Increased Siglec-9/Siglec-9L interactions on NK cells predict poor HCC prognosis and present a targetable checkpoint for immunotherapy.

BACKGROUND & AIMS: Natural killer (NK) cell-based anti-hepatocellular carcinoma (HCC) therapy is an increasingly attractive approach that warrants further study. Siglec-9 interacts with its ligand (Siglec-9L) and restrains NK cell functions, suggesting it is a potential therapeutic target. However, in situ Siglec-9/Siglec-9L interactions in HCC have not been reported, and a relevant interventional strategy is lacking. Herein, we aim to illustrate Siglec-9/Siglec-9L-mediated cell sociology and identify small-molecule inhibitors targeting Siglec-9 that could improve the efficacy of NK cell-based immunotherapy for HCC. METHODS: Multiplexed immunofluorescence staining was performed to analyze the expression pattern of Siglec-7, -9 and their ligands in HCC tissues. Then we conducted docking-based virtual screening combined with bio-layer interferometry assays to identify a potent small-molecule Siglec-9 inhibitor. The therapeutic potential was further evaluated in vitro and in hepatoma-bearing NCG mice. RESULTS: Siglec-9 expression, rather than Siglec-7, was markedly upregulated on tumor-infiltrating NK cells, which correlated significantly with reduced survival of patients with HCC. Moreover, the number of Siglec-9L+ cells neighboring Siglec-9+ NK cells was increased in HCC tissues and was also associated with tumor recurrence and reduced survival, further suggesting that Siglec-9/Siglec-9L interactions are a potential therapeutic target in HCC. In addition, we identified a small-molecule Siglec-9 inhibitor MTX-3937 which inhibited phosphorylation of Siglec-9 and downstream SHP1 and SHP2. Accordingly, MTX-3937 led to considerable improvement in NK cell function. Notably, MTX-3937 enhanced cytotoxicity of both human peripheral and tumor-infiltrating NK cells. Furthermore, transfer of MTX-3937-treated NK92 cells greatly suppressed the growth of hepatoma xenografts in NCG mice. CONCLUSIONS: Our study provides the rationale for HCC treatment by targeting Siglec-9 on NK cells and identifies a promising small-molecule inhibitor against Siglec-9 that enhances NK cell-mediated HCC surveillance. IMPACT AND IMPLICATIONS: Herein, we found that Siglec-9 expression is markedly upregulated on tumor-infiltrating natural killer (TINK) cells and correlates with reduced survival in patients with hepatocellular carcinoma (HCC). Moreover, the number of Siglec-9L+ cells neighboring Siglec-9+ NK cells was increased in HCC tissues and was also associated with tumor recurrence and reduced survival. More importantly, we identified a small-molecule inhibitor targeting Siglec-9 that augments NK cell functions, revealing a novel immunotherapy strategy for liver cancer that warrants further clinical investigation.

Identification of a small-molecule Tim-3 inhibitor to potentiate T cell-mediated antitumor immunotherapy in preclinical mouse models.

T cell immunoglobulin and mucin-containing molecule 3 (Tim-3), expressed in dysfunctional and exhausted T cells, has been widely acknowledged as a promising immune checkpoint target for tumor immunotherapy. Here, using a strategy combining virtual and functional screening, we identified a compound named ML-T7 that targets the FG-CC' cleft of Tim-3, a highly conserved binding site of phosphatidylserine (PtdSer) and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). ML-T7 enhanced the survival and antitumor activity of primary CD8+ cytotoxic T lymphocytes (CTLs) and human chimeric antigen receptor (CAR) T cells and reduced their exhaustion in vitro and in vivo. In addition, ML-T7 promoted NK cells' killing activity and DC antigen-presenting capacity, consistent with the reported activity of Tim-3. ML-T7 strengthened DCs' functions through both Tim-3 and Tim-4, which is consistent with the fact that Tim-4 contains a similar FG-CC' loop. Intraperitoneal dosing of ML-T7 showed comparable tumor inhibitory effects to the Tim-3 blocking antibody. ML-T7 reduced syngeneic tumor progression in both wild-type and Tim-3 humanized mice and alleviated the immunosuppressive microenvironment. Furthermore, combined ML-T7 and anti-PD-1 therapy had greater therapeutic efficacy than monotherapy in mice, supporting further development of ML-T7 for tumor immunotherapy. Our study demonstrates a potential small molecule for selectively blocking Tim-3 and warrants further study.

