Late-stage Autophagy Inhibitor Research Articles

Abstract 1230: Critical role of cav1 in high-throughput identification of gallic acid as a novel late-stage autophagy suppresser against invasive breast cancer

Abstract Purpose: Development of autophagy inhibitors has important clinical significance in cancer therapies. We and others demonstrated that over-expression of Caveolin-1 (Cav1), which was the major scaffolding protein of caveolae, was associated with higher tumor stage, metastasis and worse prognosis in various carcinomas, particularly in breast cancer. Furthermore, we identified critical role of Cav1 in breast cancer metastasis via modulation of lysosomal function and autophagy. Based on the above, we screened a series of 2, 645 kinds of small-molecule drugs to find out certain cav1 inhibitors which could suppress autophagy and breast cancer metastasis. Experimental Design: We conducted the 1st high-throughput screening based on surface plasmon resonance (SPR) analysis of cav-1 interaction with 2, 645 kinds of small-molecule drugs. Secondly, we launched the 2nd high-throughput screening based on CCK8 analysis to validate the anti-cancer potential of the selected compounds. We also carried out the final high-throughput screening to investigate their autophagic effects with high content image cytometry assay. The anti-cancer and anti-metastasis effects of the final drug candidate was investigated using invasive breast cancer cells MDA-MB-231 and BT-549, and its role on autophagy was further investigated by transfection of mRFP-GFP-LC3 adenoviral vector and western blot analysis of autophagic proteins. Results: 2, 645 kinds of small-molecule drugs were screened based on a series of high-throughput screening. The compound gallic acid was identified as the candidate drug owing to its strong interaction and competitive inhibition with cav1, its potent anti-proliferation potential on both invasive breast cancer cells MDA-MB-231 and BT-549, as well as its potent autophagic inhibition compared with the other 2, 644 drugs. Further experimental validation revealed that gallic acid was a potent late-stage autophagy inhibitor via inhibiting autophagosome-lysosome fusion. Conclusions: Collectively, these results demonstrated that the cav1 inhibitor gallic acid was a novel late-stage autophagy suppressor with potential clinical application for invasive breast cancer therapy. Citation Format: Neng Wang, Fengxue Zhang, Zhiyu Wang, Yifeng Zheng, Shengqi Wang, Bowen Yang. Critical role of cav1 in high-throughput identification of gallic acid as a novel late-stage autophagy suppresser against invasive breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1230.

CNOT2 Is Critically Involved in Atorvastatin Induced Apoptotic and Autophagic Cell Death in Non-Small Cell Lung Cancers.

Though Atorvastatin has been used as a hypolipidemic agent, its anticancer mechanisms for repurposing are not fully understood so far. Thus, in the current study, its apoptotic and autophagic mechanisms were investigated in non-small cell lung cancers (NSCLCs). Atorvastatin increased cytotoxicity, sub G1 population, the number of apoptotic bodies, cleaved poly (ADP-ribose) polymerase (PARP) and caspase 3 and activated p53 in H1299, H596, and H460 cells. Notably, Atorvastatin inhibited the expression of c-Myc and induced ribosomal protein L5 and L11, but depletion of L5 reduced PARP cleavages induced by Atorvastatin rather than L11 in H1299 cells. Also, Atorvastatin increased autophagy microtubule-associated protein 1A/1B-light chain 3II (LC3 II) conversion, p62/sequestosome 1 (SQSTM1) accumulation with increased number of LC3II puncta in H1299 cells. However, late stage autophagy inhibitor chloroquine (CQ) increased cytotoxicity in Atorvastatin treated H1299 cells compared to early stage autophagy inhibitor 3-methyladenine (3-MA). Furthermore, autophagic flux assay using RFP-GFP-LC3 constructs and Lysotracker Red or acridine orange-staining demonstrated that autophagosome-lysosome fusion is blocked by Atorvastatin treatment in H1299 cells. Conversely, overexpression of CCR4-NOT transcription complex subunit 2(CNOT2) weakly reversed the ability of Atorvastatin to increase cytotoxicity, sub G1 population, cleavages of PARP and caspase 3, LC3II conversion and p62/SQSTM1 accumulation in H1299 cells. In contrast, CNOT2 depletion enhanced cleavages of PARP and caspase 3, LC3 conversion and p62/SQSTM1 accumulation in Atorvastatin treated H1299 cells. Overall, these findings suggest that CNOT2 signaling is critically involved in Atorvastatin induced apoptotic and autophagic cell death in NSCLCs.

Abstract 1309: Targeting GluN2C ion channels as a promising approach in the treatment of melanoma

