NKG2D-CAR T cells eliminate senescent cells in aged mice and nonhuman primates.

Cellular senescence, characterized by stable cell cycle arrest, plays an important role in aging and age-associated pathologies. Eliminating senescent cells rejuvenates aged tissues and ameliorates age-associated diseases. Here, we identified that natural killer group 2 member D ligands (NKG2DLs) are up-regulated in senescent cells in vitro, regardless of stimuli that induced cellular senescence, and in various tissues of aged mice and nonhuman primates in vivo. Accordingly, we developed and demonstrated that chimeric antigen receptor (CAR) T cells targeting human NKG2DLs selectively and effectively diminish human cells undergoing senescence induced by oncogenic stress, replicative stress, DNA damage, or P16INK4a overexpression in vitro. Targeting senescent cells with mouse NKG2D-CAR T cells alleviated multiple aging-associated pathologies and improved physical performance in both irradiated and aged mice. Autologous T cells armed with the human NKG2D CAR effectively delete naturally occurring senescent cells in aged nonhuman primates without any observed adverse effects. Our findings establish that NKG2D-CAR T cells could serve as potent and selective senolytic agents for aging and age-associated diseases driven by senescence.

GAS6-based CAR-T cells exhibit potent antitumor activity against pancreatic cancer.

BACKGROUND: The receptor tyrosine kinases TAM family (TYRO3, AXL, and MERTK) are highly expressed in multiple forms of cancer cells and tumor-associated macrophages and promote the development of cancers including pancreatic tumor. Targeting TAM receptors could be a promising therapeutic option. METHODS: We designed a novel CAR based on the extracellular domain of growth arrest-specific protein 6 (GAS6), a natural ligand for all TAM members. The ability of CAR-T to kill pancreatic cancer cells is tested in vitro and in vivo, and the safety is evaluated in mice and nonhuman primate. RESULTS: GAS6-CAR-T cells efficiently kill TAM-positive pancreatic cancer cell lines, gemcitabine-resistant cancer cells, and cancer stem-like cells in vitro. GAS6-CAR-T cells also significantly suppressed the growth of PANC1 xenografts and patient-derived xenografts in mice. Furthermore, these CAR-T cells did not induce obvious side effects in nonhuman primate or mice although the CAR was demonstrated to recognize mouse TAM. CONCLUSIONS: Our findings indicate that GAS6-CAR-T-cell therapy may be effective for pancreatic cancers with low toxicity.

Smad4 deficiency substitutes Cdkn2b but not Cdkn2a downregulation in pancreatic cancer following induction of genetic events in adult mice.

BACKGROUND: Minor progress in pancreatic cancer treatment and prognosis implies that more reliable animal models are urgently needed to decipher its molecular mechanisms and preclinical research. We recently reported a genetically engineered adult mouse model where Cdkn2b downregulation was required together with Cdkn2a downregulation to inactivate the Rb pathway. Besides, the role of Smad4, which is mutated more frequently than Cdkn2b in human pancreatic cancer, was determined critical on the development of the pancreas tumor by some reports. However, the impact of Smad4 deficiency in combination with PDAC-relevant mutations, such as Cdkn2a when induced in adult pancreas has not been completely elucidated in mice. METHODS: Lentiviral delivered oncogene/tumor suppressors in adult pancreas. The development of pancreatic cancer was monitored. Hematoxylin and eosin staining and immunofluorescence were performed for pathological identification of the pancreatic cancer. Real-time polymerase chain reaction, immunofluorescence and western blot were used to test gene expression. RESULTS: Loss of Smad4 could cooperate with alterations of KRAS, Trp53, and Cdkn2a to induce pancreatic cancer in adult mice. The role of Smad4 was mainly in downregulating the expression of Cdkn2b and further inducing phosphorylation of the Rb1 protein. CONCLUSIONS: These findings show an essential role of Smad4 deficiency in pancreatic ductal adenocarcinoma (PDAC) formation. This model better recapitulates the adult onset, clonal origin, and genetic alterations in human PDAC and can be simply generated on a large-scale.

3D Genome of macaque fetal brain reveals evolutionary innovations during primate corticogenesis.

