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Aberrant cleavage of amyloid precursor protein (APP) by gamma-secretase is closely associated with Alzheimer's disease (AD). gamma-secretase activating protein (GSAP) specifically promotes gamma-secretase-mediated cleavage of APP. However, the underlying mechanism remains enigmatic. Here, we demonstrate that the 16-kDa C-terminal fragment of GSAP (GSAP-16K) undergoes phase separation in vitro and forms puncta-like condensates in cells. GSAP-16K exerts dual modulation on gamma-secretase cleavage; GSAP-16K in dilute phase increases APP-C-terminal 99-residue fragment (C99) cleavage toward preferred production of p-amyloid peptide 42 (A beta 42), but GSAP-16K condensates reduce APP-C99 cleavage through substrate sequestration. Notably, the A beta 42/A beta 40 ratio is markedly elevated with increasing concentrations of GSAP-16K. GSAP-16K stably associates with APP-C99 through specific sequence elements. These findings mechanistically explain GSAP-mediated modulation of gamma-secretase activity that may have ramifications on the development of potential therapeutics.

Purpose: Advances in our understanding of the contribution of aberrant glycosylation to the pro-oncogenic signaling and metastasis of tumor cells have reinvigorated the development of mucin-targeted therapies. Here, we validate the tumor-targeting ability of a novel monoclonal antibody (mAb), AR9.6, that binds MUC16 and abrogates downstream oncogenic signaling to confer a therapeutic response. Experimental Design: The in vitro and ex vivo validation of the binding of AR9.6 to MUC16 was achieved via flow cytometry, radioligand binding assay (RBA), and immunohistochemistry (IHC). The in vivo MUC16 targeting of AR9.6 was validated by creating a Zr-89-labeled radioimmunoconjugate of the mAb and utilizing immunoPET and ex vivo biodistribution studies in xenograft models of human ovarian and pancreatic cancer. Results: Flow cytometry, RBA, and IHC revealed that AR9.6 binds to ovarian and pancreatic cancer cells in an MUC16-dependent manner. The in vivo radiopharmacologic profile of Zr-89-labeled AR9.6 in mice bearing ovarian and pancreatic cancer xenografts confirmed the MUC16-dependent tumor targeting by the radioimmunoconjugate. Radioactivity uptake was also observed in the distant lymph nodes (LNs) of mice bearing xenografts with high levels of MUC16 expression (i.e., OVCAR3 and Capan-2). IHC analyses of these PET-positive LNs highlighted the presence of shed antigen as well as necrotic, phagocytized, and actively infiltrating neoplastic cells. The humanization of AR9.6 did not compromise its ability to target MUC16-expressing tumors. Conclusions: The unique therapeutic mechanism of AR9.6 combined with its excellent in vivo tumor targeting makes it a highly promising theranostic agent. huAR9.6 is poised for clinical translation to impact the management of metastatic ovarian and pancreatic cancers.

Posttransplant vaccination targeting residual disease is an immunotherapeutic strategy to improve antigen-specific immune responses and prolong disease-free survival after autologous stem cell transplantation (ASCT) for multiple myeloma (MM). We conducted a phase 1 vaccine trial to determine the safety, toxicity, and immunogenicity of autologous Langerhans-type dendritic cells (LCs) electroporated with CT7, MAGE-A3, and Wilms tumor 1 (WT1) messenger RNA (mRNA), after ASCT for MM. Ten patients received a priming immunization plus 2 boosters at 12, 30, and 90 days, respectively, after ASCT. Vaccines contained 9 x 10(6) mRNA-electroporated LCs. Ten additional patients did not receive LC vaccines but otherwise underwent identical ASCT and supportive care. At 3 months after ASCT, all patients started lenalidomide maintenance therapy. Vaccinated patients developed mild local delayed-type hypersensitivity reactions after booster vaccines, but no toxicities exceeded grade 1. At 1 and 3 months after vaccines, antigen-specific CD4 and CD8 T cells increased secretion of proinflammatory cytokines (interferon-y, interleukin-2, and tumor necrosis factor-alpha) above prevaccine levels, and also upregulated the cytotoxicity marker CD107a. CD4 and CD8 T-cell repertoire analysis showed a trend for increased clonal expansion in the vaccine cohort, which was more pronounced in the CD4 compartment. Although not powered to assess clinical efficacy, treatment responses favored the vaccine arm. Triple antigen-bearing mRNA-electroporated autologous LC vaccination initiated at engraftment after ASCT, in conjunction with standard lenalidomide maintenance therapy for MM, is safe and induces antigen-specific immune reactivity.

