TET1 mutations as a predictive biomarker for immune checkpoint inhibitors in colon adenocarcinoma.

BACKGROUND: The ten-eleven translocation 1 (TET1), which is essential for active DNA demethylation, plays a multifaceted role in the pathogenesis of colorectal cancer. The study has demonstrated the association of TET1 mutations with a high response to immune checkpoint inhibitors (ICIs) in diverse cancers. However, the relationship between TET1 mutations and the response to ICIs in colon cancer is still lacking. METHODS: The prognosis, predictive markers, immune characteristics, mutation number of DNA damage repair (DDR) pathways, pathway enrichment, and drug sensitivity conditions were all compared between TET1-mutated and wild-type patients with colon adenocarcinoma (COAD). RESULTS: The overall survival of patients with TET1 mutations in the ICI-treated cohort was significantly longer than those without (p = 0.0059). Compared with the wild-type patients, TET1-mutated patients had higher tumor mutational burden and neoantigen load, enhanced abundance of tumor-infiltrating immune cells, increased expression of immune-related genes, and mutation number of DDR pathways. Additionally, the patients with TET1 mutations were found to be more sensitive to lapatinib and 5-fluorouracil. CONCLUSION: These findings suggest that TET1 mutations may serve as a potential biomarker for the response to ICIs in COAD patients.

Comprehensive identification of FGFR1-4 alterations in 5 557 Chinese patients with solid tumors by next-generation sequencing.

Deregulation of fibroblast growth factor receptor (FGFR) network is common in cancer due to activating mutations, gene amplifications and chromosomal translocations. Currently, various FGFR inhibitors are being developed. In order to optimize their clinical applications, understanding the frequencies and types of FGFR alterations in multiple cancer types appears to be extremely important. This study characterized FGFR1-4 alterations in solid tumors by next-generation sequencing (NGS). Between Jun. 2019 and Aug. 2020, the sequencing data of 5 557 solid tumors of diverse types in the database of Simcere Diagnostics, Inc. (Nanjing, China) were retrospectively analyzed. A panel-based NGS assay was used to detect FGFR1-4 alterations in tumor samples. 9.2% of cancer cases had FGFR1-4 alterations, in which gene amplifications (51.5%) and mutations (40.7%) were frequent, whereas gene rearrangements were less common (10.0%). FGFR1 was involved in 4.6% of 5 557 cases, FGFR2 in 2.1%, FGFR3 in 1.6%, and FGFR4 in 1.4%. Of patients with FGFR1-4 alterations, TP53, MUC16, NSD3, MYC and LRP1B genes were the top 5 mutant genes. FGFR1-4 aberrations occurred in almost every type of solid tumors, with the most common tumor being endometrial carcinoma (22.2%), followed by sarcoma (17.3%), breast cancer (13.2%), gastric cancer (12.2%), and more. 0.6% of cancer cases harbored FGFR1-4 fusions, with the most common fusion partner being TACC3. Two cases of GBM harboring FGFR3-TACC3 fusions were responsive to anlotinib treatment. In conclusion, FGFR1-4 alterations are prevalent in solid tumors of diverse types, with the majority being gene amplifications and mutations. FGFR1-4 fusions only occur in a minority of cancer cases, and those with glioblastoma harboring FGFR3-TACC3 fusions may benefit from anlotinib.

Proteome and phosphoproteome profiling reveals the regulation mechanism of hibernation in a freshwater leech (Whitmania pigra).

Hibernation is an energy-saving and adaptive strategy adopted by leech, an important medicinal resource in Asia, to survive low temperature. Reversible protein phosphorylation (RPP) plays a key role in the regulation of mammalian hibernation processes but has never been documented in freshwater invertebrate such as leech. In this study, we detected the effects of hibernation on the proteome and phosphoproteome of the leech Whitmania pigra. A total of 2184 proteins and 2598 sites were quantified. Deep-hibernation resulted in 85 up-regulated and 107 down-regulated proteins and 318 up-regulated and 204 down-regulated phosphosites using a 1.5-fold threshold (P<0.05). Proteins involved in protein digestion and absorption, amino acid metabolism and N-glycan biosynthesis were significantly down-regulated during deep-hibernation. However, proteins involved in maintaining cell structure stability in hibernating animals were up-regulated. Differentially phosphorylated proteins provided the first global picture of a shift in energy metabolism, protein synthesis, cytoprotection and signaling during deep hibernation. Furthermore, AMP-activated protein kinase and protein kinase C play major roles in the regulation of these functional processes. These data significantly improve our understanding of the regulatory mechanisms of leech hibernation processes and provides substantial candidate phosphorylated proteins that could be important for functionally adapt in freshwater animals. SIGNIFICANCE: The leech Whitmania pigra as an important medicinal resource in Asia is an excellent model freshwater invertebrate for studies of environmentally-induced hibernation. The present study provides the first quantitative proteomics and phosphoproteomic analysis of leech hibernation using isobaric tag based TMT labeling and high-resolution mass spectrometry. These data significantly improve our understanding of the regulatory mechanisms when ectotherm animals face environmental stress and provides substantial candidate phosphorylated proteins that could be important for functionally adapt in freshwater animals.