Elucidating the regulatory mechanisms of human brain evolution is essential to understanding human cognition and mental disorders. We generated multi-omics profiles and constructed a high-resolution map of 3D genome architecture of rhesus macaque during corticogenesis. By comparing the 3D genomes of human, macaque, and mouse brains, we identified many human-specific chromatin structure changes, including 499 topologically associating domains (TADs) and 1,266 chromatin loops. The human-specific loops are significantly enriched in enhancer-enhancer interactions, and the regulated genes show human-specific expression changes in the subplate, a transient zone of the developing brain critical for neural circuit formation and plasticity. Notably, many human-specific sequence changes are located in the human-specific TAD boundaries and loop anchors, which may generate new transcription factor binding sites and chromatin structures in human. Collectively, the presented data highlight the value of comparative 3D genome analyses in dissecting the regulatory mechanisms of brain development and evolution.

Trio deep-sequencing does not reveal unexpected off-target and on-target mutations in Cas9-edited rhesus monkeys.

CRISPR-Cas9 is a widely-used genome editing tool, but its off-target effect and on-target complex mutations remain a concern, especially in view of future clinical applications. Non-human primates (NHPs) share close genetic and physiological similarities with humans, making them an ideal preclinical model for developing Cas9-based therapies. However, to our knowledge no comprehensive in vivo off-target and on-target assessment has been conducted in NHPs. Here, we perform whole genome trio sequencing of Cas9-treated rhesus monkeys. We only find a small number of de novo mutations that can be explained by expected spontaneous mutations, and no unexpected off-target mutations (OTMs) were detected. Furthermore, the long-read sequencing data does not detect large structural variants in the target region.

Author Correction: Loss-of-function mutations in QRICH2 cause male infertility with multiple morphological abnormalities of the sperm flagella.

In the original version of this Article, the Acknowledgements section omitted the National Key Research and Development Program of China (SQ2018YFC1003603).

A novel pancreatic cancer model originated from transformation of acinar cells in adult tree shrew, a primate-like animal.

Pancreatic cancer is one of the most lethal common cancers. The cell of origin of pancreatic ductal adenocarcinoma (PDAC) has been controversial, and recent evidence suggested acinar cells as the most probable candidate. However, the genetic alterations driving the transformation of pancreatic acinar cells in fully mature animals remain to be deciphered. In this study, lentivirus was used as a tool to introduce genetic engineering in tree shrew pancreatic acinar cells to explore the driver mutation essential for malignant transformation, establishing a novel tree shrew PDAC model, because we found that lentivirus could selectively infect acinar cells in tree shrew pancreas. Combination of oncogenic KRASG12D expression and inactivation of tumor suppressor genes Tp53, Cdkn2a and Cdkn2b could induce pancreatic cancer with full penetrance. Silencing of Cdkn2b is indispensable for Rb1 phosphorylation and tumor induction. Tree shrew PDAC possesses the main histological and molecular features of human PDAC. The gene expression profile of tree shrew PDAC was more similar to human disease than a mouse model. In conclusion, we established a novel pancreatic cancer model in tree shrew and identified driver mutations indispensable for PDAC induction from acinar cells in mature adults, demonstrating the essential roles of Cdkn2b in the induction of PDAC originating from adult acinar cells. Tree shrew could thus provide a better choice than mouse for a PDAC model derived from acinar cells in fully mature animals.

Loss-of-function mutations in QRICH2 cause male infertility with multiple morphological abnormalities of the sperm flagella.

Aberrant sperm flagella impair sperm motility and cause male infertility, yet the genes which have been identified in multiple morphological abnormalities of the flagella (MMAF) can only explain the pathogenic mechanisms of MMAF in a small number of cases. Here, we identify and functionally characterize homozygous loss-of-function mutations of QRICH2 in two infertile males with MMAF from two consanguineous families. Remarkably, Qrich2 knock-out (KO) male mice constructed by CRISPR-Cas9 technology present MMAF phenotypes and sterility. To elucidate the mechanisms of Qrich2 functioning in sperm flagellar formation, we perform proteomic analysis on the testes of KO and wild-type mice. Furthermore, in vitro experiments indicate that QRICH2 is involved in sperm flagellar development through stabilizing and enhancing the expression of proteins related to flagellar development. Our findings strongly suggest that the genetic mutations of human QRICH2 can lead to male infertility with MMAF and that QRICH2 is essential for sperm flagellar formation.

