Methylthioadenosine Metabolism Research Articles

Abstract 2395: Homozygous MTAP deletion in primary human glioblastoma is not associated with elevation of methylthioadenosine

Abstract Homozygous deletion of the CDK2NA locus frequently results in the co-deletion of methylthioadenosine phosphorylase (MTAP) in many fatal cancers such as glioblastoma multiform (GBM). In cell culture, cell lines with MTAP-deletions show elevations of its substrate metabolite, methylthioadenosine (MTA). High levels of MTA inhibit PRMT5, which sensitizes MTAP-deleted cell lines to PRMT5 and MAT2A inhibition. While extensively corroborated in vitro, the clinical efficacy of these strategies ultimately relies on equally significant accumulations of MTA in human tumors. In this work, using comprehensive metabolomic profiling, we show that MTA is primarily secreted, resulting in exceedingly high levels of extracellular MTA in vitro. We further show that primary human glioblastoma tumors minimally accumulate MTA in vivo, which is likely explained by the metabolism of MTA by MTAP-competent stromal cells. Together, these findings highlight the metabolic discrepancies between in vitro models and primary human tumors and should thus be carefully considered in the development of the precision therapies targeting MTAPhomozygous deleted GBM. Citation Format: Yasaman Barekatain, Jeffrey Ackroyd, Victoria Yan, Sunada Khadka, Ko-Chien Chen, Raghu Kalluri, John de Groot, Jason Huse, Florian muller. Homozygous MTAP deletion in primary human glioblastoma is not associated with elevation of methylthioadenosine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2395.

Homozygous MTAP deletion in primary human glioblastoma is not associated with elevation of methylthioadenosine

Homozygous deletion of methylthioadenosine phosphorylase (MTAP) in cancers such as glioblastoma represents a potentially targetable vulnerability. Homozygous MTAP-deleted cell lines in culture show elevation of MTAP’s substrate metabolite, methylthioadenosine (MTA). High levels of MTA inhibit protein arginine methyltransferase 5 (PRMT5), which sensitizes MTAP-deleted cells to PRMT5 and methionine adenosyltransferase 2A (MAT2A) inhibition. While this concept has been extensively corroborated in vitro, the clinical relevance relies on exhibiting significant MTA accumulation in human glioblastoma. In this work, using comprehensive metabolomic profiling, we show that MTA secreted by MTAP-deleted cells in vitro results in high levels of extracellular MTA. We further demonstrate that homozygous MTAP-deleted primary glioblastoma tumors do not significantly accumulate MTA in vivo due to metabolism of MTA by MTAP-expressing stroma. These findings highlight metabolic discrepancies between in vitro models and primary human tumors that must be considered when developing strategies for precision therapies targeting glioblastoma with homozygous MTAP deletion.

The Oncometabolite 5'-Deoxy-5'-Methylthioadenosine Blocks Multiple Signaling Pathways of NK Cell Activation.

Tumor cells develop various mechanisms to escape immune surveillance. In this context, oncometabolites secreted by tumor cells due to deregulated metabolic pathways, have been in the spotlight of researchers during the last years. 5′-Deoxy-5′-methylthioadenosine (MTA) phosphorylase (MTAP) deficiency in tumors results in the accumulation of MTA within the tumor microenvironment and thereby negatively influencing immune functions of various immune cells, including T and NK cells. The influence of MTA on T cell activation has been recently described in more detail, while its impact on NK cells is still largely unknown. Therefore, we aimed to illuminate the molecular mechanism of MTA-induced NK cell dysfunction. NK cell cytotoxicity against target cells was reduced in the presence of MTA in a dose-dependent manner, while NK cell viability remained unaffected. Furthermore, we revealed that MTA blocks NK cell degranulation and cytokine production upon target cell engagement as well as upon antibody stimulation. Interestingly, the immune-suppressive effect of MTA was less pronounced in healthy donors harboring an expansion of NKG2C+ NK cells. Finally, we demonstrated that MTA interferes with various signaling pathways downstream of the CD16 receptor upon NK cell activation, including the PI3K/AKT/S6, MAPK/ERK, and NF-κB pathways. In summary, we revealed that MTA blocks NK cell functions like cytotoxicity and cytokine production by interfering with the signaling cascade of activating NK cell receptors. Specific targeting of MTA metabolism in MTAP-deficient tumors therefore could offer a promising new strategy to reverse immune dysfunction of NK cells within the tumor microenvironment.

