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The nonreceptor tyrosine kinase c-Src is frequently over-expressed or hyperactivated in various human cancers and contributes to cancer progression in cooperation with up-regulated growth factor receptors. However,Src-selective anticancer drugs are still in clinical trials. To identify more effective inhibitors of c-Src-mediated cancer progression,we developed a new screening platform using Csk-deficient cells that can be transformed by c-Src. We found that purvalanol A,developed as a CDK inhibitor,potently suppressed the anchorage-independent growth of c-Src-transformed cells,indicating that the activation of CDKs contributes to the c-Src transformation. We also found that purvalanol A suppressed the c-Src activity as effectively as the Src-selective inhibitor PP2,and that it reverted the transformed morphology to a nearly normal shape with less cytotoxicity than PP2. Purvalanol A induced a strong G2-M arrest,whereas PP2 weakly acted on the G1-S transition. Furthermore,when compared with PP2,purvalanol A more effectively suppressed the growth of human colon cancer HT29 and SW480 cells,in which Src family kinases and CDKs are activated. These findings demonstrate that the coordinated inhibition of cell cycle progression and tyrosine kinase signaling by the multi-selective purvalanol A is effective in suppressing cancer progression associated with c-Src up-regulation. View Publication

New strategies that augment T cell responses are required to broaden the therapeutic arsenal against cancer. CD96,TIGIT,and CD226 are receptors that bind to a communal ligand,CD155,and transduce either inhibitory or activating signals. The function of TIGIT and CD226 is established,whereas the role of CD96 remains ambiguous. Using a panel of engineered antibodies,we discovered that the T cell stimulatory activity of anti-CD96 antibodies requires antibody cross-linking and is potentiated by Fc$\gamma$ receptors. Thus,soluble Fc silent" anti-CD96 antibodies failed to stimulate human T cells whereas the same antibodies were stimulatory after coating onto plastic surfaces. Remarkably the activity of soluble anti-CD96 antibodies was reinstated by engineering the Fc domain to a human IgG1 isotype and it was dependent on antibody trans-cross-linking by Fc$\gamma$RI. In contrast neither human IgG2 nor variants with increased Fc$\gamma$ receptor IIB binding possessed stimulatory activity. Anti-CD96 antibodies acted directly on T cells and augmented gene expression networks associated with T cell activation leading to proliferation cytokine secretion and resistance to Treg suppression. Furthermore CD96 expression correlated with survival in HPV+ head and neck squamous cell carcinoma and its cross-linking activated tumor-infiltrating T cells thus highlighting the potential of anti-CD96 antibodies in cancer immunotherapy." View Publication

Human brain organoids are emerging as translationally relevant models for the study of human brain health and disease. However,it remains to be shown whether human-specific protein processing is conserved in human brain organoids. Herein,we demonstrate that cell fate and composition of unguided brain organoids are dictated by culture conditions during embryoid body formation,and that culture conditions at this stage can be optimized to result in the presence of glia-associated proteins and neural network activity as early as three-months in vitro. Under these optimized conditions,unguided brain organoids generated from induced pluripotent stem cells (iPSCs) derived from male–female siblings are similar in growth rate,size,and total protein content,and exhibit minimal batch-to-batch variability in cell composition and metabolism. A comparison of neuronal,microglial,and macroglial (astrocyte and oligodendrocyte) markers reveals that profiles in these brain organoids are more similar to autopsied human cortical and cerebellar profiles than to those in mouse cortical samples,providing the first demonstration that human-specific protein processing is largely conserved in unguided brain organoids. Thus,our organoid protocol provides four major cell types that appear to process proteins in a manner very similar to the human brain,and they do so in half the time required by other protocols. This unique copy of the human brain and basic characteristics lay the foundation for future studies aiming to investigate human brain-specific protein patterning (e.g.,isoforms,splice variants) as well as modulate glial and neuronal processes in an in situ-like environment. View Publication

