Oncolytic strategy using new bifunctional HDACs/BRD4 inhibitors against virus-associated lymphomas.

Primary effusion lymphoma (PEL) caused by Kaposi sarcoma-associated herpesvirus (KSHV) is an aggressive malignancy with poor prognosis even under chemotherapy. Currently, there is no specific treatment for PEL therefore requiring new therapies. Both histone deacetylases (HDACs) and bromodomain-containing protein 4 (BRD4) have been found as therapeutic targets for PEL through inducing viral lytic reactivation. However, the strategy of dual targeting with one agent and potential synergistic effects have never been explored. In the current study, we first demonstrated the synergistic effect of concurrently targeting HDACs and BRD4 on KSHV reactivation by using SAHA or entinostat (HDACs inhibitors) and (+)-JQ1 (BRD4 inhibitor), which indicated dual blockage of HDACs/BRD4 is a viable therapeutic approach. We were then able to rationally design and synthesize a series of new small-molecule inhibitors targeting HDACs and BRD4 with a balanced activity profile by generating a hybrid of the key binding motifs between (+)-JQ1 and entinostat or SAHA. Upon two iterative screenings of optimized compounds, a pair of epimers, 009P1 and 009P2, were identified to better inhibit the growth of KSHV positive lymphomas compared to (+)-JQ1 or SAHA alone at low nanomolar concentrations, but not KSHV negative control cells or normal cells. Mechanistic studies of 009P1 and 009P2 demonstrated significantly enhanced viral reactivation, cell cycle arrest and apoptosis in KSHV+ lymphomas through dually targeting HDACs and BRD4 signaling activities. Importantly, in vivo preclinical studies showed that 009P1 and 009P2 dramatically suppressed KSHV+ lymphoma progression with oral bioavailability and minimal visible toxicity. These data together provide a novel strategy for the development of agents for inducing lytic activation-based therapies against these viruses-associated malignancies.

Human endogenous retrovirus type K encoded Np9 oncoprotein induces DNA damage response.

Human endogenous retrovirus sequences (HERVs) constitute up to 8% of the human genome, yet not all HERVs remain silent passengers within our genomes. Some HERVs, especially the HERV type K (HERV-K), have been found to be frequently transactivated in a variety of inflammatory diseases and human cancers. Np9, a 9-kDa HERV-K encoded protein, has been reported as an oncoprotein and found present in a variety of tumors and transformed cells. In the current study, we for the first time reported that ectopic expression of Np9 protein was able to induce DNA damage response from host cells especially through upregulation of γH2AX. Furthermore, we found that direct knockdown of Np9 by RNAi in Kaposi's Sarcoma-associated herpesvirus (KSHV) infected cells effectively reduced LANA expression, the viral major latent oncoprotein in vitro and in vivo, which may represent a novel strategy against virus-associated malignancies.

Novel LINC01923 -anaplasticlymphoma kinase rearrangement identified in advanced lung adenocarcinoma with durable response to ensartinib: a case report.

Anaplastic lymphoma kinase ( ALK ) rearrangements are one of the most common mutations in nonsmall cell lung cancer. Majority of the ALK rearrangements were sensitive to crizotinib, yet some rare fusion types may less benefit. The patient with LINC01923-ALK fusion was treated with crizotinib for 1 week and developed an adverse rash reaction. Replaced with second-line treatment with esatinib, the patient had a partial response in the primary site and achieved a complete response in the brain metastases. The patient was treated successfully with ensartinib leading to a progression-free survival of 6 months (and counting). This is the first report on one lung adenocarcinoma patient with a novel LINC01923-ALK fusion beneficial from ensartinib, which provides more knowledge for ALK fusion spectrum.

Identification of natural compounds tubercidin and lycorine HCl against small-cell lung cancer and BCAT1 as a therapeutic target.