Microbial pathway for anaerobic 5′-methylthioadenosine metabolism coupled to ethylene formation

Numerous cellular processes involving S-adenosyl-l-methionine result in the formation of the toxic by-product, 5'-methylthioadenosine (MTA). To prevent inhibitory MTA accumulation and retain biologically available sulfur, most organisms possess the "universal" methionine salvage pathway (MSP). However, the universal MSP is inherently aerobic due to a requirement of molecular oxygen for one of the key enzymes. Here, we report the presence of an exclusively anaerobic MSP that couples MTA metabolism to ethylene formation in the phototrophic bacteria Rhodospirillum rubrum and Rhodopseudomonas palustris In vivo metabolite analysis of gene deletion strains demonstrated that this anaerobic MSP functions via sequential action of MTA phosphorylase (MtnP), 5-(methylthio)ribose-1-phosphate isomerase (MtnA), and an annotated class II aldolase-like protein (Ald2) to form 2-(methylthio)acetaldehyde as an intermediate. 2-(Methylthio)acetaldehyde is reduced to 2-(methylthio)ethanol, which is further metabolized as a usable organic sulfur source, generating stoichiometric amounts of ethylene in the process. Ethylene induction experiments using 2-(methylthio)ethanol versus sulfate as sulfur sources further indicate anaerobic ethylene production from 2-(methylthio)ethanol requires protein synthesis and that this process is regulated. Finally, phylogenetic analysis reveals that the genes corresponding to these enzymes, and presumably the pathway, are widespread among anaerobic and facultatively anaerobic bacteria from soil and freshwater environments. These results not only establish the existence of a functional, exclusively anaerobic MSP, but they also suggest a possible route by which ethylene is produced by microbes in anoxic environments.

Abstract 3971: MTA-mediated inhibition of human T cells: Mechanism and MTAP overexpression as putative overcoming strategy

Abstract Metabolic changes of malignant cells lead to the secretion of tumor metabolites which contribute to the shaping of a favorable milieu for tumor immune escape facilitating cancer development and resistance to anti-tumor immunotherapy. Recently, disrupted methylthioadenosine metabolism drew interest as further putative immunoinhibitory metabolic dysregulation. A broad spectrum of tumor entities was reported to lack methylthioadenosine phosphorylase (MTAP) expression leading to elevated levels of its substrate 5’-deoxy-5’-methylthioadenosine (MTA). MTAP deficiency is reported to correlate with worse response to adjuvant interferon-alpha therapy and a higher risk for metastatic disease in malignant melanoma providing evidence for immunobiological relevance. We therefore examined the effect of the tumor metabolite MTA on human T cell responses. We could show that MTA has a strong inhibitory impact on human T cell function regarding proliferation, viability, activation, differentiation, tumor antigen-specific expansion and cytotoxicity. We therefore aimed to overexpress MTAP in human T cells by retroviral transduction as a putative strategy to overcome MTA-mediated inhibition of T cells. In addition, we investigated the effect of MTA on T cell metabolism and maturation of human monocyte derived mDC. We successfully generated stable MTAP-overexpressing primary human T cells. First studies were performed to examine the resistance to MTA after equipping the T cells with higher levels of its metabolizing enzyme MTAP. MTAPhigh T cells revealed less sensitivity against inhibitory effects of MTA on proliferation, viability or cytotoxicity compared to mock transduced controls suggesting a promising approach to strengthen the performance of T cells facing the MTA-rich milieu of MTAP-deficient tumors. Mechanistically, we found MTA to interfere with several signaling pathways. Of most importance, MTA impaired Akt phosphorylation as well as intracellular protein methylation, both crucial processes for proper T cell function. In addition, we studied the combined effect of MTA and PRMT5 inhibition on T cells. Several metabolic studies focusing on glycolysis and fatty acid metabolism could confirm our findings that MTA keeps T cells in a rather inactive, naïve state. Finally, we found MTA to interfere with DC maturation and their potential to induce cytokine secretion of T cells. Our data emphasizes the crucial role of tumor metabolites such as MTA in the tumor milieu for tumor immune escape. Additionally, the identification of potential molecular mechanisms of MTA-induced immunosuppression offer the opportunity to pharmacologically tackle MTA-mediated immunoinhibition and will help to develop more effective immune-based therapies against MTAP-deficient tumors. Citation Format: Carolin D. Strobl, Frederik Henrich, Katrin Singer, Katrin Peter, Marina Kreutz, Anita Kremer, Andreas Mackensen, Michael Aigner. MTA-mediated inhibition of human T cells: Mechanism and MTAP overexpression as putative overcoming strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3971. doi:10.1158/1538-7445.AM2017-3971

