Extracellular 5'-methylthioadenosine inhibits intracellular symmetric dimethylarginine protein methylation of FUSE-binding proteins.

Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway that converts the polyamine synthesis byproduct 5'-deoxy-5'-methylthioadenosine (MTA) into methionine. Inactivation of MTAP, often by homozygous deletion, is found in both solid and hematologic malignancies and is one of the most frequently observed genetic alterations in human cancer. Previous work established that MTAP-deleted cells accumulate MTA and contain decreased amounts of proteins with symmetric dimethylarginine (sDMA). These findings led to the hypothesis that accumulation of intracellular MTA inhibits the protein arginine methylase (PRMT5) responsible for bulk protein sDMAylation. Here, we confirm that MTAP-deleted cells have increased MTA accumulation and reduced protein sDMAylation. However, we also show that addition of extracellular MTA can cause a dramatic reduction of the steady-state levels of sDMA-containing proteins in MTAP+ cells, even though no sustained increase in intracellular MTA is found because of catabolism of MTA by MTAP. We determined that inhibition of protein sDMAylation by MTA occurs within 48 h, is reversible, and is specific. In addition, we have identified two enhancer-binding proteins, FUBP1 and FUBP3, that are differentially sDMAylated in response to MTAP and MTA. These proteins work via the far upstream element site located upstream of Myc and other promoters. Using a transcription reporter construct containing the far upstream element site, we demonstrate that MTA addition can reduce transcription, suggesting that the reduction in FUBP1 and FUBP3 sDMAylation has functional consequences. Overall, our findings show that extracellular MTA can inhibit protein sDMAylation and that this inhibition can affect FUBP function.

Quantitation of cellular metabolic fluxes of methionine.

Methionine is an essential proteogenic amino acid. In addition, it is a methyl donor for DNA and protein methylation and a propylamine donor for polyamine biosynthesis. Both the methyl and propylamine donation pathways involve metabolic cycles, and methods are needed to quantitate these cycles. Here, we describe an analytical approach for quantifying methionine metabolic fluxes that accounts for the mixing of intracellular and extracellular methionine pools. We observe that such mixing prevents isotope tracing experiments from reaching the steady state due to the large size of the media pools and hence precludes the use of standard stationary metabolic flux analysis. Our approach is based on feeding cells with (13)C methionine and measuring the isotope-labeling kinetics of both intracellular and extracellular methionine by liquid chromatography-mass spectrometry (LC-MS). We apply this method to quantify methionine metabolism in a human fibrosarcoma cell line and study how methionine salvage pathway enzyme methylthioadenosine phosphorylase (MTAP), frequently deleted in cancer, affects methionine metabolism. We find that both transmethylation and propylamine transfer fluxes amount to roughly 15% of the net methionine uptake, with no major changes due to MTAP deletion. Our method further enables the quantification of flux through the pro-tumorigenic enzyme ornithine decarboxylase, and this flux increases 2-fold following MTAP deletion. The analytical approach used to quantify methionine metabolic fluxes is applicable for other metabolic systems affected by mixing of intracellular and extracellular metabolite pools.

Specific Targeting of MTAP-Deleted Tumors with a Combination of 2'-Fluoroadenine and 5'-Methylthioadenosine.

Homozygous deletion of the methylthioadenosine phosphorylase (MTAP) gene is a frequent event in a wide variety of human cancers and is a possible molecular target for therapy. One potential therapeutic strategy to target MTAP-deleted tumors involves combining toxic purine analogues such as 6'-thioguanine (6TG) or 2'-fluoroadenine (2FA) with the MTAP substrate 5'-deoxy-5'-methylthioadenosine (MTA). The rationale is that excess MTA will protect normal MTAP+ cells from purine analogue toxicity because MTAP catalyzes the conversion of MTA to adenine, which then inhibits the conversion of purine base analogues into nucleotides. However, in MTAP- tumor cells, no protection takes place because adenine is not formed. Here, we examine the effects of 6TG and 2FA in combination with MTA in vitro and in vivoIn vitro, MTA protected against both 6TG and 2FA toxicity in an MTAP-dependent manner, shifting the IC50 concentration by one to three orders of magnitude. However, in mice, MTA protected against toxicity from 2FA but failed to protect against 6TG. Addition of 100 mg/kg MTA to 20 mg/kg 2FA entirely reversed the toxicity of 2FA in a variety of tissues and the treatment was well tolerated by mice. The 2FA+MTA combination inhibited tumor growth of four different MTAP- human tumor cell lines in mouse xenograft models. Our results suggest that 2FA+MTA may be a promising combination for treating MTAP-deleted tumors.Significance: Loss of MTAP occurs in about 15% of all human cancers; the MTAP protection strategy presented in this study could be very effective in treating these cancers. Cancer Res; 78(15); 4386-95. ©2018 AACR.

