Icaritin inhibits PD-L1 expression by Targeting Protein IκB Kinase α.

Icaritin, a small molecule currently being investigated in phase III clinical trials in China (NCT03236636 and NCT03236649) for treatment of advanced hepatocellular carcinoma (HCC), is a prenylflavonoid derivative obtained from the Epimedium genus. Previously, it was found that Icaritin decreased the expression of PD-L1, but its direct molecular targets and the underlying mechanisms have not been identified. In this study, we report the identification of IKK-α as the protein target of Icaritin by biotin-based affinity binding assay. The further mutagenesis assay has provided evidence that C46 and C178 in IKK-α were essential amino acids for Icaritin binding to IKK-α, revealing the binding sites of Icaritin to IKK-α for the first time. Functionally, Icaritin inhibited the NF-κB signalling pathway by blocking IKK complex formation, which led to decreased nuclear translocation of NF-κB p65, and subsequent downregulation of PD-L1 expression in a dose-dependent manner. More importantly, PD-L1-positive patients exhibited longer overall survival upon Icaritin therapy. Finally, Icaritin in combination with checkpoints antibodies, such as α-PD-1, has demonstrated much better efficacy than any single therapy in animal models. This is the first report that anticancer effects of Icaritin are mediated, at least in part, by impairing functions of IKK-α.

Icaritin promotes tumor T-cell infiltration and induces antitumor immunity in mice.

Icaritin, a hydrolytic product of icariin isolated from traditional Chinese herbal medicine genus Epimedium, has many pharmacological and biological activities. Here, we show that icaritin can effectively decrease tumor burden of murine B16F10 melanoma and MC38 colorectal tumors in a T-cell dependent manner. The treatment effects are associated with increased CD8 T-cell infiltration and increased effector memory T-cell frequency. In vivo depletion of CD8 T cell using an anti-CD8 monoclonal antibody abolished the antitumor effect, which supports the critical role of CD8 T cells during icaritin treatment. By analyzing immune cells in the tumor tissue, we found reduced frequency of CD11b+ Gr1+ myeloid-derived suppression cells (MDSCs) infiltration and downregulation of PD-L1 expression on MDSCs after icaritin treatment. This was not limited to MDSCs, as icaritin also decreased the expression of PD-L1 on neutrophils. Importantly, the combination of anti-PD-1/CTLA-4 and icaritin significantly enhances antitumor ability and increases the efficacy of either treatment alone. Our findings reveal that icaritin induces antitumor immunity in a CD8 T-cell-dependent way and justify further investigation of combining immune checkpoint therapy to icaritin-based antitumor therapy.

Phylogeography of the large white-bellied rat Niviventer excelsior suggests the influence of Pleistocene glaciations in the Hengduan mountains.

The Hengduan Mountains, situated in the southeastern Tibetan Plateau, have undergone dramatic geological and climatic changes over the Pleistocene epoch. Several studies have revealed that the mountains served as a refugium during the ice age. The large white-bellied rat Niviventer excelsior is a rodent endemic to the Hengduan Mountains, which makes it an appropriate species for investigating the influence of glacial movements on the genetic structure of mammals. In this study, we sequenced the partial mitochondrial DNA control region from 72 N. excelsior specimens collected from 20 localities. The results revealed very high levels of haplotype diversity (h = 0.947) and nucleotide diversity (pi = 0.101) in this species. No common haplotype was found to be shared in samples from all geographic regions. Demographic analyses suggested that N. excelsior populations had not been subject to either expansion or bottleneck. The phylogenetic relationships among the haplotypes have no correlation with their geographical origins, while topology revealed two major clades. We speculate that the populations of N. excelsior may have been restricted to two separate refugia during the Last Glacial Maximum (0.60-0.17 Mya), with one west and one east of the Shaluli Mountains. Between the two major refugia, there existed a more widely distributed network subrefugia, which conserved genetic variations in N. excelsior. These results indicated that complex topographic configuration in the Hengduan Mountains provided a network of refugia to maintain the high level of genetic diversity in Pleistocene glaciations.

Taxonomic position of Chinese voles of the tribe Arvicolini and the description of 2 new species from Xizang, China.

