Potent Autophagy Inhibitor Research Articles

CREBRF is a potent tumor suppressor of glioblastoma by blocking hypoxia-induced autophagy via the CREB3/ATG5 pathway.

Hypoxia induces protective autophagy in advanced glioblastoma cells, and targeting this process may improve the outcome for glioblastoma patients. Recent studies have suggested that the autophagic process is upregulated in glioblastoma cells in response to extensive hypoxia. Here, we describe a novel tumor suppressor in glioblastoma cells, whereby hypoxia downregulated CREBRF expression and acts as a potent inhibitor of autophagy in glioblastoma cells via the CREB3/ATG5 pathway. Our results demonstrate that CREBRF expression negatively correlates with autophagic and HIF-1α levels in different grade gliomas. Given that CREBRF is a negative regulator of CREB3, CREB3 knockdown also repressed hypoxia-induced autophagy in glioblastoma cells invitro. Collectively, our findings provide new insight into the molecular mechanisms underlying hypoxia-induced glioblastoma cell autophagy and indicate that the hypoxia/CREBRF/CREB3/ATG5 pathway plays a central role in malignant glioma progression.

Tumor acidosis enhances cytotoxic effects and autophagy inhibition by salinomycin on cancer cell lines and cancer stem cells.

Sustained autophagy contributes to the metabolic adaptation of cancer cells to hypoxic and acidic microenvironments. Since cells in such environments are resistant to conventional cytotoxic drugs, inhibition of autophagy represents a promising therapeutic strategy in clinical oncology. We previously reported that the efficacy of hydroxychloroquine (HCQ), an autophagy inhibitor under clinical investigation is strongly impaired in acidic tumor environments, due to poor uptake of the drug, a phenomenon widely associated with drug resistance towards many weak bases. In this study we identified salinomycin (SAL) as a potent inhibitor of autophagy and cytotoxic agent effective on several cancer cell lines under conditions of transient and chronic acidosis. Since SAL has been reported to specifically target cancer-stem cells (CSC), we used an established model of breast CSC and CSC derived from breast cancer patients to examine whether this specificity may be associated with autophagy inhibition. We indeed found that CSC-like cells are more sensitive to autophagy inhibition compared to cells not expressing CSC markers. We also report that the ability of SAL to inhibit mammosphere formation from CSC-like cells was dramatically enhanced in acidic conditions. We propose that the development and use of clinically suitable SAL derivatives may result in improved autophagy inhibition in cancer cells and CSC in the acidic tumor microenvironment and lead to clinical benefits.

Antitumor DNA‐Damaging C‐1748 is a New Inhibitor of Autophagy that Triggers Apoptosis in Human Pancreatic Cancer Cell Lines

Despite the enormous progress that has been made over the past decades in diagnosis, treatment and prevention of many types of tumors, survival rates in pancreatic cancer still remain poor. Pancreatic cancer is one of the most malignant and chemoresistant tumors and the profound mechanism supporting these phenomena is the constitutively activated prosurvival autophagy. The antitumor 1‐nitroacridine derivative C‐1748 (9‐(2′‐hydroxyethylamino)‐4‐methyl‐1‐nitroacridine) belongs to a new set of DNA‐damaging agents developed in our laboratory and is currently ready for phase I clinical trials. Here, we have uncovered a new role for C‐1748 as a potent inhibitor of autophagy in four tested pancreatic cancer cell lines: Panc‐1, MiaPaCa‐2, BxPC‐3 and AsPC‐1. C‐1748 treatment at 0.1 – 1 μM significantly reduced the typical hallmarks of autophagy, such as conversion of soluble LC3‐I protein to its lipid‐bound LC3‐II isoform or development of acidic vesicular organelles (AVOs), and importantly shifted pancreatic cancer cells into apoptotic cell death. Co‐treatment with C‐1748 and wortmannin, an early‐stage autophagy inhibitor, had a synergistic effect on autophagy inhibition and potentiated C‐1748‐induced apoptosis. In particular, compared with exposure to C‐1748 alone, the combination treatment increased time‐ and dose‐dependent cytotoxic activity of C‐1748 with the most profound effect observed in BxPC‐3 cells. Moreover, along with decrease in autophagy, co‐treatment with wortmannin significantly elevated dysfunction of mitochondria, as manifested by a drop in mitochondrial membrane potential (ΔΨm), increased caspase‐3 activation, PARP cleavage, DNA fragmentation into apoptotic bodies and accumulation of lipid droplets arising due to the interruption of autophagy. In all studied cell lines, the level of apoptotic cells after dual exposure with C‐1748 and wortmannin increased by about 20% as compared to C‐1748 alone. Surprisingly, simultaneous exposure of pancreatic cancer cells to C‐1748 in combination with the late‐stage autophagy inhibitor, chloroquine, neither significantly increased the cytotoxic activity of C‐1748 nor ameliorated its apoptosis‐inducing potential. The synergistic effect of C‐1748 and wortmannin on the inhibition of autophagy and lack of such effect when C‐1748 was co‐administered with chlroquine, suggests that C‐1748, in addition to its DNA‐damaging properties may also affects early stages of autophagy. Further studies are undergoing to uncover the role of C‐1748 as a new inhibitor of autophagy, which is especially effective against pancreatic cancer.Support or Funding InformationResearch Grant from the Polish Ministry of Science and Higher Education NR 13‐0133‐10/2011 to BB‐M, AS, EA, ZM; DoD‐W81XWH1110795 to AR‐P and UAMS TRI ABCRP funds to AR‐P

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.

