Ping Chi

Covalent histone modifications — miswritten, misinterpreted and mis-erased in human cancers

Post-translational modification of histones provides an important regulatory platform for processes such as gene transcription and DNA damage repair. It has become increasingly apparent that the misregulation of histone modification, which is caused by the deregulation of factors that mediate the modification installation, removal and/or interpretation, actively contributes to human cancer. In this Review, we summarize recent advances in understanding the interpretation of certain histone methylations by plant homeodomain finger-containing proteins, and how misreading, miswriting and mis-erasing of histone methylation marks can be associated with oncogenesis and progression. These observations provide us with a greater mechanistic understanding of epigenetic alterations in human cancers and might also help direct new therapeutic interventions in the future.

Synapsin dispersion and reclustering during synaptic activity.

Presynaptic modulation of synaptic transmission provides an important basis for control of synaptic function. The synapsins, a family of highly conserved proteins associated with synaptic vesicles, have long been implicated in the regulation of neurotransmitter release. However, direct physiological measurements of the molecular mechanisms have been lacking. Here we show that in living hippocampal terminals, green fluorescent protein (GFP)-labeled synapsin Ia dissociates from synaptic vesicles, disperses into axons during action potential (AP) firing, and reclusters to synapses after the cessation of synaptic activity. Using various mutated forms of synapsin Ia that prevent phosphorylation at specific sites, we performed simultaneous FM 4-64 measurements of vesicle pool mobilization along with synapsin dispersion kinetics. These studies indicate that the rate of synapsin dispersion is controlled by phosphorylation, which in turn controls the kinetics of vesicle pool turnover. Thus synapsin acts as a phosphorylation-state-dependent regulator of synaptic vesicle mobilization, and hence, neurotransmitter release.

Synaptic Vesicle Mobilization Is Regulated by Distinct Synapsin I Phosphorylation Pathways at Different Frequencies

During action potential firing, the rate of synapsin dissociation from synaptic vesicles and dispersion into axons controls the rate of vesicle availability for exocytosis at the plasma membrane. Here we show that synapsin Ias dispersion rate tracks the synaptic vesicle pool turnover rate linearly over the range 5-20 Hz and that the molecular basis for this lies in regulation at both the calcium-calmodulin-dependent kinase (CaM kinase) and the mitogen-activated protein (MAP) kinase/calcineurin sites. Our results show that CaM kinase sites control vesicle mobilization at low stimulus frequency, while MAP kinase/calcineurin sites are critical at both lower and higher stimulus frequencies. Thus, multiple signaling pathways serve to allow synapsins control of vesicle mobilization over different stimulus frequencies.

ETV1 is a lineage survival factor that cooperates with KIT in gastrointestinal stromal tumours

Gastrointestinal stromal tumour (GIST) is the most common human sarcoma and is primarily defined by activating mutations in the KIT or PDGFRA receptor tyrosine kinases1,2. KIT is highly expressed in interstitial cells of Cajal (ICCs)—the presumed cell of origin for GIST—as well as in hematopoietic stem cells, melanocytes, mast cells and germ cells2,3. Yet, families harbouring germline activating KIT mutations and mice with knock-in Kit mutations almost exclusively develop ICC hyperplasia and GIST4–7, suggesting that the cellular context is important for KIT to mediated oncogenesis. Here we show that the ETS family member ETV1 is highly expressed in the subtypes of ICCs sensitive to oncogenic KIT mediated transformation8, and is required for their development. In addition, ETV1 is universally highly expressed in GISTs and is required for growth of imatinib-sensitive and resistant GIST cell lines. Transcriptome profiling and global analyses of ETV1-binding sites suggest that ETV1 is a master regulator of an ICC-GIST-specific transcription network mainly through enhancer binding. The ETV1 transcriptional program is further regulated by activated KIT, which prolongs ETV1 protein stability and cooperates with ETV1 to promote tumourigenesis. We propose that GIST arises from ICCs with high levels of endogenous ETV1 expression that, when coupled with an activating KIT mutation, drives an oncogenic ETS transcription program. This differs from other ETS-dependent tumours such as prostate cancer, melanoma, and Ewing sarcoma where genomic translocation or amplification drives aberrant ETS expression9–11 and represents a novel mechanism of oncogenic transcription factor activation.Gastrointestinal stromal tumour (GIST) is the most common human sarcoma and is primarily defined by activating mutations in the KIT or PDGFRA receptor tyrosine kinases. KIT is highly expressed in interstitial cells of Cajal (ICCs)—the presumed cell of origin for GIST—as well as in haematopoietic stem cells, melanocytes, mast cells and germ cells. Yet, families harbouring germline activating KIT mutations and mice with knock-in Kit mutations almost exclusively develop ICC hyperplasia and GIST, suggesting that the cellular context is important for KIT to mediate oncogenesis. Here we show that the ETS family member ETV1 is highly expressed in the subtypes of ICCs sensitive to oncogenic KIT mediated transformation, and is required for their development. In addition, ETV1 is universally highly expressed in GISTs and is required for growth of imatinib-sensitive and resistant GIST cell lines. Transcriptome profiling and global analyses of ETV1-binding sites suggest that ETV1 is a master regulator of an ICC-GIST-specific transcription network mainly through enhancer binding. The ETV1 transcriptional program is further regulated by activated KIT, which prolongs ETV1 protein stability and cooperates with ETV1 to promote tumorigenesis. We propose that GIST arises from ICCs with high levels of endogenous ETV1 expression that, when coupled with an activating KIT mutation, drives an oncogenic ETS transcriptional program. This differs from other ETS-dependent tumours such as prostate cancer, melanoma and Ewing sarcoma where genomic translocation or amplification drives aberrant ETS expression. It also represents a novel mechanism of oncogenic transcription factor activation.

