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S-methyl-5'-thioadenosine phosphorylase (MTAP) deficiency is an emerging biomarker in non-small cell lung cancer (NSCLC) and beyond. The MTAP gene is located in the chromosomal region 9p21.3, which shows one of the most common homozygous deletions across all human cancers (9p21 loss). Loss of 9p21 is found in the majority of pleural mesotheliomas, where it serves as an established diagnostic marker. Until recently, fluorescence in situ hybridization (FISH) was the gold standard for the detection of 9p21 losses, but loss of MTAP expression by immunohistochemistry (IHC) gains increasing importance as an easy to apply and cost-effective diagnostic surrogate marker. Besides, MTAP loss, which has been reported in 13% of NSCLC, is becoming an emerging predictive biomarker in two different scenarios in NSCLC and other cancer types: 1) MTAP loss seems to negatively predict the response to immune checkpoint inhibitor (ICI) treatment via silencing of the tumor microenvironment, and 2) MTAP loss serves as a predictive biomarker for novel targeted treatment strategies. MTAP deficiency leads to an impaired function of the protein arginine methyltransferase 5 (PRMT5) due to its partial inhibition by MTAP's accumulating substrate methylthioadenosine (MTA). This process leaves MTAP deficient tumor cells heavily dependent on the remaining function of PRMT5, making it a perfect target for synthetic lethality. Indeed, MTA-cooperative PRMT5-inhibitors are now tested in several clinical trials with promising early results in solid malignancies. With its emergence as a predictive biomarker, the implementation of MTAP IHC into diagnostic routine for NSCLC and other tumors is likely to take place soon. In this review article, we summarize the current literature on the role of MTAP in thoracic tumors and evaluate different testing methods, including IHC, FISH and next generation sequencing., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Lukas Bubendorf reports a relationship with Astra Zeneca that includes: consulting or advisory and speaking and lecture fees. Lukas Bubendorf reports a relationship with Bristol Myers Squibb Co that includes: consulting or advisory. Lukas Bubendorf reports a relationship with Bayer AG that includes: consulting or advisory and speaking and lecture fees. Lukas Bubendorf reports a relationship with Eli Lilly and Company that includes: consulting or advisory. Lukas Bubendorf reports a relationship with Amgen Inc that includes: consulting or advisory. Lukas Bubendorf reports a relationship with Takeda Oncology that includes: consulting or advisory and speaking and lecture fees. Lukas Bubendorf reports a relationship with Thermo Fisher Scientific Inc that includes: funding grants and speaking and lecture fees. Lukas Bubendorf reports a relationship with Novartis that includes: funding grants. Lukas Bubendorf reports a relationship with Systems Oncology that includes: board membership and consulting or advisory. David Koenig reports a relationship with Astra Zeneca that includes:. David Koenig reports a relationship with MSD that includes: consulting or advisory. David Koenig reports a relationship with Novartis Pharmaceuticals Corporation that includes: consulting or advisory. David Koenig reports a relationship with Mirati Therapeutics Inc that includes: consulting or advisory. David Koenig reports a relationship with Bristol Myers Squibb Co that includes: consulting or advisory. David Koenig reports a relationship with PharmaMar SA that includes: consulting or advisory. David Koenig reports a relationship with Amgen Inc that includes: consulting or advisory. David Koenig reports a relationship with Sanofi that includes: consulting or advisory. Lukas Bubendorf reports a relationship with Janssen Pharmaceuticals Inc that includes: consulting or advisory and speaking and lecture fees. Spasenija Savic Prince reports a relationship with Merck & Co Inc that includes: consulting or advisory. Spasenija Savic Prince reports a relationship with Astra Zeneca that includes: consulting or advisory. Spasenija Savic Prince reports a relationship with Boehringer Ingelheim GmbH that includes: consulting or advisory. Spasenija Savic Prince reports a relationship with F Hoffmann-La Roche Ltd that includes: consulting or advisory. Spasenija Savic Prince reports a relationship with Pfizer that includes: consulting or advisory. Spasenija Savic Prince reports a relationship with Bristol Myers Squibb Co that includes: consulting or advisory. Spasenija Savic Prince reports a relationship with Thermo Fisher Scientific Inc that includes: consulting or advisory. Lukas Bubendorf reports a relationship with F Hoffmann-La Roche Ltd that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Loss of S-methyl-5'-thioadenosine phosphorylase (MTAP) expression is a common event in cancer leading to a critical vulnerability of cancer cells towards anti-cancer drugs. Homozygous MTAP deletions result in a complete expression loss that can be detected by immunohistochemistry (IHC). In this study, a tissue microarray containing 17,078 samples from 149 different tumor entities was analyzed by IHC, and complete MTAP loss was validated by fluorescence in situ hybridization. MTAP loss was observed in 83 of 149 tumor categories, including neuroendocrine neoplasms (up to 80%), Hodgkin lymphoma (50.0%), mesothelioma (32.0% to 36.8%), gastro-intestinal adenocarcinoma (4.0% to 40.5%), urothelial neoplasms (10.5% to 36.7%), squamous cell carcinomas (up to 38%), and various types of sarcomas (up to 20%) and non-Hodgkin lymphomas (up to 14%). Homozygous MTAP deletion was found in 90% to 100% of cases with MTAP expression loss in most tumor categories. However, neuroendocrine tumors, Hodgkin lymphomas, and other lymphomas lacked MTAP deletions. MTAP deficiency was significantly linked to unfavorable tumor phenotype in selected tumor entities and the presence of PD-L1 expression on tumor cells, absence of PD-L1 expression on immune cells, and a low density of CD8 + lymphocytes. In summary, MTAP deficiency can occur in various tumor entities and is linked to unfavorable tumor phenotype and noninflamed tumor microenvironment, but is not always related to deletions. MTAP IHC is of considerable diagnostic value for the detection of neoplastic transformation in multiple different applications., Competing Interests: Conflicts of Interest and Source of Funding: The recombinant rabbit monoclonal antibody, clone MSVA-741R was provided from MS Validated Antibodies GmbH, Hamburg, Germany (owned by a family member of G.S.). For the remaining authors none were declared., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Two Iranian patients with purine nucleoside phosphorylase (PNP) deficiency are described in terms of their clinical and molecular evaluations. PNP deficiency is a rare form of combined immunodeficiency with a profound cellular defect. Patients with PNP deficiency suffer from variable recurrent infections, hypouricemia, and neurological manifestations. Furthermore, patient 1 developed mild cortical atrophy, and patient 2 presented developmental delay, general muscular hypotonia, and food allergy. The two unrelated patients with developed autoimmune hemolytic anemia and T cells lymphopenia and eosinophilia were referred to Immunology, Asthma and Allergy Research Institute (IAARI) in 2019. After taking blood and DNA extraction, genetic analysis of patient 1 was performed by PCR and direct sequencing and whole exome sequencing was applied for patient 2 and the result was confirmed by direct sequencing in the patient and his parents. The genetic result showed two novel variants in exon 3 (c.246_285+9del) and exon 5 (c.569G>T) PNP (NM_000270.4) in the patients, respectively. These variants are considered likely pathogenic based on the American College of Medical Genetics and Genomics (ACMG) guideline. PNP deficiency has a poor prognosis; therefore, early diagnosis would be vital to receive hematopoietic stem cell transplantation (HSCT) as a prominent and successful treatment., (© 2023 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.)

