CD70-Targeted Allogeneic CAR T-Cell Therapy for Advanced Clear Cell Renal Cell Carcinoma.

Therapeutic approaches for clear cell renal cell carcinoma (ccRCC) remain limited; however, chimeric antigen receptor (CAR) T-cell therapies may offer novel treatment options. CTX130, an allogeneic CD70-targeting CAR T-cell product, was developed for the treatment of advanced or refractory ccRCC. We report that CTX130 showed favorable preclinical proliferation and cytotoxicity profiles and completely regressed RCC xenograft tumors. We also report results from 16 patients with relapsed/refractory ccRCC who received CTX130 in a phase I, multicenter, first-in-human clinical trial. No patients encountered dose-limiting toxicity, and disease control was achieved in 81.3% of patients. One patient remains in a durable complete response at 3 years. Finally, we report on a next-generation CAR T construct, CTX131, in which synergistic potency edits to CTX130 confer improved expansion and efficacy in preclinical studies. These data represent a proof of concept for the treatment of ccRCC and other CD70+ malignancies with CD70-targeted allogeneic CAR T cells. SIGNIFICANCE: Although the role of CAR T cells is well established in hematologic malignancies, the clinical experience in solid tumors has been disappointing. This clinical trial demonstrates the first complete response in a patient with RCC, reinforcing the potential benefit of CAR T cells in the treatment of solid tumors.

Shifts in the genetic counseling workforce highlight a need for laboratory fieldwork experience for genetic counseling trainees.

As the genetic counseling workforce experiences an increase in genetic counselors (GCs) in non-direct patient care roles, it is essential that genetic counseling students are trained in these settings. The Accreditation Council for Genetic Counseling (ACGC) standards regarding laboratory exposure have evolved over time, but laboratory fieldwork experience continues to remain a suggestion for a diversified setting. As more trainees seek laboratory exposure and an increasing number of new graduates opt for laboratory positions, learning firsthand from GCs employed in this setting is a valuable experience that should be available to all trainees. Historically, laboratory educational offerings consisted of onsite rotations for students from local training programs focused on understanding diagnostic testing methodologies and shadowing GCs. Through the years, multiple laboratories have expanded their curriculums to expose students to variant interpretation and report writing, research, client services, marketing, and product development. Alongside the growth of laboratory rotation curriculum grew opportunities for remote rotations. Prior to the COVID-19 pandemic, GeneDx offered remote education options including both individualized rotations and a webinar series. These offerings expanded due to the pandemic coupled with increased demand and have positive implications for future trainees. The evolution of the rotation also included conscious efforts to incorporate diversity, equity, and inclusion into the curriculum, as well as to improved accessibility to laboratory rotations. Notably, there are inconsistencies in laboratory rotation curricula and requirements, and a standardized evaluation and definition of competencies are lacking. ACGC guidelines defining common core concepts required from laboratory rotations would help ensure students receive an equitable minimum skill set, regardless of training site. Stakeholders in GC education should collaborate to enhance the experiences of future trainees and provide the skills needed by a workforce shifting to remote work and increasing numbers of non-direct patient-facing laboratory roles. Drawing upon our years of experience, GeneDx aims to actively contribute to discussions around these questions. Alongside other laboratories and training programs, we hope to foster further innovation surrounding the training needs of our future GC colleagues. This educational innovation illustrates an approach to helping genetic counseling students achieve competencies related to lab-based roles.

An innate ability: How do basal invertebrates manage their chronic exposure to microbes?

Homologs of mammalian innate immune sensing and downstream pathway proteins have been discovered in a variety of basal invertebrates, including cnidarians and sponges, as well as some single-celled protists. Although the structures of these proteins vary among the basal organisms, many of the activities found in their mammalian counterparts are conserved. This is especially true for the Toll-like receptor (TLR) and cGAS-STING pathways that lead to downstream activation of transcription factor NF-κB. In this short perspective, we describe the evidence that TLR and cGAS-STING signaling to NF-κB is also involved in immunity in basal animals, as well as in the maintenance of microbial symbionts. Different from terrestrial animals, immunity in many marine invertebrates might have a constitutively active state (to protect against continual exposure to resident or waterborne microbes), as well as a hyperactive state that can be induced by pathogens at both transcriptional and posttranscriptional levels. Research on basal immunity may be important for (1) understanding different approaches that organisms take to sensing and protecting against microbes, as well as in maintaining microbial symbionts; (2) the identification of novel antimicrobial effector genes and processes; and (3) the molecular pathways that are being altered in basal marine invertebrates in the face of the effects of a changing environment.