Salmonella enterica MTAN at 1.36 Å Resolution: A Structure-Based Design of Tailored Transition State Analogs

Accumulation of 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH) in bacteria disrupts the S-adenosylmethionine pool to alter biological methylations, synthesis of polyamines, and production of quorum-sensing molecules. Bacterial metabolism of MTA and SAH depends on MTA/SAH nucleosidase (MTAN), an enzyme not present in humans and a target for quorum sensing because MTAN activity is essential for synthesis of autoinducer-2 molecules. Crystals of Salmonella enterica MTAN with product and transition state analogs of MTA and SAH explain the structural contacts causing pM binding affinity for the inhibitor and reveal a "water-wire" channel for the catalytic nucleophile. The crystal structure shows an extension of the binding pocket filled with polyethylene glycol. We exploited this discovery by the design and synthesis of tailored modifications of the currently existing transition state analogs to fill this site. This site was not anticipated in MTAN structures. Tailored inhibitors with dissociation constants of 5 to 15 pM are characterized.

Methionine salvage and S-adenosylmethionine: essential links between sulfur, ethylene and polyamine biosynthesis

Both Met (methionine) and SAM (S-adenosylmethionine), the activated form of Met, participate in a number of essential metabolic pathways in plants. The subcellular compartmentalization of Met fluxes will be discussed in the present review with respect to regulation and communication with the sulfur assimilation pathway, the network of the aspartate-derived amino acids and the demand for production of SAM. SAM enters the ethylene, nicotianamine and polyamine biosynthetic pathways and provides the methyl group for the majority of methylation reactions required for plant growth and development. The multiple essential roles of SAM require regulation of its synthesis, recycling and distribution to sustain these different pathways. A particular focus of the present review will be on the function of recently identified genes of the Met salvage cycle or Yang cycle and the importance of the Met salvage cycle in the metabolism of MTA (5'-methylthioadenosine). MTA has the potential for product inhibition of ethylene, nicotianamine and polyamine biosynthesis which provides an additional link between these pathways. Interestingly, regulation of Met cycle genes was found to differ between plant species as shown for Arabidopsis thaliana and Oryza sativa.

Methylthioinosine Phosphorylase fromPseudomonas aeruginosa. Structure and Annotation of a Novel Enzyme in Quorum Sensing

The PA3004 gene of Pseudomonas aeruginosa PAO1 was originally annotated as a 5'-methylthioadenosine phosphorylase (MTAP). However, the PA3004 encoded protein uses 5'-methylthioinosine (MTI) as a preferred substrate and represents the only known example of a specific MTI phosphorylase (MTIP). MTIP does not utilize 5'-methylthioadenosine (MTA). Inosine is a weak substrate with a k(cat)/K(m) value 290-fold less than MTI and is the second best substrate identified. The crystal structure of P. aeruginosa MTIP (PaMTIP) in complex with hypoxanthine was determined to 2.8 Å resolution and revealed a 3-fold symmetric homotrimer. The methylthioribose and phosphate binding regions of PaMTIP are similar to MTAPs, and the purine binding region is similar to that of purine nucleoside phosphorylases (PNPs). The catabolism of MTA in P. aeruginosa involves deamination to MTI and phosphorolysis to hypoxanthine (MTA → MTI → hypoxanthine). This pathway also exists in Plasmodium falciparum, where the purine nucleoside phosphorylase (PfPNP) acts on both inosine and MTI. Three tight-binding transition state analogue inhibitors of PaMTIP are identified with dissociation constants in the picomolar range. Inhibitor specificity suggests an early dissociative transition state for PaMTIP. Quorum sensing molecules are associated with MTA metabolism in bacterial pathogens suggesting PaMTIP as a potential therapeutic target.

