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Oncotarget Mar 2023Homozygous deletion of upregulates synthesis of purine (DNSP) and increases the proliferation of neoplastic cells. This increases the sensitivity of breast cancer...
INTRODUCTION
Homozygous deletion of upregulates synthesis of purine (DNSP) and increases the proliferation of neoplastic cells. This increases the sensitivity of breast cancer cells to DNSP inhibitors such as methotrexate, L-alanosine and pemetrexed.
MATERIALS AND METHODS
7,301 cases of MBC underwent hybrid-capture based comprehensive genomic profiling (CGP). Tumor mutational burden (TMB) was determined on up to 1.1 Mb of sequenced DNA and microsatellite instability (MSI) was determined on 114 loci. Tumor cell PD-L1 expression was determined by IHC (Dako 22C3).
RESULTS
208 (2.84%) of MBC featured loss. loss patients were younger ( = 0.002) and were more frequently ER- (30% vs. 50%; < 0.0001), triple negative (TNBC) (47% vs. 27%; < 0.0001) and less frequently HER2+ (2% vs. 8%; = 0.0001) than intact MBC. Lobular histology and mutations were more frequent in intact (14%) than loss MBC ( < 0.0001). (100%) and (97%) loss (9p21 co-deletion) were significantly associated with loss ( < 0.0001). Likely associated with the increased TNBC cases, BRCA1 mutation was also more frequent in loss MBC (10% vs. 4%; < 0.0001). As for immune checkpoint inhibitors biomarkers, higher TMB >20 mut/Mb levels in the intact MBC ( < 0.0001) and higher PD-L1 low expression (1-49% TPS) in the loss ( = 0.002) were observed.
CONCLUSIONS
loss in MBC has distinct clinical features with genomic alterations (GA) affecting both targeted and immunotherapies. Further efforts are necessary to identify alternative means of targeting PRMT5 and MTA2 in -ve cancers to benefit from the high-MTA environment of -deficient cancers.
Topics: Humans; B7-H1 Antigen; Homozygote; Triple Negative Breast Neoplasms; Sequence Deletion; Purine-Nucleoside Phosphorylase; Genomics; Histone Deacetylases; Repressor Proteins; Protein-Arginine N-Methyltransferases
PubMed: 36913304
DOI: 10.18632/oncotarget.28376 -
Biomedicines Mar 2022Glioblastoma (GBM) is a lethal brain cancer exhibiting high levels of drug resistance, a feature partially imparted by tumor cell stemness. Recent work shows that...
Glioblastoma (GBM) is a lethal brain cancer exhibiting high levels of drug resistance, a feature partially imparted by tumor cell stemness. Recent work shows that homozygous deletion, a genetic alteration occurring in about half of all GBMs, promotes stemness in GBM cells. Exploiting MTAP loss-conferred deficiency in purine salvage, we demonstrate that purine blockade via treatment with L-Alanosine (ALA), an inhibitor of de novo purine synthesis, attenuates stemness of -deficient GBM cells. This ALA-induced reduction in stemness is mediated in part by compromised mitochondrial function, highlighted by ALA-induced elimination of mitochondrial spare respiratory capacity. Notably, these effects of ALA are apparent even when the treatment was transient and with a low dose. Finally, in agreement with diminished stemness and compromised mitochondrial function, we show that ALA sensitizes GBM cells to temozolomide (TMZ) in vitro and in an orthotopic GBM model. Collectively, these results identify purine supply as an essential component in maintaining mitochondrial function in GBM cells and highlight a critical role of mitochondrial function in sustaining GBM stemness. We propose that purine synthesis inhibition can be beneficial in combination with the standard of care for -deficient GBMs, and that it may be feasible to achieve this benefit without inflicting major toxicity.
PubMed: 35453502
DOI: 10.3390/biomedicines10040751 -
Chembiochem : a European Journal of... Apr 2020N-Nitroso-containing natural products are bioactive metabolites with antibacterial and anticancer properties. In particular, compounds containing the diazeniumdiolate...
N-Nitroso-containing natural products are bioactive metabolites with antibacterial and anticancer properties. In particular, compounds containing the diazeniumdiolate (N-nitrosohydroxylamine) group display a wide range of bioactivities ranging from cytotoxicity to metal chelation. Despite the importance of this structural motif, knowledge of its biosynthesis is limited. Herein we describe the discovery of a biosynthetic gene cluster in Streptomyces alanosinicus ATCC 15710 responsible for producing the diazeniumdiolate natural product l-alanosine. Gene disruption and stable isotope feeding experiments identified essential biosynthetic genes and revealed the source of the N-nitroso group. Additional biochemical characterization of the biosynthetic enzymes revealed that the non-proteinogenic amino acid l-2,3-diaminopropionic acid (l-Dap) is synthesized and loaded onto a free-standing peptidyl carrier protein (PCP) domain in l-alanosine biosynthesis, which we propose may be a mechanism of handling unstable intermediates generated en route to the diazeniumdiolate. These discoveries will facilitate efforts to determine the biochemistry of diazeniumdiolate formation.
