Abstract The RecQ DNA helicase WRN was identified as a synthetic lethal target in tumors with microsatellite instability (MSI) by several genetic screens. Despite recent advances in the treatment of MSI tumors by immune checkpoint inhibitors, a significant proportion of patients still fails to respond to or relapses after single agent anti-PD1 or combination of anti-PD1 plus anti-CTLA4 treatments. We present the biochemical, cellular and pharmacological characterization of the first potent and selective WRN helicase inhibitor, HRO761. We show that HRO761 is an allosteric WRN inhibitor that binds at the interface of the D1 and D2 helicase domains, locking WRN in an inactive conformation. Pharmacological inhibition of WRN by HRO761 recapitulates the phenotype observed by WRN genetic suppression, leading to activation of the DNA damage response and inhibition of tumor cell growth selectively in MSI but not in MSS cells. We further show that WRN inhibition leads to WRN protein degradation only in MSI tumor cells. Although HRO761 activates a p53-related response, anti-proliferative effects were observed in both p53 wild-type as well as p53 null or mutated tumor cells. HRO761 exposure in mice is linear with oral dose and results in dose-dependent in vivo DNA damage induction and tumor growth inhibition in MSI cell- and patient-derived xenograft models. Altogether these findings represent preclinical pharmacological validation of WRN as a selective therapeutic target in MSI cancers. A global first in human clinical trial with HRO761 (NCT05838768) is currently ongoing to assess the safety, tolerability and preliminary anti-tumor activity in patients with MSI colorectal cancer and other MSI solid tumors. Citation Format: Marta Cortes-Cros, Henrik Moebitz, Stephanie Barbe, Jutta Blank, Vincent Bordas, Giorgia Clementi, Ernesta Dammassa, Andrea Decker, Noemi Di Nanni, Ruben de Kanter, Marion Dourdoigne, Elena Gavioli, Stephane Ferretti, Marc Hattenberger, Christelle Hemmerlin, Jacques Hamon, Juergen Hinrichs, Isabel Jaco, Dragana Jankovic, Eloisa Jimenez Nunez, Yifang Li, Hans-Joerg Martus, Michele Moschetta, Cornelia Quadt, Markus Reschke, Vincent Romanet, Clemens Scheufler, Joseph Schoepfer, Ross Strang, Maxime Therier, Hans Voshol, Markus Wartmann, Sarah Welly, Monica Pham, Laurent Laborde. Discovery of HRO761, a novel, first-in-class clinical stage WRN inhibitor with potent and selective anti-tumor activity in cancers with microsatellite instability [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR007.
<p>List of individual shRNA scores. File containing raw data of individual shRNAs quantified by barcode sequencing of DMSO- and HDM201- treated samples. Log fold changes of individual shRNAs compared to plasmid library and of HDM201-treated samples compared to DMSO-control are also listed.</p>
<p>Insertional results from Arf-/- PB in vivo resistance screens. A and C, Tables listing all sequenced samples with their model number (i.e. X25272), the transplanted fragment number (i.e. P0M1-P1M5-P2M19, P for passage, and M for Mouse), tumor status (DR=drug resistant, or veh=vehicle treated) and treatment schedule (A) QD for daily or (C) 2QW for biweekly. Total number of reads and tumor pathology are labeled for each sample. Insertional landscapes are displayed on the right part of the table where the averaged normalized diversity sequencing counts are indicated for each Common insertion site gene (gCIS) and each tumor. B and D, List of the genes found differentially enriched for PB insertions in HDM201 resistant tumors compared to untreated tumors (B) continuous daily treatment or (D) biweekly intermittent treatment. The data in D were extracted from previous report (28) for intermittent 100 mg/kg biweekly dosing schedule, and reanalyzed with only 6 tumor models used out of 16 in previous report. Fold change, p-value and FDR were calculated for each gene in comparative analyses of untreated tumor samples vs. resistant tumor samples. PercentSample indicated the percentage of resistant tumors with insertion in that gene. The predicted function GOF (gain of function), LOF (loss of function) or uncertain is also indicated.</p>
Abstract Sphingosine‐1‐phosphate (S1P) receptors are widely expressed in the central nervous system where they are thought to regulate glia cell function. The phosphorylated version of fingolimod/FTY720 (FTY720P) is active on a broad spectrum of S1P receptors and the parent compound is currently in phase III clinical trials for the treatment of multiple sclerosis. Here, we aimed to identify which cell type(s) and S1P receptor(s) of the central nervous system are targeted by FTY720P. Using calcium imaging in mixed cultures from embryonic rat cortex we show that astrocytes are the major cell type responsive to FTY720P in this assay. In enriched astrocyte cultures, we detect expression of S1P1 and S1P3 receptors and demonstrate that FTY720P activates Gi protein‐mediated signaling cascades. We also show that FTY720P as well as the S1P1‐selective agonist SEW2871 stimulate astrocyte migration. The data indicate that FTY720P exerts its effects on astrocytes predominantly via the activation of S1P1 receptors, whereas S1P signals through both S1P1 and S1P3 receptors. We suggest that this distinct pharmacological profile of FTY720P, compared with S1P, could play a role in the therapeutic effects of FTY720 in multiple sclerosis.
