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1.  Therapeutics by cytotoxic metabolite accumulation: Pemetrexed causes ZMP accumulation, AMPK activation, and mTOR inhibition 134 
Cancer research  2009;69(13):5467-5474.
Pemetrexed represents the first antifolate cancer drug to be approved by the FDA in 20 years; it is currently in widespread use for first line therapy of mesothelioma and non- small cell lung cancer. Pemetrexed has more than one site of action; the primary site is thymidylate synthase. We now report that the secondary target is the downstream folate-dependent enzyme in de novo purine synthesis, aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART). The substrate of the AICART reaction, ZMP, accumulated in intact pemetrexed-inhibited tumor cells, identifying AICART as the step in purine synthesis which becomes rate-limiting after drug treatment. The accumulating ZMP causes an activation of AMP-activated protein kinase with subsequent inhibition of the mammalian target of rapamycin (mTOR) and hypophosphorylation of the downstream targets of mTOR that control initiation of protein synthesis and cell growth. We suggest that the activity of pemetrexed against human cancers is a reflection of its direct inhibition of folate-dependent target proteins combined with prolonged inhibition of the mTOR pathway secondary to accumulation of ZMP.
doi:10.1158/0008-5472.CAN-08-4979
PMCID: PMC2706929  PMID: 19549896
Purine synthesis; ZMP; AMP-activated protein kinase; mammalian target of rapamycin; pemetrexed
2.  Sorafenib enhances pemetrexed cytotoxicity through an autophagy -dependent mechanism in cancer cells 
Cancer research  2011;71(14):4955-4967.
Pemetrexed (ALIMTA) is a folate anti-metabolite that has been approved for the treatment of non-small cell lung cancer, and has been shown to stimulate autophagy. In the present study, we sought to further understand the role of autophagy in the response to pemetrexed and to test if combination therapy could enhance the level of toxicity through altered autophagy in tumor cells. The multi-kinase inhibitor sorafenib (NEXAVAR), used in the treatment of renal and hepatocellular carcinoma, suppresses tumor angiogenesis and promotes autophagy in tumor cells. We found that sorafenib interacted in a greater than additive fashion with pemetrexed to increase autophagy and to kill a diverse array of tumor cell types. Tumor cell types that displayed high levels of cell killing after combination treatment showed elevated levels of AKT, p70 S6K and/or phosphorylated mTOR, in addition to Class III RTKs such as PDGFRβ and VEGFR1, known in vivo targets of sorafenib. In xenograft and in syngeneic animal models of mammary carcinoma and glioblastoma, the combination of sorafenib and pemetrexed suppressed tumor growth without deleterious effects on normal tissues or animal body mass. Taken together, the data suggest that premexetred and sorafenib act synergistically to enhance tumor killing via the promotion of a toxic form of autophagy that leads to activation of the intrinsic apoptosis pathway, and predict that combination treatment represents a future therapeutic option in the treatment of solid tumors.
doi:10.1158/0008-5472.CAN-11-0898
PMCID: PMC3139015  PMID: 21622715
3.  Pemetrexed indirectly activates the metabolic kinase AMPK in human carcinomas 
Cancer research  2010;70(24):10299-10309.
The chemotherapeutic drug pemetrexed, an inhibitor of thymidylate synthase, has an important secondary target in human leukemic cells, AICART, the second folate-dependent enzyme of purine biosynthesis. The purine intermediate aminoimidazolecarboxamide ribonucleotide (ZMP), which accumulates behind this block, transmits an inhibitory signal to the mTORC1 complex via activation of the cellular energy sensor AMPK. Given that the PI3K-AKT-mTOR pathway is frequently deregulated during carcinogenesis, we asked whether the indirect activation of AMPK by pemetrexed offers an effective therapeutic strategy for carcinomas with defects in this pathway. Activation of AMPK by ZMP in pemetrexed-treated colon and lung carcinoma cells and the downstream consequences of this activation were strikingly more robust than previously seen in leukemic cells. Genetic experiments demonstrated the intermediacy of AICART inhibition and the centrality of AMPK activation in these effects. While AMPK activation resulted in marked inhibition of mTORC1, other targets of AMPK were phosphorylated that were not mTORC1-dependent. Whereas AMPK activation is thought to require AMPKα T172 phosphorylation, pemetrexed also activated AMPK in carcinoma cells null for LKB1, the predominant AMPKα T172 kinase whose deficiency is common in lung adenocarcinomas. Like rapamycin analogs, pemetrexed relieved feedback suppression of PI3K and AKT, but the prolonged accumulation of unphosphorylated 4E-BP1, a tight-binding inhibitor of cap-dependent translation, was seen following AMPK activation. Our findings indicate that AMPK activation by pemetrexed inhibits mTORC1- dependent and -independent processes that control translation and lipid metabolism, identifying pemetrexed as a targeted therapeutic agent for this pathway that differs significantly from rapamycin analogs.
doi:10.1158/0008-5472.CAN-10-1873
PMCID: PMC3059241  PMID: 21159649
pemetrexed; Non-small cell lung cancer; AMP-activated protein kinase; mTOR; Aminoimidazolecarboxamide ribonucleotide formyltransferase
4.  Sorafenib enhances pemetrexed cytotoxicity through an autophagy- dependent mechanism in cancer cells 
Autophagy  2011;7(10):1261-1262.
