Lysosome-dependent Manner Research Articles

Lysosomal-associated protein transmembrane 5 ameliorates non-alcoholic steatohepatitis by promoting the degradation of CDC42 in mice

Non-alcoholic steatohepatitis (NASH) has received great attention due to its high incidence. Here, we show that lysosomal-associated protein transmembrane 5 (LAPTM5) is associated with NASH progression through extensive bioinformatical analysis. The protein level of LAPTM5 bears a negative correlation with NAS score. Moreover, LAPTM5 degradation is mediated through its ubiquitination modification by the E3 ubquitin ligase NEDD4L. Discovered by experiments conducted on male mice, hepatocyte-specific depletion of Laptm5 exacerbates mouse NASH symptoms. In contrast, Laptm5 overexpression in hepatocytes exerts diametrically opposite effects. Mechanistically, LAPTM5 interacts with CDC42 and promotes its degradation through a lysosome-dependent manner under the stimulation of palmitic acid, thus inhibiting activation of the mitogen-activated protein kinase signaling pathway. Finally, adenovirus-mediated hepatic Laptm5 overexpression ameliorates aforementioned symptoms in NASH models.

Human Papillomavirus Genome Copy Number Is Maintained by S-Phase Amplification, Genome Loss to the Cytosol during Mitosis, and Degradation in G1 Phase.

The current model of human papillomavirus (HPV) replication is comprised of three modes of replication. Following infectious delivery, the viral genome is amplified during the establishment phase to reach up to some hundred copies per cell. The HPV genome copy number remains constant during the maintenance stage. The differentiation of infected cells induces HPV genome amplification. Using highly sensitive in situ hybridization (DNAscope) and freshly HPV16-infected as well as established HPV16-positive cell lines, we observed that the viral genome is amplified in each S phase of undifferentiated keratinocytes cultured as monolayers. The nuclear viral genome copy number is reset to pre-S-phase levels during mitosis. The majority of the viral genome fails to tether to host chromosomes and is lost to the cytosol. Cytosolic viral genomes gradually decrease during cell cycle progression. The loss of cytosolic genomes is blocked in the presence of NH4Cl or other drugs that interfere with lysosomal acidification, suggesting the involvement of autophagy in viral genome degradation. These observations were also made with HPV31 cell lines obtained from patient samples. Cytosolic viral genomes were not detected in UMSCC47 cells carrying integrated HPV16 DNA. Analyses of organotypic raft cultures derived from keratinocytes harboring episomal HPV16 revealed the presence of cytosolic viral genomes as well. We conclude that HPV maintains viral genome copy numbers by balancing viral genome amplification during S phase with the loss of viral genomes to the cytosol during mitosis. It seems plausible that restrictions to viral genome tethering to mitotic chromosomes reset genome copy numbers in each cell cycle. IMPORTANCE HPV genome maintenance is currently thought to be achieved by regulating the expression and activity of the viral replication factors E1 and E2. In addition, the E8^E2 repressor has been shown to be important for restricting genome copy numbers by competing with E1 and E2 for binding to the viral origin of replication and by recruiting repressor complexes. Here, we demonstrate that the HPV genome is amplified in each S phase. The nuclear genome copy number is reset during mitosis by a failure of the majority of the genomes to tether to mitotic chromosomes. Rather, HPV genomes accumulate in the cytoplasm of freshly divided cells. Cytosolic viral DNA is degraded in G1 in a lysosome-dependent manner, contributing to the genome copy reset. Our data imply that the mode of replication during establishment and maintenance is the same and further suggest that restrictions to genome tethering significantly contribute to viral genome maintenance.

