ERG activity is regulated by endothelial FAK coupling with TRIM25/USP9x in vascular patterning.

Precise vascular patterning is crucial for normal growth and development. The ERG transcription factor drives Delta-like ligand 4 (DLL4)/Notch signalling and is thought to act as a pivotal regulator of endothelial cell (EC) dynamics and developmental angiogenesis. However, molecular regulation of ERG activity remains obscure. Using a series of EC-specific focal adhesion kinase (FAK)-knockout (KO) and point-mutant FAK-knock-in mice, we show that loss of ECFAK, its kinase activity or phosphorylation at FAK-Y397, but not FAK-Y861, reduces ERG and DLL4 expression levels together with concomitant aberrations in vascular patterning. Rapid immunoprecipitation mass spectrometry of endogenous proteins identified that endothelial nuclear-FAK interacts with the deubiquitinase USP9x and the ubiquitin ligase TRIM25. Further in silico analysis confirms that ERG interacts with USP9x and TRIM25. Moreover, ERG levels are reduced in FAKKO ECs via a ubiquitin-mediated post-translational modification programme involving USP9x and TRIM25. Re-expression of ERG in vivo and in vitro rescues the aberrant vessel-sprouting defects observed in the absence of ECFAK. Our findings identify ECFAK as a regulator of retinal vascular patterning by controlling ERG protein degradation via TRIM25/USP9x.

Inhibition of Bromodomain Proteins Enhances Oncolytic HAdVC5 Replication and Efficacy in Pancreatic Ductal Adenocarcinoma (PDAC) Models.

Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive type of pancreatic cancer, which rapidly develops resistance to the current standard of care. Several oncolytic Human AdenoViruses (HAdVs) have been reported to re-sensitize drug-resistant cancer cells and in combination with chemotherapeutics attenuate solid tumour growth. Obstacles preventing greater clinical success are rapid hepatic elimination and limited viral replication and spread within the tumour microenvironment. We hypothesised that higher intratumoural levels of the virus could be achieved by altering cellular epigenetic regulation. Here we report on the screening of an enriched epigenetics small molecule library and validation of six compounds that increased viral gene expression and replication. The greatest effects were observed with three epigenetic inhibitors targeting bromodomain (BRD)-containing proteins. Specifically, BRD4 inhibitors enhanced the efficacy of Ad5 wild type, Ad∆∆, and Ad-3∆-A20T in 3-dimensional co-culture models of PDAC and in vivo xenografts. RNAseq analysis demonstrated that the inhibitors increased viral E1A expression, altered expression of cell cycle regulators and inflammatory factors, and attenuated expression levels of tumour cell oncogenes such as c-Myc and Myb. The data suggest that the tumour-selective Ad∆∆ and Ad-3∆-A20T combined with epigenetic inhibitors is a novel strategy for the treatment of PDAC by eliminating both cancer and associated stromal cells to pave the way for immune cell access even after systemic delivery of the virus.

Multifunctional nanoparticles co-loaded with Adriamycin and MDR-targeting siRNAs for treatment of chemotherapy-resistant esophageal cancer.

The development of multidrug resistance (MDR) during cancer chemotherapy is a major challenge in current cancer treatment strategies. Numerous molecular mechanisms, including increased drug efflux, evasion of drug-induced apoptosis, and activation of DNA repair mechanisms, can drive chemotherapy resistance. Here we have identified the major vault protein (MVP) and the B-cell lymphoma-2 (BCL2) gene as two potential factors driving MDR in esophageal squamous cell carcinoma (ESCC). We have designed a novel and versatile self-assembling nanoparticle (NP) platform on a multifunctional carboxymethyl chitosan base to simultaneously deliver Adriamycin, and siRNAs targeting MVP and BCL2 (CEAMB NPs), thus reducing drug efflux and promoting apoptosis of esophageal cancer cells. To achieve effective delivery to tumor tissues and inhibit tumor growth in vivo, carboxymethyl chitosan was engineered to contain multiple histidines for enhanced cytosol delivery, cholesterol for improved self-assembly, and epidermal growth factor receptor (EGFR) antibodies to target cancer cells. Our results indicate that these nanoparticles are efficiently synthesized with the desired chemical composition to self-assemble into cargo-containing NPs. Furthermore, we have shown that the synthesized NPs will successfully inhibit cancer cells growth and tumor development when delivered to cultured ESCC cells or to in vivo mouse xenograft models. Our engineered NPs offer a potential novel platform in treating various types of chemotherapy-resistant tumors.

