A benchmarked, high-efficiency prime editing platform for multiplexed dropout screening.

Prime editing installs precise edits into the genome with minimal unwanted byproducts, but low and variable editing efficiencies have complicated application of the approach to high-throughput functional genomics. Leveraging several recent advances, we assembled a prime editing platform capable of high-efficiency substitution editing across a set of engineered prime editing guide RNAs (epegRNAs) and corresponding target sequences (80% median intended editing). Then, using a custom library of 240,000 epegRNAs targeting >17,000 codons with 175 different substitution types, we benchmarked our platform for functional interrogation of small substitution variants (1-3 nucleotides) targeted to essential genes. Resulting data identified negative growth phenotypes for nonsense mutations targeted to ~8,000 codons, and comparing those phenotypes to results from controls demonstrated high specificity. We also observed phenotypes for synonymous mutations that disrupted splice site motifs at 3' exon boundaries. Altogether, we establish and benchmark a high-throughput prime editing approach for functional characterization of genetic variants with simple readouts from multiplexed experiments.

Improving prime editing with an endogenous small RNA-binding protein.

Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3' ends of CRISPR-Cas guide RNAs1. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3' ends of RNA polymerase III transcripts2. We found that La functionally interacts with the 3' ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.

Mapping the Genetic Interaction Network of PARP inhibitor Response.

Genetic interactions have long informed our understanding of the coordinated proteins and pathways that respond to DNA damage in mammalian cells, but systematic interrogation of the genetic network underlying that system has yet to be achieved. Towards this goal, we measured 147,153 pairwise interactions among genes implicated in PARP inhibitor (PARPi) response. Evaluating genetic interactions at this scale, with and without exposure to PARPi, revealed hierarchical organization of the pathways and complexes that maintain genome stability during normal growth and defined changes that occur upon accumulation of DNA lesions due to cytotoxic doses of PARPi. We uncovered unexpected relationships among DNA repair genes, including context-specific buffering interactions between the minimally characterized AUNIP and BRCA1-A complex genes. Our work thus establishes a foundation for mapping differential genetic interactions in mammalian cells and provides a comprehensive resource for future studies of DNA repair and PARP inhibitors.

Germline immunomodulatory expression quantitative trait loci (ieQTLs) associated with immune-related toxicity from checkpoint inhibition.

BACKGROUND: Immune checkpoint inhibition (ICI) has improved clinical outcomes for metastatic melanoma patients; however, 65-80% of patients treated with ICI experience immune-related adverse events (irAEs). Given the plausible link of irAEs with underlying host immunity, we explored whether germline genetic variants controlling the expression of 42 immunomodulatory genes were associated with the risk of irAEs in melanoma patients treated with the single-agent anti-CTLA-4 antibody ipilimumab (IPI). METHODS: We identified 42 immunomodulatory expression quantitative trait loci (ieQTLs) most significantly associated with the expression of 382 immune-related genes. These germline variants were genotyped in IPI-treated melanoma patients, collected as part of a multi-institutional collaboration. We tested the association of ieQTLs with irAEs in a discovery cohort of 95 patients, followed by validation in an additional 97 patients. RESULTS: We found that the alternate allele of rs7036417, a variant linked to increased expression of SYK, was strongly associated with an increased risk of grade 3-4 toxicity [odds ratio (OR) = 7.46; 95% confidence interval (CI) = 2.65-21.03; p = 1.43E-04]. This variant was not associated with response (OR = 0.90; 95% CI = 0.37-2.21; p = 0.82). CONCLUSION: We report that rs7036417 is associated with increased risk of severe irAEs, independent of IPI efficacy. SYK plays an important role in B-cell/T-cell expansion, and increased pSYK has been reported in patients with autoimmune disease. The association between rs7036417 and IPI irAEs in our data suggests a role of SYK overexpression in irAE development. These findings support the hypothesis that inherited variation in immune-related pathways modulates ICI toxicity and suggests SYK as a possible future target for therapies to reduce irAEs.

Mapping the genetic landscape of DNA double-strand break repair.