Abstract Ion channels are pore-forming integral membrane proteins that are known to have an impact on all hallmarks of cancer. Due to their expression on the surface of the cells, ion channels provide a distinct therapeutic target with more facilitated access from the extracellular milieu. NMDA receptors are a subtype of ligand-gated ion channels that express unique features including high calcium permeability as well as the requirement for binding of glutamate and the co-agonist glycine in order to be activated. Here we show that a glutamate ionotropic NMDA (N-methyl-D-aspartate) type ion channel, GluN2C is critically required for survival of human melanoma cells. Publicly available microarray data from Gene Expression Omnibus (GEO) were acquired in order to identify the differentially expressed ion channel genes in 127 melanoma patient samples relative to benign nevi or human epidermal melanocytes. Data exists into two major cohorts: First cohort consisted of 18 benign nevi and 45 melanoma (GSE3189), while second cohort contained 8 melanocytes and 82 melanoma (GSE29377). A bioinformatics approach was undertaken to determine commonly upregulated ion channels between the two cohorts. Subsequently, functional enrichment analysis along with pathway evaluation of these genes revealed GluN2C as a novel candidate for further investigation in this study. Upregulation of GluN2C was confirmed in a panel of melanoma cell lines and fresh melanoma isolates. Strikingly, silencing of GluN2C markedly reduced viability and clonogenicity in melanoma cells. Consistently, a pharmacological inhibitor of GluN2C potently killed melanoma cells with minimal effects on melanocytes and fibroblasts. Killing of melanoma cells by inhibition of GluN2C was not due to induction of apoptosis, since a general caspase inhibitor Z-VAD-FMK failed to rescue cells from dying. In contrast, the late stage autophagy inhibitor Bafilomycin A1 blocked the GluN2C induced cell death suggesting that killing of melanoma cells following inhibition of GluN2C is in part associated with induction of autophagy. Collectively, these results uncover a potential role for GluN2C ion channels in the pathogenesis of melanoma and suggest that these ion channels may constitute a promising target for melanoma treatment. Citation Format: Hessam Tabatabaee, Yuchen Feng, Hamed Yari, Ting La, Simonne Sherwin, Lei Jin, Rebecca Lim, Alan Brichta, Xu Dong Zhang. Targeting GluN2C ion channels as a promising approach in the treatment of melanoma [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 1309.

Autophagic flux disruption contributes to Ganoderma lucidum polysaccharide-induced apoptosis in human colorectal cancer cells via MAPK/ERK activation

Targeting autophagy may serve as a promising strategy for cancer therapy. Ganoderma lucidum polysaccharide (GLP) has been shown to exert promising anti-cancer effects. However, the underlying mechanisms remain elusive. Whether GLP regulates autophagy in cancer has never been reported. In this study, GLP induced the initiation of autophagy in colorectal cancer (CRC) HT-29 and HCT116 cells, as evidenced by enhanced level of LC3-II protein, GFP-LC3 puncta, and increased formation of double membrane vacuoles. However, GLP treatment caused marked increase of p62 expression. Addition of late stage autophagy inhibitor, chloroquine (CQ), further enhanced LC3-II and p62 level, as well as increased autophagosome accumulation, suggesting a blockage of autophagic flux by GLP in CRC cells. We then found GLP blocked autophagosome and lysosome fusion as determined by mRFP-GFP-LC3 colocalization analysis. Mechanistic study revealed that GLP-induced disruption of autophagosome-lysosome fusion is due to reduced lysosome acidification and lysosomal cathepsin activities. Cell viability and flow cytometry assays revealed that GLP-induced autophagosome accumulation is responsible for GLP-induced apoptosis in CRC cells. In line with this, inhibition of autophagy initiation by 3-methyladenine (3-MA), an early stage autophagy inhibitor, attenuated GLP-induced apoptosis. In contrast, suppression of autophagy at late stage by CQ enhanced the anti-cancer effect of GLP. Furthermore, we demonstrated that GLP-induced autophagosome accumulation and apoptosis is mediated via MAPK/ERK activation. Finally, GLP inhibited tumor growth and also inhibited autophagic flux in vivo. These results unveil new molecular mechanism underlying anti-cancer effects of GLP, suggesting that GLP is a potent autophagy inhibitor and might be useful in anticancer therapy.

Saikosaponin D suppresses enterovirus A71 infection by inhibiting autophagy

The dysregulation of autophagy, an evolutionarily conserved lysosomal degradation process, has been implicated in a wide variety of human diseases, and thus, small chemicals that modulate autophagy have therapeutic potential. Here, we assessed the ability of active components isolated from Bupleurum falcatum, a popular Chinese herb, to modulate autophagy. We found that saikosaponin D (SsD) and A (SsA) but not C (SsC) potently and reversibly inhibited the fusion of autophagosomes and lysosomes, resulting in the accumulation of autophagosomes, an increased lysosomal pH, and TFEB nuclear translocation. RAB5A knockdown or the expression of a dominant-negative RAB5 mutant significantly reduced the ability of SsD or SsA to block autophagy. Enterovirus A71 (EV-A71), the cause of hand-foot-mouth disease, has been shown to induce autophagy. We found that SsD potently inhibited EV-A71 RNA replication and subsequent viral protein synthesis, thereby preventing EV-A71-induced cell death. ATG5 knockdown inhibited EV-A71 viral protein synthesis, whereas autophagy induction by rapamycin promoted synthesis. Taken together, our data indicate that SsD and SsA are potent late-stage autophagy inhibitors that can be used to prevent EV-A71 infection.

Chloroquine Derivative Lys05 Overcomes Hypoxia-Induced Chemoresistance in Acute Myeloid Leukemia through Metabolic Disruption