A retrospective real-world study: the efficacy of immune-related combination therapies in advanced non-small cell lung cancer after resistance to EGFR-TKIs.

BACKGROUND: Whether patients with advanced non-small cell lung cancer (NSCLC) should choose an immune-combination therapy regimen after EGFR-tyrosine kinase inhibitors (EGFR-TKIs) resistance is currently unclear. METHODS: We evaluated 118 NSCLC patients treated by immune checkpoint inhibitors (ICIs) + chemotherapy (I + C), ICIs + chemotherapy + antiangiogenic therapy (I + C + A), chemotherapy + antiangiogenic therapy (C + A) after inefficacy of EGFR-TKIs. We assessed the objective remission rate (ORR), disease control rate (DCR), and progression-free survival (PFS) of these treatments. RESULTS: The ORR was 26.1% vs 38.2% vs 16.3% in the three groups (P = 0.093). The divergence in DCR was also statistically significant (65.2% vs 85.3% vs 74.4%, P = 0.209). The median PFS was no statistically significant difference in PFS (3.09 vs 6.31 vs 5.91 months, P = 0.809), but the Kaplan-Meier survival curve of 12-month-PFS indicated an apparent survival advantage in the I + C + A group (P = 0.001). In addition, the I + C/I + C + A group showed higher median PFS than the C + A group in patients with brain metastases (median PFS, 6.44 vs 4.21 months, P = 0.022). The divergence in ORR of patients in the brain group was also statistically significant (P = 0.045). The I + C + A group showed superior efficacy in patients with liver metastases (median PFS, 0.95 vs 6.44 vs 3.48 months, P < 0.0001). The Cox proportional hazard modeling analysis suggested that the age, brain metastases, and liver metastases were all connected with the prognosis. CONCLUSIONS: This study suggests that advanced NSCLC patients after resistance to EGFR-TKIs may achieve better outcomes from triple therapy. Patients with brain metastases favor ICIs-related combination therapies and patients with liver metastases prefer I + C + A therapy.

Selenium Improves Physiological Parameters and Alleviates Oxidative Stress in Strawberry Seedlings under Low-Temperature Stress.

Here, we investigated the effects of selenium (Se) applications on two strawberry varieties, Akihime and Benihoppe, under chilling stress and recovery conditions. Changes in photosynthetic parameters, antioxidant enzyme activities, ascorbate (AsA)-glutathione (GSH) cycle-related enzyme activities, and low-molecular-mass antioxidant contents were determined. Foliar spraying with Se alleviated the decline in the net photosynthetic rate and chlorophyll content and increased the malondialdehyde and hydrogen peroxide contents of strawberry seedlings’ leaves under chilling stress. As the time under chilling stress increased, the stomatal conductance decreased and intercellular CO2 concentration increased, suggesting that nonstomatal factors had major limiting effects on the net photosynthetic rate's decrease. Se applications significantly alleviated the adverse impacts of chilling stress on changes in stomatal conductance and intercellular CO2 concentration. Se, especially at lower concentrations, significantly increased superoxide dismutase, catalase, and peroxide enzyme activities during chilling stress. Approximately 5 mg·L−1 of sodium selenite solution had the greatest stress-alleviating effects. Among the AsA-GSH cycle-related enzymes, ascorbate peroxidase, glutathione reductase, dehydroascorbate reductase, and monodehydroascorbate reductase (MDHAR) treatments, coupled with an appropriate dose of Se, significantly enhanced ascorbate peroxidase and MDHAR activities, which suggested that Se applications played important roles in strawberry leaves by affecting AsA-GSH cycle-related defenses against the oxidative damage caused by chilling stress. Furthermore, MDHAR was the key enzyme required to maintain the balance between AsA consumption and regeneration that may assist in protecting strawberry seedlings in a low-temperature environment.

