Many solid tumors and hematologic malignancies lack expression of the enzyme methylthioadenosine phosphorylase (MTAP), due either to deletion of the MTAP gene or to methylation of the MTAP promoter. In cells that have MTAP, its natural substrate, methylthioadenosine (MTA), generated during polyamine biosynthesis, is cleaved to adenine and 5-methylthioribose-1-phosphate. The latter compound is further metabolized to methionine. Adenine and methionine are further metabolized and hence salvaged. In MTAP-deficient cells, however, MTA is not cleaved and the salvage pathway for adenine and methionine is absent. As a result, MTAP-deficient cells are more sensitive than MTAP-positive cells to inhibitors of de novo purine synthesis and to methionine deprivation. The challenge has been to take advantage of MTAP deficiency, and the changes in metabolism that follow, to design a strategy for targeted treatment. In this review, the frequency of MTAP-deficiency is presented and past and recent strategies to target such deficient cells are discussed, including one in which MTA is administered, followed by very high doses of a toxic purine or pyrimidine analog. In normal host cells, adenine, generated from MTA, blocks conversion of the analog to its toxic nucleotide. In MTAP-deficient tumor cells, conversion proceeds and the tumor cells are selectively killed. Successful mouse studies using this novel strategy were recently reported.
MTAP; MTA; adenine; 6-mercaptopurine; methotrexate
The thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are used in the treatment of leukaemia. Incorporation of deoxythioguanosine nucleotides (dGs) into the DNA of thiopurine-treated cells causes cell death but there is also evidence that thiopurine metabolites, particularly the 6-MP metabolite methylthioinosine monophosphate (MeTIMP), inhibit de novo purine synthesis (DNPS). The toxicity of DNPS inhibitors is influenced by methylthioadenosine phosphorylase (MTAP), a gene frequently deleted in cancers. Since the growth of MTAP-deleted tumour cells is dependent on DNPS or hypoxanthine salvage, we would predict such cells to show differential sensitivity to 6-MP and 6-TG. To test this hypothesis, sensitivity to 6-MP and 6-TG was compared in relation to MTAP status using cytotoxicity assays in two MTAP-deficient cell lines transfected to express MTAP: the T-cell acute lymphoblastic leukaemic cell line, Jurkat, transfected with MTAP cDNA under the control of a tetracycline-inducible promoter, and a lung cancer cell line (A549-MTAP−ve) transfected to express MTAP constitutively (A549-MTAP+ve). Sensitivity to 6-MP or methyl mercaptopurine riboside, which is converted intra-cellularly to MeTIMP, was markedly higher in both cell lines under MTAP−ve conditions. Measurement of thiopurine metabolites support the hypothesis that DNPS inhibition is a major cause of cell death with 6-MP, whereas dGs incorporation is the main cause of cytotoxicity with 6-TG. These data suggest that thiopurines, particularly 6-MP, may be more effective in patients with deleted MTAP.
Methylthioadenosine phosphorylase; purine synthesis; 6-mercaptopurine; 6-thioguanine; cancer; 9p-deletion
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 (MtaplacZ) could accelerate tumorigenesis development in two different mouse cancer models, Eμ-myc transgenic and Pten+/−.
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 MtaplacZ/+ and Mtap+/+ mice.
Survival in both models was significantly decreased in MtaplacZ/+ 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 MtaplacZ/+ 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 MtaplacZ/+ 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.
Our findings show that germline inactivation of a single Mtap allele alters gene expression and enhances lymphomagenesis in Eµ-myc mice.
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.
Cancer; Tumor Suppressor Gene; Methionine; Embryonic Lethal
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.
chemotherapy; mesothelioma; methionine; osteosarcoma; purine
The gene for methylthioadenosine phosphorylase (MTAP) lies on 9p21, close to the gene CDKN2A that encodes the tumor suppressor proteins p16 and p14ARF. MTAP and CDKN2A are homozygously co-deleted, with a frequency of 35 to 70%, in lung and pancreatic cancer, glioblastoma, osteosarcoma, soft-tissue sarcoma, mesothelioma, and T-cell acute lymphoblastic leukemia. In normal cells, but not in tumor cells lacking MTAP, MTAP cleaves the natural substrate, 5′-deoxy-5′-methylthioadenosine (MTA), to adenine and 5-methylthioribose-1-phosphate (MTR-1-P), which are then converted to adenine nucleotides and methionine. This distinct difference between normal MTAP-positive cells and tumor MTAP-negative cells led to several proposals for therapy. We offer a novel strategy in which both MTA and a toxic adenine analog, such as 2,6-diaminopurine (DAP), 6-methylpurine (MeP), or 2-fluoroadenine (F-Ade), are administered. In MTAP-positive cells, abundant adenine, generated from supplied MTA, competitively blocks the conversion of an analog, by adenine phosphoribosyltransferase (APRT), to its active nucleotide form. In MTAP-negative tumor cells, the supplied MTA cannot generate adenine; hence conversion of the analog is not blocked.