Perturbation of Autophagy by a Beclin 1-Targeting Stapled Peptide Induces Mitochondria Stress and Inhibits Proliferation of Pancreatic Cancer Cells.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer, with a dismal five-year survival rate of less than 10%. PDAC possesses prominent genetic alterations in the oncogene KRAS and tumor suppressors p53, SMAD4 and CDKN2A. However, efforts to develop targeted drugs against these molecules have not been successful, and novel therapeutic modalities for PDAC treatment are urgently needed. Autophagy is an evolutionarily conserved self-degradative process that turns over intracellular components in a lysosome-dependent manner. The role of autophagy in PDAC is complicated and context-dependent. Elevated basal autophagy activity has been detected in multiple human PDAC cell lines and primary tumors resected from patients. However, clinical trials using chloroquine (CQ) to inhibit autophagy failed to show therapeutic efficacy. Here we show that a Beclin 1-targeting stapled peptide (Tat-SP4) developed in our lab further enhanced autophagy in multiple PDAC cell lines possessing high basal autophagy activity. Tat-SP4 also triggered faster endolysosomal degradation of EGFR and induced significant mitochondria stress as evidenced by partial loss of Δψ, increased level of ROS and reduced OXPHOS activity. Tat-SP4 exerted a potent anti-proliferative effect in PDAC cell lines in vitro and prohibited xenograft tumor growth in vivo. Intriguingly, excessive autophagy has been reported to trigger a unique form of cell death termed autosis. Tat-SP4 does induce autosis-like features in PDAC cells, including mitochondria stress and non-apoptotic cell death. Overall, our study suggests that autophagy perturbation by a Beclin 1-targeting peptide and the resulting autosis may offer a new strategy for PDAC drug discovery.

The Iridoid Glycoside Loganin Modulates Autophagic Flux Following Chronic Constriction Injury-Induced Neuropathic Pain.

Autophagy facilitates the degradation of organelles and cytoplasmic proteins in a lysosome-dependent manner. It also plays a crucial role in cell damage. Whether loganin affects autophagy in chronic constriction injury (CCI)-induced neuropathic pain remains unclear. We investigated the neuroprotective effect of loganin on the autophagic-lysosomal pathway in the rat CCI model. Sprague-Dawley rats were divided into sham, CCI, sham + loganin, and CCI + loganin. Loganin (5 mg/kg/day) was intraperitoneally injected once daily, and rats were sacrificed on day 7 after CCI. This study focused on the mechanism by which loganin modulates autophagic flux after CCI. CCI enhanced the autophagic marker LC3B-II in the ipsilateral spinal cord. The ubiquitin-binding protein p62 binds to LC3B-II and integrates into autophagosomes, which are degraded by autophagy. CCI caused the accumulation of p62, indicating the interruption of autophagosome turnover. Loganin significantly attenuated the expression of Beclin-1, LC3B-II, and p62. Double immunofluorescence staining was used to confirm that LC3B-II and p62 were reduced by loganin in the spinal microglia and astrocytes. Loganin also lessened the CCI-increased colocalization of both proteins. Enhanced lysosome-associated membrane protein 2 (LAMP2) and pro-cathepsin D (pro-CTSD) in CCI rats were also attenuated by loganin, suggesting that loganin improves impaired lysosomal function and autophagic flux. Loganin also attenuated the CCI-increased apoptosis protein Bax and cleaved caspase-3. Loganin prevents CCI-induced neuropathic pain, which could be attributed to the regulation of neuroinflammation, neuronal autophagy, and associated cell death. These data suggest autophagy could be a potential target for preventing neuropathic pain.

Bifunctional Compounds as Molecular Degraders for Integrin-Facilitated Targeted Protein Degradation.

As effective ways to regulate protein levels, targeted protein degradation technologies have attracted great attention in recent years. Here, we established a novel integrin-facilitated lysosomal degradation (IFLD) strategy to degrade extracellular and cell membrane proteins using bifunctional compounds as molecular degraders. By conjugation of a target protein-binding ligand with an integrin-recognition ligand, the resulting molecular degrader proved to be highly efficient to induce the internalization and subsequent degradation of extracellular or cell membrane proteins in an integrin- and lysosome-dependent manner. As demonstrated in the development of BMS-L1-RGD, which is an efficient programmed death-ligand 1 (PD-L1) degrader validated both in vitro and in vivo, the IFLD strategy expands the toolbox for regulation of secreted and membrane-associated proteins and thus has great potential to be applied in chemical biology and drug discovery.

E3 ubiquitin ligase MID1 ubiquitinates and degrades type‐I interferon receptor 2

Type I interferon (IFN-I) is a common biological molecule used for the treatment of viral diseases. However, the clinical antiviral efficacy of IFN-I needs to be greatly improved. In this study, IFN-I receptor 2 (IFNAR2) was revealed to undergo degradation at the protein level in cells treated with IFN-I for long periods of time. Further studies found a physical interaction between the E3 ubiquitin ligase midline-1 (MID1) and IFNAR2. As a consequence, MID1 induced both K48- and K63-linked polyubiquitination of IFNAR2, which promoted IFNAR2 protein degradation in a lysosome-dependent manner. Conversely, knockdown of MID1 largely restricted IFN-I-induced degradation of IFNAR2. Importantly, MID1 regulated the strength of IFN-I signalling and IFN-I-induced antiviral activity. These findings reveal a regulatory mechanism of IFNAR2 ubiquitination and protein stability in IFN-I signalling, which could provide a potential target for improving the antiviral efficacy of IFN-I.

