Pharmacogenomic discovery of genetically targeted cancer therapies optimized against clinical outcomes.

Despite the clinical success of dozens of genetically targeted cancer therapies, the vast majority of patients with tumors caused by loss-of-function (LoF) mutations do not have access to these treatments. This is primarily due to the challenge of developing a drug that treats a disease caused by the absence of a protein target. The success of PARP inhibitors has solidified synthetic lethality (SL) as a means to overcome this obstacle. Recent mapping of SL networks using pooled CRISPR-Cas9 screens is a promising approach for expanding this concept to treating cancers driven by additional LoF drivers. In practice, however, translating signals from cell lines, where these screens are typically conducted, to patient outcomes remains a challenge. We developed a pharmacogenomic (PGx) approach called "Clinically Optimized Driver Associated-PGx" (CODA-PGX) that accurately predicts genetically targeted therapies with clinical-stage efficacy in specific LoF driver contexts. Using approved targeted therapies and cancer drugs with available real-world evidence and molecular data from hundreds of patients, we discovered and optimized the key screening principles predictive of efficacy and overall patient survival. In addition to establishing basic technical conventions, such as drug concentration and screening kinetics, we found that replicating the driver perturbation in the right context, as well as selecting patients where those drivers are genuine founder mutations, were key to accurate translation. We used CODA-PGX to screen a diverse collection of clinical stage drugs and report dozens of novel LoF genetically targeted opportunities; many validated in xenografts and by real-world evidence. Notable examples include treating STAG2-mutant tumors with Carboplatin, SMARCB1-mutant tumors with Oxaliplatin, and TP53BP1-mutant tumors with Etoposide or Bleomycin.

Identifying tumorigenic non-coding mutations through altered cis-regulation.

Identification of non-coding mutations driving tumorigenesis requires alternative approaches to coding mutations. Enriched associations between mutated regulatory elements and altered cis-regulation in tumors are a promising approach to stratify candidate non-coding driver mutations. Here we provide a bioinformatics pipeline to mine data from the Cancer Genomic Commons (GDC) for such associations. The pipeline integrates RNA and whole-genome sequencing with genotyping data to reveal putative non-coding driver mutations by cancer type. For complete information on the generation and use of this protocol, please refer to Cheng et al. (2021).

Lineage-specific selection and the evolution of virulence in the Candida clade.

Candida albicans is the most common cause of systemic fungal infections in humans and is considerably more virulent than its closest known relative, Candida dubliniensis. To investigate this difference, we constructed interspecies hybrids and quantified mRNA levels produced from each genome in the hybrid. This approach systematically identified expression differences in orthologous genes arising from cis-regulatory sequence changes that accumulated since the two species last shared a common ancestor, some 10 million y ago. We documented many orthologous gene-expression differences between the two species, and we pursued one striking observation: All 15 genes coding for the enzymes of glycolysis showed higher expression from the C. albicans genome than the C. dubliniensis genome in the interspecies hybrid. This pattern requires evolutionary changes to have occurred at each gene; the fact that they all act in the same direction strongly indicates lineage-specific natural selection as the underlying cause. To test whether these expression differences contribute to virulence, we created a C. dubliniensis strain in which all 15 glycolysis genes were produced at modestly elevated levels and found that this strain had significantly increased virulence in the standard mouse model of systemic infection. These results indicate that small expression differences across a deeply conserved set of metabolism enzymes can play a significant role in the evolution of virulence in fungal pathogens.

Cis-regulatory mutations with driver hallmarks in major cancers.

Despite the recent availability of complete genome sequences of tumors from thousands of patients, isolating disease-causing (driver) non-coding mutations from the plethora of somatic variants remains challenging, and only a handful of validated examples exist. By integrating whole-genome sequencing, genetic data, and allele-specific gene expression from TCGA, we identified 320 somatic non-coding mutations that affect gene expression in cis (FDR<0.25). These mutations cluster into 47 cis-regulatory elements that modulate expression of their subject genes through diverse molecular mechanisms. We further show that these mutations have hallmark features of non-coding drivers; namely, that they preferentially disrupt transcription factor binding motifs, are associated with a selective advantage, increased oncogene expression and decreased tumor suppressor expression.