Background: Despite initial responses to standard chemotherapy, most patients with acute myeloid leukemia (AML) relapse and have poor prognoses after subsequent therapy. The hypoxic bone marrow (BM) microenvironment is hypothesized to contribute to clinical resistance through the sheltering of AML blasts and leukemia stem cells (LSCs), allowing them to evade chemotherapy and reinitiate the disease. We have previously shown that AML cell lines cultured under prolonged hypoxia (1% O2) can upregulate autophagy and become resistant to cytarabine (AraC). Furthermore, the inhibition of autophagosome turnover with either Bafilomycin A1 (Baf A1) or chloroquine (CQ) can re-sensitize AML cells to AraC. Additionally, Baf A1 therapy effectively eradicated in vivo functional LSCs in a primary human AML xenotransplantation mouse model. However, clinical development of BafA1 and CQ has been limited by poor drug pharmokinetics. Lys05 is a novel water-soluble CQ derivative which exhibits 10 fold more potent autophagy inhibitory properties than CQ in vitro and is well tolerated in mouse models. Here, we tested the effects of Lys05 on human AML growth, chemoresistance under hypoxia, and in vivo leukemia burden in a human AML xenograft model. We also explored the effects of autophagosome accumulation on cellular metabolism as a new mechanism for targeting chemoresistant AML blasts and LSCs.Methods: Human AML cell line MOLM13 and CD34 positive AML patient samples were cultured under hypoxia (1% O2) or normoxia (21% O2) with cytarabine (AraC) and late-stage autophagy inhibitor Lys05 alone and in combination. After 72 hours of drug exposure, apoptosis was measured using Annexin V/propidium iodide flow cytometry. Metabolic assays were performed using a Seahorse XFe96 analyzer to measure oxygen consumption and extracellular acidification rates using the Mitochondrial or Glycolytic Stress Tests on AML cells treated with late-stage autophagy inhibitors for 48 hours. Mitochondrial mass was measured following staining with MITO-ID Green Detection Kit using flow cytometry. Human xenograft models were performed using NSG mice inoculated with MOLM13 BLIV, a luciferase-expressing human AML cell line, and treated with either vehicle or Lys05 (20 mg/kg 4 days on/3 off for 4 weeks). Tumor burden was assessed using bioluminescence and analyzed with IVIS Living Image Software.Results: Treatment of AML MOLM13 cells with Lys05 in vitro resulted in a dose-dependent decrease in cell proliferation and increased apoptosis. Lys05 treatment further displayed similar results to BafA1 and CQ, enhancing the anti-leukemic effects of AraC and overcoming hypoxia-induced chemoresistance. Seahorse XFe analysis revealed that BafA1 inhibited both basal and maximal mitochondrial respiration under both normoxic and hypoxic conditions at 24 hours. CQ and Lys05, however, had no effect on oxidative phosphorylation under normoxia, yet significantly decreased mitochondrial function under hypoxia. Interestingly, reduced function did not coincide with a decrease in mitochondrial mass, but rather increased mitochondrial mass. We also observed decreases in glycolytic function after treatment with late-stage autophagy inhibitors under hypoxia suggesting these cells may be undergoing an energy crisis. Treatment of leukemia-bearing mice with Lys05 trended toward decreased tumor burden compared to vehicle. Studies optimizing Lys05 monotherapy and testing combination treatment with AraC are underway.Conclusions: Our research shows that CQ derivative Lys05 exhibits significant anti-leukemic activity against human AML cells and restores chemosensitivity to cytarabine under hypoxia, further supporting our hypothesis that autophagosome accumulation can overcome hypoxia-induced chemoresistance. We also demonstrate that Lys05 may effectively inhibit leukemia progression in vivo. Our studies further reveal metabolic disruption through the accumulation of autophagosomes as a mechanism for restoring chemosensitivity of AML cells under hypoxia and targeting LSCs. These results establish late-stage autophagy inhibitors, specifically Lys05, as a promising new treatment approach for resistant AML and supports further study of these drugs for treatment of minimal residual disease. [Display omitted] DisclosuresGuzman:Cellectis: Research Funding. Wang:Novartis: Speakers Bureau; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Speakers Bureau; Amgen: Consultancy; Jazz: Speakers Bureau.

Phyllanthusmin Derivatives Induce Apoptosis and Reduce Tumor Burden in High-Grade Serous Ovarian Cancer by Late-Stage Autophagy Inhibition.

High-grade serous ovarian cancer (HGSOC) is a lethal gynecological malignancy with a need for new therapeutics. Many of the most widely used chemotherapeutic drugs are derived from natural products or their semi-synthetic derivatives. We have developed potent synthetic analogues of a class of compounds known as phyllanthusmins, inspired by natural products isolated from Phyllanthus poilanei Beille. The most potent analogue, PHY34, had the highest potency in HGSOC cell lines in vitro and displayed cytotoxic activity through activation of apoptosis. PHY34 exerts its cytotoxic effects by inhibiting autophagy at a late stage in the pathway, involving the disruption of lysosomal function. The autophagy activator, rapamycin, combined with PHY34 eliminated apoptosis, suggesting that autophagy inhibition may be required for apoptosis. PHY34 was readily bioavailable through intraperitoneal administration in vivo where it significantly inhibited the growth of cancer cell lines in hollow fibers, as well as reduced tumor burden in a xenograft model. We demonstrate that PHY34 acts as a late-stage autophagy inhibitor with nanomolar potency and significant antitumor efficacy as a single agent against HGSOC in vivo This class of compounds holds promise as a potential, novel chemotherapeutic and demonstrates the effectiveness of targeting the autophagic pathway as a viable strategy for combating ovarian cancer. Mol Cancer Ther; 17(10); 2123-35. ©2018 AACR.