Although small-cell lung cancer (SCLC) accounts for a small fraction of lung cancer cases (~15%), the prognosis of patients with SCLC is poor with an average overall survival period of a few months without treatment. Current treatments include standard chemotherapy, which has minimal efficacy and a newly developed immunotherapy that thus far, benefits a limited number of patients. In the current study, we screened a natural product library and identified 5 natural compounds, in particular tubercidin and lycorine HCl, that display prominent anti-SCLC activities in vitro and in vivo. Subsequent RNA-sequencing and functional validation assays revealed the anti-SCLC mechanisms of these new compounds, and further identified new cellular factors such as BCAT1 as a potential therapeutic target with clinical implication in SCLC patients. Taken together, our study provides promising new directions for fighting this aggressive lung cancer.

The Anti-COVID-19 Drug Remdesivir Promotes Oncogenic Herpesvirus Reactivation through Regulation of Intracellular Signaling Pathways.

Recently, remdesivir and molnupiravir were approved for treating COVID-19 caused by SARS-CoV-2 infection. However, little is known about the impact of these drugs on other viruses preexisted in COVID-19 patients. Here we report that remdesivir but not molnupiravir induced lytic reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), two major oncogenic herpesviruses. Remdesivir induced mature virion production from latently infected cells. Mechanistic studies showed that remdesivir induced KSHV and EBV reactivation by regulating several intracellular signaling pathways.

SARS-CoV-2 infection and lytic reactivation of herpesviruses: A potential threat in the postpandemic era?

The outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative pathogen for the coronavirus disease 2019 (COVID-19) pandemic, has greatly stressed our healthcare system. In addition to severe respiratory and systematic symptoms, several comorbidities increase the risk of fatal disease outcomes, including chronic viral infections. Increasing cases of lytic reactivation of human herpesviruses in COVID-19 patients and vaccinated people have been reported recently. SARS-CoV2 coinfection, COVID-19 treatments, and vaccination may aggravate those herpesvirus-associated diseases by reactivating the viruses in latently infected host cells. In this review, we summarize recent clinical findings and limited mechanistic studies regarding the relationship between SARS-CoV-2 and different human herpesviruses that suggest an ongoing potential threat to human health in the postpandemic era.

Developing new ceramide analogs and identifying novel sphingolipid-controlled genes against a virus-associated lymphoma.

Primary effusion lymphoma (PEL) is an aggressive malignancy with poor prognosis even under chemotherapy. Kaposi sarcoma-associated herpesvirus (KSHV), one of the human oncogenic viruses, is the principal causative agent. Currently, there is no specific treatment for PEL; therefore, developing new therapies is of great importance. Sphingolipid metabolism plays an important role in determining the fate of tumor cells. Our previous studies have demonstrated that there is a correlation between sphingolipid metabolism and KSHV+ tumor cell survival. To further develop sphingolipid metabolism-targeted therapy, after screening a series of newly synthesized ceramide analogs, here, we have identified compounds with effective anti-PEL activity. These compounds induce significant PEL apoptosis, cell-cycle arrest, and intracellular ceramide production through regulation of ceramide synthesizing or ceramide metabolizing enzymes and dramatically suppress tumor progression without visible toxicity in vivo. These new compounds also increase viral lytic gene expression in PEL cells. Our comparative transcriptomic analysis revealed their mechanisms of action for inducing PEL cell death and identified a subset of novel cellular genes, including AURKA and CDCA3, controlled by sphingolipid metabolism, and required for PEL survival with functional validation. These data provide the framework for the development of promising sphingolipid-based therapies against this virus-associated malignancy.

Role of EIF4G1 network in non-small cell lung cancers (NSCLC) cell survival and disease progression.

Although the Eukaryotic Translation Initiation Factor 4 Gamma 1 (EIF4G1) has been found overexpressed in a variety of cancers, its role in non-small cell lung cancers (NSCLC) pathogenesis especially in immunoregulatory functions, its clinical relevance and therapeutic potential remain largely unknown. By using cancer patients tissue assays, the results indicate that EIF4G1 expressional levels are much higher in NSCLC tissues than in adjacent or normal lung tissues, which are also associated with NSCLC patient survival. By using an RNA-Sequencing based pipeline, the data show that EIF4G1 has a significant association with immune checkpoint molecules such as PD-1/PD-L1 in NSCLC. EIF4G1 small-molecule inhibitors effectively repress NSCLC growth in cell culture and xenograft animal models. Protein array results identify the signature of proteins controlled by EIF4G1 in NSCLC cells, in which new candidates such as MUC1 and NRG1 are required for NSCLC survival and tumorigenesis with clinical relevance. Taken together, these results have for the first time demonstrated the immunoregulatory functions, clinical relevance and therapeutic potential of the EIF4G1 network in NSCLC, which may represent a promising and novel target to improve lung cancer treatment.