Single-cell RNA-sequencing reveals the existence of naive and primed pluripotency in pre-implantation rhesus monkey embryos.

Naive pluripotency exists in epiblast cells of mouse pre-implantation embryos. However, whether the naive pluripotency is transient or nonexistent in primate embryos remains unclear. Using RNA-seq in single blastomeres from 16-cell embryos through to hatched blastocysts of rhesus monkey, we constructed the lineage segregation roadmap in which the specification of trophectoderm, epiblast, and primitive endoderm is initiated simultaneously at the early blastocyst stage. Importantly, we uncovered the existence of distinct pluripotent states in monkey pre-implantation embryos. At the early- and middle-blastocyst stages, the epiblast cells have the transcriptome features of naive pluripotency, whereas they display a continuum of primed pluripotency characteristics at the late and hatched blastocyst stages. Moreover, we identified potential regulators that might play roles in the transition from naive to primed pluripotency. Thus, our study suggests the transient existence of naive pluripotency in primates and proposes an ideal time window for derivation of primate embryonic stem cells with naive pluripotency.

HUWE1 controls the development of non-small cell lung cancer through down-regulation of p53.

Lung cancer is the most frequent cancer type and the leading cause of tumor-associated deaths worldwide. TP53 is an important tumor suppressor gene and is frequently inactivated in lung cancer. E3 ligases targeting p53, such as MDM2, are involved in the development of lung cancer. The E3 ligase HUWE1, which targets many tumor-associated proteins including p53, has been reported to be highly expressed in lung cancer; however, its role in lung tumorigenesis is unclear. Methods: The expression of HUWE1 and p53 in lung cancer cells was modulated and the phenotypes were assessed by performing soft agar colony forming assays, cell cycle analysis, BrdU incorporation assays, and xenograft tumor growth assays. The effect on tumorigenesis in genetically-engineered mice was also analyzed. The mechanism through which HUWE1 sustained lung cancer cell malignancy was confirmed by western blotting. HUWE1 expression in clinical lung cancer was identified by immunohistochemistry and validated by analyzing lung adenocarcinoma and lung squamous carcinoma samples from the Cancer Genome Atlas (TCGA) database. Finally, we assessed the association between HUWE1 expression and patient outcome using online survival analysis software including survival information from the caBIG, GEO, and TCGA database. Results: Inactivation of HUWE1 in a human lung cancer cell line inhibited proliferation, colony-forming capacity, and tumorigenicity. Mechanistically, this phenotype was driven by increased p53, which was due to attenuated proteasomal degradation by HUWE1. Up-regulation of p53 inhibited cancer cell malignancy, mainly through the induction of p21 expression and the down-regulation of HIF1α. Huwe1 deletion completely abolished the development of EGFRVIII-induced lung cancer in Huwe1 conditional knockout mice. Furthermore, survival analysis of lung cancer patients showed that increased HUWE1 expression is significantly associated with worse prognosis. Conclusion: Our data suggest that HUWE1 plays a critical role in lung cancer and that the HUWE1-p53 axis might be a potential target for lung cancer therapy.

Depletion of endogenous germ cells in tree shrews in preparation for spermatogonial transplantation.

To achieve successful spermatogonial transplantation, endogenous germ cells must be depleted in recipient animals to allow donor germ cells to colonize efficiently. Busulfan is commonly used for the depletion of endogenous germ cells in recipient males. However, the optimal dose of busulfan is species-specific, and the optimal dose in tree shrews is yet to be determined. The current study aimed to determine the optimal dose of busulfan for effective suppression of endogenous spermatogenesis in tree shrews. Different doses (15, 20, 25, 30, 35, 40 and 45 mg/kg) of busulfan were injected into tree shrews intraperitoneally. Survival rates of the different treatment groups were calculated at 2 weeks and body weights were measured at 4, 6, 8, 10 and 28 weeks post-busulfan treatment. The testes were also removed and weighed at 4, 6, 8, 10 and 28 weeks post-treatment, and the cross and longitude diameters of the testes and diameters of the seminiferous tubules were measured and histologically evaluated. It was observed that there were no significant differences in the survival rates between the 15-35 mg/kg treatment groups and the control group (P>0.05), while the survival rate of the 40 mg/kg treatment group significantly decreased relative to the control group (P<0.05) and the survival rate of the 45 mg/kg treatment group was 0% (P<0.05 vs. control). In addition, the weight and diameters of the testes, diameters of the seminiferous tubules and proportion of normal type tubules in the 40 mg/kg group significantly decreased over 4-10 weeks relative to the control group (P<0.05), though gradually recovered with time. At 28 weeks, the recovery was significant relative to 4 weeks (P<0.05). Similarly, histological analysis indicted that recovery of abnormal tubules was delayed in the 40 mg/kg group relative to the lower dose groups as the 40 mg/kg dose tree shrews had more tubules with no spermatogenesis compared with the lower dose group at the same time points. These data indicate that a busulfan dose of 40 mg/kg is optimal for the depletion of endogenous germ cells in tree shrews. This dose led to maximum suppression of endogenous spermatogenesis while maintaining an acceptable survival rate of >50% of the lethal dose of busulfan for tree shrews.

