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, we have performed a differential chemical genetic screen in isogenic MTAP+ and MTAP− S. 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 four-fold shift in IC50 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 four of the five 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.

Background

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 transulfuration pathway by the action of cystathionine β-synthase (CBS). Here, we have examined the metabolic response in mice of a shift from a methionine-replete (M+) to a methionine-free (M−) diet.

Methods/Principle Findings

We found that shifting three-month old C57BL6 mice to a M− diet caused a transient increase in tHcy, as well as an increase in the tHcy/methionine ratio. Since CBS is a key regulator of tHcy, we examined CBS protein levels and found that within 3 days on 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 indicate 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/Significance

Our results imply that methionine deprivation induces a metabolic state in which methionine is effectively conserved in tissue by shutdown of the transsulfuration pathway via an S-adenosylmethionine-independent mechanism that signals a rapid down-regulation of CBS protein.

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 (MtaplacZ) have an embryonic lethal phenotype dying around day 8 post-conception. Mtap/MtaplacZ 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 FACS 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, and unaltered levels of methyldeoxycytidine. These studies show that Mtap is a tumor suppressor gene independent of CDKN2A and ARF.

Cystathionine β-synthase (CBS) deficiency is a recessive genetic disorder in humans characterized by elevated levels in 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 S. 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 μM. 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.