Molecular basis for substrate recruitment to the PRMT5 methylosome.

PRMT5 is an essential arginine methyltransferase and a therapeutic target in MTAP-null cancers. PRMT5 uses adaptor proteins for substrate recruitment through a previously undefined mechanism. Here, we identify an evolutionarily conserved peptide sequence shared among the three known substrate adaptors (CLNS1A, RIOK1, and COPR5) and show that it is necessary and sufficient for interaction with PRMT5. We demonstrate that PRMT5 uses modular adaptor proteins containing a common binding motif for substrate recruitment, comparable with other enzyme classes such as kinases and E3 ligases. We structurally resolve the interface with PRMT5 and show via genetic perturbation that it is required for methylation of adaptor-recruited substrates including the spliceosome, histones, and ribosomal complexes. Furthermore, disruption of this site affects Sm spliceosome activity, leading to intron retention. Genetic disruption of the PRMT5-substrate adaptor interface impairs growth of MTAP-null tumor cells and is thus a site for development of therapeutic inhibitors of PRMT5.

Engineering the IL-4/IL-13 axis for targeted immune modulation.

The structurally and functionally related interleukin-4 (IL-4) and IL-13 cytokines play pivotal roles in shaping immune activity. The IL-4/IL-13 axis is best known for its critical role in T helper 2 (Th2) cell-mediated Type 2 inflammation, which protects the host from large multicellular pathogens, such as parasitic helminth worms, and regulates immune responses to allergens. In addition, IL-4 and IL-13 stimulate a wide range of innate and adaptive immune cells, as well as non-hematopoietic cells, to coordinate various functions, including immune regulation, antibody production, and fibrosis. Due to its importance for a broad spectrum of physiological activities, the IL-4/IL-13 network has been targeted through a variety of molecular engineering and synthetic biology approaches to modulate immune behavior and develop novel therapeutics. Here, we review ongoing efforts to manipulate the IL-4/IL-13 axis, including cytokine engineering strategies, formulation of fusion proteins, antagonist development, cell engineering approaches, and biosensor design. We discuss how these strategies have been employed to dissect IL-4 and IL-13 pathways, as well as to discover new immunotherapies targeting allergy, autoimmune diseases, and cancer. Looking ahead, emerging bioengineering tools promise to continue advancing fundamental understanding of IL-4/IL-13 biology and enabling researchers to exploit these insights to develop effective interventions.

Design of cell-type-specific hyperstable IL-4 mimetics via modular de novo scaffolds.

The interleukin-4 (IL-4) cytokine plays a critical role in modulating immune homeostasis. Although there is great interest in harnessing this cytokine as a therapeutic in natural or engineered formats, the clinical potential of native IL-4 is limited by its instability and pleiotropic actions. Here, we design IL-4 cytokine mimetics (denoted Neo-4) based on a de novo engineered IL-2 mimetic scaffold and demonstrate that these cytokines can recapitulate physiological functions of IL-4 in cellular and animal models. In contrast with natural IL-4, Neo-4 is hyperstable and signals exclusively through the type I IL-4 receptor complex, providing previously inaccessible insights into differential IL-4 signaling through type I versus type II receptors. Because of their hyperstability, our computationally designed mimetics can directly incorporate into sophisticated biomaterials that require heat processing, such as three-dimensional-printed scaffolds. Neo-4 should be broadly useful for interrogating IL-4 biology, and the design workflow will inform targeted cytokine therapeutic development.

Modular complement assemblies for mitigating inflammatory conditions.

