Systematic discovery of mutation-directed neo-protein-protein interactions in cancer.

Comprehensive sequencing of patient tumors reveals genomic mutations across tumor types that enable tumorigenesis and progression. A subset of oncogenic driver mutations results in neomorphic activity where the mutant protein mediates functions not engaged by the parental molecule. Here, we identify prevalent variant-enabled neomorph-protein-protein interactions (neoPPI) with a quantitative high-throughput differential screening (qHT-dS) platform. The coupling of highly sensitive BRET biosensors with miniaturized coexpression in an ultra-HTS format allows large-scale monitoring of the interactions of wild-type and mutant variant counterparts with a library of cancer-associated proteins in live cells. The screening of 17,792 interactions with 2,172,864 data points revealed a landscape of gain of interactions encompassing both oncogenic and tumor suppressor mutations. For example, the recurrent BRAF V600E lesion mediates KEAP1 neoPPI, rewiring a BRAFV600E/KEAP1 signaling axis and creating collateral vulnerability to NQO1 substrates, offering a combination therapeutic strategy. Thus, cancer genomic alterations can create neo-interactions, informing variant-directed therapeutic approaches for precision medicine.

Acquisition of taxane resistance by p53 inactivation in ovarian cancer cells.

Ovarian cancer is one of the most common gynecologic malignancies in women and has a poor prognosis. Taxanes are a class of standard first-line chemotherapeutic agents for the treatment of ovarian cancer. However, tumor-intrinsic and acquired resistance to taxanes poses major challenges to improving clinical outcomes. Hence, there is an urgent clinical need to understand the mechanisms of resistance in order to discover potential biomarkers and therapeutic strategies to increase taxane sensitivity in ovarian cancer. Here, we report the identification of an association between the TP53 status and taxane sensitivity in ovarian cancer cells through complementary experimental and informatics approaches. We found that TP53 inactivation is associated with taxane resistance in ovarian cancer cells, supported by the evidence from (i) drug sensitivity profiling with bioinformatic analysis of large-scale cancer therapeutic response and genomic datasets and (ii) gene signature identification based on experimental isogenic cell line models. Further, our studies revealed TP53-dependent gene expression patterns, such as overexpression of ACSM3, as potential predictive biomarkers of taxane resistance in ovarian cancer. The TP53-dependent hyperactivation of the WNT/ß-catenin pathway discovered herein revealed a potential vulnerability to exploit in developing combination therapeutic strategies. Identification of this genotype-phenotype relationship between the TP53 status and taxane sensitivity sheds light on TP53-directed patient stratification and therapeutic discoveries for ovarian cancer treatment.

Disruption of the lamin A and matrin-3 interaction by myopathic LMNA mutations.

The nuclear face of the nuclear membrane is enriched with the intermediate filament protein lamin A. Mutations in LMNA, the gene encoding lamin A, lead to a diverse set of inherited conditions including myopathies that affect both the heart and skeletal muscle. To gain insight about lamin A protein interactions, binding proteins associated with the tail of lamin A were characterized. Of 130 nuclear proteins found associated with the lamin A tail, 17 (13%) were previously described lamin A binding partners. One protein not previously linked to lamin A, matrin-3, was selected for further study, because like LMNA mutations, matrin-3 has also been implicated in inherited myopathy. Matrin-3 binds RNA and DNA and is a nucleoplasmic protein originally identified from the insoluble nuclear fraction, referred to as the nuclear matrix. Anti-matrin-3 antibodies were found to co-immunoprecipitate lamin A, and the lamin-A binding domain was mapped to the carboxy-terminal half of matrin-3. Three-dimensional mapping of the lamin A-matrin-3 interface showed that the LMNA truncating mutation Δ303, which lacks the matrin-3 binding domain, was associated with an increased distance between lamin A and matrin-3. LMNA mutant cells are known to have altered biophysical properties and the matrin-3-lamin A interface is positioned to contribute to these defects.

Pan-cancer transcriptome analysis reveals long noncoding RNAs with conserved function.