Huwe1 Sustains Normal Ovarian Epithelial Cell Transformation and Tumor Growth through the Histone H1.3-H19 Cascade.

Ubiquitination-directed protein degradation is important in many cancers for tumor initiation and maintenance, and E3 ligases containing HECT domains are emerging as new therapeutic targets. In contrast to many other E3 ligases, the role of HUWE1 in ovarian cancer where HUWE1 is dysregulated has been unclear. Here we report that genetic deletion of Huwe1 in the mouse inhibits transformation of ovary surface epithelium cells without significantly affecting cell survival and apoptosis, and that Huwe1 deletion after tumors have been initiated inhibits tumor growth. In Huwe1-deficient cells, expression of histone H1.3 increased, inhibiting the expression of noncoding RNA H19H19 silencing phenocopied the effects of Huwe1 deficiency, whereas H1.3 silencing partially rescued the expression of H19 and the Huwe1-null phenotype. Inducible silencing of HUWE1 in human ovarian cancer cells produced a similar phenotype. Mechanistically, HUWE1 bound and ubiquitinated H1.3, which was consequently marked for destruction by proteasomes. Our results establish that HUWE1 plays an essential role in promoting ovarian cancer. Cancer Res; 77(18); 4773-84. ©2017 AACR.

A tree shrew glioblastoma model recapitulates features of human glioblastoma.

Tupaia belangeri (tree shrew), an animal species whose genome has significantly higher similarity to primates than rodents, has been used in biomedical research. To generate animal models that reproduce the human tumors more faithfully than rodents, we present the first report of a cancer model mimicking human tumor genetics in tree shrew. By engineering a lentiviral system for the transduction of mutant H-Ras and a shRNA against tree shrew p53, we successfully generated malignant glioma in tree shrew. The tree shrew glioma exhibited aggressive behavior and a relatively short latency, and markedly reduced animal survival. Remarkably, the biological features of human high-grade glioma (necrosis, microvascular proliferation, pseudopalisading) were all present in tree shrew glioma. Furthermore, genetic analysis of tree shrew glioma revealed that the tumors were clustered within the mesenchymal subgroup of human glioblastoma multiforme. Compared with the corresponding mouse glioma, tree shrew gliomas were markedly more similar to human glioblastoma at gene expression profile. The tree shrew glioma model provides colleagues working in the field of gliomas and cancer in general with a more accurate animal model.

Long-term propagation of tree shrew spermatogonial stem cells in culture and successful generation of transgenic offspring.

Tree shrews have a close relationship to primates and have many advantages over rodents in biomedical research. However, the lack of gene manipulation methods has hindered the wider use of this animal. Spermatogonial stem cells (SSCs) have been successfully expanded in culture to permit sophisticated gene editing in the mouse and rat. Here, we describe a culture system for the long-term expansion of tree shrew SSCs without the loss of stem cell properties. In our study, thymus cell antigen 1 was used to enrich tree shrew SSCs. RNA-sequencing analysis revealed that the Wnt/ß-catenin signaling pathway was active in undifferentiated SSCs, but was downregulated upon the initiation of SSC differentiation. Exposure of tree shrew primary SSCs to recombinant Wnt3a protein during the initial passages of culture enhanced the survival of SSCs. Use of tree shrew Sertoli cells, but not mouse embryonic fibroblasts, as feeder was found to be necessary for tree shrew SSC proliferation, leading to a robust cell expansion and long-term culture. The expanded tree shrew SSCs were transfected with enhanced green fluorescent protein (EGFP)-expressing lentiviral vectors. After transplantation into sterilized adult male tree shrew's testes, the EGFP-tagged SSCs were able to restore spermatogenesis and successfully generate transgenic offspring. Moreover, these SSCs were suitable for the CRISPR/Cas9-mediated gene modification. The development of a culture system to expand tree shrew SSCs in combination with a gene editing approach paves the way for precise genome manipulation using the tree shrew.