Topics: Alanine; Azo Compounds; Bacterial Proteins; Biosynthetic Pathways; Molecular Structure; Multigene Family; Streptomyces
PubMed: 31643127
DOI: 10.1002/cbic.201900565 -
Oncotarget Nov 2014Myxofibrosarcomas are genetically complex and involve recurrently deleted chromosome 9p, for which we characterized the pathogenically relevant target(s) using genomic...
Myxofibrosarcomas are genetically complex and involve recurrently deleted chromosome 9p, for which we characterized the pathogenically relevant target(s) using genomic profiling. In 12 of the 15 samples, we detected complete or partial losses of 9p. The only aggressiveness-associated, differentially lost region was 9p21.3, spanning the potential inactivated methylthioadenosine phosphorylase (MTAP) that exhibited homozygous (4/15) or hemizygous (3/15) deletions. In independent samples, MTAP gene status was assessed using quantitative- and methylation-specific PCR assays, and immunoexpression was evaluated. We applied MTAP reexpression or knockdown to elucidate the functional roles of MTAP and the therapeutic potential of L-alanosine in MTAP-preserved and MTAP-deficient myxofibrosarcoma cell lines and xenografts. MTAP protein deficiency (37%) was associated with MTAP gene inactivation (P < 0.001) by homozygous deletion or promoter methylation, and independently portended unfavorable metastasis-free survival (P = 0.0318) and disease-specific survival (P = 0.014). Among the MTAP-deficient cases, the homozygous deletion of MTAP predicted adverse outcome. In MTAP-deficient cells, MTAP reexpression inhibited cell migration and invasion, proliferation, and anchorage-independent colony formation and downregulated cyclin D1. This approach also attenuated the tube-forming abilities of human umbilical venous endothelial cells, attributable to the transcriptional repression of MMP-9, and abrogated the susceptibility to L-alanosine. The inhibiting effects of MTAP expression on tumor growth, angiogenesis, and the induction of apoptosis by L-alanosine were validated using MTAP-reexpressing xenografts and reverted using RNA interference in MTAP-preserved cells. In conclusion, homozygous deletion primarily accounts for the adverse prognostic impact of MTAP deficiency and confers the biological aggressiveness and susceptibility to L-alanosine in myxofibrosarcomas.
Topics: Alanine; Animals; Apoptosis; Cell Growth Processes; Cell Line, Tumor; Cyclin D1; Down-Regulation; Female; Fibrosarcoma; Heterografts; Human Umbilical Vein Endothelial Cells; Humans; Male; Mice; Mice, SCID; Middle Aged; Molecular Targeted Therapy; Purine-Nucleoside Phosphorylase; Survival Analysis
PubMed: 25426549
DOI: 10.18632/oncotarget.2552 -
Cancer Biology & Therapy Sep 2012Methylthioadenosine phosphorylase (MTAP), a key enzyme in the catabolism of 5'-deoxy-5'-methylthioadenosine (MTA), catalyzes the formation of adenine and...
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.
Topics: Adenosine; Animals; Cell Line; Cell Line, Tumor; Disease Models, Animal; Fibrosarcoma; Fluorouracil; Humans; Mesothelioma; Mice; Mice, Inbred C57BL; Neoplasms; Purine-Nucleoside Phosphorylase; Thioguanine; Thionucleosides
PubMed: 22825330
DOI: 10.4161/cbt.21115 -
Blood Feb 2006The deficiency of methylthioadenosine phosphorylase (MTAP) in T-cell acute lymphoblastic leukemia (T-ALL) and other cancers, while constitutively expressed in normal...
EFA (9-beta-D-erythrofuranosyladenine) is an effective salvage agent for methylthioadenosine phosphorylase-selective therapy of T-cell acute lymphoblastic leukemia with L-alanosine.