A one-step tRNA-CRISPR system for genome-wide genetic interaction mapping in mammalian cells.

Mapping genetic interactions in mammalian cells is limited due to technical obstacles. Here we describe a method called TCGI (tRNA-CRISPR for genetic interactions) to generate a high-efficient, barcode-free and scalable pairwise CRISPR libraries in mammalian cells for identifying genetic interactions. We have generated a genome- wide library to identify genes genetically interacting with TAZ in cell viability regulation. Validation of candidate synergistic genes reveals the screening accuracy of 85% and TAZ-MCL1 is characterized as combinational drug targets for non-small cell lung cancer treatments. TCGI has dramatically improved the current methods for mapping genetic interactions and screening drug targets for combinational therapies.

Dynamics of gene silencing during X inactivation using allele-specific RNA-seq.

BACKGROUND: During early embryonic development, one of the two X chromosomes in mammalian female cells is inactivated to compensate for a potential imbalance in transcript levels with male cells, which contain a single X chromosome. Here, we use mouse female embryonic stem cells (ESCs) with non-random X chromosome inactivation (XCI) and polymorphic X chromosomes to study the dynamics of gene silencing over the inactive X chromosome by high-resolution allele-specific RNA-seq. RESULTS: Induction of XCI by differentiation of female ESCs shows that genes proximal to the X-inactivation center are silenced earlier than distal genes, while lowly expressed genes show faster XCI dynamics than highly expressed genes. The active X chromosome shows a minor but significant increase in gene activity during differentiation, resulting in complete dosage compensation in differentiated cell types. Genes escaping XCI show little or no silencing during early propagation of XCI. Allele-specific RNA-seq of neural progenitor cells generated from the female ESCs identifies three regions distal to the X-inactivation center that escape XCI. These regions, which stably escape during propagation and maintenance of XCI, coincide with topologically associating domains (TADs) as present in the female ESCs. Also, the previously characterized gene clusters escaping XCI in human fibroblasts correlate with TADs. CONCLUSIONS: The gene silencing observed during XCI provides further insight in the establishment of the repressive complex formed by the inactive X chromosome. The association of escape regions with TADs, in mouse and human, suggests that TADs are the primary targets during propagation of XCI over the X chromosome.

Genetic conflict reflected in tissue-specific maps of genomic imprinting in human and mouse.

Genomic imprinting is an epigenetic process that restricts gene expression to either the maternally or paternally inherited allele. Many theories have been proposed to explain its evolutionary origin, but understanding has been limited by a paucity of data mapping the breadth and dynamics of imprinting within any organism. We generated an atlas of imprinting spanning 33 mouse and 45 human developmental stages and tissues. Nearly all imprinted genes were imprinted in early development and either retained their parent-of-origin expression in adults or lost it completely. Consistent with an evolutionary signature of parental conflict, imprinted genes were enriched for coexpressed pairs of maternally and paternally expressed genes, showed accelerated expression divergence between human and mouse, and were more highly expressed than their non-imprinted orthologs in other species. Our approach demonstrates a general framework for the discovery of imprinting in any species and sheds light on the causes and consequences of genomic imprinting in mammals.

Oct4 is required ~E7.5 for proliferation in the primitive streak.

