Allan B. James

Alternative Splicing Mediates Responses of the Arabidopsis Circadian Clock to Temperature Changes

The circadian clock is a timing device that allows plants to anticipate environmental changes rather than just respond to them. This work demonstrates that alternative splicing of clock gene transcripts is one of the mechanisms that regulate the clock, particularly in response to changes in temperature. Alternative splicing plays crucial roles by influencing the diversity of the transcriptome and proteome and regulating protein structure/function and gene expression. It is widespread in plants, and alteration of the levels of splicing factors leads to a wide variety of growth and developmental phenotypes. The circadian clock is a complex piece of cellular machinery that can regulate physiology and behavior to anticipate predictable environmental changes on a revolving planet. We have performed a system-wide analysis of alternative splicing in clock components in Arabidopsis thaliana plants acclimated to different steady state temperatures or undergoing temperature transitions. This revealed extensive alternative splicing in clock genes and dynamic changes in alternatively spliced transcripts. Several of these changes, notably those affecting the circadian clock genes LATE ELONGATED HYPOCOTYL (LHY) and PSEUDO RESPONSE REGULATOR7, are temperature-dependent and contribute markedly to functionally important changes in clock gene expression in temperature transitions by producing nonfunctional transcripts and/or inducing nonsense-mediated decay. Temperature effects on alternative splicing contribute to a decline in LHY transcript abundance on cooling, but LHY promoter strength is not affected. We propose that temperature-associated alternative splicing is an additional mechanism involved in the operation and regulation of the plant circadian clock.

The Circadian Clock in Arabidopsis Roots Is a Simplified Slave Version of the Clock in Shoots

The circadian oscillator in eukaryotes consists of several interlocking feedback loops through which the expression of clock genes is controlled. It is generally assumed that all plant cells contain essentially identical and cell-autonomous multiloop clocks. Here, we show that the circadian clock in the roots of mature Arabidopsis plants differs markedly from that in the shoots and that the root clock is synchronized by a photosynthesis-related signal from the shoot. Two of the feedback loops of the plant circadian clock are disengaged in roots, because two key clock components, the transcription factors CCA1 and LHY, are able to inhibit gene expression in shoots but not in roots. Thus, the plant clock is organ-specific but not organ-autonomous.

Regulation of the Neuronal Proteasome by Zif268 (Egr1)

Most forms of neuronal plasticity are associated with induction of the transcription factor Zif268 (Egr1/Krox24/NGF-IA). In a genome-wide scan, we obtained evidence for potential modulation of proteasome subunit and regulatory genes by Zif268 in neurons, a finding of significance considering emerging evidence that the proteasome modulates synaptic function. Bioinformatic analysis indicated that the candidate proteasome Zif268 target genes had a rich concentration of putative Zif268 binding sites immediately upstream of the transcriptional start sites. Regulation of the mRNAs encoding the psmb9 (Lmp2) and psme2 (PA28β) proteasome subunits, along with the proteasome-regulatory kinase serum/glucocorticoid-regulated kinase (SGK) and the proteasome-associated antigen peptide transporter subunit 1 (Tap1), was confirmed after transfection of a neuronal cell line with Zif268. Conversely, these mRNAs were upregulated in cerebral cortex tissue from Zif268 knock-out mice relative to controls, confirming that Zif268 suppresses their expression in the CNS. Transfected Zif268 reduced the activity of psmb9, SGK, and Tap1 promoter–reporter constructs. Altered psmb9, SGK, and Tap1 mRNA levels were also observed in an in vivo model of neuronal plasticity involving Zif268 induction: the effect of haloperidol administration on striatal gene expression. Consistent with these effects on proteasome gene expression, increased Zif268 expression suppressed proteasome activity, whereas Zif268 knock-out mice exhibited elevated cortical proteasome activity. Our findings reveal that Zif268 regulates the expression of proteasome and related genes in neuronal cells and provide new evidence that altered expression of proteasome activity after Zif268 induction may be a key component of long-lasting CNS plasticity.

Genomic profiling of the neuronal target genes of the plasticity-related transcription factor – Zif268

The later phases of neuronal plasticity are invariably dependent on gene transcription. Induction of the transcription factor Zif268 (Egr‐1) in neurones is closely associated with many forms of functional plasticity, yet the neuronal target genes modulated by Zif268 have not been characterized. After transfection of a neuronal cell line with Zif268 we identified genes that show altered expression using high density microarrays. Although some of the genes identified have previously been associated with forms of neuronal plasticity, the majority have not been linked with neuronal plasticity or Zif268 action. Altered expression of a representative sample of the novel target genes was confirmed in Zif268‐transfected PC12 neurones, and in in vitro and in vivo models of Zif268‐associated neuronal plasticity. In particular, altered expression of the protease inhibitor Cystatin C and the chemokine Cxcl10 was observed in striatal tissue after haloperidol administration. Surprisingly, the group of identified genes is enriched for components of the proteasome and the major histocompatibility complex. Our findings suggest that altered expression of these genes following Zif268 induction may be a key component of long lasting plasticity in the CNS.