Identification of new antiviral agents against Kaposi's sarcoma-associated herpesvirus (KSHV) by high-throughput drug screening reveals the role of histamine-related signaling in promoting viral lytic reactivation.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several human cancers, such as Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). Current treatment options for KSHV infection and virus associated diseases are sometimes ineffective, therefore, more effectively antiviral agents are urgently needed. As a herpesvirus, lytic replication is critical for KSHV pathogenesis and oncogenesis. In this study, we have established a high-throughput screening assay by using an inducible KSHV+ cell-line, iSLK.219. After screening a compound library that consisted of 1280 Food and Drug Administration (FDA)-approved drugs, 15 hit compounds that effectively inhibited KSHV virion production were identified, most of which have never been reported with anti-KSHV activities. Interestingly, 3 of these drugs target histamine receptors or signaling. Our data further confirmed that antagonists targeting different histamine receptors (HxRs) displayed excellent inhibitory effects on KSHV lytic replication from induced iSLK.219 or BCBL-1 cells. In contrast, histamine and specific agonists of HxRs promoted viral lytic replication from induced iSLK.219 or KSHV-infected primary cells. Mechanistic studies indicated that downstream MAPK and PI3K/Akt signaling pathways were required for histamine/receptors mediated promotion of KSHV lytic replication. Direct knockdown of HxRs in iSLK.219 cells effectively blocked viral lytic gene expression during induction. Using samples from a cohort of HIV+ patients, we found that the KSHV+ group has much higher levels of histamine in their plasma and saliva than the KSHV- group. Taken together, our data have identified new anti-KSHV agents and provided novel insights into the molecular bases of host factors that contribute to lytic replication and reactivation of this oncogenic herpesvirus.

The potential impacts of early secreted antigenic target of 6 kDa of Mycobacterium tuberculosis on KSHV-infected cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several human cancers, including Kaposi's sarcoma (KS) and primary effusion lymphoma, which are mostly seen in immunocompromised patients, such as human immunodefeciency virus (HIV)+ individuals. Tuberculosis (TB), caused by the bacterial pathogen Mycobacterium tuberculosis (Mtb), remains one of the deadliest infectious diseases in the world. The risk of developing TB is dramatically higher in people living with HIV than among those without HIV infection. Case reports link cutaneous or pulmonary KS in HIV+ patients with mycobacterial co-infections, however, impacts of Mtb infection or its products on KSHV-infected cells are not known. We report here that ESAT-6, a secreted Mtb virulence factor, induces viral reactivation from KSHV-infected cells. KSHV-infected pulmonary endothelial cells were resistant to ESAT-6 induced inhibition of cell growth. Our data demonstrate that Mtb virulence factors influence the biology of KSHV-infected cells, highlighting the need to study the interactions between these two pathogens commonly found in people living with HIV.

Identification of new therapeutic targets and natural compounds against diffuse intrinsic pontine glioma (DIPG).

Diffuse Intrinsic Pontine Glioma (DIPG) is a highly aggressive pediatric brainstem tumor which accounts for about 10-20% of childhood brain tumors. The survival rate for DIPG remains very poor, with a median survival of less than 1 year. The dismal prognosis associated with DIPG has been exacerbated by the failure of a large number of clinical trials to meaningfully improve survival compared with radiotherapy, the current standard of care for DIPG. In the current study, we screened a natural product library and for the first time identified 6 natural compounds displaying inhibitory effects on DIPG proliferation and anchorage-independent growth through inducing tumor cell apoptosis and cell cycle arrest. Subsequent RNA-Sequencing and functional validation revealed the molecular mechanisms of these compounds with anti-DIPG activities, and identified new cellular factors such as Fibronectin 1 (FN1) and Eukaryotic translation initiation factor 3 subunit C-like (EIF3CL), required for DIPG survival as potential therapeutic targets. Our study provides promising directions to fight against this deadly pediatric cancer.

