Biallelic pathogenic variants in the mitochondrial form of phosphoenolpyruvate carboxykinase cause peripheral neuropathy.

Phosphoenolpyruvate carboxykinase (PCK) plays a critical role in cytosolic gluconeogenesis, and defects in PCK1 cause a fasting-aggravated metabolic disease with hypoglycemia and lactic acidosis. However, there are two genes encoding PCK, and the role of the mitochondrial resident PCK (encoded by PCK2) is unclear, since gluconeogenesis is cytosolic. We identified three patients in two families with biallelic variants in PCK2. One has compound heterozygous variants (p.Ser23Ter/p.Pro170Leu), and the other two (siblings) have homozygous p.Arg193Ter variation. All three patients have weakness and abnormal gait, an absence of PCK2 protein, and profound reduction in PCK2 activity in fibroblasts, but no obvious metabolic phenotype. Nerve conduction studies showed reduced conduction velocities with temporal dispersion and conduction block compatible with a demyelinating peripheral neuropathy. To validate the association between PCK2 variants and clinical disease, we generated a mouse knockout model of PCK2 deficiency. The animals present abnormal nerve conduction studies and peripheral nerve pathology, corroborating the human phenotype. In total, we conclude that biallelic variants in PCK2 cause a neurogenetic disorder featuring abnormal gait and peripheral neuropathy.

Biallelic loss-of-function variants in RABGAP1 cause a novel neurodevelopmental syndrome.

PURPOSE: RABGAP1 is a GTPase-activating protein implicated in a variety of cellular and molecular processes, including mitosis, cell migration, vesicular trafficking, and mTOR signaling. There are no known Mendelian diseases caused by variants in RABGAP1. METHODS: Through GeneMatcher, we identified 5 patients from 3 unrelated families with homozygous variants in the RABGAP1 gene found on exome sequencing. We established lymphoblastoid cells lines derived from an affected individual and her parents and performed RNA sequencing and functional studies. Rabgap1 knockout mice were generated and phenotyped. RESULTS: We report 5 patients presenting with a common constellation of features, including global developmental delay/intellectual disability, microcephaly, bilateral sensorineural hearing loss, and seizures, as well as overlapping dysmorphic features. Neuroimaging revealed common features, including delayed myelination, white matter volume loss, ventriculomegaly, and thinning of the corpus callosum. Functional analysis of patient cells revealed downregulated mTOR signaling and abnormal localization of early endosomes and lysosomes. Rabgap1 knockout mice exhibited several features in common with the patient cohort, including microcephaly, thinning of the corpus callosum, and ventriculomegaly. CONCLUSION: Collectively, our results provide evidence of a novel neurodevelopmental syndrome caused by biallelic loss-of-function variants in RABGAP1.

X-linked myotubular myopathy is associated with epigenetic alterations and is ameliorated by HDAC inhibition.

X-linked myotubular myopathy (XLMTM) is a fatal neuromuscular disorder caused by loss of function mutations in MTM1. At present, there are no directed therapies for XLMTM, and incomplete understanding of disease pathomechanisms. To address these knowledge gaps, we performed a drug screen in mtm1 mutant zebrafish and identified four positive hits, including valproic acid, which functions as a potent suppressor of the mtm1 zebrafish phenotype via HDAC inhibition. We translated these findings to a mouse XLMTM model, and showed that valproic acid ameliorates the murine phenotype. These observations led us to interrogate the epigenome in Mtm1 knockout mice; we found increased DNA methylation, which is normalized with valproic acid, and likely mediated through aberrant 1-carbon metabolism. Finally, we made the unexpected observation that XLMTM patients share a distinct DNA methylation signature, suggesting that epigenetic alteration is a conserved disease feature amenable to therapeutic intervention.

Natural history of a mouse model of X-linked myotubular myopathy.

X-linked myotubular myopathy (XLMTM) is a severe monogenetic disorder of the skeletal muscle. It is caused by loss-of-expression/function mutations in the myotubularin (MTM1) gene. Much of what is known about the disease, as well as the treatment strategies, has been uncovered through experimentation in pre-clinical models, particularly the Mtm1 gene knockout mouse line (Mtm1 KO). Despite this understanding, and the identification of potential therapies, much remains to be understood about XLMTM disease pathomechanisms, and about the normal functions of MTM1 in muscle development. To lay the groundwork for addressing these knowledge gaps, we performed a natural history study of Mtm1 KO mice. This included longitudinal comparative analyses of motor phenotype, transcriptome and proteome profiles, muscle structure and targeted molecular pathways. We identified age-associated changes in gene expression, mitochondrial function, myofiber size and key molecular markers, including DNM2. Importantly, some molecular and histopathologic changes preceded overt phenotypic changes, while others, such as triad structural alternations, occurred coincidentally with the presence of severe weakness. In total, this study provides a comprehensive longitudinal evaluation of the murine XLMTM disease process, and thus provides a critical framework for future investigations.

