloma patient samples. In vitro NP137 did not induce apoptosis of myeloma cell lines yet enhanced the cytotoxicity of bortezomib and dexamethasone. In vivo, NP137 treatment of SCID mice with established RPMI8226 myeloma tumors led to a reduction of tumor size compared to controls. Ex vivo, NP137 lowered the plasma cells percentage in bone marrow aspirates in a fraction of the patient samples analyzed. These results suggest that netrin signaling could constitute a novel therapeutic target in multiple myeloma.

Cytotoxic and Antitumoral Activity of N-(9H-purin-6-yl) Benzamide Derivatives and Related Water-soluble Prodrugs.

BACKGROUND: The development of small molecules as cancer treatments is still of both interest and importance. OBJECTIVE: Having synthesized and identified the initial cytotoxic activity of a series of chemically related N-(9H-purin-6-yl) benzamide derivatives, we continued their evaluation on cancer cell models. We also synthesized water-soluble prodrugs of the main compound and performed in vivo experiments. METHOD: We used organic chemistry to obtain compounds of interest and prodrugs. The biological evaluation included MTT assays, synergy experiments, proliferation assays by CFSE, cell cycle distribution and in vivo antitumoral activity. RESULTS: Our results show activities on cancer cell lines ranging from 3-39 µM for the best compounds, with both induction of apoptosis and decrease in cell proliferation. Two compounds evaluated in vivo showed weak antitumoral activity. In addition, the lead compound and its prodrug had a synergistic activity with the nucleoside analogue fludarabine in vitro and in vivo. CONCLUSION: Our work allowed us to gain better knowledge on the activity of N-(9H-purin-6-yl) benzamide derivatives and showed new examples of water-soluble prodrugs. More research is warranted to decipher the molecular mechanisms of the molecules.

Variability in CD39 and CD73 protein levels in uveal melanoma patients.

Extracellular adenosine is produced from ATP by CD39 and CD73, and can modulate tumor development by acting on cancer cells or immune cells. Adenosine metabolism has been poorly studied in uveal melanoma. We studied the protein levels of CD39 and CD73 in a small, well described cohort of patients with uveal melanoma. Our results show a high variability in the levels of the two proteins, both in positivity and in intensity. Our results suggest that similar studies on larger cohorts could determine the clinical value and the druggability of these enzymes in the given clinical setting.Supplemental data for this article is available online at http://dx.doi.org/10.1080/15257770.2022.2032738.

Targeting the Nucleotide Metabolism Proteins of the NUDIX Family and SAMHD1 in Cancer.

Nucleotide metabolism has been targeted for many years and in various clinical settings, including cancer. The increased knowledge of certain enzymes involved in this metabolism and associated cellular processes accumulated over the last few years, gives important information related to the druggability of certain proteins and the use of inhibitors for others. Here, we review recent data on such enzymes with a major interest in drug development, i.e. SAMHD1 and the proteins of the NUDIX family. These include information on their roles in cancer progression, correlations with clinical outcomes in cancer patients, and the development and study of enzymatic inhibitors.

CD73 and cN-II regulate the cellular response to chemotherapeutic and hypoxic stress in lung adenocarcinoma cells.

BACKGROUND: Cytosolic 5'-nucleotidase II (cN-II) and ecto-5'-nucleotidase (CD73) are enzymes involved in the nucleotide metabolism by dephosphorylating nucleoside monophosphates. Both enzymes are involved in cancer by modifying anticancer drug activity, cancer cell biology and immune modulation. METHODS: We have modified lung cancer cells (NCI-H292) to become deficient for either or both enzymes using the CRISPR/Cas9 technique, and studied the implication of the two enzymes in the cellular response to different stress condition i.e. chemotherapeutic agents, hypoxia and nucleotide stress. RESULTS: Our results show that there is no significant role of these enzymes in cell proliferation under hypoxic stress. Similarly, cN-II and CD73 are not involved in wound healing ability under CoCl2-mediated HIF-1α stabilization. Furthermore, our results show that CD73-deficiency is associated with increased apoptosis in response to 1600 µM adenosine, decreased sensitivity to mitomycin and enhanced sensitivity to vincristine. cN-II deficiency increased in vivo tumor growth and sensitivity to vincristine and mitomycin C. CONCLUSIONS: Our study gives new insights into the biological roles of cN-II and CD73 under stress conditions in this particular cancer cell line. Further experiments will help deciphering the molecular mechanisms underlying the observed differences.