Cells repair DNA double-strand breaks (DSBs) through a complex set of pathways critical for maintaining genomic integrity. To systematically map these pathways, we developed a high-throughput screening approach called Repair-seq that measures the effects of thousands of genetic perturbations on mutations introduced at targeted DNA lesions. Using Repair-seq, we profiled DSB repair products induced by two programmable nucleases (Cas9 and Cas12a) in the presence or absence of oligonucleotides for homology-directed repair (HDR) after knockdown of 476 genes involved in DSB repair or associated processes. The resulting data enabled principled, data-driven inference of DSB end joining and HDR pathways. Systematic interrogation of this data uncovered unexpected relationships among DSB repair genes and demonstrated that repair outcomes with superficially similar sequence architectures can have markedly different genetic dependencies. This work provides a foundation for mapping DNA repair pathways and for optimizing genome editing across diverse modalities.

Tumor immunogenomic signatures improve a prognostic model of melanoma survival.

BACKGROUND: Tumor mutation burden (TMB) has been associated with melanoma immunotherapy (IT) outcomes, including survival. We explored whether combining TMB with immunogenomic signatures recently identified by The Cancer Genome Atlas (TCGA) can refine melanoma prognostic models of overall survival (OS) in patients not treated by IT. METHODS: Cox proportional-hazards (Cox PH) analysis was performed on 278 metastatic melanomas from TCGA not treated by IT. In a discovery and two validation cohorts Cox PH models assessed the interaction between TMB and 53 melanoma immunogenomic features to refine prediction of melanoma OS. RESULTS: Interferon-γ response (IFNγRes) and macrophage regulation gene signatures (MacReg) combined with TMB significantly associated with OS (p = 8.80E-14). We observed that patients with high TMB, high IFNγRes and high MacReg had significantly better OS compared to high TMB, low IFNγRes and low MacReg (HR = 2.8, p = 3.55E-08). This association was not observed in low TMB patients. CONCLUSIONS: We report a model combining TMB and tumor immune features that significantly improves prediction of melanoma OS, independent of IT. Our analysis revealed that patients with high TMB, high levels of IFNγRes and MacReg had significantly more favorable OS compared to high TMB patients with low IFNγRes and low MacReg. These findings may substantially improve current melanoma prognostic models.

Immunomodulatory germline variation associated with the development of multiple primary melanoma (MPM).

Multiple primary melanoma (MPM) has been associated with a higher 10-year mortality risk compared to patients with single primary melanoma (SPM). Given that 3-8% of patients with SPM develop additional primary melanomas, new markers predictive of MPM risk are needed. Based on the evidence that the immune system may regulate melanoma progression, we explored whether germline genetic variants controlling the expression of 41 immunomodulatory genes modulate the risk of MPM compared to patients with SPM or healthy controls. By genotyping these 41 variants in 977 melanoma patients, we found that rs2071304, linked to the expression of SPI1, was strongly associated with MPM risk reduction (OR = 0.60; 95% CI = 0.45-0.81; p = 0.0007) when compared to patients with SPM. Furthermore, we showed that rs6695772, a variant affecting expression of BATF3, is also associated with MPM-specific survival (HR = 3.42; 95% CI = 1.57-7.42; p = 0.0019). These findings provide evidence that the genetic variation in immunomodulatory pathways may contribute to the development of secondary primary melanomas and also associates with MPM survival. The study suggests that inherited host immunity may play an important role in MPM development.

Autoimmune genetic risk variants as germline biomarkers of response to melanoma immune-checkpoint inhibition.

Immune-checkpoint inhibition (ICI) treatments improve outcomes for metastatic melanoma; however, > 60% of treated patients do not respond to ICI. Current biomarkers do not reliably explain ICI resistance. Given the link between ICI and autoimmunity, we investigated if genetic susceptibility to autoimmunity modulates ICI efficacy. In 436 patients with metastatic melanoma receiving single line ICI or combination treatment, we tested 25 SNPs, associated with > 2 autoimmune diseases in recent genome-wide association studies, for modulation of ICI efficacy. We found that rs17388568-a risk variant for allergy, colitis and type 1 diabetes-was associated with increased anti-PD-1 response, with significance surpassing multiple testing adjustments (OR 0.26; 95% CI 0.12-0.53; p = 0.0002). This variant maps to a locus of established immune-related genes: IL2 and IL21. Our study provides first evidence that autoimmune genetic susceptibility may modulate ICI efficacy, suggesting that systematic testing of autoimmune risk loci could reveal personalized biomarkers of ICI response.