Purine nucleoside phosphorylase (PNP) is an important enzyme in the purine degradation and salvage pathway. PNP deficiency results in marked T lineage lymphopenia and severe immunodeficiency. Additionally, PNP-deficient patients and mice suffer from diverse non-infectious neurological abnormalities of unknown etiology. To further investigate the cause for these neurologic abnormalities, induced pluripotent stem cells (iPSC) from two PNP-deficient patients were differentiated into neurons. The iPSC-derived PNP-deficient neurons had significantly reduced soma and nuclei volumes. The PNP-deficient neurons demonstrated increased spontaneous and staurosporine-induced apoptosis, measured by cleaved caspase-3 expression, together with decreased mitochondrial membrane potential and increased cleaved caspase-9 expression, indicative of enhanced intrinsic apoptosis. Greater expression of tumor protein p53 was also observed in these neurons, and inhibition of p53 using pifithrin-α prevented the apoptosis. Importantly, treatment of the iPSC-derived PNP-deficient neurons with exogenous PNP enzyme alleviated the apoptosis. Inhibition of ribonucleotide reductase (RNR) in iPSC derived from PNP-proficient neurons with hydroxyurea or with nicotinamide and trichostatin A increased the intrinsic neuronal apoptosis, implicating RNR dysfunction as the potential mechanism for the damage caused by PNP deficiency. The findings presented here establish a potential mechanism for the neurological defects observed in PNP-deficient patients and reinforce the critical role that PNP has for neuronal viability., (© 2022. The Author(s).)

Background/aim: Methionine addiction is a fundamental and general hallmark of cancer cells, which require exogenous methionine, despite large amounts of methionine synthesized endogenously. 5-Methylthioadenosine phosphorylase (MTAP) plays a principal role as an enzyme in the methionine-salvage pathway, which produces methionine and adenine from methylthioadenosine and is deleted in 27.5% to 37.5% of osteosarcoma patients., Materials and Methods: Human osteosarcoma cell lines U2OS, SaOS2, MNNG/HOS (HOS) and 143B, were used. The MTAP gene was knocked out in U2OS with CRISPR/Cas9. 143B and HOS have an MTAP deletion and SaOS2 is positive for MTAP. MTAP was determined by western blotting. The four cell lines were compared for sensitivity to recombinant methioninase (rMETase)., Results: MTAP-deleted osteosarcoma cell lines MNNG/HOS and 143B were significantly more sensitive to rMETase than MTAP-positive osteosarcoma cell lines U2OS and SaOS2. In addition, MTAP knock-out U2OS cells were more sensitive to rMETase than the parental MTAP-positive U2OS cells., Conclusion: The present results demonstrated that the absence of MTAP sensitizes osteosarcoma cells to methionine restriction by rMETase, a promising clinical strategy., (Copyright© 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine salvage pathway. PNP deficiency, caused by the autosomal recessive mutations in the PNP gene, can lead to severe combined immunodeficiency (SCID). PNP deficiency patients typically have profound T-cell deficiency with variable B and NK cell functions. They present clinically with recurrent infections, failure to thrive, various neurological disorders, malignancies, and autoimmune diseases. Hematopoietic stem cell transplantation (HSCT) is the only available cure for patients with PNP deficiency. We present three patients, two of whom were successfully treated with HSCT. One patient died prior to HSCT due to EBV-associated lymphoma. Over the course of post-HSCT, there was no further aggravation of the patients' neurological symptoms. Although both of the patients still had mild developmental delay, new developmental milestones were achieved., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

The PRMT5•MTA complex has recently emerged as a new synthetically lethal drug target for the treatment of MTAP -deleted cancers. Here, we report the discovery of development candidate MRTX1719 . MRTX1719 is a potent and selective binder to the PRMT5•MTA complex and selectively inhibits PRMT5 activity in MTAP -deleted cells compared to MTAP -wild-type cells. Daily oral administration of MRTX1719 to tumor xenograft-bearing mice demonstrated dose-dependent inhibition of PRMT5-dependent symmetric dimethylarginine protein modification in MTAP -deleted tumors that correlated with antitumor activity. A 4-(aminomethyl)phthalazin-1(2 H )-one hit was identified through a fragment-based screen, followed by X-ray crystallography, to confirm binding to the PRMT5•MTA complex. Fragment growth supported by structural insights from X-ray crystallography coupled with optimization of pharmacokinetic properties aided the discovery of development candidate MRTX1719 .

Methylthioadenosine phosphorylase (MTAP) is a key enzyme associated with the salvage of methionine and adenine that is deficient in 20% to 30% of pancreatic cancer. Our previous study revealed that MTAP deficiency indicates a poor prognosis for patients with pancreatic ductal adenocarcinoma (PDAC). In this study, bioinformatics analysis of The Cancer Genome Atlas (TCGA) data indicated that PDACs with MTAP deficiency display a signature of elevated glycolysis. Metabolomics studies showed that that MTAP deletion-mediated metabolic reprogramming enhanced glycolysis and de novo purine synthesis in pancreatic cancer cells. Western blot analysis revealed that MTAP knockout stabilized hypoxia-inducible factor 1α (HIF1α) protein via posttranslational phosphorylation. RIO kinase 1 (RIOK1), a downstream kinase upregulated in MTAP-deficient cells, interacted with and phosphorylated HIF1α to regulate its stability. In vitro experiments demonstrated that the glycolysis inhibitor 2-deoxy-d-glucose (2-DG) and the de novo purine synthesis inhibitor l-alanosine synergized to kill MTAP-deficient pancreatic cancer cells. Collectively, these results reveal that MTAP deficiency drives pancreatic cancer progression by inducing metabolic reprogramming, providing a novel target and therapeutic strategy for treating MTAP-deficient disease. SIGNIFICANCE: This study demonstrates that MTAP status impacts glucose and purine metabolism, thus identifying multiple novel treatment options against MTAP-deficient pancreatic cancer., (©2021 American Association for Cancer Research.)

