iGWAS: Image-based genome-wide association of self-supervised deep phenotyping of retina fundus images.

Existing imaging genetics studies have been mostly limited in scope by using imaging-derived phenotypes defined by human experts. Here, leveraging new breakthroughs in self-supervised deep representation learning, we propose a new approach, image-based genome-wide association study (iGWAS), for identifying genetic factors associated with phenotypes discovered from medical images using contrastive learning. Using retinal fundus photos, our model extracts a 128-dimensional vector representing features of the retina as phenotypes. After training the model on 40,000 images from the EyePACS dataset, we generated phenotypes from 130,329 images of 65,629 British White participants in the UK Biobank. We conducted GWAS on these phenotypes and identified 14 loci with genome-wide significance (p<5×10-8 and intersection of hits from left and right eyes). We also did GWAS on the retina color, the average color of the center region of the retinal fundus photos. The GWAS of retina colors identified 34 loci, 7 are overlapping with GWAS of raw image phenotype. Our results establish the feasibility of this new framework of genomic study based on self-supervised phenotyping of medical images.

Gene augmentation therapy to rescue degenerative photoreceptors in a Cwc27 mutant mouse model.

Previous reports have demonstrated that defects in the spliceosome-associated protein CWC27 can lead to the degeneration of retinal cells in Cwc27 mutant mouse models. However, it is unknown whether gene replacement therapy can rescue this phenotype. The purpose of this study was to evaluate whether AAV based gene therapy could rescue the retinal degeneration observed in Cwc27 mutant mice. By 6 months of age, Cwc27 mutant mice show a retinal degenerative phenotype, including morphological and functional abnormalities, primarily driven by the death of photoreceptors. We hypothesize that subretinal injection of AAV8 to drive exogenous CWC27 protein expression will improve the retinal phenotype. We evaluated these improvements after gene therapy with electroretinography (ERG) and histology, either hematoxylin and eosin (H&E) or immunostaining. In this study, we demonstrated that subretinal injection of AAV8-GRK-Cwc27-FLAG in mutant mice can improve the functionality and morphology of the retina. Immunostaining analyses revealed a notable decrease in photoreceptor degeneration, including cone cell degeneration, in the AAV-injected eyes compared to the PBS-injected eyes. Based on these results, gene replacement therapy could be a promising method for treating retinal degeneration caused by mutations in Cwc27.

Ceramide compensation by ceramide synthases preserves retinal function and structure in a retinal dystrophy mouse model.

Increasing evidence has supported the role of ceramide as a mediator of photoreceptor dysfunction or cell death in ceramide accumulation and deficiency contexts. TLCD3B, a non-canonical ceramide synthase, was previously identified in addition to the six canonical ceramide synthases (CerSs), and the Tlcd3b-/- mouse model exhibited both retinal dysfunction and degeneration. As previous canonical CerS-deficient mouse models failed to display retinal degeneration, the mechanisms of how TLCD3B interacts with CerSs have not been investigated. Additionally, as the ceramide profile of each CerS is distinct, it is unclear whether the overall level or the homeostasis of different ceramide species plays a critical role in photoreceptor degeneration. Interactions between TLCD3B with canonical CerSs expressed in the retina were examined by subretinally injecting recombinant adeno-associated virus 8 vectors containing the Cers2 (rAAV8-CerS2), Cers4 (rAAV8-CerS4) and Cers5 (rAAV8-CerS5) genes. Injection of all three rAAV8-CerS vectors restored retinal functions as indicated by improved electroretinogram responses, but only rAAV8-CerS5 successfully retained retinal morphology in Tlcd3b-/- mice. CerSs and TLCD3B played partially redundant roles. Additionally, rather than acting as an integral entity, different ceramide species had different impacts on retinal cells, suggesting that the maintenance of the overall ceramide profile is critical for retinal function.

Single-cell multiomics of the human retina reveals hierarchical transcription factor collaboration in mediating cell type-specific effects of genetic variants on gene regulation.

