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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.
Das adaptive Immunsystem CRISPR (engl. „clustered regularly interspaced short palindromic repeats“) revolutioniert die medizinische Grundlagenforschung. Die Einfachheit, Präzision und Vielseitigkeit der CRISPR-Technologie ermöglicht es nicht nur, Gene gezielt aus- oder einzuschalten, sondern auch zu korrigieren. Die Hoffnung richtet sich auf eine CRISPR-vermittelte Gentherapie, um krebsverursachende Mutationen gezielt zu korrigieren und somit Tumorwachstum zu verhindern oder therapieren zu können. Technisch ist dies zeitnah vorstellbar, doch ethische und rechtliche Rahmenbedingungen sollten dringend vorab geklärt werden. Die durch Gene Editing aufgeworfenen ethischen und rechtlichen Fragen werden zwar schon seit vielen Jahren diskutiert; durch die nun eingetretene rapide technische Entwicklung stellen sie sich jedoch in neuer Dringlichkeit. Eine umfassende ethische Bewertung der Erforschung und möglichen Anwendung ist daher geboten, einschließlich Fragen der Wissenschaftsethik und -kultur sowie längerfristiger potenzieller sozialer Konsequenzen der CRISPR-Technologie. Rechtlich unterliegt die Gentherapie den allgemeinen arzneimittelrechtlichen Regelungen, die Keimbahntherapie dagegen ist in Deutschland verboten. Auf Dauer und angesichts der erwartbaren weltweiten Entwicklung ist dieses Verbot jedoch zu hinterfragen. In der vorliegenden Arbeit erläutern die Autoren technische, ethische und rechtliche Aspekte des Gene Editing in der Krebsforschung und -therapie und diskutieren die daraus resultierenden Fragen: „Was kann, soll und darf gemacht werden?“.
The transcriptional regulator far upstream binding protein 1 (FUBP1) is essential for fetal and adult hematopoietic stem cell (HSC) self-renewal, and the constitutive absence of FUBP1 activity during early development leads to embryonic lethality in homozygous mutant mice. To investigate the role of FUBP1 in murine embryonic stem cells (ESCs) and in particular during differentiation into hematopoietic lineages, we generated Fubp1 knockout (KO) ESC clones using CRISPR/Cas9 technology. Although FUBP1 is expressed in undifferentiated ESCs and during spontaneous differentiation following aggregation into embryoid bodies (EBs), absence of FUBP1 did not affect ESC maintenance. Interestingly, we observed a delayed differentiation of FUBP1-deficient ESCs into the mesoderm germ layer, as indicated by impaired expression of several mesoderm markers including Brachyury at an early time point of ESC differentiation upon aggregation to EBs. Coculture experiments with OP9 cells in the presence of erythropoietin revealed a diminished differentiation capacity of Fubp1 KO ESCs into the erythroid lineage. Our data showed that FUBP1 is important for the onset of mesoderm differentiation and maturation of hematopoietic progenitor cells into the erythroid lineage, a finding that is supported by the phenotype of FUBP1-deficient mice.
During early G1 phase, Rb is exclusively mono-phosphorylated by cyclin D:Cdk4/6, generating 14 different isoforms with specific binding patterns to E2Fs and other cellular protein targets. While mono-phosphorylated Rb is dispensable for early G1 phase progression, interfering with cyclin D:Cdk4/6 kinase activity prevents G1 phase progression, questioning the role of cyclin D:Cdk4/6 in Rb inactivation. To dissect the molecular functions of cyclin D:Cdk4/6 during cell cycle entry, we generated a single cell reporter for Cdk2 activation, RB inactivation and cell cycle entry by CRISPR/Cas9 tagging endogenous p27 with mCherry. Through single cell tracing of Cdk4i cells, we identified a time-sensitive early G1 phase specific Cdk4/6-dependent phosphorylation gradient that regulates cell cycle entry timing and resides between serum-sensing and cyclin E:Cdk2 activation. To reveal the substrate identity of the Cdk4/6 phosphorylation gradient, we performed whole proteomic and phospho-proteomic mass spectrometry, and identified 147 proteins and 82 phospho-peptides that significantly changed due to Cdk4 inhibition in early G1 phase. In summary, we identified novel (non-Rb) cyclin D:Cdk4/6 substrates that connects early G1 phase functions with cyclin E:Cdk2 activation and Rb inactivation by hyper-phosphorylation.