A Cancer Research UK first time in human phase I trial of IMA950 (novel multipeptide therapeutic vaccine) in patients with newly diagnosed glioblastoma

Purpose: To perform a two-cohort, phase I safety and immunogenicity study of IMA950 in addition to standard chemoradiotherapy and adjuvant temozolomide in patients with newly diagnosed glioblastoma. IMA950 is a novel glioblastoma-specific therapeutic vaccine containing 11 tumor-associated peptides (TUMAP), identified on human leukocyte antigen (HLA) surface receptors in primary human glioblastoma tissue. Experimental Design: Patients were HLA-A*02–positive and had undergone tumor resection. Vaccination comprised 11 intradermal injections with IMA950 plus granulocyte macrophage colony-stimulating factor (GM-CSF) over a 24-week period, beginning 7 to 14 days prior to initiation of chemoradiotherapy (Cohort 1) or 7 days after chemoradiotherapy (Cohort 2). Safety was assessed according to NCI CTCAE Version 4.0 and TUMAP-specific T-cell immune responses determined. Secondary observations included progression-free survival (PFS), pretreatment regulatory T cell (Treg) levels, and the effect of steroids on T-cell responses. Results: Forty-five patients were recruited. Related adverse events included minor injection site reactions, rash, pruritus, fatigue, neutropenia and single cases of allergic reaction, anemia and anaphylaxis. Two patients experienced grade 3 dose-limiting toxicity of fatigue and anaphylaxis. Of 40 evaluable patients, 36 were TUMAP responders and 20 were multi-TUMAP responders, with no important differences between cohorts. No effect of pretreatment Treg levels on IMA950 immunogenicity was observed, and steroids did not affect TUMAP responses. PFS rates were 74% at 6 months and 31% at 9 months. Conclusions: IMA950 plus GM-CSF was well-tolerated with the primary immunogenicity endpoint of observing multi-TUMAP responses in at least 30% of patients exceeded. Further development of IMA950 is encouraged. Clin Cancer Res; 22(19); 4776–85. ©2016 AACR. See related commentary by Lowenstein and Castro, p. 4760

A high quality Arabidopsis transcriptome for accurate transcript-level analysis of alternative splicing

Abstract Alternative splicing generates multiple transcript and protein isoforms from the same gene and thus is important in gene expression regulation. To date, RNA-sequencing (RNA-seq) is the standard method for quantifying changes in alternative splicing on a genome-wide scale. Understanding the current limitations of RNA-seq is crucial for reliable analysis and the lack of high quality, comprehensive transcriptomes for most species, including model organisms such as Arabidopsis, is a major constraint in accurate quantification of transcript isoforms. To address this, we designed a novel pipeline with stringent filters and assembled a comprehensive Reference Transcript Dataset for Arabidopsis (AtRTD2) containing 82,190 non-redundant transcripts from 34 212 genes. Extensive experimental validation showed that AtRTD2 and its modified version, AtRTD2-QUASI, for use in Quantification of Alternatively Spliced Isoforms, outperform other available transcriptomes in RNA-seq analysis. This strategy can be implemented in other species to build a pipeline for transcript-level expression and alternative splicing analyses.

Thermoplasticity in the plant circadian clock How plants tell the time-perature

In the March 2012 issue of The Plant Cell we describe extensive alternative splicing (AS) of Arabidopsis circadian clock genes. Notably these distinct post-transcriptional events associate with different steady-state temperatures and also with plants undergoing temperature transitions leading us to propose that temperature-associated AS is an additional mechanism involved in the operation and control of the plant circadian clock. Here we show that temperature associated AS also extends to REVEILLE 8 (RVE8), demonstrating a hitherto unrecognized link between the expression of this clock associated gene and temperature. Finally we discuss our observations of the plastic nature of clock gene expression at the post-transcriptional level in the context of the ongoing fascination of how plants respond to temperature.

