SUMMARY Human genetic studies have identified the neuronal RNA binding protein, Rbfox1, as a candidate gene for autism spectrum disorders. While Rbfox1 functions as a splicing regulator in the nucleus, it is also alternatively spliced to produce cytoplasmic isoforms. To investigate the function of cytoplasmic Rbfox1, we knocked down Rbfox proteins in mouse neurons and rescued with cytoplasmic or nuclear Rbfox1. Transcriptome profiling showed that nuclear Rbfox1 rescued splicing changes, whereas cytoplasmic Rbfox1 rescued changes in mRNA levels. iCLIP-seq of subcellular fractions revealed that Rbfox1 bound predominantly to introns in nascent RNA, while cytoplasmic Rbox1 bound to 3′ UTRs. Cytoplasmic Rbfox1 binding increased target mRNA stability and translation, and Rbfox1 and miRNA binding sites overlapped significantly. Cytoplasmic Rbfox1 target mRNAs were enriched in genes involved in cortical development and autism. Our results uncover a new Rbfox1 regulatory network and highlight the importance of cytoplasmic RNA metabolism to cortical development and disease.

SUMMARY Alternative splicing contributes to diverse aspects of cancer pathogenesis including altered cellular metabolism, but the specificity of the process or its consequences are not well understood. We characterized genome-wide alternative splicing induced by the activating EGFRvIII mutation in glioblastoma (GBM). EGFRvIII upregulates the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 splicing factor, promoting glycolytic gene expression and conferring significantly shorter survival in patients. HnRNPA1 promotes splicing of a transcript encoding the Myc-interacting partner Max, generating Delta Max, an enhancer of Myc-dependent transformation. Delta Max, but not full length Max, rescues Myc-dependent glycolytic gene expression upon induced EGFRvIII loss, and correlates with hnRNPA1 expression and downstream Myc-dependent gene transcription in patients. Finally, Delta Max is shown to promote glioma cell proliferation in vitro and augment EGFRvIII expressing GBM growth in vivo. These results demonstrate an important role for alternative splicing in GBM and identify Delta Max as a mediator of Myc-dependent tumor cell metabolism.

BackgroundBrain metastases are common in patients with metastatic melanoma. With increasing numbers of melanoma patients on anti-PD-1 therapy, we sought to evaluate the safety and initial response of brain metastases treated with concurrent pembrolizumab and radiation therapy.MethodsFrom an institutional database, we retrospectively identified patients with melanoma brain metastases treated with radiation therapy (RT) who received concurrent pembrolizumab. Concurrent treatment was defined as RT during pembrolizumab administration period and up to 4 months after most recent pembrolizumab treatment. Response was categorized by change in maximum diameter on first scheduled follow-up MRI. Lesion and patient specific outcomes including response, lesion control, brain control and overall survival were recorded and descriptively compared to contemporary treatments with RT and concurrent ipilimumab or RT without immunotherapy.ResultsFrom January 2014 through December 2015, we identified 21 patients who received concurrent radiation therapy and pembrolizumab for brain metastases or resection cavities that had at least one scheduled follow-up MRI. Eleven underwent stereotactic radiosurgery (SRS), 7 received hypofractionated radiation and 3 had whole brain treatment (WBRT). All treatments were well tolerated with no observed Grade 4 or 5 toxicities; Grade 3 edema and confusion occurred in 1 patient treated with WBRT after prior SRS. For metastases treated with SRS, at first scheduled follow-up MRI (median 57 days post SRS), 70% (16/23) exhibited complete (CR, n = 8) or partial response (PR, n = 8). The intracranial response rates (CR/PR) for patients treated with SRS and concurrent ipilimumab and SRS without concurrent immunotherapy was 32% and 22%, respectively.ConclusionsConcurrent pembrolizumab with brain RT appears safe in patients with metastatic melanoma, and SRS in particular is effective in markedly reducing the size of brain metastases at the time of first follow-up MRI. These results compare favorably to SRS in combination with ipilimumab and SRS without concurrent immunotherapy.

Hsf1p, the heat-shock transcription factor from Saccharomyces cerevisiae, has a low level of constitutive transcriptional activity and is kept in this state through negative regulation. In an effort to understand this negative regulation, we developed a novel genetic selection that detects altered expression from the HSP26 promoter. Using this reporter strain, we identified mutations and dosage compensators in the Ras/ cAMP signaling pathway that decrease cAMP levels and increase expression from the HSP26 promoter. In yeast, low cAMP levels reduce the catalytic activity of the cAMP-dependent kinase PKA. Previous studies had proposed that the stress response transcription factors Msn2p/4p, but not Hsf1p, are repressed by PKA. However, we found that reduction or elimination of PKA activity strongly derepresses transcription of the small heat-shock genes HSP26 and HSP12, even in the absence of MSN2/4. In a strain deleted for MSN2/4 and the PKA catalytic subunits, expression of HSP12 and HSP26 depends on HSF1 expression. Our findings indicate that Hsf1p functions downstream of PKA and suggest that PKA might be involved in negative regulation of Hsf1p activity. These results represent a major change in our understanding of how PKA signaling influences the heat-shock response and heat-shock protein expression.

Background-The role of radiotherapy (RT) in anaplastic thyroid cancer (ATC) for local tumor control is critical as mortality is often secondary to complications of tumor volume rather than metastatic disease. Here we report the long-term outcomes of RT for ATC.Methods-We identified 104 patients with histologically confirmed ATC presenting to our institution between 1984-2017 who received curative-intent or post-operative RT. Locoregional progression free survival (LPFS), overall survival (OS), and distant metastasis free survival (DMFS) were assessed.Results-Median age was 63.5 years. Median follow-up was 5.9 months (IQR 2.7-17.0) for the entire cohort and 10.6 months (IQR 5.3-40.0) in surviving patients. Thirty-one (29.8%) patients had metastatic disease prior to the start of RT. Concurrent chemoradiation was administered in 99 (95.2%) patients and trimodal therapy in 53 (51.0%) patients. Systemic therapy included doxorubicin (73.7%), paclitaxel with or without pazopanib (24.3%), and other systemic agents (2.0%). One-year OS and LPFS were 34.4% and 74.4% respectively. On multivariate analysis, RT dose60 Gy was associated with improved LPFS (HR=0.135, p=0.001) and improved OS (HR=0.487, p=0.004), and trimodal therapy was associated with improved LPFS (HR=0.060, p=0.017). Most commonly observed acute Grade 3 adverse events (AE) included dermatitis (20%) and mucositis (13%), with no observed Grade 4 subacute or late AE's.