Chemical and genetic screening approaches for the discovery of novel treatments

<p dir="ltr">To discover novel treatments, a variety of methods are used. In our laboratory we focus on the discovery of novel drug candidates through chemical high- throughput phenotypic screening. After identifying new compounds with therapeutic potential, the characterization of the properties of these drugs as well as elucidate their mechanism of action (MoA) becomes an important task. In addition to identifying novel therapies, and with the help of genome-wide CRISPR- Cas9 screens, we also try to discover mutations that modulate its effects, which can help to identify patients most likely to respond, or resist, to the drug.</p><p dir="ltr">In <b>Paper I</b>, we conducted a high-throughput image based chemical screen to identify compounds which could upregulate human leukocyte antigen I (HLA-I) on the surface of cancer cells and thus improve the recognition of tumor cells by cytotoxic T cells. In the screen we utilized cells with an inactive IFNy signaling pathway achieved by knocking out STAT1, since mutations in this pathway are one of the main causes of resistance to cancer immunotherapies. We thus explored whether it is possible to upregulate HLA-I expression with drugs independently of the JAK/STAT signaling pathway. The compound library comprised of more than 5.000 chemicals, including medically approved drugs and several agents in advanced preclinical development, targeting several different cell signaling pathways. Unfortunately, we could not identify any significant hit compound, indicating that drug-dependent modulation of HLA-I expression is strictly dependent on intact IFNy signaling.</p><p dir="ltr">In <b>Paper II</b>, we performed a genome-wide CRISPR screen to identify genetic modulators of the toxicity of RNA Polymerase I (Pol I) inhibitors, which are being explored as novel agents in the treatment of cancer. To do so, we studied the antibiotic Actinomycin D (ActD), which is commonly used in cancer therapy, and the pre-clinical tested small molecule BMH21, both known to inhibit Pol I and induce nucleolar stress. While mutations in genes known to regulate the nucleolar stress checkpoint such as TP53 and RB led to resistance to both compounds, mutations that enhanced PI3K/mTOR signalling lead to an increased sensitivity to Pol I inhibition. Interestingly, we could identify mutations which lead to resistance to one drug but sensitivity to the other, suggesting that the drugs have distinct effects on the cells despite both being used as Pol I inhibitors. Overall, our research provides an overview on genetic modulators of nucleolar stress inducing compounds and could therefore help to identify patients who could benefit the most from Pol I targeted therapies based on their tumor genetics.</p><p dir="ltr">In <b>Paper III</b>, we discovered a novel molecule with antiviral properties by conducting a high-throughput phenotypic screen using a pseudotype virus expressing the SARS-CoV-2 spike protein. Subsequence experiments using the molecule, which we named virapinib, demonstrated its ability to limit an infection by SARS-CoV-2 as well as other viruses, such as mpox and tick-borne encephalitis virus (TBEV). Additional mechanistic studies showed that virapinib inhibits macropinocytosis, limiting the entry for viruses using this route of infection of host cells. Virapinib presented no significant toxic effect on the host cells which further highlights its potential as an alternative treatment to prevent viral infections.</p><h3>List of scientific papers</h3><p dir="ltr">I. <b>Myriam Barz</b>*, Bartlomiej Porebski*, Pranauti Panshikar, Maria Häggbladd, Daniela Hühn, Oscar Fernandez-Capetillo. 2023. 'A chemical screen underscores the essential role of STAT1- dependent IFNy signaling to regulate HLA-I expression in cancer cells'. microPublication Biology. <a href="https://doi.org/10.17912/micropub.biology.000697" rel="noreferrer" target="_blank">https://doi.org/10.17912/micropub.biology.000697</a>.</p><p dir="ltr">II. <b>Myriam Barz</b>*, Alba Corman*, Maria Häggbladd, Alicia González- Serrano, Louise Lidemalm, Matilde Murga, Daniela Hühn, Oscar Fernandez-Capetillo. 2025. 'The PI3K/mTOR axis modulates the response to nucleolar stress'. [Manuscript]</p><p dir="ltr">III. Bartlomiej Porebski, Wanda Christ, Alba Corman, Martin Haraldsson, <b>Myriam Barz</b>, Louise Lidemalm, Maria Häggbladd, Juliana Ilmain, Shane C Wright, Matilde Murga, Jan Schlegel, Malin Jarvius, Maris Lapins, Erdinc Sezgin, Gira Bhabha, Volker M Lauschke, Jordi Carreras-Puigvert, Miguel Lafarga, Jonas Klingström, Daniela Hühn, Oscar Fernandez-Capetillo. 2024. 'Discovery of a novel inhibitor of macropinocytosis with antiviral activity' Mol Ther. 2024 Jul 2:S1525-0016(24)00455-6. <a href="https://doi.org/10.1016/j.ymthe.2024.06.038" target="_blank">https://doi.org/10.1016/j.ymthe.2024.06.038</a><br><br>*These authors contributed equally</p>