ETS factors reprogram the androgen receptor cistrome and prime prostate tumorigenesis in response to PTEN loss

Studies of ETS-mediated prostate oncogenesis have been hampered by a lack of suitable experimental systems. Here we describe a new conditional mouse model that shows robust, homogenous ERG expression throughout the prostate. When combined with homozygous Pten loss, the mice developed accelerated, highly penetrant invasive prostate cancer. In mouse prostate tissue, ERG markedly increased androgen receptor (AR) binding. Robust ERG-mediated transcriptional changes, observed only in the setting of Pten loss, included the restoration of AR transcriptional output and upregulation of genes involved in cell death, migration, inflammation and angiogenesis. Similarly, ETS variant 1 (ETV1) positively regulated the AR cistrome and transcriptional output in ETV1-translocated, PTEN-deficient human prostate cancer cells. In two large clinical cohorts, expression of ERG and ETV1 correlated with higher AR transcriptional output in PTEN-deficient prostate cancer specimens. We propose that ETS factors cause prostate-specific transformation by altering the AR cistrome, priming the prostate epithelium to respond to aberrant upstream signals such as PTEN loss.

PRC2 is recurrently inactivated through EED or SUZ12 loss in malignant peripheral nerve sheath tumors

Malignant peripheral nerve sheath tumors (MPNSTs) represent a group of highly aggressive soft-tissue sarcomas that may occur sporadically, in association with neurofibromatosis type I (NF1 associated) or after radiotherapy. Using comprehensive genomic approaches, we identified loss-of-function somatic alterations of the Polycomb repressive complex 2 (PRC2) components (EED or SUZ12) in 92% of sporadic, 70% of NF1-associated and 90% of radiotherapy-associated MPNSTs. MPNSTs with PRC2 loss showed complete loss of trimethylation at lysine 27 of histone H3 (H3K27me3) and aberrant transcriptional activation of multiple PRC2-repressed homeobox master regulators and their regulated developmental pathways. Introduction of the lost PRC2 component in a PRC2-deficient MPNST cell line restored H3K27me3 levels and decreased cell growth. Additionally, we identified frequent somatic alterations of CDKN2A (81% of all MPNSTs) and NF1 (72% of non-NF1-associated MPNSTs), both of which significantly co-occur with PRC2 alterations. The highly recurrent and specific inactivation of PRC2 components, NF1 and CDKN2A highlights their critical and potentially cooperative roles in MPNST pathogenesis.

Roscovitine: a novel regulator of P/Q-type calcium channels and transmitter release in central neurons

Roscovitine is widely used for inhibition of cdk5, a cyclin‐dependent kinase expressed predominantly in the brain. A novel function of roscovitine, i.e. an effect on Ca2+ channels and transmitter release in central neurons, was studied by whole‐cell voltage‐clamp recordings and time‐lapse fluorescence imaging techniques. Extracellular application of roscovitine markedly enhanced the tail calcium current following repolarization from depolarized voltages. This effect was rapid, reversible and dose dependent. Roscovitine dramatically slowed the deactivation kinetics of calcium channels. The deactivation time constant was increased 3‐ to 6‐fold, suggesting that roscovitine could prolong the channel open state and increase the calcium influx. The potentiation of tail calcium currents caused by roscovitine and by the L‐channel activator Bay K 8644 was not occluded but additive. Roscovitine‐induced potentiation of tail calcium currents was significantly blocked by the P/Q‐channel blocker CgTx‐MVIIC, indicating that the major target of roscovitine is the P/Q‐type calcium channel. In mutant mice with targeted deletion of p35, a neuronal specific activator of cdk5, roscovitine regulated calcium currents in a manner similar to that observed in wild‐type mice. Moreover, intracellular perfusion of roscovitine failed to modulate calcium currents. These results suggest that roscovitine acts on extracellular site(s) of calcium channels via a cdk5‐independent mechanism. Roscovitine potentiated glutamate release at presynaptic terminals of cultured hippocampal neurons detected with the vesicle trafficking dye FM1–43, consistent with the positive effect of roscovitine on the P/Q‐type calcium channel, the major mediator of action potential‐evoked transmitter release in the mammalian CNS.