Complement protein C3dg, a key linkage between innate and adaptive immunity, is capable of stimulating both humoral and cell-mediated immune responses, leading to considerable interest in its use as a molecular adjuvant. However, the potential of C3dg as an adjuvant is limited without ways of controllably assembling multiple copies of it into vaccine platforms. Here, we report a strategy to assemble C3dg into supramolecular nanofibers with excellent compositional control, using ß-tail fusion tags. These assemblies were investigated as therapeutic active immunotherapies, which may offer advantages over existing biologics, particularly toward chronic inflammatory diseases. Supramolecular assemblies based on the Q11 peptide system containing ß-tail-tagged C3dg, B cell epitopes from TNF, and the universal T cell epitope PADRE raised strong antibody responses against both TNF and C3dg, and prophylactic immunization with these materials significantly improved protection in a lethal TNF-mediated inflammation model. Additionally, in a murine model of psoriasis induced by imiquimod, the C3dg-adjuvanted nanofiber vaccine performed as well as anti-TNF monoclonal antibodies. Nanofibers containing only ß-tail-C3dg and lacking the TNF B cell epitope also showed improvements in both models, suggesting that supramolecular C3dg, by itself, played an important therapeutic role. We observed that immunization with ß-tail-C3dg caused the expansion of an autoreactive C3dg-specific T cell population, which may act to dampen the immune response, preventing excessive inflammation. These findings indicate that molecular assemblies displaying C3dg warrant further development as active immunotherapies.

Carbon-11-Labeled Methionine PET/CT in Patients With FDG-Occult Multiple Myeloma: A Prospective Pilot Study.

In a pilot study (ClinicalTrials.gov NCT02646085), seven patients with treated multiple myeloma and negative FDG PET/CT underwent amino acid imaging with 11C-methionine PET/CT. In five participants, 11C-methionine PET/CT showed focal uptake corresponding with lytic lesions; two to 18 lesions were found (SUVmax, 2.8-6.4). Findings indicated a potential role for 11C-methionine PET/CT in detecting residual disease after negative FDG PET/CT, thereby guiding further treatment.

Second Harmonic Generation Microscopy as a Novel Intraoperative Assessment of Rat Median Nerve Injury.

PURPOSE: Nerves that are functionally injured but appear macroscopically intact pose the biggest clinical dilemma. Second Harmonic Generation (SHG) Microscopy may provide a real-time assessment of nerve damage, with the ultimate goal of allowing surgeons to accurately quantify the degree of nerve damage present. The aim of this study was to demonstrate the utility of SHG microscopy to detect nerve damage in vivo in an animal model. METHODS: Ten Sprague-Dawley rats were anesthetized and prepared for surgery. After surgical exposure and using a custom-made stretch applicator, the right median nerves were stretched by 20%, corresponding to a high strain injury, and held for 5 minutes. The left median nerve served as a sham control (SC), only being placed in the applicator for 5 minutes with no stretch. A nerve stimulator was used to assess the amount of stimulation required to induce a flicker and contraction of the paw. Nerves were then imaged using a multiphoton laser scanning microscope. RESULTS: Immediately after injury (day 0), SHG images of SC median nerves exhibited parallel collagen fibers with linear, organized alignment. In comparison with SC nerves, high strain nerves demonstrated artifacts indicative of nerve damage consisting of wavy, undulating fibers with crossing fibers and tears, as well as a decrease in the linear organization, which correlated with an increase in the mean stimulation required to induce a flicker and contraction of the paw. CONCLUSIONS: Second Harmonic Generation microscopy may provide the ability to detect an acute neural stretch injury in the rat median nerve. Epineurial collagen disorganization correlated with the stimulation required for nerve function. CLINICAL RELEVANCE: In the future, SHG may provide the ability to visualize nerve damage intraoperatively, allowing for better clinical decision-making. However, this is currently a research tool and requires further validation before translating to the clinical setting.

An Analysis of Profundus Tendon Repairs After Distal Phalanx Amputation in a Cadaveric Model of Little Finger Superficialis Deficiency.

