Robert K. Bradley

Integrative clinical genomics of advanced prostate cancer

Toward development of a precision medicine framework for metastatic, castration-resistant prostate cancer (mCRPC), we established a multi-institutional clinical sequencing infrastructure to conduct prospective whole-exome and transcriptome sequencing of bone or soft tissue tumor biopsies from a cohort of 150 mCRPC affected individuals. Aberrations of AR, ETS genes, TP53, and PTEN were frequent (40%-60% of cases), with TP53 and AR alterations enriched in mCRPC compared to primary prostate cancer. We identified new genomic alterations in PIK3CA/B, R-spondin, BRAF/RAF1, APC, β-catenin, and ZBTB16/PLZF. Moreover, aberrations of BRCA2, BRCA1, and ATM were observed at substantially higher frequencies (19.3% overall) compared to those in primary prostate cancers. 89% of affected individuals harbored a clinically actionable aberration, including 62.7% with aberrations in AR, 65% in other cancer-related genes, and 8% with actionable pathogenic germline alterations. This cohort study provides clinically actionable information that could impact treatment decisions for these affected individuals.

Braveheart, a Long Noncoding RNA Required for Cardiovascular Lineage Commitment

Long noncoding RNAs (lncRNAs) are often expressed in a development-specific manner, yet little is known about their roles in lineage commitment. Here, we identified Braveheart (Bvht), a heart-associated lncRNA in mouse. Using multiple embryonic stem cell (ESC) differentiation strategies, we show that Bvht is required for progression of nascent mesoderm toward a cardiac fate. We find that Bvht is necessary for activation of a core cardiovascular gene network and functions upstream of mesoderm posterior 1 (MesP1), a master regulator of a common multipotent cardiovascular progenitor. We also show that Bvht interacts with SUZ12, a component of polycomb-repressive complex 2 (PRC2), during cardiomyocyte differentiation, suggesting that Bvht mediates epigenetic regulation of cardiac commitment. Finally, we demonstrate a role for Bvht in maintaining cardiac fate in neonatal cardiomyocytes. Together, our work provides evidence for a long noncoding RNA with critical roles in the establishment of the cardiovascular lineage during mammalian development.

Fast Statistical Alignment

We describe a new program for the alignment of multiple biological sequences that is both statistically motivated and fast enough for problem sizes that arise in practice. Our Fast Statistical Alignment program is based on pair hidden Markov models which approximate an insertion/deletion process on a tree and uses a sequence annealing algorithm to combine the posterior probabilities estimated from these models into a multiple alignment. FSA uses its explicit statistical model to produce multiple alignments which are accompanied by estimates of the alignment accuracy and uncertainty for every column and character of the alignment—previously available only with alignment programs which use computationally-expensive Markov Chain Monte Carlo approaches—yet can align thousands of long sequences. Moreover, FSA utilizes an unsupervised query-specific learning procedure for parameter estimation which leads to improved accuracy on benchmark reference alignments in comparison to existing programs. The centroid alignment approach taken by FSA, in combination with its learning procedure, drastically reduces the amount of false-positive alignment on biological data in comparison to that given by other methods. The FSA program and a companion visualization tool for exploring uncertainty in alignments can be used via a web interface at http://orangutan.math.berkeley.edu/fsa/, and the source code is available at http://fsa.sourceforge.net/.

SRSF2 Mutations Contribute to Myelodysplasia by Mutant-Specific Effects on Exon Recognition

Mutations affecting spliceosomal proteins are the most common mutations in patients with myelodysplastic syndromes (MDS), but their role in MDS pathogenesis has not been delineated. Here we report that mutations affecting the splicing factor SRSF2 directly impair hematopoietic differentiation in vivo, which is not due to SRSF2 loss of function. By contrast, SRSF2 mutations alter SRSF2s normal sequence-specific RNA binding activity, thereby altering the recognition of specific exonic splicing enhancer motifs to drive recurrent mis-splicing of key hematopoietic regulators. This includes SRSF2 mutation-dependent splicing of EZH2, which triggers nonsense-mediated decay, which, in turn, results in impaired hematopoietic differentiation. These data provide a mechanistic link between a mutant spliceosomal protein, alterations in the splicing of key regulators, and impaired hematopoiesis.