BACKGROUND: Systematic characterization of how  genetic variation modulates gene regulation in a cell type-specific context is essential for understanding complex traits. To address this question, we profile gene expression and chromatin accessibility in cells from healthy retinae of 20 human donors through single-cell multiomics and genomic sequencing. RESULTS: We map eQTL, caQTL, allelic-specific expression, and allelic-specific chromatin accessibility in major retinal cell types. By integrating these results, we identify and characterize regulatory elements and genetic variants effective on gene regulation in individual cell types. The majority of identified sc-eQTLs and sc-caQTLs display cell type-specific effects, while the cis-elements containing genetic variants with cell type-specific effects are often accessible in multiple cell types. Furthermore, the transcription factors whose binding sites are perturbed by genetic variants tend to have higher expression levels in the cell types where the variants exert their effects, compared to the cell types where the variants have no impact. We further validate our findings with high-throughput reporter assays. Lastly, we identify the enriched cell types, candidate causal variants and genes, and cell type-specific regulatory mechanism underlying GWAS loci. CONCLUSIONS: Overall, genetic effects on gene regulation are highly context dependent. Our results suggest that cell type-dependent genetic effect is driven by precise modulation of both trans-factor expression and chromatin accessibility of cis-elements. Our findings indicate hierarchical collaboration among transcription factors plays a crucial role in mediating cell type-specific effects of genetic variants on gene regulation.

Integrated multi-omics single cell atlas of the human retina.

Single-cell sequencing has revolutionized the scale and resolution of molecular profiling of tissues and organs. Here, we present an integrated multimodal reference atlas of the most accessible portion of the mammalian central nervous system, the retina. We compiled around 2.4 million cells from 55 donors, including 1.4 million unpublished data points, to create a comprehensive human retina cell atlas (HRCA) of transcriptome and chromatin accessibility, unveiling over 110 types. Engaging the retina community, we annotated each cluster, refined the Cell Ontology for the retina, identified distinct marker genes, and characterized cis-regulatory elements and gene regulatory networks (GRNs) for these cell types. Our analysis uncovered intriguing differences in transcriptome, chromatin, and GRNs across cell types. In addition, we modeled changes in gene expression and chromatin openness across gender and age. This integrated atlas also enabled the fine-mapping of GWAS and eQTL variants. Accessible through interactive browsers, this multimodal cross-donor and cross-lab HRCA, can facilitate a better understanding of retinal function and pathology.

AAV8-Mediated Gene Therapy Rescues Retinal Degeneration Phenotype in a Tlcd3b Knockout Mouse Model.

Purpose: The purpose of this study was to assess the therapeutic efficacy of rAAV8-hGRK1-Tlcd3b in a Tlcd3b-/- mouse model of retinal generation and validate TLCD3B's role as a ceramide synthase in vivo. Methods: Using Tlcd3b-/- mice as an inherited retinal disease animal model, we performed subretinal injection of rAAV8-hGRK1-Tlcd3b and evaluated the efficacy of gene replacement therapy. Tlcd3b-/- mice were treated at two time points: postnatal day 21 (P21) and postnatal day 120 (P120) with various dosages. Results: Tlcd3b overexpression rescued retinal degeneration in the mutant mice, as indicated by significantly improved photoreceptor function and preservation of photoreceptor cells over the course of 1 year. Although Tlcd3b is expressed in all cell types in the retina, photoreceptor cell-specific expression of Tlcd3b is sufficient to rescue the phenotype, indicating the primary function of TLCD3B is in photoreceptors. Consistent with the idea that TLCD3B is a ceramide synthase, mass spectrometry analyses of the mutant retina indicate the reduction of C16-, C18-, and C20-ceramides in the retina, which are restored with Tlcd3b overexpression. Conclusions: Our findings demonstrated the therapeutic efficacy of gene therapy in treating Tlcd3b mutant retina, laying the foundation for developing future therapy for TLCD3B retinopathy.

Spata7 is required for maintenance of the retinal connecting cilium.