MRI-guided focused ultrasound enhances drug delivery in experimental diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) is a surgically unresectable and devasting tumour in children. To date, there are no effective chemotherapeutics despite a myriad of clinical trials. The intact blood-brain barrier (BBB) is likely responsible for the limited clinical response to chemotherapy. MRI-guided focused ultrasound (MRgFUS) is a promising non-invasive method for treating CNS tumours. Moreover, MRgFUS allows for the temporary and repeated disruption of the BBB. Our group previously reported the feasibility of temporary BBB opening within the normal murine brainstem using MRgFUS following intravenous (IV) administration of microbubbles. In the current study, we set out to test the effectiveness of targeted chemotherapy when paired with MRgFUS in murine models of DIPG. Doxorubicin was selected from a drug screen consisting of conventional chemotherapeutics tested on patient-derived cell lines. We studied the RCAS/Tv-a model where RCAS-Cre, RCAS-PDGFB, and RCAS-H3.3K27M were used to drive tumourigenesis upon injection in the pons. We also used orthotopically injected SU-DIPG-6 and SU-DIPG-17 xenografts which demonstrated a diffusely infiltrative tumour growth pattern similar to human DIPG. In our study, SU-DIPG-17 xenografts were more representative of human DIPG with an intact BBB. Following IV administration of doxorubicin, MRgFUS-treated animals exhibited a 4-fold higher concentration of drug within the SU-DIPG-17 brainstem tumours compared to controls. Moreover, the volumetric tumour growth rate was significantly suppressed in MRgFUS-treated animals whose tumours also exhibited decreased Ki-67 expression. Herein, we provide evidence for the ability of MRgFUS to enhance drug delivery in a mouse model of DIPG. These data provide critical support for clinical trials investigating MRgFUS-mediated BBB opening, which may ameliorate DIPG chemotherapeutic approaches in children.

Inhibition of TRPM7 with waixenicin A reduces glioblastoma cellular functions.

Glioblastoma (GBM) is the most common malignant primary brain tumour originating in the CNS. Median patient survival is <15 months with standard treatment which consists of surgery alongside radiation therapy and temozolomide chemotherapy. However, because of the aggressive nature of GBM, and the significant toxicity of these adjuvant therapies, long-term therapeutic effects are unsatisfactory. Thus, there is urgency to identify new drug targets for GBM. Recent evidence shows that the transient receptor potential melastatin 7 (TRPM7) cation channel is aberrantly upregulated in GBM and its inhibition leads to reduction of GBM cellular functions. This suggests that TRPM7 may be a potential drug target for GBM treatment. In this study, we assessed the effects of the specific TRPM7 antagonist waixenicin A on human GBM cell lines U87 or U251 both in vitro and in vivo. First, we demonstrated in vitro that application of waixenicin A reduced TRPM7 protein expression and inhibited the TRPM7-like currents in GBM cells. We also observed reduction of GBM cell viability, migration, and invasion. Using an intracranial xenograft GBM mouse model, we showed that with treatment of waixenicin A, there was increased cleaved caspase 3 activity, alongside reduction in Ki-67, cofilin, and Akt activity in vivo. Together, these data demonstrate higher GBM cell apoptosis, and lower proliferation, migration, invasion and survivability following treatment with waixenicin A.

Mouse model of severe recessive RYR1-related myopathy.

