The Novel Phosphatase Domain Mutations Q171R and Y65S Switch PTEN from Tumor Suppressor to Oncogene

Phosphatase and tensin homolog deleted on chromosome 10, or PTEN, is a well-characterized tumor suppressor with both lipid and protein phosphatase activities. PTEN is often downregulated by epigenetic mechanisms such as hypermethylation, which leads to constitutive activation of the PI3K–Akt pathway. Large datasets from next-generation sequencing, however, revealed that mutations in PTEN may not only hamper protein function but may also affect interactions with downstream effectors, leading to variable oncogenic readouts. Here, two novel PTEN mutations, Q171R and Y65S, identified in Filipino colorectal cancer patients, were phenotypically characterized in NIH3T3 and HCT116 cells, alongside the C124S canonical mutant and wild-type controls. The novel mutants increased cellular proliferation, resistance to apoptosis and migratory capacity. They induced gross morphological changes including cytoplasmic shrinkage, increased cellular protrusions and extensive cytoskeletal reorganization. The mutants also induced a modest increase in Akt phosphorylation. Further mechanistic studies will help determine the differential oncogenic potencies of these mutants, and resolve whether the structural constraints imposed by the mutations may have altered associations with downstream effectors.

Molecular associations, clinical, and prognostic implications of PTPN11 mutations in acute myeloid leukemia (Alliance)

Prognostic factors associated with chemotherapy outcomes in patients with acute myeloid leukemia (AML) are extensively reported, and one gene whose mutation is recognized as conferring resistance to several newer targeted therapies is protein tyrosine phosphatase non-receptor type 11 (PTPN11). The broader clinical implications of PTPN11 mutations in AML are still not well understood. The objective of this study was to determine which cytogenetic abnormalities and gene mutations co-occur with PTPN11 mutations and how PTPN11 mutations impact outcomes of patients treated with intensive chemotherapy. We studied 1,725 newly diagnosed AML patients (excluding acute promyelocytic leukemia) enrolled onto the Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology trials. In 140 PTPN11-mutated patient samples, PTPN11 most commonly co-occurred with mutations in NPM1, DNMT3A, and TET2. PTPN11 mutations were relatively common in patients with an inv(3)(q21q26)/t(3;3)(q21;q26) and a normal karyotype but were very rare in patients with typical complex karyotype and core-binding factor AML. Mutations in the N-terminal SH2 domain of PTPN11 were associated with a higher early death rate than those in the phosphatase domain. PTPN11 mutations did not affect outcomes of NPM1-mutated patients, but these patients were less likely to have co-occurring kinase mutations (i.e., FLT3-ITD), suggesting activation of overlapping signaling pathways. However, in AML patients with wild-type NPM1, PTPN11 mutations were associated with adverse patient outcomes providing a rationale to study the biology and treatment approaches in this molecular group.

Tensin Phosphatase-Like System of Hantavirus Facilitates Membrane Fusion to Disrupt Vascular permeability

Increased vascular permeability is a characteristic of Hantavirus illness, for which there is now no treatment. We employed the domain search method to investigate the Hantavirus protein in this present work. The results indicated that the membrane glycoprotein E protein (containing Gn-Gc) of Hantavirus had lipid phosphatase and C2-like domains. The E protein was a tensin phosphatase-like (PTEN) enzyme that could shuttle in the cytoplasm and cell membrane. In an acidic endosomal environment, Gn dissociates, exposing Gc's autophosphorylation region to complete autophosphorylation and activating the C2 domain. The C2 domain facilitates Gc's conformational transition, which is followed by Gc binding to the endosomal membrane. After being inserted into the endosomal membrane, the phosphatase domain of Gc phosphorylates PI(3,4,5)P3 on the endosomal membrane. Then converted PI(3,4,5)P3 to PI(4,5)P2 . PI(4,5)P2 bound to the N-terminal of Gc, completely anchoring the tetramer-shaped Gc to the endosomal membrane and forming a fusion hole. Then analogous to PTEN, phosphorylation of PI(3,4,5)P3 directly induced the disintegration of Gc tetramer. The enlargement of the fusion pore speeded up the fusion of the viral and endosomal membranes. Through the fusion hole, the virus's intracellular material was swiftly discharged into the cytoplasm. The C2 domain promoted the PKC signaling route during Hantavirus membrane fusion, whereas the phosphatase inhibited the PI3K signaling pathway. E protein's PTEN-like action impaired lipid metabolism and endothelial cell remodeling, increasing blood vessel permeability and resulting in renal and cardiac syndromes. Additionally, E protein inhibited the immune system and Akt-mediated eNOS activation, resulting in a cascade of consequences.

