Mapping genetic interactions in cancer: a road to rational combination therapies.

The discovery of synthetic lethal interactions between poly (ADP-ribose) polymerase (PARP) inhibitors and BRCA genes, which are involved in homologous recombination, led to the approval of PARP inhibition as a monotherapy for patients with BRCA1/2-mutated breast or ovarian cancer. Studies following the initial observation of synthetic lethality demonstrated that the reach of PARP inhibitors is well beyond just BRCA1/2 mutants. Insights into the mechanisms of action of anticancer drugs are fundamental for the development of targeted monotherapies or rational combination treatments that will synergize to promote cancer cell death and overcome mechanisms of resistance. The development of targeted therapeutic agents is premised on mapping the physical and functional dependencies of mutated genes in cancer. An important part of this effort is the systematic screening of genetic interactions in a variety of cancer types. Until recently, genetic-interaction screens have relied either on the pairwise perturbations of two genes or on the perturbation of genes of interest combined with inhibition by commonly used anticancer drugs. Here, we summarize recent advances in mapping genetic interactions using targeted, genome-wide, and high-throughput genetic screens, and we discuss the therapeutic insights obtained through such screens. We further focus on factors that should be considered in order to develop a robust analysis pipeline. Finally, we discuss the integration of functional interaction data with orthogonal methods and suggest that such approaches will increase the reach of genetic-interaction screens for the development of rational combination therapies

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing

The novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 2.3 million people, killed over 160,000, and caused worldwide social and economic disruption1,2. There are currently no antiviral drugs with proven clinical efficacy, nor are there vaccines for its prevention, and these efforts are hampered by limited knowledge of the molecular details of SARS-CoV-2 infection. To address this, we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), identifying 332 high-confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (29 FDA-approved drugs, 12 drugs in clinical trials, and 28 preclinical compounds). Screening a subset of these in multiple viral assays identified two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the Sigma1 and Sigma2 receptors. Further studies of these host factor targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19

60S Ribosomal Subunit Assembly in Yeast Involves Major Conformational Changes Facilitated by Sequential Binding of Individual Ribosomal Proteins

<p>Ribosome assembly is a dynamic process that involves the concurrent folding and processing of pre-rRNA upon association of ribosomal proteins (r-proteins), and the binding, function and release of assembly factors. These remodeling events exert their function as timers and switches for the progression of ribosome assembly. Using combined approaches of molecular genetics, proteomics and structural biology, we were able to address several major questions currently faced by those who study ribosome assembly in eukaryotes. The crystal structure of mature yeast ribosomes revealed the exact locations of r-proteins, especially how they interact with rRNA. However, this snapshot of the final product did not provide information on the timing of association of r-proteins with pre-ribosomes and the roles for their rRNA interactions in assembly. In Chapter 2, I describe experiments that establish the hierarchy of ribosome assembly by analyzing the depletion phenotypes of eight r-proteins from the large subunit, as representatives of experiments conducted on 32 large subunit r-proteins. Our results indicate that the solvent interface of the pre-ribosome is stabilized first, followed by the polypeptide exit tunnel, and finally, the central protuberance and subunit-interface. Besides r-proteins, about 75 assembly factors were identified to mediate assembly of 60S ribosomal subunits. In order to dissect the functions of interactions between assembly factors and rRNA or r-proteins, we needed highresolution structural data showing the exact locations of assembly factors in v pre-ribosomes. In Chapter 3, I analyze the near atomic resolution cryo-EM structures of Nog2-associated pre-ribosomes. Besides revealing the locations of about 30 assembly factors, these structures enabled visualization of the three major remodeling events that occur during late nuclear stages of ribosome assembly. One striking feature revealed by the crystal structure of mature ribosomes is that many r-proteins contain eukaryote-specific extensions. Although the functions of these extensions were not clear, their intrinsic properties, such as structural flexibility and ability to reach long distances in ribosomes, suggested their involvement in the sequential binding of r-proteins to pre-ribosomes. Previous ribosome reconstitution studies pointed to sequential binding of r-proteins, where upon initial contact, r-proteins and rRNA form encounter complexes that are stabilized later, as assembly proceeds. In Chapter 4, I establish that portions of r-proteins are stabilized at different stages of assembly, based on the rRNA domain with which they interact. In addition, through the clues provided by the cryo-EM structures, I suggest a communication paradigm mediated by the eukaryote-specific N-terminal extension of L8 during late nuclear stages of assembly. Finally, we and others identified a drastic remodeling event, an ~180° rotation of the central protuberance, that happens shortly before nuclear export of pre-ribosomes. The mechanism of this rotation was not known. In Chapter 5, I provide multiple testable hypotheses on the mechanism of rotation of the central protuberance, based on interactions that are broken or established upon rotation.</p

Mapping genetic interactions in cancer: a road to rational combination therapies.

