Biology subjects

Levin-Konigsberg, R.

Publications and source records attributed to Levin-Konigsberg, R..

7 recordsLinked to original sources

bioRxiv2025

RIG-I-dependent tumor-intrinsic type I interferon signaling restricts growth in breast cancer 3D culture

Discovery of key growth drivers that can be targeted for therapy is a central goal in cancer research. While high-throughput CRISPR screens have revolutionized our ability to identify gene dependencies in cancer, most large-scale screens are conducted in two-dimensional (2D) culture systems that fail to recapitulate tumor organization and behavior. To uncover architecture-dependent vulnerabilities in breast cancer, we performed parallel CRISPR interference (CRISPRi) screens in 2D and three-dimensional (3D) cultures of MCF7 cells, an estrogen receptor-positive (ER+) breast cancer model representative of a high risk of relapse, luminal subtype. Knockdown of IFNAR2 and TYK2 conferred a growth advantage in 3D cultures, implicating type I interferon signaling as a tumor-intrinsic suppressor of proliferation in 3D spheroids. Transcriptomic and functional analyses demonstrated that type I IFN signaling is endogenously activated in 3D spheroids via RIG-I-mediated sensing of cytosolic double-stranded RNA, leading to TBK1 activation and induction of interferon-stimulated genes (ISGs). This tumor-intrinsic IFN response slowed proliferation in 3D culture, independent of exogenous stimuli or the presence of immune cells. Analysis of bulk, single-cell, and spatial transcriptomic datasets from breast cancer patients revealed that a subset of tumors exhibit elevated IFN signaling in cancer cells, including in immune-depleted tumor cores, consistent with a tumor-intrinsic IFN signature. Our findings uncover an IFN-mediated growth-suppressive program shaped by 3D tumor architecture, and contribute towards a better understanding of the role of tumor-intrinsic IFN activity.

cancer biology↗

bioRxiv2023

Drug Discovery in Low Data Regimes: Leveraging a Computational Pipeline for the Discovery of Novel SARS-CoV-2 Nsp14-MTase Inhibitors

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has led to significant global morbidity and mortality. A crucial viral protein, the non-structural protein 14 (nsp14), catalyzes the methylation of viral RNA and plays a critical role in viral genome replication and transcription. Due to the low mutation rate in the nsp region among various SARS-CoV-2 variants, nsp14 has emerged as a promising therapeutic target. However, discovering potential inhibitors remains a challenge. In this work, we introduce a computational pipeline for the rapid and efficient identification of potential nsp14 inhibitors by leveraging virtual screening and the NCI open compound collection, which contains 250,000 freely available molecules for researchers worldwide. The introduced pipeline provides a cost-effective and efficient approach for early-stage drug discovery by allowing researchers to evaluate promising molecules without incurring synthesis expenses. Our pipeline successfully identified seven promising candidates after experimentally validating only 40 compounds. Notably, we discovered NSC620333, a compound that exhibits a strong binding affinity to nsp14 with a dissociation constant of 427 {+/-} 84 nM. In addition, we gained new insights into the structure and function of this protein through molecular dynamics simulations. We identified new conformational states of the protein and determined that residues Phe367, Tyr368, and Gln354 within the binding pocket serve as stabilizing residues for novel ligand interactions. We also found that metal coordination complexes are crucial for the overall function of the binding pocket. Lastly, we present the solved crystal structure of the nsp14-MTase complexed with SS148 (PDB:8BWU), a potent inhibitor of methyltransferase activity at the nanomolar level (IC50 value of 70 {+/-} 6 nM). Our computational pipeline accurately predicted the binding pose of SS148, demonstrating its effectiveness and potential in accelerating drug discovery efforts against SARS-CoV-2 and other emerging viruses.

biochemistry↗

bioRxiv2023

SLC12A9 is a lysosome-detoxifying ammonium - chloride co-transporter

Ammonia is a ubiquitous, toxic by-product of cell metabolism. Its high membrane permeability and proton affinity causes ammonia to accumulate inside acidic lysosomes in its poorly membrane-permeant form: ammonium (NH4+). Ammonium buildup compromises lysosomal function, suggesting the existence of mechanisms that protect cells from ammonium toxicity. Here, we identified SLC12A9 as a lysosomal ammonium exporter that preserves lysosomal homeostasis. SLC12A9 knockout cells showed grossly enlarged lysosomes and elevated ammonium content. These phenotypes were reversed upon removal of the metabolic source of ammonium or dissipation of the lysosomal pH gradient. Lysosomal chloride increased in SLC12A9 knockout cells and chloride binding by SLC12A9 was required for ammonium transport. Our data indicate that SLC12A9 is a chloride-driven ammonium co-transporter that is central in an unappreciated, fundamental mechanism of lysosomal physiology that may have special relevance in tissues with elevated ammonia, such as tumors.

cell biology↗

bioRxiv2023

VirtualFlow 2.0 - The Next Generation Drug Discovery Platform Enabling Adaptive Screens of 69 Billion Molecules