The methionine salvage pathway compound 4-methylthio-2-oxobutanate causes apoptosis independent of down-regulation of ornithine decarboxylase.

4-Methylthio-2-oxobutanoic acid (MTOB) is the final compound of the methionine salvage pathway that converts the polyamine byproduct methylthioadenosine to adenine and methionine. Here we find that MTOB inhibits growth of several human cell lines in a dose-dependent manner. Growth inhibition was specific for MTOB as we did not observe any inhibition with other chemically related compounds. MTOB treatment causes apoptosis and reduction of ornithine decarboxylase (ODC) activity but not ODC mRNA. To determine if MTOB exerts its effects primarily via ODC inhibition, we compared the effects of MTOB with the ODC-specific inhibitor difluoromethylornithine (DFMO). We found that MTOB was a more potent inducer of apoptosis than DFMO, lacked activation of caspase 3/7, and was able to induce apoptosis in cells lacking p53. Our results show that MTOB-induced growth inhibition and apoptosis is not simply secondary due to ODC inhibition and implies that MTOB activates apoptosis via other mechanisms.

Loss of methylthioadenosine phosphorylase and elevated ornithine decarboxylase is common in pancreatic cancer.

PURPOSE: Loss of the methylthioadenosine phosphorylase (MTAP) gene at 9p21 is observed frequently in a variety of human cancers. We have shown previously that MTAP can act as a tumor suppressor gene and that its tumor suppressor function is related to its effect on polyamine homeostasis. Ornithine decarboxylase is a key enzyme in the regulation of polyamine metabolism. The aim of this study is to analyze MTAP and ornithine decarboxylase (ODC) expression in primary pancreatic tumor specimens. EXPERIMENTAL DESIGN: We measured MTAP and ODC activity in protein extracts derived from 30 surgically resected tumor samples and eight normal pancreas samples. In a subset of six samples, we also examined MTAP DNA using interphase fluorescence in situ hybridization. In addition, we examined the effect of the ODC inhibitor difluoromethylornithine on two pancreatic adenocarcinoma-derived cell lines. RESULT: MTAP activity was 2.8-fold reduced in adenocarcinomas and 6.3-fold reduced in neuroendocrine tumors compared with control pancreas. Conversely, ODC activity was 3.6-fold elevated in adenocarcinomas and 3.9-fold elevated in neuroendocrine tumors compared with control pancreas. Using interphase fluorescence in situ hybridization, we found in tumor samples that 43 to 75% of the nuclei had lost at least one copy of MTAP locus, indicating that loss of MTAP activity was at least partially because of deletion of the MTAP locus. We also show that inhibition of ODC by difluoromethylornithine caused decreased cell growth and increased apoptosis in two MTAP-deleted pancreatic adenocarcinoma-derived cell lines. CONCLUSIONS: MTAP activity is frequently lost, and ODC activity is frequently elevated in both pancreatic adenocarcinoma and neuroendocrine tumors. Inhibition of ODC activity caused decreased cell growth and increased apoptosis in pancreatic tumor-derived cell lines. These findings suggest that MTAP and polyamine metabolism could be potential therapeutic targets in the treatment of pancreatic cancer.

Expression of MTAP inhibits tumor-related phenotypes in HT1080 cells via a mechanism unrelated to its enzymatic function.

Methylthioadenosine Phosphorylase (MTAP) is a tumor suppressor gene that is frequently deleted in human cancers and encodes an enzyme responsible for the catabolism of the polyamine byproduct 5'deoxy-5'-methylthioadenosine (MTA). To elucidate the mechanism by which MTAP inhibits tumor formation, we have reintroduced MTAP into MTAP-deleted HT1080 fibrosarcoma cells. Expression of MTAP resulted in a variety of phenotypes, including decreased colony formation in soft-agar, decreased migration, decreased in vitro invasion, increased matrix metalloproteinase production, and reduced ability to form tumors in severe combined immunodeficiency mice. Microarray analysis showed that MTAP affected the expression of genes involved in a variety of processes, including cell adhesion, extracellular matrix interaction, and cell signaling. Treatment of MTAP-expressing cells with a potent inhibitor of MTAP's enzymatic activity (MT-DADMe-ImmA) did not result in a MTAP- phenotype. This finding suggests that MTAP's tumor suppressor function is not the same as its known enzymatic function. To confirm this, we introduced a catalytically inactive version of MTAP, D220A, into HT1080 cells and found that this mutant was fully capable of reversing the soft agar colony formation, migration, and matrix metalloproteinase phenotypes. Our results show that MTAP affects cellular phenotypes in HT1080 cells in a manner that is independent of its known enzymatic activity.