Fused heterocycles bearing bridgehead nitrogen as potent HIV-1 NNRTIs. Part 3: optimization of [1,2,4]triazolo[1,5-a]pyrimidine core via structure-based and physicochemical property-driven approaches.

In our arduous efforts to develop new potent HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs), novel piperidine-linked [1,2,4]triazolo[1,5-a]pyrimidine derivatives were designed, synthesized and evaluated for their antiviral activities in MT-4 cell cultures. Biological results showed that all of the title compounds displayed moderate to excellent activities against wild-type (wt) HIV-1 strain (IIIB) with EC50 values ranging from 8.1 nM to 2284 nM in a cell-based assay. Among them, the most promising analog 7d possessed an EC50 value of 8.1 nM against wt HIV-1, which was much more potent than the reference drugs DDI, 3 TC, NVP and DLV. Additionally, 7d demonstrated weak activity against the double mutant HIV-1 strain (K103N + Y181C), and was more efficient than NVP in a RT inhibition assay. Besides, some measured and calculated physicochemical properties of 7d, like log P and water solubility, as well as the structure-activity relationships (SARs) analysis have been discussed in detail. Furthermore, the binding mode of the active compound 7d was rationalized by molecular simulation studies.

Distinct states of methionyl-tRNA synthetase indicate inhibitor binding by conformational selection.

To guide development of new drugs targeting methionyl-tRNA synthetase (MetRS) for treatment of human African trypanosomiasis, crystal structure determinations of Trypanosoma brucei MetRS in complex with its substrate methionine and its intermediate product methionyl-adenylate were followed by those of the enzyme in complex with high-affinity aminoquinolone inhibitors via soaking experiments. Drastic changes in conformation of one of the two enzymes in the asymmetric unit allowed these inhibitors to occupy an enlarged methionine pocket and a new so-called auxiliary pocket. Interestingly, a small low-affinity compound caused the same conformational changes, removed the methionine without occupying the methionine pocket, and occupied the previously not existing auxiliary pocket. Analysis of these structures indicates that the binding of the inhibitors is the result of conformational selection, not induced fit.

Fstl1 antagonizes BMP signaling and regulates ureter development.

Bone morphogenetic protein (BMP) signaling pathway plays important roles in urinary tract development although the detailed regulation of its activity in this process remains unclear. Here we report that follistatin-like 1 (Fstl1), encoding a secreted extracellular glycoprotein, is expressed in developing ureter and antagonizes BMP signaling activity. Mouse embryos carrying disrupted Fstl1 gene displayed prominent hydroureter arising from proximal segment and ureterovesical junction defects. These defects were associated with significant reduction in ureteric epithelial cell proliferation at E15.5 and E16.5 as well as absence of subepithelial ureteral mesenchymal cells in the urinary tract at E16.5 and E18.5. At the molecular level, increased BMP signaling was found in Fstl1 deficient ureters, indicated by elevated pSmad1/5/8 activity. In vitro study also indicated that Fstl1 can directly bind to ALK6 which is specifically expressed in ureteric epithelial cells in developing ureter. Furthermore, Sonic hedgehog (SHH) signaling, which is crucial for differentiation of ureteral subepithelial cell proliferation, was also impaired in Fstl1(-/-) ureter. Altogether, our data suggest that Fstl1 is essential in maintaining normal ureter development by antagonizing BMP signaling.

Follistatin-like 1 (Fstl1) is a bone morphogenetic protein (BMP) 4 signaling antagonist in controlling mouse lung development.