Tumourigenesis and cancer progression require enhanced global protein translation(1-3). Such enhanced translation is caused by oncogenic and tumour-suppressive events that drive the synthesis and activity of translational machinery(4,5). Here we report the surprising observation that leucyl-tRNA synthetase (LARS) becomes repressed during mammary cell transformation and in human breast cancer. Monoallelic genetic deletion of LARS in mouse mammary glands enhanced breast cancer tumour formation and proliferation. LARS repression reduced the abundance of select leucine tRNA isoacceptors, leading to impaired leucine codon-dependent translation of growth suppressive genes, including epithelial membrane protein 3 (EMP3) and gamma-glutamyltransferase 5 (GGT5). Our findings uncover a tumour-suppressive tRNA synthetase and reveal that dynamic repression of a specific tRNA synthetase-along with its downstream cognate tRNAs-elicits a downstream codon-biased translational gene network response that enhances breast tumour formation and growth.

Inward rectifier K+(Kir) channels regulate cell membrane potential. Different Kir channels respond to unique ligands, but all are regulated by phosphatidylinositol 4,5bisphosphate (PI(4,5)P-2). Using planar lipid bilayers, we show that Kir2.2 exhibits bursts of openings separated by long quiescent interburst periods. Increasing PI(4,5)P-2 concentration shortens the Kir2.2 interburst duration and lengthens the burst duration without affecting dwell times within a burst. From this, we propose that burst and interburst durations correspond to the cytoplasmic domain (CTD)-docked and CTDundocked conformations observed in the presence and absence of PI(4,5)P-2 in atomic structures. We also studied the effect of different phosphatidylinositol lipids on Kir2.2 activation and conclude that the 5' phosphate is essential to Kir2.2 pore opening. Other phosphatidylinositol lipids can compete with PI(4,5)P-2 but cannot activate Kir2.2 without the 5' phosphate. PI(4)P, which is directly interconvertible to and from PI(4,5)P-2, might thus be a regulator of Kir channels in the plasma membrane.

Decrypting the B cell ontogeny of HIV-1 broadly neutralizing antibodies (bNAbs) is paramount for vaccine design. Here, we characterized IgA and IgG bNAbs of three distinct B cell lineages in a viremic controller, two of which comprised only IgG(+) or IgA(+) blood memory B cells; the third combined both IgG and IgA clonal variants. 7-269 bNAb in the IgA-only lineage displayed the highest neutralizing capacity despite limited somatic mutation, and delayed viral rebound in humanized mice. bNAbs in all three lineages targeted the N332 glycan supersite. The 2.8-angstrom resolution cryo-EM structure of 7-269-BG505 SOSIP.664 complex showed a similar pose as 2G12, on an epitope mainly composed of sugar residues comprising the N332 and N295 glycans. Binding and cryo-EM structural analyses showed that antibodies from the two other lineages interact mostly with glycans N332 and N386. Hence, multiple B cell lineages of IgG and IgA bNAbs focused on a unique HIV-1 site of vulnerability can codevelop in HIV-1 viremic controllers.

Therapeutic human IgG antibodies are routinely tested in mouse models of oncologic, infectious, and autoimmune diseases. However, assessing the efficacy and safety of long-term administration of these agents has been limited by endogenous anti-human IgG immune responses that act to clear human IgG from serum and relevant tissues, thereby reducing their efficacy and contributing to immune complex-mediated pathologies, confounding evaluation of potential toxicity. For this reason, human antibody treatment in mice is generally limited in duration and dosing, thus failing to recapitulate the potential clinical applications of these therapeutics. Here, we report the development of a mouse model that is tolerant of chronic human antibody administration. This model combines both a human IgG1 heavy chain knock-in and a full recapitulation of human Fc receptor (Fc gamma R) expression, providing a unique platform for in vivo testing of human monoclonal antibodies with relevant receptors beyond the short term. Compared to controls, hIgG1 knock-in mice mount minimal anti-human IgG responses, allowing for the persistence of therapeutically active circulating human IgG even in the late stages of treatment in chronic models of immune thrombocytopenic purpura and metastatic melanoma.

