Search

The COVID-19 pandemic has triggered concerns about the emergence of more infectious and pathogenic viral strains. As a public health measure, efficient screening methods are needed to determine the functional effects of new sequence variants. Here we show that structural modeling of SARS-CoV-2 Spike protein binding to the human ACE2 receptor, the first step in host-cell entry, predicts many novel variant combinations with enhanced binding affinities. By focusing on natural variants at the Spike-hACE2 interface and assessing over 700 mutant complexes, our analysis reveals that high-affinity Spike mutations (including N440K, S443A, G476S, E484R, G502P) tend to cluster near known human ACE2 recognition sites (K31 and K353). These Spike regions are structurally flexible, allowing certain mutations to optimize interface interaction energies. Although most human ACE2 variants tend to weaken binding affinity, they can interact with Spike mutations to generate high-affinity double mutant complexes, suggesting variation in individual susceptibility to infection. Applying structural analysis to highly transmissible variants, we find that circulating point mutations S477N, E484K and N501Y form high-affinity complexes (~40% more than wild-type). By combining predicted affinities and available antibody escape data, we show that fast-spreading viral variants exploit combinatorial mutations possessing both enhanced affinity and antibody resistance, including S477N/E484K, E484K/N501Y and K417T/E484K/N501Y. Thus, three-dimensional modeling of the Spike/hACE2 complex predicts changes in structure and binding affinity that correlate with transmissibility and therefore can help inform future intervention strategies.

Tomato is the most economically important horticultural crop used as a model to study plant biology and particularly fruit development. Knowledge obtained from tomato research initiated improvements in tomato and, being transferrable to other such economically important crops, has led to a surge of tomato-related research and published literature. We developed DES-TOMATO knowledgebase (KB) for exploration of information related to tomato. Information exploration is enabled through terms from 26 dictionaries and combination of these terms. To illustrate the utility of DES-TOMATO, we provide several examples how one can efficiently use this KB to retrieve known or potentially novel information. DES-TOMATO is free for academic and nonprofit users and can be accessed at http://cbrc.kaust.edu.sa/des_tomato/, using any of the mainstream web browsers, including Firefox, Safari and Chrome.

The transcription factor EB (TFEB) was identified as a master regulator of autophagy and lysosomal biogenesis. We recently demonstrated that TFEB is aberrantly regulated in pancreatic cancer (PDAC) cells. Interference with TFEB impairs PDAC cell growth supporting an important role for TFEB in maintaining PDAC cell phenotype. Given the limited impact of chemotherapeutic drugs in the context of PDAC, we aimed at testing whether the aberrantly regulated TFEB in PDAC cells could confer resistance to DNA damage agents, commonly used in PDAC therapeutic regimens. Methods: Experiments were performed in PDAC cells (MIA PaCa2, PANC1). The DNA damage agents gemcitabine, 5-FU, cisplatin and doxorubicin were used. Autophagic flux was measured upon 4-hours treatment with bafilomycin A1. Results: Treatment with DNA damage agents 1) increased the autophagic flux in PDAC cells. This autophagic signal was associated with 2) the dephosphorylation and nuclear accumulation of TFEB. The mTORC1 pathway is well-known to regulate TFEB phosphorylation and nuclear localization of TFEB. 3) However, the impact of the DNA damage agents on TFEB appeared independent of the mTORC1 pathway since no modulation in the phosphorylation of the mTORC1 target S6K1 was observed. To delineate whether TFEB influenced the response to DNA damage agents, we generated stable populations of PDAC cells expressing a non-targeting or shRNA targeting TFEB. Interfering with TFEB function 4) prevented the autophagic response upon treatment with DNA damage agents. This correlated with 5) increased accumulation of DNA damage and 6) increased sensitivity to the DNA damage agents. Conclusion: Our results suggest that DNA damage agents can induce a pro-survival autophagic response involving TFEB. Interfering with TFEB function limits the autophagic response and leads to increased sensitivity to DNA damage agents. Altogether, our results suggest that the aberrantly expressed TFEB in PDAC cells could confer resistance to DNA damage agents by, among others, promoting autophagy and limiting DNA damage accumulation. Citation Format: Benoit Marchand, Marie-Josée Boucher. DNA damage agents promote TFEB function and induce pro-survival autophagy signals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2619.