Identification of compound CA-5f as a novel late-stage autophagy inhibitor with potent anti-tumor effect against non-small cell lung cancer

ABSTRACTCurrently, particular focus is placed on the implication of autophagy in a variety of human diseases, including cancer. Discovery of small-molecule modulators of autophagy as well as their potential use as anti-cancer therapeutic agents would be of great significance. To this end, a series of curcumin analogs previously synthesized in our laboratory were screened. Among these compounds, (3E,5E)-3-(3,4-dimethoxybenzylidene)-5-[(1H-indol-3-yl)methylene]-1-methylpiperidin-4-one (CA-5f) was identified as a potent late-stage macroautophagy/autophagy inhibitor via inhibiting autophagosome-lysosome fusion. We found that CA-5f neither impaired the hydrolytic function nor the quantity of lysosomes. Use of an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic screen in combination with bioinformatics analysis suggested that treatment of human umbilical vein endothelial cells (HUVECs) with CA-5f for 1 h suppressed the levels of cytoskeletal proteins and membrane traffic proteins. Subsequent studies showed that CA-5f exhibited strong cytotoxicity against A549 non-small cell lung cancer (NSCLC) cells, but low cytotoxicity to normal human umbilical vein endothelial cells (HUVECs), by increasing mitochondrial-derived reactive oxygen species (ROS) production. Moreover, CA-5f effectively suppressed the growth of A549 lung cancer xenograft as a single agent with an excellent tolerance in vivo. Results from western blot, immunofluorescence, and TdT-mediated dUTP nick end labeling (TUNEL) assays showed that CA-5f inhibited autophagic flux, induced apoptosis, and did not affect the level of CTSB (cathepsin B) and CTSD (cathepsin D) in vivo, which were consistent with the in vitro data. Collectively, these results demonstrated that CA-5f is a novel late-stage autophagy inhibitor with potential clinical application for NSCLC therapy.Abbreviations: 3-MA, 3-methyladenine; ANXA5, annexin A5; ATG, autophagy related; CA-5f, (3E,5E)-3-(3,4-dimethoxybenzylidene)-5-[(1H-indol-3-yl)methylene]-1-methylpiperidin-4-one; CQ, chloroquine; CTSB, cathepsin B; CTSD, cathepsin D; DMSO, dimethyl sulfoxide; DNM2, dynamin 2; EBSS, Earle’s balanced salt solution; GFP, green fluorescent protein; HCQ, hydroxyl CQ; HEK293, human embryonic kidney 293; HUVEC, human umbilical vein endothelial cells; LAMP1, lysosomal associated membrane protein 1; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; LDH, lactic acid dehydrogenase; LMO7, LIM domain 7; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; NAC, N-acetyl cysteine; MYO1E, myosin IE; NSCLC, non-small cell lung cancer; PARP1, poly(ADP-ribose) polymerase 1; PI, propidium iodide; RFP, red fluorescent protein; ROS, reactive oxygen species; SQSTM1, sequestosome 1; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

Abstract 2864: Inhibition of the late stages of autophagy overcomes hypoxia-induced chemoresistance and targets leukemic stem cells in acute myeloid leukemia

Abstract Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy which resides within an inherently hypoxic bone marrow (BM) microenvironment. Hypoxia has been linked to chemoresistance, particularly of leukemia stem cells (LSCs) responsible for relapsed disease. We hypothesized that upregulation of autophagy promotes survival and chemoresistance in AML cells (specifically LSCs) within the hypoxic BM. Targeted inhibition of specific stages of autophagy therefore may represent a novel means of eradicating disease. Methods: Human AML cell lines (HEL-Luc, HL60, MOLM13) were treated with cytarabine (AraC) and multiple autophagy inhibitors (Spautin-1, MRT68921, chloroquine (CQ), bafilomycin (BafA1)) under normoxia (21% O2) or hypoxia (1% O2) and evaluated for apoptosis. MOLM13 cells were infected with lentivirus containing shRNAs against LC3B, Atg7 or control. Autophagy was measured using CytoID dye and immunoblotting for LC3 and p62. SCID mice systematically engrafted with HEL-Luc cells were treated with CQ (25 mg/kg ip for 5 days/week for 3 weeks) and evaluated for leukemia burden by bioluminescent imaging and time to morbidity. Irradiated NSG mice were injected with primary AML cells and treated with vehicle or Baf A1 (1mg/kg ip twice a week for 4 weeks). BM was then harvested and transplanted into secondary NSG recipients without additional treatment. Leukemia engraftment in mice was measured by flow cytometry for hCD45. Results: Autophagy was upregulated in multiple human AML cell lines after prolonged exposure to hypoxia as well as in a subset of primary AML samples. Inhibiting the early stages of autophagy pharmacologically with Spautin-1 or MRT68921 or shRNA knockdown of autophagy proteins Atg7 or LC3B had no effect on chemosensitivity under hypoxia. However, inhibiting fusion of the autophagosome with the lysosome (late stages) with either CQ or Baf A1 did overcome hypoxia-induced resistance to AraC. CQ also reduced tumor burden in a systemic xenograft model of AML as measured by bioluminescent imaging and resulted in a slight overall increase in survival (17.5 vs. 19 days, p=0.02). In the in vivo LSC model, Baf A1 had no effect on leukemic burden in the primary recipients; however, mice that received secondary transplants from Baf A1-treated mice demonstrated significantly reduced leukemic burden as compared to the control, consistent with LSC specific targeting. Conclusions: Our results demonstrate that treatment with the late stage autophagy inhibitors (chloroquine, bafilomycin A1) can both overcome hypoxia-induced chemoresistance and preferentially inhibit LSC growth in vivo. Taken together, these data support the development of late stage autophagy inhibitors for the treatment of AML and prevention of relapsed disease. In vivo studies to assess the efficacy of AraC/Baf A1 combination therapy are currently underway. Citation Format: Kaitlyn M. Dykstra, Dirkje W. Hanekamp, Matthew Johnson, Monica L. Guzman, Eunice S. Wang. Inhibition of the late stages of autophagy overcomes hypoxia-induced chemoresistance and targets leukemic stem cells in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2864.

