SARS-CoV-2, the etiological agent of COVID-19, has so far resulted in >6.1 million deaths worldwide. The spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor.

To maintain genome stability, regulators of chromosome segregation must be expressed in coordination with mitotic events. Expression of these late cell cycle genes is regulated by cyclin-dependent kinase (Cdk1), which phosphorylates a network of conserved transcription factors (TFs). However, the effects of Cdk1 phosphorylation on many key TFs are not known. We find that elimination of Cdk1-mediated phosphorylation of four S-phase TFs decreases expression of many late cell cycle genes, delays mitotic progression, and reduces fitness in budding yeast. Blocking phosphorylation impairs degradation of all four TFs. Consequently, phosphorylation-deficient mutants of the repressors Yox1 and Yhp1 exhibit increased promoter occupancy and decreased expression of their target genes. Interestingly, although phosphorylation of the transcriptional activator Hcm1 on its N-terminus promotes its degradation, phosphorylation on its C-terminus is required for its activity, indicating that Cdk1 both activates and inhibits a single TF. We conclude that Cdk1 promotes gene expression by both activating transcriptional activators and inactivating transcriptional repressors. Furthermore, our data suggest that coordinated regulation of the TF network by Cdk1 is necessary for faithful cell division.

The transcription factor Hcm1 is a key regulator of chromosome segregation and genome stability. The phosphatase calcineurin directly inactivates Hcm1 in response to environmental stress, which inhibits proliferation. Hcm1 functions as a rheostat, whose phosphorylation state affects the rate of proliferation.

Protein degradation during the cell cycle is controlled by the opposing activities of ubiquitin ligases and deubiquitinating enzymes (DUBs). Although the functions of ubiquitin ligases in the cell cycle have been studied extensively, the roles of DUBs in this process are less well understood. Here, we used an overexpression screen to examine the specificities of each of the 21 DUBs in budding yeast for 37 cell cycle-regulated proteins. We find that DUBs up-regulate specific subsets of proteins, with five DUBs regulating the greatest number of targets. Overexpression of Ubp10 had the largest effect, stabilizing 15 targets and delaying cells in mitosis. Importantly, UBP10 deletion decreased the stability of the cell cycle regulator Dbf4, delayed the G1/S transition, and slowed proliferation. Remarkably, deletion of UBP10 together with deletion of four additional DUBs restored proliferation to near-wild-type levels. Among this group, deletion of the proteasome-associated DUB Ubp6 alone reversed the G1/S delay and restored the stability of Ubp10 targets in ubp10Δ cells. Similarly, deletion of UBP14, another DUB that promotes proteasomal activity, rescued the proliferation defect in ubp10Δ cells. Our results suggest that DUBs function through a complex genetic network in which their activities are coordinated to facilitate accurate cell cycle progression.

The Spike (S)-protein of SARS-CoV-2 binds host-cell receptor ACE2 and requires proteolytic “priming” (S1/S2) and “fusion-activation” (S2’) for viral entry. The S-protein furin-like motifs PRRAR685↓ and KPSKR815↓ indicated that proprotein convertases promote virus entry. We demonstrate that furin and PC5A induce cleavage at both sites, ACE2 enhances S2’ processing, and their pharmacological inhibition (BOS-inhibitors) block endogenous cleavages. S1/S2-mutations (μS1/S2) limit S-protein-mediated cell-to-cell fusion, similarly to BOS-inhibitors. Unexpectedly, TMPRSS2 does not cleave at S1/S2 or S2’, but it can: (i) cleave/inactivate S-protein into S2a/S2b; (ii) shed ACE2; (iii) cleave S1-subunit into secreted S1’, activities inhibited by Camostat. In lung-derived Calu-3 cells, BOS-inhibitors and µS1/S2 severely curtail “pH-independent” viral entry, and BOS-inhibitors alone/with Camostat potently reduce infectious viral titer and cytopathic effects. Overall, our results show that: furin plays a critical role in generating fusion-competent S-protein, and indirectly, TMPRSS2 promotes viral entry, supporting furin and TMPRSS2 inhibitors as potential antivirals against SARS-CoV-2.