MicroRNA-143 enhances chemosensitivity of Quercetin through autophagy inhibition via target GABARAPL1 in gastric cancer cells.

MicroRNAs have emerged as fundamental regulators in gene expression through silencing gene expression at the post-transcriptional and translational levels. In this study, miR-143 expression and biological functions in AGS/MNK28 cell lines was investigated. Results indicated that the expression of miR-143 was significantly down-regulated in cancer tissues and in gastric cancer (GC) cell lines. Target prediction algorithms (Target Scan and miRanda) showed that GABARAPL1 was a potential target gene of miR-143. GABARAPL1, also regarded as autophagy-related protein 8 (Atg8) is a ubiquitin-like protein required for the formation of autophagosomal membranes. Then, several different assays were conducted to detect autophagy in AGS/MNK28 after transfected with miR-143. In the present study, miR-143 was firstly identified as a autophagy inhibitor in GC cells via targeting GABARAPL1. Quercetin is one of the most prominent dietary antioxidants in human diet and lately it is grabbing some serious attention as a potentially powerful cancer fighter. However, the effect of Quercetin was unexpected decreased in GC cells on account of the appearance of Quercetin-induced autophagy. Therefore, applicable autophagy inhibitors might enhance the chemosensitivity of Quercetin. Furthermore, the therapeutic response of Quercetin in the combination of miR-143 was evaluated by MTT, Hochest and western blot, results suggesting that the chemosensitivity of Quercetin was enhanced when in combination with miR-143 in AGS/MNK28 cells. In conclusion, we determined miR-143 as a potent inhibitor of autophagy via targeting GABARAPL1 and miR-143 could improve the efficacy of Quercetin though autophagy inhibition in GC cell lines, thus representing a novel potential therapeutic target for gastric cancer.

Abstract 2902: The effectiveness of autophagy inhibition in sensitizing triple-negative breast cancer cells to chemotherapy

Abstract Introduction: High recurrence rates, drug resistance after initial response to chemotherapy, and overall poor prognosis along with the limited treatment options make triple-negative breast cancers (TNBCs) a major clinical challenge. Autophagy, an evolutionary conserved degradation and recycling process, has been shown to function as an adaptive survival response to chemotherapy. Previous studies have indicated higher expression of autophagy markers in TNBCs compared to other breast cancer subtypes, as well as their dependence on autophagy for survival. Our laboratory has also shown in xenograft models an enhanced effectiveness of chemotherapy for the treatment of TNBC when given in combination with autophagy inhibition (AI). These results support TNBCs as a good candidate for AI to improve efficacy of existing therapeutic regimens. However, currently available agents for AI in cancer patients have limited effectiveness, and development of more potent autophagy inhibitors (AIs) is underway. Objective: Develop and test new tools for more potent AI in vivo. Experimental Design: We are employing in vitro models using TNBC lines MDA-MB-231 and SUM159PT, as well as their derivatives R8 and R75, resistant to Epirubicin (EPI) and other anthracyclines. We are evaluating effects of various AIs, including lysosomotropic agents HCQ and lys05, siRNAs and shRNAs targeting autophagy-related (Atg) proteins, and small molecule inhibitors (under development) of ATG4B protein. In vivo xenograft mouse models of MDA-MB-231 and R8 are being used to evaluate the effects of combinatorial therapy with EPI and AI. Methods: For the assessment of autophagy levels before and after AI we used autophagy flux (degradative completion of autophagy) assays. We evaluated the effects of chemotherapy alone and in combination with AIs on parent and resistant sub-lines by assessing their proliferation. For in vivo studies, TNBC cells are injected subcutaneously in Rag2M mice. Treatment with EPI, AI, or their combination is administered after tumor formation. Treatment efficacy is evaluated by tumor volume measurements; tumors are also assessed for the expression of autophagy markers. Results: Our in vitro experiments showed enhanced cytotoxicity of lys05 compared to HCQ either alone or in combination with EPI. However, the use of lys05 in vivo gives contradictory results and requires further evaluation. AI targeting ATG4B, using shRNA-inducible monoclonal cell lines derived from MDA-MB-231 cells and novel small molecule inhibitors of ATG4B, significantly affected cancer cell proliferation in vitro, and is currently being investigated in vivo. Conclusion: Novel approaches to AI may serve as useful tools to assess the effects of AI in vitro and in vivo. Our preliminary results suggest that more potent AIs may improve the effectiveness of treatment of TNBC. Supported by CIHR GPG102167 and CIHR/AVON OBC127216. Citation Format: Svetlana Bortnik, Suganthi Chittaranjan, Jing Xu, Wieslawa H. Dragowska, Jianghong An, Adrienne Kyle, Nancy E. Go, Lubomir Vezenkov, Courtney Choutka, Amy Leung, Suzana Kovacic, Damien Bosc, Karen Gelmon, Marcel Bally, Steven Jones, Robert Young, Sharon Gorski. The effectiveness of autophagy inhibition in sensitizing triple-negative breast cancer cells to chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2902. doi:10.1158/1538-7445.AM2015-2902

