Large-scale Functional Screening Research Articles

Establishment and application of a root wounding-immersion method for efficient virus-induced gene silencing in plants.

In the post-genomic era, virus-induced gene silencing (VIGS) has played an important role in research on reverse genetics in plants. Commonly used Agrobacterium-mediated VIGS inoculation methods include stem scratching, leaf infiltration, use of agrodrench, and air-brush spraying. In this study, we developed a root wounding-immersion method in which 1/3 of the plant root (length) was cut and immersed in a tobacco rattle virus (TRV)1:TRV2 mixed solution for 30 min. We optimized the procedure in Nicotiana benthamiana and successfully silenced N. benthamiana, tomato (Solanum lycopersicum), pepper (Capsicum annuum L.), eggplant (Solanum melongena), and Arabidopsis thaliana phytoene desaturase (PDS), and we observed the movement of green fluorescent protein (GFP) from the roots to the stem and leaves. The silencing rate of PDS in N. benthamiana and tomato was 95-100%. In addition, we successfully silenced two disease-resistance genes, SITL5 and SITL6, to decrease disease resistance in tomatoes (CLN2037E). The root wounding-immersion method can be used to inoculate large batches of plants in a short time and with high efficiency, and fresh bacterial infusions can be reused several times. The most important aspect of the root wounding-immersion method is its application to plant species susceptible to root inoculation, as well as its ability to inoculate seedlings from early growth stages. This method offers a means to conduct large-scale functional genome screening in plants.

The Functional Circular RNA Screening via RfxCas13d/BSJ-gRNA System.

Although discovered decades ago, functions of circular RNAs (circRNAs) produced from exon(s) back-splicing of pre-mRNAs have only been unveiled recently. As circRNAs share overlapping sequences with their cognate linear RNAs, except for the back-splicing junction sites, it is difficult to distinguish circRNAs from cognate mRNAs in functional studies. In this chapter, we describe a programmable method for the large-scale functional circRNA screening based on the RNA-guided, RNA-targeting CRISPR-Cas13 (RfxCas13d) system. This method can be applied both in vivo and in cell to explore highly expressed circRNAs that may influence cell growth, either under natural conditions or in response to environmental stimulation, without disturbing cognate linear mRNAs.

Abstract B143: HRO761, a first-in-class, clinical stage WRN inhibitor with potent preclinical anti-tumor activity in MSIhigh models

Abstract Colorectal carcinoma (CRC) as well as other indications that have lost competence in mismatch repair and have a phenotype known as microsatellite instability (MSI), remain a significant unmet medical need. Large-scale functional genomics screens across cell line panels have identified the Werner Syndrome RecQ helicase (WRN) as being synthetic lethal with MSIhigh cancers. Thus, WRN inhibitors may offer a new therapeutic avenue in MSIhigh cancers. We report the preclinical characteristics of HRO761, a first-in-class, highly potent and selective inhibitor of WRN helicase, with optimized drug-like properties. We evaluated the pharmacokinetics and pharmacodynamics of HRO761 when administered orally once daily in MSIhigh and also assessed the efficacy as single agent across cell (CDX) and patient-derived (PDX) xenografts as well as in combination with irinotecan, a prodrug derivative of camptothecin which acts as topoisomerase I inhibitor. Daily oral administration of HRO761 was highly efficacious across the CDX and PDX models evaluated with a disease control rate of 70% across all indications. In the SW48 CDX model, tumor regression was sustained for 2 months at the highest dose followed by slow relapse. HRO761 also induced WRN degradation, activated the DNA damage response and induced p53 target genes in a dose dependent manner. In vitro combination with irinotecan deepened sensitivity to HRO761 with a more sustained response in MSIhigh CRC cell lines. Combining administration of HRO761 daily with irinotecan weekly was highly efficacious and beneficial over single agent in CDX and PDX models. In CDX models, the combination showed a robust benefit, translating into homogeneous, sustained regression leading to a dose-dependent tumor growth delay even after treatment discontinuation. Overall, we demonstrate that the novel, selective, first-in-class WRN inhibitor HRO761 is a promising therapeutic approach for the treatment of MSIhigh cancer patients either as single agent or in combination with irinotecan. Clinical development is currently ongoing to assess the safety, tolerability and preliminary anti-tumor activity in patients with MSIhigh colorectal cancer and other MSIhigh solid tumors [clinicaltrials.gov NCT05838768]. Citation Format: Stephane Ferretti, Isabel Jaco, Andrea Decker, Christelle Hemmerlin, Clemens Scheufler, Cornelia Quadt, Dario Sterker, Elena Gavioli, Eloisa Jimenez Nunez, Ernesta Dammassa, Fanny Schaeffer, Genevieve Albrecht, Giorgia Clementi, Hansjoerg Martus, Jacques Hamon, Juergen Hinrichs, Laurent Laborde, Marion Dourdoigne, Michele Moschetta, Ramona Stump, Rita Andraos-Rey, Sarah Welly, Stephanie Barbe, Vincent Romanet, Markus Reschke, Ruben De Kanter, Michael Jensen, Henrik Moebitz, Marta Cortes Cros. HRO761, a first-in-class, clinical stage WRN inhibitor with potent preclinical anti-tumor activity in MSIhigh models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B143.