SARS-CoV-2 infection is benign in most individuals but, in around 10% of cases, it triggers hypoxaemic COVID-19 pneumonia, which leads to critical illness in around 3% of cases. The ensuing risk of death (approximately 1% across age and gender) doubles every five years from childhood onwards and is around 1.5 times greater in men than in women. Here we review the molecular and cellular determinants of critical COVID-19 pneumonia. Inborn errors of type I interferons (IFNs), including autosomal TLR3 and X-chromosome-linked TLR7 deficiencies, are found in around 1-5% of patients with critical pneumonia under 60 years old, and a lower proportion in older patients. Pre-existing auto-antibodies neutralizing IFN alpha, IFN beta and/or IFN omega, which are more common in men than in women, are found in approximately 15-20% of patients with critical pneumonia over 70 years old, and a lower proportion in younger patients. Thus, at least 15% of cases of critical COVID-19 pneumonia can be explained. The TLR3- and TLR7-dependent production of type I IFNs by respiratory epithelial cells and plasmacytoid dendritic cells, respectively, is essential for host defence against SARS-CoV-2. In ways that can depend on age and sex, insufficient type I IFN immunity in the respiratory tract during the first few days of infection may account for the spread of the virus, leading to pulmonary and systemic inflammation.

Background Microsatellite instability-high (MSI-H)/mismatch repair deficiency (dMMR) is a biomarker for responses to immune checkpoint inhibitors (ICIs). Whether mechanisms underlying microsatellite instability alter responses to ICIs is unclear. This article reports data from a prospective phase 2 pilot study of pembrolizumab in patients with recurrent MSI-H endometrial cancer (EC) analyzed by whole exome sequencing (WES) and potential mechanisms of primary/secondary ICI resistance (NCT02899793). Methods Patients with measurable MSI-H/dMMR EC confirmed by polymerase chain reaction/immunohistochemistry were evaluated by WES and received 200 mg of pembrolizumab every 3 weeks for <= 2 years. The primary end point was the objective response rate (ORR). Secondary end points included progression-free survival (PFS) and overall survival (OS). Results Twenty-five patients (24 evaluable) were treated. Six patients (25%) harbored Lynch/Lynch-like tumors, whereas 18 (75%) had sporadic EC. The tumor mutation burden was higher in Lynch-like tumors (median, 2939 mutations/megabase [Mut/Mb]; interquartile range [IQR], 867-5108 Mut/Mb) than sporadic tumors (median, 604 Mut/Mb; IQR, 411-798 Mut/Mb; P = .0076). The ORR was 100% in Lynch/Lynch-like patients but only 44% in sporadic patients (P = .024). The 3-year PFS and OS proportions were 100% versus 30% (P = .017) and 100% versus 43% (P = .043), respectively. Conclusions This study suggests prognostic significance of Lynch-like cancers versus sporadic MSI-H/dMMR ECs for ORR, PFS, and OS when patients are treated with pembrolizumab. Larger confirmatory studies in ECs and other MSI-H/dMMR tumors are necessary. Defective antigen processing/presentation and deranged induction in interferon responses serve as mechanisms of resistance in sporadic MSI-H ECs. Oligoprogression in MSI-H/dMMR patients appears salvageable with surgical resection and/or local treatment and the continuation of pembrolizumab off study. Clinical studies evaluating separate MSI-H/dMMR EC subtypes treated with ICIs are warranted.

Background: Frontal fibrosing alopecia (FFA) is a progressive, scarring alopecia of the frontotemporal scalp that poses a substantial burden on quality of life. Large-scale global profiling of FFA is lacking, preventing the development of effective therapeutics. Objective: To characterize FFA compared to normal and alopecia areata using broad molecular profiling and to identify biomarkers linked to disease severity. Methods: This cross-sectional study assessed 33,118 genes in scalp using RNA sequencing and 350 proteins in serum using OLINK high-throughput proteomics. Disease biomarkers were also correlated with clinical severity and a fibrosis gene set. Results: Genes differentially expressed in lesional FFA included markers related to Th1 (IFN gamma/CXCL9/CXCL10), T-cell activation (CD2/CD3/CCL19/ICOS), fibrosis (CXCR3/FGF14/FGF22/VIM/FN1), T-regulatory (FOXP3/TGFB1/TGFB3), and Janus kinase/JAK (JAK3/STAT1/STAT4) (Fold changes [FCH]>1.5, FDR<.05 for all). Only one protein, ADM, was differentially expressed in FFA serum compared to normal (FCH>1.3, FDR>.05). Significant correlations were found between scalp biomarkers (IL-36RN/IL-25) and FFA severity, as well as between JAK/STAT and fibrosis gene-sets (r>6; P<.05). Limitations: This study was limited by a small sample size and predominantly female FFA patients. Conclusion: Our data characterize FFA as an inflammatory condition limited to scalp, involving Th1/JAK skewing, with associated fibrosis and elevated T-regulatory markers, suggesting the potential for disease reversibility with JAK/STAT inhibition.