Homozygous deletion of methylthioadenosine phosphorylase (MTAP) in cancers such as glioblastoma represents a potentially targetable vulnerability. Homozygous MTAP-deleted cell lines in culture show elevation of MTAP's substrate metabolite, methylthioadenosine (MTA). High levels of MTA inhibit protein arginine methyltransferase 5 (PRMT5), which sensitizes MTAP-deleted cells to PRMT5 and methionine adenosyltransferase 2A (MAT2A) inhibition. While this concept has been extensively corroborated in vitro, the clinical relevance relies on exhibiting significant MTA accumulation in human glioblastoma. In this work, using comprehensive metabolomic profiling, we show that MTA secreted by MTAP-deleted cells in vitro results in high levels of extracellular MTA. We further demonstrate that homozygous MTAP-deleted primary glioblastoma tumors do not significantly accumulate MTA in vivo due to metabolism of MTA by MTAP-expressing stroma. These findings highlight metabolic discrepancies between in vitro models and primary human tumors that must be considered when developing strategies for precision therapies targeting glioblastoma with homozygous MTAP deletion., (© 2021. The Author(s).)

Intestinal epithelial cells are subject to attack by a diverse array of microbes, including intracellular as well as extracellular pathogens. While defense in epithelial cells can be triggered by pattern recognition receptor-mediated detection of microbe-associated molecular patterns, there is much to be learned about how they sense infection via perturbations of host physiology, which often occur during infection. A recently described host defense response in the nematode C. elegans called the Intracellular Pathogen Response (IPR) can be triggered by infection with diverse natural intracellular pathogens, as well as by perturbations to protein homeostasis. From a forward genetic screen, we identified the C. elegans ortholog of purine nucleoside phosphorylase pnp-1 as a negative regulator of IPR gene expression, as well as a negative regulator of genes induced by extracellular pathogens. Accordingly, pnp-1 mutants have resistance to both intracellular and extracellular pathogens. Metabolomics analysis indicates that C. elegans pnp-1 likely has enzymatic activity similar to its human ortholog, serving to convert purine nucleosides into free bases. Classic genetic studies have shown how mutations in human purine nucleoside phosphorylase cause immunodeficiency due to T-cell dysfunction. Here we show that C. elegans pnp-1 acts in intestinal epithelial cells to regulate defense. Altogether, these results indicate that perturbations in purine metabolism are likely monitored as a cue to promote defense against epithelial infection in the nematode C. elegans., Competing Interests: The authors have declared that no competing interests exist.

Background: Purine nucleoside phosphorylase (PNP) deficiency accounts for about 4% of severe combined immunodeficiency diseases. PNP deficiency is a variable disease with recurrent infections and neurodevelopmental delay. Autoimmunity and malignancy can still occur in one-third of patients., Methods: Case report., Case Presentation: An 8-year-old Saudi female who was apparently healthy presented at the age of 7 years with confirmed systemic lupus erythematosus (SLE) and lupus nephritis that were poorly controlled with conventional therapy. She also had frequent sinopulmonary and varicella infections. Preliminary immunological workup showed severe lymphopenia and depressed lymphocyte proliferation assay. The uric acid was within normal levels at 179 μmol/L (normal range, 150 to 350 μmol/L) 6 weeks after blood transfusion. Genetic study revealed a homozygous missense mutation c.265G>A in the PNP gene, resulting in a substitution of glutamic acid to lysine at amino acid 89 of the encoded protein (E89K). The PNP serum level was 798 nmol/h/mg (normal level 1354 ± 561 nmol/h/mg) 6 weeks after blood transfusion. Hematopoietic stem cell transplantation (HSCT) was planned from a matched unrelated donor; however, she developed an EBV and varicella meningoencephalitis. Atypical malignant cells suggestive of lymphoma were discovered, likely induced by EBV, and suspicious lesions were shown on brain MRI and PET scan. Unfortunately, she passed away before HSCT due to multiorgan failure., Conclusion: This report emphasizes the challenges in recognizing PNP deficiency in a patient suffering from SLE.

Introduction: Complete or near complete absence of the purine nucleoside phosphorylase (PNP) enzyme causes a profound T cell immunodeficiency and neurological abnormalities that are often lethal in infancy and early childhood. We hypothesized that patients with partial PNP deficiency, characterized by a late and mild phenotype due to residual PNP enzyme, would provide important information about the minimal PNP activity needed for normal development. Methods: Three siblings with a homozygous PNP gene mutation (c.769C>G, p.His257Asp) resulting in partial PNP deficiency were investigated. PNP activity was semi-quantitively assayed by the conversion of [14C]inosine in hemolysates, mononuclear cells, and lymphoblastoid B cells. PNP protein expression was determined by Western Blotting in lymphoblastoid B cells. DNA repair was quantified by measuring viability of lymphoblastoid B cells following ionizing irradiation. Results: A 21-year-old female was referred for recurrent sino-pulmonary infections while her older male siblings, aged 25- and 28- years, did not suffer from significant infections. Two of the siblings had moderately reduced numbers of T, B, and NK cells, while the other had near normal lymphocyte subset numbers. T cell proliferations were normal in the two siblings tested. Hypogammaglobulinemia was noted in two siblings, including one that required immunoglobulin replacement. All siblings had typical (normal) neurological development. PNP activity in various cells from two patients were 8-11% of the normal level. All siblings had normal blood uric acid and increased PNP substrates in the urine. PNP protein expression in cells from the two patients examined was similar to that observed in cells from healthy controls. The survival of lymphoblastoid B cells from 2 partial PNP-deficient patients after irradiation was similar to that of PNP-proficient cells and markedly higher than the survival of cells from a patient with absent PNP activity or a patient with ataxia telangiectasia. Conclusions: Patients with partial PNP deficiency can present in the third decade of life with mild-moderate immune abnormalities and typical development. Near-normal immunity might be achieved with relatively low PNP activity., (Copyright © 2020 Grunebaum, Campbell, Leon-Ponte, Xu and Chapdelaine.)

To investigate the predictive value of methylthioadenosine phosphorylase (MTAP) on treatment response and survival in advanced lung adenocarcinoma. MTAP expression was detected by immunohistochemistry. Treatment response and survival were compared according to MTAP expression level. The results indicated MTAP-low expression was observed in 61.2% (101/165) of all patients. The objective response rate and disease control rate improved in the MTAP-low group (64.4% vs 46.9%, p = 0.035; 92.1% vs. 79.7%, p = 0.03; respectively). The median progression-free survival and survival time in the MTAP-low group were significantly lower than that in the MTAP-high group (8.1 vs. 13.1 months, p = 0.002; 22 vs. 32 months, p = 0.044). Multivariate analysis demonstrated that brain metastasis (HR 1.55, p = 0.046), thoracic radiation (HR 0.52, p = 0.026), and MTAP-low expression (HR 1.36, p = 0.038) were independent factors on survival. It is concluded that MTAP-low expression could predict improved treatment response but worsened survival in advanced lung adenocarcinoma.

Purpose: Purine nucleoside phosphorylase (PNP) is a known yet rare cause of combined immunodeficiency with a heterogeneous clinical presentation. We aim to add to the expanding clinical spectrum of disease, and to summarize the available data on bone marrow transplant for this condition., Methods: Data was collected from patient files retrospectively. A review of the literature of hematopoietic stem cell transplantation (HSCT) for PNP deficiency was conducted., Results: Four patients were treated in two centers in Israel. One patient died of EBV-related lymphoma with CNS involvement prior to transplant. The other three patients underwent successful HSCT with good immune reconstitution post-transplant (follow-up 8-108 months) and excellent neurological outcomes., Conclusion: PNP is a variable immunodeficiency and should be considered in various clinical contexts, with or without neurological manifestations. HSCT offers a good treatment option, with excellent clinical outcomes, when preformed in a timely manner.