Visual monitoring of the mitochondrial pH changes during mitophagy with a NIR fluorescent probe and its application in tumor imaging

Mitophagy, a mitochondria-selective autophagy process, plays critical roles in maintaining intracellular homeostasis by removing the damaged mitochondria and recycling the nutrients in a lysosome-dependent manner. Mitophagy process could result in the changes of mitochondrial pH. So fluorescent probes for detecting mitochondrial pH during mitophagy are highly needed for exploring the functions of mitochondria. Herein, a series of near-infrared pH probes were designed based on the rhodamine framework. The probes showed high sensitivity for pH with the tunable pKa from 4.74 to 6.54. Particularly, for probe 5 (with the pKa of 6.54), a linear relationship between fluorescence intensity and pH in the range of 5.6–7.2 was observed, which was suitable for mitochondrial pH detection. The probe displayed excellent mitochondria-targeting ability. It was applied to monitor pH changes during mitophagy caused by starvation. Besides, in vivo non-invasive visualization of tumor pH variations was achieved via the fluorescence imaging in the near-infrared region. We anticipate that the probe may be a useful tool for revealing essential information about mitophagy-related research and clinical tumor diagnosis.

Lipopolysaccharide Accelerates Neuropilin-1 Protein Degradation by Activating the Large GTPase Dynamin-1 in Macrophages.

Neuropilin-1 (Nrp1) is highly expressed in macrophages and plays a critical role in acute and chronic inflammation-associated diseases, such as sepsis, type II diabetes, and metabolic syndrome. Therefore, it is of importance to understand the regulation of Nrp1. It is known that lipopolysaccharide (LPS) downregulates Nrp1 mRNA levels through the NF-κB signaling in macrophages. However, whether and how LPS regulates Nrp1 protein degradation remain unknown. Here, we show that LPS promotes Nrp1 protein decay through a lysosome-dependent manner. Liver kinase B1 (LKB1)-Rab7 does not mediate this process. However, the large GTPase dynamin-1 (Dyn1) but not Dyn2 is involved in LPS-accelerated Nrp1 degradation. Mechanistically, LPS activates Dyn1 by attenuating p-Dyn1 (Ser774) levels, implying increased Nrp1 endocytosis and consequent degradation. As a result, blocking Nrp1 degradation by Dyn1 siRNA attenuates LPS-induced inflammatory response. Collectively, our study shows that LPS promotes Nrp1 protein degradation via a Dyn1-dependent pathway, revealing a previously uncovered role of Dyn1 in LPS-promoted Nrp1 protein decay.

Dual roles of AMAP1 in the transcriptional regulation and intracellular trafficking of carbonic anhydrase IX

BackgroundThe cell-surface enzyme carbonic anhydrase IX (CAIX/CA9) promotes tumor growth, survival, invasion, and metastasis, mainly via its pH-regulating functions. Owing to its tumor-specific expression, CAIX-targeting antibodies/chemicals are utilized for therapeutic and diagnostic purposes. However, mechanisms of CAIX trafficking, which affects such CAIX-targeting modalities remain unclear. In this study, roles of the AMAP1-PRKD2 pathway, which mediates integrin recycling of invasive cancer cells, in CAIX trafficking were investigated. MethodsUsing highly invasive MDA-MB-231 breast cancer cells, the physical association and colocalization of endogenous proteins were analyzed by immunoprecipitation and immunofluorescence, protein/mRNA levels were quantified by western blotting/qPCR, and cell-surface transport and intracellular/extracellular pH regulation were measured by biotin-labeling and fluorescent dye-based assays, respectively. The correlation between mRNA levels and patients’ prognoses was analyzed using a TCGA breast cancer dataset. ResultsAMAP1 associated with the CAIX protein complex, and they colocalized at the plasma membrane and tubulovesicular structures. AMAP1 knockdown reduced total/surface CAIX, induced its lysosomal accumulation and degradation, and affected intracellular/extracellular pH. PRKD2 knockdown excluded AMAP1 from the CAIX complex and reduced total CAIX in a lysosome-dependent manner. Unexpectedly, AMAP1 knockdown also reduced CAIX mRNA. AMAP1 interacted with PIAS3, which stabilizes HIF-1α, a transcriptional regulator of CA9. AMAP1 knockdown inhibited the PIAS3-HIF-1α interaction and destabilized the HIF-1α protein. High-ASAP1 (AMAP1-encoding gene) together with high-PIAS3 correlated with high-CA9 and an unfavorable prognosis in breast cancer. ConclusionThe AMAP1-PRKD2 pathway regulates CAIX trafficking, and modulates its total/surface expression. The AMAP1-PIAS3 interaction augments CA9 transcription by stabilizing HIF-1α, presumably contributing to an unfavorable prognosis.