Abstract 5315: Hydroxychloroquine inhibits proliferation and S6 phosphorylation in human renal carcinoma cells

Abstract mTOR inhibitors are used to treat metastatic renal cell cancer (RCC), but most patients eventually become resistant. One possible mechanism for resistance is upregulation of autophagy, a pathway which helps recycle intracellular proteins and promotes cell survival. Hydroxychloroquine (HCQ), a potent autophagy inhibitor used to treat malaria and autoimmune disorders, is currently being studied in the context of cancer treatment. Here, we have investigated the effects of HCQ on three different renal carcinoma derived cell lines. We found that HCQ treatment inhibits RCC cell growth, promotes apoptosis, inhibits mitochondrial oxygen consumption, and increases glycolysis rates. To understand the molecular mechanism behind these effects, we examined various nodes in the mTOR pathway and compared the effects of HCQ with the effects of the mTOR inhibitor RAD001. A key downstream readout of the pathway, phospho-S6 protein, was inhibited by both HCQ and RAD001. However, the upstream kinase, P70S6K, was only inhibited by RAD001 and not HCQ, suggesting that the block by HCQ was downstream of the p70S6K. Treatment with the proteasome inhibitor bortezomib, restored phospho-S6 levels, suggesting that the reduction of phospho S6 is caused by increased degradation of phospho, but not total S6. Surprisingly, depletion of the key autophagy regulator Atg7 did not mimic the effects of HCQ, indicating that HCQ regulates S6 phosphorylation by ATG7-independent mechanism. Our finding suggests that HCQ may be useful in the treatment of renal cell carcinoma. Citation Format: Hyung-Ok Lee, Aladdin Mustafa, Gary R. Hudes, Warren D. Kruger. Hydroxychloroquine inhibits proliferation and S6 phosphorylation in human renal carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5315. doi:10.1158/1538-7445.AM2015-5315

Hydroxychloroquine Destabilizes Phospho-S6 in Human Renal Carcinoma Cells.

mTOR inhibitors are used to treat metastatic renal cell cancer (RCC), but most patients eventually become resistant. One possible mechanism for resistance is upregulation of autophagy, a pathway that helps recycle intracellular proteins and promotes cell survival. Hydroxychloroquine (HCQ), a potent autophagy inhibitor used to treat malaria and autoimmune disorders, is currently being studied in the context of cancer treatment. Here, we have investigated the effects of HCQ on three different renal carcinoma derived cell lines. We found that HCQ treatment inhibits RCC cell growth, promotes apoptosis, inhibits mitochondrial oxygen consumption, and increases rates of glycolysis. To understand the molecular mechanism behind these effects, we examined various nodes in the mTOR pathway and compared the effects of HCQ with the effects of the mTOR inhibitor RAD001. A key downstream readout of the pathway, phospho-S6 protein, was inhibited by both HCQ and RAD001. However, the upstream kinase, P70S6K was only inhibited by RAD001 and not HCQ, suggesting that the block by HCQ was downstream of P70S6K. Treatment with the proteasome inhibitor bortezomib restored phospho-S6 levels, suggesting that the reduction of phospho-S6 is caused by increased degradation of phospho-S6, but not total S6. Surprisingly, treatment with other autophagy inhibitors did not exhibit the same effects. Our findings suggest that HCQ causes the down-regulation of phospho-S6 in RCC cell lines via a novel mechanism that is not shared with other autophagy inhibitors.

Abstract IA25: Autophagy: A potentially druggable resistance mechanism to PI3K/mTOR inhibitors

Abstract Inhibition of PI3K/mTOR signaling in cancer cells activates autophagy. Numerous preclinical models have demonstrated therapy-induced autophagy in advanced cancer promotes tumor cell survival and is a targetable resistance mechanism. Clinical trials involving the lysosomal autophagy inhibitor hydroxychloroquine (HCQ) have been launched for patients with refractory malignancies. Currently there are nearly 30 early phase clinical trials involving HCQ for a variety of malignancies in development or actively accruing patients in the United States and abroad. The results of the first 6 HCQ clinical trials (5 in humans, 1 in dogs) that represent the first deliberate attempt to modulate autophagy therapeutically in patients will be presented. This includes a phase I trial of the mTOR inhibitor temsirolimus, and hydroxychloroquine in patients with advanced melanoma which produced a low rate of grade 3-4 toxicities and a 74 % stable disease rate. The development of novel translatable pharmacodynamic assays to characterize autophagy modulation in peripheral blood cells and tumor tissue using electron microscopy, and newer serum-based approaches based on a proteomics discovery effort will be highlighted. Our efforts to synthesize a more potent second generation autophagy inhibitor Lys05, and our plans to move a Lys05 derivative into the clinic will be discussed. Citation Format: Ravi Amaravadi. Autophagy: A potentially druggable resistance mechanism to PI3K/mTOR inhibitors. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr IA25.