Type I protein arginine methyltransferases (PRMTs) catalyze asymmetric dimethylation of arginines on proteins. Type I PRMTs and their substrates have been implicated in human cancers, suggesting inhibition of type I PRMTs may offer a therapeutic approach for oncology. The current report describes GSK3368715 (EPZ019997), a potent, reversible type I PRMT inhibitor with anti-tumor effects in human cancer models. Inhibition of PRMT5, the predominant type II PRMT, produces synergistic cancer cell growth inhibition when combined with GSK3368715. Interestingly, deletion of the methylthioadenosine phosphorylase gene (MTAP) results in accumulation of the metabolite 2-methylthioadenosine, an endogenous inhibitor of PRMT5, and correlates with sensitivity to GSK3368715 in cell lines. These data provide rationale to explore MTAP status as a biomarker strategy for patient selection., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Systemic juvenile idiopathic arthritis (sJIA) is an inflammatory condition that presents with fever, rash and arthritis. At onset systemic features are predominant and the diagnosis may be a challenge. Secondary hemophagocytic lymphohistiocytosis (sHLH) forms may be associated with different disorders, including rheumatic diseases, and this form is called macrophage activation syndrome (MAS). CXCL9 levels, a chemokine induced by IFNγ, are significantly elevated in patients with sHLH or MAS and are correlated with laboratory features of disease activity. High levels of IL-18 have been reported in patients with MAS during sJIA, as well as in some patients with sHLH and IL-18 is indeed known to induce IFNγ production., Findings: We report a patient with a clinical presentation highly suggestive for systemic juvenile idiopathic arthritis (sJIA) onset complicated by MAS, and was later diagnosed with purine nucleoside phosphorylase (PNP)-deficiency with HLH. Some unusual features appeared when HLH was controlled and further investigations provided the correct diagnosis. Serum CXCL9 and IL-18 levels were found markedly elevated at disease onset, during the active phase of MAS and decreased progressively during the course., Conclusion: The reported case underlines the potential difficulties in discriminating sJIA from other causes of systemic inflammation. Furthermore, this supports the notion that especially in young children with a sJIA-like disease other mimicking conditions should be actively sought for. CXCL9 and IL-18 levels suggested that patients with PNP-deficiency may have a subclinical activation of the IFNγ pathway and indeed they are predisposed to develop sHLH.

Purine nucleoside phosphorylase (PNP) deficiency is a rare autosomal recessive primary immunodeficiency disorder characterized by decreased numbers of T-cells, variable B-cell abnormalities, decreased amount of serum uric acid and PNP enzyme activity. The affected patients usually present with recurrent infections, neurological dysfunction and autoimmune phenomena. In this study, whole-exome sequencing was used to detect mutation in the case suspected of having primary immunodeficiency. We found a homozygous mutation in PNP gene in a girl who is the third case from the national Iranian registry. She had combined immunodeficiency, autoimmune hemolytic anemia and a history of recurrent infections. She developed no neurological dysfunction. She died at the age of 11 after a severe chicken pox infection. PNP deficiency should be considered in late-onset children with recurrent infections, autoimmune disorders without typical neurologic impairment.

Despite a wealth of potential applications inside target cells, protein-based therapeutics are largely limited to extracellular targets due to the inability of proteins to readily cross biological membranes and enter the cytosol. Bacterial toxins, which deliver a cytotoxic enzyme into cells as part of their intoxication mechanism, hold great potential as platforms for delivering therapeutic protein cargo into cells. Diphtheria toxin (DT) has been shown to be capable of delivering an array of model proteins of varying sizes, structures, and stabilities into mammalian cells as amino-terminal fusions. Here, seeking to expand the utility of DT as a delivery vector, we asked whether an active human enzyme, purine nucleoside phosphorylase (PNP), could be delivered by DT into cells to rescue PNP deficiency. Using a series of biochemical and cellular readouts, we demonstrate that PNP is efficiently delivered into target cells in a receptor- and translocation-dependent manner. In patient-derived PNP-deficient lymphocytes and pluripotent stem cell-differentiated neurons, we show that human PNP is efficiently translocated into target cells by DT, where it is able to restore intracellular hypoxanthine levels. Further, through replacement of the native receptor-binding moiety of DT with single-chain variable fragments that were selected to bind mouse HBEGF, we show that PNP can be retargeted into mouse splenocytes from PNP-deficient mice, resulting in restoration of the proliferative capacity of T-cells. These findings highlight the versatility of the DT delivery platform and provide an attractive approach for the delivery of PNP as well as other cytosolic enzymes implicated in disease.