Inhibition of 5’-methylthioadenosine metabolism in the Yang cycle alters polyamine levels, and impairs seedling growth and reproduction in Arabidopsis

The methionine or Yang cycle recycles Met from 5'-methylthioadenosine (MTA) which is produced from S-adenosyl-L-methionine (SAM) as a by-product of ethylene, polyamines, and nicotianamine (NA) synthesis. MTA nucleosidase is encoded by two genes in Arabidopsis thaliana, MTN1 and MTN2. Analysis of T-DNA insertion mutants and of wt revealed that MTN1 provides approximately 80% of the total MTN activity. Severe knock down of MTN enzyme activity in the mtn1-1 and mtn1-2 allelic lines resulted in accumulation of SAM/dSAM (decarboxylated SAM) and of MTA in seedlings grown on MTA as sulfur source. While ethylene and NA synthesis were not altered in mtn1-1 and mtn1-2 seedlings grown on MTA, putrescine and spermine were elevated. By contrast, mtn2-1 and mtn2-2 seedlings with near wt enzyme activity had wt levels of SAM/dSAM, MTA, and polyamines. In addition to the metabolic phenotypes, mtn1-1 and mtn1-2 seedlings were growth retarded, while seedlings of wt, mtn2-1, and mtn2-2 showed normal growth on 500 microm MTA. The double knock down mutant mtn1-1/mtn2-1 was sterile. In conclusion, the data presented identify MTA as a crucial metabolite that acts as a regulatory link between the Yang cycle and polyamine biosynthesis and identifies MTA nucleosidase as a crucial enzyme of the Yang cycle.

Enzymatic Transition State Theory and Transition State Analogue Design

Enzymatic Transition State Theory and Transition State Analogue Design

Methylthioadenosine and polyamine biosynthesis in a Saccharomyces cerevisiae meu1Δ mutant

As part of our studies on polyamine biosynthesis in yeast, the metabolism of methylthioadenosine was studied in a mutant that lacks methylthioadenosine phosphorylase ( meu1Δ). The nucleoside accumulates in this mutant and is mainly excreted into the culture medium. Intracellular accumulation of the nucleoside is enough to account for the inhibition of spermidine synthase and thus to indirectly regulate the polyamine content of the meu1Δ cells. By comparing the results with this mutant with a meu1Δ spe2Δ mutant that cannot synthesize spermidine or spermine, we showed that >98% of methylthioadenosine is produced as a byproduct of polyamine synthesis (i.e., from decarboxylated S-adenosylmethionine). In contrast, in MEU1 + SPE2 + cells methylthioadenosine does not accumulate and is metabolized through the methionine salvage pathway. Using a met15Δ mutant we show that this pathway (i.e., involving polyamine biosynthesis and methylthioadenosine metabolism) is a significant factor in the metabolism of methionine, accounting for 15% of the added methionine.

Bayesian phylogenetic analysis reveals two-domain topology of S-adenosylhomocysteine hydrolase protein sequences

