Design principles for cyclin K molecular glue degraders.

Molecular glue degraders are an effective therapeutic modality, but their design principles are not well understood. Recently, several unexpectedly diverse compounds were reported to deplete cyclin K by linking CDK12-cyclin K to the DDB1-CUL4-RBX1 E3 ligase. Here, to investigate how chemically dissimilar small molecules trigger cyclin K degradation, we evaluated 91 candidate degraders in structural, biophysical and cellular studies and reveal all compounds acquire glue activity via simultaneous CDK12 binding and engagement of DDB1 interfacial residues, in particular Arg928. While we identify multiple published kinase inhibitors as cryptic degraders, we also show that these glues do not require pronounced inhibitory properties for activity and that the relative degree of CDK12 inhibition versus cyclin K degradation is tuneable. We further demonstrate cyclin K degraders have transcriptional signatures distinct from CDK12 inhibitors, thereby offering unique therapeutic opportunities. The systematic structure-activity relationship analysis presented herein provides a conceptual framework for rational molecular glue design.

Antiviral signalling by a cyclic nucleotide activated CRISPR protease.

CRISPR defence systems such as the well-known DNA-targeting Cas9 and the RNA-targeting type III systems are widespread in prokaryotes1,2. The latter orchestrates a complex antiviral response that is initiated through the synthesis of cyclic oligoadenylates after recognition of foreign RNA3-5. Among the large set of proteins that are linked to type III systems and predicted to bind cyclic oligoadenylates6,7, a CRISPR-associated Lon protease (CalpL) stood out to us. CalpL contains a sensor domain of the SAVED family7 fused to a Lon protease effector domain. However, the mode of action of this effector is unknown. Here we report the structure and function of CalpL and show that this soluble protein forms a stable tripartite complex with two other proteins, CalpT and CalpS, that are encoded on the same operon. After activation by cyclic tetra-adenylate (cA4), CalpL oligomerizes and specifically cleaves the MazF homologue CalpT, which releases the extracytoplasmic function σ factor CalpS from the complex. Our data provide a direct connection between CRISPR-based detection of foreign nucleic acids and transcriptional regulation. Furthermore, the presence of a SAVED domain that binds cyclic tetra-adenylate in a CRISPR effector reveals a link to the cyclic-oligonucleotide-based antiphage signalling system.

IKKß primes inflammasome formation by recruiting NLRP3 to the trans-Golgi network.

The NLRP3 inflammasome plays a central role in antimicrobial defense as well as in the context of sterile inflammatory conditions. NLRP3 activity is governed by two independent signals: the first signal primes NLRP3, rendering it responsive to the second signal, which then triggers inflammasome formation. Our understanding of how NLRP3 priming contributes to inflammasome activation remains limited. Here, we show that IKKß, a kinase activated during priming, induces recruitment of NLRP3 to phosphatidylinositol-4-phosphate (PI4P), a phospholipid enriched on the trans-Golgi network. NEK7, a mitotic spindle kinase that had previously been thought to be indispensable for NLRP3 activation, was redundant for inflammasome formation when IKKß recruited NLRP3 to PI4P. Studying iPSC-derived human macrophages revealed that the IKKß-mediated NEK7-independent pathway constitutes the predominant NLRP3 priming mechanism in human myeloid cells. Our results suggest that PI4P binding represents a primed state into which NLRP3 is brought by IKKß activity.

Myoglobin regulates fatty acid trafficking and lipid metabolism in mammary epithelial cells.

Myoglobin (MB) is known to bind and deliver oxygen in striated muscles at high expression levels. MB is also expressed at much reduced levels in mammary epithelial cells, where the protein´s function is unclear. In this study, we aim to determine whether MB impacts fatty acid trafficking and facilitates aerobic fatty acid ß-oxidation in mammary epithelial cells. We utilized MB-wildtype versus MB-knockout mice and human breast cancer cells to examine the impact of MB and its oxygenation status on fatty acid metabolism in mouse milk and mammary epithelia. MB deficient cells were generated through CRISPR/Cas9 and TALEN approaches and exposed to various oxygen tensions. Fatty acid profiling of milk and cell extracts were performed along with cell labelling and immunocytochemistry. Our findings show that MB expression in mammary epithelial cells promoted fatty acid oxidation while reducing stearyl-CoA desaturase activity for lipogenesis. In cells and milk product, presence of oxygenated MB significantly elevated indices of limited fatty acid ß-oxidation, i.e., the organelle-bound removal of a C2 moiety from long-chain saturated or monounsaturated fatty acids, thus shifting the composition toward more saturated and shorter fatty acid species. Presence of the globin also increased cytoplasmic fatty acid solubility under normoxia and fatty acid deposition to lipid droplets under severe hypoxia. We conclude that MB can function in mammary epithelia as intracellular O2-dependent shuttle of oxidizable fatty acid substrates. MB's impact on limited oxidation of fatty acids could generate inflammatory mediator lipokines, such as 7-hexadecenoate. Thus, the novel functions of MB in breast epithelia described herein range from controlling fatty acid turnover and homeostasis to influencing inflammatory signalling cascade. Future work is needed to analyse to what extent these novel roles of MB also apply to myocytic cell physiology and malignant cell behaviour, respectively.