Recent evidences suggest that the activity of glycogen synthase kinase-3 (GSK3) contributes to the tumorigenic potential of pancreatic cancer cells through modulation of cell proliferation and survival. However, further investigations are needed to identify GSK3-dependent mechanisms involved in the control of pancreatic cancer cell proliferation and survival. This study was undertaken to provide further support for a role of GSK3 in pancreatic cancer cell growth as well as to identify new cellular and molecular mechanisms involved. Herein, we demonstrate that prolonged inhibition of GSK3 triggers an apoptotic response only in human pancreatic cancer cells but not in human non-transformed pancreatic epithelial cells. We show that prolonged inhibition of GSK3 activity increases Bim messenger RNA and protein expressions. Moreover, we provide evidence that activation of the c-jun N-terminal kinase (JNK) pathway is necessary for the GSK3 inhibition-mediated increase in Bim expression and apoptotic response. Finally, we demonstrate that concomitant inhibition of GSK3 potentiates the death ligand-induced apoptotic response in pancreatic cancer cells but not in non-transformed pancreatic epithelial cells and that this effect also requires JNK activity. Considering that different approaches leading to stimulation of death receptor signaling are under clinical trials for treatment of unresectable or metastatic pancreatic cancer, inhibition of GSK3 could represent an attractive new avenue to improve their effectiveness.

Pancreatic cancer (PC) is the fourth leading cause of cancer-related death worldwide and has the highest fatality rate of all cancers. The chemoresistance of PC cells contributes to the poor prognosis of PC patients which raises the need for the identification and development of new effective therapy. Studies have revealed that the glycogen synthase kinase 3 (GSK3) is overexpressed in human pancreatic adenocarcinoma as compared to normal pancreas and its inhibition leads to the growth arrest of established tumor xenografts thus suggesting a tumor promoting role for GSK3 in PC cells. Considering that GSK3 inhibitors are currently under clinical trial for treatment of different disorders such as neurodegenerative diseases and diabetes, inhibition of GSK3 activity might represent an attractive therapeutic option for PC patients. However, mechanisms under the control of GSK3 activity in PC cells need to be clarified. We have previously demonstrated that prolonged inhibition of GSK3 activity induces JNK-dependent cell death in human PC cells. Recently, it was suggested in prostate cancer cells that GSK3 promotes both apoptosis and autophagy, the latter playing a protective role against cell death. Interestingly, inhibition of autophagy was shown to sensitize diverse cancer cells to a wide array of stress conditions. The AIM of the study was to evaluate whether GSK3 controls autophagy in PC cells and determine the role of autophagy with regard to PC cell survival. METHODS. Experiments were performed using the human PC cell lines PANC1 and MIAPaCa2. GSK3 activity was inhibited by treatment with a specific GSK3 inhibitor SB216763 (20μM). Apoptosis was measured by assessment of PARP cleavage and caspase 3 and 7 activities. Autophagy was evaluated by the detection of the membrane-bound LC3B-II isoform. The autophagy inhibitor 3-methyladenine 3-MA (10mM) and the JNK inhibitor SP600125 (25μM) were also used. RESULTS. 1) Prolonged inhibition of GSK3 activity (48-72h) in PC cells induced both an apoptotic and autophagic responses. Treatment with 3-MA 2) elicited an apoptotic response and 3) strongly promoted the SB216763-induced apoptosis. 4) Mechanistically, inhibition of GSK3 activity led to a rapid and persistent activation of the JNK-cJun pathway. 5) Interestingly, blockade of the JNK pathway prevented the SB216763-induced apoptosis but not the SB216763-induced autophagy. CONCLUSION. Our results provide evidences that GSK3 regulates both apoptosis and autophagy in PC cells. On one hand, inhibition of GSK3 activity triggers apoptosis through JNK-dependent mechanisms. On the other hand, inhibition of GSK3, independently of the JNK pathway, promotes autophagy, a process that appears to protect PC cells against cell death. Inhibition of autophagy might represent an attractive avenue to promote cytotoxic activity of potential therapeutic agents such as GSK3 inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2874. doi:10.1158/1538-7445.AM2011-2874