Oct4 is a widely recognized pluripotency factor as it maintains Embryonic Stem (ES) cells in a pluripotent state, and, in vivo, prevents the inner cell mass (ICM) in murine embryos from differentiating into trophectoderm. However, its function in somatic tissue after this developmental stage is not well characterized. Using a tamoxifen-inducible Cre recombinase and floxed alleles of Oct4, we investigated the effect of depleting Oct4 in mouse embryos between the pre-streak and headfold stages, ~E6.0-E8.0, when Oct4 is found in dynamic patterns throughout the embryonic compartment of the mouse egg cylinder. We found that depletion of Oct4 ~E7.5 resulted in a severe phenotype, comprised of craniorachischisis, random heart tube orientation, failed turning, defective somitogenesis and posterior truncation. Unlike in ES cells, depletion of the pluripotency factors Sox2 and Oct4 after E7.0 does not phenocopy, suggesting that ~E7.5 Oct4 is required within a network that is altered relative to the pluripotency network. Oct4 is not required in extraembryonic tissue for these processes, but is required to maintain cell viability in the embryo and normal proliferation within the primitive streak. Impaired expansion of the primitive streak occurs coincident with Oct4 depletion ∼E7.5 and precedes deficient convergent extension which contributes to several aspects of the phenotype.

Identification of imprinted loci by transcriptome sequencing.

Enabled by high-throughput technologies that are capable of generating millions of sequencing reads, transcriptome sequencing is emerging as an important approach for mapping allelic imbalance (AI), where transcription is biased toward one allele in a diploid system. AI is identified by counting sequencing reads that map to genomic regions containing heterozygous SNPs, where the base identity of the SNP is used to distinguish allelic origin. Genomic imprinting is a special case of AI where bias is toward parental sex and can be identified by transcriptome sequencing of systems that represent reciprocally inherited loci. The focus of this protocol is on experimental design, analysis, and interpretation of genomic imprint discovery using whole transcriptome sequencing.

Global analysis of the evolution and mechanism of echinocandin resistance in Candida glabrata.

The evolution of drug resistance has a profound impact on human health. Candida glabrata is a leading human fungal pathogen that can rapidly evolve resistance to echinocandins, which target cell wall biosynthesis and are front-line therapeutics for Candida infections. Here, we provide the first global analysis of mutations accompanying the evolution of fungal drug resistance in a human host utilizing a series of C. glabrata isolates that evolved echinocandin resistance in a patient treated with the echinocandin caspofungin for recurring bloodstream candidemia. Whole genome sequencing identified a mutation in the drug target, FKS2, accompanying a major resistance increase, and 8 additional non-synonymous mutations. The FKS2-T1987C mutation was sufficient for echinocandin resistance, and associated with a fitness cost that was mitigated with further evolution, observed in vitro and in a murine model of systemic candidemia. A CDC6-A511G(K171E) mutation acquired before FKS2-T1987C(S663P), conferred a small resistance increase. Elevated dosage of CDC55, which acquired a C463T(P155S) mutation after FKS2-T1987C(S663P), ameliorated fitness. To discover strategies to abrogate echinocandin resistance, we focused on the molecular chaperone Hsp90 and downstream effector calcineurin. Genetic or pharmacological compromise of Hsp90 or calcineurin function reduced basal tolerance and resistance. Hsp90 and calcineurin were required for caspofungin-dependent FKS2 induction, providing a mechanism governing echinocandin resistance. A mitochondrial respiration-defective petite mutant in the series revealed that the petite phenotype does not confer echinocandin resistance, but renders strains refractory to synergy between echinocandins and Hsp90 or calcineurin inhibitors. The kidneys of mice infected with the petite mutant were sterile, while those infected with the HSP90-repressible strain had reduced fungal burden. We provide the first global view of mutations accompanying the evolution of fungal drug resistance in a human host, implicate the premier compensatory mutation mitigating the cost of echinocandin resistance, and suggest a new mechanism of echinocandin resistance with broad therapeutic potential.

Critical evaluation of imprinted gene expression by RNA-Seq: a new perspective.