Rapid Identification of High-Temperature Responsive Genes Using Large-Scale Yeast Functional Screening System in Potato.

As the third largest global food crop, potato plays an important role in ensuring food security. However, it is particularly sensitive to high temperatures, which seriously inhibits its growth and development, thereby reducing yield and quality and severely limiting its planting area. Therefore, rapid, and high-throughput screening for high-temperature response genes is highly significant for analyzing potato high-temperature tolerance molecular mechanisms and cultivating new high-temperature-tolerant potato varieties. We screened genes that respond to high temperature by constructing a potato cDNA yeast library. After high-temperature treatment at 39 °C, the yeast library was subjected to high-throughput sequencing, and a total of 1931 heat resistance candidate genes were screened. Through GO and KEGG analysis, we found they were mainly enriched in "photosynthesis" and "response to stimuli" pathways. Subsequently, 12 randomly selected genes were validated under high temperature, drought, and salt stress using qRT-PCR. All genes were responsive to high temperature, and most were also induced by drought and salt stress. Among them, five genes ectopically expressed in yeast enhance yeast's tolerance to high temperatures. We provide numerous candidate genes for potato response to high temperature stress, laying the foundation for subsequent analysis of the molecular mechanism of potato response to high temperature.

Cancer lineage-specific regulation of YAP responsive elements revealed through large-scale functional epigenomic screens

YAP is a key transcriptional co-activator of TEADs, it regulates cell growth and is frequently activated in cancer. In Malignant Pleural Mesothelioma (MPM), YAP is activated by loss-of-function mutations in upstream components of the Hippo pathway, while, in Uveal Melanoma (UM), YAP is activated in a Hippo-independent manner. To date, it is unclear if and how the different oncogenic lesions activating YAP impact its oncogenic program, which is particularly relevant for designing selective anti-cancer therapies. Here we show that, despite YAP being essential in both MPM and UM, its interaction with TEAD is unexpectedly dispensable in UM, limiting the applicability of TEAD inhibitors in this cancer type. Systematic functional interrogation of YAP regulatory elements in both cancer types reveals convergent regulation of broad oncogenic drivers in both MPM and UM, but also strikingly selective programs. Our work reveals unanticipated lineage-specific features of the YAP regulatory network that provide important insights to guide the design of tailored therapeutic strategies to inhibit YAP signaling across different cancer types.

An unbiased AAV-STARR-seq screen revealing the enhancer activity map of genomic regions in the mouse brain in vivo

Enhancers are important cis-regulatory elements controlling cell-type specific expression patterns of genes. Furthermore, combinations of enhancers and minimal promoters are utilized to construct small, artificial promoters for gene delivery vectors. Large-scale functional screening methodology to construct genomic maps of enhancer activities has been successfully established in cultured cell lines, however, not yet applied to terminally differentiated cells and tissues in a living animal. Here, we transposed the Self-Transcribing Active Regulatory Region Sequencing (STARR-seq) technique to the mouse brain using adeno-associated-viruses (AAV) for the delivery of a highly complex screening library tiling entire genomic regions and covering in total 3 Mb of the mouse genome. We identified 483 sequences with enhancer activity, including sequences that were not predicted by DNA accessibility or histone marks. Characterizing the expression patterns of fluorescent reporters controlled by nine candidate sequences, we observed differential expression patterns also in sparse cell types. Together, our study provides an entry point for the unbiased study of enhancer activities in organisms during health and disease.