Comparison of NF-κB from the protists Capsaspora owczarzaki and Acanthoeca spectabilis reveals extensive evolutionary diversification of this transcription factor.

We provide a functional characterization of transcription factor NF-κB in protists and provide information about the evolution and diversification of this biologically important protein. We characterized NF-κB in two protists using phylogenetic, cellular, and biochemical techniques. NF-κB of the holozoan Capsaspora owczarzaki (Co) has an N-terminal DNA-binding domain and a C-terminal Ankyrin repeat (ANK) domain, and its DNA-binding specificity is more similar to metazoan NF-κB proteins than to Rel proteins. Removal of the ANK domain allows Co-NF-κB to enter the nucleus, bind DNA, and activate transcription. However, C-terminal processing of Co-NF-κB is not induced by IκB kinases in human cells. Overexpressed Co-NF-κB localizes to the cytoplasm in Co cells. Co-NF-κB mRNA and DNA-binding levels differ across three Capsaspora life stages. RNA-sequencing and GO analyses identify possible gene targets of Co-NF-κB. Three NF-κB-like proteins from the choanoflagellate Acanthoeca spectabilis (As) contain conserved Rel Homology domain sequences, but lack C-terminal ANK repeats. All three As-NF-κB proteins constitutively enter the nucleus of cells, but differ in their DNA-binding abilities, transcriptional activation activities, and dimerization properties. These results provide a basis for understanding the evolutionary origins of this key transcription factor and could have implications for the origins of regulated immunity in higher taxa.

Molecular and Biochemical Approaches to Study the Evolution of NF-κB Signaling in Basal Metazoans.

Extensive genomic and transcriptomic sequencing over the past decade has revealed NF-κB signaling pathway homologs in organisms basal to insects, for example, in members of the phyla Cnidaria (e.g., sea anemones, corals, hydra, jellyfish) and Porifera (sponges), and in several single-celled protists (e.g., Capsaspora owczarzaki, some choanoflagellates). Therefore, methods are required to study the function of NF-κB and its pathway members in early branching organisms, many of which do not have histories as model organisms. Here, we describe a combination of cellular, molecular, and biochemical techniques that have been used for studying NF-κB, and related pathway proteins, in some of these basal organisms. These methods are useful for studying the evolution of NF-κB signaling, and may be adaptable to the study of NF-κB in other non-model organisms.

Looking Down on NF-κB.

The diversified NF-κB transcription factor family has been extensively characterized in organisms ranging from flies to humans. However, homologs of NF-κB and many upstream signaling components have recently been characterized in basal phyla, including Cnidaria (sea anemones, corals, hydras, and jellyfish), Porifera (sponges), and single-celled protists, including Capsaspora owczarzaki and some choanoflagellates. Herein, we review what is known about basal NF-κBs and how that knowledge informs on the evolution and conservation of key sequences and domains in NF-κB, as well as the regulation of NF-κB activity. The structures and DNA-binding activities of basal NF-κB proteins resemble those of mammalian NF-κB p100 proteins, and their posttranslational activation appears to have aspects of both canonical and noncanonical pathways in mammals. Several studies suggest that the single NF-κB proteins found in some basal organisms have dual roles in development and immunity. Further research on NF-κB in invertebrates will reveal information about the evolutionary roots of this major signaling pathway, will shed light on the origins of regulated innate immunity, and may have relevance to our understanding of the responses of ecologically important organisms to changing environmental conditions and emerging pathogen-based diseases.

Transcription factor NF-κB in a basal metazoan, the sponge, has conserved and unique sequences, activities, and regulation.