Ryanodine receptor type I (RYR1)-related myopathies (RYR1 RM) are a clinically and histopathologically heterogeneous group of conditions that represent the most common subtype of childhood onset non-dystrophic muscle disorders. There are no treatments for this severe group of diseases. A major barrier to therapy development is the lack of an animal model that mirrors the clinical severity of pediatric cases of the disease. To address this, we used CRISPR/Cas9 gene editing to generate a novel recessive mouse model of RYR1 RM. This mouse (Ryr1TM/Indel) possesses a patient-relevant point mutation (T4706M) engineered into 1 allele and a 16 base pair frameshift deletion engineered into the second allele. Ryr1TM/Indel mice exhibit an overt phenotype beginning at 14 days of age that consists of reduced body/muscle mass and myofibre hypotrophy. Ryr1TM/Indel mice become progressively inactive from that point onward and die at a median age of 42 days. Histopathological assessment shows myofibre hypotrophy, increased central nuclei and decreased triad number but no clear evidence of metabolic cores. Biochemical analysis reveals a marked decrease in RYR1 protein levels (20% of normal) as compared to only a 50% decrease in transcript. Functional studies at end stage show significantly reduced electrically evoked Ca2+ release and force production. In summary, Ryr1TM/Indel mice exhibit a post-natal lethal recessive form of RYR1 RM that pheno-copies the severe congenital clinical presentation seen in a subgroup of RYR1 RM children. Thus, Ryr1TM/Indel mice represent a powerful model for both establishing the pathomechanisms of recessive RYR1 RM and pre-clinical testing of therapies for efficacy.

Tamoxifen therapy in a murine model of myotubular myopathy.

Myotubular myopathy (MTM) is a severe X-linked disease without existing therapies. Here, we show that tamoxifen ameliorates MTM-related histopathological and functional abnormalities in mice, and nearly doubles survival. The beneficial effects of tamoxifen are mediated primarily via estrogen receptor signaling, as demonstrated through in vitro studies and in vivo phenotypic rescue with estradiol. RNA sequencing and protein expression analyses revealed that rescue is mediated in part through post-transcriptional reduction of dynamin-2, a known MTM modifier. These findings demonstrate an unexpected ability of tamoxifen to improve the murine MTM phenotype, providing preclinical evidence to support clinical translation.

Characterization of a Clival Chordoma Xenograft Model Reveals Tumor Genomic Instability.

Patient-derived xenografts retain the genotype of the parent tumors more readily than tumor cells maintained in culture. The two previously reported clival chordoma xenografts were derived from recurrent tumors after radiation. To study the genetics of clival chordoma in the absence of prior radiation exposure we established a patient-derived xenograft at primary resection of a clival chordoma. Epicranial grafting of clival chordoma collected during surgery was performed. Tumor growth was established in a nonobese diabetic/severe combined immunodeficiency mouse and tumors have been passaged serially for seven generations. Physaliferous cell architecture was shown in the regenerated tumors, which stained positive for Brachyury, cytokeratin, and S100 protein. The tumors showed bone invasion. Single-nucleotide polymorphism analysis of the tumor xenograft was compared with the parental tumor. Copy number gain of the T gene (brachyury) and heterozygous loss of cyclin dependent kinase inhibitor 2A (CDKN2A) was observed. Heterozygous loss of the tumor-suppressor fragile histidine triad (FHIT) gene also was observed, although protein expression was preserved. Accumulation of copy number losses and gains as well as increased growth rate was observed over three generations. The patient-derived xenograft reproduces the phenotype of clival chordoma. This model can be used in the future to study chordoma biology and to assess novel treatments.

Brainstem blood brain barrier disruption using focused ultrasound: A demonstration of feasibility and enhanced doxorubicin delivery.