The ferroptosis inducer erastin enhances sensitivity of acute myeloid leukemia cells to chemotherapeutic agents

Acute myeloid leukemia (AML) is the most common type of leukemia in adults. Development of resistance to chemotherapeutic agents is a major hurdle in the effective treatment of patients with AML. The quinazolinone derivative erastin was originally identified in a screen for small molecules that exhibit synthetic lethality with expression of the RAS oncogene. This lethality was subsequently shown to occur by induction of a novel form of cell death termed ferroptosis. In this study we demonstrate that erastin enhances the sensitivity of AML cells to chemotherapeutic agents in an RAS-independent manner. Erastin dose-dependently induced mixed types of cell death associated with ferroptosis, apoptosis, necroptosis, and autophagy in HL-60 cells (AML, NRAS_Q61L), but not Jurkat (acute T-cell leukemia, RAS wild type), THP-1 (AML, NRAS_G12D), K562 (chronic myelogenous leukemia, RAS wild type), or NB-4 (acute promyelocytic leukemia M3, KRAS_A18D) cells. Treatment with ferrostatin-1 (a potent ferroptosis inhibitor) or necrostatin-1 (a potent necroptosis inhibitor), but not with Z-VAD-FMK (a general caspase inhibitor) or chloroquine (a potent autophagy inhibitor), prevented erastin-induced growth inhibition in HL-60 cells. Moreover, inhibition of c-JUN N-terminal kinase and p38, but not of extracellular signal-regulated kinase activation, induced resistance to erastin in HL-60 cells. Importantly, low-dose erastin significantly enhanced the anticancer activity of 2 first-line chemotherapeutic drugs (cytarabine/ara-C and doxorubicin/adriamycin) in HL-60 cells. Collectively, the induction of ferroptosis and necroptosis contributed to erastin-induced growth inhibition and overcame drug resistance in AML cells.

MiRNA-30a-mediated autophagy inhibition sensitizes renal cell carcinoma cells to sorafenib

Chemotherapy-induced autophagy activation often contributes to cancer resistance. MiRNA-30a (miR-30a) is a potent inhibitor of autophagy by downregulating Beclin-1. In this study, we characterized the role of miR-30a in sorafenib-induced activity in renal cell carcinoma (RCC) cells. We found that expression of miR-30a was significantly downregulated in several human RCC tissues and in RCC cell lines. Accordingly, its targeted gene Beclin-1 was upregulated. Sorafenib activated autophagy in RCC cells (786-0 and A489 lines), evidenced by p62 degradation, Beclin-1/autophagy protein 5 (ATG-5) upregulation and light chain (LC)3B-I/-II conversion. Exogenously expressing miR-30a in 786-0 or A489 cells inhibited Beclin-1 expression and enhanced sorafenib-induced cytotoxicity. In contrast, knockdown of miR-30a by introducing antagomiR-30a increased Beclin-1 expression, and inhibited sorafenib-induced cytotoxicity against RCC cells. Autophagy inhibitors, including chloroquine, 3-methyaldenine or Bafliomycin A1, enhanced sorafenib activity, causing substantial cell apoptosis. Meanwhile, knockdown of Beclin-1 or ATG-5 by targeted siRNAs also increased sorafenib-induced cytotoxicity in above RCC cells. These findings indicate that dysregulation of miR-30a in RCC may interfere with the effectiveness of sorafenib-mediated apoptosis by an autophagy-dependent pathway, thus representing a novel potential therapeutic target for RCC.

Hypoxia-induced autophagy contributes to radioresistance via c-Jun-mediated Beclin1 expression in lung cancer cells.