Germline Mutations in Mtap Cooperate with Myc to Accelerate Tumorigenesis in Mice.

OBJECTIVE: The gene encoding the methionine salvage pathway methylthioadenosine phosphorylase (MTAP) is a tumor suppressor gene that is frequently inactivated in a wide variety of human cancers. In this study, we have examined if heterozygosity for a null mutation in Mtap (Mtap(lacZ)) could accelerate tumorigenesis development in two different mouse cancer models, Eµ-myc transgenic and Pten(+/-) . METHODS: Mtap Eµ-myc and Mtap Pten mice were generated and tumor-free survival was monitored over time. Tumors were also examined for a variety of histological and protein markers. In addition, microarray analysis was performed on the livers of Mtap(lacZ/+) and Mtap (+/+) mice. RESULTS: Survival in both models was significantly decreased in Mtap(lacZ/+) compared to Mtap(+/+) mice. In Eµ-myc mice, Mtap mutations accelerated the formation of lymphomas from cells in the early pre-B stage, and these tumors tended to be of higher grade and have higher expression levels of ornithine decarboxylase compared to those observed in control Eµ-myc Mtap(+/+) mice. Surprisingly, examination of Mtap status in lymphomas in Eµ-myc Mtap(lacZ/+) and Eµ-myc Mtap(+/+) animals did not reveal significant differences in the frequency of loss of Mtap protein expression, despite having shorter latency times, suggesting that haploinsufficiency of Mtap may be playing a direct role in accelerating tumorigenesis. Consistent with this idea, microarray analysis on liver tissue from age and sex matched Mtap(+/+) and Mtap(lacZ/+) animals found 363 transcripts whose expression changed at least 1.5-fold (P<0.01). Functional categorization of these genes reveals enrichments in several pathways involved in growth control and cancer. CONCLUSION: Our findings show that germline inactivation of a single Mtap allele alters gene expression and enhances lymphomagenesis in Eµ-myc mice.

Increasing the therapeutic index of 5-fluorouracil and 6-thioguanine by targeting loss of MTAP in tumor cells.

Methylthioadenosine phosphorylase (MTAP), a key enzyme in the catabolism of 5'-deoxy-5'-methylthioadenosine (MTA), catalyzes the formation of adenine and 5-methylthioribose-1-phosphate. MTAP is expressed in all cells throughout the body, but a significant percentage of human tumors have lost MTAP expression, thereby making MTAP-loss a potential therapeutic target. Here, we have tested an MTAP-targeting strategy based on the idea that MTAP-expressing cells can be protected from toxic purine and uracil analogs by addition of MTA, but MTAP-deleted tumor cells cannot. Addition of as little as 10 µM MTA could entirely protect isogenic MTAP (+) , but not MTAP (-) , HT1080 cells from toxicity caused by the chemotherapy agents 6-thioguanine (6TG) or 5-fluorouracil (5FU). Inhibitor studies showed that MTA protection requires functional MTAP activity. Addition of adenine protected both MTAP (+) and MTAP (-) cells from 6TG and 5FU, consistent with the idea that adenine produced from the MTAP reaction competes with 6TG and 5FU for a rate limiting pool of phosphoribosyl-1-pyrophosphate (PRPP), which is required for the conversion of purine and uracil bases into nucleotides. Extracellular MTA can also protect mouse mesothelioma cells from killing by 6-TG or the drug L-alanosine in an MTAP-dependent manner. In addition, MTA can protect non-transformed MTAP (+) mouse embryo fibroblasts from 6TG toxicity. Taken together, our data suggest that the addition of MTA to anti-purine-based chemotherapy may greatly increase the therapeutic index of this class of drugs if used specifically to treat MTAP (-) tumors.

Chemical genetic screening for compounds that preferentially inhibit growth of methylthioadenosine phosphorylase (MTAP)-deficient Saccharomyces cerevisiae.

