A new class of antibodies that overcomes a steric barrier to cross-group neutralization of influenza viruses.

Antibody titers that inhibit the influenza virus hemagglutinin (HA) from engaging its receptor are the accepted correlate of protection from infection. Many potent antibodies with broad, intra-subtype specificity bind HA at the receptor binding site (RBS). One barrier to broad H1-H3 cross-subtype neutralization is an insertion (133a) between positions 133 and 134 on the rim of the H1 HA RBS. We describe here a class of antibodies that overcomes this barrier. These genetically unrestricted antibodies are abundant in the human B cell memory compartment. Analysis of the affinities of selected members of this class for historical H1 and H3 isolates suggest that they were elicited by H3 exposure and broadened or diverted by later exposure(s) to H1 HA. RBS mutations in egg-adapted vaccine strains cause the new H1 specificity of these antibodies to depend on the egg adaptation. The results suggest that suitable immunogens might elicit 133a-independent, H1-H3 cross neutralization by RBS-directed antibodies.

Structural basis of paralog-specific KDM2A/B nucleosome recognition.

The nucleosome acidic patch is a major interaction hub for chromatin, providing a platform for enzymes to dock and orient for nucleosome-targeted activities. To define the molecular basis of acidic patch recognition proteome wide, we performed an amino acid resolution acidic patch interactome screen. We discovered that the histone H3 lysine 36 (H3K36) demethylase KDM2A, but not its closely related paralog, KDM2B, requires the acidic patch for nucleosome binding. Despite fundamental roles in transcriptional repression in health and disease, the molecular mechanisms governing nucleosome substrate specificity of KDM2A/B, or any related JumonjiC (JmjC) domain lysine demethylase, remain unclear. We used a covalent conjugate between H3K36 and a demethylase inhibitor to solve cryogenic electron microscopy structures of KDM2A and KDM2B trapped in action on a nucleosome substrate. Our structures show that KDM2-nucleosome binding is paralog specific and facilitated by dynamic nucleosomal DNA unwrapping and histone charge shielding that mobilize the H3K36 sequence for demethylation.

Multivalent DNA and nucleosome acidic patch interactions specify VRK1 mitotic localization and activity.

A key role of chromatin kinases is to phosphorylate histone tails during mitosis to spatiotemporally regulate cell division. Vaccinia-related kinase 1 (VRK1) is a serine-threonine kinase that phosphorylates histone H3 threonine 3 (H3T3) along with other chromatin-based targets. While structural studies have defined how several classes of histone-modifying enzymes bind to and function on nucleosomes, the mechanism of chromatin engagement by kinases is largely unclear. Here, we paired cryo-electron microscopy with biochemical and cellular assays to demonstrate that VRK1 interacts with both linker DNA and the nucleosome acidic patch to phosphorylate H3T3. Acidic patch binding by VRK1 is mediated by an arginine-rich flexible C-terminal tail. Homozygous missense and nonsense mutations of this acidic patch recognition motif in VRK1 are causative in rare adult-onset distal spinal muscular atrophy. We show that these VRK1 mutations interfere with nucleosome acidic patch binding, leading to mislocalization of VRK1 during mitosis, thus providing a potential new molecular mechanism for pathogenesis.

A phase 1 study with dose expansion of the CDK inhibitor dinaciclib (SCH 727965) in combination with epirubicin in patients with metastatic triple negative breast cancer.

PURPOSE: Low molecular weight cyclin E (LMW-E) isoforms, overexpressed in a majority (~70 %) of triple-negative breast cancers (TNBC), were found in preclinical models to mediate tumorigenesis through binding and activation of CDK2. CDK1/CDK2 inhibitors, such as dinaciclib, combined with anthracyclines, were synergistic in decreasing viability of TNBC cell lines. Based on this data, a phase 1 study was conducted to determine the maximum tolerated dose of dinaciclib in combination with epirubicin in patients with metastatic TNBC. METHODS: Cohorts of at least 2 patients were treated with escalating doses of dinaciclib given on day 1 followed by standard dose of epirubicin given on day 2 of a 21 day cycle. No intra-patient dose escalation was allowed. An adaptive accrual design based upon toxicity during cycle 1 determined entry into therapy cohorts. The target acceptable dose limiting toxicity (DLT) to advance to the next treatment level was 30 %. RESULTS: Between 9/18/2012 and 7/18/2013, 9 patients were enrolled and treated at MD Anderson Cancer Center. DLTs included febrile neutropenia (grade 3, n = 2), syncope (grade 3, n = 2) and vomiting (grade 3, n = 1). Dose escalation did not proceed past the second cohort due to toxicity. After further accrual, the first dose level was also found to be too toxic. No treatment responses were noted, median time to progression was 5.5 weeks (range 3-12 weeks). Thus, accrual was stopped rather than explore the -1 dose level. CONCLUSION: The combination of dinaciclib and epirubicin is associated with substantial toxicities and does not appear to be an effective treatment option for TNBC.