Cancer spectrum in TP53-deficient golden Syrian hamsters: A new model for Li-Fraumeni syndrome.

BACKGROUND: The TP53 tumor suppressor gene is the most commonly mutated gene in human cancers. Humans who inherit mutant TP53 alleles develop a wide range of early onset cancers, a disorder called Li-Fraumeni Syndrome (LFS). Trp53-deficient mice recapitulate most but not all of the cancer phenotypes observed in TP53-deficient human cancers, indicating that new animal models may complement current mouse models and better inform on human disease development. MATERIALS AND METHODS: The recent application of CRISPR/Cas9 genetic engineering technology has permitted the emergence of golden Syrian hamsters as genetic models for wide range of diseases, including cancer. Here, the first cancer phenotype of TP53 knockout golden Syrian hamsters is described. RESULTS: Hamsters that are homozygous for TP53 mutations become moribund on average ~ 139 days of age, while hamsters that are heterozygous become moribund at ~ 286 days. TP53 homozygous knockout hamsters develop a wide range of cancers, often synchronous and metastatic to multiple tissues, including lymphomas, several sarcomas, especially hemangiosarcomas, myeloid leukemias and several carcinomas. TP53 heterozygous mutants develop a more restricted tumor spectrum, primarily lymphomas. CONCLUSIONS: Overall, hamsters may provide insights into how TP53 deficiency leads to cancer in humans and can become a new model to test novel therapies.

Transient Inhibition of PI3Kδ Enhances the Therapeutic Effect of Intravenous Delivery of Oncolytic Vaccinia Virus.

Tumor-targeting oncolytic viruses such as vaccinia virus (VV) are attractive cancer therapeutic agents that act through multiple mechanisms to provoke both tumor lysis and anti-tumor immune responses. However, delivery of these agents remains restricted to intra-tumoral administration, which prevents effective targeting of inaccessible and disseminated tumor cells. In the present study we have identified transient pharmacological inhibition of the leukocyte-enriched phosphoinositide 3-kinase δ (PI3Kδ) as a novel mechanism to potentiate intravenous delivery of oncolytic VV to tumors. Pre-treatment of immunocompetent mice with the PI3Kδ-selective inhibitor IC87114 or the clinically approved idelalisib (CAL-101), prior to intravenous delivery of a tumor-tropic VV, dramatically improved viral delivery to tumors. This occurred via an inhibition of viral attachment to, but not internalization by, systemic macrophages through perturbation of signaling pathways involving RhoA/ROCK, AKT, and Rac. Pre-treatment using PI3Kδ-selective inhibitors prior to intravenous delivery of VV resulted in enhanced anti-tumor efficacy and significantly prolonged survival compared to delivery without PI3Kδ inhibition. These results indicate that effective intravenous delivery of oncolytic VV may be clinically achievable and could be useful in improving anti-tumor efficacy of oncolytic virotherapy.

Disrupted structure and aberrant function of CHIP mediates the loss of motor and cognitive function in preclinical models of SCAR16.