The Front Cover shows the initial screening strategy for hit identification on WRN helicase. The main challenge was to understand the type of false positives which came out of standard DNA unwinding or ATPase assays. Beyond DNA binders, direct interferences of compounds with WRN protein were the major source of artefacts as illustrated by the extensive characterization of inhibitors (ML216 and NSC617145) wrongly considered as WRN probes. Some learnings on how to minimize and triage out those in vitro artefacts are shared for best hit discovery on WRN helicase. More information can be found in the Research Article by Alisia Heuser, Henrik Möbitz, Jacques Hamon et al.
Abstract The Werner Syndrome RecQ helicase (WRN) is a synthetic lethal target of interest for the treatment of cancers with microsatellite instability (MSI). Different hit finding approaches were initially tested. The identification of WRN inhibitors proved challenging due to a high propensity for artefacts via protein interference, i. e., hits inhibiting WRN enzymatic activities through multiple, unspecific mechanisms. Previously published WRN Helicase inhibitors (ML216, NSC19630 or NSC617145) were characterized in an extensive set of biochemical and biophysical assays and could be ruled out as specific WRN helicase probes. More innovative screening strategies need to be developed for successful drug discovery of non‐covalent WRN helicase inhibitors.
Abstract Radioligand therapy (RLT) is emerging as a safe and effective targeted approach for treating several types of cancers. Lutathera® (177Lu-Dotatate) and Pluvicto® (177Lu-PSMA-617) are two examples of FDA-approved 177Lu-based RLT drugs for the treatment of somatostatin receptor-positive gastroenteropancreatic neuroendocrine tumors (GEP-NETs) and of PSMA-positive metastatic castration-resistant prostate cancer (mCRPC), respectively. Despite the clinical success of 177Lu-RLT, there are still patients lacking complete, durable responses. Arguably, elucidating the cellular responses to 177Lu-RLT and its mechanism of action could reveal potential opportunities for the improvement of therapy and outcome for patients. We have investigated the phenotypes and responses of multiple 177Lu- RLT-treated cell lines and observed that 177Lu-RLT induces a plethora of DNA Damage Response (DDR) markers, especially those indicative of double-strand break (DSB) repair by either non-homologous end-joining (NHEJ) or homologous recombination (HR). Furthermore, 177Lu-RLT leads to cell-cycle alterations, accumulation of micronuclei and cell death. In agreement with published reports, we have observed that either the knock-out or inhibition of NHEJ core factors such as the catalytic subunit of DNA-PK (PRKDC) renders cells sensitive to 177Lu-RLT and the combination of both the 177Lu-RLT and DNA-PK inhibitor provides a beneficial tumor growth inhibition in vivo. To further unravel the mechanism of action of 177Lu-RLT, we have conducted ‘DDR-ome’ targeted genetic screens, which also suggest that DSBs are the most cytotoxic form of 177Lu-RLT-induced DNA damage. In addition to DNA-PK, we have identified novel DDR targets that could sensitize to 177Lu-RLT. We are currently testing whether inhibiting these targets in combination with certain 177Lu-RLT could, in addition to potentially achieving greater antitumor response, allow for lower dosing to reduce radiation exposure. Taken together, our work provides better understanding of the cellular responses to 177Lu-based RLT and pinpoints NHEJ as a critical pathway promoting survival to this treatment, which could set the basis for novel combination therapies. Citation Format: Marco Ranzani, Sravanth Hindupur, Alessandro Cicconi, Guillermo Sastre-Moreno, Alexander Kristian, Barbara Schacher Engstler, Benika Pinch, Charlene Hartnagel, Delphine Gorses, Elise Simon, Emeline Mandon, Emilien Schramm, Fanny Schaeffer, Josefine Reber, Louise Barys, Luisa Deberle, Milene Walter, Ralph Bessey, Riccardo Destefani, Xavier Miot, Elias Elinati, Nan Shao, Marie Boursier, Joseph Barlow, Asmita Thapa, Alessandro Galbiati, Diego Grande, Eeson Rajendra, Niall Martin, Graeme Smith, Tobias Schmelzle, Markus Reschke, Helen Robinson, Marta Cortes-Cros. Elucidating the cellular responses and mechanism of action of 177Lu-based radioligand therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB222.
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