PURPOSE: The flexor digitorum superficialis tendon to the little finger (FDS-5) has been observed to have a higher degree of functional and structural variation than the FDS of other digits. FDS-5-deficient individuals necessarily rely on the flexor digitorum profundus tendon to the little finger (FDP-5) for flexion in their little fingers. FDS-5 deficient patients who experience a considerable injury to their FDP-5 are therefore at a risk of losing substantial little finger flexion. The purpose of this study was to evaluate the degree of flexion of the little finger at the metacarpophalangeal and proximal interphalangeal (PIP) joints in a cadaveric model of FDS-5 deficiency following amputation of the distal phalanx. METHODS: Ten fresh-frozen cadaveric upper extremities with no prior trauma were used. Loads were applied to the FDP-5. Flexion at the PIP and metacarpophalangeal joints was measured in degrees with a goniometer. Little finger flexion testing was conducted under 5 different conditions: "baseline," "FDS-deficient," "no repair," "bone anchor" repair, and "A4 pulley" repair. RESULTS: The results were as follows: (1) no significant differences in the flexion between baseline and FDS-deficient conditions; (2) a significant decline in PIP flexion in the no repair condition after FDP-5 division compared with the FDS-deficient condition; (3) a significant restoration in PIP flexion in both surgical repair groups compared with the no repair group; and (4) no significant differences in PIP flexion between the A4 pulley and bone anchor groups. CONCLUSIONS: The bone anchor repair and the A4 pulley repair demonstrate similar abilities to restore flexion of the little finger at the PIP joint to baseline levels in this cadaveric model. CLINICAL RELEVANCE: A clinical protocol is yet to be established for the surgical treatment in FDS-5-deficient patients requiring amputation of the distal phalanx of the little finger. This study aims to address this area of uncertainty by comparing the little finger flexion after 2 different approaches to profundus tendon reattachment that may be applicable in this clinical scenario.

Tendon progenitor cells as biological augmentation improve functional gait and reduce scar formation after rotator cuff repair.

BACKGROUND: High rates of structural failure are reported after rotator cuff repairs due to inability to recreate the native enthesis during healing. The development of biological augmentation methods that mitigate scar formation and regenerate the enthesis is still an unmet need. Since neonatal enthesis is capable of regeneration after injury, this study tested whether delivery of neonatal tendon progenitor cells (TPCs) into the adult injured environment can enhance functional and structural supraspinatus enthesis and tendon healing. METHODS: TPCs were isolated from Ai14 Rosa26-TdTomato mouse Achilles tendons and labeled using adenovirus-Cre. Fifty-two CB57BL/6J mice underwent detachment and acute repair of the supraspinatus tendon and received either a fibrin-only or TPC-fibrin gel. Immunofluorescence analysis was carried out to determine cellularity (DAPI), fibrocartilage (SOX9), macrophages (F4/80), myofibroblasts (α-smooth muscle actin), and scar (laminin). Assays for function (gait and biomechanical testing) and structure (micro-computed tomography imaging, picrosirius red/Alcian Blue staining, type I and III collagen staining) were carried out. RESULTS: Analysis of TdTomato cells after injury showed minimal retention of TPCs by day 7 and day 14, with detected cells localized near the bursa and deltoid rather than the enthesis/tendon. However, TPC delivery led to significantly increased %Sox9+ cells in the enthesis at day 7 after injury and decreased laminin intensity across almost all time points compared to fibrin-only treatment. Similarly, TPC-treated mice showed gait recovery by day 14 (paw area and stride length) and day 28 (stance time), while fibrin-treated mice failed to recover gait parameters. Despite improved gait, biomechanical testing showed no differences between groups. Structural analysis by micro-computed tomography suggests that TPC application improves cortical thickness after surgery compared to fibrin. Superior collagen alignment at the neo-enthesis was also observed in the TPC-augmented group at day 28, but no difference was detected in type I and III collagen intensity. CONCLUSION: We found that neonatal TPCs improved and restored functional gait by reducing overall scar formation, improving enthesis collagen alignment, and altering bony composition response after supraspinatus tendon repair. TPCs did not appear to integrate into the healing tissue, suggesting improved healing may be due to paracrine effects at early stages. Future work will determine the factors secreted by TPCs to develop translational targets.

Characterizing the association between CSF biomarkers and risk for ventriculoperitoneal shunt following posterior fossa tumor resection in a case-control study.