LAMP-Seq enables sensitive, multiplexed COVID-19 diagnostics using molecular barcoding.

Frequent testing of large population groups combined with contact tracing and isolation measures will be crucial for containing Coronavirus Disease 2019 outbreaks. Here we present LAMP-Seq, a modified, highly scalable reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. Unpurified biosamples are barcoded and amplified in a single heat step, and pooled products are analyzed en masse by sequencing. Using commercial reagents, LAMP-Seq has a limit of detection of ~2.2 molecules per µl at 95% confidence and near-perfect specificity for severe acute respiratory syndrome coronavirus 2 given its sequence readout. Clinical validation of an open-source protocol with 676 swab samples, 98 of which were deemed positive by standard RT-qPCR, demonstrated 100% sensitivity in individuals with cycle threshold values of up to 33 and a specificity of 99.7%, at a very low material cost. With a time-to-result of fewer than 24 h, low cost and little new infrastructure requirement, LAMP-Seq can be readily deployed for frequent testing as part of an integrated public health surveillance program.

Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo-electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.

Diverse enzymatic activities mediate antiviral immunity in prokaryotes.

Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses.

Highly Parallel Profiling of Cas9 Variant Specificity.

Determining the off-target cleavage profile of programmable nucleases is an important consideration for any genome editing experiment, and a number of Cas9 variants have been reported that improve specificity. We describe here tagmentation-based tag integration site sequencing (TTISS), an efficient, scalable method for analyzing double-strand breaks (DSBs) that we apply in parallel to eight Cas9 variants across 59 targets. Additionally, we generated thousands of other Cas9 variants and screened for variants with enhanced specificity and activity, identifying LZ3 Cas9, a high specificity variant with a unique +1 insertion profile. This comprehensive comparison reveals a general trade-off between Cas9 activity and specificity and provides information about the frequency of generation of +1 insertions, which has implications for correcting frameshift mutations.

Optical Pooled Screens in Human Cells.

Genetic screens are critical for the systematic identification of genes underlying cellular phenotypes. Pooling gene perturbations greatly improves scalability but is not compatible with imaging of complex and dynamic cellular phenotypes. Here, we introduce a pooled approach for optical genetic screens in mammalian cells. We use targeted in situ sequencing to demultiplex a library of genetic perturbations following image-based phenotyping. We screened a set of 952 genes across millions of cells for involvement in nuclear factor κB (NF-κB) signaling by imaging the translocation of RelA (p65) to the nucleus. Screening at a single time point across 3 cell lines recovered 15 known pathway components, while repeating the screen with live-cell imaging revealed a role for Mediator complex subunits in regulating the duration of p65 nuclear retention. These results establish a highly multiplexed approach to image-based screens of spatially and temporally defined phenotypes with pooled libraries.

RNA-guided DNA insertion with CRISPR-associated transposases.

CRISPR-Cas nucleases are powerful tools for manipulating nucleic acids; however, targeted insertion of DNA remains a challenge, as it requires host cell repair machinery. Here we characterize a CRISPR-associated transposase from cyanobacteria Scytonema hofmanni (ShCAST) that consists of Tn7-like transposase subunits and the type V-K CRISPR effector (Cas12k). ShCAST catalyzes RNA-guided DNA transposition by unidirectionally inserting segments of DNA 60 to 66 base pairs downstream of the protospacer. ShCAST integrates DNA into targeted sites in the Escherichia coli genome with frequencies of up to 80% without positive selection. This work expands our understanding of the functional diversity of CRISPR-Cas systems and establishes a paradigm for precision DNA insertion.

The DNA Inflammasome in Human Myeloid Cells Is Initiated by a STING-Cell Death Program Upstream of NLRP3.