A growing number of gene-centric studies have highlighted the emerging significance of lncRNAs in cancer. However, these studies primarily focus on a single cancer type. Therefore, we conducted a pan-cancer analysis of lncRNAs comparing tumor and matched normal expression levels using RNA-Seq data from ∼ 3,000 patients in 8 solid tumor types. While the majority of differentially expressed lncRNAs display tissue-specific expression we discovered 229 lncRNAs with outlier or differential expression across multiple cancers, which we refer to as 'onco-lncRNAs'. Due to their consistent altered expression, we hypothesize that these onco-lncRNAs may have conserved oncogenic and tumor suppressive functions across cancers. To address this, we associated the onco-lncRNAs in biological processes based on their co-expressed protein coding genes. To validate our predictions, we experimentally confirmed cell growth dependence of 2 novel oncogenic lncRNAs, onco-lncRNA-3 and onco-lncRNA-12, and a previously identified lncRNA CCAT1. Overall, we discovered lncRNAs that may have broad oncogenic and tumor suppressor roles that could significantly advance our understanding of cancer lncRNA biology.

TMPRSS2 and SARS-CoV-2 SPIKE interaction assay for uHTS.

SARS-CoV-2, the coronavirus that causes the disease COVID-19, has claimed millions of lives over the past 2 years. This demands rapid development of effective therapeutic agents that target various phases of the viral replication cycle. The interaction between host transmembrane serine protease 2 (TMPRSS2) and viral SPIKE protein is an important initial step in SARS-CoV-2 infection, offering an opportunity for therapeutic development of viral entry inhibitors. Here, we report the development of a time-resolved fluorescence/Förster resonance energy transfer (TR-FRET) assay for monitoring the TMPRSS2-SPIKE interaction in lysate from cells co-expressing these proteins. The assay was configured in a 384-well-plate format for high-throughput screening with robust assay performance. To enable large-scale compound screening, we further miniaturized the assay into 1536-well ultrahigh-throughput screening (uHTS) format. A pilot screen demonstrated the utilization of the assay for uHTS. Our optimized TR-FRET uHTS assay provides an enabling platform for expanded screening campaigns to discover new classes of small-molecule inhibitors that target the SPIKE and TMPRSS2 protein-protein interaction.

A time-resolved fluorescence resonance energy transfer screening assay for discovery of protein-protein interaction modulators.

Protein-protein interactions (PPIs) have emerged as promising yet challenging therapeutic targets. A robust bioassay is required for rapid PPI modulator discovery. Here, we present a time-resolved Förster's (fluorescence) resonance energy transfer assay protocol for PPI modulator screening in a 1536-well plate format. We use hypomorph SMAD4R361H-SMAD3 PPI as an example to illustrate the application of the protocol for screening of variant-directed PPI inducers. This platform can be readily adapted for the discovery of both small-molecule PPI inducers and inhibitors. For complete details on the use and execution of this protocol, please refer to Tang et al. (2020).

Presentation of treatment effect in glioblastoma after dose-escalation radiation therapy.

Glioblastoma is the most aggressive primary brain tumor in adults. Limited treatment options and the intense nature of therapy make determining the appropriate treatment course for each patient difficult. The appearance of transient worsening of imaging findings, known as treatment effect, after chemoradiation further complicates clinical decision-making. Accurately differentiating treatment effects from true progression is critical as subsequent treatment decisions are based largely on radiographic evidence of tumor progression. As chemoradiation can cause worsening of imaging findings, it is possible that the use of new treatments and modified chemoradiation regimens may alter the presentation of treatment effect. Therefore, physicians should be aware that atypical presentations of treatment effects can occur, and may be more likely, when treatment regimens are modified. Here, we present the case of a patient with isocitrate dehydrogenase 1 wild type, O-6-methylguanine-DNA methyltransferase-methylated glioblastoma who underwent dose-escalation radiation therapy (to 75 Gy) and exhibited worsened imaging findings at 8 months post-radiation.