Reduced radiosensitivity of lung cancer cells represents a pivotal obstacle in clinical oncology. The hypoxia-inducible factor (HIF)-1α plays a crucial role in radiosensitivity, but the detailed mechanisms remain elusive. A relationship has been suggested to exist between hypoxia and autophagy recently. In the current study, we studied the effect of hypoxia-induced autophagy on radioresistance in lung cancer cell lines. A549 and H1299 cells were cultured under normoxia or hypoxia, followed by irradiation at dosage ranging from 0 to 8 Gy. Clonogenic assay was performed to calculate surviving fraction. EGFP-LC3 plasmid was stably transfected into cells to monitor autophagic processes. Western blotting was used to evaluate the protein expression levels of HIF-1α, c-Jun, phosphorylated c-Jun, Beclin 1, LC3 and p62. The mRNA levels of Beclin 1 were detected by qRT-PCR. We found that under hypoxia, both A549 and H1299 cells were radio-resistant compared with normoxia. Hypoxia-induced elevated HIF-1α protein expression preferentially triggered autophagy, accompanied by LC3 induction, EGFP-LC3 puncta and p62 degradation. In the meantime, HIF-1α increased downstream c-Jun phosphorylation, which in turn upregulated Beclin 1 mRNA and protein expression. The upregulation of Beclin 1 expression, instead of HIF-1α, could be blocked by SP600125 (a specific inhibitor of c-Jun NH2-terminal kinase), followed by suppression of autophagy. Under hypoxia, combined treatment of irradiation and chloroquine (a potent autophagy inhibitor) significantly decreased the survival potential of lung cancer cells in vitro and in vivo. In conclusion, hypoxia-induced autophagy through evaluating Beclin1 expression may be considered as a target to reverse the radioresistance in cancer cells.

Phase I trial of hydroxychloroquine with dose-intense temozolomide in patients with advanced solid tumors and melanoma

Blocking autophagy with hydroxychloroquine (HCQ) augments cell death associated with alkylating chemotherapy in preclinical models. This phase I study evaluated the maximum tolerated dose (MTD), safety, preliminary activity, pharmacokinetics, and pharmacodynamics of HCQ in combination with dose-intense temozolomide (TMZ) in patients with advanced solid malignancies. Forty patients (73% metastatic melanoma) were treated with oral HCQ 200 to 1200 mg daily with dose-intense oral TMZ 150 mg/m2 daily for 7/14 d. This combination was well tolerated with no recurrent dose-limiting toxicities observed. An MTD was not reached for HCQ and the recommended phase II dose was HCQ 600 mg twice daily combined with dose-intense TMZ. Common toxicities included grade 2 fatigue (55%), anorexia (28%), nausea (48%), constipation (20%), and diarrhea (20%). Partial responses and stable disease were observed in 3/22 (14%) and 6/22 (27%) patients with metastatic melanoma. In the final dose cohort 2/6 patients with refractory BRAF wild-type melanoma had a near complete response, and prolonged stable disease, respectively. A significant accumulation in autophagic vacuoles (AV) in peripheral blood mononuclear cells was observed in response to combined therapy. Population pharmacokinetics (PK) modeling, individual PK simulations, and PK-pharmacodynamics (PD) analysis identified a threshold HCQ peak concentration that predicts therapy-associated AV accumulation. This study indicates that the combination of high-dose HCQ and dose-intense TMZ is safe and tolerable, and is associated with autophagy modulation in patients. Prolonged stable disease and responses suggest antitumor activity in melanoma patients, warranting further studies of this combination, or combinations of more potent autophagy inhibitors and chemotherapy in melanoma.

Abstract 298: Effects of Long-Term Autophagy Inhibition With Chloroquine on the Development and Progression of Experimental Abdominal Aortic Aneurysms

Abdominal aortic aneurysms (AAA) are a significant cause of worldwide mortality and morbidity. Although several mechanisms have been put forth, the central determinants of progressive AAA development appear to involve heightened vascular smooth muscle cell (VSMC) apoptosis, with increased degradation of the extracellular matrix, in the setting of chronic inflammation. Recent evidence suggests that autophagy genes are deregulated in human AAA and that autophagy may modulate VSMC fate and plasticity. We sought to determine if long-term autophagy inhibition with chloroquine attenuates AAA progression and mortality in an experimental model of AAA. ApoE-/- mice were implanted with miniosmotic pumps containing either saline (n=15) or angiotensin II (Ang II; 1.44 mg/kg/day) (n=27). Mice from each group were subdivided and administered either the potent autophagy inhibitor chloroquine (50 mg/kg/day, i.p.) or saline for 8 weeks. Hemodynamics, abdominal aortic diameter (by ultrasound) and mortality were monitored. Necroscopy evaluations, histological and immunohistological staining for markers of autophagy, apoptosis and inflammation were conducted. Kaplan-Meier estimates were compared between the four groups over the 8-week follow-up. Ang II infusion was associated with a cumulative mortality of 38%. Long-term chloroquine treatment did not alter Ang II-associated mortality (43%, p>0.05 vs. saline-treated group). When rupture-related AAA mortality was specifically evaluated, no difference between the Ang II groups was observed. Maximal luminal diameter of the abdominal aorta was similar between the Ang II-infused groups (Saline: 1.88±0.13 mm vs. Chloroquine: 1.97± 0.17 mm, p>0.05). Ang II infusion also significantly increased MAP, an effect that was unaffected by chloroquine treatment (Saline: 140±16 mmHg vs. Chloroquine: 135±11 mmHg, p>0.05). Long term chloroquine administration does not alter the development and/or natural history of experimental AAA. These data suggest that a global and non-specific autophagy inhibition strategy is unlikely to represent a target for intervention for AAA. Whether cell- and/or gene-specific targeting of the autophagy machinery improves AAA outcomes remains to be determined.