Abstract 2672: Phyllanthusmins induce apoptosis and reduce tumor burden in high grade serous ovarian cancer by late-stage autophagy inhibition

Abstract High grade serous ovarian cancer (HGSOC) is a lethal gynecological malignancy with a need for new therapeutics. Many of the most widely used chemotherapeutic drugs are derived from natural products or their semi-synthetic derivatives. We developed potent synthetic analogues of a class of compounds known as the phyllanthusmins, inspired by natural products isolated from Phyllanthus poilanei Beille. The most potent analogue, PHY34, had the highest potency in HGSOC cell lines in vitro and displayed cytotoxic activity through activation of apoptosis. PHY34 exerts its effects by initially inhibiting autophagy at a late stage in the pathway, involving the disruption of lysosomal function. The autophagy activator, rapamycin, combined with PHY34 eliminated apoptosis, suggesting that autophagy inhibition was required for apoptosis. PHY34 was readily bioavailable through intraperitoneal administration in vivo where it significantly reduced cancer cell lines grown in hollow fibers as well as ovarian tumor burden. We show that PHY34 is a new late-stage autophagy inhibitor with nanomolar potency and significant antitumor efficacy as a single-agent against HGSOC in vivo. This class of compounds holds promise as a potential, novel chemotherapeutic and demonstrates the effectiveness of targeting the autophagic pathway as a viable strategy for combating the disease. Citation Format: Alexandria N. Young, Denisse Herrera, Andrew Huntsman, Daniel D. Lantvit, Melissa A. Korkmaz, Leslie N. Aldrich, A. Douglas Kinghorn, James R. Fuchs, Joanna E. Burdette. Phyllanthusmins induce apoptosis and reduce tumor burden in high grade serous ovarian cancer by late-stage autophagy inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2672.

Abstract 1431: Autophagic degradation of the endoplasmic reticulum leads to reduced phospholipid biosynthesis in mutant isocitrate dehydrogenase 1 gliomas

Abstract Introduction: Cancer cells up-regulate phospholipid biosynthesis in order to meet the demands of uncontrolled cell proliferation. Unusually, mutant isocitrate dehydrogenase 1 (IDHmut) gliomas down-regulate levels of the phospholipid precursors phosphocholine and phosphoethanolamine relative to wild-type IDH1 (IDHwt) gliomas. The goal of this study was therefore to investigate whether the phospholipids phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdE) are also down-regulated in IDHmut gliomas. Methods: Immortalized normal human astrocytes and U87 cells expressing IDHwt or IDHmut enzyme were studied. Steady-state phospholipid levels were measured by 31P-magnetic resonance spectroscopy (MRS) of cell extracts. De novo phospholipid biosynthesis was quantified by 13C-MRS of cells labeled with 56μM [1,2-13C]-choline and [1,2-13C]-ethanolamine. ER area was measured by STORM imaging of calnexin fluorescence. Results: Steady-state PtdCho and PtdE levels and de novo phospholipid biosynthesis were significantly reduced in IDHmut cells relative to IDHwt. Concomitantly, the activities of CTP:PC cytidylyltransferase (CCT) and CTP:PE cytidylyltransferase (ECT), the rate-limiting enzymes for phospholipid biosynthesis, were significantly reduced in IDHmut cells. CCT and ECT are localized to the endoplasmic reticulum (ER), the site of phospholipid biosynthesis. We therefore measured ER area by super-resolution STORM imaging and found a significant reduction in IDHmut cells relative to IDHwt while cell size remained unchanged. Autophagy of the ER (ER-phagy) can regulate ER area and we found that IDHmut cells showed increased expression of FAM134b, the ER-phagy receptor and higher autophagic flux. These results suggest that IDHmut cells undergo ER-phagy, leading to reduced phospholipid biosynthesis. Silencing FAM134b or the autophagy proteins Atg5 and Atg7 restored ER area and phospholipid biosynthesis and abrogated clonogenicity of IDHmut cells, linking ER-phagy to reduced phospholipid biosynthesis and pointing to a therapeutic opportunity. In the absence of specific ER-phagy inhibitors, we used the late-stage autophagy inhibitor chloroquine (CQ) to test proof-of-principle and found that CQ significantly inhibited orthotopic IDHmut glioma growth in vivo. Importantly, we observed ER-phagy and down-regulation of phospholipid levels in IDHmut glioma patient biopsies, highlighting the translational validity of our findings. Conclusions: We find that down-regulation of phospholipid biosynthesis is a unique metabolic outcome of ER-phagy in IDHmut gliomas. While autophagy has been linked to glucose and glutamine metabolism, this study is the first to link autophagy to phospholipid biosynthesis. Importantly, our study identifies a rationale for the development of novel therapies targeting ER-phagy in IDHmut gliomas. Citation Format: Pavithra Viswanath, Russell O. Pieper, Joanna J. Phillips, Sabrina M. Ronen. Autophagic degradation of the endoplasmic reticulum leads to reduced phospholipid biosynthesis in mutant isocitrate dehydrogenase 1 gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1431.

Temporal Pattern and Crosstalk of Necroptosis Markers with Autophagy and Apoptosis Associated Proteins in Ischemic Hippocampus.