Methylthioadenosine phosphorylase (MTAP), a key enzyme in the adenine and methionine salvage pathways, catalyzes the hydrolysis of methylthioadenosine (MTA), a compound suggested to affect pivotal cellular processes in part through the regulation of protein methylation. MTAP is expressed in a wide range of cell types and tissues, and its deletion is common to cancer cells and in liver injury. The aim of this study was to investigate the proteome and methyl proteome alterations triggered by MTAP deficiency in liver cells to define novel regulatory mechanisms that may explain the pathogenic processes of liver diseases. iTRAQ analysis resulted in the identification of 216 differential proteins (p < 0.05) that suggest deregulation of cellular pathways as those mediated by ERK or NFκB. R-methyl proteome analysis led to the identification of 74 differentially methylated proteins between SK-Hep1 and SK-Hep1+ cells, including 116 new methylation sites. Restoring normal MTA levels in SK-Hep1+ cells parallels the specific methylation of 56 proteins, including KRT8, TGF, and CTF8A, which provides a novel regulatory mechanism of their activity with potential implications in carcinogenesis. Inhibition of RNA-binding proteins methylation is especially relevant upon accumulation of MTA. As an example, methylation of quaking protein in Arg(242) and Arg(256) in SK-Hep1+ cells may play a pivotal role in the regulation of its activity as indicated by the up-regulation of its target protein p27(kip1) The phenotype associated with a MTAP deficiency was further verified in the liver of MTAP± mice. Our data support that MTAP deficiency leads to MTA accumulation and deregulation of central cellular pathways, increasing proliferation and decreasing the susceptibility to chemotherapeutic drugs, which involves differential protein methylation. Data are available via ProteomeXchange with identifier PXD002957 (http://www.ebi.ac.uk/pride/archive/projects/PXD002957)., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Homozygous deletions of p16/CDKN2A are prevalent in cancer, and these mutations commonly involve co-deletion of adjacent genes, including methylthioadenosine phosphorylase (MTAP). Here, we used shRNA screening and identified the metabolic enzyme, methionine adenosyltransferase II alpha (MAT2A), and the arginine methyltransferase, PRMT5, as vulnerable enzymes in cells with MTAP deletion. Metabolomic and biochemical studies revealed a mechanistic basis for this synthetic lethality. The MTAP substrate methylthioadenosine (MTA) accumulates upon MTAP loss. Biochemical profiling of a methyltransferase enzyme panel revealed that MTA is a potent and selective inhibitor of PRMT5. MTAP-deleted cells have reduced PRMT5 methylation activity and increased sensitivity to PRMT5 depletion. MAT2A produces the PRMT5 substrate S-adenosylmethionine (SAM), and MAT2A depletion reduces growth and PRMT5 methylation activity selectively in MTAP-deleted cells. Furthermore, this vulnerability extends to PRMT5 co-complex proteins such as RIOK1. Thus, the unique biochemical features of PRMT5 create an axis of targets vulnerable in CDKN2A/MTAP-deleted cancers., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Purine nucleoside phosphorylase (PNP) deficiency has been recently introduced in the newborn screening program in Tuscany. In order to improve the PNP screening efficiency, we developed a 2nd tier test to quantify PNP primary markers deoxyguanosine (dGuo) and deoxyinosine (dIno)., Methods: Dried blood spots (DBS) samples were extracted with 200 μL of methanol and 100 μL of water (by two steps). Internal standards were added at a final concentration of 10 μmol/L. After extraction, samples were analysed by LC-MS/MS. The chromatographic run was performed in gradient mode by using a Synergi Fusion column., Results: The assay was linear over a concentration range of 0.05-50 μmol/L (R2>0.999) for dGuo and 0.5-50 μmol/L (R2>0.998) for dIno. Intra- and interassay imprecision (mean CVs) for dIno and dGuo ranged from 2.9% to 12%. Limit of quantitaion (LOQ) were found to be 0.05 μmol/L and 0.5 μmol/L for dGuo and dIno, respectively. The reference ranges, obtained by measuring dGuo and dIno concentrations on DBS, were close to zero for both biomarkers. Moreover, DBS samples from seven patients with confirmed PNP were retrospectively evaluated and correctly identified., Conclusions: The LC-MS/MS method can reliably measure dIno and dGuo in DBS for the diagnosis of PNP. Validation data confirm the present method is characterised by good reproducibility, accuracy and imprecision for the quantitation of dIno and dGuo. The assay also appears suitable for use in monitoring treatment of PNP patients.

Background: The cause of primary immunodeficiency has expanded to nearly 200 distinct disorders. An improved understanding of these disorders has resulted in decreased morbidity and mortality with reciprocal improved life expectancy. Obstetricians should have knowledge of primary immunodeficiency, as more women with these disorders will reach reproductive age., Case: 21-year-old G1P0 with purine nucleoside phosphorylase (PNP) deficiency delivered a viable infant vaginally at 37 weeks. Although the patient's diagnosis and pregnancy placed her at increased risk for infection, she remained asymptomatic and infection-free throughout pregnancy., Conclusion: The management of pregnancy complicated by PNP deficiency requires strict immune surveillance and regimented immunoglobulin replacement.

Identification of cancer-associated genes by genomic profiling contributes to the elucidation of tumor development and progression. The methylthioadenosine phosphorylase (MTAP) gene, located at chromosome 9p21, plays a critical role in tumorigenicity and disease progression in a wide variety of cancers. However, the prognostic impact of MTAP in patients with nasopharyngeal carcinoma (NPC) remains obscured. Through data mining from published transcriptomic database, MTAP was first identified as a differentially downregulated gene in NPC. In this study, our aim was to evaluate the expression of MTAP in NPC and to clarify its prognostic significance.MTAP immunohistochemistry was retrospectively performed and analyzed in biopsy specimens from 124 NPC patients who received standard treatment without distant metastasis at initial diagnosis. The immunoexpression status was correlated with the clinicopathological variables, disease-specific survival (DSS), distant metastasis-free survival (DMFS), and local recurrence-free survival (LRFS). Real-time quantitative polymerase chain reaction (PCR) was used to measure MTAP gene dosage. In some cases, we also performed methylation-specific PCR and pyrosequencing to assess the status of promoter methylation.MTAP deficiency was significantly associated with advanced tumor stages (P = 0.023) and univariately predictive of adverse outcomes for DSS, DMFS, and LRFS. In the multivariate comparison, MTAP deficiency still remained prognostically independent to portend worse DSS (P = 0.021, hazard ratio = 1.870) and DMFS (P = 0.009, hazard ratio = 2.154), together with advanced AJCC stages III to IV. Homozygous deletion or promoter methylation of MTAP gene were identified to be significantly associated with MTAP protein deficiency (P < 0.001).MTAP deficiency was correlated with an aggressive phenotype and independently predictive of worse DSS and DMFS, suggesting its role in disease progression and as an independent prognostic biomarker of NPC, which potentially offers new strategy of targeted treatment for patients lacking MTAP expression., Competing Interests: The authors have no conflicts of interest to disclose.

Purpose: To investigate the effectiveness of a combination of 6-thioguanine (6-TG) and pralatrexate (PDX) in methylthioadenosine phosphorylase (MTAP)-deficient T-cell acute lymphoblastic leukemia (T-cell ALL)., Methods: CCRF-CEM (MTAP(-/-)) and Molt4 (MTAP(+/+)) T-cell ALL cell lines were treated with 6-TG or PDX and evaluated for efficacy 72 h later. NOD/SCID gamma mice bearing CEM or Molt4 xenografts were treated with 6-TG and PDX alone or in combination to evaluate antitumor effects., Results: CEM cells were more sensitive to 6-TG and PDX in vitro than Molt4. In vivo, CEM cells were very sensitive to PDX and 6-TG, whereas Molt4 cells were highly resistant to 6-TG. A well-tolerated combination of PDX and 6-TG achieved significant tumor regression in CEM xenografts., Conclusions: The loss of MTAP expression may be therapeutically exploited in T-cell ALL. The combination of 6-TG and PDX, with the inclusion of leucovorin rescue, allows for a safe and effective regimen in MTAP-deficient T-cell ALL.