The Expression of Anti-Müllerian Hormone Type II Receptor (AMHRII) in Non-Gynecological Solid Tumors Offers Potential for Broad Therapeutic Intervention in Cancer.

The anti-Müllerian hormone (AMH) belongs to the TGF-ß family and plays a key role during fetal sexual development. Various reports have described the expression of AMH type II receptor (AMHRII) in human gynecological cancers including ovarian tumors. According to qRT-PCR results confirmed by specific In-Situ Hybridization (ISH) experiments, AMHRII mRNA is expressed in an extremely restricted number of normal tissues. By performing ISH on tissue microarray of solid tumor samples AMHRII mRNA was unexpectedly detected in several non-gynecological primary cancers including lung, breast, head and neck, and colorectal cancers. AMHRII protein expression, evaluated by immunohistochemistry (IHC) was detected in approximately 70% of epithelial ovarian cancers. Using the same IHC protocol on more than 900 frozen samples covering 18 different cancer types we detected AMHRII expression in more than 50% of hepato-carcinomas, colorectal, lung, and renal cancer samples. AMHRII expression was not observed in neuroendocrine lung tumor samples nor in non-Hodgkin lymphoma samples. Complementary analyses by immunofluorescence and flow cytometry confirmed the detection of AMHRII on a panel of ovarian and colorectal cancers displaying comparable expression levels with mean values of 39,000 and 50,000 AMHRII receptors per cell, respectively. Overall, our results suggest that this embryonic receptor could be a suitable target for treating AMHRII-expressing tumors with an anti-AMHRII selective agent such as murlentamab, also named 3C23K or GM102. This potential therapeutic intervention was confirmed in vivo by showing antitumor activity of murlentamab against AMHRII-expressing colorectal cancer and hepatocarcinoma Patient-Derived tumor Xenografts (PDX) models.

Enhancing the activity of platinum-based drugs by improved inhibitors of ERCC1-XPF-mediated DNA repair.

PURPOSE: The ERCC1-XPF 5'-3' DNA endonuclease complex is involved in the nucleotide excision repair pathway and in the DNA inter-strand crosslink repair pathway, two key mechanisms modulating the activity of chemotherapeutic alkylating agents in cancer cells. Inhibitors of the interaction between ERCC1 and XPF can be used to sensitize cancer cells to such drugs. METHODS: We tested recently synthesized new generation inhibitors of this interaction and evaluated their capacity to sensitize cancer cells to the genotoxic activity of agents in synergy studies, as well as their capacity to inhibit the protein-protein interaction in cancer cells using proximity ligation assay. RESULTS: Compound B9 showed the best activity being synergistic with cisplatin and mitomycin C in both colon and lung cancer cells. Also, B9 abolished the interaction between ERCC1 and XPF in cancer cells as shown by proximity ligation assay. Results of different compounds correlated with values from our previously obtained in silico predictions. CONCLUSION: Our results confirm the feasibility of the approach of targeting the protein-protein interaction between ERCC1 and XPF to sensitize cancer cells to alkylating agents, thanks to the improved binding affinity of the newly synthesized compounds.

Transcriptional and Metabolic Investigation in 5'-Nucleotidase Deficient Cancer Cell Lines.

Enzymes of nucleoside and nucleotide metabolism regulate important cellular processes with potential impacts on nucleotide-unrelated parameters. We have used a set of CRISPR/Cas9-modified cell models expressing both, one, or none of the 5'-nucleotidases cN-II and CD73, together with RNA sequencing and targeted metabolomics, to decipher new regulatory roles of these proteins. We observed important transcriptional modifications between models as well as upon exposure to adenosine. Metabolite content varied differently between cell models in response to adenosine exposure but was rather similar in control conditions. Our original cell models allowed us to identify a new unobvious link between proteins in the nucleotide metabolism and other cellular pathways. Further analyses of our models, including additional experiments, could help us to better understand some of the roles played by these enzymes.