The deficiency of methylthioadenosine phosphorylase (MTAP) in T-cell acute lymphoblastic leukemia (T-ALL) and other cancers, while constitutively expressed in normal cells, allows for selective therapy using L-alanosine, an inhibitor of de novo AMP synthesis. We demonstrate that MTAP- T-ALL cells obtained at relapse are as sensitive to L-alanosine toxicity as diagnosis samples. The therapeutic index of L-alanosine can be increased by the use of a MTAP substrate, which protects MTAP+ normal cells. Since MTAP substrates MTA and 5'deoxyadenosine are prone to toxicities associated with adenosine, we synthesized and evaluated a potentially nontoxic MTAP substrate, 9-beta-D-erythrofuranosyladenine (EFA). The cytotoxicity of EFA to hematopoietic progenitors erythroid burst-forming units (BFU-Es) and granulocyte-macrophage colony-forming units (CFU-GMs) was at least 26- to 41-fold less than that of MTA. In addition, EFA selectively rescued MTAP+ MOLT-4 cells from L-alanosine toxicity at 25 microM with negligible toxicity even at 100 microM. As for MTA, significant, albeit incomplete, rescue was achieved at 12.5 microM, but higher concentrations were toxic. EFA at 20 microM or less rescued primary MTAP+ T-ALL cells and normal lymphocytes from L-alanosine toxicity. Collectively, these data indicate that EFA is an effective agent for salvaging MTAP+ cells from L-alanosine toxicity and is superior to MTA due to lower cytotoxicity.
Topics: Adenine; Alanine; Antibiotics, Antineoplastic; Cell Line, Tumor; Deoxyadenosines; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Enzyme Inhibitors; Erythroid Precursor Cells; Furans; Granulocyte Precursor Cells; Humans; Leukemia-Lymphoma, Adult T-Cell; Purine-Nucleoside Phosphorylase; Salvage Therapy; Thionucleosides
PubMed: 16234352
DOI: 10.1182/blood-2005-06-2430 -
American Journal of Physiology. Cell... Oct 2000Intracellular ATP depletion is a hallmark event in ischemic injury. It has been extensively characterized in models of chemical anoxia in vitro. In contrast, the fate of...
Intracellular ATP depletion is a hallmark event in ischemic injury. It has been extensively characterized in models of chemical anoxia in vitro. In contrast, the fate of GTP during ischemia remains unknown. We used LLC-PK proximal tubular cells to measure GTP and ATP changes during anoxia. In 45 min, antimycin A decreased ATP and GTP to 8% and 2% of controls, respectively. Ischemia in vivo resulted in comparable reductions in GTP and ATP. After 2 h of recovery, GTP levels in LLC-PK cells increased to 65% while ATP increased to 29%. We also investigated steady-state models of selective ATP or GTP depletion. Combinations of antimycin A and mycophenolic acid selectively reduced GTP to 51% or 25% of control. Similarly, alanosine selectively reduced ATP to 61% or 26% of control. Selective GTP depletion resulted in significant apoptosis. Selective ATP depletion caused mostly necrosis. These models of ATP or GTP depletion can prove useful in dissecting the relative contribution of the two nucleotides to the ischemic phenotype.
Topics: Adenosine Triphosphate; Alanine; Animals; Antibiotics, Antineoplastic; Antimycin A; Apoptosis; Cell Hypoxia; Cells, Cultured; Deoxyglucose; Enzyme Inhibitors; Guanosine; Guanosine Triphosphate; Ischemia; Kidney Cortex; Kidney Tubules, Proximal; LLC-PK1 Cells; Male; Models, Biological; Mycophenolic Acid; Necrosis; Oxidative Phosphorylation; Rats; Rats, Sprague-Dawley; Swine
PubMed: 11003607
DOI: 10.1152/ajpcell.2000.279.4.C1270 -
Blood Oct 1996Methylthioadenosine phosphorylase (MTAP), an enzyme essential for the salvage of adenine and methionine, is deficient in a variety of cancers, including acute... (Comparative Study)
Comparative Study
Methylthioadenosine phosphorylase (MTAP), an enzyme essential for the salvage of adenine and methionine, is deficient in a variety of cancers, including acute lymphoblastic leukemia (ALL). Because the MTAP gene is located adjacent to the tumor-suppressor gene p16 on chromosome 9p21 and more than 60% of T-cell ALL (T-ALL) patients have deletion in the p16 gene, we examined the status of the MTAP gene in T-ALL patients. Quantitative polymerase chain reaction amplification of exon 8 of MTAP showed a deletion in 16 of 48 (33.3%) patients at diagnosis and in 13 of 33 (39.4%) patients at relapse. Southern blot analysis showed that, in addition to deletion of the entire MTAP gene, a common break point was between exons 4 and 5, resulting in deletion of exons 5 through 8. The finding of frequent deficiency of MTAP in T-ALL offers the possibility of an enzyme targeted therapy for T-ALL. MTAP(-) T-ALL-derived cell line, CEM cells were very sensitive to methionine deprivation, with cell viability at 50% of control as early as 48 hours after methionine deprivation. In contrast, methionine deprivation had little effect on the viability of normal lymphocytes or on their proliferative response to phytohemagglutinin. Alanosine, an inhibitor of AMP synthesis, inhibited the growth of both MTAP(+) (Molt-4 and Molt-16) and MTAP(-) (CEM and HSB2) cell lines. However, the addition of methylthioadenosine, the substrate of MTAP, protected the MTAP(+) cells but not the MTAP(-) cells from alanosine toxicity. These findings suggest the possibility of targeting MTAP for selective therapy of T-ALL.