The discovery of synthetic lethal interactions between poly (ADP-ribose) polymerase (PARP) inhibitors and BRCA genes, which are involved in homologous recombination, led to the approval of PARP inhibition as a monotherapy for patients with BRCA1/2-mutated breast or ovarian cancer. Studies following the initial observation of synthetic lethality demonstrated that the reach of PARP inhibitors is well beyond just BRCA1/2 mutants. Insights into the mechanisms of action of anticancer drugs are fundamental for the development of targeted monotherapies or rational combination treatments that will synergize to promote cancer cell death and overcome mechanisms of resistance. The development of targeted therapeutic agents is premised on mapping the physical and functional dependencies of mutated genes in cancer. An important part of this effort is the systematic screening of genetic interactions in a variety of cancer types. Until recently, genetic-interaction screens have relied either on the pairwise perturbations of two genes or on the perturbation of genes of interest combined with inhibition by commonly used anticancer drugs. Here, we summarize recent advances in mapping genetic interactions using targeted, genome-wide, and high-throughput genetic screens, and we discuss the therapeutic insights obtained through such screens. We further focus on factors that should be considered in order to develop a robust analysis pipeline. Finally, we discuss the integration of functional interaction data with orthogonal methods and suggest that such approaches will increase the reach of genetic-interaction screens for the development of rational combination therapies

The Landscape of Human Cancer Proteins Targeted by SARS-CoV-2.

The mapping of SARS-CoV-2 human protein-protein interactions by Gordon and colleagues revealed druggable targets that are hijacked by the virus. Here, we highlight several oncogenic pathways identified at the host-virus interface of SARS-CoV-2 to enable cancer biologists to apply their knowledge for rapid drug repurposing to treat COVID-19, and help inform the response to potential long-term complications of the disease

The Landscape of Human Cancer Proteins Targeted by SARS-CoV-2.

The mapping of SARS-CoV-2 human protein-protein interactions by Gordon and colleagues revealed druggable targets that are hijacked by the virus. Here, we highlight several oncogenic pathways identified at the host-virus interface of SARS-CoV-2 to enable cancer biologists to apply their knowledge for rapid drug repurposing to treat COVID-19, and help inform the response to potential long-term complications of the disease

A protein interaction landscape of breast cancer

Cancers have been associated with a diverse array of genomic alterations. To help mechanistically understand such alterations in breast-invasive carcinoma, we applied affinity purification–mass spectrometry to delineate comprehensive biophysical interaction networks for 40 frequently altered breast cancer (BC) proteins, with and without relevant mutations, across three human breast cell lines. These networks identify cancer-specific protein-protein interactions (PPIs), interconnected and enriched for common and rare cancer mutations, that are substantially rewired by the introduction of key BC mutations. Our analysis identified BPIFA1 and SCGB2A1 as PIK3CA-interacting proteins, which repress PI3K-AKT signaling, and uncovered USP28 and UBE2N as functionally relevant interactors of BRCA1. We also show that the protein phosphatase 1 regulatory subunit spinophilin interacts with and regulates dephosphorylation of BRCA1 to promote DNA double-strand break repair. Thus, PPI landscapes provide a powerful framework for mechanistically interpreting disease genomic data and can identify valuable therapeutic targets

Interpretation of cancer mutations using a multiscale map of protein systems.

A major goal of cancer research is to understand how mutations distributed across diverse genes affect common cellular systems, including multiprotein complexes and assemblies. Two challenges—how to comprehensively map such systems and how to identify which are under mutational selection—have hindered this understanding. Accordingly, we created a comprehensive map of cancer protein systems integrating both new and published multi-omic interaction data at multiple scales of analysis. We then developed a unified statistical model that pinpoints 395 specific systems under mutational selection across 13 cancer types. This map, called NeST (Nested Systems in Tumors), incorporates canonical processes and notable discoveries, including a PIK3CA-actomyosin complex that inhibits phosphatidylinositol 3-kinase signaling and recurrent mutations in collagen complexes that promote tumor proliferation. These systems can be used as clinical biomarkers and implicate a total of 548 genes in cancer evolution and progression. This work shows how disparate tumor mutations converge on protein assemblies at different scales