Influence of physiological and environmental factors on the diurnal variation in emissions of biogenic volatile compounds from Pinus tabuliformis.

Biological volatile organic compounds (BVOCs) have a large influence on atmospheric environmental quality, climate change and the carbon cycle. This study assesses the composition and diurnal variation in emission rates of BVOCs from Pinus tabuliformis, using an enclosure technique. Environmental parameters (temperature and light intensity) and physiological parameters (net photosynthetic rate, Pn; stomatal conductance, gs; intercellular CO2 concentration, Ci; and transpiration rate, Tr) that may affect emission behavior were continuously monitored. The 10 most abundant compound groups emitted by P. tabuliformis were classified by gas chromatography-mass spectrometry. The dominant monoterpenoid compounds emitted were α-pinene, ß-myrcene, α-farnesene and limonene. The diurnal emission rate of BVOCs changed with temperature and light intensity, with dynamic analysis of BVOCs emissions revealing that their emission rates were more affected by temperature than light. The variation in monoterpene emission rates was consistent with estimates of Pn, gs and Tr. Basal emission rates (at 30 °C,) of the main BVOCs ranged from 0.006 to 0.273 µg  -1/(hr g), while the basal ER standardization coefficients ranged from 0.049 to 0.144 °C-1. Overall, these results provide a detailed reference for the effective selection and configuration of tree species to effectively prevent and control atmospheric pollution.

[Multi Spectral Detection of Ethanol Content in Gasoline Based on SiPLS Feature Extraction and Information Fusion].

The ethanol content in ethanol gasoline was detected with ultraviolet/visible(UV/vis) and near-infrared (NIR) spectroscopy while information fusion technology and synergy interval PLS(SiPLS) algorithm were used as the feature extraction method with the establishment of partial least squares(PLS) regression model. Using the information fusion theory, UV/vis and NIR spectra were used for data fusion, the data level fusion (Low level data fusion, LLDF) and feature level fusion(Mid-level data fusion, MLDF) model were established. The results were compared with the single source modelwith low level data fusion before vector normalization(LLDF-VN1) selected for the optimal model. Finally, the optimal model was tested using the spectral data collected from the samples of high ethanol content and commercial gasoline. The results showed that both UV/vis and NIR can be used to detect and provide good prediction results, whereas direct fusion of the UV/vis and NIR spectral data provided the best results in the regression model based on the calibration set, with the highest correlation coefficient rc, the smallest Biasc and RMSECV values, as 0.999 9, 0.125 8 and 0.000 6, respectively. And the prediction effect of the model of LLDF-VN1(low level data fusion before vector normalization) was the best, r(p)=0.999 1，Bias(p)=0.352 7，RMSEP=-0.073 8. In the verification of the optimal model (LLDF-VN1) by the self distribution solution, rp=0.999 7, Bias(p)=0.102 2, RMSEP=0.329 1; and that for gasoline sold on market, r(p)=0.990 1, RMSEP=0.675 1, Bias(p)=0.892 7, respectively. It showed that the data level fusion based on UV/vis and NIR spectral information could be used to detect the content of ethanol in ethanol-gasoline quickly and accurately, achieving a wide range of ethanol concentration detection, which laid a foundation for further realization of the rapid detection of substances in the blended fuel oil.

Identification of new microtubule small-molecule inhibitors and microtubule-associated genes against triple negative breast cancer.