In contrast to existing estimates of approximately 200 murine imprinted genes, recent work based on transcriptome sequencing uncovered parent-of-origin allelic effects at more than 1,300 loci in the developing brain and two adult brain regions, including hundreds present in only males or females. Our independent replication of the embryonic brain stage, where the majority of novel imprinted genes were discovered and the majority of previously known imprinted genes confirmed, resulted in only 12.9% concordance among the novel imprinted loci. Further analysis and pyrosequencing-based validation revealed that the vast majority of the novel reported imprinted loci are false-positives explained by technical and biological variation of the experimental approach. We show that allele-specific expression (ASE) measured with RNA-Seq is not accurately modeled with statistical methods that assume random independent sampling and that systematic error must be accounted for to enable accurate identification of imprinted expression. Application of a robust approach that accounts for these effects revealed 50 candidate genes where allelic bias was predicted to be parent-of-origin-dependent. However, 11 independent validation attempts through a range of allelic expression biases confirmed only 6 of these novel cases. The results emphasize the importance of independent validation and suggest that the number of imprinted genes is much closer to the initial estimates.

A quantitative atlas of polyadenylation in five mammals.

We developed PolyA-seq, a strand-specific and quantitative method for high-throughput sequencing of 3' ends of polyadenylated transcripts, and used it to globally map polyadenylation (polyA) sites in 24 matched tissues in human, rhesus, dog, mouse, and rat. We show that PolyA-seq is as accurate as existing RNA sequencing (RNA-seq) approaches for digital gene expression (DGE), enabling simultaneous mapping of polyA sites and quantitative measurement of their usage. In human, we confirmed 158,533 known sites and discovered 280,857 novel sites (FDR < 2.5%). On average 10% of novel human sites were also detected in matched tissues in other species. Most novel sites represent uncharacterized alternative polyA events and extensions of known transcripts in human and mouse, but primarily delineate novel transcripts in the other three species. A total of 69.1% of known human genes that we detected have multiple polyA sites in their 3'UTRs, with 49.3% having three or more. We also detected polyadenylation of noncoding and antisense transcripts, including constitutive and tissue-specific primary microRNAs. The canonical polyA signal was strongly enriched and positionally conserved in all species. In general, usage of polyA sites is more similar within the same tissues across different species than within a species. These quantitative maps of polyA usage in evolutionarily and functionally related samples constitute a resource for understanding the regulatory mechanisms underlying alternative polyadenylation.

Systematic detection of polygenic cis-regulatory evolution.

The idea that most morphological adaptations can be attributed to changes in the cis-regulation of gene expression levels has been gaining increasing acceptance, despite the fact that only a handful of such cases have so far been demonstrated. Moreover, because each of these cases involves only one gene, we lack any understanding of how natural selection may act on cis-regulation across entire pathways or networks. Here we apply a genome-wide test for selection on cis-regulation to two subspecies of the mouse Mus musculus. We find evidence for lineage-specific selection at over 100 genes involved in diverse processes such as growth, locomotion, and memory. These gene sets implicate candidate genes that are supported by both quantitative trait loci and a validated causality-testing framework, and they predict a number of phenotypic differences, which we confirm in all four cases tested. Our results suggest that gene expression adaptation is widespread and that these adaptations can be highly polygenic, involving cis-regulatory changes at numerous functionally related genes. These coordinated adaptations may contribute to divergence in a wide range of morphological, physiological, and behavioral phenotypes.

NSR-seq transcriptional profiling enables identification of a gene signature of Plasmodium falciparum parasites infecting children.

Malaria caused by Plasmodium falciparum results in approximately 1 million annual deaths worldwide, with young children and pregnant mothers at highest risk. Disease severity might be related to parasite virulence factors, but expression profiling studies of parasites to test this hypothesis have been hindered by extensive sequence variation in putative virulence genes and a preponderance of host RNA in clinical samples. We report here the application of RNA sequencing to clinical isolates of P. falciparum, using not-so-random (NSR) primers to successfully exclude human ribosomal RNA and globin transcripts and enrich for parasite transcripts. Using NSR-seq, we confirmed earlier microarray studies showing upregulation of a distinct subset of genes in parasites infecting pregnant women, including that encoding the well-established pregnancy malaria vaccine candidate var2csa. We also describe a subset of parasite transcripts that distinguished parasites infecting children from those infecting pregnant women and confirmed this observation using quantitative real-time PCR and mass spectrometry proteomic analyses. Based on their putative functional properties, we propose that these proteins could have a role in childhood malaria pathogenesis. Our study provides proof of principle that NSR-seq represents an approach that can be used to study clinical isolates of parasites causing severe malaria syndromes as well other blood-borne pathogens and blood-related diseases.