Herein, we characterize transcription factor NF-κB from the demosponge Amphimedon queenslandica (Aq). Aq-NF-κB is most similar to NF-κB p100/p105 among vertebrate proteins, with an N-terminal DNA-binding domain, a C-terminal Ankyrin (ANK) repeat domain, and a DNA binding-site profile akin to human NF-κB proteins. Like mammalian NF-κB p100, C-terminal truncation allows nuclear translocation of Aq-NF-κB and increases its transcriptional activation activity. Expression of IκB kinases (IKKs) induces proteasome-dependent C-terminal processing of Aq-NF-κB in human cells, and processing requires C-terminal serines in Aq-NF-κB. Unlike NF-κB p100, C-terminal sequences of Aq-NF-κB do not inhibit its DNA-binding activity. Tissue of a black encrusting demosponge contains NF-κB site DNA-binding activity, as well as nuclear and processed NF-κB. Treatment of sponge tissue with LPS increases both DNA-binding activity and processing of NF-κB. A. queenslandica transcriptomes contain homologs to upstream NF-κB pathway components. This is first functional characterization of NF-κB in sponge, the most basal multicellular animal.

A conserved Toll-like receptor-to-NF-κB signaling pathway in the endangered coral Orbicella faveolata.

Herein, we characterize the Toll-like receptor (TLR)-to-NF-κB innate immune pathway of Orbicella faveolata (Of), which is an ecologically important, disease-susceptible, reef-building coral. As compared to human TLRs, the intracellular TIR domain of Of-TLR is most similar to TLR4, and it can interact in vitro with the human TLR4 adapter MYD88. Treatment of O. faveolata tissue with lipopolysaccharide, a ligand for mammalian TLR4, resulted in gene expression changes consistent with NF-κB pathway mobilization. Biochemical and cell-based assays revealed that Of-NF-κB resembles the mammalian non-canonical NF-κB protein p100 in that C-terminal truncation results in translocation of Of-NF-κB to the nucleus and increases its DNA-binding and transcriptional activation activities. Moreover, human IκB kinase (IKK) and Of-IKK can both phosphorylate conserved residues in Of-NF-κB in vitro and induce C-terminal processing of Of-NF-κB in vivo. These results are the first characterization of TLR-to-NF-κB signaling proteins in an endangered coral, and suggest that these corals have conserved innate immune pathways.

Sea anemone model has a single Toll-like receptor that can function in pathogen detection, NF-κB signal transduction, and development.

In organisms from insects to vertebrates, Toll-like receptors (TLRs) are primary pathogen detectors that activate downstream pathways, specifically those that direct expression of innate immune effector genes. TLRs also have roles in development in many species. The sea anemone Nematostella vectensis is a useful cnidarian model to study the origins of TLR signaling because its genome encodes a single TLR and homologs of many downstream signaling components, including the NF-κB pathway. We have characterized the single N. vectensis TLR (Nv-TLR) and demonstrated that it can activate canonical NF-κB signaling in human cells. Furthermore, we show that the intracellular Toll/IL-1 receptor (TIR) domain of Nv-TLR can interact with the human TLR adapter proteins MAL and MYD88. We demonstrate that the coral pathogen Vibrio coralliilyticus causes a rapidly lethal disease in N. vectensis and that heat-inactivated V. coralliilyticus and bacterial flagellin can activate a reconstituted Nv-TLR-to-NF-κB pathway in human cells. By immunostaining of anemones, we show that Nv-TLR is expressed in a subset of cnidocytes and that many of these Nv-TLR-expressing cells also express Nv-NF-κB. Additionally, the nematosome, which is a Nematostella-specific multicellular structure, expresses Nv-TLR and many innate immune pathway homologs and can engulf V. coralliilyticus Morpholino knockdown indicates that Nv-TLR also has an essential role during early embryonic development. Our characterization of this primitive TLR and identification of a bacterial pathogen for N. vectensis reveal ancient TLR functions and provide a model for studying the molecular basis of cnidarian disease and immunity.

Transcription factor NF-κB is modulated by symbiotic status in a sea anemone model of cnidarian bleaching.