Development of potent autophagy inhibitors that sensitize oncogenic BRAF V600E mutant melanoma tumor cells to vemurafenib

Autophagy is a dynamic cell survival mechanism by which a double-membrane vesicle, or autophagosome, sequesters portions of the cytosol for delivery to the lysosome for recycling. This process can be inhibited using the antimalarial agent chloroquine (CQ), which impairs lysosomal function and prevents autophagosome turnover. Despite its activity, CQ is a relatively inadequate inhibitor that requires high concentrations to disrupt autophagy, highlighting the need for improved small molecules. To address this, we screened a panel of antimalarial agents for autophagy inhibition and chemically synthesized a novel series of acridine and tetrahydroacridine derivatives. Structure-activity relationship studies of the acridine ring led to the discovery of VATG-027 as a potent autophagy inhibitor with a high cytotoxicity profile. In contrast, the tetrahydroacridine VATG-032 showed remarkably little cytotoxicity while still maintaining autophagy inhibition activity, suggesting that both compounds act as autophagy inhibitors with differential effects on cell viability. Further, knockdown of autophagy-related genes showed no effect on cell viability, demonstrating that the ability to inhibit autophagy is separate from the compound cytotoxicity profiles. Next, we determined that both inhibitors function through lysosomal deacidification mechanisms and ultimately disrupt autophagosome turnover. To evaluate the genetic context in which these lysosomotropic inhibitors may be effective, they were tested in patient-derived melanoma cell lines driven by oncogenic BRAF (v-raf murine sarcoma viral oncogene homolog B). We discovered that both inhibitors sensitized melanoma cells to the BRAF V600E inhibitor vemurafenib. Overall, these autophagy inhibitors provide a means to effectively block autophagy and have the potential to sensitize mutant BRAF melanomas to first-line therapies.

Abstract A15: HDAC6 inhibitor C1A abrogates the recruitment of the autophagic machinery and synergizes with proteasome, src kinase, and PI3K-mTOR inhibition.

Abstract The histone deacetylase (HDAC) family of proteins plays a profound role in transcriptional silencing and intracellular protein function, and HDAC inhibitors have shown promise for the treatment of human malignancies. HDAC6 activity is altered in tumorigenesis and its selective inhibition is likely to cause less toxicity compared to broad spectrum inhibitors. An important function of HDAC6 is the processing of misfolded proteins within cells during autophagy and formation of aggresomes. Prosurvival autophagy may represent a major impediment to successful cancer therapy and more potent and specific inhibitors of autophagy are needed. This study evaluated whether targeting HDAC6 could be used to impair autophagy and restore chemosensitivity. Treatment of colon cancer HCT-116 cells and osteosarcoma U2OS-LC3-GFP cells with HDAC6 inhibitor C1A for 24 hours (or tubastatin A, another HDAC6 inhibitor) caused a dose dependent increase in expression of lipid associated LC3 II, suggesting autophagosome formation or blockage. Using a pH-sensitive, double tagged mCherry-GFP-LC3 reporter, we could further demonstrate impairment of autophagosome-lysosome fusion. We hypothesized that the effect of autophagy tool compounds will be modulated by C1A. Increased LC3 II/p62 expression induced by chloroquine, 3-MA, bortezomib or BEZ-235 were inhibited by C1A (also seen with tubastatin A). Combination of C1A with bortezomib or BEZ-235 induced a greater proportion of cells undergoing apoptosis as depicted by an increase of caspase 3/7 activity in HCT-116 cells. Treatment with C1A (or tubastatin A) also impaired the formation of LC3 II following treatment with src inhibitor, dasatinib (or PP2, another src inhibitor but not PP3, a negative control compound); synergy as assessed by caspase 3/7 activity was seen in lung cancer A549 cells. Based on combination index values, we demonstrated that C1A synergistically enhanced proteasome, src kinase and PI3K-mTOR growth inhibition. In vivo, the anti-tumor activity of C1A in combination with bortezomib or BEZ-235 was confirmed in HCT-116 tumor bearing mice. Tumor growth was significantly delayed in animals treated with combination therapy. A similar observation was seen in A549 tumor bearing mice treated with C1A in combination with dasatinib. The efficacy of the combination C1A and BEZ-235 was also predicted as early as 48 h with [18F]fluorothymidine positron emission tomography ([18F]FLT-PET) in HCT-116 tumor bearing mice. These results provide a rationale for combination therapy with HDAC6 inhibitors and chemotherapy associated with pro-survival autophagy in the treatment of solid tumors. Imaging of cell proliferation with [18F]FLT-PET detected the mechanistic validity of this combination treatment approach and, thus, warrants further investigation in pre-clinical models and patients. This work was supported in part by Cancer Research U.K.-Engineering and Physical Sciences Research Council Grant C2536/A10337. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A15. Citation Format: Maciej Kaliszczak, Olivier E. Pardo, Michael J. Seckl, Eric O. Aboagye. HDAC6 inhibitor C1A abrogates the recruitment of the autophagic machinery and synergizes with proteasome, src kinase, and PI3K-mTOR inhibition. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A15.

