The small molecules thalidomide, lenalidomide, and pomalidomide induce the ubiquitination and proteasomal degradation of the transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) by recruiting a ...Cys
-His
(C2H2) zinc finger domain to Cereblon (CRBN), the substrate receptor of the CRL4
E3 ubiquitin ligase. We screened the human C2H2 zinc finger proteome for degradation in the presence of thalidomide analogs, identifying 11 zinc finger degrons. Structural and functional characterization of the C2H2 zinc finger degrons demonstrates how diverse zinc finger domains bind the permissive drug-CRBN interface. Computational zinc finger docking and biochemical analysis predict that more than 150 zinc fingers bind the drug-CRBN complex in vitro, and we show that selective zinc finger degradation can be achieved through compound modifications. Our results provide a rationale for therapeutically targeting transcription factors that were previously considered undruggable.
Cell-based therapies are emerging as effective agents against cancer and other diseases. As autonomous "living drugs," these therapies lack precise control. Chimeric antigen receptor (CAR) T cells ...effectively target hematologic malignancies but can proliferate rapidly and cause toxicity. We developed ON and OFF switches for CAR T cells using the clinically approved drug lenalidomide, which mediates the proteasomal degradation of several target proteins by inducing interactions between the CRL4
E3 ubiquitin ligase and a C2H2 zinc finger degron motif. We performed a systematic screen to identify "super-degron" tags with enhanced sensitivity to lenalidomide-induced degradation and used these degradable tags to generate OFF-switch degradable CARs. To create an ON switch, we engineered a lenalidomide-inducible dimerization system and developed split CARs that required both lenalidomide and target antigen for activation. Subtherapeutic lenalidomide concentrations controlled the effector functions of ON- and OFF-switch CAR T cells. In vivo, ON-switch split CARs demonstrated lenalidomide-dependent antitumor activity, and OFF-switch degradable CARs were depleted by drug treatment to limit inflammatory cytokine production while retaining antitumor efficacy. Together, the data showed that these lenalidomide-gated switches are rapid, reversible, and clinically suitable systems to control transgene function in diverse gene- and cell-based therapies.
Measles–we've become inured to its cruel, insidious impact as it kills over 100,000 children yearly because of suboptimal vaccination coverage. It does not have to be this way. A familiar, safe, ...exceptionally effective measles vaccine saves lives and permanent, global measles eradication is within reach. But now we need to be clever and courageously explore new strategies to save lives. Firstly, let us enable people to vaccinate themselves, not with a needle and syringe, but with a quick inhaled puff of dry powder vaccine. Secondly, let us provide micro-payments using digital currency to incentivize those who vaccinate themselves. Thirdly, let us leverage learnings from how our social networks guide our behaviors to further encourage self-vaccination. Fourthly, let us inspire friendly regional competition among communities vying for the highest proportion of citizens who show measles neutralizing antibodies in spot saliva samples. With global cooperation and relentless determination, we eradicated smallpox. Next up? Measles.
Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that have mutations associated with increased transmission and antibody escape have arisen over the course of the ...current pandemic. Although the current vaccines have largely been effective against past variants, the number of mutations found on the Omicron (B.1.1.529) spike protein appear to diminish the protection conferred by preexisting immunity. Using vesicular stomatitis virus (VSV) pseudoparticles expressing the spike protein of several SARS-CoV-2 variants, we evaluated the magnitude and breadth of the neutralizing antibody response over time in individuals after infection and in mRNA-vaccinated individuals. We observed that boosting increases the magnitude of the antibody response to wild-type (D614), Beta, Delta, and Omicron variants; however, the Omicron variant was the most resistant to neutralization. We further observed that vaccinated healthy adults had robust and broad antibody responses, whereas responses may have been reduced in vaccinated pregnant women, underscoring the importance of learning how to maximize mRNA vaccine responses in pregnant populations. Findings from this study show substantial heterogeneity in the magnitude and breadth of responses after infection and mRNA vaccination and may support the addition of more conserved viral antigens to existing SARS-CoV-2 vaccines.
Molecular glue compounds induce protein-protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation
. Unlike traditional enzyme inhibitors, these molecular glue ...degraders act substoichiometrically to catalyse the rapid depletion of previously inaccessible targets
. They are clinically effective and highly sought-after, but have thus far only been discovered serendipitously. Here, through systematically mining databases for correlations between the cytotoxicity of 4,518 clinical and preclinical small molecules and the expression levels of E3 ligase components across hundreds of human cancer cell lines
, we identify CR8-a cyclin-dependent kinase (CDK) inhibitor
-as a compound that acts as a molecular glue degrader. The CDK-bound form of CR8 has a solvent-exposed pyridyl moiety that induces the formation of a complex between CDK12-cyclin K and the CUL4 adaptor protein DDB1, bypassing the requirement for a substrate receptor and presenting cyclin K for ubiquitination and degradation. Our studies demonstrate that chemical alteration of surface-exposed moieties can confer gain-of-function glue properties to an inhibitor, and we propose this as a broader strategy through which target-binding molecules could be converted into molecular glues.