Transcription factor NF-κB plays a central role in immunity from fruit flies to humans, and NF-κB activity is altered in many human diseases. To investigate a role for NF-κB in immunity and disease on a broader evolutionary scale we have characterized NF-κB in a sea anemone (Exaiptasia pallida; called Aiptasia herein) model for cnidarian symbiosis and dysbiosis (i.e., "bleaching"). We show that the DNA-binding site specificity of Aiptasia NF-κB is similar to NF-κB proteins from a broad expanse of organisms. Analyses of NF-κB and IκB kinase proteins from Aiptasia suggest that non-canonical NF-κB processing is an evolutionarily ancient pathway, which can be reconstituted in human cells. In Aiptasia, NF-κB protein levels, DNA-binding activity, and tissue expression increase when loss of the algal symbiont Symbiodinium is induced by heat or chemical treatment. Kinetic analysis of NF-κB levels following loss of symbiosis show that NF-κB levels increase only after Symbiodinium is cleared. Moreover, introduction of Symbiodinium into naïve Aiptasia larvae results in a decrease in NF-κB expression. Our results suggest that Symbiodinium suppresses NF-κB in order to enable establishment of symbiosis in Aiptasia. These results are the first to demonstrate a link between changes in the conserved immune regulatory protein NF-κB and cnidarian symbiotic status.

Methods for imaging individual cilia in living echinoid embryos.

The embryos of echinoids (sea urchins and sand dollars) serve as excellent models for studying cilia differentiation and stages of the cilia life cycle including ciliogenic initiation, growth, maintenance, and retraction. Early in echinoid development, uniform motile cilia form on all cells simultaneously but then rapidly differentiate into multiple cilia types that differ in morphology, motility, and signaling sensitivity. Metal ion treatments that shift germ layer boundaries and thereby "animalize" or "vegetalize" embryos can be used to enrich for low-abundance cilia types rendering those specialized cilia and the differentiation processes they exhibit much easier to study. The experimental advantages of having robust cilia growth and differentiation is tempered by the challenge of restraining ciliated embryos well enough to view the process of ciliogenesis live. We have developed four observation chambers as modifications of the Kiehart chamber for long-term light microscopic imaging of ciliated echinoid embryos. One of these systems employs paramagnetic beads to render ciliated larvae magnetic so they can be gently and reversibly trapped directly under the objective lens. With this magnetic trapping system, the larva can be positioned and repositioned until they achieve the orientation with the clearest view of any cilia of interest. These methods of gentle embryo restraint allow normal embryo development and the normal ciliogenic cycle and ciliary differentiation processes to continue in direct view. Sequential image series can then be collected and analyzed to quantitatively study the wide spectrum of cilia behaviors and properties that arise in developing echinoid embryos.

Conserved and distinct modes of CREB/ATF transcription factor regulation by PP2A/B56gamma and genotoxic stress.

Activating transcription factor 1 (ATF1) and the closely related proteins CREB (cyclic AMP resonse element binding protein) and CREM (cyclic AMP response element modulator) constitute a subfamily of bZIP transcription factors that play critical roles in the regulation of cellular growth, metabolism, and survival. Previous studies demonstrated that CREB is phosphorylated on a cluster of conserved Ser residues, including Ser-111 and Ser-121, in response to DNA damage through the coordinated actions of the ataxia-telangiectasia-mutated (ATM) protein kinase and casein kinases 1 and 2 (CK1/2). Here, we show that DNA damage-induced phosphorylation by ATM is a general feature of CREB and ATF1. ATF1 harbors a conserved ATM/CK cluster that is constitutively and stoichiometrically phosphorylated by CK1 and CK2 in asynchronously growing cells. Exposure to DNA damage further induced ATF1 phosphorylation on Ser-51 by ATM in a manner that required prior phosphorylation of the upstream CK residues. Hyperphosphorylated ATF1 showed a 4-fold reduced affinity for CREB-binding protein. We further show that PP2A, in conjunction with its targeting subunit B56gamma, antagonized ATM and CK1/2-dependent phosphorylation of CREB and ATF1 in cellulo. Finally, we show that CK sites in CREB are phosphorylated during cellular growth and that phosphorylation of these residues reduces the threshold of DNA damage required for ATM-dependent phosphorylation of the inhibitory Ser-121 residue. These studies define overlapping and distinct modes of CREB and ATF1 regulation by phosphorylation that may ensure concerted changes in gene expression mediated by these factors.

Non-specific in vivo inhibition of CK1 by the pyridinyl imidazole p38 inhibitors SB 203580 and SB 202190.