Magnetic Resonance Image-guided Focused Ultrasound (MRgFUS) has been used to achieve transient blood brain barrier (BBB) opening without tissue injury. Delivery of a targeted ultrasonic wave causes an interaction between administered microbubbles and the capillary bed resulting in enhanced vessel permeability. The use of MRgFUS in the brainstem has not previously been shown but could provide value in the treatment of tumours such as Diffuse Intrinsic Pontine Glioma (DIPG) where the intact BBB has contributed to the limited success of chemotherapy. Our primary objective was to determine whether the use of MRgFUS in this eloquent brain region could be performed without histological injury and functional deficits. Our secondary objective was to select an effective chemotherapeutic against patient derived DIPG cell lines and demonstrate enhanced brainstem delivery when combined with MRgFUS in vivo. Female Sprague Dawley rats were randomised to one of four groups: 1) Microbubble administration but no MRgFUS treatment; 2) MRgFUS only; 3) MRgFUS + microbubbles; and 4) MRgFUS + microbubbles + cisplatin. Physiological assessment was performed by monitoring of heart and respiratory rates. Motor function and co-ordination were evaluated by Rotarod and grip strength testing. Histological analysis for haemorrhage (H&E), neuronal nuclei (NeuN) and apoptosis (cleaved Caspase-3) was also performed. A drug screen of eight chemotherapy agents was conducted in three patient-derived DIPG cell lines (SU-DIPG IV, SU-DIPG XIII and SU-DIPG XVII). Doxorubicin was identified as an effective agent. NOD/SCID/GAMMA (NSG) mice were subsequently administered with 5 mg/kg of intravenous doxorubicin at the time of one of the following: 1) Microbubbles but no MRgFUS; 2) MRgFUS only; 3) MRgFUS + microbubbles and 4) no intervention. Brain specimens were extracted at 2 h and doxorubicin quantification was conducted using liquid chromatography mass spectrometry (LC/MS). BBB opening was confirmed by contrast enhancement on T1-weighted MR imaging and positive Evans blue staining of the brainstem. Normal cardiorespiratory parameters were preserved. Grip strength and Rotarod testing demonstrating no decline in performance across all groups. Histological analysis showed no evidence of haemorrhage, neuronal loss or increased apoptosis. Doxorubicin demonstrated cytotoxicity against all three cell lines and is known to have poor BBB permeability. Quantities measured in the brainstem of NSG mice were highest in the group receiving MRgFUS and microbubbles (431.5 ng/g). This was significantly higher than in mice who received no intervention (7.6 ng/g). Our data demonstrates both the preservation of histological and functional integrity of the brainstem following MRgFUS for BBB opening and the ability to significantly enhance drug delivery to the region, giving promise to the treatment of brainstem-specific conditions.

PIK3C2B inhibition improves function and prolongs survival in myotubular myopathy animal models.

Myotubular myopathy (MTM) is a devastating pediatric neuromuscular disorder of phosphoinositide (PIP) metabolism resulting from mutations of the PIP phosphatase MTM1 for which there are no treatments. We have previously shown phosphatidylinositol-3-phosphate (PI3P) accumulation in animal models of MTM. Here, we tested the hypothesis that lowering PI3P levels may prevent or reverse the MTM disease process. To test this, we targeted class II and III PI3 kinases (PI3Ks) in an MTM1-deficient mouse model. Muscle-specific ablation of Pik3c2b, but not Pik3c3, resulted in complete prevention of the MTM phenotype, and postsymptomatic targeting promoted a striking rescue of disease. We confirmed this genetic interaction in zebrafish, and additionally showed that certain PI3K inhibitors prevented development of the zebrafish mtm phenotype. Finally, the PI3K inhibitor wortmannin improved motor function and prolonged lifespan of the Mtm1-deficient mice. In all, we have identified Pik3c2b as a genetic modifier of Mtm1 mutation and demonstrated that PIK3C2B inhibition is a potential treatment strategy for MTM. In addition, we set the groundwork for similar reciprocal inhibition approaches for treating other PIP metabolic disorders and highlight the importance of modifier gene pathways as therapeutic targets.

A role for activated Cdc42 in glioblastoma multiforme invasion.

Cdc42 is a Rho-GTPase which plays a major role in regulating cell polarity and migration by specifying the localization of filopodia. However, the role of Cdc42 in GBM invasion has not been thoroughly investigated. We generated stable doxycycline-inducible clones expressing wild type (WT)-, constitutively active (CA)-, and dominant negative (DN)-Cdc42 in three different human glioma cell lines. Expression of CA-Cdc42 significantly increased the migration and invasive properties of malignant glioma cells compared to WT and DN-Cdc42 cell clones, and this was accompanied by a greater number of filopodia and focal adhesion structures which co-localize with phosphorylated focal adhesion kinase (FAK). By mass spectrometry and immunoprecipitation studies, we demonstrated that activated Cdc42 binds to IQGAP1. When implanted orthotopically in mice, the CA-Cdc42 expressing glioma cells exhibited enhanced local migration and invasion, and led to larger tumors, which significantly reduced survival. Using the Cancer Genome Atlas dataset, we determined that high Cdc42 expression is associated with poorer progression free survival, and that Cdc42 expression is highest in the proneural and neural subgroups of GBM. In summary, our studies demonstrate that activated Cdc42 is a critical determinant of the migratory and invasive phenotype of malignant gliomas, and that its effect may be mediated, at least in part, through its interaction with IQGAP1 and phosphorylated FAK.

