In many cancers, high proliferation rates correlate with elevation of rRNA and tRNA levels, and nucleolar hypertrophy. However, the underlying mechanisms linking increased nucleolar transcription and tumorigenesis are only minimally understood. Here we show that IMP dehydrogenase-2 (IMPDH2), the rate-limiting enzyme for de novo guanine nucleotide biosynthesis, is overexpressed in the highly lethal brain cancer, glioblastoma (GBM). This leads to increased rRNA and tRNA synthesis, stabilization of the nucleolar GTP-binding protein, Nucleostemin, and enlarged, malformed nucleoli. Pharmacological or genetic inactivation of IMPDH2 in GBM reverses these effects and inhibits cell proliferation, whereas untransformed glia cells are unaffected by similar IMPDH2 perturbations. Impairment of IMPDH2 activity triggers nucleolar stress and growth arrest of GBM cells even in the absence of functional p53. Our results reveal that upregulation of IMPDH2 is a prerequisite for aberrant nucleolar function and increased anabolic processes in GBM, which constitutes a primary event in gliomagenesis.

Purpose: In neurofibromatosis type 1 (NF1) and in highly aggressive malignant peripheral nerve sheath tumors (MPNSTs), constitutively active RAS-GTP and increased MAPK signaling are important in tumorigenesis. Dual specificity phosphatases (DUSPs) are negative regulators of MAPK signaling that dephosphorylate p38, JNK, and ERK in different settings. Although often acting as tumor suppressors, DUSPs may also act as oncogenes, helping tumor cells adapt to high levels of MAPK signaling. We hypothesized that inhibiting DUSPs might be selectively toxic to cells from NF1-driven tumors.Experimental Design: We examined DUSP gene and protein expression in neurofibroma and MPNSTs. We used small hairpin RNA (shRNA) to knock down DUSP1 and DUSP6 to evaluate cell growth, downstream MAPK signaling, and mechanisms of action. We evaluated the DUSP inhibitor, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), in MPNST cell lines and in cell-line and patient-derived MPNST xenografts. Results: DUSP1 and DUSP6 are expressed in NF1-deleted tumors. Knockdown of DUSP1 and DUSP6, alone or in combination, reduced MPNST cell growth and led to ERK and JNK hyperactivation increasing downstream TP53 and p-ATM. The DUSP inhibitor, BCI, diminished the survival of NF1-deleted Schwann cells and MPNST cell lines through activation of JNK. In vivo, treatment of an established cellline xenograft or a novel patient-derived xenograft (PDX) of MPNSTs with BCI increased ERK and JNK activation, caused tumor necrosis and fibrosis, and reduced tumor volume in one model. Conclusions: Targeting DUSP1 and DUSP6 genetically or with BCI effectively inhibits MPNST cell growth and promotes cell death, in vitro and in xenograft models. The data support further investigation of DUSP inhibition in MPNSTs.

Reactive oxygen species (ROS) are implicated in amphetamine-initiated neurodegeneration, but the mechanism is unclear. Here, we show that amphetamines are bioactivated by CNS prostaglandin H synthase (PHS) to free radical intermediates that cause ROS formation and neurodegenerative oxidative DNA damage. In vitro incubations of purified PHS-1 with 3,4-methylenedioxyamphetamine (MDA) and methamphetamine (METH) demonstrated PHS-catalyzed time- and concentration-dependent formation of an amphetamine carbon- and/or nitrogen-centered free radical intermediate, and stereoselective oxidative DNA damage, evidenced by 8-oxo-2'-deoxyguanosine (8-oxo-dG) formation. Similarly in vivo, MDA and METH caused dose- and time-dependent DNA oxidation in multiple brain regions, remarkably dependent on the regional PHS levels, including the striatum and substantia nigra, wherein neurodegeneration of dopaminergic nerve terminals was evidenced by decreased immunohistochemical staining of tyrosine hydroxylase. Motor impairment using the rotarod test was evident within 3 wk after the last drug dose, and persisted for at least 6 months. Pretreatment with the PHS inhibitor acetylsalicylic acid blocked MDA-initiated DNA oxidation and protected against functional motor impairment for at least 1.5 months after drug treatment. This is the first direct evidence for PHS-catalyzed bioactivation of amphetamines causing temporal and regional differences in CNS oxidative DNA damage directly related to structural and functional neurodegenerative consequences.

The role of prostaglandin H synthase-1 (PHS-1) and a related model enzyme, horseradish peroxidase (HRP), in catalyzing the bioactivation of dopamine (DA) and epinephrine and their precursors and metabolites to potential neurodegenerative free radical intermediates was examined. To determine the potential contribution of PHS-dependent reactive oxygen species (ROS) formation, the neurotransmitter DA or its precursor and metabolites were incubated in vitro with purified ovine PHS-1 and calf thymus DNA. DA, its L-dihydroxyphenylalanine (L-DOPA), precursor, and its dihydroxyphenylacetic acid (DOPAC) metabolite were excellent PHS-1 substrates, resulting in PHS-1-dependent ROS formation that initiated oxidative DNA damage, selectively quantified as 8-oxo-2'-deoxyguanosine. Most substrates generated isotropic electron spin resonance (ESR) spectra with a resolved hyperfine structure attributable to ortho-semiquinone free radical intermediates upon autoxidation at pH 6, with up to a 18-fold increase via HRP-catalyzed oxidation. Remarkably, HRP-mediated oxidation of DOPAC and dihydroxymandelic acid (DHMA) produced asymmetric ESR spectra characteristic of an immobilized radical, possibly due to free radical intermediates and melanin or melanin-like polymers. These results show that the precursors and metabolites of endogenous neurotransmitters, while inactive in receptor binding assays, may actually play an important role in free radical formation. Additionally, ROS generated by PHS-catalyzed bioactivation produce oxidative DNA damage in the central nervous system, which may initiate neurodegeneration associated with aging.