Genotype Phenotype Correlation in Dent Disease 2 and Review of the Literature: OCRL Gene Pleiotropism or Extreme Phenotypic Variability of Lowe Syndrome?

Dent disease is a rare X-linked renal tubulopathy due to CLCN5 and OCRL (DD2) mutations. OCRL mutations also cause Lowe syndrome (LS) involving the eyes, brain and kidney. DD2 is frequently described as a mild form of LS because some patients may present with extra-renal symptoms (ESs). Since DD2 is a rare disease and there are a low number of reported cases, it is still unclear whether it has a clinical picture distinct from LS. We retrospectively analyzed the phenotype and genotype of our cohort of 35 DD2 males and reviewed all published DD2 cases. We analyzed the distribution of mutations along the OCRL gene and evaluated the type and frequency of ES according to the type of mutation and localization in OCRL protein domains. The frequency of patients with at least one ES was 39%. Muscle findings are the most common ES (52%), while ocular findings are less common (11%). Analysis of the distribution of mutations revealed (1) truncating mutations map in the PH and linker domain, while missense mutations map in the 5-phosphatase domain, and only occasionally in the ASH-RhoGAP module; (2) five OCRL mutations cause both DD2 and LS phenotypes; (3) codon 318 is a DD2 mutational hot spot; (4) a correlation was found between the presence of ES and the position of the mutations along OCRL domains. DD2 is distinct from LS. The mutation site and the mutation type largely determine the DD2 phenotype.

Identification and validation of tissue or ctDNA PTPRD phosphatase domain deleterious mutations as prognostic and predictive biomarkers for immune checkpoint inhibitors in non-squamous NSCLC

Background With the revolutionary progress of immune checkpoint inhibitors (ICIs) achieved in non-small cell lung cancers (NSCLC), identifying patients benefiting from ICIs becomes critical and urgent. The associations of genomic alterations in protein tyrosine phosphatase receptor-type (PTPRs) and ICIs responses are unknown. Methods Whole-exome sequencing (WES) of 73 advanced NSCLC tumors sampled before anti-PD-(L)1 therapy was carried out with corresponding clinical data collected as a discovery cohort to find the associations of PTPR mutations and ICI responses. Three validation cohorts consolidated by 7 public cohorts of 1920 NSCLC patients with WES or target sequencing data of tumor tissue-derived DNA or circulating tumor DNA (ctDNA) and relevant clinical data were applied as validation cohorts. The lung adenocarcinoma (LUAD) cohort (n=586) in The Cancer Genome Atlas (TCGA) database was used for analyzing the potential anti-tumor immunologic mechanisms. Results With the highest mutation frequency among all PTPRs, PTPRD mutations in non-squamous NSCLC (ns-NSCLC) were linked to longer progression-free survivals (PFS, 324 vs 63 days, hazard ratio (HR)=0.36, p= 0.0152) and higher objective response rate (ORR, p=0.0099). In validation cohort 1 (n=377), ns-NSCLC patients with tissue PTPRD mutations had favorable PFS (9.10 vs 4.33 months, HR=0.62, p=0.0184) and ORR (p=0.013). In validation cohort 2 (n=406), ns-NSCLC patients with tissue PTPRD mutations had favorable overall survivals (OS, over 40 vs 11.94 months, HR=0.57, p=0.011). In validation cohort 3 (n=1137), ns-NSCLC patients with ctDNA PTPRD mutations had longer PFS (6.97 vs 2.73 months, HR=0.63, p=0.028) and higher ORR (p=0.047). Moreover, it was deleterious mutations in phosphatase domains (phosphatase-mut), rather than other mutations (other-mut), that were responsible of PTPRD’s prediction efficiency. In addition, in validation cohort 3, ctDNA phosphatase-mut also functioned as a predictive biomarker helping identify patients benefiting more from ICIs than chemotherapy (interaction P for PFS=0.0506, for OS=0.04). Univariate and multivariate regression analysis revealed that phosphatase-mut was independent on PD-L1 expression and tumor mutation burden (TMB) to predict. In silico analysis based on TCGA LUAD cohort discovered enhanced anti-tumor immunity in phosphatase-mut patients. Conclusions Tissue or ctDNA PTPRD phosphatase domain deleterious mutations might function as a both prognostic and predictive biomarker predicting clinical outcomes of ICIs in ns-NSCLC patients, independent on TMB or PD-L1 expression.

