The purine nucleoside phosphorylase pnp-1 regulates epithelial cell resistance to infection in C. elegans.

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.

The Broad Clinical Spectrum and Transplant Results of PNP Deficiency.

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.

A Case with Purine Nucleoside Phosphorylase Deficiency Suffering from Late-Onset Systemic Lupus Erythematosus and Lymphoma.

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.

The First Purine Nucleoside Phosphorylase Deficiency Patient Resembling IgA Deficiency and a Review of the Literature.

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.

Intracellular Delivery of Human Purine Nucleoside Phosphorylase by Engineered Diphtheria Toxin Rescues Function in Target Cells.

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 (MTA) Regulates Liver Cells Proteome and Methylproteome: Implications in Liver Biology and 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).

Development and validation of a 2nd tier test for identification of purine nucleoside phosphorylase deficiency patients during expanded newborn screening by liquid chromatography-tandem mass spectrometry.

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.

The First Report of a Pregnancy in a Patient with Purine Nucleoside Phosphorylase Deficiency.

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.

A successful unrelated peripheral blood stem cell transplantation with reduced intensity-conditioning regimen in a patient with late-onset purine nucleoside phosphorylase deficiency.

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.

Diagnosis of immunodeficiency caused by a purine nucleoside phosphorylase defect by using tandem mass spectrometry on dried blood spots.

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.

Nucleotide degradation and ribose salvage in yeast.

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.

Prevalence of S-methyl-5'-thioadenosine Phosphorylase (MTAP) Deficiency in Human Cancer: A Tissue Microarray Study on 13,067 Tumors From 149 Different Tumor Types.

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.

Purine Nucleoside Phosphorylase Deficiency in Two Unrelated Patients with Autoimmune Hemolytic Anemia and Eosinophilia: Two Novel Mutations.

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.

Cerebellar abnormalities in purine nucleoside phosphorylase deficient mice.

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.

The diagnostic value of immunohistochemically detected methylthioadenosine phosphorylase deficiency in malignant pleural mesotheliomas.

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.

Combined immunodeficiency due to purine nucleoside phosphorylase deficiency: Outcome of three patients.

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.

Purine nucleoside phosphorylase deficiency induces p53-mediated intrinsic apoptosis in human induced pluripotent stem cell-derived neurons.

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.

Fragment-Based Discovery of MRTX1719, a Synthetic Lethal Inhibitor of the PRMT5•MTA Complex for the Treatment of MTAP-Deleted Cancers.

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(2H)-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.

Deletion of MTAP Highly Sensitizes Osteosarcoma Cells to Methionine Restriction With Recombinant Methioninase.

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.