Large-scale microRNA functional high-throughput screening identifies miR-515-3p and miR-519e-3p as inducers of human cardiomyocyte proliferation

Ischemic cardiomyopathy, driven by loss of cardiomyocytes and inadequate proliferative response, persists to be a major global health problem. Using a functional high-throughput screening, we assessed differential proliferative potential of 2019 miRNAs after transient hypoxia by transfecting both miR-inhibitor and miR-mimic libraries in human iPSC-CM. Whereas miR-inhibitors failed to enhance EdU uptake, overexpression of 28 miRNAs substantially induced proliferative activity in hiPSC-CM, with an overrepresentation of miRNAs belonging to the primate-specific C19MC-cluster. Two of these miRNAs, miR-515-3p and miR-519e-3p, increased markers of early and late mitosis, indicative of cell division, and substantially alter signaling pathways relevant for cardiomyocyte proliferation in hiPSC-CM.

Multi-omics reveals the key and specific miRNA-mRNA modules underlying salt tolerance in wild emmer wheat (Triticum dicoccoides L.)

BackgroundSalt stress is one of the most destructive environmental factors limiting crop growth and development. MicroRNAs (miRNAs) are a class of conserved endogenous small non-coding RNAs, playing the crucial role in regulating salt response and tolerance in plants. However, the miRNAs in wild emmer wheat, especially the key and specific salt-responsive miRNAs are not well studied.ResultsHere, we performed small RNA, transcriptome, and degradome sequencing of both of salt-tolerance (ST) and salt-sensitive (SS) wild emmer genotypes to identify the miRNA-mRNA modules associating with salt tolerance. Totally, 775 miRNAs, including 361 conserved known miRNAs and 414 novel miRNAs were detected. Differential expression analysis identified 93 salt-responsive miRNAs under salt stress. Combined with RNA-seq and degradome sequencing analysis, 224 miRNA-mRNA modules displayed the complete opposite expression trends between ST and SS genotypes, most of which functionally enriched into ROS homeostasis maintaining, osmotic pressure modulating, and root growth and development. Finally, the qRT-PCR and a large-scale yeast functional screening were also performed to initially validate the expression pattern and function of candidate genes.ConclusionsThis study reported the key and specific miRNA-mRNA modules associated with salt tolerance in wild emmer, which lay the foundation for improving the salt tolerance in cultivated emmer and bread wheat through miRNA engineering approach.

WT1 and DNMT3A play essential roles in the growth of certain patient AML cells in mice

WT1 and DNMT3A play essential roles in the growth of certain patient AML cells in mice

A large-scale functional high-throughput screening identifies miR-515 and miR-519e as potent inducers of human iPSC-cardiomyocyte proliferation