We show that this combination treatment – adenine analog plus MTA – kills MTAP-negative A549 lung tumor cells, while MTAP-positive human fibroblasts (HF) are protected. In co-cultures of the breast tumor cell line, MCF-7, and HF cells, MCF-7 is inhibited or killed, while HF cells proliferate robustly. 5-fluorouracil (5-FU) and 6-thioguanine (6-TG) may also be used with our strategy. Though neither analog is activated by APRT, in MTAP-positive cells, adenine produced from supplied MTA blocks conversion of 5-FU and 6-TG to their toxic nucleotide forms by competing for 5-phosphoribosyl-1-pyrophosphate (PRPP). The combination of MTA with 5-FU or 6-TG, in the treatment of MTAP-negative tumors, may produce a significantly improved therapeutic index.
We describe a selective strategy to kill tumor cells lacking MTAP.
Human 5′-methylthioadenosine phosphorylase (MTAP) links the polyamine biosynthetic and S-adenosyl-L-methionine salvage pathways and is a target for anticancer drugs. p-Cl-PhT-DADMe-ImmA is a 10 pM, slow-onset tight-binding transition state analogue inhibitor of the enzyme. Titration of homotrimeric MTAP with this inhibitor established equivalent binding and independent catalytic function of the three catalytic sites. Thermodynamic analysis of MTAP with tight-binding inhibitors revealed entropic-driven interactions with small enthalpic penalties. A large negative heat capacity change of −600 cal/mol•K upon inhibitor binding to MTAP is consistent with the loss of hydrophobic interactions and release of water. Crystal structures of apo MTAP and MTAP in complex with p-Cl-PhT-DADMe-ImmA were determined at 1.9 A and 2.0 A resolution, respectively. Inhibitor binding caused condensation of the enzyme active site, reorganization at the trimer interfaces, the release of water from the active sites and subunit interfaces, and compaction of the trimeric structure. These structural changes cause the entropy-favored binding of transition state analogues. Homotrimeric human MTAP is contrasted to the structurally related homotrimeric human purine nucleoside phosphorylase. p-Cl-PhT-DADMe- ImmA binding to MTAP involves a favorable entropy term of −17.6 kcal/mol with unfavorable enthalpy of 2.6 kcal/mol. In contrast, binding of an 8.5 pM transition state analogue to human PNP has been shown to exhibit the opposite behavior, with an unfavorable entropy term of 3.5 kcal/mol and a favorable enthalpy of −18.6 kcal/mol. Transition state analogue interactions reflect protein architecture near the transition state and the profound thermodynamic differences for MTAP and PNP suggest dramatic differences in contributions to catalysis from protein architecture.
The methionine salvage pathway is widely distributed among some eubacteria, yeast, plants and animals and recycles the sulfur-containing metabolite 5-methylthioadenosine (MTA) to methionine. In eukaryotic cells, the methionine salvage pathway takes place in the cytosol and usually involves six enzymatic activities: MTA phosphorylase (MTAP, EC 126.96.36.199), 5′-methylthioribose-1-phosphate isomerase (mtnA, EC 188.8.131.52), 5′-methylthioribulose-1-phosphate dehydratase (mtnB, EC: 184.108.40.206), 2,3-dioxomethiopentane-1-phosphate enolase/phosphatase (mtnC, EC 220.127.116.11), aci-reductone dioxygenase (mtnD, EC 18.104.22.168) and 4-methylthio-2-oxo-butanoate (MTOB) transaminase (EC 2.6.1.-). The aim of this study was to complete the available information on the methionine salvage pathway in human by identifying the enzyme responsible for the dehydratase step. Using a bioinformatics approach, we propose that a protein called APIP could perform this role. The involvement of this protein in the methionine salvage pathway was investigated directly in HeLa cells by transient and stable short hairpin RNA interference. We show that APIP depletion specifically impaired the capacity of cells to grow in media where methionine is replaced by MTA. Using a Shigella mutant auxotroph for methionine, we confirm that the knockdown of APIP specifically affects the recycling of methionine. We also show that mutation of three potential phosphorylation sites does not affect APIP activity whereas mutation of the potential zinc binding site completely abrogates it. Finally, we show that the N-terminal region of APIP that is missing in the short isoform is required for activity. Together, these results confirm the involvement of APIP in the methionine salvage pathway, which plays a key role in many biological functions like cancer, apoptosis, microbial proliferation and inflammation.
