Protein tyrosine phosphatases in glioma biology

Gliomas are a diverse group of brain tumors of glial origin. Most are characterized by diffuse infiltrative growth in the surrounding brain. In combination with their refractive nature to chemotherapy this makes it almost impossible to cure patients using combinations of conventional therapeutic strategies. The drastically increased knowledge about the molecular underpinnings of gliomas during the last decade has elicited high expectations for a more rational and effective therapy for these tumors. Most studies on the molecular pathways involved in glioma biology thus far had a strong focus on growth factor receptor protein tyrosine kinase (PTK) and phosphatidylinositol phosphatase signaling pathways. Except for the tumor suppressor PTEN, much less attention has been paid to the PTK counterparts, the protein tyrosine phosphatase (PTP) superfamily, in gliomas. PTPs are instrumental in the reversible phosphorylation of tyrosine residues and have emerged as important regulators of signaling pathways that are linked to various developmental and disease-related processes. Here, we provide an overview of the current knowledge on PTP involvement in gliomagenesis. So far, the data point to the potential implication of receptor-type (RPTPδ, DEP1, RPTPμ, RPTPζ) and intracellular (PTP1B, TCPTP, SHP2, PTPN13) classical PTPs, dual-specific PTPs (MKP-1, VHP, PRL-3, KAP, PTEN) and the CDC25B and CDC25C PTPs in glioma biology. Like PTKs, these PTPs may represent promising targets for the development of novel diagnostic and therapeutic strategies in the treatment of high-grade gliomas

Iterative one-tube-only SDM using large combinations of primers targeting the same residue.

<p>Iterative one-tube-only SDM using large combinations of primers targeting the same residue.</p

SDM using mutagenic primers of different length, GC content, and Tm.

<p>SDM using mutagenic primers of different length, GC content, and Tm.</p

Standardized SDM using mutagenic primers of 29-mer length and different GC content and Tm.

<p>Standardized SDM using mutagenic primers of 29-mer length and different GC content and Tm.</p

Data plots representing the obtaining (extraction) of all different individual mutations (samples to be extracted) from a given mix of mutations, assuming a stochastic distribution.

<p>The <i>x</i> axis indicates the number of different mutations included in the one-tube-only mutagenesis reaction, and the <i>y</i> axis indicates the number of samples (bacteria colonies) to be analyzed to obtain at least one of each mutation with a certain probability (95%, in red; 90%, in blue; 85%, in green). Dots indicate data obtained from a computer-assisted simulation, up to a mix of 15 different mutations. Solid lines indicate the probability distribution plots (Theory) up to a mix of 6 different mutations. Numeric values (non-brackets, simulation; brackets, theory) are indicated in the inserted table up to a mix of 6 different mutations.</p

Schematic depiction of the different one-tube-only SDM approaches used in this study.

<p>The cDNAs are represented as lines divided in 3-mer base codons. <b>(A)</b> Strategy for the simultaneous introduction of a mutation (in red) in several background plasmids by a mixed templates SDM reaction. Wild type residue is indicated as 1, and mutated residue as 1’. The 13+<u>3</u>+13 design of our standardized mutagenic primer pairs is indicated. Plasmids are indicated as A and B. <b>(B)</b> Strategy for one-tube parallel substitution of sequential amino acids (scanning mutagenesis) by mixing overlapping mutagenic primers targeting adjacent residues in the SDM reaction. Wild type residues are indicated as 1 to 8, and mutated residues (in red and blue) as 1’ to 8’. Note that individual mutations are obtained when primer pair overlap is more than five codons, whereas multiple mutations are obtained when the overlap is smaller. <b>(C)</b> Strategy for the simultaneous substitution of one residue to a collection of distinct residues (single site-multiple mutagenesis) by mixing distinct mutagenic primers targeting the same residue in the SDM reaction. Wild type residue is indicated as 1, and mutated residues (in red) as 1’ to 1”“.</p

One-tube-only SDM using combinations of template plasmids.

<p>One-tube-only SDM using combinations of template plasmids.</p

Identification of a novel inactivating mutation in Isocitrate Dehydrogenase 1 (IDH1-R314C) in a high grade astrocytoma

The majority of low-grade and secondary high-grade gliomas carry heterozygous hotspot mutations in cytosolic isocitrate dehydrogenase 1 (IDH1) or the mitochondrial variant IDH2. These mutations mostly involve Arg132 in IDH1, and Arg172 or Arg140 in IDH2. Whereas IDHs convert isocitrate to alpha-ketoglutarate (alpha-KG) with simultaneous reduction of NADP(+) to NADPH, these IDH mutants reduce alpha-KG to D-2-hydroxyglutarate (D-2-HG) while oxidizing NADPH. D-2-HG is a proposed oncometabolite, acting via competitive inhibition of alpha-KG-dependent enzymes that are involved in metabolism and epigenetic regulation. However, much less is known about the implications of the metabolic stress, imposed by decreased alpha-KG and NADPH production, for tumor biology. We here present a novel heterozygous IDH1 mutation, IDH1(R314C), which was identified by targeted next generation sequencing of a high grade glioma from which a mouse xenograft model and a cell line were generated. IDH1(R314C) lacks isocitrate-to-alpha-KG conversion activity due to reduced affinity for NADP(+), and differs from the IDH1(R132) mutants in that it does not produce D-2-HG. Because IDH1(R314C) is defective in producing alpha-KG and NADPH, without concomitant production of the D-2-HG, it represents a valuable tool to study the effects of IDH1-dysfunction on cellular metabolism in the absence of this oncometabolit