Developmental Regulation of Small-Conductance Ca²⁺-Activated K⁺ Channel Expression and Function in Rat Purkinje Neurons

Calcium transients play an important role in the early and later phases of differentiation and maturation of single neurons and neuronal networks. Small-conductance calcium-activated potassium channels of the SK type modulate membrane excitability and are important determinants of the firing properties of central neurons. Increases in the intracellular calcium concentration activate SK channels, leading to a hyperpolarization of the membrane potential, which in turn reduces the calcium inflow into the cell. This feedback mechanism is ideally suited to regulate the spatiotemporal occurrence of calcium transients. However, the role of SK channels in neuronal development has not been addressed so far. We have concentrated on the ontogenesis and function of SK channels in the developing rat cerebellum, focusing particularly on Purkinje neurons. Electrophysiological recordings combined with specific pharmacological tools have revealed for the first time the presence of an afterhyperpolarizing current (I_{AHP}) in immature Purkinje cells in rat cerebellar slices. The channel subunits underlying this current were identified as SK2 and localized by in situ hybridization and subunit-specific antibodies. Their expression level was shown to be high at birth and subsequently to decline during the first 3 weeks of postnatal life, both at the mRNA and protein levels. This developmental regulation was tightly correlated with the expression of I_{AHP} and the prominent role of SK2 channels in shaping the spontaneous firing pattern in young, but not in adult, Purkinje neurons. These results provide the first evidence of the developmental regulation and function of SK channels in central neurons

Systemic epidermal nevus with involvement of the oral mucosa due to FGFR3 mutation

Our results show that activating FGFR3 mutations can also affect the oral mucosa and that extracutaneous manifestations of EN syndrome can be subtle. We highlight the theoretical risk of the patient having an offspring with thanatophoric dysplasia as gonadal mosaicism for the R248C mutation cannot be excluded

Activation of the PI3K/AKT Pathway in Merkel Cell Carcinoma

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with an increasing incidence. The understanding of the molecular carcinogenesis of MCC is limited. Here, we scrutinized the PI3K/AKT pathway, one of the major pathways activated in human cancer, in MCC. Immunohistochemical analysis of 41 tumor tissues and 9 MCC cell lines revealed high levels of AKT phosphorylation at threonine 308 in 88% of samples. Notably, the AKT phosphorylation was not correlated with the presence or absence of the Merkel cell polyoma virus (MCV). Accordingly, knock-down of the large and small T antigen by shRNA in MCV positive MCC cells did not affect phosphorylation of AKT. We also analyzed 46 MCC samples for activating PIK3CA and AKT1 mutations. Oncogenic PIK3CA mutations were found in 2/46 (4%) MCCs whereas mutations in exon 4 of AKT1 were absent. MCC cell lines demonstrated a high sensitivity towards the PI3K inhibitor LY-294002. This finding together with our observation that the PI3K/AKT pathway is activated in the majority of human MCCs identifies PI3K/AKT as a potential new therapeutic target for MCC patients

The clinical trial landscape in oncology and connectivity of somatic mutational profiles to targeted therapies

BACKGROUND: Precision medicine in oncology relies on rapid associations between patient-specific variations and targeted therapeutic efficacy. Due to the advancement of genomic analysis, a vast literature characterizing cancer-associated molecular aberrations and relative therapeutic relevance has been published. However, data are not uniformly reported or readily available, and accessing relevant information in a clinically acceptable time-frame is a daunting proposition, hampering connections between patients and appropriate therapeutic options. One important therapeutic avenue for oncology patients is through clinical trials. Accordingly, a global view into the availability of targeted clinical trials would provide insight into strengths and weaknesses and potentially enable research focus. However, data regarding the landscape of clinical trials in oncology is not readily available, and as a result, a comprehensive understanding of clinical trial availability is difficult. RESULTS: To support clinical decision-making, we have developed a data loader and mapper that connects sequence information from oncology patients to data stored in an in-house database, the JAX Clinical Knowledgebase (JAX-CKB), which can be queried readily to access comprehensive data for clinical reporting via customized reporting queries. JAX-CKB functions as a repository to house expertly curated clinically relevant data surrounding our 358-gene panel, the JAX Cancer Treatment Profile (JAX CTP), and supports annotation of functional significance of molecular variants. Through queries of data housed in JAX-CKB, we have analyzed the landscape of clinical trials relevant to our 358-gene targeted sequencing panel to evaluate strengths and weaknesses in current molecular targeting in oncology. Through this analysis, we have identified patient indications, molecular aberrations, and targeted therapy classes that have strong or weak representation in clinical trials. CONCLUSIONS: Here, we describe the development and disseminate system methods for associating patient genomic sequence data with clinically relevant information, facilitating interpretation and providing a mechanism for informing therapeutic decision-making. Additionally, through customized queries, we have the capability to rapidly analyze the landscape of targeted therapies in clinical trials, enabling a unique view into current therapeutic availability in oncology