Castrate resistant prostate cancer (CRPC) and neuroendocrine carcinoma of the prostate are invariably fatal diseases for which only palliative therapies exist. As part of a prostate tumour sequencing program, a patient tumour was analyzed using Illumina genome sequencing and a matched renal capsule tumour xenograft was generated. Both tumour and xenograft had a homozygous 9p21 deletion spanning the MTAP, CDKN2 and ARF genes. It is rare for this deletion to occur in primary prostate tumours yet approximately 10% express decreased levels of MTAP mRNA. Decreased MTAP expression is a prognosticator for poor outcome. Moreover, it appears that this deletion is more common in CRPC than in primary prostate cancer. We show for the first time that treatment with methylthioadenosine and high dose 6-thioguanine causes marked inhibition of a patient derived neuroendocrine xenograft growth while protecting the host from 6-thioguanine toxicity. This therapeutic approach can be applied to other MTAP-deficient human cancers since deletion or hypermethylation of the MTAP gene occurs in a broad spectrum of tumours at high frequency. The combination of genome sequencing and patient-derived xenografts can identify candidate therapeutic agents and evaluate them for personalized oncology.
massively parallel sequencing; MTAP; patient-derived xenograft; genitourinary cancers: prostate; animal models of cancer; gene expression profiling; functional genomics; xenograft models
5′-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is a dual substrate bacterial enzyme involved in S-adenosylmethionine (SAM)-related quorum sensing pathways that regulates virulence in many bacterial species. MTANs from many bacteria are directly involved in the quorum sensing mechanism by regulating the synthesis of autoinducer molecules that are used by bacterial communities to communicate. In humans, 5′-methylthioadenosine phosphorylase (MTAP) is involved in polyamine biosynthesis as well as in purine and SAM salvage pathways and thus has been identified as an anticancer target. Previously we have described the synthesis and biological activity of several aza-C-nucleoside mimics with a sulfur atom at the 5′ position that are potent E. coli MTAN and human MTAP inhibitors. Because of the possibility that the sulfur may affect bioavailability we were interested in synthesizing “sulfur-free” analogues. Herein we describe the preparation of a series of “sulfur-free” transition state analogues inhibitors, of E. coli MTAN and human MTAP that have low nano- to pico-molar dissociation constants and are potentially novel bacterial anti-infective and anti-cancer drug candidates.
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’-methylthioadcnosine 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.
5′-methylthioadenosine (MTA) is a natural purine that is metabolized by methylthioadenosine phosphorylase (MTAP, E.C 22.214.171.124) in Eukarya and Archaea but generally not in bacteria. In this work, Rv0535, which has been annotated as a probable MTAP in M. tuberculosis, was expressed in and purified from E. coli BL21 (DE3). The purified protein displayed properties of a phosphorylase and MTA was the preferred substrate. Adenosine and S-adenosyl-L-homocysteine were poor substrates and no activity was detected with 5′-methylthioinosine, the other natural purines or the natural pyrimidines. Kinetic analysis of M. tuberculosis MTAP showed that the Km value for MTA was 9.1 μM. Rv0535 was estimated as a 30 kDa protein on a denaturing SDS-PAGE gel, which agreed with the molecular mass predicted by its gene sequence. Using gel filtration chromatography, the native molecular mass of the enzyme was determined to be 60 ± 4 kDa, and thus indicates that M. tuberculosis MTAP is a dimer. Differences in active site between mycobacterial and human MTAPs were identified by homology modeling based on the crystal of the human enzyme. A complete structure activity relationship analysis could identify differences in substrate specificity between the two enzymes to aid in the development of purine-based, anti-tuberculosis drugs.