Pemetrexed (ALIMTA) is a folate anti-metabolite that has been approved for the treatment of non-small cell lung cancer, and has been shown to stimulate autophagy. In the present study, we sought to further understand the role of autophagy in the response to pemetrexed and to test if combination therapy could enhance the level of toxicity through altered autophagy in tumor cells. The multikinase inhibitor sorafenib (NEXAVAR), used in the treatment of renal and hepatocellular carcinoma, suppresses tumor angiogenesis and promotes autophagy in tumor cells. We found that sorafenib interacted in a greater than additive fashion with pemetrexed to increase autophagy and to kill a diverse array of tumor cell types. Tumor cell types that displayed high levels of cell killing after combination treatment showed elevated levels of AKT, p70 S6K and/or phosphorylated mTOR, in addition to class III RTKs such as PDGFRβ and VEGFR1, known in vivo targets of sorafenib. In xenograft and in syngeneic animal models of mammary carcinoma and glioblastoma, the combination of sorafenib and pemetrexed suppressed tumor growth without deleterious effects on normal tissues or animal body mass. Taken together, the data suggest that premexetred and sorafenib act synergistically to enhance tumor killing via the promotion of a toxic form of autophagy that leads to activation of the intrinsic apoptosis pathway, and predict that combination treatment represents a future therapeutic option in the treatment of solid tumors.
doi:10.4161/auto.7.10.17029
PMCID: PMC3210312  PMID: 21814046
pemetrexed; sorafenib; autophagy; apoptosis; PDGFR; ZMP; AMP; thymidylate synthase
5.  Concentration-Dependent Processivity of Multiple Glutamate Ligations Catalyzed by Folylpoly-γ-glutamate Synthetase 
Biochemistry  2008;47(34):9040-9050.
Folylpoly-γ-glutamate synthetase (FPGS, EC 6.3.2.17) is an ATP-dependent ligase that catalyzes formation of poly-γ-glutamate derivatives of reduced folates and anti-folates such as methotrexate and 5,10-dideaza-5,6,7,8-tetrahydrofolate (DDAH4PteGlu1). While the chemical mechanism of the reaction catalyzed by FPGS is known, it is unknown whether single or multiple glutamate residues are added following each folate binding event. A very sensitive high performance liquid chromatography method has been used to analyze the multiple ligation reactions onto radiolabeled DDAH4PteGlu1 catalyzed by FPGS in order to distinguish between distributive or processive mechanisms of catalysis. Reaction time courses, substrate trapping, and pulse-chase experiments were used to measure folate release during multiple glutamate additions. Together, the results of these experiments indicate that hFPGS can catalyze the processive addition of approximately four glutamate residues onto DDAH4PteGlu1. The degree of processivity was determined to be dependent on the concentration of the folate substrate, thus suggesting a mechanism for the regulation of folate polyglutamate synthesis in cells.
doi:10.1021/bi800406w
PMCID: PMC2805413  PMID: 18672898
6.  A Mouse Gene That Coordinates Epigenetic Controls and Transcriptional Interference To Achieve Tissue-Specific Expression▿ † 
Molecular and Cellular Biology  2007;28(2):836-848.
The mouse fpgs gene uses two distantly placed promoters to produce functionally distinct isozymes in a tissue-specific pattern. We queried how the P1 and P2 promoters were differentially controlled. DNA methylation of the CpG-sparse P1 promoter occurred only in tissues not initiating transcription at this site. The P2 promoter, which was embedded in a CpG island, appeared open to transcription in all tissues by several criteria, including lack of DNA methylation, yet was used only in dividing tissues. The patterns of histone modifications over the two promoters were very different: over P1, histone activation marks (acetylated histones H3 and H4 and H3 trimethylated at K4) reflected transcriptional activity and apparently reinforced the effects of hypomethylated CpGs; over P2, these marks were present in tissues whether P2 was active, inactive, or engaged in assembly of futile initiation complexes. Since P1 transcriptional activity coexisted with silencing of P2, we sought the mechanism of this transcriptional interference. We found RNA polymerase II, phosphorylated in a pattern consistent with transcriptional elongation, and only minimal levels of initiation factors over P2 in liver. We concluded that mouse fpgs uses DNA methylation to control tissue-specific expression from a CpG-sparse promoter, which is dominant over a downstream promoter masked by promoter occlusion.
doi:10.1128/MCB.01088-07
PMCID: PMC2223435  PMID: 17998333
7.  Transducing Particles of Staphylococcus aureus Pathogenicity Island SaPI1 Are Comprised of Helper Phage-Encoded Proteins▿ †  
Journal of Bacteriology  2007;189(20):7520-7524.
The relationship between the composition of SaPI1 transducing particles and those of helper phage 80α was investigated by direct comparison of virion proteins. Twelve virion proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry; all were present in both 80α and SaPI1 virions, and all were encoded by 80α. No SaPI1-encoded proteins were detected. This confirms the prediction that SaPI1 is encapsidated in a virion assembled from helper phage-encoded proteins.
doi:10.1128/JB.00738-07
PMCID: PMC2168463  PMID: 17693489

Results 1-7 (7)