Abstract Introduction Ischemic heart failure persists as a global health problem despite optimized medical and adjunctive device therapies. Loss of cardiomyocytes in the absence of a proliferative response comprise a major contributor to pathological remodeling and death in this patient population. Experimental studies have shown that microRNAs (miRNAs) may be used as a therapeutic option to reinduce adult cardiomyocyte proliferation. Purpose This study thought to evaluate proliferative potential in human cardiomyocytes after overexpression and inhibition of 2019 miRNAs. Methods To identify miRNAs that regulate cardiomyocyte proliferation, we performed functional high-throughput screenings in human iPSC-derived cardiomyocytes (hiPSC-CM) after transient hypoxia. Herein, 2019 miRNA-mimics for overexpression and 2019 anti-miRs for inhibition were individually transfected to examine EdU-incorporation in hiPSC-CM. MiR-mimic-515 and miR-mimic-519e that induced the highest EdU-uptake, were further assessed by immunostaining and molecular methods for markers indicative of early and late mitosis. In addition, RNA-Sequencing in hiPSC-CM after overexpression of miR-515 and miR-519e was performed to examine differential gene expression and miRNA-modulated pathways involved in cardiomyocyte proliferation. Results Using a functional high-throughput screening, we assessed differential proliferative potential of 2019 miRNAs after transient hypoxia by transfecting both miR-inhibitor and miR-mimic libraries in human iPSC-derived cardiomyocytes (hiPSC-CM). Overexpression of 28 miRNAs substantially induced proliferative activity in hiPSC-CM, with an overrepresentation of miRNAs belonging to the C19MC-cluster and adjacent miR-371–373 family. Two of these miRNAs, miR-515 and miR-519e increased markers of early and late mitosis, with an additive cardiomyocyte turnover after transient hypoxia and substantially increased Aurora B-kinase activity in midbodies, indicative of cell division. These findings were supported by molecular studies using qRT-PCR, Western blot, and RNA-Sequencing after overexpression of miR-515 and miR-519e showing substantial alterations of signaling pathways relevant for cardiomyocytes proliferation in human iPSC-CM. Conclusion Collectively, these results support a critical role of miR-515 and miR-519e for induction of proliferation in human cardiomyocytes under hypoxic conditions, such as present in patients with ischemia-driven cardiomyopathy. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): This work was supported by the German Centre for Cardiovascular Research (DZHK), Deutsche Stiftung für Herzforschung (DSHF) and OPO Foundation.

Long non-coding RNA DARS-AS1 promotes tumor progression by directly suppressing PACT-mediated cellular stress

Cancer cells evolve various mechanisms to overcome cellular stresses and maintain progression. Protein kinase R (PKR) and its protein activator (PACT) are the initial responders in monitoring diverse stress signals and lead to inhibition of cell proliferation and cell apoptosis in consequence. However, the regulation of PACT-PKR pathway in cancer cells remains largely unknown. Herein, we identify that the long non-coding RNA (lncRNA) aspartyl-tRNA synthetase antisense RNA 1 (DARS-AS1) is directly involved in the inhibition of the PACT-PKR pathway and promotes the proliferation of cancer cells. Using large-scale CRISPRi functional screening of 971 cancer-associated lncRNAs, we find that DARS-AS1 is associated with significantly enhanced proliferation of cancer cells. Accordingly, knocking down DARS-AS1 inhibits cell proliferation of multiple cancer cell lines and promotes cancer cell apoptosis in vitro and significantly reduces tumor growth in vivo. Mechanistically, DARS-AS1 directly binds to the activator domain of PACT and prevents PACT-PKR interaction, thereby decreasing PKR activation, eIF2α phosphorylation and inhibiting apoptotic cell death. Clinically, DARS-AS1 is broadly expressed across multiple cancers and the increased expression of this lncRNA indicates poor prognosis. This study elucidates the lncRNA DARS-AS1 directed cancer-specific modulation of the PACT-PKR pathway and provides another target for cancer prognosis and therapeutic treatment.

Characterisation of a Novel Cell Line (ICR-SS-1) Established from a Patient-Derived Xenograft of Synovial Sarcoma.

Synovial sarcoma is a rare translocation-driven cancer with poor survival outcomes, particularly in the advanced setting. Previous synovial sarcoma preclinical studies have relied on a small panel of cell lines which suffer from the limitation of genomic and phenotypic drift as a result of being grown in culture for decades. Patient-derived xenografts (PDX) are a valuable tool for preclinical research as they retain many histopathological features of their originating human tumour; however, this approach is expensive, slow, and resource intensive, which hinders their utility in large-scale functional genomic and drug screens. To address some of these limitations, in this study, we have established and characterised a novel synovial sarcoma cell line, ICR-SS-1, which is derived from a PDX model and is amenable to high-throughput drug screens. We show that ICR-SS-1 grows readily in culture, retains the pathognomonic SS18::SSX1 fusion gene, and recapitulates the molecular features of human synovial sarcoma tumours as shown by proteomic profiling. Comparative analysis of drug response profiles with two other established synovial sarcoma cell lines (SYO-1 and HS-SY-II) finds that ICR-SS-1 harbours intrinsic resistance to doxorubicin and is sensitive to targeted inhibition of several oncogenic pathways including the PI3K-mTOR pathway. Collectively, our studies show that the ICR-SS-1 cell line model may be a valuable preclinical tool for studying the biology of anthracycline-resistant synovial sarcoma and identifying new salvage therapies following failure of doxorubicin.