5′-methylthioadenosine phosphorylase; Rv0535; purine metabolism; Mycobacterium tuberculosis
Kinetic isotope effects (KIEs) and computer modeling using density functional theory were used to approximate the transition state of human 5′-methylthioadenosine phosphorylase (MTAP). KIEs were measured on the arsenolysis of 5′-methylthioadenosine (MTA) catalyzed by MTAP and were corrected for the forward commitment to catalysis. Intrinsic KIEs were obtained for [1′-3H], [1′-14C], [2′-3H], [4′-3H], [5′-3H], [9-15N] and [Me-3H3] MTAs. The primary intrinsic KIEs (1′-14C and 9-15N) suggest that MTAP has a dissociative SN1 transition state with cationic center at the anomeric carbon and insignificant bond order to the leaving group. The 9-15N intrinsic KIE of 1.039 also establishes an anionic character to the adenine leaving group, whereas the α-primary 1′-14C KIE of 1.031 indicates significant nucleophilic participation at the transition state. Computational matching of the calculated EIEs to the intrinsic isotope effects places the oxygen nucleophile 2.0 Å from the anomeric carbon. The 4′-3H KIE is sensitive to the polarization of the 3′-OH group. Calculations suggest that a 4′-3H KIE of 1.047 is consistent with ionization of the 3′-OH group, indicating formation of a zwitterion at the transition state. The transition state has cationic character at the anomeric carbon and is anionic at the 3′-OH oxygen, with an anionic leaving group. The isotope effects predicted a 3′-endo conformation for the ribosyl zwitterion corresponding to a H1′-C1′-C2′-H2′ torsional angle of 33°. The [Me-3H3] and [5′-3H] KIEs arise predominantly from the negative hyperconjugation of the lone pairs of sulphur with the σ* (C-H) antibonding orbitals. Human MTAP is characterized by a late, SN1 transition state with significant participation of the phosphate nucleophile.
5′-methylthioadenosine; kinetic isotope effects; 5′-methylthioadenosine phosphorylase; polyamines; MTAP; transition state; zwitterionic transition state; transition state structure
Males homozygous for the mouse male sterility and histoincompatibility (mshi) mutation exhibit small testes and produce no sperm. In addition, mshi generates an “antigen-loss” histoincompatibility barrier, such that homozygous mutants reject skin grafts from wild-type co-isogenic BALB/cByJ donors. To facilitate the molecular characterization of the pleiotropic mshi mutation, we genetically mapped mshi into a 0.68 megabasepair region which contains fewer than 10 candidate genes. Complementation testing showed that one of these, Mtap7, is disrupted in mshi mice. Sequence analysis has revealed a 13 kilobasepair deletion in BALB/cByJ-mshi/J mice that begins in Intron 10–11 of Mtap7, and ends less than 2,000 base pairs downstream of the wild type gene. Analysis of the mutant cDNA predicts that Mtap7mshi encodes a 457 amino acid protein, the first 423 of which are identical to wild type, and the last 34 of which are due to aberrant mRNA splicing with two cryptic exons in the Mtap7 to P04Rik intergenic region. This molecular assignment for the mshi mutation further supports an essential role for microtubule stabilization in spermatogenesis and indicates a new role in allograft transplantation.
Spermatogenesis; E-MAP-115; Positional cloning; Complementation testing
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 kcat/Km 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 three-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.
We compared the proteins which associate with middle T antigen (MT) of polyomavirus in human cells infected with Ad5(pymT), a recombinant adenovirus which directs the overexpression of MT, with the MT-associated proteins (MTAPs) previously identified in murine fibroblasts expressing MT. MTAPs of 27, 29, 36, and 63 kilodaltons (kDa) appeared to be fairly well conserved between the two species, as judged by comigration on two-dimensional gels. Several 61-kDa MTAP species detected in MT immunoprecipitates from both cell sources also comigrated on these gels. However, no protein comigrating precisely with the murine 85-kDa MTAP could be detected in the human cells. Furthermore, two proteins of 72 and 74 kDa associated with wild-type MT in the infected human cells but not in murine fibroblasts expressing MT. It had been previously reported for murine cells that the 70-kDa heat shock protein associates with a particular mutant MT but not with wild-type MT (G. Walter, A. Carbone, and W.J. Welch, J. Virol. 61:405-410, 1987). By the criteria of comigration on two-dimensional gels, tryptic peptide mapping, and immunoblotting, we showed that the 72- and 74-kDa proteins that associate with wild-type MT in human cells are the major human 70-kDa heat shock proteins.