Epigenetic regulation of autophagy: A key modification in cancer cells and cancer stem cells.

Aberrant epigenetic alterations play a decisive role in cancer initiation and propagation via the regulation of key tumor suppressor genes and oncogenes or by modulation of essential signaling pathways. Autophagy is a highly regulated mechanism required for the recycling and degradation of surplus and damaged cytoplasmic constituents in a lysosome dependent manner. In cancer, autophagy has a divergent role. For instance, autophagy elicits tumor promoting functions by facilitating metabolic adaption and plasticity in cancer stem cells (CSCs) and cancer cells. Moreover, autophagy exerts pro-survival mechanisms to these cancerous cells by influencing survival, dormancy, immunosurveillance, invasion, metastasis, and resistance to anti-cancer therapies. In addition, recent studies have demonstrated that various tumor suppressor genes and oncogenes involved in autophagy, are tightly regulated via different epigenetic modifications, such as DNA methylation, histone modifications and non-coding RNAs. The impact of epigenetic regulation of autophagy in cancer cells and CSCs is not well-understood. Therefore, uncovering the complex mechanism of epigenetic regulation of autophagy provides an opportunity to improve and discover novel cancer therapeutics. Subsequently, this would aid in improving clinical outcome for cancer patients. In this review, we provide a comprehensive overview of the existing knowledge available on epigenetic regulation of autophagy and its importance in the maintenance and homeostasis of CSCs and cancer cells.

PVHL promotes lysosomal degradation of YAP in lung adenocarcinoma

Yes-associated protein (YAP) is a vital transcriptional co-activator that activates cell proliferation and evasion of apoptosis for the promotion of tumorigenesis. The von Hippel-Lindau tumor suppressor protein (pVHL), as a critical component of E3 ubiquitin ligase, targets various substrates to regulate tumor progression. However, the precise molecular mechanisms of pVHL during tumorigenesis remain largely unclear. Herein, we found that there was a significant negative correlation between pVHL and YAP at protein level in the TCGA-LUAD dataset and our cohort. Over-expression of pVHL decreased YAP protein expression and reduced its transcriptional activity. Further study indicated that pVHL did not affect YAP mRNA level but decreased YAP protein stability in a lysosome-dependent manner. In addition, the pVHL-mediated degradation of YAP inhibited cellular proliferation, migration, and enhanced chemosensitivity to cisplatin in lung adenocarcinoma cells. Interestingly, the pVHL-mediated YAP degradation was blocked by elevated O-GlcNAcylation. Collectively, our findings demonstrate that pVHL modulates the lysosomal degradation of YAP, and may provide more clues to better understanding the tumor suppressive effects of pVHL.

Zebrafish RBM47 Promotes Lysosome-Dependent Degradation of MAVS to Inhibit IFN Induction.

IFN is essential for hosts to defend against viral invasion, whereas it must be tightly regulated to prevent hyperimmune responses. Fish mitochondrial antiviral signaling protein (MAVS) is a vital factor for IFN production, but until now, there have been few studies on the regulation mechanisms of fish MAVS enabling IFN to be properly controlled. In this study, we show that zebrafish RNA-binding motif protein 47 (RBM47) promotes MAVS degradation in a lysosome-dependent manner to suppress IFN production. First, the transcription of IFN activated by polyinosinic/polycytidylic acid (poly I:C), spring viremia of carp virus, or retinoic acid-inducible gene I (RIG-I)-like receptor pathway components were significantly suppressed by RBM47. Second, RBM47 interacted with MAVS and promoted lysosome-dependent degradation of MAVS, changing the cellular location of MAVS from the cytoplasm to the lysosome region. Finally, RBM47 inhibited downstream MITA and IRF3/7 activation, impairing the host antiviral response. Collectively, these data suggest that zebrafish RBM47 negatively regulates IFN production by promoting lysosome-dependent degradation of MAVS, providing insights into the role of RBM47 in the innate antiviral immune response in fish.