Breast cancer represents the leading cancer type and leading cause of cancer-related death among women in the world. Triple-negative breast cancer (TNBC) is a subset of breast cancer with the poorest prognosis and still lacking of effective therapeutic options. We recently screened a natural product library and identified 3 new hit compounds with selective and prominent anti-TNBC activities on different subtype of TNBC cell lines. Interestingly, all of these 3 hit compounds belong to "cytoskeletal drugs" that target tubulin and microtubule function. Our data also showed that these hit compounds showed consistently effective on TNBC cells which are resistant to those currently used antimicrotubule agents such as Paclitaxel. RNA-Sequencing analyses revealed the anti-TNBC mechanisms of these hit compounds and identified a subset of new cellular factors commonly affected by hit compounds in different subtypes of TNBC cells. Among them, we demonstrated AHCYL1 and SPG21 as new microtubule-associated proteins, which were required for TNBC cell survival with clinical implication through tissue array analysis. Our studies provide new insights into the mechanisms of TNBC pathogenesis and offer promising therapeutic directions for this aggressive breast cancer.

Lytic infection of Kaposi's sarcoma-associated herpesvirus induces DNA double-strand breaks and impairs non-homologous end joining.

Kaposi's sarcoma-associated herpesvirus (KSHV) has been associated with the development of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease. Cytogenetic studies have revealed chromosome abnormalities in KS tissues, including recurring copy number changes in chromosomes and the loss of chromosomes. Unfaithful DNA repair may contribute to the genomic instability that is one of the most common hallmarks of tumours. We found that lytic infection of KSHV can cause severe DNA double-strand breaks (DSBs) and impair non-homologous end joining (NHEJ) in host cells. Processivity factor 8 (PF-8) of KSHV was identified as interacting with Ku70 and Ku86, and the interaction was dependent on DSBs and DNA. Overexpression of PF-8 in HeLa cells impaired NHEJ by blocking the interaction between the Ku complex and the DNA-dependent protein kinase catalytic subunit. These results suggest that KSHV lytic replication may contribute to tumorigenesis by causing DNA DSBs and interfering with the repair of DSBs.

Development of humanized rabbit monoclonal antibodies against vascular endothelial growth factor receptor 2 with potential antitumor effects.

Vascular endothelial growth factor-A (VEGF-A) plays a critical role in physiologic and pathologic angiogenesis through its receptors especially through VEGFR2. The lack of cross-reactivity of monoclonal antibodies with human VEGFR2/mouse Flk-1 is a major obstacle in preclinical developments. In this study, using a unique hybridoma technique, we generated a panel of 30 neutralization anti-VEGFR2 rabbit monoclonal antibodies (RabMAbs) either blocking VEGF/VEGFR2 interaction or inhibiting VEGF-stimulated VEGFR2 tyrosine kinase phosphorylation. Among 18 RabMAbs with human/mouse VEGFR2 cross-reactivity, we humanized one lead candidate RabMAb by Mutational Lineage Guided (MLG) method and further demonstrated its potent inhibition of tumor growth in xenograft mouse model. Our study suggests that RabMAbs are highly relevant for therapeutic applications.

Role of Histamine and Related Signaling in Kaposi's Sarcoma-Associated Herpesvirus Pathogenesis and Oncogenesis.

Although Kaposi's sarcoma-associated herpesvirus (KSHV) has been reported to cause several human cancers including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), the mechanisms of KSHV-induced tumorigenesis, especially virus-host interaction network, are still not completely understood, which therefore hinders the development of effective therapies. Histamine, together with its receptors, plays an important role in various allergic diseases by regulating different inflammation and immune responses. Our previous data showed that antagonists targeting histamine receptors effectively repressed KSHV lytic replication. In the current study, we determined that histamine treatment increased cell proliferation and anchorage-independent growth abilities of KSHV-infected cells. Furthermore, histamine treatment affected the expression of some inflammatory factors from KSHV-infected cells. For clinical relevance, several histamine receptors were highly expressed in AIDS-KS tissues when compared to normal skin tissues. We determined that histamine treatment promoted KSHV-infected lymphoma progression in immunocompromised mice models. Therefore, besides viral replication, our data indicate that the histamine and related signaling are also involved in other functions of KSHV pathogenesis and oncogenesis.