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The Spike (S)-protein of SARS-CoV-2 binds host-cell receptor ACE2 and requires proteolytic "priming" at PRRAR685↓ into S1 and S2 (cleavage at S1/S2), and "fusion-activation" at KPSKR815↓ (cleavage at S2′) for viral entry. Both cleavages occur at Furin-like motifs suggesting that proprotein convertases might promote virus entry. In vitro Furin cleaved peptides mimicking the S1/S2 cleavage site more efficiently than S2′, whereas TMPRSS2 cleaved at both sites. In HeLa cells endogenous Furin-like enzymes cleave mainly at S1/S2 during intracellular protein trafficking, as confirmed by mutagenesis. We also mapped the S2′ cleavage site by proteomics and further showed that S2′-processing by Furin, while limited, was strongly enhanced in the presence of ACE2. In contrast, the S2′ KRRKR815↓ mutant (μS2′) was considerably better cleaved by Furin, whereas individual/double KR815AA mutants are retained in the endoplasmic reticulum (ER). Pharmacological inhibitors of convertases (Boston Pharmaceuticals - BOS-inhibitors) effectively blocked endogenous S-protein processing in HeLa cells. However, under co-expression the S-protein was prematurely cleaved by TMPRSS2 into ER-retained, non-O-glycosylated S2 and S2′ products. Quantitative analysis of cell-to-cell fusion and Spike processing using Hela cells revealed the key importance of the Furin sites for syncytia formation and unveiled the enhanced fusogenic potential of the α- and δ-variants of the S-protein of SARS-CoV-2. Our fusion assay indicated that TMPRSS2 enhances S2′ formation, especially in the absence of Furin cleavage, as well as ACE2 shedding. Furthermore, we provide evidence using pseudoparticles that while entry by a "pH-dependent" endocytosis pathway in HEK293 cells did not require Furin processing at S1/S2, a "pH-independent" viral entry in lung-derived Calu-3 cells was sensitive to inhibitors of Furin and TMPRSS2. Consistently, in Calu-3 cells BOS-inhibitors or Camostat potently reduce infectious viral titer and cytopathic effects and this outcome was enhanced when both compounds were combined. Overall, our results show that Furin and TMPRSS2 play synergistic roles in generating fusion-competent S-protein, and promote viral entry, supporting the combination of Furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2.

Our multiplexed cell viability platform, PRISM (profiling relative inhibition simultaneously in mixtures), enables screening of potential cancer therapeutics at an unprecedented scale. We routinely assess the effects of perturbations against more than 900 cancer cell lines concurrently through the use of unique oligonucleotide barcodes stably transduced into individual cancer cell lines. Following barcode transduction, individual cell lines are pooled together in groups of 20-25 based on growth rate similarity, then thawed into 384-well assay-ready plates containing compounds of interest. After 5 days of growth, isolated mRNA is used to detect transcribed barcode abundance of each individual cancer cell line to measure relative viability. We leverage the baseline cellular features (e.g., gene expression, cell lineage, mutation, copy number, metabolomics, proteomics, genome-wide RNAi and CRISPR dependencies) of each cell line to interpret viability profiles, enabling identification of drivers of differential sensitivity and potential biomarkers of compound response. Critically, the scale and throughput of PRISM has enabled the generation of large, publicly available datasets and the rapid characterization of emerging therapeutic targets and classes (e.g., isoform-selective RAS inhibitors and degraders). Although the read-out of PRISM is relative cell line viability, the platform can be used to answer a multitude of scientific questions. For example, PRISM data can be used to identify potential patient populations who would most benefit from treatment with a specific compound, uncover unexpected off-target toxicities, and validate mechanism of action hypotheses on a more holistic scale. Ultimately, PRISM offers a large-scale and comprehensive platform for the testing and validation of anti-cancer compounds to aid in the search for new oncology therapeutics. Citation Format: Ellen Nguyen, Shiker Nair, John Davis, Antonella Masciotti, Connor Mochi, John Finn, Cole Ponsi, Brienne Engel, Claudine Mapa, Mustafa Kocak, Melissa Ronan, Matthew G. Rees, Jennifer A. Roth. Identifying therapeutic mechanism of action and new potential patient populations using PRISM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2748.