SPATA7, an early onset LCA3 retinal disease gene, encodes a putative scaffold protein that is essential for the proper assembly of the connecting cilium (CC) complex in photoreceptors. Previous studies have shown that SPATA7 interacts with other photoreceptor-specific ciliary proteins, such as RPGR and RPGRIP1, and maintains the integrity of CC integrity. However, although it is known that Spata7 is required for early formation of the CC, it is unclear if Spata7 is also required for the maintenance of the CC. To investigate Spata7 function in the retina at the adult stage, loss of function was induced in the adult retina upon tamoxifen induction of an inducible Spata7 knockout allele (Spata7flox/-; UbcCreERT2/+). The phenotype of mutant retina was characterized by a combination of histology, immunobiochemistry, and electroretinography (ERG). Our results demonstrated that Spata7 is also essential for maintaining the integrity of the mature retinal CC. Loss of Spata7 in adults caused phenotypes similar to those seen in germline mutant mice, including photoreceptor cell degeneration and defective ERG responses. Close examination of the CC revealed significantly shortened NPHP1 length as a result of Spata7 deletion. Furthermore, mislocalization of rhodopsin, leading to ER stress-mediated apoptosis, was observed in the retinal layers. Our results indicate that Spata7 is required not only for the establishment but also for the maintenance of the CC of photoreceptors.

Inhibition of Ubiquitin-Specific Protease 14 Suppresses Cell Proliferation and Synergizes with Chemotherapeutic Agents in Neuroblastoma.

Neuroblastoma is the most common extracranial malignant solid tumor in children, and drug resistance is a major reason for poor outcomes. Elevated proteasome activity plays an important role in neuroblastoma tumor development and resistance to conventional chemotherapy. Ubiquitin-specific protease 14 (USP14), one of three deubiquitinases associated with the regulatory subunit of the proteasome, is emerging as a potential therapeutic target in multiple tumor types. However, the role of USP14 in neuroblastoma is yet to be elucidated. We found that USP14 inhibition in neuroblastoma via knockdown or a specific inhibitor such as b-AP15 suppressed cell proliferation by inducing cell apoptosis. Furthermore, b-AP15 significantly inhibited neuroblastoma tumor growth in NGP and SH-SY5Y xenograft mouse models. For combination treatment, b-AP15 plus conventional chemotherapeutic agents such as doxorubicin or VP-16 resulted in synergistic antitumor effects on neuroblastoma. Our study demonstrates that USP14 is required for cell viability and is a novel therapeutic target in neuroblastoma. Moreover, USP14 inhibition may add value in combination therapy due to its powerful synergistic effects in treating neuroblastoma.

Delanzomib, a novel proteasome inhibitor, sensitizes breast cancer cells to doxorubicin-induced apoptosis.

BACKGROUND: Delanzomib, a novel proteasome inhibitor, has demonstrated promising efficacy and antitumor ability in human multiple myeloma cell lines and patient-derived cells. However, the potential therapeutic effects of delanzomib on breast cancer remain unknown. In this study, we show that delanzomib has antitumor effects and synergizes with doxorubicin (Dox) in human breast cancer cell lines. METHODS: Cell proliferation assay and flow cytometry were used to evaluate cell viability and apoptosis in eight human breast cancer cell lines after treatment with delanzomib or Dox. Essential molecules of the p53, MAPK, and apoptosis signaling pathways were analyzed by Western blotting. RESULTS: Delanzomib induced cell death and demonstrated synergism with Dox in all tested breast cancer cell lines. In addition, delanzomib enhanced the Dox-induced phosphorylation of p38/JNK and the expression of transcriptional target proteins of p53, such as p21, p27, NOXA, and PUMA. CONCLUSION: The combined regimen of the proteasome inhibitor delanzomib with Dox chemotherapy may become an effective strategy for breast cancer therapy.

Small molecule inhibitor agerafenib effectively suppresses neuroblastoma tumor growth in mouse models via inhibiting ERK MAPK signaling.

Neuroblastoma (NB) is the most common extracranial solid tumor in early childhood. Despite intensive multimodal therapy, nearly half of children with high-risk disease will relapse with therapy-resistant tumors. Dysregulation of MAPK pathway has been implicated in the pathogenesis of relapsed and refractory NB patients, which underscores the possibility of targeting MAPK signaling cascade as a novel therapeutic strategy. In this study, we found that high expressions of RAF family kinases correlated with advanced tumor stage, high-risk disease, tumor progression, and poor overall survival. Targeted inhibition of RAF family kinases with the novel small molecule inhibitor agerafenib abrogated the activation of ERK MAPK pathway in NB cells. Agerafenib significantly inhibited the cell proliferation and colony formation ability of NB cells in vitro, and its combination with traditional chemotherapy showed a synergistic pro-apoptotic effect. More importantly, agerafenib exhibited a favorable toxicity profile, potently suppressed tumor growth, and prolonged survival in NB mouse models. In conclusion, our preclinical data suggest that agerafenib might be an effective therapeutic agent for NB treatment, both as a single-agent and in combination with chemotherapy.