AtRTD – a comprehensive reference transcript dataset resource for accurate quantification of transcript‐specific expression in Arabidopsis thaliana

Summary RNA‐sequencing (RNA‐seq) allows global gene expression analysis at the individual transcript level. Accurate quantification of transcript variants generated by alternative splicing (AS) remains a challenge. We have developed a comprehensive, nonredundant Arabidopsis reference transcript dataset (AtRTD) containing over 74 000 transcripts for use with algorithms to quantify AS transcript isoforms in RNA‐seq. The AtRTD was formed by merging transcripts from TAIR10 and novel transcripts identified in an AS discovery project. We have estimated transcript abundance in RNA‐seq data using the transcriptome‐based alignment‐free programmes Sailfish and Salmon and have validated quantification of splicing ratios from RNA‐seq by high resolution reverse transcription polymerase chain reaction (HR RT‐PCR). Good correlations between splicing ratios from RNA‐seq and HR RT‐PCR were obtained demonstrating the accuracy of abundances calculated for individual transcripts in RNA‐seq. The AtRTD is a resource that will have immediate utility in analysing Arabidopsis RNA‐seq data to quantify differential transcript abundance and expression.

1057PDFINAL RESULTS FROM A CANCER RESEARCH UK FIRST IN MAN PHASE I TRIAL OF IMA950 (A NOVEL MULTI PEPTIDE VACCINE) PLUS GM-CSF IN PATIENTS WITH NEWLY DIAGNOSED GLIOBLASTOMA

ABSTRACT Background IMA950 is a novel multi-peptide glioblastoma (GBM) specific vaccine that contains 11 HLA binding tumour associated peptides (TUMAPs), identified on human leukocyte antigen (HLA) surface receptors in primary human GBM tissue, and one viral (HBV) marker peptide. The TUMAPs are designed to activate TUMAP-specific CD8+ cytotoxic and CD4+ helper T lymphocytes, which then recognise cognate TUMAPs presented by GBM tumour cells and effect a targeted immune response. Methods Patients (pts) must be eligible for standard treatment of newly diagnosed GBM (maximal safe tumour resection, concomitant chemoradiotherapy (CRT) and adjuvant temozolomide (TMZ)) and be HLA-A*02 positive with no history of autoimmune disease. Vaccination comprises IMA950 plus GM-CSF injected intradermally at 11 time points over a 24 week period. Up to 45 pts will be entered into one of two cohorts with similar schedules. In Cohort 1 vaccination begins 7 to 14 days prior to initial CRT; in Cohort 2 it begins at least 7 days post CRT and 28 days prior to adjuvant TMZ. Safety is assessed according to NCI CTCAE Version 4.0. Immune response is determined by HLA-multimer analysis of vaccine-induced Tcell response in PBMC samples. Secondary objectives include observation of any anti-tumour effects, measurement of pre-treatment regulatory T-cell levels and evaluation of the effect of steroid dose on observed T-cell responses. Results As of 21 May-12, 25 pts (12 in Cohort 1 and 13 in Cohort 2) have been recruited. Adverse events related to either IMA950 or GM-CSF have been restricted to minor injection site reactions, a single distant allergic rash and a case of isolated asymptomatic neutropenia. Eleven pts have been analysed for immune response with 10 being evaluable (6 from Cohort 1 and 4 from Cohort 2). Eight pts responded to the HBV marker peptide, 8 pts to at least one TUMAP and 4 pts to multiple TUMAPs; 100% of pts in Cohort 2 responded to at least one TUMAP and 50% to multiple TUMAPs. Conclusion IMA950 plus GMCSF given alongside standard treatment for GBM has been well tolerated to date and these results already give encouragement for further development of this vaccine. Disclosure H. Singh-Jasuja: Harpreet Singh-Jasuja is a Founder and the Chief Scientific Officer of Immatics Biotechnologies GmbH. He also has stock or ownership stakes to disclose. All other authors have declared no conflicts of interest.

Sequential TPF chemotherapy followed by concurrent chemoradiotherapy in locally advanced head and neck cancer--a retrospective analysis of toxicity and outcomes.

Background and aims Phase III trials have shown that the addition of a taxane to cisplatin/5FU-based induction chemotherapy (TPF) improves response rates and overall survival in unresectable stage III/IV head and neck cancer. We sought to assess the tolerability, compliance and clinical outcomes of this treatment regime. Methods A retrospective study of patients treated within a single centre between September 2007 and November 2010. Toxicities were graded according to CTCAE version 3.0. Survival, distant metastasis and local control rates are expressed as percentages at two years using the Kaplan–Meier method. Results A total of 100 patients were identified (11% stage III, 86% stage IV) and 32% of patients were admitted as an emergency after TPF. The rate of neutropenic fever was 31%, this number fell to 9% when prophylactic G-CSF was used. In addition, 89% of patients underwent radical chemoradiation. Of these, 96% completed the full radiotherapy course. However, only 64% of patients received a minimum of two cycles of concurrent platinum chemotherapy. The two-year overall survival, metastasis free survival and local control rates were 62.6%, 88.5% and 73.3%, respectively. Conclusions TPF chemotherapy can be delivered safely in a non-trial cohort of patients. There is, however, a significant reduction in concurrent chemotherapy dose intensity. The long-term impact of this remains unclear.