The extrafollicular (EF) plasmablast response to self-antigens that contain Toll-like receptor (TLR) ligands is prominent in murine lupus models and some bacterial infections,but the inhibitors and activators involved have not been fully delineated. Here,we used two conventional dendritic cell (cDC) depletion systems to investigate the role of cDCs on a classical TLR-dependent autoreactive EF response elicited in rheumatoid-factor B cells by DNA-containing immune complexes. Contrary to our hypothesis,cDC depletion amplified rather than dampened the EF response in Fas-intact but not Fas-deficient mice. Further,we demonstrated that cDC-dependent regulation requires Fas and Fas ligand (FasL) expression by T cells,but not Fas expression by B cells. Thus,cDCs activate FasL-expressing T cells that regulate Fas-expressing extrafollicular helper T (Tefh) cells. These studies reveal a regulatory role for cDCs in B cell plasmablast responses and provide a mechanistic explanation for the excess autoantibody production observed in Fas deficiency. View Publication

Cancer stem cells (CSCs) are one of the main reasons behind cancer recurrence due to their resistance to conventional anti-cancer therapies. Thus,many efforts are being devoted to developing CSC-targeted therapies to overcome the resistance of CSCs to conventional anti-cancer therapies and decrease cancer recurrence. Differentiation therapy is one potential approach to achieve CSC-targeted therapies. This method involves inducing immature cancer cells with stem cell characteristics into more mature or differentiated cancer cells. In this study,we found that a CDK4 inhibitor sensitized MDA-MB-231 cells but not MCF7 cells to irradiation. This difference appeared to be associated with the relative percentage of CSC-population between the two breast cancer cells. The CDK4 inhibitor induced differentiation and reduced the cancer stem cell activity of MDA-MB-231 cells,which are shown by multiple marker or phenotypes of CSCs. Thus,these results suggest that radiosensitization effects may be caused by reducing the CSC-population of MDA-MB-231 through the use of the CDK4 inhibitor. Thus,further investigations into the possible application of the CDK4 inhibitor for CSC-targeted therapy should be performed to enhance the efficacy of radiotherapy for breast cancer. View Publication

Autophagy is characterized by the sequestration of bulk cytoplasm,including damaged proteins and organelles,and delivery of the cargo to lysosomes for degradation. Although the autophagic pathway is also linked to tumour suppression activity,the mechanism is not yet clear. Here we report that cytosolic FoxO1,a forkhead O family protein,is a mediator of autophagy. Endogenous FoxO1 was required for autophagy in human cancer cell lines in response to oxidative stress or serum starvation,but this process was independent of the transcriptional activity of FoxO1. In response to stress,FoxO1 was acetylated by dissociation from sirtuin-2 (SIRT2),a NAD(+)-dependent histone deacetylase,and the acetylated FoxO1 bound to Atg7,an E1-like protein,to influence the autophagic process leading to cell death. This FoxO1-modulated cell death is associated with tumour suppressor activity in human colon tumours and a xenograft mouse model. Our finding links the anti-neoplastic activity of FoxO1 and the process of autophagy. View Publication

Chronic myelomonocytic leukemia (CMML) is a clinically heterogeneous neoplasm in which JAK2 inhibition has demonstrated reductions in inflammatory cytokines and promising clinical activity. We hypothesize that annotation of inflammatory cytokines may uncover mutation-independent cytokine subsets associated with novel CMML prognostic features. A Luminex cytokine profiling assay was utilized to profile cryopreserved peripheral blood plasma from 215 CMML cases from three academic centers,along with center-specific,age-matched plasma controls. Significant differences were observed between CMML patients and healthy controls in 23 out of 45 cytokines including increased cytokine levels in IL-8,IP-10,IL-1RA,TNF-alpha$,IL-6,MCP-1/CCL2,hepatocyte growth factor (HGF),M-CSF,VEGF,IL-4,and IL-2RA. Cytokine associations were identified with clinical and genetic features,and Euclidian cluster analysis identified three distinct cluster groups associated with important clinical and genetic features in CMML. CMML patients with decreased IL-10 expression had a poor overall survival when compared to CMML patients with elevated expression of IL-10 (P = 0.017),even when adjusted for ASXL1 mutation and other prognostic features. Incorporating IL-10 with the Mayo Molecular Model statistically improved the prognostic ability of the model. These established cytokines,such as IL-10,as prognostically relevant and represent the first comprehensive study exploring the clinical implications of the CMML inflammatory state. View Publication