Lung morphogenesis is a well orchestrated, tightly regulated process through several molecular pathways, including TGF-ß/bone morphogenetic protein (BMP) signaling. Alteration of these signaling pathways leads to lung malformation. We investigated the role of Follistatin-like 1 (Fstl1), a secreted follistatin-module-containing glycoprotein, in lung development. Deletion of Fstl1 in mice led to postnatal lethality as a result of respiratory failure. Analysis of the mutant phenotype showed that Fstl1 is essential for tracheal cartilage formation and alveolar maturation. Deletion of the Fstl1 gene resulted in malformed tracheal rings manifested as discontinued rings and reduced ring number. Fstl1-deficient mice displayed septal hypercellularity and end-expiratory atelectasis, which were associated with impaired differentiation of distal alveolar epithelial cells and insufficient production of mature surfactant proteins. Mechanistically, Fstl1 interacted directly with BMP4, negatively regulated BMP4/Smad1/5/8 signaling, and inhibited BMP4-induced surfactant gene expression. Reducing BMP signaling activity by Noggin rescued pulmonary atelectasis of Fstl1-deficient mice. Therefore, we provide in vivo and in vitro evidence to demonstrate that Fstl1 modulates lung development and alveolar maturation, in part, through BMP4 signaling.

Follistatin-like 1 suppresses sensory afferent transmission by activating Na+,K+-ATPase.

Excitatory synaptic transmission is modulated by inhibitory neurotransmitters and neuromodulators. We found that the synaptic transmission of somatic sensory afferents can be rapidly regulated by a presynaptically secreted protein, follistatin-like 1 (FSTL1), which serves as a direct activator of Na(+),K(+)-ATPase (NKA). The FSTL1 protein is highly expressed in small-diameter neurons of the dorsal root ganglion (DRG). It is transported to axon terminals via small translucent vesicles and secreted in both spontaneous and depolarization-induced manners. Biochemical assays showed that FSTL1 binds to the α1 subunit of NKA and elevates NKA activity. Extracellular FSTL1 induced membrane hyperpolarization in cultured cells and inhibited afferent synaptic transmission in spinal cord slices by activating NKA. Genetic deletion of FSTL1 in small DRG neurons of mice resulted in enhanced afferent synaptic transmission and sensory hypersensitivity, which could be reduced by intrathecally applied FSTL1 protein. Thus, FSTL1-dependent activation of NKA regulates the threshold of somatic sensation.

NDST-1 modulates BMPR and PTHrP signaling during endochondral bone formation in a gene knockout model.

GlcNAc N-deacetylase/N-sulfotransferase-1 (NDST-1), a member of the enzyme family catalyzing the first modification step in the biosynthesis of heparan sulfate (HS), was knocked out in mice to investigate its role in embryonic development. NDST-1 null mice exhibited delayed endochondral bone formation including shortened calcified zones in limbs, delayed chondrocyte and osteogenetic differentiation, and increased chondrocyte proliferation. In situ HS binding assay revealed that the binding ability of bone morphogenetic protein (BMP) -2, -4, and -6 to endogenous HS was decreased in mutant phalanges, while that of fibroblast growth factor-1 (FGF-1) was not affected. Up-regulation of BMPR-IA, Phospho-Smad1 (P-Smad1) and parathyroid-hormone related protein (PTHrP), but not the Indian hedgehog, Gli1, Gli3, Patched, and FGFR-3, was observed. Furthermore, block of BMPR signaling with noggin rescued the delayed chondrocyte hypertrophic differentiation in NDST-1 (-/-) mice and recovered the expression of both P-Smad1 and PTHrP proteins. These results suggested that NDST-1-dependent heparan sulfate might negatively modulate BMP and its downstream PTHrP signaling, and thus affect endochondral bone development.

hNRAGE, a human neurotrophin receptor interacting MAGE homologue, regulates p53 transcriptional activity and inhibits cell proliferation.

hNRAGE, a neurotrophin receptor p75 interacting MAGE homologue, is cloned from a human placenta cDNA library. hNRAGE can inhibit the colony formation of and arrest cell proliferation at the G1/S and G2/M stages in hNRAGE overexpressing cells. Interestingly, hNRAGE also increases the p53 protein level as well as its phosphorylation (Ser392). Further studies demonstrated that hNRAGE does not affect the proliferation of mouse p53-/- embryonic fibroblasts, suggesting that p53 function is required for hNRAGE induced cell cycle arrest. Moreover, the cell cycle inhibiting protein p21(WAF) is induced by hNRAGE in a p53 dependent manner. The data provide original evidence that hNRAGE arrests cell growth through a p53 dependent pathway.