Anagrus dmitrievi sp. n. (Hymenoptera, Mymaridae), an egg parasitoid of Zyginidia eremita (Hemiptera, Cicadellidae), a pest of maize in Xinjiang, China.

A new Palaearctic species of Anagrus Haliday (Hymenoptera, Mymaridae), A. (Anagrus) dmitrievi Triapitsyn & Hu, sp. n., is described, diagnosed, and illustrated from Xinjiang Uyghur Autonomous Region of China. It was reared from parasitized eggs of the leafhopper Zyginidia (Zyginidia) eremita Zachvatkin (Hemiptera, Cicadellidae) on leaves of maize. A key to the 21 named species of Anagrus known from China is provided. Zyginidia eremita is an important economic pest in Xinjiang, occurring in approximately 90% of maize fields. The phenology and life cycle of Z. eremita is summarized. Parasitism of its eggs by A. dmitrievi in maize fields was 12-75 % (average about 38 %), thus making it a rather effective natural enemy.

MELK is a novel therapeutic target in high-risk neuroblastoma.

Maternal embryonic leucine zipper kinase (MELK) is known to modulate intracellular signaling and control cellular processes. However, the role of MELK in oncogenesis is not well defined. In this study, using two microarray datasets of neuroblastoma (NB) patients, we identified that MELK expression is significantly correlated to poor overall survival, unfavorable prognosis, and high-risk status. We found that MELK is a direct transcription target of MYCN and MYC in NB, and MYCN increases MELK expression via direct promoter binding. Interestingly, knockdown of MELK expression significantly reduced the phosphorylation of target protein Retinoblastoma (pRb) and inhibited NB cell growth. Furthermore, pharmacological inhibition of MELK activity by small-molecule inhibitor OTSSP167 significantly inhibited cell proliferation, anchorage-independent colony formation, blocked cell cycle progression, and induced apoptosis in different NB cell lines including a drug-resistant cell line. Additionally, OTSSP167 suppressed NB tumor growth in an orthotopic xenograft mouse model. Overall, our data suggest that MELK is a novel therapeutic target for NB and its inhibitor OTSSP167 is a promising drug for further clinical development.

TAK1 inhibitor 5Z-7-oxozeaenol sensitizes cervical cancer to doxorubicin-induced apoptosis.

Aberrant activation of nuclear factor-κB (NF-κB) allows cancer cells to escape chemotherapy-induced cell death and acts as one of the major mechanisms of acquired chemoresistance in cervical cancer. TAK1, a crucial mediator that upregulates NF-κB activation in response to cellular genotoxic stress, is required for tumor cell viability and survival. Herein, we examined whether TAK1 inhibition is a potential therapeutic strategy for treating cervical cancer. We found that TAK1 inhibitor 5Z-7-oxozeaenol significantly augmented the cytotoxic effects of Dox in a panel of cervical cancer cell lines. Treatment with 5Z-7-oxozeaenol hindered Dox-induced NF-κB activation and promoted Dox-induced apoptosis in cervical cancer cells. Moreover, 5Z-7-oxozeaenol showed similar effects in both positive and negative human papillomavirus-infected cervical cancer cells. Taken together, our results provide evidence that TAK1 inhibition significantly sensitizes cervical cancer cells to chemotherapy-induced cell death and supports the use of TAK1 inhibitor with current chemotherapies in the clinic for patients with refractory cervical cancer.

Novel proteasome inhibitor delanzomib sensitizes cervical cancer cells to doxorubicin-induced apoptosis via stabilizing tumor suppressor proteins in the p53 pathway.