We report a genome-wide association study of melanoma conducted by the GenoMEL consortium based on 317k tagging SNPs for 1650 genetically-enriched cases (from Europe and Australia) and 4336 controls and subsequent replication in 1149 genetically-enriched cases and 964 controls and a population-based case-control study of 1163 cases and 903 controls. The genome-wide screen identified five regions with genotyped or imputed SNPs reaching p < 5×10−7; three regions were replicated: 16q24 encompassing MC1R (overall p=2.54×10−27 for rs258322), 11q14-q21 encompassing TYR (p=2.41×10−14 for rs1393350) and 9p21 adjacent to MTAP and flanking CDKN2A (p=4.03×10−7 for rs7023329). MC1R and TYR are associated with pigmentation, freckling and cutaneous sun sensitivity, well-recognised melanoma risk factors, while the 9p21 locus is novel for common variants associated with melanoma. Despite wide variation in allele frequency, these genetic variants show notable homogeneity of effect across populations of European ancestry living at different latitudes and contribute independently to melanoma risk.
Superficial spreading melanoma (SSM) and nodular melanoma (NM) are believed to represent sequential phases of linear progression from radial to vertical growth. Several lines of clinical, pathological and epidemiologic evidence suggest, however, that SSM and NM might be the result of independent pathways of tumor development. We utilized an integrative genomic approach that combines single nucleotide polymorphism array (SNP 6.0, Affymetrix) with gene expression array (U133A 2.0, Affymetrix) to examine molecular differences between SSM and NM. Pathway analysis of the most differentially expressed genes between SSM and NM (N=114) revealed significant differences related to metabolic processes. We identified 8 genes (DIS3, FGFR1OP, G3BP2, GALNT7, MTAP, SEC23IP, USO1, ZNF668) in which NM/SSM-specific copy number alterations correlated with differential gene expression (P<0.05, Spearman’s rank). SSM-specific genomic deletions in G3BP2, MTAP, and SEC23IP were independently verified in two external data sets. Forced overexpression of metabolism-related gene methylthioadenosine phosphorylase (MTAP) in SSM resulted in reduced cell growth. The differential expression of another metabolic related gene, aldehyde dehydrogenase 7A1 (ALDH7A1), was validated at the protein level using tissue microarrays of human melanoma. In addition, we show that the decreased ALDH7A1 expression in SSM may be the result of epigenetic modifications. Our data reveal recurrent genomic deletions in SSM not present in NM, which challenge the linear model of melanoma progression. Furthermore, our data suggest a role for altered regulation of metabolism-related genes as a possible cause of the different clinical behavior of SSM and NM.
melanoma; nodular; genomics; SNP array; DNA copy number
Survival and evolution of aneuploid cells after an asymmetric segregation of chromosomes at mitosis may be the common initiating event and underlying cause of the genetic diversity and adaptability of cancers. We hypothesize that mechanisms exist to detect impending aneuploidy and prevent it before completion of an aberrant mitosis.
The distribution of isoforms of C19ORF5, an interactive partner with mitochondria-associated LRPPRC and tumor suppressor RASSF1A, state of spindle microtubules and mitochondrial aggregation was analyzed in synchronized mitotic cells and cells stalled in mitosis after treatment with paclitaxel.
C19ORF5 distributed broadly across the mitotic spindle and reversibly accumulated during reversible mitotic arrest. Prolonged stabilization of microtubules caused an accumulation of a C19ORF5 product with dual MAP and MtAP properties that caused irreversible aggregation of mitochondria and death of mitotic cells.
Dual function microtubule-associated (MAP) and mitochondria-associated (MtAP) proteins generated by prolonged mitotic arrest trigger mitochondrial-induced mitotic cell death. This is a potential mechanism to prevent minimal survivable aneuploidy resulting from an aberrant cell division and cancers in general at their earliest common origin.
Aneuploidy; C19ORF5; genetic instability; LRPPRC; microtubule dynamics; mitochondrial dynamics; RASSF1A; paclitaxel; tumor suppression; mitochondria aggregation
The characterization of modifier genes can provide insights into disease pathways and identify novel therapeutic targets. This study identifies Mtap1a as a modifier gene of photoreceptor loss in Tulp1 and Tub mutant mice, which are models of retinal degeneration.