Necroptosis, a novel type of programmed cell death, has been recently implicated as a possible mechanism for cerebral ischemia-reperfusion (I/R) injury. We herein studied time-dependent changes of necroptosis markers along with apoptosis- and autophagy-associated proteins in rat hippocampus at 1, 3, 6, 12, 24, and 48h after global cerebral I/R injury. Furthermore, to determine the cross talk between autophagy and necroptosis, we examined the effects of pretreatment with bafilomycin-A1 (Baf-A1), as a late-stage autophagy inhibitor, on necroptosis. Highest levels of receptor-interacting protein 1 and 3 (RIP1 and RIP3), as key mediators of necroptosis, were observed at 24h after reperfusion. Alongside, activity of glutamate dehydrogenase (GLUD1), downstream enzyme of RIP3, was increased. Peak time of necroptosis was subsequent to caspase-3-dependent cell death that peaked at 12h of reperfusion but concurrent with autophagy. Administration of Baf-A1 could attenuate necroptosis, verified by decrease in RIP1 and RIP3 protein levels, as well as GLUD1 activity. However, there was no significant change in caspase-3-dependent cell death. Taken together, our results highlight that global cerebral I/R activates necroptosis that could be triggered by autophagy and interacts reversely with caspase-3-dependent apoptosis.

Lithocholic acid induces endoplasmic reticulum stress, autophagy and mitochondrial dysfunction in human prostate cancer cells.

Lithocholic acid (LCA) is a secondary bile acid that is selectively toxic to human neuroblastoma, breast and prostate cancer cells, whilst sparing normal cells. We previously reported that LCA inhibited cell viability and proliferation and induced apoptosis and necrosis of androgen-dependent LNCaP and androgen-independent PC-3 human prostate cancer cells. In the present study, we investigated the roles of endoplasmic reticulum (ER) stress, autophagy and mitochondrial dysfunction in the toxicity of LCA in PC-3 and autophagy deficient, androgen-independent DU-145 cells. LCA induced ER stress-related proteins, such as CCAAT-enhancer-binding protein homologous protein (CHOP), and the phosphorylation of eukaryotic initiation factor 2-alpha (p-eIF2α) and c-Jun N-terminal kinases (p-JNK) in both cancer cell-types. The p53 upregulated modulator of apoptosis (PUMA) and B cell lymphoma-like protein 11 (BIM) levels were decreased at overtly toxic LCA concentrations, although PUMA levels increased at lower LCA concentrations in both cell lines. LCA induced autophagy-related conversion of microtubule-associated proteins 1A/1B light chain 3B (LC3BI–LC3BII), and autophagy-related protein ATG5 in PC-3 cells, but not in autophagy-deficient DU-145 cells. LCA (>10 µM) increased levels of reactive oxygen species (ROS) concentration-dependently in PC-3 cells, whereas ROS levels were not affected in DU-145 cells. Salubrinal, an inhibitor of eIF2α dephosphorylation and ER stress, reduced LCA-induced CHOP levels slightly in PC-3, but not DU-145 cells. Salubrinal pre-treatment increased the cytotoxicity of LCA in PC-3 and DU-145 cells and resulted in a statistically significant loss of cell viability at normally non-toxic concentrations of LCA. The late-stage autophagy inhibitor bafilomycin A1 exacerbated LCA toxicity at subtoxic LCA concentrations in PC-3 cells. The antioxidant α-tocotrienol strongly inhibited the toxicity of LCA in PC-3 cells, but not in DU-145 cells. Collectively, although LCA induces autophagy and ER stress in PC-3 cells, these processes appear to be initially of protective nature and subsequently consequential to, but not critical for the ROS-mediated mitochondrial dysfunction and cytotoxicity of LCA. The full mechanism of LCA-induced mitochondrial dysfunction and cytotoxicity in the similarly sensitive DU-145 cells remains to be elucidated.

Modulation of Autophagy by a Thioxanthone Decreases the Viability of Melanoma Cells.

(1) Background: Our previous studies unveiled the hit thioxanthone TXA1 as an inhibitor of P-glycoprotein (drug efflux pump) and of human tumor cells growth, namely of melanoma cells. Since TXA1 is structurally similar to lucanthone (an autophagy inhibitor and apoptosis inducer) and to N10-substituted phenoxazines (isosteres of thioxanthones, and autophagy inducers), this study aimed at further assessing its cytotoxic mechanism and evaluating its potential as an autophagy modulator in A375-C5 melanoma cells; (2) Methods: Flow cytometry with propidium iodide (PI) for cell cycle profile analysis; Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, flow cytometry with Annexin V/PI labeling and Western blot for apoptosis analysis were conducted. A pharmacophore approach was used for mapping TXA1 onto pharmacophores for autophagy induction. Autophagy analyses included transmission electron microscopy for visualization of autophagic structures, fluorescence microscopy for observation of monodansylcadaverine (MDC) staining, pattern of LC3 expression in the cells and acridine orange staining, and Western blot for autophagic proteins expression; (3) Results: TXA1 induced autophagy of melanoma cells at the GI50 concentration (3.6 μM) and apoptosis at twice that concentration. Following treatment with TXA1, autophagic structures were observed, together with the accumulation of autophagosomes and the formation of autophagolysosomes. An increase in LC3-II levels was also observed, which was reverted by 3-methyladenine (3-MA) (an early stage autophagy-inhibitor) but further increased by E-64d/pepstatin (late-stage autophagy inhibitors). Finally, 3-MA also reverted the effect of TXA1 in cellular viability; (4) Conclusion: TXA1 decreases the viability of melanoma cells by modulation of autophagy and may, therefore, serve as a lead compound for the development of autophagy modulators with antitumor activity.