Identifying potent lead molecules for specific targets remains a major bottleneck in drug discovery. As structural information about proteins becomes increasingly available, ultra-large virtual screenings (ULVSs) which computationally evaluate billions of molecules offer a powerful way to accelerate early-stage drug discovery. Here, we introduce AdaptiveFlow, an open-source platform designed to make ULVSs more accessible, scalable, and efficient. AdaptiveFlow provides free access to a screening-ready version of the Enamine REAL Space (1), the largest library of ready-to-dock, drug-like molecules, containing 69 billion compounds that we prepared using the ligand preparation module of the platform. A key innovation of the platform is its use of a multi-dimensional grid of molecular properties, which helps researchers explore and prioritize chemical space more effectively and reduce the computational costs by a factor of approximately 1000. This grid forms the basis of a new method for identifying promising regions of chemical space, enabling systematic exploration and prioritization of compound libraries. An optional active learning component can further accelerate this process by adaptively steering the search toward molecules most likely to bind a given target. To support a broad range of applications, AdaptiveFlow is compatible with over 1,500 docking protocols. The platform achieves near-linear scaling on up to 5.6 million CPUs in the AWS Cloud, setting a new benchmark for large-scale cloud computing in drug discovery. Using this approach, we identified nanomolar inhibitors of two disease-relevant targets: ferroptosis suppressor protein 1 (FSP1) and poly(ADP-ribose) polymerase 1 (PARP-1) (2, 3). By leveraging newly solved crystal structures of FSP1 in complex with NAD+, FAD, and coenzyme Q1, we validated these hits experimentally and determined the co-crystal structures of FSP1 bound to small-molecule inhibitors, enabling insights into inhibitor binding mechanisms previously unknown. With its high scalability, flexibility, and open accessibility, AdaptiveFlow offers a powerful new resource for discovering and optimizing drug candidates at an unprecedented scale and speed.

bioinformatics↗

bioRxiv2022

A lysosomal lipid transport pathway that enables cell survival under cholinelimitation

Lysosomes degrade macromolecules and recycle their nutrient content to support cell function and survival over a broad range of metabolic conditions. Yet, the machineries involved in lysosomal recycling of many essential nutrients remain to be discovered, with a notable example being choline, an essential metabolite liberated in large quantities within the lysosome via the degradation of choline-containing lipids. To identify critical lysosomal choline transport pathways, we engineered metabolic dependency on lysosome-derived choline in pancreatic cancer cells. We then exploited this dependency to perform an endolysosome-focused CRISPR-Cas9 negative selection screen for genes mediating lysosomal choline recycling. Our screen identified the orphan lysosomal transmembrane protein SPNS1, whose loss leads to neurodegeneration-like disease in animal models, as critical for cell survival under free choline limitation. We find that SPNS1 loss leads to massive accumulation of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) within the lysosome. Mechanistically, we revealed that SPNS1 is required for the efflux of LPC species from the lysosome to enable their reesterification into choline-containing phospholipids in the cytosol. Using cell-based lipid uptake assays, we determine that SPNS1 functions as a proton gradient-dependent transporter of LPC. Collectively, our work defines a novel lysosomal phospholipid salvage pathway that is required for cell survival under conditions of choline limitation, and more broadly, provides a robust platform to deorphan lysosomal gene functions.

cell biology↗

bioRxiv2022

TORC1, BORC, and ARL-8 cycling drive vesiculation of cell corpse phagolysosomes

The dynamics of phagolysosomes leading to cargo clearance are important to provide cells with metabolites and avoid auto-immune responses, but little is known about how phagolysosomes finally resolve cell corpses and cell debris. We previously discovered that polar body phagolysosomes tubulate into small vesicles to facilitate corpse clearance within 1.5 hours in C. elegans. Here, we show that vesiculation depends on activating TORC1 through amino acid release by the solute transporter SLC-36.1. Downstream of TORC1, BLOC-1-related complex (BORC) recruits the Arf-like GTPase ARL-8 to the phagolysosome for tubulation by kinesin-1. We find that disrupting the regulated GTP-GDP cycle of ARL-8 reduces tubulation, delays corpse clearance, and mislocalizes ARL-8 away from lysosomes. We also demonstrate that mammalian phagocytes use BORC to promote phagolysosomal degradation, confirming the conserved importance of this pathway. Finally, we show that HOPS is required for rapid degradation of the small phagolysosomal vesicles. Thus, by observing single phagolysosomes over time, we identified the molecular pathway regulating phagolysosome vesiculation that promotes efficient resolution.

cell biology↗

bioRxiv2020

Phosphatidylinositol-4-kinase IIα licenses phagosomes for TLR4 signaling and MHC-II presentation in dendritic cells

Toll like receptor (TLR) recruitment to phagosomes in dendritic cells (DCs) and downstream TLR signaling are essential to initiate antimicrobial immune responses. However, the mechanisms underlying TLR localization to phagosomes are poorly characterized. We show herein that phosphatidylinositol-4-kinase II (PI4KII) plays a key role in initiating phagosomal TLR4 responses in murine DCs by generating a phosphatidylinositol-4-phosphate (PtdIns4P) platform conducive to the binding of the TLR sorting adaptor TIRAP. PI4KII is recruited to LPS-containing phagosomes in an adaptor protein AP-3 dependent manner, and both PI4KII and PtdIns4P are also detected on phagosomal membrane tubules. Knockdown of PI4KII - but not of the related PI4KII{beta} - impairs TIRAP and TLR4 localization to phagosomes, reduces proinflammatory cytokine secretion, and impairs phagosomal tubule formation and MHC-II presentation. Phagosomal TLR responses in PI4KII-deficient DCs are restored by re-expression of wild-type PI4KII, but not of variants lacking kinase activity or AP-3 binding. Our data indicate that PI4KII is an essential regulator of phagosomal TLR signaling in DCs by ensuring optimal TIRAP recruitment to phagosomes.

cell biology↗