Gene knockout of Zmym3 in mice arrests spermatogenesis at meiotic metaphase with defects in spindle assembly checkpoint.

ZMYM3, a member of the MYM-type zinc finger protein family and a component of a LSD1-containing transcription repressor complex, is predominantly expressed in the mouse brain and testis. Here, we show that ZMYM3 in the mouse testis is expressed in somatic cells and germ cells until pachytene spermatocytes. Knockout (KO) of Zmym3 in mice using the CRISPR-Cas9 system resulted in adult male infertility. Spermatogenesis of the KO mice was arrested at the metaphase of the first meiotic division (MI). ZMYM3 co-immunoprecipitated with LSD1 in spermatogonial stem cells, but its KO did not change the levels of LSD1 or H3K4me1/2 or H3K9me2. However, Zmym3 KO resulted in elevated numbers of apoptotic germ cells and of MI spermatocytes that are positive for BUB3, which is a key player in spindle assembly checkpoint. Zmym3 KO also resulted in up-regulated expression of meiotic genes in spermatogonia. These results show that ZMYM3 has an essential role in metaphase to anaphase transition during mouse spermatogenesis by regulating the expression of diverse families of genes.

MicroRNA-202 maintains spermatogonial stem cells by inhibiting cell cycle regulators and RNA binding proteins.

miRNAs play important roles during mammalian spermatogenesis. However, the function of most miRNAs in spermatogenesis and the underlying mechanisms remain unknown. Here, we report that miR-202 is highly expressed in mouse spermatogonial stem cells (SSCs), and is oppositely regulated by Glial cell-Derived Neurotrophic Factor (GDNF) and retinoic acid (RA), two key factors for SSC self-renewal and differentiation. We used inducible CRISPR-Cas9 to knockout miR-202 in cultured SSCs, and found that the knockout SSCs initiated premature differentiation accompanied by reduced stem cell activity and increased mitosis and apoptosis. Target genes were identified with iTRAQ-based proteomic analysis and RNA sequencing, and are enriched with cell cycle regulators and RNA-binding proteins. Rbfox2 and Cpeb1 were found to be direct targets of miR-202 and Rbfox2 but not Cpeb1, is essential for the differentiation of SSCs into meiotic cells. Accordingly, an SSC fate-regulatory network composed of signaling molecules of GDNF and RA, miR-202 and diverse downstream effectors has been identified.

Retinoic Acid Is Sufficient for the In Vitro Induction of Mouse Spermatocytes.

Meiosis is the key step in gametogenesis. However, the mechanism of mammalian meiosis remains poorly understood due to the lack of an in vitro model. Here, we report that retinoic acid (RA) is sufficient for inducing leptotene/zygotene spermatocytes from cultured mouse spermatogonial stem cells. Multiple genes regulated by RA were identified by RNA sequencing. RA in combination with pup Sertoli cell co-culture resulted in a higher induction efficiency of 28%. Comparisons in the transcriptomic profiles of the induced spermatogenic cells and the isolated ones revealed the progressive induction of the germ cells. Using this model, we showed that Stra8, Agpat3, Fam57a, Wdr91, and Sox30 contributed to the proliferation and meiosis initiation differentially. In conclusion, we have efficiently generated spermatocytes using an RA/pup Sertoli cell-based in vitro model and provided proof-of-concept evidence for its application in identifying genes involved in mammalian meiosis.

Transcription Factor RFX2 Is a Key Regulator of Mouse Spermiogenesis.