Neuregulin-1 Attenuates Doxorubicin-induced Autophagy in Neonatal Rat Cardiomyocytes

Recombinant human neuregulin-1 (rhNRG-1) improves cardiac function in animal models of doxorubicin (DOX)-induced cardiomyopathy, but the underlying mechanism remains largely unknown. Here, we confirm a role for rhNRG-1 in attenuating DOX-induced autophagy and define the signaling pathways through which it mediates some of its effects. Neonatal rat ventricular myocytes were subjected to different treatments both to induce autophagy and to determine the effects of rhNRG-1 on the process. The rhNRG-1 inhibited DOX-induced autophagy, reduced reactive oxygen species production and increased protein expression of Bcl-2, effects that were recapitulated when the cells were treated with the antioxidant N-acetylcysteine. These effects were blocked by the phosphatidylinositol 3-kinase inhibitor LY294002, pointing to the involvement of the Akt pathway in mediating the process. Inhibition of Bcl-2 expression with small interfering RNA silencing also inhibited rhNRG-1's ability to attenuate DOX-induced autophagy. The rhNRG-1 is a potent inhibitor of DOX-induced autophagy and multiple signaling pathways, including Akt and activation of reactive oxygen species, play important roles in the anti-autophagy effect. The rhNRG-1 is a novel drug that may be effectively therapeutically in protecting further damage in DOX-induced damaged myocardium.

Abstract 2041: Inhibition of autophagy in melanoma: using cell lines to model chloroquine sensitivity.

Abstract Metastatic melanoma offers an ongoing clinical challenge. According to the American Cancer Society, the 5-year survival rate in 2012 for stage IV melanoma is only about 15% even with therapies. To improve outcomes, it is important to investigate new pathways involved in the regulation of metastasis as possible targets for prevention or treatment, such as autophagy. Autophagy (self-eating pathway) destroys a cell's damaged proteins and organelles through lysosomal degradation and has been shown to play roles in a wide variety of normal physiological processes including energy metabolism, nutrient or stress responses, growth regulation, and aging. The role of dysregulated autophagy in melanoma has not been well characterized. Preliminary research found cutaneous malignant melanoma cells display high levels of autophagy suggesting that this cellular process may provide an active metabolic state for invasive and metastatic melanoma. Chloroquine (CLQ), a classic antimalarial agent, is a potent inhibitor of autophagy and has been used safely in human patients for decades. This study is designed to dissect the role of autophagy in melanoma using melanoma cell lines (e.g. SK-Mel 19, 103, and 147), and primary melanocytes as controls to determine whether these cell types respond to CLQ as measured by cell viability . Similar to pancreatic and prostate cancer cell lines, our preliminary results indicate that metastatic melanoma cells require autophagy for growth and show high sensitivity to CLQ at doses as low as 20uM which resulted in greater than 60% decreases in cell viability. These findings suggest that metastatic melanoma cell lines rely on lysosomal degradation to a greater extent than what has been reported for other cell types. In addition, both N-ras mutation and androgen deprivation mitigate the effects of CLQ on melanoma cells. Ongoing experiments will confirm the expression of autophagy related genes, as well as imaging to determine the rate of autophagy in these cell lines. Finally, we will introduce stripped hormonal factors including vitamin D and estrogen, to evaluate whether this addition will progressively restore the original chloroquine sensitive phenotype. Findings from this work have the potential to be immediately translatable to patients as CLQ is already approved for therapeutic use in other human diseases. Citation Format: Kirsten A. White, Salina M. Torres, Natalia J. Gurule, Marianne Berwick, Chien-An A. Hu. Inhibition of autophagy in melanoma: using cell lines to model chloroquine sensitivity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2041. doi:10.1158/1538-7445.AM2013-2041

Abstract 1683: Autophagy inhibition for cancer therapy.