Primary Melanoma Histologic Subtype: Impact on Survival and Response to Therapy.

BACKGROUND: Two primary histologic subtypes, superficial spreading melanoma (SSM) and nodular melanoma (NM), comprise the majority of all cutaneous melanomas. NM is associated with worse outcomes, which have been attributed to increased thickness at presentation, and it is widely expected that NM and SSM would exhibit similar behavior once metastasized. Herein, we tested the hypothesis that primary histologic subtype is an independent predictor of survival and may impact response to treatment in the metastatic setting. METHODS: We examined the most recent Surveillance, Epidemiology, and End Results (SEER) cohort (n = 118 508) and the New York University (NYU) cohort (n = 1621) with available protocol-driven follow-up. Outcomes specified by primary histology were studied in both the primary and metastatic settings with respect to BRAF-targeted therapy and immunotherapy. We characterized known driver mutations and examined a 140-gene panel in a subset of NM and SSM cases using next-generation sequencing. All statistical tests were two-sided. RESULTS: NM was an independent risk factor for death in both the SEER (hazard ratio [HR] = 1.55, 95% confidence interval [CI] = 1.41 to 1.70, P < .001) and NYU (HR = 1.47, 95% CI = 1.05, 2.07, P = .03) cohorts, controlling for thickness, ulceration, stage, and other variables. In the metastatic setting, NM remained an independent risk factor for death upon treatment with BRAF-targeted therapy (HR = 3.33, 95% CI = 1.06 to 10.47, P = .04) but showed no statistically significant difference with immune checkpoint inhibition. NM was associated with a higher rate of NRAS mutation (P < .001), and high-throughput sequencing revealed NM-specific genomic alterations in NOTCH4, ANK3, and ZNF560, which were independently validated. CONCLUSIONS: Our data reveal distinct clinical and biological differences between NM and SSM that support revisiting the prognostic and predictive impact of primary histology subtype in the management of cutaneous melanoma.

Baseline antibody profiles predict toxicity in melanoma patients treated with immune checkpoint inhibitors.

BACKGROUND: Immune checkpoint inhibitors (anti-CTLA-4, anti-PD-1, or the combination) enhance anti-tumor immune responses, yielding durable clinical benefit in several cancer types, including melanoma. However, a subset of patients experience immune-related adverse events (irAEs), which can be severe and result in treatment termination. To date, no biomarker exists that can predict development of irAEs. METHODS: We hypothesized that pre-treatment antibody profiles identify a subset of patients who possess a sub-clinical autoimmune phenotype that predisposes them to develop severe irAEs following immune system disinhibition. Using a HuProt human proteome array, we profiled baseline antibody levels in sera from melanoma patients treated with anti-CTLA-4, anti-PD-1, or the combination, and used support vector machine models to identify pre-treatment antibody signatures that predict irAE development. RESULTS: We identified distinct pre-treatment serum antibody profiles associated with severe irAEs for each therapy group. Support vector machine classifier models identified antibody signatures that could effectively discriminate between toxicity groups with > 90% accuracy, sensitivity, and specificity. Pathway analyses revealed significant enrichment of antibody targets associated with immunity/autoimmunity, including TNFα signaling, toll-like receptor signaling and microRNA biogenesis. CONCLUSIONS: Our results provide the first evidence supporting a predisposition to develop severe irAEs upon immune system disinhibition, which requires further independent validation in a clinical trial setting.

Are Fireworms Venomous? Evidence for the Convergent Evolution of Toxin Homologs in Three Species of Fireworms (Annelida, Amphinomidae).