Discovery of novel resorcinol diphenyl ether-based PROTAC-like molecules as dual inhibitors and degraders of PD-L1

Novel resorcinol diphenyl ether-based PROTACs (PROteolysis TArgeting Chimeras) were designed and evaluated for their inhibitory activity against the programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) pathway and their ability to degrade PD-L1 protein. Most of the compounds displayed excellent inhibitory activities against PD-1/PD-L1, as assessed by the homogenous time-resolved fluorescence (HTRF) binding assay, with IC50 values ranging from 25 nM to 200 nM. Among them, compound P22 is one of the best with an IC50 value of 39.2 nM. In addition to inhibiting PD-1/PD-L1 interaction, P22 also significantly restored the immunity repressed in a co-culture model of Hep3B/OS-8/hPD-L1 and CD3 T cells. Furthermore, flow cytometry (FCM) and western-blot data demonstrated that P22 could moderately reduce the protein levels of PD-L1 in a lysosome-dependent manner, which may contribute to its immune effects. Preliminary FCM and western-blot data suggest that it is possible to build PD-L1-targeting PROTAC-like molecules based on PD-1/PD-L1 small molecule inhibitors, though these compounds showed only modest degradation efficiencies. Collectively, this work suggests that P22 may serve as a starting point for exploring the degradation of PD-L1 by PROTAC-like strategy.

Yeast β-D-glucan exerts antitumour activity in liver cancer through impairing autophagy and lysosomal function, promoting reactive oxygen species production and apoptosis

Autophagy is an evolutionarily conserved catabolic process that recycles proteins and organelles in a lysosome-dependent manner and is induced as an alternative source of energy and metabolites in response to diverse stresses. Inhibition of autophagy has emerged as an appealing therapeutic strategy in cancer. However, it remains to be explored whether autophagy inhibition is a viable approach for the treatment of hepatocellular carcinoma (HCC). Here, we identify that water-soluble yeast β-D-glucan (WSG) is a novel autophagy inhibitor and exerts significant antitumour efficacy on the inhibition of HCC cells proliferation and metabolism as well as the tumour growth in vivo. We further reveal that WSG inhibits autophagic degradation by increasing lysosomal pH and inhibiting lysosome cathepsins (cathepsin B and cathepsin D) activities, which results in the accumulation of damaged mitochondria and reactive oxygen species (ROS) production. Furthermore, WSG sensitizes HCC cells to apoptosis via the activation of caspase 8 and the transfer of truncated BID (tBID) into mitochondria under nutrient deprivation condition. Of note, administration of WSG as a single agent achieves a significant antitumour effect in xenograft mouse model and DEN/CCl4 (diethylnitrosamine/carbon tetrachloride)-induced primary HCC model without apparent toxicity. Our studies reveal, for the first time, that WSG is a novel autophagy inhibitor with significant antitumour efficacy as a single agent, which has great potential in clinical application for liver cancer therapy.

Heat Shock Protein 90 Chaperone Regulates the E3 Ubiquitin-Ligase Hakai Protein Stability.

The E3 ubiquitin-ligase Hakai binds to several tyrosine-phosphorylated Src substrates, including the hallmark of the epithelial-to-mesenchymal transition E-cadherin, and signals for degradation of its specific targets. Hakai is highly expressed in several human cancers, including colon cancer, and is considered as a drug target for cancer therapy. Here, we report a link between Hakai and the heat shock protein 90 (Hsp90) chaperone complex. Hsp90 participates in the correct folding of its client proteins, allowing them to maintain their stability and activity. Hsp90 inhibitors specifically interfere with the association with its Hsp90 client proteins, and exhibit potent anti-cancer properties. By immunoprecipitation, we present evidence that Hakai interacts with Hsp90 chaperone complex in several epithelial cells and demonstrate that is a novel Hsp90 client protein. Interestingly, by overexpressing and knocking-down experiments with Hakai, we identified Annexin A2 as a Hakai-regulated protein. Pharmacological inhibition of Hsp90 with geldanamycin results in the degradation of Hakai in a lysosome-dependent manner. Interestingly, geldanamycin-induced Hakai degradation is accompanied by an increased expression of E-cadherin and Annexin A2. We also show that geldanamycin suppresses cell motility at least in part through its action on Hakai expression. Taken together, our results identify Hakai as a novel Hsp90 client protein and shed light on the regulation of Hakai stability. Our results open the possibility to the potential use of Hsp90 inhibitors for colorectal cancer therapy through its action on Hakai client protein of Hsp90.

