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Drug resistance is a commonly unavoidable consequence of cancer treatment that results in therapy failure and disease relapse. Intrinsic (pre-existing) or acquired resistance mechanisms can be drug-specific or be applicable to multiple drugs, resulting in multidrug resistance. The presence of drug resistance is, however, tightly coupled to changes in cellular homeostasis, which can lead to resistance-coupled vulnerabilities. Unbiased gene perturbations through RNAi and CRISPR technologies are invaluable tools to establish genotype-to-phenotype relationships at the genome scale. Moreover, their application to cancer cell lines can uncover new vulnerabilities that are associated with resistance mechanisms. Here, we discuss targeted and unbiased RNAi and CRISPR efforts in the discovery of drug resistance mechanisms by focusing on first-in-line chemotherapy and their enforced vulnerabilities, and we present a view forward on which measures should be taken to accelerate their clinical translation. info:eu-repo/semantics/publishedVersion

PICH (Plk1-interacting checkpoint helicase), a DNA-dependent ATPase, was identified as a binding partner and substrate of Plk1. During mitosis, PICH localizes to the centromere/KTs of condensed mitotic chromosomes and to ultra-fine DNA bridges (UFBs) during anaphase. Upon depletion or chemical inhibition of Plk1, PICH localizes to the chromosome arms, suggesting that the kinase activity of Plk1 regulates the dynamic localization of PICH. In addition, PICH (bound to Plk1) has been proposed to function in prometaphase chromosome arm architecture and cohesion. Here, two questions were asked: how is the dynamic localization of PICH regulated and what is the mitotic function of PICH? In the first part of this thesis, we identify the ATPase activity of PICH as being essential for its correct subcellular localization and show that ATPase-dead mutants of PICH localize to the chromosome arms. Rescue experiments with a mutant of PICH that is unable to interact with Plk1 imply that the kinase activity of Plk1 is only indirectly required to delocalize PICH from the chromosome arms. This suggests that an unknown Plk1 substrate regulates the localization of PICH. PICH-immunoprecipitation and mass spectrometry identified the uncharacterized protein BEND3 as a binding partner. BEND3 localizes to mitotic chromosomes and its depletion results in the loss of PICH and Plk1 from the centromere/KTs. BEND3 depleted cells are unable to align their chromosomes at the metaphase plate and undergo apoptosis within a short time, suggesting that BEND3 regulates not only the localization of PICH and Plk1 but has additional functions. In the second part of this thesis, we demonstrate that PICH does not function in the spindle assembly checkpoint. Instead, we show that neutralization or depletion of PICH results in chromatin bridges during anaphase. We identify the ATPase activity of PICH as indispensable to prevent the formation of these bridges and show that they most likely arise from non-centromeric chromatin. PICH was previously shown to interact with the BTR complex (composed of BLM, TOP3A and RMI1) that has been linked to the faithful separation of chromosomes during mitosis. It is of great interest, therefore, that we discovered an association of PICH with Rif1, which has recently been shown to provide a DNA-binding interface for the BTR complex. In contrast to the colocalization of PICH and the BTR complex to non-centromeric UFBs, Rif1 localizes to centromeric UFBs in a PICH-dependent manner. Thus, we speculate that PICH, Rif1 and the BTR complex act together to prevent chromatin bridges during mitosis.

Funder: - Cancer Center Support Grant 5P30CA045508, Funder: - Medical Research Council (MRC_MC_UU_12022/6), Funder: - Max Planck Society - European Research Council (ERC) Consolidator Grant EMERGE (773047) - European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie action (814316) - Deutsche Forschungsgemeinschaft (SFB 834) - Cardio-Pulmonary Institute (EXC 2026, Project ID: 390649896) - DZHK (German Center for Cardiovascular Research) - Foundation Leducq Transatlantic Network - Stiftung Charité - European Molecular BiologyOrganization (EMBO) Young Investigator Programme, Endothelial cells (ECs) adapt their metabolism to enable the growth of new blood vessels, but little is known how ECs regulate metabolism to adopt a quiescent state. Here, we show that the metabolite S-2-hydroxyglutarate (S-2HG) plays a crucial role in the regulation of endothelial quiescence. We find that S-2HG is produced in ECs after activation of the transcription factor forkhead box O1 (FOXO1), where it limits cell cycle progression, metabolic activity and vascular expansion. FOXO1 stimulates S-2HG production by inhibiting the mitochondrial enzyme 2-oxoglutarate dehydrogenase. This inhibition relies on branched-chain amino acid catabolites such as 3-methyl-2-oxovalerate, which increase in ECs with activated FOXO1. Treatment of ECs with 3-methyl-2-oxovalerate elicits S-2HG production and suppresses proliferation, causing vascular rarefaction in mice. Our findings identify a metabolic programme that promotes the acquisition of a quiescent endothelial state and highlight the role of metabolites as signalling molecules in the endothelium.