Detection of cytosolic DNA constitutes a central event in the context of numerous infectious and sterile inflammatory conditions. Recent studies have uncovered a bipartite mode of cytosolic DNA recognition, in which the cGAS-STING axis triggers antiviral immunity, whereas AIM2 triggers inflammasome activation. Here, we show that AIM2 is dispensable for DNA-mediated inflammasome activation in human myeloid cells. Instead, detection of cytosolic DNA by the cGAS-STING axis induces a cell death program initiating potassium efflux upstream of NLRP3. Forward genetics identified regulators of lysosomal trafficking to modulate this cell death program, and subsequent studies revealed that activated STING traffics to the lysosome, where it triggers membrane permeabilization and thus lysosomal cell death (LCD). Importantly, the cGAS-STING-NLRP3 pathway constitutes the default inflammasome response during viral and bacterial infections in human myeloid cells. We conclude that targeting the cGAS-STING-LCD-NLRP3 pathway will ameliorate pathology in inflammatory conditions that are associated with cytosolic DNA sensing.

MAPK Signaling and Inflammation Link Melanoma Phenotype Switching to Induction of CD73 during Immunotherapy.

Evolution of tumor cell phenotypes promotes heterogeneity and therapy resistance. Here we found that induction of CD73, the enzyme that generates immunosuppressive adenosine, is linked to melanoma phenotype switching. Activating MAPK mutations and growth factors drove CD73 expression, which marked both nascent and full activation of a mesenchymal-like melanoma cell state program. Proinflammatory cytokines like TNFα cooperated with MAPK signaling through the c-Jun/AP-1 transcription factor complex to activate CD73 transcription by binding to an intronic enhancer. In a mouse model of T-cell immunotherapy, CD73 was induced in relapse melanomas, which acquired a mesenchymal-like phenotype. We also detected CD73 upregulation in melanoma patients progressing under adoptive T-cell transfer or immune checkpoint blockade, arguing for an adaptive resistance mechanism. Our work substantiates CD73 as a target to combine with current immunotherapies, but its dynamic regulation suggests limited value of CD73 pretreatment expression as a biomarker to stratify melanoma patients. Cancer Res; 77(17); 4697-709. ©2017 AACR.

Disruptive non-disruptive applications of CRISPR/Cas9.

The bacterial type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR Associated (Cas) systems, and in particular Streptococcus pyogenes CRISPR-Cas9, have been broadly applied to edit the genome of bacterial and eukaryotic cells. Cas9, which is an RNA-guided programmable nuclease, is a powerful tool for disrupting protein-coding genes. Cas9 cleaves target sites to generate a double-strand break (DSB) that is repaired via an error-prone repair process, leading to insertion/deletion mutations and gene knockouts. However, Cas9 can also be used to modulate genome function without gene disruption, enabling base editing, transcriptional and epigenetic reprogramming, genome imaging, cellular barcoding, genetic recording, and genetic computation.

The Second-Generation Exportin-1 Inhibitor KPT-8602 Demonstrates Potent Activity against Acute Lymphoblastic Leukemia.

Purpose: Human exportin-1 (XPO1) is the key nuclear-cytoplasmic transport protein that exports different cargo proteins out of the nucleus. Inducing nuclear accumulation of these proteins by inhibiting XPO1 causes cancer cell death. First clinical validation of pharmacological inhibition of XPO1 was obtained with the Selective Inhibitor of Nuclear Export (SINE) compound selinexor (KPT-330) demonstrating activity in phase-II/IIb clinical trials when dosed 1 to 3 times weekly. The second-generation SINE compound KPT-8602 shows improved tolerability and can be dosed daily. Here, we investigate and validate the drug-target interaction of KPT-8602 and explore its activity against acute lymphoblastic leukemia (ALL).Experimental Design: We examined the effect of KPT-8602 on XPO1 function and XPO1-cargo as well as on a panel of leukemia cell lines. Mutant XPO1 leukemia cells were designed to validate KPT-8602's drug-target interaction. In vivo, anti-ALL activity was measured in a mouse ALL model and patient-derived ALL xenograft models.Results: KPT-8602 induced caspase-dependent apoptosis in a panel of leukemic cell lines in vitro Using CRISPR/Cas9 genome editing, we demonstrated the specificity of KPT-8602 for cysteine 528 in the cargo-binding groove of XPO1 and validated the drug target interaction. In vivo, KPT-8602 showed potent anti-leukemia activity in a mouse ALL model as well as in patient-derived T- and B-ALL xenograft models without affecting normal hematopoiesis.Conclusions: KPT-8602 is highly specific for XPO1 inhibition and demonstrates potent anti-leukemic activity supporting clinical application of the second-generation SINE compound for the treatment of ALL. Clin Cancer Res; 23(10); 2528-41. ©2016 AACR.

CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism.