BACKGROUND: Recalcitrant hydrocephalus necessitating permanent cerebrospinal fluid (CSF) diversion is a known complication after resection of a posterior fossa tumor (PFT). Various CSF contents, such as protein and other markers, have been variably correlated with the need for permanent CSF diversion. This study aims to evaluate which CSF laboratory values are associated with permanent CSF diversion following PFT resection in adults. METHODS: This study queried our multi-institutional database (Central Nervous System Tumor Outcome Registry at Emory; CTORE) consisting of 617 adult patients with PFT resections from 2006 to 2021. Retrospective data was collected from the 89 patients of this cohort that required EVD placement. Patients were stratified into two groups: those that required a shunt following EVD removal (n = 30) and those that did not (n = 40). CSF variables collected included glucose, protein, nucleated cell count, and presence of infection. An unadjusted logistic regression was performed to assess exposures associated with shunt requirement and unadjusted odds ratios (ORs) and their associated 95 % confidence intervals (CIs) were obtained. RESULTS: Immediately following surgery, no CSF variables were significantly associated with shunt placement. Except for post-operative CSF being not-clear (OR: 4.15 (1.47-12.56), p = 0.009) and CSF glucose (OR: 0.97 (1.03-1.07), p = 0.031) all other variables were not significantly associated with shunt at time point 2. CONCLUSION: In our retrospective analysis, most routinely collected CSF values were not associated with permanent CSF diversion via a ventriculoperitoneal shunt following PFT resection. Further research is needed to identify other potential predictive markers.

Solitary metastasis to the skull as the first sign of hepatocellular carcinoma in a patient in long-term remission.

Background: Hepatocellular carcinoma (HCC) is a common malignant tumor with a 5-year survival rate of 10%, presenting with extrahepatic metastases in 15-17% of patients. HCC-bone metastases represent approximately one-quarter of all HCC metastases, most frequently in the spine, pelvis, ribs, or femur. HCC-skull metastases, however, make up 0.4-1.6% of all HCC- bone metastases. Furthermore, solitary HCC-skull metastasis without known active primary HCC is an unusual presentation warranting further review and consideration. Case Description: Here, the authors report a unique case of a solitary HCC-skull metastasis in a patient without known active cancer but in long-term remission for HCC. The patient is a 69-year-old male with past HCC who presented with a nontender skull mass. A computed tomography scan showed a heterogeneously enhancing mass centered in the high left parietal bone with intracranial extension. There was a noted mass effect on the left posterior frontoparietal region without worrisome midline shift. Pathology ultimately revealed the mass to be metastatic HCC. To aid in the understanding and clinical management of this rare presentation, we reviewed the literature regarding clinical presentation, radiological features, pathology, and outcome. Conclusion: Ultimately, early detection of the primary source of cancer is pivotal to successful treatment and prognosis, and skull lesions such as these must include HCC in the differential diagnosis.

Recurrent Neisseria cinerea bacteremia secondary to cardiovascular implantable electronic device infection.

We present the first case of cardiac implantable electronic device (CIED) infection due to Neisseria cinerea in a 64-year-old woman from Panama. She had a history of splenectomy, aortic valve stenosis requiring transcatheter aortic valve replacement (TAVR), and permanent pacemaker placement. She presented with relapsing N. cinerea bacteremia over a 3-month period. Transesophageal echocardiography revealed a lead vegetation in the superior vena cava. She was successfully treated with pacemaker removal and 2 weeks of IV antibiotic therapy. N. cinerea is an aerobic gram-negative commensal diplococcus typically found in the human nasopharynx. Infection in humans is rare with few case reports in the literature.

Bone Disease in Multiple Myeloma: Biologic and Clinical Implications.

Multiple Myeloma (MM) is a hematologic malignancy characterized by the proliferation of monoclonal plasma cells localized within the bone marrow. Bone disease with associated osteolytic lesions is a hallmark of MM and develops in the majority of MM patients. Approximately half of patients with bone disease will experience skeletal-related events (SREs), such as spinal cord compression and pathologic fractures, which increase the risk of mortality by 20-40%. At the cellular level, bone disease results from a tumor-cell-driven imbalance between osteoclast bone resorption and osteoblast bone formation, thereby creating a favorable cellular environment for bone resorption. The use of osteoclast inhibitory therapies with bisphosphonates, such as zoledronic acid and the RANKL inhibitor denosumab, have been shown to delay and lower the risk of SREs, as well as the need for surgery or radiation therapy to treat severe bone complications. This review outlines our current understanding of the molecular underpinnings of bone disease, available therapeutic options, and highlights recent advances in the management of MM-related bone disease.