Abstract
Children living in war-torn and geographically remote regions often die from measles due to undervaccination. Protective community immunity could be safely improved through the comprehensive ...use of small, inexpensive, easy-to-use, dry-powder aerosolized measles vaccination inhalers. Influential local community members could be engaged to provide risk counseling and inform their peers of measles risks to inspire vaccine uptake. Vaccination by inhaled live attenuated measles vaccine has been shown to be safe and protective among several million research subjects and omits (1) needles, syringes, glass vials, and specialized disposal systems; (2) deadly vaccine reconstitution errors; (3) cold chain technology to protect temperature-sensitive vaccine; (4) vaccine wastage associated with suboptimal use of multidose vials; (5) trained vaccinators; (6) food, housing, and transportation costs associated with centralized vaccination campaigns; and (7) risk of violence to vaccinators and associated staff. All elements for inhaler-based measles vaccination are readily available. Dry-powder measles vaccine inhalers can be assembled and distributed to save lives.
Hematologic malignancies are driven by combinations of genetic lesions that have been difficult to model in human cells. We used CRISPR/Cas9 genome engineering of primary adult and umbilical cord ...blood CD34+ human hematopoietic stem and progenitor cells (HSPCs), the cells of origin for myeloid pre-malignant and malignant diseases, followed by transplantation into immunodeficient mice to generate genetic models of clonal hematopoiesis and neoplasia. Human hematopoietic cells bearing mutations in combinations of genes, including cohesin complex genes, observed in myeloid malignancies generated immunophenotypically defined neoplastic clones capable of long-term, multi-lineage reconstitution and serial transplantation. Employing these models to investigate therapeutic efficacy, we found that TET2 and cohesin-mutated hematopoietic cells were sensitive to azacitidine treatment. These findings demonstrate the potential for generating genetically defined models of human myeloid diseases, and they are suitable for examining the biological consequences of somatic mutations and the testing of therapeutic agents.
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•CD34+ cells can be edited with multiple tumor suppressor lesions with >70% efficiency•Edited CD34+ cells expand in vivo after transplantation into immunodeficient mice•CD34+ cells engineered with combinations of mutations model clonal dynamics in vivo•Engineered cells model genotype-specific therapeutic liabilities of myeloid neoplasia
Testing novel therapies for hematologic malignancies requires human cell models that reflect the combinatorial complexity of genetic mutations observed in patients. Ebert and colleagues use multiplex CRISPR/Cas9-based targeting of human hematopoietic stem and progenitor cells to model the genetics of clonal hematopoiesis and myeloid neoplasms in vivo.
Pharmacologic agents that modulate ubiquitin ligase activity to induce protein degradation are a major new class of therapeutic agents, active in a number of hematologic malignancies. However, we ...currently have a limited understanding of the determinants of activity of these agents and how resistance develops. We developed and used a novel quantitative, targeted mass spectrometry (MS) assay to determine the relative activities, kinetics, and cell-type specificity of thalidomide and 4 analogs, all but 1 of which are in clinical use or clinical trials for hematologic malignancies. Thalidomide analogs bind the CRL4CRBN ubiquitin ligase and induce degradation of particular proteins, but each of the molecules studied has distinct patterns of substrate specificity that likely underlie the clinical activity and toxicities of each drug. Our results demonstrate that the activity of molecules that induce protein degradation depends on the strength of ligase-substrate interaction in the presence of drug, the levels of the ubiquitin ligase, and the expression level of competing substrates. These findings highlight a novel mechanism of resistance to this class of drugs mediated by competition between substrates for access to a limiting pool of the ubiquitin ligase. We demonstrate that increased expression of a nonessential substrate can lead to decreased degradation of other substrates that are critical for antineoplastic activity of the drug, resulting in drug resistance. These studies provide general rules that govern drug-dependent substrate degradation and key differences between thalidomide analog activity in vitro and in vivo.
•Sensitivity to thalidomide analogs is determined by an interplay between the level of CRBN and multiple potential substrates.•Substrates compete for access to a limiting pool of the CRBN E3 ligase substrate adaptor in the presence of thalidomide analogs.
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A damaging inflammatory response is implicated in the pathogenesis of severe coronavirus disease 2019 (COVID-19), but mechanisms contributing to this response are unclear. In two prospective cohorts, ...early non-neutralizing, afucosylated immunoglobulin G (IgG) antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were associated with progression from mild to more severe COVID-19. To study the biology of afucosylated IgG immune complexes, we developed an in vivo model that revealed that human IgG-Fc-gamma receptor (FcγR) interactions could regulate inflammation in the lung. Afucosylated IgG immune complexes isolated from patients with COVID-19 induced inflammatory cytokine production and robust infiltration of the lung by immune cells. In contrast to the antibody structures that were associated with disease progression, antibodies that were elicited by messenger RNA SARS-CoV-2 vaccines were highly fucosylated and enriched in sialylation, both modifications that reduce the inflammatory potential of IgG. Vaccine-elicited IgG did not promote an inflammatory lung response. These results show that human IgG-FcγR interactions regulate inflammation in the lung and define distinct lung activities mediated by the IgG that are associated with protection against, or progression to, severe COVID-19.
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