CHIP (carboxyl terminus of heat shock 70-interacting protein) has long been recognized as an active member of the cellular protein quality control system given the ability of CHIP to function as both a co-chaperone and ubiquitin ligase. We discovered a genetic disease, now known as spinocerebellar autosomal recessive 16 (SCAR16), resulting from a coding mutation that caused a loss of CHIP ubiquitin ligase function. The initial mutation describing SCAR16 was a missense mutation in the ubiquitin ligase domain of CHIP (p.T246M). Using multiple biophysical and cellular approaches, we demonstrated that T246M mutation results in structural disorganization and misfolding of the CHIP U-box domain, promoting oligomerization, and increased proteasome-dependent turnover. CHIP-T246M has no ligase activity, but maintains interactions with chaperones and chaperone-related functions. To establish preclinical models of SCAR16, we engineered T246M at the endogenous locus in both mice and rats. Animals homozygous for T246M had both cognitive and motor cerebellar dysfunction distinct from those observed in the CHIP null animal model, as well as deficits in learning and memory, reflective of the cognitive deficits reported in SCAR16 patients. We conclude that the T246M mutation is not equivalent to the total loss of CHIP, supporting the concept that disease-causing CHIP mutations have different biophysical and functional repercussions on CHIP function that may directly correlate to the spectrum of clinical phenotypes observed in SCAR16 patients. Our findings both further expand our basic understanding of CHIP biology and provide meaningful mechanistic insight underlying the molecular drivers of SCAR16 disease pathology, which may be used to inform the development of novel therapeutics for this devastating disease.

Genomic Variations in Pancreatic Cancer and Potential Opportunities for Development of New Approaches for Diagnosis and Treatment.

Human pancreatic cancer has a very poor prognosis with an overall five-year survival rate of less than 5% and an average median survival time of six months. This is largely due to metastatic disease, which is already present in the majority of patients when diagnosed. Although our understanding of the molecular events underlying multi-step carcinogenesis in pancreatic cancer has steadily increased, translation into more effective therapeutic approaches has been inefficient in recent decades. Therefore, it is imperative that novel and targeted approaches are designed to facilitate the early detection and treatment of pancreatic cancer. Presently, there are numerous ongoing studies investigating the types of genomic variations in pancreatic cancer and their impact on tumor initiation and growth, as well as prognosis. This has led to the development of therapeutics to target these genetic variations for clinical benefit. Thus far, there have been minimal clinical successes directly targeting these genomic alterations; however research is ongoing to ultimately discover an innovative approach to tackle this devastating disease. This review will discuss the genomic variations in pancreatic cancer, and the resulting potential diagnostic and therapeutic implications.

Haploinsufficiency of Hand1 improves mice survival after acute myocardial infarction through preventing cardiac rupture.

Previous studies have demonstrated a significantly lower level of Hand1 in ischemic cardiomyopathy than in normal heart tissue. The role of decreased Hand1 in myocardial infarction remains unclear. This study was designed to investigate the effects of haploinsufficiency of Hand1 on mouse heart after myocardial infarction. 8-10 weeks old male heterozygous Hand1-deficient (Hand1(+/-)) mice and wild-type littermates (control) were subjected to sham operation or ligation of the left anterior descending coronary artery to induce acute myocardial infarction (AMI). Hand1(+/-) mice have low incidence of left ventricular free wall rupture in the first week after operation than control mice. Then we found lower MMP9 activity and less cardiomyocytes apoptosis in Hand1(+/-) than in control mice. All of these contribute to the protection role of haploinsufficiency of Hand1 after AMI.

Efficiently editing the vaccinia virus genome by using the CRISPR-Cas9 system.

Vaccinia virus (VACV) continues to be used in immunotherapy for the prevention of infectious diseases and treatment of cancer since its use for the eradication of smallpox. However, the current method of editing the VACV genome is not efficient. Here, we demonstrate that the CRISPR-Cas9 system can be used to edit the VACV genome rapidly and efficiently. Additionally, a set of 8,964 computationally designed unique guide RNAs (gRNAs) targeting all VACV genes will be valuable for the study of VACV gene functions.

Exome capture sequencing identifies a novel CCM1 mutation in a Chinese family with multiple cerebral cavernous malformations.