Cervical cancer, the third most commonly occurring cancer, is the second leading cause of cancer related mortality among women. Aberrant ubiquitination and proteasome activity, both human papillomavirus and tumor derived, have been shown to contribute to tumor angiogenesis, proliferation, and invasion in many cancers, including cervical cancer. Thus, small molecule proteasome inhibitors are a potential and strategic treatment option for cervical cancer. In this study, novel proteasome inhibitor delanzomib (CEP-18770) exhibited potent pro-apoptotic and cytotoxic effects on a panel of cervical cancer cell lines by blocking proteasomal activity. Delanzomib also significantly sensitized cervical cancer cells to treatment of doxorubicin (Dox), a traditional chemotherapeutic agent. Furthermore, proteasome inhibition revealed stabilization of p53 and p53 transcriptional targets and induction of p38/JNK phosphorylation. Additionally, delanzomib worked synergistically with Dox to further upregulate p53 and its downstream targets and enhanced Dox-induced p38 phosphorylation. Our study strongly supports the 26S proteasome as a potential therapeutic target in cervical cancer and proteasome inhibition by delanzomib may be a potential treatment strategy for cervical cancer patients.

Novel multiple tyrosine kinase inhibitor ponatinib inhibits bFGF-activated signaling in neuroblastoma cells and suppresses neuroblastoma growth in vivo.

Neuroblastoma (NB) is one of the most common pediatric malignancies in children. Abnormal activation of receptor tyrosine kinases contributes to the pathological development of NB. Therefore, targeting tyrosine kinase receptors to cure NB is a promising strategy. Here, we report that a multi-targeted tyrosine kinase inhibitor ponatinib inhibited NB cell proliferation and induced NB cell apoptosis in a dose-dependent manner. In addition, ponatinib suppressed the colony formation ability of NB cells. Mechanistically, ponatinib effectively inhibited the FGFR1-activated signaling pathway. Ponatinib also enhanced the cytotoxic effects of doxorubicin on NB cells. Furthermore, ponatinib demonstrated anti-tumor efficacy in vivo by inhibiting tumor growth in an orthotopic xenograft NB mouse model. In summary, our results showed that ponatinib inhibited NB growth both in vitro and in vivo.

Novel multi-targeted ErbB family inhibitor afatinib blocks EGF-induced signaling and induces apoptosis in neuroblastoma.

Neuroblastoma is the most common extracranial solid tumor in children. The ErbB family of proteins is a group of receptor tyrosine kinases that promote the progression of various malignant cancers including neuroblastoma. Thus, targeting them with small molecule inhibitors is a promising strategy for neuroblastoma therapy. In this study, we investigated the anti-tumor effect of afatinib, an irreversible inhibitor of members of the ErbB family, on neuroblastoma. We found that afatinib suppressed the proliferation and colony formation ability of neuroblastoma cell lines in a dose-dependent manner. Afatinib also induced apoptosis and blocked EGF-induced activation of PI3K/AKT/mTOR signaling in all neuroblastoma cell lines tested. In addition, afatinib enhanced doxorubicin-induced cytotoxicity in neuroblastoma cells, including the chemoresistant LA-N-6 cell line. Finally, afatinib exhibited antitumor efficacy in vivo by inducing apoptosis in an orthotopic xenograft neuroblastoma mouse model. Taken together, these results show that afatinib inhibits neuroblastoma growth both in vitro and in vivo by suppressing EGFR-mediated PI3K/AKT/mTOR signaling. Our study supports the idea that EGFR is a potential therapeutic target in neuroblastoma. And targeting ErbB family protein kinases with small molecule inhibitors like afatinib alone or in combination with doxorubicin is a viable option for treating neuroblastoma.

The second-generation ALK inhibitor alectinib effectively induces apoptosis in human neuroblastoma cells and inhibits tumor growth in a TH-MYCN transgenic neuroblastoma mouse model.