The complex signaling pathways downstream of KRAS constitute collateral compensatory mechanisms that contribute to PDAC maintenance and therapy resistance. We used a mouse model with a dual recombinase system, which allows a secondary genetic deletion of Pdk1 in KrasG12D-driven tumors and thereby leads to inactivation of PI3K signaling. By combining this system with multi-directional genome-wide CRISPR screens, we delineate key effectors in PDAC signaling and identify major targetable nodes for combination therapies in addition to unexpected resistance mechanisms to PI3K inhibition. Die komplexen Signalwege ausgehend von KRAS stellen zentrale Mechanismen dar, die zur PDAC-Aufrechterhaltung und Therapieresistenz beitragen. Wir verwendeten ein Mausmodell mit einem dualen Rekombinasesystem, das eine sekundäre genetische Deletion von Pdk1 in KrasG12D-gesteuerten Tumoren ermöglicht und dadurch eine PI3K-Signalübertragung inaktiviert. Durch die Kombination dieses Systems mit multidirektionalen genomweiten CRISPR-Screens fanden wir Schlüsseleffektoren in der PDAC-Signalübertragung und identifizierten wichtige Zielmoleküle für Kombinationstherapien und Resistenzmechanismen der PI3K-Hemmung.

© 2021 Springer Nature Limited. Open Access. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/., Endothelial cells (ECs) adapt their metabolism to enable the growth of new blood vessels, but little is known how ECs regulate metabolism to adopt a quiescent state. Here, we show that the metabolite S-2-hydroxyglutarate (S-2HG) plays a crucial role in the regulation of endothelial quiescence. We find that S-2HG is produced in ECs after activation of the transcription factor forkhead box O1 (FOXO1), where it limits cell cycle progression, metabolic activity and vascular expansion. FOXO1 stimulates S-2HG production by inhibiting the mitochondrial enzyme 2-oxoglutarate dehydrogenase. This inhibition relies on branched-chain amino acid catabolites such as 3-methyl-2-oxovalerate, which increase in ECs with activated FOXO1. Treatment of ECs with 3-methyl-2-oxovalerate elicits S-2HG production and suppresses proliferation, causing vascular rarefaction in mice. Our findings identify a metabolic programme that promotes the acquisition of a quiescent endothelial state and highlight the role of metabolites as signalling molecules in the endothelium., Research in the M.P. laboratory was supported by the Max Planck Society, the European Research Council (ERC) Consolidator Grant EMERGE (773047), the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Project-ID 75732319 – SFB 834, the Leducq Foundation, the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie action (814316), the Excellence Cluster Cardio-Pulmonary Institute (EXC 2026, project ID 390649896), the DZHK (German Centre for Cardiovascular Research), the Stiftung Charité, and the European Molecular Biology Organization (EMBO) Young Investigator Programme. Work in the T.B. laboratory was supported by the DFG – Project-ID 268555672 – SFB 1213. Work in the C.F. laboratory was supported by the Medical Research Council (MRC_MC_UU_12022/6). This work was performed with assistance from the CSHL Mass Spectrometry Shared Resource, which is supported by the Cancer Center Support Grant 5P30CA045508.

Ubiquitination of invading Salmonella Typhimurium triggers autophagy of cytosolic bacteria and restricts their spread in epithelial cells. Ubiquitin (Ub) chains recruit autophagy receptors such as p62/SQSTM1, NDP52/CALCOCO and optineurin (OPTN), which initiate the formation of double-membrane autophagosomal structures and lysosomal destruction in a process known as xenophagy. Besides this, the functional consequences and mechanistic regulation of differentially linked Ub chains at the host-Salmonella interface have remained unexplored. Here, we show, for the first time, that distinct Ub chains on cytosolic S. Typhimurium serve as a platform triggering further signalling cascades. By using single-molecule localization microscopy, we visualized the balance and nanoscale distribution pattern of linear (M1-linked) Ub chain formation at the surface of cytosolic S. Typhimurium. In addition, we identified the deubiquitinase OTULIN as central regulator of these M1-linked Ub chains on the bacterial coat. OTULIN depletion leads to enhanced formation of linear Ub chains, resulting in local recruitment of NEMO, activation of IKKα/IKKβ and ultimately NF-κB, which in turn promotes secretion of pro-inflammatory cytokines and restricts bacterial proliferation. Our results establish a role for the linear Ub coat around cytosolic S. Typhimurium as the local NF-κB signalling platform and provide insights into the function of OTULIN in NF-κB activation during bacterial pathogenesis.