Methylthioadenosine phosphorylase (MTAP), a key enzyme in the methionine salvage pathway, is inactivated in a variety of human cancers. Since all human tissues express MTAP, it would be of potential interest to identify compounds that selectively inhibit the growth of MTAP-deficient cells. To determine if MTAP inactivation could be targeted, the authors have performed a differential chemical genetic screen in isogenic MTAP(+) and MTAP(-) Saccharomyces cerevisiae. A low molecular weight compound library containing 30,080 unique compounds was screened for those that selectively inhibit growth of MTAP(-) yeast using a differential growth assay. One compound, containing a 1,3,4-thiadiazine ring, repeatedly showed a differential dose response, with MTAP(-) cells exhibiting a 4-fold shift in IC(50) compared to MTAP(+) cells. Several structurally related derivatives of this compound also showed enhanced growth inhibition in MTAP(-) yeast. These compounds were also examined for growth inhibition of isogenic MTAP(+) and MTAP(-) HT1080 fibrosarcoma cells, and 4 of the 5 compounds exhibited evidence of modest but significant increased potency in MTAP(-) cells. In summary, these studies show the feasibility of differential growth screening technology and have identified a novel class of compounds that can preferentially inhibit growth of MTAP(-) cells.

Methionine-deficient diet induces post-transcriptional downregulation of cystathionine ß-synthase.

OBJECTIVE: Elevated plasma total homocysteine (tHcy) is a risk factor for a variety of human diseases. Homocysteine is formed from methionine and has two primary metabolic fates: remethylation to form methionine or commitment to the transsulfuration pathway by the action of cystathionine ß-synthase (CBS). We have examined the metabolic response in mice of a shift from a methionine-replete to a methionine-free diet. METHODS AND RESULTS: We found that shifting 3-mo-old C57BL6 mice to a methionine-free diet caused a transient increase in tHcy and an increase in the tHcy/methionine ratio. Because CBS is a key regulator of tHcy, we examined CBS protein levels and found that within 3 d on the methionine-deficient diet, animals had a 50% reduction in the levels of liver CBS protein and enzyme activity. Examination of CBS mRNA and studies of transgenic animals that express CBS from a heterologous promoter indicated that this reduction is occurring post-transcriptionally. Loss of CBS protein was unrelated to intracellular levels of S-adenosylmethionine, a known regulator of CBS activity and stability. CONCLUSION: Our results imply that methionine deprivation induces a metabolic state in which methionine is effectively conserved in tissue by shutdown of the transsulfuration pathway by an S-adenosylmethionine-independent mechanism that signals a rapid downregulation of CBS protein.

Mice heterozygous for germ-line mutations in methylthioadenosine phosphorylase (MTAP) die prematurely of T-cell lymphoma.

Large homozygous deletions of 9p21 that inactivate CDKN2A, ARF, and MTAP are common in a wide variety of human cancers. The role for CDKN2A and ARF in tumorigenesis is well established, but whether MTAP loss directly affects tumorigenesis is unclear. MTAP encodes the enzyme methylthioadenosine phosphorylase, a key enzyme in the methionine salvage pathway. To determine if loss of MTAP plays a functional role in tumorigenesis, we have created an MTAP-knockout mouse. Mice homozygous for a MTAP null allele (Mtap(lacZ)) have an embryonic lethal phenotype dying around day 8 postconception. Mtap/Mtap(lacZ) heterozygotes are born at Mendelian frequencies and appear indistinguishable from wild-type mice during the first year of life, but they tend to die prematurely with a median survival of 585 days. Autopsies on these animals reveal that they have greatly enlarged spleens, altered thymic histology, and lymphocytic infiltration of their livers, consistent with lymphoma. Immunohistochemical staining and fluorescence-activated cell sorting analysis indicate that these lymphomas are primarily T-cell in origin. Lymphoma-infiltrated tissues tend to have reduced levels of Mtap mRNA and MTAP protein in addition to unaltered levels of methyldeoxycytidine. These studies show that Mtap is a tumor suppressor gene independent of CDKN2A and ARF.

Expression of mutant human cystathionine beta-synthase rescues neonatal lethality but not homocystinuria in a mouse model.