Genetic validation of whole-transcriptome sequencing for mapping expression affected by cis-regulatory variation.

BACKGROUND: Identifying associations between genotypes and gene expression levels using microarrays has enabled systematic interrogation of regulatory variation underlying complex phenotypes. This approach has vast potential for functional characterization of disease states, but its prohibitive cost, given hundreds to thousands of individual samples from populations have to be genotyped and expression profiled, has limited its widespread application. RESULTS: Here we demonstrate that genomic regions with allele-specific expression (ASE) detected by sequencing cDNA are highly enriched for cis-acting expression quantitative trait loci (cis-eQTL) identified by profiling of 500 animals in parallel, with up to 90% agreement on the allele that is preferentially expressed. We also observed widespread noncoding and antisense ASE and identified several allele-specific alternative splicing variants. CONCLUSION: Monitoring ASE by sequencing cDNA from as little as one sample is a practical alternative to expression genetics for mapping cis-acting variation that regulates RNA transcription and processing.

Global survey of escape from X inactivation by RNA-sequencing in mouse.

X inactivation equalizes the dosage of gene expression between the sexes, but some genes escape silencing and are thus expressed from both alleles in females. To survey X inactivation and escape in mouse, we performed RNA sequencing in Mus musculus x Mus spretus cells with complete skewing of X inactivation, relying on expression of single nucleotide polymorphisms to discriminate allelic origin. Thirteen of 393 (3.3%) mouse genes had significant expression from the inactive X, including eight novel escape genes. We estimate that mice have significantly fewer escape genes compared with humans. Furthermore, escape genes did not cluster in mouse, unlike the large escape domains in human, suggesting that expression is controlled at the level of individual genes. Our findings are consistent with the striking differences in phenotypes between female mice and women with a single X chromosome--a near normal phenotype in mice versus Turner syndrome and multiple abnormalities in humans. We found that escape genes are marked by the absence of trimethylation at lysine 27 of histone H3, a chromatin modification associated with genes subject to X inactivation. Furthermore, this epigenetic mark is developmentally regulated for some mouse genes.

A systematic characterization of Cwc21, the yeast ortholog of the human spliceosomal protein SRm300.

Cwc21 (complexed with Cef1 protein 21) is a 135 amino acid yeast protein that shares homology with the N-terminal domain of human SRm300/SRRM2, a large serine/arginine-repeat protein shown previously to associate with the splicing coactivator and 3'-end processing stimulatory factor, SRm160. Proteomic analysis of spliceosomal complexes has suggested a role for Cwc21 and SRm300 at the core of the spliceosome. However, specific functions for these proteins have remained elusive. In this report, we employ quantitative genetic interaction mapping, mass spectrometry of tandem affinity-purified complexes, and microarray profiling to investigate genetic, physical, and functional interactions involving Cwc21. Combined data from these assays support multiple roles for Cwc21 in the formation and function of splicing complexes. Consistent with a role for Cwc21 at the core of the spliceosome, we observe strong genetic, physical, and functional interactions with Isy1, a protein previously implicated in the first catalytic step of splicing and splicing fidelity. Together, the results suggest multiple functions for Cwc21/SRm300 in the splicing process, including an important role in the activation of splicing in association with Isy1.

Digital transcriptome profiling using selective hexamer priming for cDNA synthesis.

We developed a procedure for the preparation of whole transcriptome cDNA libraries depleted of ribosomal RNA from only 1 microg of total RNA. The method relies on a collection of short, computationally selected oligonucleotides, called 'not-so-random' (NSR) primers, to obtain full-length, strand-specific representation of nonribosomal RNA transcripts. In this study we validated the technique by profiling human whole brain and universal human reference RNA using ultra-high-throughput sequencing.