Methylthio-DADMe-immucillin-A (MT-DADMeCellular proliferation is associated with increased levels of polyamine biosynthesis and polyamine pools (1). Target validation for the polyamine pathway as an anticancer approach has come from ␣-difluoromethylornithine (DFMO), 3 a suicide inhibitor of ornithine decarboxylase (ODC) and the committed step of polyamine biosynthesis (2, 3). ODC is a difficult cancer target because of its rapid turnover and the dose-limiting toxicity of anti-ODC agents (4). Because of these difficulties, DFMO has not gained wide use. But the polyamine pathway, through its close interaction with S-adenosylmethionine (AdoMet) recycling, remains a target for cancer therapy. We investigated the possibility that feedback inhibition by 5Ј-methylthioadenosine (MTA), induced by a transition state analogue inhibitor of 5Ј-methylthioadenosine phosphorylase (MTAP), could be used to block this pathway and initiate anticancer effects. The results indicate that blocking MTA recycling with transition state analogues of MTAP induces apoptosis through specific epigenetic changes in specific cultured cancer cell lines. Inhibition of MTAP is effective in treating a xenograft model of head and neck cancer in mice.MTA is a product of both spermidine and spermine synthases and provides product inhibition at two sequential sites in the polyamine pathway (Fig. 1A). In humans, MTA is degraded exclusively by MTAP (EC 2.4.2.28), expressed from a single gene locus at 9p21. MTAP produces adenine and 5-methylthio-␣-D-ribose-1-phosphate (Fig. 1B), and these products are recycled to AdoMet. Inhibitors of MTAP are therefore expected to increase intracellular MTA, cause feedback inhibition of polyamine biosynthesis, prevent AdoMet recycling, and disrupt AdoMet-dependent methylation activity. One or more of these activities is expected to be associated with antiproliferative activity (5-8).The transition state structure of human MTAP has been established by kinetic isotope effects and quantum chemical calculations. It is characterized by a late transition state with weak participation of the phosphate nucleophile, similar to that of human purine nucleoside phosphorylase but slightly more advanced (Fig. 1B) (9 -15). Analogues of the human MTAP transition state have been synthesized and are powerful and specific inhibitors (16 -18). Methylthio-DADMe-Immucillin-A (MT-DADMe-ImmA) is a chemically stable transition state analogue of human MTAP and is a slow onset tightly binding inhibitor with a dissociation constant of 86 pM (18). * This work was supported by National Institutes of Health Grants GM41916and CA85953 and a pilot project award from P30 CA013330. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Diaphyseal medullary stenosis with malignant fibrous histiocytoma (DMS-MFH) is an autosomal-dominant syndrome characterized by bone dysplasia, myopathy, and bone cancer. We previously mapped the DMS-MFH tumor-suppressing-gene locus to chromosomal region 9p21-22 but failed to identify mutations in known genes in this region. We now demonstrate that DMS-MFH results from mutations in the most proximal of three previously uncharacterized terminal exons of the gene encoding methylthioadenosine phosphorylase, MTAP. Intriguingly, two of these MTAP exons arose from early and independent retroviral-integration events in primate genomes at least 40 million years ago, and since then, their genomic integration has gained a functional role. MTAP is a ubiquitously expressed homotrimeric-subunit enzyme critical to polyamine metabolism and adenine and methionine salvage pathways and was believed to be encoded as a single transcript from the eight previously described exons. Six distinct retroviral-sequence-containing MTAP isoforms, each of which can physically interact with archetype MTAP, have been identified. The disease-causing mutations occur within one of these retroviral-derived exons and result in exon skipping and dysregulated alternative splicing of all MTAP isoforms. Our results identify a gene involved in the development of bone sarcoma, provide evidence of the primate-specific evolution of certain parts of an existing gene, and demonstrate that mutations in parts of this gene can result in human disease despite its relatively recent origin.
Transition state structures can be derived from kinetic isotope effects and computational chemistry. Molecular electrostatic potential maps of transition states serve as blueprints to guide synthesis of transition state analogue inhibitors of target enzymes. 5'- Methylthioadenosine phosphorylase (MTAP) functions in the polyamine pathway by recycling methylthioadenosine (MTA) and maintaining cellular S-adenosylmethionine (SAM). Its transition state structure was used to guide synthesis of MT-DADMe-ImmA, a picomolar inhibitor that shows anticancer effects against solid tumors. Biochemical and genomic analysis suggests that MTAP inhibition acts by altered DNA methylation and gene expression patterns. A related bacterial enzyme, 5'-methylthioadenosine nucleosidase (MTAN), functions in pathways of quorum sensing involving AI-1 and AI-2 molecules. Transition states have been solved for several bacterial MTANs and used to guide synthesis of powerful inhibitors with dissociation constants in the femtomolar to picomolar range. BuT-DADMe-ImmA blocks quorum sensing in Vibrio cholerae without changing bacterial growth rates. Transition state analogue inhibitors show promise as anticancer and antibacterial agents.
Hereditary cancer syndromes represent a powerful and tractable biologic system for identifying cancer-causing genes. Though the syndromes themselves may be rare, their study can provide insights into the basis of the more common sporadic forms of the cancer. Diaphyseal medullary stenosis with malignant fibrous histiocytoma (DMS-MFH) is an autosomal dominant bone dysplasia / bone cancer syndrome. This hereditary cancer syndrome is characterized by bone infarctions, cortical growth abnormalities, and pathologic fractures. Most notably, 35% of affected individuals develop bone MFH, a sarcoma that in its sporadic form accounts for 6% of all bone cancers and is believed to be etiologically related to osteosarcoma. Indeed, one of our affected family members developed histologically-proven osteosarcoma, thus further supporting a genetic link between these tumor types. Using a linkage based approach, we previously mapped the DMS-MFH tumor suppressor gene locus to chromosome 9p21-22 (1) and then through LOH analysis demonstrated that hereditary and sporadic forms of MFH most likely share a single underlying genetic etiology (2). We now demonstrate that DMS-MFH results from mutations in the most proximal of three previously unrecognized terminal exons of the methylthioadenosine phosphorylase (MTAP) gene. MTAP is a ubiquitously expressed homotrimeric-subunit enzyme critical to polyamine metabolism and adenine/methionine salvage pathways and was believed to be encoded as a single transcript from the eight previously described exons. Intriguingly, two of the novel MTAP exons arose from early and independent retroviral integration events in primate genomes at least 40 MYR ago and since their genomic integration have gained a functional role. Six distinct retroviral-sequence containing MTAP isoforms, each of which can physically interact with archetype MTAP (i.e., exons 1-8), are identified. The disease-causing / cancer-associated mutations occur within one of these retroviral-derived exons. The mutations result in exon skipping and dysregulated alternative splicing of all MTAP isoforms. Based on these findings in a hereditary form of bone sarcoma, we then analyzed the expression of these MTAP isoforms in a sample set (n=16) of sporadic osteosarcoma samples. All tumor samples expressed similar levels of the archetype MTAP RNA sequence but the expression pattern of the splice variants varied markedly between nearly all the samples. The majority of samples did not express SV1 (n=11/16) and nearly half did not express SV6 (n=9/16). Taken together, these results identify the first gene involved in the development of bone MFH / osteosarcoma and have potential implications for the treatment of this human cancer. References: 1. Am J Hum Genet. 64:801-7; 1999. 2. Genes Chromosomes Cancer. 27:191-5; 2000. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-89. doi:1538-7445.AM2012-LB-89
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