Small-molecule inhibitors of protein kinases have contributed immensely to our understanding of biological signaling pathways and have been exploited therapeutically for the treatment of cancers and other disease states. The pyridinyl imidazole compounds SB 203580 and SB 202190 were identified as ATP competitive antagonists of the p38 stress-activated protein kinases and have been widely used to elucidate p38-dependent cellular processes. Here, we identify SB 203580 and SB 202190 as potent inhibitors of stress-induced CREB phosphorylation on Serine 111 (Ser-111) in intact cells. Unexpectedly, we found that the inhibitory activity of SB 203580 and SB 202190 on CREB phosphorylation was independent of p38, but instead correlated with inhibition of casein kinase 1 (CK1) in vitro. The inhibition of CK1-mediated CREB phosphorylation by concentrations of pyridinyl imidazoles commonly employed to suppress p38, suggests that in some cases conclusions of p38-dependence derived solely from the use of these inhibitors may be invalid.

Potentiation of amyotrophic lateral sclerosis (ALS)-associated TDP-43 aggregation by the proteasome-targeting factor, ubiquilin 1.

TDP-43 (43-kDa TAR DNA-binding domain protein) is a major constituent of ubiquitin-positive cytoplasmic aggregates present in neurons of patients with fronto-temporal lobular dementia and amyotrophic lateral sclerosis (ALS). The pathologic significance of TDP-43 aggregation is not known; however, dominant mutations in TDP-43 cause a subset of ALS cases, suggesting that misfolding and/or altered trafficking of TDP-43 is relevant to the disease process. Here, we show that the presenilin-binding protein ubiquilin 1 (UBQLN) plays a role in TDP-43 aggregation. TDP-43 interacted with UBQLN both in yeast and in vitro, and the carboxyl-terminal ubiquitin-associated domain of UBQLN was both necessary and sufficient for binding to polyubiquitylated forms of TDP-43. Overexpression of UBQLN recruited TDP-43 to detergent-resistant cytoplasmic aggregates that colocalized with the autophagosomal marker, LC3. UBQLN-dependent aggregation required the UBQLN UBA domain, was mediated by non-overlapping regions of TDP-43, and was abrogated by a mutation in UBQLN previously linked to Alzheimer disease. Four ALS-associated alleles of TDP-43 also coaggregated with UBQLN, and the extent of aggregation correlated with in vitro UBQLN binding affinity. Our findings suggest that UBQLN is a polyubiquitin-TDP-43 cochaperone that mediates the autophagosomal delivery and/or proteasome targeting of TDP-43 aggregates.

Coregulated ataxia telangiectasia-mutated and casein kinase sites modulate cAMP-response element-binding protein-coactivator interactions in response to DNA damage.

The cyclic AMP-response element-binding protein (CREB) is a bZIP family transcription factor implicated as an oncoprotein and neuron survival factor. CREB is activated in response to cellular stimuli, including cAMP and Ca(2+), via phosphorylation of Ser-133, which promotes interaction between the kinase-inducible domain (KID) of CREB and the KID-interacting domain of CREB-binding protein (CBP). We previously demonstrated that the interaction between CREB and CBP is inhibited by DNA-damaging stimuli through a mechanism whereby CREB is phosphorylated by the ataxia telangiectasia-mutated (ATM) protein kinase. We now show that the ATM phosphorylation sites in CREB are functionally intertwined with a cluster of coregulated casein kinase (CK) sites. We demonstrate that DNA damage-induced phosphorylation of CREB occurs in three steps. The initial event in the CREB phosphorylation cascade is the phosphorylation of Ser-111, which is carried out by CK1 and CK2 under basal conditions and by ATM in response to ionizing radiation. The phosphorylation of Ser-111 triggers the CK2-dependent phosphorylation of Ser-108 and the CK1-dependent phosphorylation of Ser-114 and Ser-117. The phosphorylation of Ser-114 and Ser-117 by CK1 then renders CREB permissive for ATM-dependent phosphorylation on Ser-121. Mutation of Ser-121 alone abrogates ionizing radiation-dependent repression of CREB-CBP complexes, which can be recapitulated using a CK1 inhibitor. Our findings outline a complex mechanism of CREB phosphorylation in which coregulated ATM and CK sites control CREB transactivation potential by modulating its CBP-binding affinity. The coregulated ATM and CK sites identified in CREB may constitute a signaling motif that is common to other DNA damage-regulated substrates.