The regulatory factor X (RFX) family of transcription factors is crucial for ciliogenesis throughout evolution. In mice, Rfx1-4 are highly expressed in the testis where flagellated sperm are produced, but the functions of these factors in spermatogenesis remain unknown. Here, we report the production and characterization of the Rfx2 knockout mice. The male knockout mice were sterile due to the arrest of spermatogenesis at an early round spermatid step. The Rfx2-null round spermatids detached from the seminiferous tubules, forming large multinucleated giant cells that underwent apoptosis. In the mutants, formation of the flagellum was inhibited at its earliest stage. RNA-seq analysis identified a large number of cilia-related genes and testis-specific genes that were regulated by RFX2. Many of these genes were direct targets of RFX2, as revealed by chromatin immunoprecipitation-PCR assays. These findings indicate that RFX2 is a key regulator of the post-meiotic development of mouse spermatogenic cells.

Rapid Generation of Human-Like Neutralizing Monoclonal Antibodies in Urgent Preparedness for Influenza Pandemics and Virulent Infectious Diseases.

BACKGROUND: The outbreaks of emerging infectious diseases caused by pathogens such as SARS coronavirus, H5N1, H1N1, and recently H7N9 influenza viruses, have been associated with significant mortality and morbidity in humans. Neutralizing antibodies from individuals who have recovered from an infection confer therapeutic protection to others infected with the same pathogen. However, survivors may not always be available for providing plasma or for the cloning of monoclonal antibodies (mAbs). METHODOLOGY/PRINCIPAL FINDINGS: The genome and the immunoglobulin genes in rhesus macaques and humans are highly homologous; therefore, we investigated whether neutralizing mAbs that are highly homologous to those of humans (human-like) could be generated. Using the H5N1 influenza virus as a model, we first immunized rhesus macaques with recombinant adenoviruses carrying a synthetic gene encoding hemagglutinin (HA). Following screening an antibody phage display library derived from the B cells of immunized monkeys, we cloned selected macaque immunoglobulin heavy chain and light chain variable regions into the human IgG constant region, which generated human-macaque chimeric mAbs exhibiting over 97% homology to human antibodies. Selected mAbs demonstrated potent neutralizing activities against three clades (0, 1, 2) of the H5N1 influenza viruses. The in vivo protection experiments demonstrated that the mAbs effectively protected the mice even when administered up to 3 days after infection with H5N1 influenza virus. In particular, mAb 4E6 demonstrated sub-picomolar binding affinity to HA and superior in vivo protection efficacy without the loss of body weight and obvious lung damage. The analysis of the 4E6 escape mutants demonstrated that the 4E6 antibody bound to a conserved epitope region containing two amino acids on the globular head of HA. CONCLUSIONS/SIGNIFICANCE: Our study demonstrated the generation of neutralizing mAbs for potential application in humans in urgent preparedness against outbreaks of new influenza infections or other virulent infectious diseases.

Comparative immunogenicity of recombinant adenovirus-vectored vaccines expressing different forms of hemagglutinin (HA) proteins from the H5 serotype of influenza A viruses in mice.

Recent outbreaks of highly pathogenic avian influenza (HPAI) H5N1 viruses in poultry and their subsequent transmission to humans have highlighted an urgent need to develop preventive vaccines in the event of a pandemic. In this paper we constructed recombinant adenovirus (rAd)-vectored influenza vaccines expressing different forms of H5 hemagglutinin (HA) from the A/Vietnam/1194/04 (VN/1194/04) virus, a wild-type HA, a sequence codon-optimized HA and a transmembrane (TM) domain-truncated HA. Compared to the rAd vectors expressing the wild-type HA (rAd-04wtHA) and the TM-truncated form of HA (rAd-04optHA-dTM), the rAd vectored vaccine with the sequence codon-optimized HA (rAd-04optHA) showed a tendency to induce much higher hemagglutinin inhibition (HI) antibody titers in mice immunized with a prime-boost vaccine. Furthermore, administration of the rAd-04optHA vaccine to mice could elicit cross-reactive immune responses against the antigenically distinct HK/482/97 virus. Additionally, we constructed another vector containing the codon-optimized HA of the A/Hong Kong/482/97 (HK/482/97) virus. Administration of a bivalent immunization formulation including the rAd-04optHA and rAd-97optHA vaccines to mice induced a stronger immune response against HK/482/97 virus than the monovalent formulation. Taken together, these findings may have some implications for the development of rAd-vectored vaccines in the event of the pandemic spread of HPAI.