PURPOSE: Cerebral cavernous malformations (CCMs) are vascular anomalies predominantly in the central nervous system but may include lesions in other tissues, such as the retina, skin and liver. The main clinical manifestations include seizures, hemorrhage, recurrent headaches and focal neurological deficits. Previous studies of familial CCMs (FCCMs) have mainly reported in Hispanic and Caucasian cases. Here, we report on FCCMs in a Chinese family further characterized by a novel CCM1 gene mutation. MATERIALS AND METHODS: We investigated clinical and neuroradiological features of a Chinese family of 30 members. Furthermore, we used exome capture sequencing to identify the causing gene. The CCM1 mRNA expression level in three patients of the family and 10 wild-type healthy individuals were detected by real-time quantitative polymerase chain reaction (real-time RT-PCR). RESULTS: Brain magnetic resonance imaging demonstrated multiple intracranial lesions in seven members. The clinical manifestation of CCM was found in five of these cases, including recurrent headaches, weakness, hemorrhage and seizures. Moreover, we identified a novel nonsense mutation c.1159G>T (p. E387*) in the CCM1 gene in the pedigree. Based on real-time RT-PCR results, we have found that the CCM1 mRNA expression level in three patients was reduced by 35% than that in wild-type healthy individuals. CONCLUSIONS: Our finding suggests that the novel nonsense mutation c.1159G>T in CCM1 gene is associated with FCCM, and that CCM1 haploinsufficiency may be the underlying mechanism of CCMs. Furthermore, it also demonstrates that exome capture sequencing is an efficient and direct diagnostic tool to identify causes of genetically heterogeneous diseases.

STAT1 interaction with E3-14.7K in monocytes affects the efficacy of oncolytic adenovirus.

Oncolytic viruses based on adenovirus type 5 (Ad5) have been developed as a new class of therapeutic agents for cancers that are resistant to conventional therapies. Clinical experience shows that these agents are safe, but virotherapy alone has not achieved long-term cure in cancer patients. The vast majority of oncolytic adenoviruses used in clinical trials to date have deletion of the E3B genes. It has been demonstrated that the antitumor potency of the E3B-deleted mutant (dl309) is inferior to adenovirus with E3B genes intact. Tumors treated with dl309 show markedly greater macrophage infiltration than E3B-intact adenovirus. However, the functional mechanisms for this were not previously known. Here, we demonstrate that deletion of E3B genes increases production of chemokines by monocytes after adenovirus infection and increases monocyte migration. The E3B 14,700-Da protein (E3B-14.7K) inhibits STAT1 function by preventing its phosphorylation and nuclear translocation. The STAT1 inhibitor, fludarabine, rescues the effect of E3B-14.7K deletion by downregulating target chemokine expression in human and murine monocytes and results in an enhanced antitumor efficacy with dl309 in vivo. These findings have important implications for clinical use of E3B-deleted oncolytic adenovirus and other E3B-deleted adenovirus vector-based therapy.

Vascular endothelial growth factor A promotes vaccinia virus entry into host cells via activation of the Akt pathway.

Vaccinia virus (VV) is an enveloped DNA virus from the poxvirus family and has played a crucial role in the eradication of smallpox. It continues to be used in immunotherapy for the prevention of infectious diseases and treatment of cancer. However, the mechanisms of poxvirus entry, the host factors that affect viral virulence, and the reasons for its natural tropism for tumor cells are incompletely understood. By studying the effect of hypoxia on VV infection, we found that vascular endothelial growth factor A (VEGF-A) augments oncolytic VV cytotoxicity. VEGF derived from tumor cells acts to increase VV internalization, resulting in increased replication and cytotoxicity in an AKT-dependent manner in both tumor cells and normal respiratory epithelial cells. Overexpression of VEGF also enhances VV infection within tumor tissue in vivo after systemic delivery. These results highlight the importance of VEGF expression in VV infection and have potential implications for the design of new strategies to prevent poxvirus infection and the development of future generations of oncolytic VV in combination with conventional or biological therapies.

The dilemmas and possible solutions for CAR-T cell therapy application in solid tumors.