S-Adenosylhomocysteine hydrolase (SahH) is involved in the degradation of the compound which inhibits methylation reactions. Using a Bayesian approach and other methods, we reconstructed a phylogenetic tree of amino acid sequences of this protein originating from all three major domains of living organisms. The SahH sequences formed two major branches: one composed mainly of Archaea and the other of eukaryotes and majority of bacteria, clearly contradicting the three-domain topology shown by small subunit rRNA gene. This topology suggests the occurrence of lateral transfer of this gene between the domains. Poor resolution of eukaryotes and bacteria excluded an ultimate conclusion in which out of the two domains this gene appeared first, however, the congruence of the secondary branches with SS rRNA and/or concatenated ribosomal protein datasets phylogenies suggested an “early” acquisition by some bacterial and eukaryotic phyla. Similarly, the branching pattern of Archaea reflected the phylogenies shown by SS rRNA and ribosomal proteins. SahH is widespread in Eucarya, albeit, due to reductive evolution, it is missing in the intracellular parasite Encephalitozoon cuniculi. On the other hand, the lack of affinity to the sequences from the α- Proteobacteria and cyanobacteria excludes a possibility of its acquisition in the course of mitochondrial or chloroplast endosymbioses. Unlike Archaea, most bacteria carry MTA/SAH nucleosidase, an enzyme involved also in metabolism of methylthioadenosine. However, the double function of MTA/SAH nucleosidase may be a barrier to ensure the efficient degradation of S-adenosylhomocysteine, specially when the intensity of methylation processes is high. This would explain the presence of S-adenosylhomocysteine hydrolase in the bacteria that have more complex metabolism. On the other hand, majority of obligate pathogenic bacteria due to simpler metabolism rely entirely on MTA/SAH nucleosidase. This could explain the observed phenetic pattern in which bacteria with larger (>6 Mb—million base pairs) genomes carry SAH hydrolase, whereas bacteria that have undergone reductive evolution usually carry MTA/SAH nucleosidase. This suggests that the presence or acquisition of S-adenosylhomocysteine hydrolase in bacteria may predispose towards higher metabolic, and in consequence, higher genomic complexity. The good examples are the phototrophic bacteria all of which carry this gene, however, the SahH phylogeny shows lack of congruence with SSU rRNA and photosyntethic genes, implying that the acquisition was independent and presumably preceded the acquisition of photosyntethic genes. The majority of cyanobacteria acquired this gene from Archaea, however, in some species the sahH gene was replaced by a copy from the β- or γ- Proteobacteria.

METABOLISM OF 5′-METHYLTHIOADENOSINE IN METHIONINE DEPENDENT AND INDEPENDENT CELLS: 33

Methionine (Met) dependency was investigated in a Met-dependent (CCL 39) and in a Met-independent (Raji) cell line. Met dependency was similarly observed with two Met precursors, L-homocysteine and 5'-deoxy-5'-methylthioadenosine (MTA), suggesting that this phenomenon represents, in fact, the inability of a cell to grow on a metabolic precursor of Met. The study of MTA metabolism in CCL 39 and Raji cells indicates that Met dependency is not due to the inability to synthetize Met from MTA, nor to incorporate newly synthetized Met into proteins, nor into S-adenosylmethionine (SAM). However, there are differences in SAM metabolism and disposition between CCL 39 and Raji cells. The synthesis of SAM from labelled MTA is higher in CCL 39 than in Raji cells, but SAM is much more excreted by CCL 39 than by Raji cells, with ratios of extracellular to intracellular SAM of 20.3 and 5.1, respectively. However, when the cells are incubated with labelled Met, the synthesis of SAM is similar in both types of cells, and the ratios of extracellular to intracellular SAM are much lower than in the previous case (0.8 and 1.0 for CCL 39 and Raji cells, respectively). These results, and the fact that exogenous unlabelled Met moderately affects the incorporation of labelled MTA into intracellular SAM by CCL 39 cells, and profoundly depresses it in Raji cells, support the hypothesis of a metabolic compartmentation of exogenous and endogenous Met in Met-dependent cells.