Managing Gastrointestinal Symptoms Resulting from Treatment with Trofinetide for Rett Syndrome: Caregiver and Nurse Perspectives.

Rett syndrome (RTT) is a rare genetic neurodevelopmental disorder mainly affecting female individuals. Trofinetide was recently approved as the first treatment for RTT, largely on the basis of results from the phase 3 LAVENDER trial, in which trofinetide showed improvements in core symptoms of RTT compared with placebo. However, gastrointestinal (GI) symptoms such as diarrhea and vomiting were commonly reported side effects, and taste was also a reported issue. The objective of this article is to describe the perspectives of five caregivers of girls in trofinetide clinical trials as well as those of three nurse trial coordinators, with a focus on management of GI symptoms of trofinetide treatment.Audio Abstract available for this article. Audio Abstract: Jane Lane provides an overview and discusses key findings of the article titled "Managing Gastrointestinal Symptoms Resulting from Treatment with Trofinetide for Rett Syndrome: Caregiver and Nurse Perspectives." (MP4 83274 KB).

A protective and broadly binding antibody class engages the influenza virus hemagglutinin head at its stem interface.

Influenza infection and vaccination impart strain-specific immunity that protects against neither seasonal antigenic variants nor the next pandemic. However, antibodies directed to conserved sites can confer broad protection. Here we identify and characterize a class of human antibodies that engage a previously undescribed, conserved epitope on the influenza hemagglutinin (HA) protein. Prototype antibody S8V1-157 binds at the normally occluded interface between the HA head and stem. Antibodies to this HA head-stem interface epitope are non-neutralizing in vitro but protect against lethal influenza infection in mice. Antibody isotypes that direct clearance of infected cells enhance this protection. Head-stem interface antibodies bind to most influenza A serotypes and seasonal human variants, and are present at low frequencies in the memory B cell populations of multiple human donors. Vaccines designed to elicit these antibodies might contribute to "universal" influenza immunity.

Time Resolved-Fluorescence Resonance Energy Transfer platform for quantitative nucleosome binding and footprinting.

Quantitative analysis of chromatin protein-nucleosome interactions is essential to understand regulation of genome-templated processes. However, current methods to measure nucleosome interactions are limited by low throughput, low signal-to-noise, and/or the requirement for specialized instrumentation. Here, we report a Lanthanide Chelate Excite Time-Resolved Fluorescence Resonance Energy Transfer (LANCE TR-FRET) assay to efficiently quantify chromatin protein-nucleosome interactions. The system makes use of commercially available reagents, offers robust signal-to-noise with minimal sample requirements, uses a conventional fluorescence microplate reader, and can be adapted for high-throughput workflows. We determined the nucleosome-binding affinities of several chromatin proteins and complexes, which are consistent with measurements obtained through orthogonal biophysical methods. We also developed a TR-FRET competition assay for high-resolution footprinting of chromatin protein-nucleosome interactions. Finally, we set up a TR-FRET competition assay using the LANA peptide to quantitate nucleosome acidic patch binding. We applied this assay to establish a proof-of-principle for regulation of nucleosome acidic patch binding by methylation of chromatin protein arginine anchors. Overall, our TR-FRET assays allow facile, high-throughput quantification of chromatin interactions and are poised to complement mechanistic chromatin biochemistry, structural biology, and drug discovery programs.

The discovery of novel calcium sensing receptor negative allosteric modulators.

The design and profile of a series of zwitterionic calcium sensing receptor negative allosteric modulators is described. Evaluation of key analogues using a rat model demonstrate a robust response, significantly improved potency over ronacaleret and have the potential as an oral, anabolic treatment for osteoporosis.

BMP4 supports self-renewal of embryonic stem cells by inhibiting mitogen-activated protein kinase pathways.

The fate of pluripotent stem cells is tightly controlled during early embryonic development. Both the derivation and the maintenance of embryonic stem cells (ES cells) in vitro depend on feeder cell-derived growth factors that are largely unidentified. To dissect the mechanisms governing pluripotency, we conducted a screen to identify factors that are produced by mouse embryonic fibroblast STO cells and are required to maintain the pluripotency of ES cells. One of the factors is bone morphogenetic protein 4 (BMP4). Unexpectedly, the major effect of BMP4 on the self-renewal of ES cells is accomplished by means of the inhibition of both extracellular receptor kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) pathways, and inhibitors of ERK and p38 MAPKs mimic the effect of BMP4 on ES cells. Importantly, inhibition of the p38 MAPK pathway by SB203580 overcomes the block in deriving ES cells from blastocysts lacking a functional Alk3, the BMP type IA receptor. These results uncover a paradigm for BMP signaling in the biology of pluripotent stem cells.