Amphinomids, more commonly known as fireworms, are a basal lineage of marine annelids characterized by the presence of defensive dorsal calcareous chaetae, which break off upon contact. It has long been hypothesized that amphinomids are venomous and use the chaetae to inject a toxic substance. However, studies investigating fireworm venom from a morphological or molecular perspective are scarce and no venom gland has been identified to date, nor any toxin characterized at the molecular level. To investigate this question, we analyzed the transcriptomes of three species of fireworms-Eurythoe complanata, Hermodice carunculata, and Paramphinome jeffreysii-following a venomics approach to identify putative venom compounds. Our venomics pipeline involved de novo transcriptome assembly, open reading frame, and signal sequence prediction, followed by three different homology search strategies: BLAST, HMMER sequence, and HMMER domain. Following this pipeline, we identified 34 clusters of orthologous genes, representing 13 known toxin classes that have been repeatedly recruited into animal venoms. Specifically, the three species share a similar toxin profile with C-type lectins, peptidases, metalloproteinases, spider toxins, and CAP proteins found among the most highly expressed toxin homologs. Despite their great diversity, the putative toxins identified are predominantly involved in three major biological processes: hemostasis, inflammatory response, and allergic reactions, all of which are commonly disrupted after fireworm stings. Although the putative fireworm toxins identified here need to be further validated, our results strongly suggest that fireworms are venomous animals that use a complex mixture of toxins for defense against predators.

Apolipoprotein L1 risk variants associate with prevalent atherosclerotic disease in African American systemic lupus erythematosus patients.

OBJECTIVE: Atherosclerosis is exaggerated in African American (AA) systemic lupus erythematosus (SLE) patients, with doubled cardiovascular disease (CVD) risk compared to White patients. The extent to which common Apolipoprotein L1 (APOL1) risk alleles (RA) contribute to this trend is unknown. This retrospective cohort study assessed prevalent atherosclerotic disease across APOL1 genotypes in AA SLE patients. METHODS: One hundred thirteen AA SLE subjects were APOL1-genotyped and stratified as having: zero risk alleles, one risk allele, or two risk alleles. Chart review assessed CVD manifestations including abdominal aortic aneurysm, angina, carotid artery disease, coronary artery disease, myocardial infarction, peripheral vascular disease, stroke, and vascular calcifications. Associations between the genotypes and a composite endpoint defined as one or more CVD manifestations were calculated using logistic regression. Symptomatic atherosclerotic disease, excluding incidental vascular calcifications, was also assessed. RESULTS: The 0-risk-allele, 1-risk-allele and 2-risk-allele groups, respectively, comprised 34%, 53%, and 13% of the cohort. Respectively, 13.2%, 41.7%, and 60.0% of the 0-risk allele, 1-risk-allele, and 2-risk-allele groups met the composite endpoint of atherosclerotic CVD (p = 0.001). Adjusting for risk factors-including smoking, ESRD, BMI >25 and hypertension-we observed an association between carrying one or more RA and atherosclerotic CVD (OR = 7.1; p = 0.002). For symptomatic disease, the OR was 3.5 (p = 0.02). In a time-to-event analysis, the proportion of subjects free from the composite primary endpoint, symptomatic atherosclerotic CVD, was higher in the 0-risk-allele group compared to the 1-risk-allele and 2-risk-allele groups (χ2 = 6.5; p = 0.04). CONCLUSIONS: Taken together, the APOL1 RAs associate with prevalent atherosclerotic CVD in this cohort of AA SLE patients, perhaps reflecting a potentiating effect of SLE on APOL1-related cardiovascular phenotypes.

From Mollusks to Medicine: A Venomics Approach for the Discovery and Characterization of Therapeutics from Terebridae Peptide Toxins.

Animal venoms comprise a diversity of peptide toxins that manipulate molecular targets such as ion channels and receptors, making venom peptides attractive candidates for the development of therapeutics to benefit human health. However, identifying bioactive venom peptides remains a significant challenge. In this review we describe our particular venomics strategy for the discovery, characterization, and optimization of Terebridae venom peptides, teretoxins. Our strategy reflects the scientific path from mollusks to medicine in an integrative sequential approach with the following steps: (1) delimitation of venomous Terebridae lineages through taxonomic and phylogenetic analyses; (2) identification and classification of putative teretoxins through omics methodologies, including genomics, transcriptomics, and proteomics; (3) chemical and recombinant synthesis of promising peptide toxins; (4) structural characterization through experimental and computational methods; (5) determination of teretoxin bioactivity and molecular function through biological assays and computational modeling; (6) optimization of peptide toxin affinity and selectivity to molecular target; and (7) development of strategies for effective delivery of venom peptide therapeutics. While our research focuses on terebrids, the venomics approach outlined here can be applied to the discovery and characterization of peptide toxins from any venomous taxa.