Abstract In tumor but not normal cells, the stress-inducible heat shock protein 70 (HSP70) is abundantly expressed and becomes incorporated into lysosome membranes, where it serves to stabilize and protect lysosome function. Inhibition of HSP70 thus results in defective lysosome function, along with impaired autophagy. We previously reported that the HSP70 inhibitor phenylethynesulfonamide (PES) is a potent and effective autophagy inhibitor (1). PES shows marked cytotoxicity to tumor cells, but minimal effects in normal, non-transformed cells. We previously reported that PES binds to both HSP70 and to the constitutively-expressed family member HSC70; these are critical co-chaperones for HSP90, and we previously reported that treatment of tumor cells with PES leads to decreased function of HSP90 client proteins like HER2, AKT and CDK4 (2). We have recently performed a preliminary structure-activity relationship for PES. These studies revealed a novel PES analogue that we call PES-Cl, which shows 10-fold decreased IC50 for tumor cells, minimal cytotoxicity to normal cells, and a greatly enhanced ability to inhibit autophagy. In vitro we show that PES-Cl is cytotoxic to melanoma cells, including those with both intrinsic and acquired resistance to BRAF inhibitors, but shows minimal cytotoxicity to primary melanocytes. In a pre-clinical model for B-cell lymphoma (Eu-myc transgenic mouse), we show that PES-Cl demonstrates significant ability to extend the life of mice (p=0.000006), with no evidence for liver pathology or toxicity. We report that PES binds to the substrate-binding domain of HSP70, and requires the C-terminal helical ‘lid’ of this protein (amino acids 573-616) in order to bind. Using molecular modeling and in silico docking, we have identified a candidate binding site for PES in HSP70, and we identify point mutants that fail to interact with this compound. Our cell cycle analyses of PES-Cl-treated cells show that this compound induces G2/M arrest. Interestingly, we also show that this HSP70 inhibitor impairs the activity of the Anaphase Promoting Complex/Cyclosome (APC/C) in cell-free extracts. PES-Cl is thus a promising new HSP70 inhibitor that binds to the C-terminal ‘lid’ of HSP70, and multiple mechanisms of action: inhibition of autophagy, inhibition of HSP90 function, and inhibition of the Anaphase Promoting Complex/Cyclosome (APC/C). 1. Leu et al, Molecular Cell, 6(1):15-27, 2009 2. Leu et al, Molecular Cancer Research, 9(7):936-47, 2011 Citation Format: Gregor Balaburski, Julia I-Ju Leu, Neil Beeharry, Seth Hayik, Mark D. Andrake, Gao Zhang, Meenhard Herlyn, Jessie Villanueva, Roland L. Dunbrack, Tim Yen, Donna L. George, Maureen E. Murphy. Autophagy inhibition for cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1683. doi:10.1158/1538-7445.AM2013-1683

Therapeutic implications of autophagy modulation in prostate cancer

Autophagy is an evolutionarily conserved process implicated in degradation and recycling of proteins, carbohydrates, and lipids, in order to adapt cell metabolism to energy needs and lead to elimination of exhausted or unwanted organelles. This process is particularly relevant for removal of non-functional mitochondria, important source of reactive oxygen species (ROS) which can damage lipids, proteins, and DNA. When autophagy occurs, double-membrane vesicles named autophagosomes sequester cytosolic substrates initiating their lysosomic degradation (1). The role of autophagy in tumorigenesis is known to be much complex and context-dependent, leading to both cancer suppression and cancer progression. It may mediate tumor suppression possibly by removing damaged organelles which generate ROS and promote genotoxic stress. By contrast, autophagy may provide a survival advantage to tumor cells by helping to overcome the metabolic stress inherently present in the tumor microenvironment (2). Therefore, accurate modulation of autophagy appears to be a new paradigm in cancer therapeutics, opening new possibilities towards the identification of novel therapeutic approaches. For prostate cancer treatment, it is well known that androgen removal induces apoptosis and tumor regression being the rational of hormone ablation therapy (3). Therefore, the commonly used treatment of advanced prostate cancer is the inhibition of androgen production and/or androgen function. Although most patients (up to 50%) respond initially (4), the therapeutic effects often last only for a short period. In fact, cancer cells gradually develop towards an androgen-independent phenotype and survive by several mechanisms, including autophagy. Remarkably, two recent studies published in August 2012 by Kaini et al. and Bennett et al. demonstrate that the pharmacological autophagy blockade in the presence of androgen deprivation causes 40% cell death of epithelial prostate cancer LNCaP cells as compared to control conditions. Such effect has been obtained both through chloroquine (a well-known late-stage autophagy inhibitor, already tested in humans for different disorders) (5) and 3-methyladenine (an early-stage autophagy inhibitor) (6). Although in vivo studies on autophagy inhibition combined with androgen deprivation are still lacking, these new results sustain the potential therapeutic value of combining autophagy-modulation with conventional hormonal therapy in vivo. We have recently published that autophagic inhibition is also able to potentiate the apoptotic effect of tumor necrosis factor (TNF)-α (7). Increased apoptosis after TNF-α/3-methyladenine combined treatment was associated with reduced levels of the apoptotic inhibitor named c-Flip (8-10). Such result, while highlighting the importance of the autophagy inhibitor as potential therapeutic agent, further confirms the cross-talk existing between autophagy and apoptosis, through a shared mediator. Altogether, these considerations make clear that targeting autophagy may provide new opportunities for prostate cancer treatment, while there is an urgent need of novel potent and specific inhibitors of autophagy. High-throughput screening of chemical libraries as well as synthesis of new autophagy inhibitors impairing tumor growth in vivo are ongoing with encouraging results (11). Another promising field is now opening and arises from a deep analysis of the pathogens proteome, since some viruses have evolved strategies to interfere, escape or even exploit the autophagic machinery. We believe that these new approaches may help in rational drug design leading to new effective ways to modulate autophagy, ultimately potentiating cancer treatment.