China has 26 species in the tribe Arvicolini. The taxonomic status of these voles remains controversial despite much effort. Herein, we evaluate the taxonomic position of 22 species plus 2 unidentified taxa using mitochondrial DNA gene sequences (cytb + CO1). We also evaluate 18 species and 2 unidentified taxa using morphological data. Phylogenetic analyses of cytb resolve monophyly for the genera Alexandromys, Lasiopodomys, Microtus, Neodon, Proedromys, and Volemys with strong support. Stenocranius clusters with Chionomys but with very weak support. Analyses of concatenated cytb + CO1 resolve the same genera with strong support, but the topology of the tree differs from that of cytb in that Chionomys roots at the base of the tree independent of Stenocranius, which forms the sister-group of Lasiopodomys in a more terminal position. The matrilineal genealogy excludes the type species Arvicola amphibius from the rest of the Arvicolini. This species forms the sister-group of Ondatra with high support. Neodon includes N. irene, N. linzhiensis, N. fuscus, N. leucurus, N. sikimensis, Microtus clarkei, and 2 unidentified specimens. Alexandromys includes the former species Microtus oeconomus, M. kikuchii, M. limnophilus, M. fortis, and M. maximowiczii. Finally, Microtus has the subgenera Blanfordimys, Microtus, Mynomes, Pedomys, Pitymys, and Terricola, which includes the Chinese species M. agrestis, M. arvalis, and Blanfordimys juldaschi. General mixed Yule-coalescent species delimitation modeling demarcates 6 currently recognized species and 2 new species of Neodon. A principal component analysis of the morphological data among 7 matrilines shows that all variables have positive loadings of high magnitude on the 1st component. Canonical discriminant analysis for Neodon (including M. clarkei and 2 unidentified species) correctly classifies 93.0% of specimens. Overall, our analyses support the recognition of Alexandromys, Lasiopodomys, Microtus, Neodon, Proedromys, and Volemys as genera. Stenocranius includes Microtus gregalis, and the genealogical position of Stenocranius remains uncertain. The status of Arvicola requires further study. We assign M. clarkei to Neodon and describe 2 new species of Neodon.

Complete mitochondrial genome of the Chinese oriental vole Eothenomys chinensis (Rodentia: Arvicolinae).

The Chinese oriental vole (Eothenomys chinensis) belongs to subfamily Arvicolinae, which is endemic to the mountains in southwest China. E. chinensis and other Arvicoline species display a number of features that make them ideal for evolutionary studies of speciation and the role of Quaternary glacial cycles on diversification. In this study, the complete mitochondrial genome of E. chinensis was sequenced. It was determined to be 16,362 bases. The nucleotide sequence data of 12 heavy-strand protein-coding genes of E. chinensis and other 19 rodents were used for phylogenetic analyses. Trees constructed using three different phylogenetic methods (Bayesian, maximum parsimony, and maximum likelihood) showed a similar topology demonstrating that E. chinensis was clustered in subfamily arvicolinae--formed a solid monophyletic group being sister to the subfamily Cricetinae. And the trees also suggested that E. chinensis is a sister to the genus Microtus and Proedromys.

Complete mitochondrial genome of a new vole Proedromys liangshanensis (Rodentia: Cricetidae) and phylogenetic analysis with related species: are there implications for the validity of the genus Proedromys?

AIM. The complete mitochondrial genome sequence of a newly discovered vole, Proedromys liangshanensis (Rodentia: Cricetidae: Arvicolinae), was determined. RESULTS. The mitogenome of P. liangshanensis is 16,296 bp in length. As with most other mammals, it contains the same gene order and an identical number of genes or regions, including 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and one putative control region. The A+T content of the control region is 58.2%, the lowest proportion detected so far in Myomorpha. To confirm the phylogenetic position of P. liangshanensis, we carried out phylogenetic analyses based on complete mitochondrial genomic data using Bayesian, maximum parsimony, and maximum likelihood methods. CONCLUSION. All results revealed that P. liangshanenis is sister to Microtus. Although the results do not bear light on the validity of the genus Proedromys, based on the morphological characters, we suggest that Proedromys is an independent genus of equal rank to the genus Microtus.