Targeting Notch Signaling and Autophagy Increases Cytotoxicity in Glioblastoma Neurospheres.

Glioblastomas are highly aggressive tumors that contain treatment resistant stem-like cells. Therapies targeting developmental pathways such as Notch eliminate many neoplastic glioma cells, including those with stem cell features, but their efficacy can be limited by various mechanisms. One potential avenue for chemotherapeutic resistance is the induction of autophagy, but little is known how it might modulate the response to Notch inhibitors. We used the γ-secretase inhibitor MRK003 to block Notch pathway activity in glioblastoma neurospheres and assessed its effects on autophagy. A dramatic, several fold increase of LC3B-II/LC3B-I autophagy marker was noted on western blots, along with the emergence of punctate LC3B immunostaining in cultured cells. By combining the late stage autophagy inhibitor chloroquine (CQ) with MRK003, a significant induction in apoptosis and reduction in growth was noted as compared to Notch inhibition alone. A similar beneficial effect on inhibition of cloogenicity in soft agar was seen using the combination treatment. These results demonstrated that pharmacological Notch blockade can induce protective autophagy in glioma neurospheres, resulting in chemoresistance, which can be abrogated by combination treatment with autophagy inhibitors.

Pan-Bcl-2 inhibitor Obatoclax is a potent late stage autophagy inhibitor in colorectal cancer cells independent of canonical autophagy signaling.

BackgroundColorectal cancer is the third most common malignancy in humans and novel therapeutic approaches are urgently needed. Autophagy is an evolutionarily highly conserved cellular process by which cells collect unnecessary organelles or misfolded proteins and subsequently degrade them in vesicular structures in order to refuel cells with energy. Dysregulation of the complex autophagy signaling network has been shown to contribute to the onset and progression of cancer in various models. The Bcl-2 family of proteins comprises central regulators of apoptosis signaling and has been linked to processes involved in autophagy. The antiapoptotic members of the Bcl-2 family of proteins have been identified as promising anticancer drug targets and small molecules inhibiting those proteins are in clinical trials.MethodsFlow cytometry and colorimetric assays were used to assess cell growth and cell death. Long term 3D cell culture was used to assess autophagy in a tissue mimicking environment in vitro. RNA interference was applied to modulate autophagy signaling. Immunoblotting and q-RT PCR were used to investigate autophagy signaling. Immunohistochemistry and fluorescence microscopy were used to detect autophagosome formation and autophagy flux.ResultsThis study demonstrates that autophagy inhibition by obatoclax induces cell death in colorectal cancer (CRC) cells in an autophagy prone environment. Here, we demonstrate that pan-Bcl-2 inhibition by obatoclax causes a striking, late stage inhibition of autophagy in CRC cells. In contrast, ABT-737, a Mcl-1 sparing Bcl-2 inhibitor, failed to interfere with autophagy signaling. Accumulation of p62 as well as Light Chain 3 (LC3) was observed in cells treated with obatoclax. Autophagy inhibition caused by obatoclax is further augmented in stressful conditions such as starvation. Furthermore, our data demonstrate that inhibition of autophagy caused by obatoclax is independent of the essential pro-autophagy proteins Beclin-1, Atg7 and Atg12.ConclusionsThe objective of this study was to dissect the contribution of Bcl-2 proteins to autophagy in CRC cells and to explore the potential of Bcl-2 inhibitors for autophagy modulation. Collectively, our data argue for a Beclin-1 independent autophagy inhibition by obatoclax. Based on this study, we recommend the concept of autophagy inhibition as therapeutic strategy for CRC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1929-y) contains supplementary material, which is available to authorized users.

MTR-10PHARMACOLOGICAL NOTCH BLOCKADE IN GLIOMAS INDUCES AUTOPHAGY AND COMBINATION TREATMENT WITH AN AUTOPHAGY INHIBITOR INCREASES TUMOR CELL DEATH

BACKGROUND: Induction of autophagy has been identified as a potential mechanism for chemotherapeutic resistance, and its manipulation has increasingly been explored as a treatment strategy in many cancers. However, there is minimal data on the effects of the Notch pathway on autophagy in cancer, and none in gliomas. We examined if Notch pathway blockade using a gamma-secretase inhibitor (GSI) MRK003 would induce autophagy in glioma neurosphere lines and studied the effects of chloroquine (CQ), a late-stage autophagy inhibitor, in combination with the GSI. METHODS: 500,000 cells from 2 neurosphere lines (JHH520 and HSR-GBM1), maintained in Neural Stem Cell medium with EGF and FGF, were treated with DMSO, 0.5-5 µM levels of MRK003, and/or 10 µM CQ for 24-72 hours. Western blotting was performed to detect alterations in protein levels of LC3B, pAkt, Akt, p62 and cleaved PARP. MTS, Ki-67, Annexin V, cell cycle and soft agar assays were performed to look at the combinatorial effects of MRK003 and CQ. RESULTS: An LC3B-II band, suggesting induction of autophagy, was observed after Notch pathway blockade in a dose dependent manner in the 2 neurosphere lines. Reduction of pAkt was seen in GBM1, suggesting mTORC2 inhibition. Decreased growth (MTS assay both p <0.001), proliferation (Ki-67 assay: both p <0.05), increased apoptosis (increased sub-G1 population: JHH520 p <0.005, HSR-GBM1 p <0.05; increased Annexin V percentage: JHH520 p <0.05, HSR-GBM1 p <0.01, increased cleaved PARP protein expression), and decreased clonogenicity (soft agar assay) were seen after combination MRK003 and CQ treatment compared to DMSO. CONCLUSIONS: Induction of autophagy, possibly through mTORC2 inhibition, occurs after Notch inhibition in glioma neurospheres using a GSI. Inhibition of autophagy using CQ caused increased cytotoxicity. These results suggest a possible resistance mechanism to Notch inhibition in malignant gliomas and pave the way for a new treatment strategy.