Current technologies used to generate CRISPR/Cas gene perturbation reagents are labor intense and require multiple ligation and cloning steps. Furthermore, increasing gRNA sequence diversity negatively affects gRNA distribution, leading to libraries of heterogeneous quality. Here, we present a rapid and cloning-free mutagenesis technology that can efficiently generate covalently-closed-circular-synthesized (3Cs) CRISPR/Cas gRNA reagents and that uncouples sequence diversity from sequence distribution. We demonstrate the fidelity and performance of 3Cs reagents by tailored targeting of all human deubiquitinating enzymes (DUBs) and identify their essentiality for cell fitness. To explore high-content screening, we aimed to generate the largest up-to-date gRNA library that can be used to interrogate the coding and noncoding human genome and simultaneously to identify genes, predicted promoter flanking regions, transcription factors and CTCF binding sites that are linked to doxorubicin resistance. Our 3Cs technology enables fast and robust generation of bias-free gene perturbation libraries with yet unmatched diversities and should be considered an alternative to established technologies.
Angiogenesis, the process by which endothelial cells (ECs) form new blood vessels from existing ones, is intimately linked to the tissue’s metabolic milieu and often occurs at nutrient-deficient sites. However, ECs rely on sufficient metabolic resources to support growth and proliferation. How endothelial nutrient acquisition and usage are regulated is unknown. Here we show that these processes are instructed by Yes-associated protein 1 (YAP)/WW domain-containing transcription regulator 1 (WWTR1/TAZ)-transcriptional enhanced associate domain (TEAD): a transcriptional module whose function is highly responsive to changes in the tissue environment. ECs lacking YAP/TAZ or their transcriptional partners, TEAD1, 2 and 4 fail to divide, resulting in stunted vascular growth in mice. Conversely, activation of TAZ, the more abundant paralogue in ECs, boosts proliferation, leading to vascular hyperplasia. We find that YAP/TAZ promote angiogenesis by fuelling nutrient-dependent mTORC1 signalling. By orchestrating the transcription of a repertoire of cell-surface transporters, including the large neutral amino acid transporter SLC7A5, YAP/TAZ-TEAD stimulate the import of amino acids and other essential nutrients, thereby enabling mTORC1 activation. Dissociating mTORC1 from these nutrient inputs—elicited by the loss of Rag GTPases—inhibits mTORC1 activity and prevents YAP/TAZ-dependent vascular growth. Together, these findings define a pivotal role for YAP/TAZ-TEAD in controlling endothelial mTORC1 and illustrate the essentiality of coordinated nutrient fluxes in the vasculature.
Targeted protein degradation is a drug modality represented by compounds that recruit a target to an E3 ubiquitin ligase to promote target ubiquitination and proteasomal degradation. Historically, the field distinguishes monovalent degraders from bifunctional degraders (PROTACs) that connect target and ligase via separate binding ligands joined via a linker1–4. Here, we elucidate the mechanism of action of a PROTAC-like degrader of the transcriptional coactivator BRD4, composed of a BRD4 ligand linked to a ligand for the E3 ligase CRL4DCAF15. Using orthogonal CRISPR/Cas9 screens we identify the degrader activity is independent of DCAF15, and relies on a different CRL4 substrate receptor, DCAF16. We demonstrate an intrinsic affinity between BRD4 and DCAF16, which is dependent on the tandem bromodomains of BRD4 and further increased by the degrader without physically engaging DCAF16 in isolation. Structural characterization of the resulting ternary complex reveals both BRD4 bromodomains are bivalently engaged in cis by the degrader and are bound to DCAF16 through several interfacial BRD4-DCAF16 and degrader-DCAF16 contacts. Our findings demonstrate that intramolecularly bridging domains can confer glue-type stabilization of intrinsic target-E3 interactions, and we propose this as a general strategy to modulate the surface topology of target proteins to nucleate co-opting of E3 ligases or other cellular effector proteins for effective proximity-based pharmacology.