Correction for: The combination of lonafarnib and sorafenib induces cyclin D1 degradation via ATG3-mediated autophagic flux in hepatocellular carcinoma cells.

Combination treatment is a promising strategy to improve prognosis of hepatocellular carcinoma (HCC). Sorafenib is a traditional first-line agent approved for the treatment of advanced HCC, though with limited efficacy. Previously, we reported that lonafarnib, an orally bioavailable non-peptide inhibitor targeting farnesyltransferase, synergizes with sorafenib against the growth of HCC cells. In the present study, we aim to clarify the underlying mechanism of this combination strategy. Initially, using in vitro HCC cell model, we confirmed that synergistic treatment of lonafarnib and sorafenib suppressed cell viability and colony formation, and induced cell death. We then found conversion of LC3-I to LC3-II via combination the treatment and observed formation of autophagosomes by electron microscopy. Knockdown of ATG3 inhibited the autophagic flux induced by the combination treatment. Furthermore, we demonstrated that drug-eliciting autophagy selectively promoted the degradation of cyclin D1 in a lysosome-dependent manner and subsequently inhibited DNA synthesis through downregulating the phosphorylation of Rb protein. In conclusion, our results provide a deeper insight into the mechanism for the combination treatment of lonafarnib and sorafenib in HCC therapy.

The combination of lonafarnib and sorafenib induces cyclin D1 degradation via ATG3-mediated autophagic flux in hepatocellular carcinoma cells.

Combination treatment is a promising strategy to improve prognosis of hepatocellular carcinoma (HCC). Sorafenib is a traditional first-line agent approved for the treatment of advanced HCC, though with limited efficacy. Previously, we reported that lonafarnib, an orally bioavailable non-peptide inhibitor targeting farnesyltransferase, synergizes with sorafenib against the growth of HCC cells. In the present study, we aim to clarify the underlying mechanism of this combination strategy. Initially, using in vitro HCC cell model, we confirmed that synergistic treatment of lonafarnib and sorafenib suppressed cell viability and colony formation, and induced cell death. We then found conversion of LC3-I to LC3-II via combination the treatment and observed formation of autophagosomes by electron microscopy. Knockdown of ATG3 inhibited the autophagic flux induced by the combination treatment. Furthermore, we demonstrated that drug-eliciting autophagy selectively promoted the degradation of cyclin D1 in a lysosome-dependent manner and subsequently inhibited DNA synthesis through downregulating the phosphorylation of Rb protein. In conclusion, our results provide a deeper insight into the mechanism for the combination treatment of lonafarnib and sorafenib in HCC therapy.

Covalent targeting of the vacuolar H+-ATPase activates autophagy via mTORC1 inhibition.

Autophagy is a lysosomal degradation pathway that eliminates aggregated proteins and damaged organelles to maintain cellular homeostasis. A major route for activating autophagy involves inhibition of the mTORC1 kinase, but current mTORC1-targeting compounds do not allow complete and selective mTORC1 blockade. Here, we have coupled screening of a covalent ligand library with activity-based protein profiling to discover EN6, a small-molecule in vivo activator of autophagy that covalently targets cysteine 277 in the ATP6V1A subunit of the lysosomal v-ATPase, which activates mTORC1 via the Rag guanosine triphosphatases. EN6-mediated ATP6V1A modification decouples the v-ATPase from the Rags, leading to inhibition of mTORC1 signaling, increased lysosomal acidification, and activation of autophagy. Consistently, EN6 clears TDP-43 aggregates, a causative agent in frontotemporal dementia, in a lysosome-dependent manner. Our results provide insight into how the v-ATPase regulates mTORC1, and reveal a unique approach for enhancing cellular clearance based on covalent inhibition of lysosomal mTORC1 signaling.