Screening circular RNAs with functional potential using the RfxCas13d/BSJ-gRNA system.

Circular RNAs (circRNAs) are covalently enclosed, single-stranded RNAs produced by back-splicing of pre-mRNA exons that have recently emerged as an important class of molecules in gene expression regulation. circRNAs share overlapping sequences with their cognate linear mRNAs except the back-splicing junction (BSJ) sites. This feature makes it difficult to discriminate between the functions of circRNAs and their cognate mRNAs. We previously reported that the programmable RNA-guided, RNA-targeting CRISPR-Cas13 (RfxCas13d) system effectively and specifically discriminates circRNAs from mRNAs by using guide RNAs (gRNAs) targeting sequences across BSJ sites. Here, we describe a detailed protocol based on this RfxCas13d/BSJ-gRNA system for large-scale functional circRNA screening in human cell lines. The protocol includes gRNA library design, construction and transduction, analysis of screening results and validation of functional circRNA candidates. In total, it takes ~3-4 months of collaborative work between a well-trained molecular biologist and a bioinformatic expert. This protocol can be applied both in cells and in vivo to identify highly expressed circRNAs affecting cell growth, either in unperturbed conditions or under environmental stimulation, without disturbing their cognate linear mRNAs.

Targeting the DNA damage response beyond poly(ADP-ribose) polymerase inhibitors: novel agents and rational combinations.

Poly(ADP-ribose) polymerase (PARP) inhibitors have transformed treatment paradigms in multiple cancer types defined by homologous recombination deficiency (HRD) and have become the archetypal example of synthetic lethal targeting within the DNA damage response (DDR). Despite this success, primary and acquired resistance to PARP inhibition inevitability threaten the efficacy and durability of response to these drugs. Beyond PARP inhibitors, recent advances in large-scale functional genomic screens have led to the identification of a steadily growing list of genetic dependencies across the DDR landscape. This has led to a wide array of novel synthetic lethal targets and corresponding inhibitors, which hold promise to widen the application of DDR inhibitors beyond HRD and potentially address PARP inhibitor resistance. In this review, we describe key synthetic lethal interactions that have been identified across the DDR landscape, summarize the early phase clinical development of the most promising DDR inhibitors, and highlight relevant combinations of DDR inhibitors with chemotherapy and other novel cancer therapies, which are anticipated to make an impact in rationally selected patient populations. The DDR landscape holds multiple opportunities for synthetic lethal targeting with multiple novel DDR inhibitors being evaluated on early phase clinical trials. Key challenges remain in optimizing the therapeutic window of ATR and WEE1 inhibitors as monotherapy and in combination approaches.

Abstract 4035: Driver-gene dependencies reveal clinically actionable drug repositioning opportunities

Abstract Cancer cell lines have numerous characteristics that make them favorable pre-clinical research models, yet they are notoriously poor at predicting drug response in the clinic. Here we sought to investigate the utility of synthetic lethality (SL) interactions discovered from large-scale CRISPR functional screens (i.e. the BROAD and Sanger Cancer Dependency Maps or "DepMap") as predictors of targets that validate in patients. Mutual exclusivity, the phenomenon where two genes are rarely mutated together in the same tumor, is a powerful clinical-stage readout that can be caused by synthetic lethality. We found that SL interactions discovered in DepMap are significantly more likely to be mutually exclusive in TCGA when they include a driver (tumor-suppressor/oncogene). These SL interactions represent high-value targeting opportunities with the advantage of clear patient selection criteria based on their driver mutation status. In an effort to identify drugs that target these proteins as potential repurposing opportunities, we found that pharmacogenomic inhibition rarely invokes the same target dependencies as a genetic deletion of the drug target. Nonetheless, we identified several dozen "clean" drugs with potential for repositioning and validated the top candidates in PDx. Although tumours are more heterogenous than cancer cell lines, we show that cell line viability readouts linked to single-gene/drug perturbations can yield accurate predictions of clinical efficacy when tied to tumor-driver biology. Citation Format: Tomas Babak, Michael Vermeulen, Doris Coto Villa, Andrew Craig. Driver-gene dependencies reveal clinically actionable drug repositioning opportunities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4035.