Topics: Adenosine Monophosphate; Alanine; Antimetabolites, Antineoplastic; Chromosomes, Human, Pair 9; DNA Mutational Analysis; DNA, Neoplasm; Exons; Genes; Genes, Tumor Suppressor; Humans; Leukemia-Lymphoma, Adult T-Cell; Methionine; Neoplasm Proteins; Polymerase Chain Reaction; Purine-Nucleoside Phosphorylase; Sequence Deletion; T-Lymphocytes; Tumor Cells, Cultured
PubMed: 8874207
DOI: No ID Found -
Biochemical and Biophysical Research... May 1996Nucleophosmin/B23 (NPM) is a nucleolar phosphoprotein which shifts from nucleoli to the nucleoplasm in cells treated with certain cytotoxic agents (NPM-translocation)....
Nucleophosmin/B23 (NPM) is a nucleolar phosphoprotein which shifts from nucleoli to the nucleoplasm in cells treated with certain cytotoxic agents (NPM-translocation). NPM requires GTP for localization into nucleoli (J. Biol. Chem. 268, 5823-5827, 1993). To understand more about NPM's dynamic localization, the effects of lowering ATP on NPM-translocation and rRNA synthesis were studied. When the ATP level in HeLa cells was reduced by sodium azide, NPM-translocation was blocked. Similar results were obtained when ATP was depleted by other agents, suggesting that ATP depletion was responsible for the blocking of NPM-translocation. It was found that newly synthesized rRNA accumulated in the nuclei during ATP-depletion. Significantly larger than normal nucleoli were also observed. These results indicate that NPM may be involved in the transportation of newly synthesized ribosomes.
Topics: 2,4-Dinitrophenol; Adenosine Triphosphate; Alanine; Antibiotics, Antineoplastic; Azides; Cell Nucleus; Dinitrophenols; HeLa Cells; Humans; Kinetics; Mycophenolic Acid; Nuclear Proteins; Nucleolus Organizer Region; Nucleophosmin; RNA, Ribosomal; Ribosomes; Sodium Azide
PubMed: 8670243
DOI: 10.1006/bbrc.1996.0782 -
Biochimica Et Biophysica Acta Apr 1995GTP and ATP are necessary for glucose-induced insulin secretion; however, the biosynthetic pathways of purine nucleotides have not been studied in pancreatic islets. The...
GTP and ATP are necessary for glucose-induced insulin secretion; however, the biosynthetic pathways of purine nucleotides have not been studied in pancreatic islets. The present work examines the cytosolic pathways of purine nucleotide synthesis using intact rat islets cultured overnight in RPMI 1640 medium containing either [14C]glycine (to label the de novo pathway) or [3H]hypoxanthine (to mark the salvage pathway), with or without mycophenolic acid or L-alanosine (selective inhibitors of cytosolic GTP and ATP synthesis, respectively). Addition of mycophenolic acid decreased total GTP content (mass) by 73-81%; although the incorporation of labeled hypoxanthine into GTP also fell by 87%, the incorporation of glycine did not change. Similarly, L-alanosine decreased ATP mass by 26-33% in the presence of either label; whereas the incorporation of hypoxanthine into ATP fell 59%, the incorporation of glycine was again not significantly decreased. Thus, both the de novo and salvage purine nucleotide biosynthetic pathways are present in rat islets; however, the salvage pathway appears to be quantitatively the more important source of nucleotides. This conclusion was supported by additional studies of the effects on nucleotide content and insulin secretion of various site-specific inhibitors of purine synthesis. These findings have potential relevance to the processes of mitogenesis, cell proliferation and differentiation of islet cells, as well as for the control of insulin secretion.
Topics: Adenosine Triphosphate; Alanine; Animals; Cytosol; Guanosine Triphosphate; Insulin; Islets of Langerhans; Male; Mercaptopurine; Mycophenolic Acid; Rats; Rats, Sprague-Dawley
PubMed: 7718617
DOI: 10.1016/0167-4889(94)00235-7