Prenatal phenotype of PNKP-related primary microcephaly associated with variants in the FHA and Phosphatase domain

Biallelic PNKP variants cause heterogeneous disorders ranging from neurodevelopmental disorder with microcephaly/seizures to adult-onset Charcot-Marie-Tooth disease. To date, only postnatal descriptions exist. We present the first prenatal diagnosis of PNKP-related primary microcephaly. Detailed pathological examination of a male fetus revealed micrencephaly with extracerebral malformations and thus presumed syndromic microcephaly. A recessive disorder was suspected because of previous pregnancy termination for similar abnormalities in a sibling fetus. Prenatal trio exome sequencing identified compound-heterozygosity for the PNKP variants c.498G>A, p.[(=),0?] and c.302C>T, p.(Pro101Leu). Segregation confirmed both variants in the sibling fetus. Through RNA analyses, we characterized skipping of exon 4 affecting the PNKP Forkhead-associated (FHA) and Phosphatase domains (p.Leu67_Lys166del) as the predominant effect of the c.498G>A variant. We retrospectively investigated two unrelated individuals diagnosed with biallelic PNKP-variants to compare prenatal/postnatal phenotypes. Both carry the same splice-donor variant c.1029+2T>C in trans with a variant in the FHA domain (c.311T>C, p.(Leu104Pro) and c.151G>C, p.(Val51Leu), respectively). RNA-seq showed complex splicing events for c.1029+2T>C and c.151G>C. Computational modelling and structural analysis revealed significant clustering of missense variants in the FHA domain, with some variants potentially generating structural damage. Our detailed clinical description extends the PNKP-continuum to the prenatal stage. Investigating possible PNKP-variant effects using RNA and structural modelling, we highlight the mutational complexity and exemplify a framework for variant characterization in this multi-domain protein.

Novel pathogenic OCRL mutations and genotype–phenotype analysis of Chinese children affected by oculocerebrorenal syndrome: two cases and a literature review

Background Oculocerebrorenal syndrome of Lowe is a rare X-linked disorder characterized by congenital cataracts, mental retardation, and proximal tubulopathy. This condition is caused by a mutation of OCRL gene (located at chromosome Xq26.1), which encodes an inositol polyphosphate 5-phosphatase. Case presentation We identified two novel OCRL mutations in two unrelated Chinese boys, each with a severe phenotype of Lowe syndrome. A novel de novo deletion (hemizygous c.659_662delAGGG, p.E220Vfs*29) was present in patient 1 and a novel splicing mutation (hemizygous c.2257-2A > T) that was maternally inherited was present in patient 2. A renal biopsy in patient 2 indicated mild mesangial proliferative glomerulonephritis, mild focal mononuclear cells infiltration, and interstitial focal fibrosis. Moreover, renal expression of OCRL-1 protein in patient 2 was significantly reduced compared to a control patient with thin basement membrane disease. Conclusions This study reports two novel OCRL variants associated with severe ocular and neurologic deficiency, despite only mild renal dysfunction. Based on our two patients and a literature review, the genotype–phenotype correlation of OCRL mutations with this severe phenotype of Lowe syndrome suggest a possible clustering of missense, deletion, and nonsense mutations in the 5-phosphatase domain and Rho-GAP domain in the Chinese population.