In This Issue

In This Issue

An In Vivo CRISPR Screening Platform to Identify New Therapeutic Targets in AML

First-generation, large-scale functional genomic screens have revealed hundreds of potential genetic vulnerabilities in acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because these large-scale genetic screens were primarily performed in vitro in established AML cell lines, their translational relevance has been debated. Therefore, we established a protocol for CRISPR screening in orthotopic xenograft models of human AML, including patient-derived-xenograft (PDX) models that are tractable for CRISPR-editing.We first defined experimental conditions necessary for an optimal in vivo screen via barcoding experiments. We determined that sub-lethal irradiation was necessary for improved barcode representation in bone marrow and to reduce mouse-to-mouse variation. Moreover, it was critical to combine samples from multiple mice to achieve complete in vivo library representation. Next, using the Broad DepMap and other publicly available functional genomic screen data, we identified 200 genes that were stronger dependencies in AML cell lines compared to all other cancer types screened. Using this list, we created a secondary library and performed parallel in vivo and in vitro screens using the MV4-11 and U937 cell lines and a PDX model. In vitro and in vivo hits were surprisingly well correlated, although a modest number of targets did not score well in vivo. Notably, dependencies identified across AML cell line models were strongly recapitulated in the PDX model, validating the application of AML cell lines for dependency discovery.Our in vivo screens nominated the mitochondria-localized RING-type ubiquitin E3 ligase MARCH5 as a potential therapeutic target in AML. Using CRISPR, we first validated this in vitro dependency on MARCH5 and determined that MARCH5 is a critical guardian to prevent apoptosis in AML. MARCH5 depletion activates the canonical mitochondrial apoptosis pathway in a BAX/BAK1-dependent manner. Multiple genome-wide screens revealed that a dependency on MARCH5 is strongly correlated with a dependency on MCL1, but not other anti-apoptotic BCL2-family members, across the AML cell lines in the screen. As observed with MCL1 inhibition, MARCH5 depletion sensitized AML cells to venetoclax, a BCL2-specific inhibitor FDA-approved in combination with a hypomethylating agent for the treatment of older adults with AML. Importantly, MARCH5 depletion diminished the venetoclax resistance induced by MCL1 overexpression but not that caused by BCLXL overexpression. Altogether, these results suggest that MARCH5 is required for maintaining MCL1 activity specifically.Since there are no small molecule inhibitors directed against MARCH5, we deployed a dTAG system as an approximation of pharmacological inhibition. This approach uses a hetero-bifunctional small molecule that binds the FKBP12 F36V-fused MARCH5 and the E3 ligase VHL, leading to the ubiquitination and proteasome-mediated degradation of the fusion protein. dTAG-MARCH5 cells were established via deleting endogenous MARCH5 by CRISPR and expressing exogenous FKBP-tagged MARCH5 protein. MARCH5 degradation with the dTAG molecule dTAG V-1 markedly impaired cell growth in vitro. Additionally, we demonstrated the utility of dTAG system in vivo using a PDX model. The combination treatment of dTAG V-1 and venetoclax elicited a much stronger anti-leukemic effect compared to the treatment with only venetoclax or dTAG V-1, further highlighting MARCH5 as a promising synergistic target for enhancing the efficacy of venetoclax in AML.Taken together, our in vivo screening approach, coupled with CRISPR-competent PDX models and dTAG-directed protein degradation, constitute a useful platform for prioritizing AML targets emerging from in vitro screens to serve as the starting point for therapy development. DisclosuresDharia: Genentech: Current Employment. Piccioni: Merck Research Laboratories: Current Employment. Stegmaier: Bristol Myers Squibb: Consultancy; KronosBio: Consultancy; AstraZeneca: Consultancy; Auron Therapeutics: Consultancy, Current equity holder in publicly-traded company; Novartis: Research Funding.