Study of the biodistribution of fluorescein in glioma-infiltrated mouse brain and histopathological correlation of intraoperative findings in high-grade gliomas resected under fluorescein fluorescence guidance.

OBJECT: Intravenous fluorescein sodium has been used during resection of high-grade gliomas to help the surgeon visualize tumor margins. Several studies have reported improved rates of gross-total resection (GTR) using high doses of fluorescein sodium under white light. The recent introduction of a fluorescein-specific camera that allows for high-quality intraoperative imaging and use of very low dose fluorescein has drawn new attention to this fluorophore. However, the ability of fluorescein to specifically stain glioma cells is not yet well understood. METHODS: The authors designed an in vitro model to assess fluorescein uptake in normal human astrocytes and U251 malignant glioma cells. An in vivo experiment was also subsequently designed to study fluorescein uptake by intracranial U87 malignant glioma xenografts in male nonobese diabetic/severe combined immunodeficient mice. A genetically induced mouse glioma model was used to adjust for the possible confounding effect of an inflammatory response in the xenograft model. To assess the intraoperative application of this technology, the authors prospectively enrolled 12 patients who underwent fluorescein-guided resection of their high-grade gliomas using low-dose intravenous fluorescein and a microscope-integrated fluorescence module. Intraoperative fluorescent and nonfluorescent specimens at the tumor margins were randomly analyzed for histopathological correlation. RESULTS: The in vitro and in vivo models suggest that fluorescein demarcation of glioma-invaded brain is the result of distribution of fluorescein into the extracellular space, most likely as a result of an abnormal blood-brain barrier. Glioblastoma tumor cell-specific uptake of fluorescein was not observed, and tumor cells appeared to mostly exclude fluorescein. For the 12 patients who underwent resection of their high-grade gliomas, the histopathological analysis of the resected specimens at the tumor margin confirmed the intraoperative fluorescent findings. Fluorescein fluorescence was highly specific (up to 90.9%) while its sensitivity was 82.2%. False negatives occurred due to lack of fluorescence in areas of diffuse, low-density cellular infiltration. Margins of contrast enhancement based on intraoperative MRI-guided StealthStation neuronavigation correlated well with fluorescent tumor margins. GTR of the contrast-enhancing area as guided by the fluorescent signal was achieved in 100% of cases based on postoperative MRI. CONCLUSIONS: Fluorescein sodium does not appear to selectively accumulate in astrocytoma cells but in extracellular tumor cell-rich locations, suggesting that fluorescein is a marker for areas of compromised blood-brain barrier within high-grade astrocytoma. Fluorescein fluorescence appears to correlate intraoperatively with the areas of MR enhancement, thus representing a practical tool to help the surgeon achieve GTR of the enhancing tumor regions.

A preclinical study demonstrating the efficacy of nilotinib in inhibiting the growth of pediatric high-grade glioma.

Solid tumors arising from malignant transformation of glial cells are one of the leading causes of central nervous system tumor-related death in children. Recurrence in spite of rigorous surgical and chemoradiation therapies remains a major hurdle in management of these tumors. Here, we investigate the efficacy of the second-generation receptor tyrosine kinase inhibitor nilotinib as a therapeutic option for the management of pediatric gliomas. We have utilized two independent pediatric high-grade glioma cell lines with either high platelet-derived growth factor receptor alpha (PDGFRα) or high PDGFRß expression in in vitro assays to investigate the specific downstream effects of nilotinib treatment. Using in vitro cell-based assays we show that nilotinib inhibits PDGF-BB-dependent activation of PDGFRα. We further show that nilotinib is able to decrease cell proliferation and anchorage-independent growth via suppression of AKT and ERK1/2 signaling pathways. Our results suggest that nilotinib may be effective for management of a PDGFRα-dependent group of pediatric gliomas.

Foretinib is effective therapy for metastatic sonic hedgehog medulloblastoma.