Abstract 4815: Novel Globo H targeting antibody-drug conjugate with binding specificity and anti-tumor efficacy in multiple cancer types

Abstract Background: Globo H, a hexasaccharide, has been reported to be highly expressed in multiple cancers types, but not express or, if at all, express to a lesser extent in normal tissue. Therefore, Globo H may be a potential target for cancer immunotherapy. OBI-999 is an antibody-drug conjugate (ADC) which consists of a Globo H-specific monoclonal antibody OBI-888, conjugated with monomethyl auristatin E (MMAE), a synthetic antineoplastic agent. The present study investigates antigen specificity, drug internalization, anti-tumor efficacy, and pharmacokinetics profiles of OBI-999. Methods: Globo H expression was surveyed in various human cancer cell lines. The binding specificity and internalization of OBI-999 were determined by flow cytometry and confocal microscopy, respectively. In vivo anti-tumor efficacy was studied in conventional xenograft and patient-derived xenograft models. Pharmacokinetic parameters were evaluated in normal and tumor bearing xenograft mice. Results: We confirmed positive Globo H expression on the cell surface of multiple cancer cell lines ranging from breast, gastric, lung, to pancreatic cancer. From flow cytometry, OBI-999 showed binding selectivity to the aforementioned Globo H expressing cell lines. In contrast, OBI-999 did not bind to non-Globo H expressing cell lines. When bound to the antigen, OBI-999 was internalized and trafficked to endosome and lysosome within 2.5 to 5 hours, suggesting that the drug payload, MMAE, was cleaved in a lysosome dependent manner. OBI-999 showed significant tumor inhibition in breast, gastric, lung, and pancreatic cancer xenograft and PDX models in dose-dependent manner. At 1 mg/kg of OBI-999, tumor growth inhibition in breast, gastric, and lung cancer model were 77%, 89%, and 68% respectively. At 10 mg/kg of OBI-999, complete tumor growth inhibition in pancreatic cancer model was observed. In vitro serum stability studies revealed that OBI-999 has comparable stabilities to Adcetris® in mouse, rat, monkey, and human sera. Tissue distribution study revealed that OBI-999 is accumulated gradually at the tumor site while the level of OBI-999 showed a time dependent decrease in blood-rich organs. Accumulation of OBI-999 at the tumor site reached its maximum level at 168 hour post treatment. Level of MMAE in tumor mass was approximately 25 folds higher than that in other organs and 250 folds higher than that in serum, suggesting that OBI-999 targets and releases its payload at tumor region. Conclusion: The preclinical studies of OBI-999 demonstrated its targeting specificity and anti-tumor efficacy in multiple Globo H positive cancer models. OBI-999 also demonstrated a longer retention at tumor site. The preclinical results provided the fundamental basis for OBI-999’s clinical application as targeted cytotoxicity therapy for solid tumors. Citation Format: Ming-Chen Yang, Yu-Jung Chen, Chi-Sheng Shia, Hui-Wen Chang, Wan-Fen Li, Cheng-Der Tony Yu, I-Ju Chen. Novel Globo H targeting antibody-drug conjugate with binding specificity and anti-tumor efficacy in multiple cancer types [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 4815.

Zebrafish MVP Recruits and Degrades TBK1 To Suppress IFN Production.

IFN production is crucial for hosts to defend against viral infection, yet it must be tightly controlled to maintain immune homeostasis. TANK-binding kinase 1 (TBK1) is a pivotal kinase in the IFN induction signaling pathway, but it is negatively regulated by multiple molecules to avoid the excessive expression of IFN in mammals. However, the identified TBK1 suppressors and the mechanisms are rare in fish. In this study, we show that zebrafish major vault protein (MVP) recruits and degrades TBK1 in a lysosome-dependent manner to inhibit IFN production. Through viral infection, polyinosinic:polycytidylic acid and RIG-I-like receptor factor stimulation upregulated IFN expression, but overexpression of MVP significantly subverted these inductions. On the protein level, MVP interacted with TBK1, and interestingly, MVP recruited TBK1 from a uniformly distributed state in the cytoplasm to an aggregated state. Finally, MVP mediated the lysosome-dependent degradation of TBK1 and decreased the IFN response and IFN-stimulated genes expression. Our findings reveal that zebrafish MVP is a negative regulator of IFN production by restricting the activation of TBK1, supplying evidence of the balanced mechanisms of IFN expression in lower vertebrates.