Cytoprotective role of autophagy against BH3 mimetic gossypol in ATG5 knockout cells generated by CRISPR-Cas9 endonuclease

Previously, we demonstrated the association between autophagy and gossypol-induced growth inhibition of mutant BRAF melanoma cells. Here, we investigate the role of autophagy in ATG5 knockout cell lines generated by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas-mediated genome editing. The MTT assay revealed that the inhibitory effect of gossypol was weaker on ATG5 knockout cells than that on the wild type (WT) cells. The conversion of non-autophagic LC3-I to autophagic LC3-II and RT-PCR confirmed the functional gene knockout. However, Cyto-ID autophagy assay revealed that gossypol induced ATG5- and LC3-independent autophagy in ATG5 knockout cells. Moreover, gossypol acts as an autophagy inducer in ATG5 knockout cells while blocking the later stages of the autophagy process in WT cells, which was determined by measuring autophagic flux after co-treatment of gossypol with chloroquine (late-stage autophagy inhibitor). On the other hand, inhibition of autophagy with 3-MA or Beclin-1 siRNA caused a partial increase in the sensitivity to gossypol in ATG5 knockout cells, but not in the WT cells. Together, our findings suggest that the resistance to gossypol in ATG5 knockout cells is associated with increased cytoprotective autophagy, independent of ATG5.

Combined efficacy of cediranib and quinacrine in glioma is enhanced by hypoxia and causally linked to autophagic vacuole accumulation.

We have previously reported that the in vivo anti-glioma efficacy of the anti-angiogenic receptor tyrosine kinase inhibitor cediranib is substantially enhanced via combination with the late-stage autophagy inhibitor quinacrine. The current study investigates the role of hypoxia and autophagy in combined cediranib/quinacrine efficacy. EF5 immunostaining revealed a prevalence of hypoxia in mouse intracranial 4C8 glioma, consistent with high-grade glioma. MTS cell viability assays using 4C8 glioma cells revealed that hypoxia potentiated the efficacy of combined cediranib/quinacrine: cell viability reductions induced by 1 µM cediranib +2.5 µM quinacrine were 78±7% (hypoxia) vs. 31±3% (normoxia), p<0.05. Apoptosis was markedly increased for cediranib/quinacrine/hypoxia versus all other groups. Autophagic vacuole biomarker LC3-II increased robustly in response to cediranib, quinacrine, or hypoxia. Combined cediranib/quinacrine increased LC3-II further, with the largest increases occurring with combined cediranib/quinacrine/hypoxia. Early stage autophagy inhibitor 3-MA prevented LC3-II accumulation with combined cediranib/quinacrine/hypoxia and substantially attenuated the associated reduction in cell viability. Combined efficacy of cediranib with bafilomycin A1, another late-stage autophagy inhibitor, was additive but lacked substantial potentiation by hypoxia. Substantially lower LC3-II accumulation was observed with bafilomycin A1 in comparison to quinacrine. Cediranib and quinacrine each strongly inhibited Akt phosphoryation, while bafilomycin A1 had no effect. Our results provide compelling evidence that autophagic vacuole accumulation plays a causal role in the anti-glioma cytotoxic efficacy of combined cediranib/quinacrine. Such accumulation is likely related to stimulation of autophagosome induction by hypoxia, which is prevalent in the glioma tumor microenvironment, as well as Akt signaling inhibition from both cediranib and quinacrine. Quinacrine's unique ability to inhibit both Akt and autophagic vacuole degradation may enhance its ability to drive cytotoxic autophagic vacuole accumulation. These findings provide a rationale for a clinical evaluation of combined cediranib/quinacrine therapy for malignant glioma.

Critical roles for nitric oxide and ERK in the completion of prosurvival autophagy in 4OHTAM-treated estrogen receptor-positive breast cancer cells

Autophagy is a mechanism of tamoxifen (TAM) resistance in ER-positive (ER+) breast cancer cells. In this study, we showed in ER+ MCF7 cells that 4-hydroxytamoxifen (4OHTAM) induced cellular nitric oxide (NO) that negatively regulates cellular superoxide (O2−) and cytotoxicity. 4OHTAM stimulated LC3 lipidation and formation of monodansylcadaverine (MDC)-labeled autophagic vesicles dependent on O2−. Depletion of NO increased O2− and LC3 lipidation, yet reduced formation of MDC-labeled autophagic vesicles. Instead, NO-depleted cells formed remarkably large vacuoles with rims decorated by LC3. The vacuoles were not labeled by MDC or the acidic lysosome-specific fluorescence dye acridine orange (AO). The vacuoles were increased by the late stage autophagy inhibitor chloroquine, which also increased LC3 lipidation. These results suggest NO is required for proper autophagic vesicle formation or maturation at a step after LC3 lipidation. In addition, 4OHTAM induced O2−-dependent activation of ERK, inhibition of which destabilized lysosomes/autolysosomes upon 4OHTAM treatment and together with depletion of NO led to necrotic cell death. These results suggest an essential role for endogenous NO and ERK activation in the completion of pro-survival autophagy.