Binding Site Turnover Produces Pervasive Quantitative Changes in Transcription Factor Binding between Closely Related Drosophila Species

Genome-wide comparison of transcription factor binding between related Drosophila species highlights how sequence changes affect the biochemical events that underlie animal development.

Modulation of splicing catalysis for therapeutic targeting of leukemia with mutations in genes encoding spliceosomal proteins.

Mutations in genes encoding splicing factors (which we refer to as spliceosomal genes) are commonly found in patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). These mutations recurrently affect specific amino acid residues, leading to perturbed normal splice site and exon recognition. Spliceosomal gene mutations are always heterozygous and rarely occur together with one another, suggesting that cells may tolerate only a partial deviation from normal splicing activity. To test this hypothesis, we engineered mice to express a mutated allele of serine/arginine-rich splicing factor 2 (Srsf2P95H)—which commonly occurs in individuals with MDS and AML—in an inducible, hemizygous manner in hematopoietic cells. These mice rapidly succumbed to fatal bone marrow failure, demonstrating that Srsf2-mutated cells depend on the wild-type Srsf2 allele for survival. In the context of leukemia, treatment with the spliceosome inhibitor E7107 (refs. 7,8) resulted in substantial reductions in leukemic burden, specifically in isogenic mouse leukemias and patient-derived xenograft AMLs carrying spliceosomal mutations. Whereas E7107 treatment of mice resulted in widespread intron retention and cassette exon skipping in leukemic cells regardless of Srsf2 genotype, the magnitude of splicing inhibition following E7107 treatment was greater in Srsf2-mutated than in Srsf2-wild-type leukemia, consistent with the differential effect of E7107 on survival. Collectively, these data provide genetic and pharmacologic evidence that leukemias with spliceosomal gene mutations are preferentially susceptible to additional splicing perturbations in vivo as compared to leukemias without such mutations. Modulation of spliceosome function may thus provide a new therapeutic avenue in genetically defined subsets of individuals with MDS or AML.

Hepatocyte growth factor (HGF) autocrine activation predicts sensitivity to MET inhibition in glioblastoma

Because oncogene MET and EGF receptor (EGFR) inhibitors are in clinical development against several types of cancer, including glioblastoma, it is important to identify predictive markers that indicate patient subgroups suitable for such therapies. We investigated in vivo glioblastoma models characterized by hepatocyte growth factor (HGF) autocrine or paracrine activation, or by MET or EGFR amplification, for their susceptibility to MET inhibitors. HGF autocrine expression correlated with high phospho-MET levels in HGF autocrine cell lines, and these lines showed high sensitivity to MET inhibition in vivo. An HGF paracrine environment may enhance glioblastoma growth in vivo but did not indicate sensitivity to MET inhibition. EGFRvIII amplification predicted sensitivity to EGFR inhibition, but in the same tumor, increased copies of MET from gains of chromosome 7 did not result in increased MET activity and did not predict sensitivity to MET inhibitors. Thus, HGF autocrine glioblastoma bears an activated MET signaling pathway that may predict sensitivity to MET inhibitors. Moreover, serum HGF levels may serve as a biomarker for the presence of autocrine tumors and their responsiveness to MET therapeutics.