PNP deficiency is a rare combined immunodeficiency with autosomal recessive mode of inheritance. The immunodeficiency is progressive with normal immune functions at birth, but then, T-cell deficiency with variable B-cell functions usually presents by the age of two yr. The only curative treatment for PNP deficiency is hematopoietic stem cell transplantation. Here, we present a 13-yr-old girl with late-onset PNP deficiency. Despite many complications of infections, she was successfully transplanted with a reduced intensity-conditioning regimen from an HLA-identical unrelated donor., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Purine nucleoside phosphorylase (PNP) deficiency is a rare form of autosomal recessive combined primary immunodeficiency caused by a enzyme defect leading to the accumulation of inosine, 2'-deoxy-inosine (dIno), guanosine, and 2'-deoxy-guanosine (dGuo) in all cells, especially lymphocytes. Treatments are available and curative for PNP deficiency, but their efficacy depends on the early approach. PNP-combined immunodeficiency complies with the criteria for inclusion in a newborn screening program., Objective: This study evaluate whether mass spectrometry can identify metabolite abnormalities in dried blood spots (DBSs) from affected patients, with the final goal of individuating the disease at birth during routine newborn screening., Methods: DBS samples from 9 patients with genetically confirmed PNP-combined immunodeficiency, 10,000 DBS samples from healthy newborns, and 240 DBSs from healthy donors of different age ranges were examined. Inosine, dIno, guanosine, and dGuo were tested by using tandem mass spectrometry (TMS). T-cell receptor excision circle (TREC) and kappa-deleting recombination excision circle (KREC) levels were evaluated by using quantitative RT-PCR only for the 2 patients (patients 8 and 9) whose neonatal DBSs were available., Results: Mean levels of guanosine, inosine, dGuo, and dIno were 4.4, 133.3, 3.6, and 3.8 μmol/L, respectively, in affected patients. No indeterminate or false-positive results were found. In patient 8 TREC levels were borderline and KREC levels were abnormal; in patient 9 TRECs were undetectable, whereas KREC levels were normal., Conclusion: TMS is a valid method for diagnosis of PNP deficiency on DBSs of affected patients at a negligible cost. TMS identifies newborns with PNP deficiency, whereas TREC or KREC measurement alone can fail., (Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Purpose of the Review: To review the recent advances in the understanding and management of the immune and nonimmune effects of inherited adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiencies., Recent Findings: Abnormal thymocyte development and peripheral T-cell activation in ADA-deficient and PNP-deficient patients cause increased susceptibility to infections and immune dysregulation. The impaired purine homeostasis also damages many other cell types and tissues. Animal studies suggest that defects in surfactant metabolism by alveolar macrophages cause the pulmonary alveolar proteinosis commonly seen in ADA-deficient infants, while toxicity of purine metabolites to cerebellar Purkinje cells may lead to the ataxia frequently observed in PNP deficiency. Patients' outcome with current treatments including enzyme replacement and stem cell transplantations are inferior to those achieved in most severe immunodeficiency conditions. New strategies, including intracellular enzyme replacement, gene therapy and innovative protocols for stem cell transplantations hold great promise for improved outcomes in ADA and PNP deficiency. Moreover, newborn screening and early diagnosis will allow prompt application of these novel treatment strategies, further improving survival and reducing morbidity., Summary: Better understanding of the complex immune and nonimmune effects of ADA and PNP deficiency holds great promise for improved patients' outcome.

Purine nucleoside phosphorylase (PNP) is one of the most important enzymes of the purine metabolism, wich promotes the recycling of purine bases. Nowadays is the actual to search for effective inhibitors of this enzyme which is necessary for creation T-cell immunodeficient status of the organism in the organs and tissues transplantation, and chemotherapy of a number pathologies as well. For their successful practical application necessary to conduct in-depth and comprehensive study of the enzyme, namely a structure, functions, and an affinity of the reaction mechanism. In the review the contemporary achievements in the study of PNP from various biological objects are presented. New data describing the structure of PNP are summarised and analysed. The physiological role of the enzyme is discussed. The enzyme basic reaction mechanisms and actions are considered. The studies on enzyme physicochemical, kinetic, and catalytic research are presented.

Nucleotide degradation is a universal metabolic capability. Here we combine metabolomics, genetics and biochemistry to characterize the yeast pathway. Nutrient starvation, via PKA, AMPK/SNF1, and TOR, triggers autophagic breakdown of ribosomes into nucleotides. A protein not previously associated with nucleotide degradation, Phm8, converts nucleotide monophosphates into nucleosides. Downstream steps, which involve the purine nucleoside phosphorylase, Pnp1, and pyrimidine nucleoside hydrolase, Urh1, funnel ribose into the nonoxidative pentose phosphate pathway. During carbon starvation, the ribose-derived carbon accumulates as sedoheptulose-7-phosphate, whose consumption by transaldolase is impaired due to depletion of transaldolase's other substrate, glyceraldehyde-3-phosphate. Oxidative stress increases glyceraldehyde-3-phosphate, resulting in rapid consumption of sedoheptulose-7-phosphate to make NADPH for antioxidant defense. Ablation of Phm8 or double deletion of Pnp1 and Urh1 prevent effective nucleotide salvage, resulting in metabolite depletion and impaired survival of starving yeast. Thus, ribose salvage provides means of surviving nutrient starvation and oxidative stress.

Purine nucleoside phosphorylase (PNP) deficiency is an autosomal recessive genetic disorder of the purine salvage pathway, associated with a variable extent of immunodeficiency. Here, we report a PNP-deficient patient who presented early in life with clinical and laboratory characteristics of severe combined immunodeficiency, including severe infections, marked T-and B-cell deficiency, lack of lymphocyte response to mitogenic stimulation, monoclonal T-cell receptors representation and the absence of T-cell receptor excision circles and Kappa-receptor excision circles. The patient carried homozygote mutation at the PNP gene that putatively led to aberrant splicing, allowing normal and abnormally spliced products from the mutant alleles. We suggest that the aberrant slice site was used preferentially over the normal slice site in some cells correlating with the severity of disease.