Chimeric antigen receptor T (CAR-T) cell therapy, as an adoptive immunotherapy, is playing an increasingly important role in the treatment of malignant tumors. CAR-T cells are referred to as "living drugs" as they not only target tumor cells directly, but also induce long-term immune memory that has the potential to provide long-lasting protection. CD19.CAR-T cells have achieved complete response rates of over 90 % for acute lymphoblastic leukemia and over 60 % for non-Hodgkin's lymphoma. However, the response rate of CAR-T cells in the treatment of solid tumors remains extremely low and the side effects potentially severe. In this review, we discuss the limitations that the solid tumor microenvironment poses for CAR-T application and the solutions that are being developed to address these limitations, in the hope that in the near future, CAR-T cell therapy for solid tumors can attain the same success rates as are now being seen clinically for hematological malignancies.

Oncolytic virus Ad-TD-nsIL-12 inhibits glioma growth and reprograms the tumor immune microenvironment.

AIMS: Gliomas are the most common central nervous system malignancies, with limited therapeutic options and poor prognosis, which are primarily attributed to the "immune desert" microenvironment. Previously, we constructed a three-gene-deleted oncolytic adenovirus (Ad-TD) loaded with non-secreting interleukin-12 (nsIL-12), which could be amplified in tumor cells and induce immunity to suppress tumors. However, the effects of this oncolytic virus on gliomas and their immune microenvironment remain unclear. There is an urgent need for further research. MATERIALS AND METHODS: We constructed a Syrian hamster brain tumor model and demonstrated the efficacy and mechanism of the novel oncolytic virus in treating brain tumors through a series of in vitro and in vivo experiments. We investigated the efficacy and safety (the number of hamsters in each group is either 5 or 10) of the oncolytic virus treatment in Syrian hamsters using a virus-treated group, a control virus-treated group, and a blank control group. KEY FINDINGS: In vitro assays showed that Ad-TD-nsIL-12 could specifically proliferate in brain tumor cells which induce tumor cell apoptosis and intracellular expression of interleukin (IL)-12. Moreover, in vivo experiments demonstrated that Ad-TD-nsIL-12 could effectively inhibit the progression of brain tumors and prolong survival. Ad-TD-nsIL-12 significantly enhanced T-cell infiltration in the brain tumor microenvironment. SIGNIFICANCE: Ad-TD-nsIL-12 can inhibit glioma progression and increase T-cell infiltration in the tumor tissue, particularly infiltration by cytotoxic T cells (CD8+). Ad-TD-nsIL-12 can amplify and produce IL-12, inducing anti-glioma immune responses to inhibit tumor progression.

Low-Dose Colchicine Ameliorates Doxorubicin Cardiotoxicity Via Promoting Autolysosome Degradation.

BACKGROUND: The only clinically approved drug that reduces doxorubicin cardiotoxicity is dexrazoxane, but its application is limited due to the risk of secondary malignancies. So, exploring alternative effective molecules to attenuate its cardiotoxicity is crucial. Colchicine is a safe and well-tolerated drug that helps reduce the production of reactive oxygen species. High doses of colchicine have been reported to block the fusion of autophagosomes and lysosomes in cancer cells. However, the impact of colchicine on the autophagy activity within cardiomyocytes remains inadequately elucidated. Recent studies have highlighted the beneficial effects of colchicine on patients with pericarditis, postprocedural atrial fibrillation, and coronary artery disease. It remains ambiguous how colchicine regulates autophagic flux in doxorubicin-induced heart failure. METHODS AND RESULTS: Doxorubicin was administered to establish models of heart failure both in vivo and in vitro. Prior studies have reported that doxorubicin impeded the breakdown of autophagic vacuoles, resulting in damaged mitochondria and the accumulation of reactive oxygen species. Following the administration of a low dose of colchicine (0.1 mg/kg, daily), significant improvements were observed in heart function (left ventricular ejection fraction: doxorubicin group versus treatment group=43.75%±3.614% versus 57.07%±2.968%, P=0.0373). In terms of mechanism, a low dose of colchicine facilitated the degradation of autolysosomes, thereby mitigating doxorubicin-induced cardiotoxicity. CONCLUSIONS: Our research has shown that a low dose of colchicine is pivotal in restoring the autophagy activity, thereby attenuating the cardiotoxicity induced by doxorubicin. Consequently, colchicine emerges as a promising therapeutic candidate to improve doxorubicin cardiotoxicity.