Cystathionine beta-synthase (CBS) deficiency is a recessive genetic disorder in humans characterized by elevated levels of total plasma homocysteine (tHcy) and frequent thrombosis in humans. The I278T mutation is the most common mutation found in human CBS-deficient patients. The T424N mutation was identified as a mutation in human CBS that could restore function to I278T in Saccharomyces cerevisiae. In this report, we have engineered mice that express human I278T and I278T/T424N proteins from a metallotheinein-driven transgene. These transgene-containing mice were then bred to CBS knockout animals (Cbs-) to generate mice that express only human I278T or I278T/T424N protein. Both the I278T and the I278T/T424N transgenes are able to entirely rescue the previously described neonatal mortality phenotype despite the animals having a mean tHcy of 250 microm. The transgenic Cbs-/- animals exhibit facial alopecia, have moderate liver steatosis and are slightly smaller than heterozygous littermates. In contrast to human CBS deficiency, these mice do not exhibit extreme methioninemia. The mutant proteins are stable in the liver, kidney and colon, and liver extracts have only 2-3% of the CBS enzyme activity found in wild-type mice. Surprisingly, the I278T/T424N enzyme had exactly the same activity as the I278T enzyme indicating that T424N is unable to suppress I278T in mice. Our results show that elevated tHcy per se is not responsible for the neonatal lethality observed in Cbs-/- animals and suggests that CBS protein may have a function in addition to its role in homocysteine catabolism. These transgenic animals should be useful in the study of homocysteine related human disease.

Methylthioadenosine phosphorylase regulates ornithine decarboxylase by production of downstream metabolites.

The gene encoding methylthioadenosine phosphorylase (MTAP), the initial enzyme in the methionine salvage pathway, is deleted in a variety of human tumors and acts as a tumor suppressor gene in cell culture (Christopher, S. A., Diegelman, P., Porter, C. W., and Kruger, W. D. (2002) Cancer Res. 62, 6639-6644). Overexpression of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) is frequently observed in tumors and has been shown to be tumorigenic in vitro and in vivo. In this paper, we demonstrate a novel regulatory pathway in which the methionine salvage pathway products inhibit ODC activity. We show that in Saccharomyces cerevisiae the MEU1 gene encodes MTAP and that Meu1delta cells have an 8-fold increase in ODC activity, resulting in large elevations in polyamine pools. Mutations in putative salvage pathway genes downstream of MTAP also cause elevated ODC activity and elevated polyamines. The addition of the penultimate salvage pathway compound 4-methylthio-2-oxobutanoic acid represses ODC levels in both MTAP-deleted yeast and human tumor cell lines, indicating that 4-methylthio-2-oxobutanoic acid acts as a negative regulator of polyamine biosynthesis. Expression of MTAP in MTAP-deleted MCF-7 breast adenocarcinoma cells results in a significant reduction of ODC activity and reduction in polyamine levels. Taken together, our results show that products of the methionine salvage pathway regulate polyamine biosynthesis and suggest that MTAP deletion may lead to ODC activation in human tumors.

Hepatitis B viremia is associated with increased risk of hepatocellular carcinoma in chronic carriers.

The role of quantitative viral load in development of hepatocellular carcinoma (HCC) among chronic hepatitis B virus (HBV) carriers was evaluated using real-time PCR (TaqMan PCR), a highly sensitive method for quantitative detection of HBV DNA. Serum samples collected at study entry from HCC cases and matched controls were chosen separately from ongoing prospective cohort studies in Senegal, West Africa, and Haimen City, China. For 14 HCC cases and 28 controls from Senegal, the relative risk (RR, 95% CI) of HCC was 15.6 (2.0-124.3) for those positive by the TaqMan PCR assay. Average length of follow-up (study entry to death from HCC) among cases was 2.8 (+/-1.6) years. The paired median difference between cases and controls was 3.8 x 10(4) virions/ml, with cases higher (P = 0.09). In order to clarify the relationship with lower-titer viremia, we selected 55 cases and 55 matched controls from the Chinese cohort all negative for serum HBV DNA by conventional dot blot hybridization. In this group, the RR associated with HBV DNA positivity by TaqMan PCR was 3.1 (1.1-9.2), with an average duration of follow-up of 3.3 (+/-2.1) years. The median difference in quantitative viremia between cases and controls was 6.0 x 10(4) virions/ml, with cases higher (P < 0.0001). Increased risk appeared to be confined to subjects with viral loads >2.3 x 10(4) virions/ml. In conclusion, HBV viremia, except perhaps at extremely low levels, is associated with increased risk for HCC in prospective studies of chronic carriers in two disparate populations.