Unlabelled: The majority of peripheral T-cell lymphomas were found to lack methylthioadenosine phosphorylase, an enzyme that is essential for the salvage of adenine from methylthioadenosine, a product of polyamine synthesis. Importantly, tumors that lack this enzyme have been shown to be more sensitive to inhibitors of de novo purine synthesis (6-thioguanine, methotrexate)., Background: T-cell lymphomas, in particular peripheral T-cell lymphoma (PTCL), angioimmunoblastic T-cell lymphoma (AITL), and anaplastic large cell lymphoma (ALCL), have only limited and noncurative treatment options., Patients and Methods: We report here that a high percentage of PTCL, AITL, and ALCL lack the enzyme methylthioadenosine phosphorylase (MTAP), as do T-cell leukemia and T-cell lymphoblastic leukemia. MTAP-deficient cells cannot cleave endogenous methylthioadenosine to adenine and 5-methylthioribose-1-phosphate, a precursor of methionine, and as a result have enhanced sensitivity to inhibitors of de novo purine biosynthesis. A recently introduced antifolate, pralatrexate, which has been shown to inhibit de novo purine biosynthesis, has been approved for treatment of PTCL and may have an increasing role in therapy. An alternative strategy involving coadministration of methylthioadenosine and high-dose 6-thioguanine has been proposed and may prove to be selectively toxic to MTAP-deficient uncommon lymphomas., Conclusion: Thus the consequences of MTAP deficiency suggest that new therapeutic interventions for T-cell lymphoma may be feasible., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Inherited defects in purine nucleoside phosphorylase (PNP) cause severe T cell immunodeficiency and progressive neurological dysfunction, yet little is known about the effects of PNP deficiency on the brain. PNP-KO mice display metabolic and immune anomalies similar to those observed in patients. Our objectives were to characterize brain abnormalities in PNP-KO mice and determine whether restoring PNP activity prevents these abnormalities. We analyzed structural brain defects in PNP-KO mice by magnetic resonance imaging, while assessing motor deficits using the accelerating rotarod and stationary balance beam tests. We detected morphological abnormalities and apoptosis in the cerebellum of PNP-KO mice by hematoxylin and eosin, electron microscopy, TUNEL and activated caspase 3 staining. We treated PNP-KO mice with PNP fused to the HIV-TAT protein transduction domain (TAT-PNP) from birth or from 4 weeks of age. Magnetic resonance imaging revealed a smaller than normal cerebellum in PNP-KO mice. PNP-KO mice displayed motor abnormalities including rapid fall from the rotating rod and frequent slips from the balance beam. The cerebellum of PNP-KO mice contained reduced purkinje cells (PC), which were irregular in shape and had degenerated dendrites. PC from the cerebellum of PNP-KO mice, expanded ex vivo, demonstrated increased apoptosis, which could be corrected by supplementing cultures with TAT-PNP. TAT-PNP injections restored PNP activity in the cerebellum of PNP-KO mice. TAT-PNP from birth, but not treatment initiated at 4 weeks of age, prevented the cerebellar PC damage and motor deficits. We conclude that PNP deficiency cause cerebellar abnormalities, including PC damage and progressive motor deficits. TAT-PNP treatment from birth can prevent the neurological abnormalities in PNP-KO mice., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Aims:   Malignant pleural mesothelioma (MPM) often causes diagnostic difficulties for pathologists. We assessed whether loss of methylthioadenosine phosphorylase (MTAP), a key enzyme in the intracellular recycling of adenosine triphosphate (ATP) often deleted in MPM, could be detected with immunohistochemistry (IHC) and used as a diagnostic marker for MPM., Methods and Results:   We used IHC to detect MTAP in a cohort of 99 MPMs and 39 reactive mesothelial proliferations (RP) (reactive mesothelial hyperplasia n = 33, reactive pleural fibrosis n = 6). MTAP staining was assessed by an H score. The median H score of the RP cohort was set as a reference point. Cases with H scores below this reference point were considered to have decreased MTAP expression. We found that 64 of 99 (65%) of the investigated MPMs had decreased MTAP expression, while this was only true for nine of 39 (23%) of the RPs (P = 0.001). We further evaluated MTAP expression in a cohort of coagulated pleural effusions from 14 patients with MPM and 20 patients with RP by using a double-staining technique with Wilms tumour 1 (WT1) as a mesothelial marker. In these samples, decreased MTAP expression diagnosed MPM with a sensitivity of 71% and a specificity of 90%., Conclusions:   Decreased MTAP expression could potentially be useful in combination with other markers in the diagnosis of MPM., (© 2012 Blackwell Publishing Ltd.)

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 tumor sequencing program, a patient tumor was analyzed using Illumina genome sequencing and a matched renal capsule tumor xenograft was generated. Both tumor 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 tumors, yet approximately 10% express decreased levels of methylthioadenosine phosphorylase (MTAP) mRNA. Decreased MTAP expression is a prognosticator for poor outcome. Moreover, it seems 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 as deletion or hypermethylation of the MTAP gene occurs in a broad spectrum of tumors at high frequency. The combination of genome sequencing and patient-derived xenografts can identify candidate therapeutic agents and evaluate them for personalized oncology.

The authors report a case of purine nucleoside phosphorylase (PNP) deficiency for the first time from India. The case presented with recurrent severe infections, developmental delays, seizures and progressive neurological deterioration. The diagnosis of primary immunodeficiency disorder was delayed in spite of recurrent infection due to predominant neurological symptoms. Sequencing of the PNP gene revealed a novel mutation resulting in a premature stop codon.

Purine nucleoside phosphorylase (PNPase) deficiency is an autosomal recessive disorder affecting purine degradation and salvage pathways. Clinically, patients typically present with severe immunodeficiency, neurological dysfunction, and autoimmunity. Biochemically, PNPase deficiency may be suspected in the presence of hypouricemia. We report biochemical and genetic data on a cohort of seven patients from six families identified as PNPase deficient. In all patients, inosine, deoxyinosine, guanosine, and deoxyguanosine were elevated in urine, and mutation analysis revealed seven different mutations of which three were novel. The mutation c.770A>G resulted in the substitution p.His257Arg. A second novel mutation c.257A>G (p.His86Arg) was identified in two siblings and a third novel mutation, c.199C>T (p.Arg67X), was found in a 2-year-old female with delayed motor milestones and recurrent respiratory infections. A review of the literature identified 67 cases of PNPase deficiency from 49 families, including the cases from our own laboratory. PNPase deficiency was confirmed in 30 patients by genotyping and 24 disease causing mutations, including the three novel mutations described in this paper, have been reported to date. In five of the seven patients, plasma uric acid was found to be within the pediatric normal range, suggesting that PNPase deficiency should not be ruled out in the absence of hypouricemia.

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.

The thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG), are used in the treatment of leukemia. Incorporation of deoxythioguanosine nucleotides (dG(s)) 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. Because the growth of MTAP-deleted tumor 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 leukemic cell line, Jurkat, transfected with MTAP cDNA under the control of a tetracycline-inducible promoter, and a lung cancer cell line (A549-MTAP(-)) transfected to express MTAP constitutively (A549-MTAP(+)). Sensitivity to 6-MP or methyl mercaptopurine riboside, which is converted intracellularly to MeTIMP, was markedly higher in both cell lines under MTAP(-) conditions. Measurement of thiopurine metabolites support the hypothesis that DNPS inhibition is a major cause of cell death with 6-MP, whereas dG(s) 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., (©2011 AACR)

Purines and pyrimidines, regarded for a long time only as building blocks for nucleic acid synthesis and intermediates in the transfer of metabolic energy, gained increasing attention since genetically determined aberrations in their metabolism were associated clinically with various degrees of mental retardation and/or unexpected and often devastating neurological dysfunction. In most instances the molecular mechanisms underlying neurological symptoms remain undefined. This suggests that nucleotides and nucleosides play fundamental but still unknown roles in the development and function of several organs, in particular central nervous system. Alterations of purine and pyrimidine metabolism affecting brain function are spread along both synthesis (PRPS, ADSL, ATIC, HPRT, UMPS, dGK, TK), and breakdown pathways (5NT, ADA, PNP, GCH, DPD, DHPA, TP, UP), sometimes also involving pyridine metabolism. Explanations for the pathogenesis of disorders may include both cellular and mitochondrial damage: e.g. deficiency of the purine salvage enzymes hypoxanthine-guanine phosphoribosyltransferase and deoxyguanosine kinase are associated to the most severe pathologies, the former due to an unexplained adverse effect exerted on the development and/or differentiation of dopaminergic neurons, the latter due to impairment of mitochondrial functions. This review gathers the presently known inborn errors of purine and pyrimidine metabolism that manifest neurological syndromes, reporting and commenting on the available hypothesis on the possible link between specific enzymatic alterations and brain damage. Such connection is often not obvious, and though investigated for many years, the molecular basis of most dysfunctions of central nervous system associated to purine and pyrimidine metabolism disorders are still unexplained.