Strategies for Glycoengineering Therapeutic Proteins.

Almost all therapeutic proteins are glycosylated, with the carbohydrate component playing a long-established, substantial role in the safety and pharmacokinetic properties of this dominant category of drugs. In the past few years and moving forward, glycosylation is increasingly being implicated in the pharmacodynamics and therapeutic efficacy of therapeutic proteins. This article provides illustrative examples of drugs that have already been improved through glycoengineering including cytokines exemplified by erythropoietin (EPO), enzymes (ectonucleotide pyrophosphatase 1, ENPP1), and IgG antibodies (e.g., afucosylated Gazyva®, Poteligeo®, Fasenra™, and Uplizna®). In the future, the deliberate modification of therapeutic protein glycosylation will become more prevalent as glycoengineering strategies, including sophisticated computer-aided tools for "building in" glycans sites, acceptance of a broad range of production systems with various glycosylation capabilities, and supplementation methods for introducing non-natural metabolites into glycosylation pathways further develop and become more accessible.

Multiepitope supramolecular peptide nanofibers eliciting coordinated humoral and cellular antitumor immune responses.

Subunit vaccines inducing antibodies against tumor-specific antigens have yet to be clinically successful. Here, we use a supramolecular α-helical peptide nanofiber approach to design epitope-specific vaccines raising simultaneous B cell, CD8+ T cell, and CD4+ T cell responses against combinations of selected epitopes and show that the concurrent induction of these responses generates strong antitumor effects in mice, with significant improvements over antibody or CD8+ T cell-based vaccines alone, in both prophylactic and therapeutic subcutaneous melanoma models. Nanofiber vaccine-induced antibodies mediated in vitro tumoricidal antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). The addition of immune checkpoint and phagocytosis checkpoint blockade antibodies further improved the therapeutic effect of the nanofiber vaccines against murine melanoma. These findings highlight the potential clinical benefit of vaccine-induced antibody responses for tumor treatments, provided that they are accompanied by simultaneous CD8+ and CD4+ responses, and they illustrate a multiepitope cancer vaccine design approach using supramolecular nanomaterials.

A versatile design platform for glycoengineering therapeutic antibodies.

Manipulation of glycosylation patterns, i.e., glycoengineering, is incorporated in the therapeutic antibody development workflow to ensure clinical safety, and this approach has also been used to modulate the biological activities, functions, or pharmacological properties of antibody drugs. Whereas most existing glycoengineering strategies focus on the canonical glycans found in the constant domain of immunoglobulin G (IgG) antibodies, we report a new strategy to leverage the untapped potential of atypical glycosylation patterns in the variable domains, which naturally occur in 15% to 25% of IgG antibodies. Glycosylation sites were added to the antigen-binding regions of two functionally divergent, interleukin-2-binding monoclonal antibodies. We used computational tools to rationally install various N-glycosylation consensus sequences into the antibody variable domains, creating "glycovariants" of these molecules. Strikingly, almost all the glycovariants were successfully glycosylated at their newly installed N-glycan sites, without reduction of the antibody's native function. Importantly, certain glycovariants exhibited modified activities compared to the parent antibody, showing the potential of our glycoengineering strategy to modulate biological function of antibodies involved in multi-component receptor systems. Finally, when coupled with a high-flux sialic acid precursor, a glycovariant with two installed glycosylation sites demonstrated superior in vivo half-life. Collectively, these findings validate a versatile glycoengineering strategy that introduces atypical glycosylation into therapeutic antibodies in order to improve their efficacy and, in certain instances, modulate their activity early in the drug development process.

Systematic profiling of conditional degron tag technologies for target validation studies.