Modification of the early gene enhancer-promoter improves the oncolytic potency of adenovirus 11.

Oncolytic adenoviruses based on serotype 5 (Ad5) have several shortcomings, including the downregulation of its receptor in cancer cells, high prevalence of neutralizing antibodies and hepatotoxicity. Another adenoviral serotype, Ad11, could overcome these obstacles. Here, we show that human cancer cell lines express higher levels of the Ad11 receptor CD46, resulting in much better infectivity than Ad5. Surprisingly, only 36% (9/25) of the cell lines were more sensitive to Ad11- than to Ad5-mediated cytotoxicity. Investigations revealed that it was the transcription of Ad11 E1A, not CD46 expression or virus infectivity, which determined the cell's sensitivity to Ad11 killing. Ad11 E1A mRNA levels have an effect on viral DNA replication, structural protein synthesis and infectious particle production. To test the hypothesis that increased E1A transcription would lead to improved Ad11 replication in Ad5-sensitive (but Ad11-less sensitive) cells, two Ad11 mutants (Ad11-Ad5-P and Ad11-Ad5-EP) were constructed where either the E1A promoter or enhancer-promoter, respectively, was replaced by that of Ad5. Ad11-Ad5-EP demonstrated increased E1A mRNA levels and replication, together with enhanced oncolytic potency in vitro and in vivo. This effect was found in both the Ad5-sensitive and Ad11-sensitive cancer cells, broadening the range of tumors that could be effectively killed by Ad11-Ad5-EP.

Clinical features of Danon disease and insights gained from LAMP-2 deficiency models.

Danon disease (DD) is an X-linked multisystem disorder with clinical features characterized by the triad of hypertrophic cardiomyopathy, skeletal muscle weakness, and mental retardation. Cardiac involvement can be fatal in the absence of an effective treatment option such as heart transplantation. Molecular studies have proved that LAMP-2 protein deficiency, mainly LAMP-2B isoform, resulting from LAMP2 gene mutation, is the culprit for DD. Autophagy impairment due to LAMP-2 deficiency mediated the accumulation of abnormal autophagic vacuoles in cells. While it is not ideal for mimicking DD phenotypes in humans, the emergence of LAMP-2-deficient animal models and induced pluripotent stem cells from DD patients provided powerful tools for exploring DD mechanism. In both in vitro and in vivo studies, much evidence has demonstrated that mitochondria dysfunction and fragmentation can result in DD pathology. Fundamental research contributes to the therapeutic transformation. By targeting the molecular core, several potential therapies have demonstrated promising results in partial phenotypes improvement. Among them, gene therapies anticipate inaugurate a class of symptom control and prevention drugs as their in vivo effects are promising, and one clinical trial is currently underway.

Syrian hamster as an ideal animal model for evaluation of cancer immunotherapy.

Cancer immunotherapy (CIT) has emerged as an exciting new pillar of cancer treatment. Although benefits have been achieved in individual patients, the overall response rate is still not satisfactory. To address this, an ideal preclinical animal model for evaluating CIT is urgently needed. Syrian hamsters present similar features to humans with regard to their anatomy, physiology, and pathology. Notably, the histological features and pathological progression of tumors and the complexity of the tumor microenvironment are equivalent to the human scenario. This article reviews the current tumor models in Syrian hamster and the latest progress in their application to development of tumor treatments including immune checkpoint inhibitors, cytokines, adoptive cell therapy, cancer vaccines, and oncolytic viruses. This progress strongly advocates Syrian hamster as an ideal animal model for development and assessment of CIT for human cancer treatments. Additionally, the challenges of the Syrian hamster as an animal model for CIT are also discussed.