Emerging common themes in regulation of PIKKs and PI3Ks

Phosphatidylinositol-3 kinase-related kinases (PIKKs) comprise a family of protein kinases that respond to various stresses, including DNA damage, blocks in DNA replication, availability of nutrients and errors in mRNA splicing. PIKKs are characterized by the presence of a conserved kinase domain (KD), whose activity is regulated by two C-terminal regions, referred to as PIKK-regulatory domain (PRD) and FRAP-ATM-TRRAP-C-terminal (FATC), respectively. Here, we review functional and structural data that implicate the PRD and FATC domains in regulation of PIKK activity, drawing parallels to phosphatidylinositol-3 kinases (PI3K), lipid kinases that have sequence similarity to PIKKs. The PI3K C-terminus, which we propose to be equivalent to the PRD and FATC domains of PIKKs, is in close proximity to the activation loop of the KD, suggesting that in PIKKs, the PRD and FATC domains may regulate kinase activity by targeting the activation loop

Recurrent somatic mutations in POLR2A define a distinct subset of meningiomas

RNA polymerase II mediates the transcription of all protein-coding genes in eukaryotic cells, a process that is fundamental to life. Genomic mutations altering this enzyme have not previously been linked to any pathology in humans, which is a testament to its indispensable role in cell biology. On the basis of a combination of next-generation genomic analyses of 775 meningiomas, we report that recurrent somatic p.Gln403Lys or p.Leu438_His439del mutations in POLR2A, which encodes the catalytic subunit of RNA polymerase II (ref. 1), hijack this essential enzyme and drive neoplasia. POLR2A mutant tumors show dysregulation of key meningeal identity genes including WNT6 and ZIC1/ZIC4. In addition to mutations in POLR2A, NF2, SMARCB1, TRAF7, KLF4, AKT1, PIK3CA, and SMO4 we also report somatic mutations in AKT3, PIK3R1, PRKAR1A, and SUFU in meningiomas. Our results identify a role for essential transcriptional machinery in driving tumorigenesis and define mutually exclusive meningioma subgroups with distinct clinical and pathological features

FGFR3, HRAS, KRAS, NRAS and PIK3CA Mutations in Bladder Cancer and Their Potential as Biomarkers for Surveillance and Therapy

Background: Fifty percent of patients with muscle-invasive bladder cancer (MI-BC) die from their disease and current chemotherapy treatment only marginally increases survival. Novel therapies targeting receptor tyrosine kinases or activated oncogenes may improve outcome. Hence, it is necessary to stratify patients based on mutations in relevant oncogenes. Patients with non-muscle-invasive bladder cancer (NMI-BC) have excellent survival, however two-thirds develop recurrences. Tumor specific mutations can be used to detect recurrences in urine assays, presenting a more patient-friendly diagnostic procedure than cystoscopy. Methodology/Principal Findings: To address these issues, we developed a mutation assay for the simultaneous detection of 19 possible mutations in the HRAS, KRAS, and NRAS genes. With this assay and mutation assays for the FGFR3 and PIK3CA oncogenes, we screened primary bladder tumors of 257 patients and 184 recurrences from 54 patients. Additionally, in primary tumors p53 expression was obtained by immunohistochemistry. Of primary tumors 64% were mutant for FGFR3, 11% for RAS, 24% for PIK3CA, and 26% for p53. FGFR3 mutations were mutually exclusive with RAS mutations (p = 0.001) and co-occurred with PIK3CA mutations (p = 0.016). P53 overexpression was mutually exclusive with PIK3CA and FGFR3 mutations (p≤0.029). Mutations in the RAS and PIK3CA genes were not predictors for recurrence-free, progression-free and disease-specific survival. In patients presenting with NMI-BC grade 3 and MI-BC, 33 and 36% of the primary tumors were mutant. In patients with low-grade NMI-BC, 88% of the primary tumors carried a mutation and 88% of the recurrences were mutant. Conclusions/Significance: The mutation assays present a companion diagnostic to define patients for targeted therapies. In addition, the assays are a potential biomarker to detect recurrences during surveillance. We showed that 88% of patients presenting with low-grade NMI-BC are eligible for such a follow-up. This may contribute to a reduction in the number of cystoscopical examinations