Conditional degron tags (CDTs) are a powerful tool for target validation that combines the kinetics and reversible action of pharmacological agents with the generalizability of genetic manipulation. However, successful design of a CDT fusion protein often requires a prolonged, ad hoc cycle of construct design, failure, and re-design. To address this limitation, we report here a system to rapidly compare the activity of five unique CDTs: AID/AID2, IKZF3d, dTAG, HaloTag, and SMASh. We demonstrate the utility of this system against 16 unique protein targets. We find that expression and degradation are highly dependent on the specific CDT, the construct design, and the target. None of the CDTs leads to efficient expression and/or degradation across all targets; however, our systematic approach enables the identification of at least one optimal CDT fusion for each target. To enable the adoption of CDT strategies more broadly, we have made these reagents, and a detailed protocol, available as a community resource.

Insights into the anticancer mechanisms of interleukin-15 from engineered cytokine therapies.

Innovative approaches in the field of cytokine engineering are revolutionizing the cancer therapeutic landscape. The IL-15 cytokine is particularly enticing as a cancer immunotherapy due to its natural propensity for stimulating the proliferation and activation of NK and CD8+ T cells. In a recent IL-15 engineering approach, the cytokine was conjugated to polyethylene glycol, and the resulting molecule (NKTR-255) exhibited potent antitumor activities. In this issue of the JCI, Robinson et al. mechanistically explored NKTR-255 and compared its immune profile to that of the unconjugated IL-15 cytokine. The authors found that NKTR-255 employs distinct activities on NK compared with CD8+ T cells. NKTR-255 signaling also showed less dependence on the expression of the IL-15 receptor-α (IL-15Rα) chain compared with unconjugated IL-15. Collectively, these findings will advance IL-15-based clinical therapies and, more generally, benefit the field of cancer immunotherapy.

Discovery of a First-in-Class Inhibitor of the PRMT5-Substrate Adaptor Interaction.

PRMT5 and its substrate adaptor proteins (SAPs), pICln and Riok1, are synthetic lethal dependencies in MTAP-deleted cancer cells. SAPs share a conserved PRMT5 binding motif (PBM) which mediates binding to a surface of PRMT5 distal to the catalytic site. This interaction is required for methylation of several PRMT5 substrates, including histone and spliceosome complexes. We screened for small molecule inhibitors of the PRMT5-PBM interaction and validated a compound series which binds to the PRMT5-PBM interface and directly inhibits binding of SAPs. Mode of action studies revealed the formation of a covalent bond between a halogenated pyridazinone group and cysteine 278 of PRMT5. Optimization of the starting hit produced a lead compound, BRD0639, which engages the target in cells, disrupts PRMT5-RIOK1 complexes, and reduces substrate methylation. BRD0639 is a first-in-class PBM-competitive inhibitor that can support studies of PBM-dependent PRMT5 activities and the development of novel PRMT5 inhibitors that selectively target these functions.

LUCS: a high-resolution nucleic acid sequencing tool for accurate long-read analysis of individual DNA molecules.

Nucleic acid sequence analyses are fundamental to all aspects of biological research, spanning aging, mitochondrial DNA (mtDNA) and cancer, as well as microbial and viral evolution. Over the past several years, significant improvements in DNA sequencing, including consensus sequence analysis, have proven invaluable for high-throughput studies. However, all current DNA sequencing platforms have limited utility for studies of complex mixtures or of individual long molecules, the latter of which is crucial to understanding evolution and consequences of single nucleotide variants and their combinations. Here we report a new technology termed LUCS (Long-molecule UMI-driven Consensus Sequencing), in which reads from third-generation sequencing are aggregated by unique molecular identifiers (UMIs) specific for each individual DNA molecule. This enables in-silico reconstruction of highly accurate consensus reads of each DNA molecule independent of other molecules in the sample. Additionally, use of two UMIs enables detection of artificial recombinants (chimeras). As proof of concept, we show that application of LUCS to assessment of mitochondrial genomes in complex mixtures from single cells was associated with an error rate of 1X10-4 errors/nucleotide. Thus, LUCS represents a major step forward in DNA sequencing that offers high-throughput capacity and high-accuracy reads in studies of long DNA templates and nucleotide variants in heterogenous samples.