Long non-coding RNAs (lncRNAs) contribute to cardiac (patho)physiology. Aging is the major risk factor for cardiovascular disease with cardiomyocyte apoptosis as one underlying cause. Here, we report the identification of the aging-regulated lncRNA Sarrah (ENSMUST00000140003) that is anti-apoptotic in cardiomyocytes. Importantly, loss of SARRAH (OXCT1-AS1) in human engineered heart tissue results in impaired contractile force development. SARRAH directly binds to the promoters of genes downregulated after SARRAH silencing via RNA-DNA triple helix formation and cardiomyocytes lacking the triple helix forming domain of Sarrah show an increase in apoptosis. One of the direct SARRAH targets is NRF2, and restoration of NRF2 levels after SARRAH silencing partially rescues the reduction in cell viability. Overexpression of Sarrah in mice shows better recovery of cardiac contractile function after AMI compared to control mice. In summary, we identified the anti-apoptotic evolutionary conserved lncRNA Sarrah, which is downregulated by aging, as a regulator of cardiomyocyte survival.
Highlights
• USP32 deubiquitinates the Ragulator complex subunit LAMTOR1 at lysine (K) 20
• LAMTOR1 K20 ubiquitination impairs its binding to the vacuolar H+-ATPase
• USP32 knockout reduces mTORC1 activity and elevates autophagic flux
• Depletion of USP32 in Caenorhabditis elegans inhibits mTOR and induces autophagy
Summary
The endosomal-lysosomal system is a series of organelles in the endocytic pathway that executes trafficking and degradation of proteins and lipids and mediates the internalization of nutrients and growth factors to ensure cell survival, growth, and differentiation. Here, we reveal regulatory, non-proteolytic ubiquitin signals in this complex system that are controlled by the enigmatic deubiquitinase USP32. Knockout (KO) of USP32 in primary hTERT-RPE1 cells results among others in hyperubiquitination of the Ragulator complex subunit LAMTOR1. Accumulation of LAMTOR1 ubiquitination impairs its interaction with the vacuolar H+-ATPase, reduces Ragulator function, and ultimately limits mTORC1 recruitment. Consistently, in USP32 KO cells, less mTOR kinase localizes to lysosomes, mTORC1 activity is decreased, and autophagy is induced. Furthermore, we demonstrate that depletion of USP32 homolog CYK-3 in Caenorhabditis elegans results in mTOR inhibition and autophagy induction. In summary, we identify a control mechanism of the mTORC1 activation cascade at lysosomes via USP32-regulated LAMTOR1 ubiquitination.
The small GTPases H, K, and NRAS are molecular switches that are indispensable for proper regulation of cellular proliferation and growth. Mutations in this family of proteins are associated with cancer and result in aberrant activation of signaling processes caused by a deregulated recruitment of downstream effector proteins. In this study, we engineered novel variants of the Ras-binding domain (RBD) of the kinase CRAF. These variants bound with high affinity to the effector binding site of active Ras. Structural characterization showed how the newly identified mutations cooperate to enhance affinity to the effector binding site compared to RBDwt. The engineered RBD variants closely mimic the interaction mode of naturally occurring Ras effectors and as dominant negative affinity reagent block their activation. Experiments with cancer cells showed that expression of these RBD variants inhibits Ras signaling leading to a reduced growth and inductions of apoptosis. Using the optimized RBD variants, we stratified patient-derived colorectal cancer organoids according to Ras dependency, which showed that the presence of Ras mutations was insufficient to predict sensitivity to Ras inhibition.
