A randomized, non-comparative phase 2 study of neoadjuvant immune-checkpoint blockade in retroperitoneal dedifferentiated liposarcoma and extremity/truncal undifferentiated pleomorphic sarcoma.

Based on the demonstrated clinical activity of immune-checkpoint blockade (ICB) in advanced dedifferentiated liposarcoma (DDLPS) and undifferentiated pleomorphic sarcoma (UPS), we conducted a randomized, non-comparative phase 2 trial ( NCT03307616 ) of neoadjuvant nivolumab or nivolumab/ipilimumab in patients with resectable retroperitoneal DDLPS (n = 17) and extremity/truncal UPS (+ concurrent nivolumab/radiation therapy; n = 10). The primary end point of pathologic response (percent hyalinization) was a median of 8.8% in DDLPS and 89% in UPS. Secondary end points were the changes in immune infiltrate, radiographic response, 12- and 24-month relapse-free survival and overall survival. Lower densities of regulatory T cells before treatment were associated with a major pathologic response (hyalinization > 30%). Tumor infiltration by B cells was increased following neoadjuvant treatment and was associated with overall survival in DDLPS. B cell infiltration was associated with higher densities of regulatory T cells before treatment, which was lost upon ICB treatment. Our data demonstrate that neoadjuvant ICB is associated with complex immune changes within the tumor microenvironment in DDLPS and UPS and that neoadjuvant ICB with concurrent radiotherapy has significant efficacy in UPS.

Morphologic and Molecular Characteristics of De Novo AML With JAK2 V617F Mutation.

Background:JAK2 V617F mutation (mut) in acute myeloid leukemia (AML) is rare. We describe the clinicopathologic findings of a single-institution series of 11 de novo AML cases with JAK2 V617. Methods: We identified cases of de novo AML with JAK2 V617F over a 10-year period. We reviewed diagnostic peripheral blood and bone marrow (BM) morphologic, cytogenetic, and molecular studies, including next-generation sequencing. The control group consisted of 12 patients with JAK2 wild-type (wt) AML matched for age, sex, and diagnosis. Results: We identified 11 patients (0.5%) with JAK2 V617F, with a median age at diagnosis of 72.5 years (range, 36-90 years). Ten neoplasms were classified as AML with myelodysplasia-related changes and 1 as AML with t(8;21)(q22;q22). All JAK2mut AML cases showed at least bilineage dysplasia, 7 of 11 showed fibrosis, 8 of 11 had an abnormal karyotype, and 5 had deletions or monosomy of chromosomes 5 and 7. Using the European LeukemiaNet (ELN) classification, 9 patients (82%) with JAK2mut AML were intermediate-2 and adverse risk. Cases of JAK2mut AML did not have mutations in other activating signaling pathways (P=.013); 7 (64%) showed additional mutations in at least one gene involving DNA methylation and/or epigenetic modification. Patients with JAK2mut AML had a significantly higher median BM granulocyte percentage (12% vs 3.5%; P=.006) and a higher frequency of ELN intermediate-2 and adverse risk cytogenetics (P=.04) compared with those with JAK2wt AML. JAK2mut AML showed higher circulating blasts, but this difference was not significant (17% vs 5.5%; P=not significant). No difference was seen in the median overall survival rate of patients with JAK2mut AML versus those with JAK2wt AML (14 vs 13.5 months, respectively). Conclusions: De novo JAK2mut AML is rare and frequently found in patients with dysplasia, BM fibrosis, and abnormal karyotype with intermediate- or high-risk features; gene mutations in DNA methylation and epigenetic-modifying pathways; and absence of gene mutations in activating signaling pathways.

The Transmembrane Domain of a Bicomponent ABC Transporter Exhibits Channel-Forming Activity.

Pseudomonas aeruginosa is an opportunistic pathogen that expresses two unique forms of lipopolysaccharides (LPSs) on its bacterial surface, the A- and B-bands. The A-band polysaccharides (A-band PSs) are thought to be exported into the periplasm via a bicomponent ATP-binding cassette (ABC) transporter located within the inner membrane. This ABC protein complex consists of the transmembrane (TMD) Wzm and nucleotide-binding (NBD) Wzt domain proteins. Here, we were able to probe ∼1.36 nS-average conductance openings of the Wzm-based protein complex when reconstituted into a lipid membrane buffered by a 200 mM KCl solution, demonstrating the large-conductance, channel-forming ability of the TMDs. In agreement with this finding, transmission electron microscopy (TEM) imaging revealed the ring-shaped structure of the transmembrane Wzm protein complex. As hypothesized, using liposomes, we demonstrated that Wzm interacts with Wzt. Further, the Wzt polypeptide indeed hydrolyzed ATP but exhibited a ∼75% reduction in the ATPase activity when its Walker A domain was deleted. The distribution and average unitary conductance of the TMD Wzm protein complex were altered by the presence of the NBD Wzt protein, confirming the regulatory role of the latter polypeptide. To our knowledge, the large-conductance, channel-like activity of the Wzm protein complex, although often hypothesized, has not previously been demonstrated. These results constitute a platform for future structural, biophysical, and functional explorations of this bicomponent ABC transporter.

Global redesign of a native ß-barrel scaffold.