Medulloblastoma is the most common malignant pediatric brain tumor, with metastases present at diagnosis conferring a poor prognosis. Mechanisms of dissemination are poorly understood and metastatic lesions are genetically divergent from the matched primary tumor. Effective and less toxic therapies that target both compartments have yet to be identified. Here, we report that the analysis of several large nonoverlapping cohorts of patients with medulloblastoma reveals MET kinase as a marker of sonic hedgehog (SHH)-driven medulloblastoma. Immunohistochemical analysis of phosphorylated, active MET kinase in an independent patient cohort confirmed its correlation with increased tumor relapse and poor survival, suggesting that patients with SHH medulloblastoma may benefit from MET-targeted therapy. In support of this hypothesis, we found that the approved MET inhibitor foretinib could suppress MET activation, decrease tumor cell proliferation, and induce apoptosis in SHH medulloblastomas in vitro and in vivo. Foretinib penetrated the blood-brain barrier and was effective in both the primary and metastatic tumor compartments. In established mouse xenograft or transgenic models of metastatic SHH medulloblastoma, foretinib administration reduced the growth of the primary tumor, decreased the incidence of metastases, and increased host survival. Taken together, our results provide a strong rationale to clinically evaluate foretinib as an effective therapy for patients with SHH-driven medulloblastoma.

Gene-expression profiling elucidates molecular signaling networks that can be therapeutically targeted in vestibular schwannoma.

OBJECT: Vestibular schwannomas (VS) are common benign tumors of the vestibular nerve that cause significant morbidity. The current treatment strategies for VS include surgery or radiation, with each treatment option having associated complications and side effects. The transcriptional landscape of schwannoma remains largely unknown. METHODS: In this study the authors performed gene-expression profiling of 49 schwannomas and 7 normal control vestibular nerves to identify tumor-specific gene-expression patterns. They also interrogated whether schwannomas comprise several molecular subtypes using several transcription-based clustering strategies. The authors also performed in vitro experiments testing therapeutic inhibitors of over-activated pathways in a schwannoma cell line, namely the PI3K/AKT/mTOR pathway. RESULTS: The authors identified over 4000 differentially expressed genes between controls and schwannomas with network analysis, uncovering proliferation and anti-apoptotic pathways previously not implicated in VS. Furthermore, using several distinct clustering technologies, they could not reproducibly identify distinct VS subtypes or significant differences between sporadic and germline NF2-associated schwannomas, suggesting that they are highly similar entities. The authors identified overexpression of PI3K/AKT/mTOR signaling networks in their gene-expression study and evaluated this pathway for therapeutic targeting. Testing the compounds BEZ235 and PKI-587, both novel dual inhibitors of PI3K and mTOR, attenuated tumor growth in a preclinical cell line model of schwannoma (HEI-293). In vitro findings demonstrated that pharmacological inhibition of the PI3K/AKT/mTOR pathway with next-generation compounds led to decreased cell viability and increased cell death. CONCLUSIONS: These findings implicate aberrant activation of the PI3K/AKT/mTOR pathway as a molecular mechanism of pathogenesis in VS and suggest inhibition of this pathway as a potential treatment strategy.

Transcriptional profiling of GBM invasion genes identifies effective inhibitors of the LIM kinase-Cofilin pathway.

Malignant gliomas are highly proliferative and invasive neoplasms where total surgical resection is often impossible and effective local radiation therapy difficult. Consequently, there is a need to develop a greater understanding of the molecular events driving invasion and to identify novel treatment targets. Using microarray analysis comparing normal brain samples and mesenchymal glioblastoma multiforme (GBM), we identified over 140 significant genes involved in cell migration and invasion. The cofilin (CFL) pathway, which disassembles actin filaments, was highly up-regulated compared to normal brain. Up-regulation of LIM domain kinase 1 and 2 (LIMK1/2), that phosphorylates and inactivates cofilin, was confirmed in an additional independent data set comparing normal brain to GBM. We identified and utilized two small molecule inhibitors BMS-5 and Cucurbitacin I directed against the cofilin regulating kinases, LIMK1 and LIMK2, to target this pathway. Significant decreases in cell viability were observed in glioma cells treated with BMS-5 and Cucurbitacin I, while no cytotoxic effects were seen in normal astrocytes that lack LIMK. BMS-5 and Cucurbitacin I promoted increased adhesion in GBM cells, and decreased migration and invasion. Collectively, these data suggest that use of LIMK inhibitors may provide a novel way to target the invasive machinery in GBM.

ATM regulates 3-methylpurine-DNA glycosylase and promotes therapeutic resistance to alkylating agents.