The current global COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a public health crisis with more than 168 million cases reported globally and more than 4.5 million deaths at the time of writing. In addition to the direct impact of the disease,the economic impact has been significant as public health measures to contain or reduce the spread have led to country wide lockdowns resulting in near closure of many sectors of the economy. Antibodies are a principal determinant of the humoral immune response to COVID-19 infections and may have the potential to reduce disease and spread of the virus. The development of monoclonal antibodies (mAbs) represents a therapeutic option that can be produced at large quantity and high quality. In the present study,a mAb combination mixture therapy was investigated for its capability to specifically neutralize SARS-CoV-2. We demonstrate that each of the antibodies bind the spike protein and neutralize the virus,preventing it from infecting cells in an in vitro cell-based assay,including multiple viral variants that are currently circulating in the human population. In addition,we investigated the effects of two different mutations in the Fc portion (YTE and LALA) of the antibody on Fc effector function and the ability to alleviate potential antibody-dependent enhancement of disease. These data demonstrate the potential of a combination of two mAbs that target two different epitopes on the SARS-CoV2 spike protein to provide protection against SARS-CoV-2 infection in humans while extending serum half-life and preventing antibody-dependent enhancement of disease. View Publication

Diabetic keratopathy (DK),a significant complication of diabetes,often leads to corneal damage and vision impairment. Effective models are essential for studying DK pathogenesis and evaluating potential therapeutic interventions. This study developed a novel biomimetic full-thickness corneal model for the first time,incorporating corneal epithelial cells,stromal cells,endothelial cells,and nerves to simulate DK conditions in vitro. By exposing the model to a high-glucose (HG) environment,the pathological characteristics of DK,including nerve bundle disintegration,compromised barrier integrity,increased inflammation,and oxidative stress,were successfully replicated. Transcriptomic analysis revealed that HG downregulated genes associated with axon and synapse formation while upregulating immune response and oxidative stress pathways,with C-C Motif Chemokine Ligand 5 (CCL5) identified as a key hub gene in DK pathogenesis. The therapeutic effects of Lycium barbarum glycopeptide (LBGP) were evaluated using this model and validated in db/db diabetic mice. LBGP promoted nerve regeneration,alleviated inflammation and oxidative stress in both in vitro and in vivo models. Notably,LBGP suppressed the expression of CCL5,highlighting its potential mechanism of action. This study establishes a robust biomimetic platform for investigating DK and other corneal diseases,and identifies LBGP as a promising therapeutic candidate for DK. These findings provide valuable insights into corneal disease mechanisms and pave the way for future translational research and clinical applications. Graphical abstractImage 1 Highlights•A full-thickness biomimetic corneal model containing corneal epithelium,nerves,stroma,and endothelium was constructed.•Using this model,the pathological characteristics of diabetic keratopathy were successfully replicated in vitro.•Lycium barbarum glycopeptide (LBGP) alleviated high-glucose-induced damage in vitro and in vivo models.•CCL5 plays an important role in the pathogenesis of diabetic keratopathy. View Publication

The development of immunocompetent skin models marks a significant advancement in in vitro methods for detecting skin sensitizers while adhering to the 3R principles,which aim to reduce,refine,and replace animal testing. This study introduces for the first time an advanced immunocompetent skin model constructed entirely from induced pluripotent stem cell (iPSC)-derived cell types,including fibroblasts (iPSC-FB),keratinocytes (iPSC-KC),and fully integrated dendritic cells (iPSC-DC). To evaluate the skin model’s capacity,the model was treated topically with a range of well-characterized skin sensitizers varying in potency. The results indicate that the iPSC-derived immunocompetent skin model successfully replicates the physiological responses of human skin,offering a robust and reliable alternative to animal models for skin sensitization testing,allowing detection of extreme and even weak sensitizers. By addressing critical aspects of immune activation and cytokine signaling,this model provides an ethical,comprehensive tool for regulatory toxicology and dermatological research. View Publication