Studies on the metabolic effects of methylthioformycin

1. 5′-Methylthioformycin, a structural analog of 5′-methylthioadenosine in which the N-C glycosidic bond is substituted by a C-C bond, has been synthesized by a newly developed procedure. 2. Membrane permeability of the molecule has been compared to that of methylthioadenosine in intact human erythrocytes and Friend erythroleukemia cells. The formycinyl compound is taken up with a rate significantly lower than that of 5′-methylthioadenosine and is not metabolized by the cells. 3. 5′-Methylthioformycin inhibits Friend erythroleukemia cells' growth: the effect is dose-dependent, fully reversible and not caused by cytotoxicity. 4. Several enzymes related to methylthioadenosine metabolism are inhibited by methylthioformycin. Rat liver methylthioadenosine phosphorylase is competitively inhibited with a K i value of 2 μM. Among the propylamine transferases tested only rat brain spermine synthase is significantly inhibited, while rat brain spermidine synthase is less sensitive. Rat liver S-adenosylhomocysteine hydrolase is irreversibly inactivated with 50% inhibition at 400 μM methylthioformycin. 5′-Methylthioformycin does not exert any significant effect on protein carboxyl- O-methyltransferase. Inferences about the mechanism of the antiproliferative effect of the drug have been drawn from the above results.

Transport and metabolism of 5′-methylthioadenosine in human erythrocytes

The transport and metabolism of 5′- deoxy-5′-S- methylthioadenosine have been studied in intact human erythrocytes. The sulfur nucleoside is rapidly accumulated into red cells and the extent of uptake largely exceeds the theoretical equilibrium between inner and outer compartment owing to its conversion into a non-permeable compound, namely 5-methylthioribose 1-phosphate. To characterize the nucleoside transport, phosphate-depleted erythrocytes, in which the methylthioadenosine metabolism is negligible, have been employed. The results indicate that: (i) the transport occurs via a facilitated-diffusion mechanism; (ii) the process is not energy-dependent and (iii) no specific cation is required. The kinetic analyses of both the transport and the metabolism shows that the uptake of methylthioadenosine is a result of the tandem action of a transport step of high capacity ( V max = 604 ± 51 pmol/10 6 cells per min ) and low affinity ( K m = 3270 ± 321 μ M ) followed by a metabolic step of low capacity ( V max = 6.6 pmol/10 6 cells per min ) and high affinity ( K m = 30 μ M ). Furthermore, a substrate inhibition exerted by methylthioadenosine at high concentration (over 200 μM) on its specific phosphorylase is reported for the first time. Experiments performed with several analogs of the thioether indicate that the adenine amino group and the hydrophobic substituent at the 5′-position are critical for the transport carrier recognition. Adenine is the most powerful inhibitor of methylthioadenosine transport.

Methylthioadenosine metabolism in malignant murine cells

An ether-extractable product formed from 5′-methylthioadenosine by extracts of malignant murine lymphocytic cells is shown to be 2-keto-4-methylthiobutyric acid. When 5′-methylthio [U- 14C]adenosine was used as substrate, the product was labelled, confirming earlier reports that carbons of the keto acid are derived from carbons of the ribose. When hydroxylamine was added to the reaction mixture, the ketomethylthiobutyric acid was trapped as the oxime. When glutamine was added, the main product was methionine.

5′-Methylthioadenosine metabolism and methionine synthesis in mammalian cells grown in culture

The biosynthesis of methionine from 5′-methylthioadenosine was examined in a number of human and mouse cell lines. 5′-Methylthioadenosine added to the culture medium was rapidly converted to methionine, accumulating in cell protein. J111 cells and mouse spleen fibroblasts grew significantly in a medium in which 5′-methylthioadenosine replaced methionine. L1210 cells, which lack 5′-methylthioadenosine phosphorylase, did not grow in this medium, and human breast fibroblasts did not grow either, even though these cells have normal levels of 5′-methylthioadenosine phosphorylase.

Uptake and utilization of 5'-methylthioadenosine by cultured baby-hamster kidney cells

5'-Methylthioadenosine was taken up and immediately metabolized further by cultured baby-hamster kidney cells during the exponential phase of growth. The adenine moiety supplied the purine-nucleotide pool via the salvage pathway and was efficiently incorporated into nucleic acids. Catabolites of methylthioadenosine excreted by the cells included adenine, purinic compounds and metabolites of the ribose portion. 5'-Methylthiotubercidin had no significant effect on the cellular metabolism of methyl-thioadenosine, but greatly inhibited its uptake. erythro-9-(2-Hydroxy-3-nonyl)adenine had no effect on the uptake, but markedly interfered with the further utilization of methylthioadenosine after cleavage in the cells.