UNLABELLED: Alkylating agents are a first-line therapy for the treatment of several aggressive cancers, including pediatric glioblastoma, a lethal tumor in children. Unfortunately, many tumors are resistant to this therapy. We sought to identify ways of sensitizing tumor cells to alkylating agents while leaving normal cells unharmed, increasing therapeutic response while minimizing toxicity. Using an siRNA screen targeting over 240 DNA damage response genes, we identified novel sensitizers to alkylating agents. In particular, the base excision repair (BER) pathway, including 3-methylpurine-DNA glycosylase (MPG), as well as ataxia telangiectasia mutated (ATM), were identified in our screen. Interestingly, we identified MPG as a direct novel substrate of ATM. ATM-mediated phosphorylation of MPG was required for enhanced MPG function. Importantly, combined inhibition or loss of MPG and ATM resulted in increased alkylating agent-induced cytotoxicity in vitro and prolonged survival in vivo. The discovery of the ATM-MPG axis will lead to improved treatment of alkylating agent-resistant tumors. SIGNIFICANCE: Inhibition of ATM and MPG-mediated BER cooperate to sensitize tumor cells to alkylating agents, impairing tumor growth in vitro and in vivo with no toxicity to normal cells, providing an ideal therapeutic window.

Conditional knockout of pik3c3 causes a murine muscular dystrophy.

Abnormalities in phosphoinositide metabolism are an emerging theme in human neurodegenerative disease. Myotubular myopathy is a prototypical disorder of phosphoinositide dysregulation that is characterized by profound muscle pathology and weakness and that is caused by mutations in MTM1, which encodes a phosphatase that targets 3-position phosphoinositides, including phosphatidylinositol 3-phosphate. Although the association between MTM1 and muscle disease has become increasingly clarified, the normal role(s) of phosphatidylinositol 3-phosphate metabolism in muscle development and homeostasis remain poorly understood. To begin to address the function of phosphatidylinositol 3-phosphate in skeletal muscle, we focused on the primary kinase responsible for its production, and created a muscle-specific conditional knockout of the class III phosphatidylinositol 3-kinase, Pik3c3. Muscle-specific deletion of Pik3c3 did not disturb embryogenesis or early postnatal development, but resulted in progressive disease characterized by reduced activity and death by 2 months of age. Histopathological analysis demonstrated changes consistent with a murine muscular dystrophy. Examination for cellular mechanism(s) responsible for the dystrophic phenotype revealed significant alterations in the autophagolysosomal pathway with mislocation of known dystrophy proteins to the lysosomal compartment. In all, we present the first analysis of Pik3c3 in skeletal muscle, and report a novel association between deletion of Pik3c3 and muscular dystrophy.

Analysis of IDH mutation, 1p/19q deletion, and PTEN loss delineates prognosis in clinical low-grade diffuse gliomas.

BACKGROUND: Grades II and III gliomas have unpredictable rates of progression, making management decisions difficult. Currently, several clinical and radiological characteristics are utilized to predict progression and survival but collectively are suboptimal. METHODS: In this study, we analyzed a set of 108 nonenhancing hemispheric grade II-III gliomas. Demographic variables, including patient age, tumor diameter, extent of resection, and performance status, were combined with molecular data (IDH mutation status [mIDH], 1p/19q codeletion, PTEN deletion, and EGFR amplification). A complete dataset for all variables was compiled for 70 of the 108 patients. Both univariable and multivariable analyses were performed to determine whether the molecular data singly or in combination offer advantages over tumor type and grade for prediction of overall survival (OS) and/or progression-free rate (PFR). RESULTS: Patient age, clinical variables (tumor diameter, extent of resection, performance status), and pathology (tumor type and grade) were not predictive of OS or PFR. IDH mutation status alone was predictive of longer OS and PFR for the entire group of tumors; 1p/19q deletion alone was predictive of OS but not PFR. In the multivariable analysis, none of the clinical or demographic factors were predictive of OS or PFR. IDH mutation status, 1p/19q codeletion, and PTEN deletion were predictive of OS (P = .003, P = .005, P = .02, respectively). Both mIDH (P < .001) and the interaction term of 1p/19q and PTEN (P < .001) were found to be predictive of PFR. CONCLUSIONS: We conclude that the combination of mIDH, 1p/19q codeletion, and PTEN deletion may be particularly effective in discriminating good prognosis from poor prognosis hemispheric gliomas. We propose that such a scheme merits testing on larger prospective cohorts. Should our findings be confirmed, routine clinical analysis of hemispheric gliomas for mIDH, 1p/19q codeletion, and PTEN deletion would be justified.