Early embryonic development and transplantation in tree shrews.

As a novel experimental animal model, tree shrews have received increasing attention in recent years. Despite this, little is known in regards to the time phases of their embryonic development. In this study, surveillance systems were used to record the behavior and timing of copulations; embryos at different post-copulation stages were collected and cultured in vitro; and the developmental characteristics of both early-stage and in vitro cultured embryos were determined. A total of 163 females were collected following effective copulation, and 150 were used in either unilateral or bilateral oviduct embryo collections, with 307 embryos from 111 females obtained (conception rate=74%). Among them, 237 embryos were collected from 78 females, bilaterally, i.e., the average embryo number per female was 3.04; 172 fertilized eggs collected from 55 females, bilaterally, were cultured for 24-108 h in vitro for developmental observations; finally, 65 embryos from 23 bilateral cases and 70 embryos from 33 unilateral cases were used in embryo transplantation.

Wnt pathway is involved in pleomorphic adenomas induced by overexpression of PLAG1 in transgenic mice.

Pleomorphic adenoma gene 1 (PLAG1) was found frequently rearranged and activated in human salivary gland pleomorphic adenomas. It encodes a developmentally regulated transcription factor. Ectopic overexpression of PLAG1 has been proposed to play a crucial role in tumorigenesis of salivary gland pleomorphic adenomas. It was reported that PLAG1 can activate the transcription of insulin-like growth factor 2 (IGF2), functioning as a protooncogene. In this report, we show that the salivary gland tumors developed in PLAG1 transgenic mice share major histopathologic features with human pleomorphic adenomas. It was found that beta-catenin, the key component of Wnt signaling pathway, was upregulated at transcriptional level in tumors developed in 3 independent transgenic mouse lines. Immunohistochemical staining revealed that expression of beta-catenin as well as c-myc, downstream of beta-catenin in Wnt signaling pathway, was highly upregulated with overexpression of PLAG1 transgene in tumor and normal transgenic salivary gland tissues. Moreover, we found that PLAG1 can activate the transcription of mouse but not human beta-catenin in the 3T3 cells cotransfected with reporter constructs. Sequence analysis shows there are 4 PLAG1 consensus binding sites in mouse beta-catenin promoter region but not in human. Our findings provide the first in vivo evidence for the oncogenic activity of PLAG1 in pleomorphic adenoma tumorigenesis, reveal a valued animal model for human salivary gland tumors and suggest that Wnt signaling pathway may also contribute to the development of pleomorphic adenomas in transgenic mice.

[Generation of transgenic mice for hygromycin and neomycin resistance genes and studies on transgene expression].

To generate transgenic mice in which both hygromycin (hyg) and neomycin (neo) resistance genes are expressed in murine fibroblast cells (MEFs), which are required for conditional gene knock-out and screening of drug resistant ES cell clones. To construct HygR-neoR expression vector, pTK-hygR-pA and PGK-neoR-pA were cloned into pBluescript vector. DNA fragments of tandem genes ( 4245bp ) were prepared by Kpn I and Xba I digestion and transgene was microinjected into pronucleus of zygotes to generate transgenic mice. Transgenic mice were identified by PCR and Southern blot; expression of hygR and neoR gene transcripts were detected by RT-PCR. 7 founder mice carrying hyg-neo resistant genes were obtained and 6 transgenic mouse lines were successfully established. The hygR and neoR gene transcripts were detected in the liver and/or ovary of transgenic mice from hn30, hn33, hn66 and hn67 mouse lines. In MEFs isolated from the mice of line hn66 and hn30, expression of hyg and neo resistant genes was also detectable. Transgenic mouse lines expressing two anti-drug genes have been established. The hyg and neo resistant gene transcripts were detected in the MEFs of two transgenic mouse lines.