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, the authors have performed a differential chemical genetic screen in isogenic MTAP(+) and MTAP(-) Saccharomyces 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 4-fold shift in IC(50) 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 4 of the 5 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.

Purine nucleoside phosphorylase (PNP) deficiency results in severe T cell dysfunction and hypouricemia. An assay to measure PNP activity in dried blood spots was developed using reversed-phase HPLC. The assay was linear with reaction times between 5 and 12.5 minutes, and protein concentrations ranging from 0.4 to 1.8 mg/ml. The intra-assay CV and the inter-assay CV for the complete assay was <3.6%. The PNP activity in a control blood spot, stored at 4 degrees C, remained stable for at least one year. In a patient suffering from a PNP deficiency, the residual PNP activity was only 0.3% compared to that observed in controls (1431 +/- 238 nmol/mg/h, n = 114). The PNP activity (483 +/- 35 nmol/mg/h, n = 3) in heterozygotes for the c.614A > C mutation (p.E205A) in the PNP gene was 34% compared to controls. Thus, the analysis of the PNP activity in blood spots can readily detect patients with a PNP deficiency.

Purine nucleoside phosphorylase (PNP) deficiency is a rare combined immunodeficiency disorder presenting with clinically recurrent infections, failure to thrive, various neurological disorders, malignancies, and autoimmune diseases. Here, we report two sisters with a fatal course of PNP deficiency due to delay in diagnosis. The first patient developed a liver abscess by Aspergillus fumigatus and the second patient developed Mycobacterium tuberculosis complex lymphadenitis and probable pulmonary tuberculosis due to disseminated BCG infection. The patients also suffered from sclerosing cholangitis. Mutation analysis of the PNP gene from both sisters revealed a homozygous mutation for a G>A at nucleotide 349 (349 G>A transition), which changes alanine 117 to theronine in exon 4 (A117T). An increased awareness of early signs, symptoms, and abnormal laboratory findings of PNP deficiency will establish the early prognosis and treatment.

Purine nucleoside phosphorylase (PNP) deficiency is a rare autosomal recessive metabolic disorder that results in combined immunodeficiency, neurologic dysfunction and autoimmunity. PNP deficiency has never been reported from Saudi Arabia or in patients with an Arabic ethnic background. We report on two Saudi girls with PNP deficiency. Both showed severe lymphopenia and neurological involvement. Sequencing of the PNP gene of one girl revealed a novel missense mutation Pro146>Leu in exon 4 due to a change in the codon from CCT>CTT. Expression of PNP (146L) cDNA in E coli indicated that the mutation greatly reduced, but did not completely eliminate PNP activity.

Objective: Methylthioadenosine phosphorylase (MTAP)-deficient tumors are dependent on the de novo purine synthesis pathway. These cancers are potential targets for selective chemotherapy with inhibitors of de novo adenine synthesis such as L-alanosine [L-2-amino-3-(N-hydroxy-N-nitrosamino) propionic acid]. This phase II study was designed to evaluate the efficacy and safety of L-alanosine in patients with MTAP-deficient solid tumors., Methods: Patients with mesothelioma, non-small cell lung cancer (NSCLC), soft tissue sarcoma, osteosarcoma, or pancreatic cancer whose tumors were MTAP deficient by immunohistochemistry were eligible. Patients received L-alanosine at a starting dose of 80 mg/m(2) by continuous intravenous infusion daily for 5 days every 21 days. Computed tomography scans or magnetic resonance imaging were performed every 3 cycles., Results: 65 patients (16 mesothelioma, 13 NSCLC, 15 soft tissue sarcoma, 7 osteosarcoma, 14 pancreatic cancer) were enrolled at 19 centers; 55 were evaluable for response. There were no objective responses; 24% had s disease, including 2 patients with mesothelioma who had prolonged stable disease lasting 7.5 and 15.2 months, respectively. Grade 3/4 toxicities included mucositis 11%, fatigue 6%, nausea 3%, and renal failure 1.5%., Conclusion: At this dose and schedule, L-alanosine was ineffective in patients with advanced MTAP-deficient tumors.

Methylthioadenosine phosphorylase (MTAP) is involved in the metabolism of purines and converts methylthioadenosine (MTA) to adenine. It is abundant in all normal tissues but is deficient in various tumors. Here, we investigated MTAP deficiency in clinical samples of lung cancer using immunohistochemistry (IHC), and compared these results with those obtained by real-time PCR. Seventy-five samples were obtained from patients who underwent operations for non-small cell lung cancer (NSCLC). MTAP genetic analysis, using real-time PCR, and IHC were carried out on the samples. Methylation-specific primers were used to analyze methylation of the MTAP promoter, using DNA treated with sodium bisulfite. Sixty-nine of 75 samples were compared using both IHC and real-time PCR. The IHC results were consistent with those of real-time PCR in 56 samples. Of 62 positive samples tested by real-time PCR, only 49 (79%) were MTAP-positive by IHC. Seven samples were MTAP-negative by real-time PCR and IHC. In 13 samples of PCR (+) and IHC (-), six samples showed that the promoter region of MTAP was methylated. IHC is an accurate and useful diagnostic method for detecting MTAP deficiency in NSCLC, and the frequency of MTAP deficiency was found to be relatively high. The metabolic alterations diagnosed by IHC could be exploited for selective chemotherapy.

Autoimmune manifestations are increasingly being recognized as a component of several forms of primary immunodeficiencies (PID). Defects in purging of self-reactive T and B cells, impaired Fas-mediated apoptosis, abnormalities in development and/or function of regulatory T cells, and persistence of immune activation as a result of inability to clear infections have been shown to account for this association. Among autoimmune manifestations in patients with PID, cytopenias are particularly common. Up to 80% of patients with autoimmune lymphoproliferative syndrome (ALPS) have autoantibodies, and autoimmune hemolytic anemia and immune thrombocytopenia have been reported in 23% and 51% of ALPS patients, and may even mark the onset of the disease. ALPS-associated cytopenias are often refractory to conventional treatment and represent a therapeutic challenge. Autoimmune manifestations occur in 22% to 48% of patients with common variable immunodeficiencies (CVIDs), and are more frequent among CVID patients with splenomegaly and granulomatous disease. Finally, autoimmune cytopenias have been reported also in patients with combined immunodeficiency. In particular, autoimmune hemolytic anemia is very common among infants with nucleoside phosphorylase deficiency. While immune suppression may be beneficial in these cases, full resolution of the autoimmune manifestations ultimately depends on immune reconstitution, which is typically provided by hematopoietic cell transplantation.