The site-specific insertion of heterologous genetic material into genomes provides a powerful means to study gene function. Here we describe a modular system entitled CRISPaint (CRISPR-assisted insertion tagging) that allows precise and efficient integration of large heterologous DNA cassettes into eukaryotic genomes. CRISPaint makes use of the CRISPR-Cas9 system to introduce a double-strand break (DSB) at a user-defined genomic location. A universal donor DNA, optionally provided as minicircle DNA, is cleaved simultaneously to be integrated at the genomic DSB, while processing the donor plasmid at three possible positions allows flexible reading-frame selection. Applying this system allows to create C-terminal tag fusions of endogenously encoded proteins in human cells with high efficiencies. Knocking out known DSB repair components reveals that site-specific insertion is completely dependent on canonical NHEJ (DNA-PKcs, XLF and ligase-4). A large repertoire of modular donor vectors renders CRISPaint compatible with a wide array of applications.

Human Monocytes Engage an Alternative Inflammasome Pathway.

Interleukin-1ß (IL-1ß) is a cytokine whose bioactivity is controlled by activation of the inflammasome. However, in response to lipopolysaccharide, human monocytes secrete IL-1ß independently of classical inflammasome stimuli. Here, we report that this constituted a species-specific response that is not observed in the murine system. Indeed, in human monocytes, lipopolysaccharide triggered an "alternative inflammasome" that relied on NLRP3-ASC-caspase-1 signaling, yet was devoid of any classical inflammasome characteristics including pyroptosome formation, pyroptosis induction, and K(+) efflux dependency. Genetic dissection of the underlying signaling pathway in a monocyte transdifferentiation system revealed that alternative inflammasome activation was propagated by TLR4-TRIF-RIPK1-FADD-CASP8 signaling upstream of NLRP3. Importantly, involvement of this signaling cascade was limited to alternative inflammasome activation and did not extend to classical NLRP3 activation. Because alternative inflammasome activation embraces both sensitivity and promiscuity of TLR4, we propose a pivotal role for this signaling cascade in TLR4-driven, IL-1ß-mediated immune responses and immunopathology in humans.

Designer Nuclease-Mediated Generation of Knockout THP1 Cells.

Recent developments in the field of designer nucleases allow the efficient and specific manipulation of genomic architectures in eukaryotic cell lines. To this end, it has become possible to introduce DNA double strand breaks (DSBs) at user-defined genomic loci. If located in critical coding regions of genes, thus induced DSBs can lead to insertions or deletions (indels) that result in frameshift mutations and thereby the knockout of the target gene. In this chapter, we describe a step-by-step workflow for establishing knockout cell clones of the difficult-to-transfect suspension cell line THP1. The here described protocol encompasses electroporation, cell cloning, and a deep sequencing-based genotyping step that allows the in-parallel analysis of 96 cell clones per gene of interest. Furthermore, we describe the use of the analysis tool OutKnocker that allows rapid identification of cell clones with all-allelic frameshift mutations.

A Genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Screen Identifies NEK7 as an Essential Component of NLRP3 Inflammasome Activation.

Inflammasomes are high molecular weight protein complexes that assemble in the cytosol upon pathogen encounter. This results in caspase-1-dependent pro-inflammatory cytokine maturation, as well as a special type of cell death, known as pyroptosis. The Nlrp3 inflammasome plays a pivotal role in pathogen defense, but at the same time, its activity has also been implicated in many common sterile inflammatory conditions. To this effect, several studies have identified Nlrp3 inflammasome engagement in a number of common human diseases such as atherosclerosis, type 2 diabetes, Alzheimer disease, or gout. Although it has been shown that known Nlrp3 stimuli converge on potassium ion efflux upstream of Nlrp3 activation, the exact molecular mechanism of Nlrp3 activation remains elusive. Here, we describe a genome-wide CRISPR/Cas9 screen in immortalized mouse macrophages aiming at the unbiased identification of gene products involved in Nlrp3 inflammasome activation. We employed a FACS-based screen for Nlrp3-dependent cell death, using the ionophoric compound nigericin as a potassium efflux-inducing stimulus. Using a genome-wide guide RNA (gRNA) library, we found that targeting Nek7 rescued macrophages from nigericin-induced lethality. Subsequent studies revealed that murine macrophages deficient in Nek7 displayed a largely blunted Nlrp3 inflammasome response, whereas Aim2-mediated inflammasome activation proved to be fully intact. Although the mechanism of Nek7 functioning upstream of Nlrp3 yet remains elusive, these studies provide a first genetic handle of a component that specifically functions upstream of Nlrp3.