To identify genes that modify photoreceptor cell loss in the retinas of homozygous Tulp1tm1Pjn and Tubtub mice, which exhibit juvenile retinitis pigmentosa.
Modifier loci were identified by genetic quantitative trait locus analysis. F2 Tulp1tm1Pjn/tm1Pjn mutant mice from a B6-Tulp1tm1Pjn/tm1Pjn × AKR/J intercross were genotyped with a panel of single nucleotide polymorphism markers and phenotyped by histology for photoreceptor nuclei remaining at 9 weeks of age. Genotype and phenotype data were correlated and examined with Pseudomarker 2.02 using 128 imputations to map modifier loci. Thresholds for the 63%, 10%, 5%, and 1% significance levels were obtained from 100 permutations. A significant, protective candidate modifier was identified by bioinformatic analysis and confirmed by crossing transgenic mice bearing a protective allele of this gene with Tulp1- and Tub-deficient mice.
A significant, protective modifier locus on chromosome 2 and a suggestive locus on chromosome 13 that increases photoreceptor loss were identified in a B6-Tulp1tm1Pjn/tm1Pjn × AKR/J intercross. The chromosome 2 locus mapped near Mtap1a, which encodes a protein associated with microtubule-based intracellular transport and synapse function. The protective Mtap1a129P2/OlaHsd allele was shown to reduce photoreceptor loss in both Tulp1tm1Pjn/tm1Pjn and Tubtub/tub mice.
It was demonstrated that the gene Mtap1a, which modifies hearing loss in Tubtub/tub mice, also modifies retinal degeneration in Tubtub/tub and Tulp1tm1Pjn/tm1Pjn mice. These results suggest that functionally nonredundant members of the TULP family (TUB and TULP1) share a common functional interaction with MTAP1A.
Genome-wide association studies (GWAS) have linked common single nucleotide polymorphisms (SNPs) on chromosome 9p21 near the INK4/ARF (CDKN2A/B) tumor suppressor locus with risk of atherosclerotic diseases and type 2 diabetes mellitus. To explore the mechanism of this association, we investigated whether expression of proximate transcripts (p16INK4a, p15INK4b, ARF, ANRIL and MTAP) correlate with genotype of representative 9p21 SNPs.
We analyzed expression of 9p21 transcripts in purified peripheral blood T-cells (PBTL) from 170 healthy donors. Samples were genotyped for six selected disease-related SNPs spanning the INK4/ARF locus. Correlations among these variables were determined by univariate and multivariate analysis. Significantly reduced expression of all INK4/ARF transcripts (p15INK4b, p16INK4a, ARF and ANRIL) was found in PBTL of individuals harboring a common SNP (rs10757278) associated with increased risk of coronary artery disease, stroke and aortic aneurysm. Expression of MTAP was not influenced by rs10757278 genotype. No association of any these transcripts was noted with five other tested 9p21 SNPs.
Genotypes of rs10757278 linked to increased risk of atherosclerotic diseases are also associated with decreased expression in PBTL of the INK4/ARF locus, which encodes three related anti-proliferative transcripts of known importance in tumor suppression and aging.
It has recently been shown that there are highly significant associations for common single nucleotide polymorphisms (SNPs) near the CDKN2B-AS1 gene region at the 9p21 locus with primary open angle glaucoma (POAG), a leading cause of irreversible blindness. This gene region houses the CDKN2B/p15INK4B,
CDKN2A/p16INK4A and p14ARF (rat equivalent, p19ARF) tumour suppressor genes and is adjacent to the S-methyl-5′-thioadenosine phosphorylase (MTAP) gene. In order to understand the ocular function of these genes and, therefore, how they may be involved in the pathogenesis of POAG, we studied the distribution patterns of each of their products within human and rat ocular tissues. MTAP mRNA was detected in the rat retina and optic nerve and its protein product was localised to the corneal epithelium, trabecular meshwork and retinal glial cells in both human and rat eyes. There was a very low level of p16INK4A mRNA present within the rat retina and slightly more in the optic nerve, although no protein product could be detected in either rat or human eyes with any of the antibodies tested. P19ARF mRNA was likewise only present at very low levels in rat retina and slightly higher levels in the optic nerve. However, no unambiguous evidence was found to indicate expression of specific P19ARF/p14ARF proteins in either rat or human eyes, respectively. In contrast, p15INK4B mRNA was detected in much higher amounts in both retina and optic nerve compared with the other genes under analysis. Moreover, p15INK4B protein was clearly localised to the retinal inner nuclear and ganglion cell layers and the corneal epithelium and trabecular meshwork in rat and human eyes. The presented data provide the basis for future studies that can explore the roles that these gene products may play in the pathogenesis of glaucoma and other models of optic nerve damage.