Histone H3K36 mutations promote sarcomagenesis through altered histone methylation landscape

An oncohistone deranges inhibitory chromatin Missense mutations (that change one amino acid for another) in histone H3 can produce a so-called oncohistone and are found in a number of pediatric cancers. For example, the lysine-36–to-methionine (K36M) mutation is seen in almost all chondroblastomas. Lu et al. show that K36M mutant histones are oncogenic, and they inhibit the normal methylation of this same residue in wild-type H3 histones. The mutant histones also interfere with the normal development of bone-related cells and the deposition of inhibitory chromatin marks. Science, this issue p. 844 The lysine-36–to–methionine mutation in histone H3 is oncogenic and interferes with inhibitory chromatin marks. Several types of pediatric cancers reportedly contain high-frequency missense mutations in histone H3, yet the underlying oncogenic mechanism remains poorly characterized. Here we report that the H3 lysine 36–to–methionine (H3K36M) mutation impairs the differentiation of mesenchymal progenitor cells and generates undifferentiated sarcoma in vivo. H3K36M mutant nucleosomes inhibit the enzymatic activities of several H3K36 methyltransferases. Depleting H3K36 methyltransferases, or expressing an H3K36I mutant that similarly inhibits H3K36 methylation, is sufficient to phenocopy the H3K36M mutation. After the loss of H3K36 methylation, a genome-wide gain in H3K27 methylation leads to a redistribution of polycomb repressive complex 1 and de-repression of its target genes known to block mesenchymal differentiation. Our findings are mirrored in human undifferentiated sarcomas in which novel K36M/I mutations in H3.1 are identified.

Alternative transcription initiation leads to expression of a novel ALK isoform in cancer

Activation of oncogenes by mechanisms other than genetic aberrations such as mutations, translocations, or amplifications is largely undefined. Here we report a novel isoform of the anaplastic lymphoma kinase (ALK) that is expressed in ∼11% of melanomas and sporadically in other human cancer types, but not in normal tissues. The novel ALK transcript initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19, and was termed ALKATI. In ALKATI-expressing tumours, the ATI site is enriched for H3K4me3 and RNA polymerase II, chromatin marks characteristic of active transcription initiation sites. ALKATI is expressed from both ALK alleles, and no recurrent genetic aberrations are found at the ALK locus, indicating that the transcriptional activation is independent of genetic aberrations at the ALK locus. The ALKATI transcript encodes three proteins with molecular weights of 61.1, 60.8 and 58.7 kilodaltons, consisting primarily of the intracellular tyrosine kinase domain. ALKATI stimulates multiple oncogenic signalling pathways, drives growth-factor-independent cell proliferation in vitro, and promotes tumorigenesis in vivo in mouse models. ALK inhibitors can suppress the kinase activity of ALKATI, suggesting that patients with ALKATI-expressing tumours may benefit from ALK inhibitors. Our findings suggest a novel mechanism of oncogene activation in cancer through de novo alternative transcription initiation.

OncoKB: A Precision Oncology Knowledge Base

PURPOSE With prospective clinical sequencing of tumors emerging as a mainstay in cancer care, there is an urgent need for a clinical support tool that distills the clinical implications associated with specific mutation events into a standardized and easily interpretable format. To this end, we developed OncoKB, an expert-guided precision oncology knowledge base. METHODS OncoKB annotates the biological and oncogenic effect and the prognostic and predictive significance of somatic molecular alterations. Potential treatment implications are stratified by the level of evidence that a specific molecular alteration is predictive of drug response based on US Food and Drug Administration (FDA) labeling, National Comprehensive Cancer Network (NCCN) guidelines, disease-focused expert group recommendations and the scientific literature. RESULTS To date, over 3000 unique mutations, fusions, and copy number alterations in 418 cancer-associated genes have been annotated. To test the utility of OncoKB, we annotated all genomic events in 5983 primary tumor samples in 19 cancer types. Forty-one percent of samples harbored at least one potentially actionable alteration, of which 7.5% were predictive of clinical benefit from a standard treatment. OncoKB annotations are available through a public web resource (http://oncokb.org/) and are also incorporated into the cBioPortal for Cancer Genomics to facilitate the interpretation of genomic alterations by physicians and researchers. CONCLUSION OncoKB, a comprehensive and curated precision oncology knowledge base, offers oncologists detailed, evidence-based information about individual somatic mutations and structural alterations present in patient tumors with the goal of supporting optimal treatment decisions.