Activating germline mutations of anaplastic lymphoma kinase (ALK) occur in most cases of hereditary neuroblastoma (NB) and the constitutively active kinase activity of ALK promotes cell proliferation and survival in NB. Therefore, ALK kinase is a potential therapeutic target for NB. In this study, we show that the novel ALK inhibitor alectinib effectively suppressed cell proliferation and induces apoptosis in NB cell lines with either wild-type ALK or mutated ALK (F1174L and D1091N) by blocking ALK-mediated PI3K/Akt/mTOR signaling. In addition, alectinib enhanced doxorubicin-induced cytotoxicity and apoptosis in NB cells. Furthermore, alectinib induced apoptosis in an orthotopic xenograft NB mouse model. Also, in the TH-MYCN transgenic mouse model, alectinib resulted in decreased tumor growth and prolonged survival time. These results indicate that alectinib may be a promising therapeutic agent for the treatment of NB.

Second-generation proteasome inhibitor carfilzomib sensitizes neuroblastoma cells to doxorubicin-induced apoptosis.

Neuroblastoma (NB), which accounts for about 15% of cancer-related mortality in children, is the most common extracranial malignant neoplasm in children. Elevated level of proteasome activity promotes cancer development and the inhibition of proteasome activity is a promising strategy for cancer treatment. Therefore, targeting proteasome by small molecule inhibitors may be a viable option for NB therapy. Here in this study, we show that a novel proteasome inhibitor Carfilzomib (CFZ) exerts anti-tumor effect on NB. CFZ caused decreased cell viability and attenuated colony formation ability of a subset of NB cell lines. CFZ induced cell apoptosis in NB cells. Moreover, CFZ enhanced the cytotoxic effect of doxorubicin (Dox) on NB cells and Dox-induced p38 and JNK phosphorylation. In addition, CFZ inhibited Dox-induced NF-κB activation by stabilizing the protein level of IκBα. Furthermore, CFZ induced apoptosis and augmented Dox-induced apoptosis in NB tumor cells in orthotopic xenograft mouse models. In summary, our study suggests that proteasome is a therapeutic target in NB and proteasome inhibition by CFZ is a potential therapeutic strategy for treating NB patients.

Multiple CDK inhibitor dinaciclib suppresses neuroblastoma growth via inhibiting CDK2 and CDK9 activity.

Neuroblastoma (NB), the most common extracranial solid tumor of childhood, is responsible for approximately 15% of cancer-related mortality in children. Aberrant activation of cyclin-dependent kinases (CDKs) has been shown to contribute to tumor cell progression in many cancers including NB. Therefore, small molecule inhibitors of CDKs comprise a strategic option in cancer therapy. Here we show that a novel multiple-CDK inhibitor, dinaciclib (SCH727965, MK-7965), exhibits potent anti-proliferative effects on a panel of NB cell lines by blocking the activity of CDK2 and CDK9. Dinaciclib also significantly sensitized NB cell lines to the treatment of chemotherapeutic agents such as doxorubicin (Dox) and etoposide (VP-16). Furthermore, dinaciclib revealed in vivo antitumor efficacy in an orthotopic xenograft mouse model of two NB cell lines and blocked tumor development in the TH-MYCN transgenic NB mouse model. Taken together, this study suggests that CDK2 and CDK9 are potential therapeutic targets in NB and that abrogating CDK2 and CDK9 activity by small molecules like dinaciclib is a promising strategy and a treatment option for NB patients.

Novel MDM2 inhibitor SAR405838 (MI-773) induces p53-mediated apoptosis in neuroblastoma.

Neuroblastoma (NB), which accounts for about 15% of cancer-related mortality in children, is the most common childhood extracranial malignant tumor. In NB, somatic mutations of the tumor suppressor, p53, are exceedingly rare. Unlike in adult tumors, the majority of p53 downstream functions are still intact in NB cells with wild-type p53. Thus, restoring p53 function by blocking its interaction with p53 suppressors such as MDM2 is a viable therapeutic strategy for NB treatment. Herein, we show that MDM2 inhibitor SAR405838 is a potent therapeutic drug for NB. SAR405838 caused significantly decreased cell viability of p53 wild-type NB cells and induced p53-mediated apoptosis, as well as augmenting the cytotoxic effects of doxorubicin (Dox). In an in vivo orthotopic NB mouse model, SAR405838 induced apoptosis in NB tumor cells. In summary, our data strongly suggest that MDM2-specific inhibitors like SAR405838 may serve not only as a stand-alone therapy, but also as an effective adjunct to current chemotherapeutic regimens for treating NB with an intact MDM2-p53 axis.