Widespread intron retention diversifies most cancer transcriptomes

BackgroundSomatic mutations affecting components of the RNA splicing machinery occur with high frequencies across many tumor types. These mutations give rise to distinct alterations in normal splice site and exon recognition, such as unusual 3′ splice site preferences, that likely contribute to tumorigenesis.MethodsWe analyzed genome-wide patterns of RNA splicing across 805 matched tumor and normal control samples from 16 distinct cancer types to identify signals of abnormal cancer-associated splicing.ResultsWe found that abnormal RNA splicing, typified by widespread intron retention, is common across cancers even in the absence of mutations directly affecting the RNA splicing machinery. Almost all liquid and solid cancer types exhibited frequent retention of both alternative and constitutive introns relative to control normal tissues. The sole exception was breast cancer, where intron retention typified adjacent normal rather than cancer tissue. Different introns were preferentially retained in specific cancer types, although a small subset of introns enriched for genes encoding RNA splicing and export factors exhibited frequent retention across diverse cancers. The extent of intron retention correlated with the presence of IDH1 and IDH2 mutations in acute myeloid leukemia and across molecular subtypes in breast cancer. Many introns that were preferentially retained in primary cancers were present at high levels in the cytoplasmic mRNA pools of cancer cell lines.ConclusionsOur data indicate that abnormal RNA splicing is a common characteristic of cancers even in the absence of mutational insults to the splicing machinery, and suggest that intron-containing mRNAs contribute to the transcriptional diversity of many cancers.

Massive Mitochondrial Gene Transfer in a Parasitic Flowering Plant Clade

Recent studies have suggested that plant genomes have undergone potentially rampant horizontal gene transfer (HGT), especially in the mitochondrial genome. Parasitic plants have provided the strongest evidence of HGT, which appears to be facilitated by the intimate physical association between the parasites and their hosts. A recent phylogenomic study demonstrated that in the holoparasite Rafflesia cantleyi (Rafflesiaceae), whose close relatives possess the worlds largest flowers, about 2.1% of nuclear gene transcripts were likely acquired from its obligate host. Here, we used next-generation sequencing to obtain the 38 protein-coding and ribosomal RNA genes common to the mitochondrial genomes of angiosperms from R. cantleyi and five additional species, including two of its closest relatives and two host species. Strikingly, our phylogenetic analyses conservatively indicate that 24%–41% of these gene sequences show evidence of HGT in Rafflesiaceae, depending on the species. Most of these transgenic sequences possess intact reading frames and are actively transcribed, indicating that they are potentially functional. Additionally, some of these transgenes maintain synteny with their donor and recipient lineages, suggesting that native genes have likely been displaced via homologous recombination. Our study is the first to comprehensively assess the magnitude of HGT in plants involving a genome (i.e., mitochondria) and a species interaction (i.e., parasitism) where it has been hypothesized to be potentially rampant. Our results establish for the first time that, although the magnitude of HGT involving nuclear genes is appreciable in these parasitic plants, HGT involving mitochondrial genes is substantially higher. This may represent a more general pattern for other parasitic plant clades and perhaps more broadly for angiosperms.

Horizontal transfer of expressed genes in a parasitic flowering plant

BackgroundRecent studies have shown that plant genomes have potentially undergone rampant horizontal gene transfer (HGT). In plant parasitic systems HGT appears to be facilitated by the intimate physical association between the parasite and its host. HGT in these systems has been invoked when a DNA sequence obtained from a parasite is placed phylogenetically very near to its host rather than with its closest relatives. Studies of HGT in parasitic plants have relied largely on the fortuitous discovery of gene phylogenies that indicate HGT, and no broad systematic search for HGT has been undertaken in parasitic systems where it is most expected to occur.ResultsWe analyzed the transcriptomes of the holoparasite Rafflesia cantleyi Solms-Laubach and its obligate host Tetrastigma rafflesiae Miq. using phylogenomic approaches. Our analyses show that several dozen actively transcribed genes, most of which appear to be encoded in the nuclear genome, are likely of host origin. We also find that hundreds of vertically inherited genes (VGT) in this parasitic plant exhibit codon usage properties that are more similar to its host than to its closest relatives.ConclusionsOur results establish for the first time a substantive number of HGTs in a plant host-parasite system. The elevated rate of unidirectional host-to- parasite gene transfer raises the possibility that HGTs may provide a fitness benefit to Rafflesia for maintaining these genes. Finally, a similar convergence in codon usage of VGTs has been shown in microbes with high HGT rates, which may help to explain the increase of HGTs in these parasitic plants.