Several acyclic hydroxy-methylthio-amines with 3 to 5 carbon atoms were prepared and coupled via a methylene link to 9-deazaadenine. The products were tested for inhibition against human MTAP and E. coli and N. meningitidis MTANs and gave Ki values as low as 0.23 nM. These results were compared to those obtained with 1st and 2nd generation inhibitors (1S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-methylthio-d-ribitol (MT-Immucillin-A, 3) and (3R,4S)-1-[9-deazaadenin-9-yl)methyl]3-hydroxy-4-methylthiomethylpyrrolidine (MT-DADMe-Immucillin-A, 4). The best inhibitors were found to exhibit binding affinities of approximately 2- to 4-fold those of 3 but were significantly weaker than 4. Cleavage of the 2,3 carbon–carbon bond in MT-Immucillin-A (3) gave an acyclic product (79) with a 21,500 fold loss of activity against E. coli MTAN. In another case, N-methylation of a side chain secondary amine resulted in a 250-fold loss of activity against the same enzyme [(±)-65 vs (±)-68]. The inhibition results were also contrasted with those acyclic derivatives previously prepared as inhibitors for a related enzyme, purine nucleoside phosphorylase (PNP), where some inhibitors in the latter case were found to be more potent than their cyclic counterparts.
Human MTAP; Bacterial MTANs; Ribooxacarbenium ion mimics; Inhibitors; Acyclic hydroxy-methylthio-amines
Chordoma is a rare tumor arising in the sacrum, clivus, or vertebrae. It is often not completely resectable and shows a high incidence of recurrence and progression with shortened patient survival and impaired quality of life. Chemotherapeutic options are limited to investigational therapies at present. Therefore, adjuvant therapy for control of tumor recurrence and progression is of great interest, especially in skull base lesions where complete tumor resection is often not possible because of the proximity of cranial nerves. To understand the extent of genetic instability and associated chromosomal and gene losses or gains in skull base chordoma, we undertook whole-genome single-nucleotide polymorphism microarray analysis of flash frozen surgical chordoma specimens, 21 from the clivus and 1 from C1 to C2 vertebrae. We confirm the presence of a deletion at 9p involving CDKN2A, CDKN2B, and MTAP but at a much lower rate (22%) than previously reported for sacral chordoma. At a similar frequency (21%), we found aneuploidy of chromosome 3. Tissue microarray immunohistochemistry demonstrated absent or reduced fragile histidine triad (FHIT) protein expression in 98% of sacral chordomas and 67%of skull base chordomas. Our data suggest that chromosome 3 aneuploidy and epigenetic regulation of FHIT contribute to loss of the FHIT tumor suppressor in chordoma. The finding that FHIT is lost in a majority of chordomas provides new insight into chordoma pathogenesis and points to a potential new therapeutic target for this challenging neoplasm.
Advantages offered by canine population substructure, combined with clinical presentations similar to human disorders, makes the dog an attractive system for studies of cancer genetics. Cancers that have been difficult to study in human families or populations are of particular interest. Histiocytic sarcoma is a rare and poorly understood neoplasm in humans that occurs in 15–25% of Bernese Mountain Dogs (BMD).
Genomic DNA was collected from affected and unaffected BMD in North America (NA) and Europe. Both independent and combined genome wide association studies (GWAS) were used to identify cancer-associated loci. Fine mapping and sequencing narrowed the primary locus to a single gene region.
Both populations shared the same primary locus, which features a single haplotype spanning MTAP and part of CDKN2A and is present in 96% of affected BMD. The haplotype is within the region homologous to human chromosome 9p21, which has been implicated in several types of cancer.
We present the first GWAS for HS in any species. The data identify an associated haplotype in the highly cited tumor suppressor locus near CDKN2A. These data demonstrate the power of studying distinctive malignancies in highly predisposed dog breeds.
Here, we establish a naturally-occurring model of cancer susceptibility due to CDKN2 dysregulation, thus providing insight regarding this cancer-associated, complex, and poorly understood genomic region.
Genome Wide Association Study; Dogs; p16; Cancer; Histiocytic sarcoma