Echinomycin as a promising therapeutic agent against KSHV-related malignancies.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of several human cancers, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), which preferentially arise in immunocompromised patients while lack of effective therapeutic options. Oncoproteins Myc and hypoxia-inducible factor-1α (HIF1α) have been found closely related to KSHV infection, replication and oncogenesis. However, the strategies of dual targeting these two oncoproteins have never been developed and tested for treatments of KSHV-related malignancies. In the current study, we report that treatment of echinomycin dramatically regresses cell growth both in vitro-cultured KSHV + tumor cells and in vivo KS or PEL xenograft mice models, through simultaneously inhibiting Myc and HIF1α expression. Echinomycin treatment also induces viral lytic gene expression whereas not increasing infectious virions production from KSHV + tumor cells. Our comparative transcriptomic analysis has identified a bunch of new Echinomycin-regulated, Myc- and HIF1α-related genes contributed to KSHV pathogenesis, including KDM4B and Tau, which are required for the survival of KSHV + tumor cells with functional validation. These data together reveal that dual targeting Myc and HIF1α such as using Echinomycin may represent a new and promising option for treatments of these virus-associated malignancies.

COVID-19 CT image segmentation method based on swin transformer.

Owing to its significant contagion and mutation, the new crown pneumonia epidemic has caused more than 520 million infections worldwide and has brought irreversible effects on the society. Computed tomography (CT) images can clearly demonstrate lung lesions of patients. This study used deep learning techniques to assist doctors in the screening and quantitative analysis of this disease. Consequently, this study will help to improve the diagnostic efficiency and reduce the risk of infection. In this study, we propose a new method to improve U-Net for lesion segmentation in the chest CT images of COVID-19 patients. 750 annotated chest CT images of 150 patients diagnosed with COVID-19 were selected to classify, identify, and segment the background area, lung area, ground glass opacity, and lung parenchyma. First, to address the problem of a loss of lesion detail during down sampling, we replaced part of the convolution operation with atrous convolution in the encoder structure of the segmentation network and employed convolutional block attention module (CBAM) to enhance the weighting of important feature information. Second, the Swin Transformer structure is introduced in the last layer of the encoder to reduce the number of parameters and improve network performance. We used the CC-CCII lesion segmentation dataset for training and validation of the model effectiveness. The results of ablation experiments demonstrate that this method achieved significant performance gain, in which the mean pixel accuracy is 87.62%, mean intersection over union is 80.6%, and dice similarity coefficient is 88.27%. Further, we verified that this model achieved superior performance in comparison to other models. Thus, the method proposed herein can better assist doctors in evaluating and analyzing the condition of COVID-19 patients.

Activation of IL1 signaling molecules by Kaposi's sarcoma-associated herpesvirus.

Objective: Kaposi's Sarcoma-associated Herpesvirus (KSHV) is the etiologic agent of several human cancers, including Kaposi's sarcoma (KS) and Primary effusion lymphoma (PEL), which are usually seen in immunocompromised patients while lack of effective therapeutic options. Interleukin1 (IL1) family is a major mediator for inflammation response and has functional role in both innate and adaptive immunity. In contrast to the well-studied IL1 molecules, the activation and functional role of IL1 receptor/co-receptor and other related ligands, such as the IL1 receptor accessory protein (IL1RAP), in KSHV pathogenesis and tumorigenesis remain almost unknown. Methods: In the current study, a series of KSHV negative and positive primary or tumor cells, as well as AIDS-KS tumor samples from cohort HIV+ patients were used to compare and determine the activation status of IL1 signaling molecules, and their functional roles in KSHV pathogenesis. Results: We reported the high activation of multiple IL1 signaling molecules, including IL1, IL36, IL1R1, IL1RAP and IRAKs, during KSHV latent and lytic stages, as well as in clinical samples from patients with KSHV-related malignancies. Directly targeting these molecules especially IL1R1 and IL1RAP significantly impaired the survival and growth of KSHV+ tumor cells, as well as their colony formation on 3-D culture. Conclusion: Our data indicate the importance of IL1 signaling molecules in KSHV pathogenesis and tumorigenesis, which may represent attractive therapeutic targets against these virus-associated diseases.