Abstract 886: Prediction of outcome in Li-Fraumeni Syndrome: Development of an integrative function-based prognostic model

Abstract Li-Fraumeni Syndrome (LFS) is a highly penetrant familial cancer disorder caused by germline TP53 mutations. Hundreds of different germline TP53 mutations with a wide gradient of functional consequences have been found in association with LFS, which has a remarkably diverse clinical spectrum in terms of tumor type, age of onset, penetrance, and survival rates. Clinical tools for prognostication and risk stratification are needed to guide clinical decision making. We performed an integrated analysis combining five large-scale mutant p53 functional screens to identify three functionally distinct groups of mutations. Combined functional scores measuring multiple parameters (loss-of-function, dominant negative activity, growth inhibition, oligomeric status, and transcriptional activity) were used to classify mutations as high, intermediate, or low pathogenicity. Patients identified with germline TP53 mutations at The Hospital for Sick Children and other institutes (174 patients from 50 families) provided the discovery set. An external validation cohort included 1674 patients from 1522 families with germline TP53 mutations collected from the International Agency for Research on Cancer database. Our mutation classifications stratified carriers into three distinct risk groups with significantly different overall survival outcomes in the discovery (P=0.02) and validation cohorts (P<0.0001). In the discovery dataset, patients in the high-risk group had considerably shorter median life expectancy (28 years; n=21) compared to the intermediate-risk group (61 years; n=51) and low-risk group (median survival not reached; n=10). On average, 92% (24/26) of patients in the high-risk group developed tumors compared to only 63% (32/51) in the low-risk group (P=0.005). This model remained significant when applied to the validation cohort. In conclusion, patients at increased risk of cancer mortality can be identified to guide treatment and cancer surveillance strategies. Citation Format: Nicholas Fischer, David Malkin. Prediction of outcome in Li-Fraumeni Syndrome: Development of an integrative function-based prognostic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 886.

Improving the design of an oxidative stress sensing biosensor in yeast

ABSTRACTTranscription factor (TF)-based biosensors have proven useful for increasing biomanufacturing yields, large-scale functional screening, and in environmental monitoring. Most yeast TF-based biosensors are built from natural promoters, resulting in large DNA parts retaining considerable homology to the host genome, which can complicate biological engineering efforts. There is a need to explore smaller, synthetic biosensors to expand the options for regulating gene expression in yeast. Here, we present a systematic approach to improving the design of an existing oxidative stress sensing biosensor in Saccharomyces cerevisiae based on the Yap1 transcription factor. Starting from a synthetic core promoter, we optimized the activity of a Yap1-dependent promoter through rational modification of a minimalist Yap1 upstream activating sequence. Our novel promoter achieves dynamic ranges of activation surpassing those of the previously engineered Yap1-dependent promoter, while reducing it to only 171 base pairs. We demonstrate that coupling the promoter to a positive-feedback-regulated TF further improves the biosensor by increasing its dynamic range of activation and reducing its limit of detection. We have illustrated the robustness and transferability of the biosensor by reproducing its activity in an unconventional probiotic yeast strain, Saccharomyces boulardii. Our findings can provide guidance in the general process of TF-based biosensor design.

Oncogenic lncRNA LINC00973 promotes Warburg effect by enhancing LDHA enzyme activity

Aerobic glycolysis, also known as the Warburg effect, is a hallmark of cancer and essential for metabolism in malignancies, but its regulation and modulation in cancer cells remain poorly understood. Here, using large-scale functional screening, we identified a tumor-associated and broadly expressed oncogenic long noncoding RNA LINC00973. Notably, knocking down LINC00973 significantly inhibits the proliferation of multiple types of cancer cells and reduces tumor growth in vivo. Mechanistically, LINC00973 directly binds to lactate dehydrogenase A (LDHA), an essential glycolytic enzyme, and enhances its enzymatic activity, thereby promoting glycolysis. Clinically, high expression of LINC00973 is significantly associated with poor prognosis in many types of human cancers. This work demonstrates that LINC00973 modulates cancer-specific regulation of the Warburg effect, and may represent a potential target for broad-acting anti-cancer therapies.