One persistent challenge in membrane protein design is accomplishing extensive modifications of proteins without impairing their functionality. A truncation derivative of the ferric hydroxamate uptake component A (FhuA), which featured the deletion of the 160-residue cork domain and five large extracellular loops, produced the conversion of a non-conductive, monomeric, 22-stranded ß-barrel protein into a large-conductance protein pore. Here, we show that this redesigned ß-barrel protein tolerates an extensive alteration in the internal surface charge, encompassing 25 negative charge neutralizations. By using single-molecule electrophysiology, we noted that a commonality of various truncation FhuA protein pores was the occurrence of 33% blockades of the unitary current at very high transmembrane potentials. We determined that these current transitions were stimulated by their interaction with an external cationic polypeptide, which occurred in a fashion dependent on the surface charge of the pore interior as well as the polypeptide characteristics. This study shows promise for extensive engineering of a large monomeric ß-barrel protein pore in molecular biomedical diagnosis, therapeutics, and biosensor technology.

Determination of the quaternary structure of a bacterial ATP-binding cassette (ABC) transporter in living cells.

Pseudomonas aeruginosa is a pathogenic Gram-negative bacterium that affects patients with cystic fibrosis and immunocompromised individuals. This bacterium coexpresses two unique forms of lipopolysaccharides (LPSs) on its surface, the A- and B-band LPS, which are among the main virulence factors that contribute to its pathogenicity. The polysaccharides in A-band LPSs are synthesized in the cytoplasm and translocated into the periplasm via an ATP-binding cassette (ABC) transporter consisting of a transmembrane protein, Wzm, and a cytoplasmic nucleotide-binding protein, Wzt. Most of the biochemical studies of A-band PSs in Pseudomonas aeruginosa are focused on the stages of the synthesis and ligation of PS, leaving the export stage involving the ABC transporter mostly unexplored. This difficulty is compounded by the fact that the subunit composition and structure of this bi-component ABC transporter are still unknown. Here we propose a simple but powerful method, based on Förster Resonance Energy Transfer (FRET) and optical micro-spectroscopy technology, to probe the structure of dynamic (as opposed to static) protein complexes in living cells. We use this method to determine the association stoichiometry and quaternary structure of the Wzm-Wzt complex in living cells. It is found that Wzt forms a rhombus-shaped homo-tetramer which becomes a square upon co-expression with Wzm, and that Wzm forms a square-shaped homo-tetramer both in the presence and absence of Wzt. Based on these results, we propose a structural model for the double-tetramer complex formed by the bi-component ABC transporter in living cells. An understanding of the structure and behavior of this ABC transporter will help develop antibiotics targeting the biosynthesis of the A-band LPS endotoxin.

Protein sensing with engineered protein nanopores.

The use of nanopores is a powerful new frontier in single-molecule sciences. Nanopores have been used effectively in exploring various biophysical features of small polypeptides and proteins, such as their folding state and structure, ligand interactions, and enzymatic activity. In particular, the α-hemolysin (αHL) protein pore has been used extensively for the detection, characterization, and analysis of polypeptides because this protein nanopore is highly robust, versatile, and tractable under various experimental conditions. Inspired by the mechanisms of protein translocation across the outer membrane translocases of mitochondria, we have shown the ability to use nanopore-probe techniques in controlling a single protein using engineered αHL pores. Here, we provide a detailed protocol for the preparation of αHL protein nanopores. Moreover, we demonstrate that placing attractive electrostatic traps is instrumental in tackling single-molecule stochastic sensing of folded proteins.

Excursion of a single polypeptide into a protein pore: simple physics, but complicated biology.

Despite its fundamental and critical importance in molecular biology and practical medical biotechnology, how a polypeptide interacts with a transmembrane protein pore is not yet comprehensively understood. Here, we employed single-channel electrical recordings to reveal the interactions of short polypeptides and small folded proteins with a robust beta-barrel protein pore. The short polypeptides were approximately 25 residues in length, resembling positively charged targeting presequences involved in protein import. The proteins were consisted of positively charged pre-cytochrome b2 fragments (pb2) fused to the small ribonuclease barnase (approximately 110 residues, Ba). Single-molecule experiments exploring the interaction of a folded pb2-Ba protein with a single beta-barrel pore, which contained negatively charged electrostatic traps, revealed the complexity of a network of intermolecular forces, including driving and electrostatic ones. In addition, the interaction was dependent on other factors, such as the hydrophobic content of the interacting polypeptide, the location of the electrostatic trap, the length of the pb2 presequence and temperature. This single-molecule approach together with protein design of either the interacting polypeptide or the pore lumen opens new opportunities for the exploration of the polypeptide-pore interaction at high temporal resolution. Such future studies are also expected to unravel the advantages and limitations of the nanopore technique for the detection and exploration of individual polypeptides.

Catalyzing the translocation of polypeptides through attractive interactions.

Facilitated translocation of polypeptides through a protein pore is a ubiquitous and fundamental process in biology. Several translocation systems possess various well-defined binding sites within the pore lumen, but a clear mechanistic understanding of how the interaction of the polypeptides with the binding site alters the underlying kinetics is still missing. Here, we employed rational protein design and single-channel electrical recordings to obtain detailed kinetic signatures of polypeptide translocation through the staphylococcal alpha-hemolysin (alphaHL) transmembrane pore, a robust, tractable, and versatile beta-barrel protein. Acidic binding sites composed of rings of negatively charged aspartic acid residues, engineered at strategic positions within the beta barrel, produced dramatic changes in the functional properties of the alphaHL protein, facilitating the transport of cationic polypeptides from one side of the membrane to the other. When two electrostatic binding sites were introduced, at the entry and exit of the beta barrel, both the rate constants of association and dissociation increased substantially, diminishing the free energy barrier for translocation. By contrast, more hydrophobic polypeptides exhibited a considerable decrease in the rate constant of association to the pore lumen, having to overcome a greater energetic barrier because of the hydrophilic nature of the pore interior.