Autophagy Inhibition In Combination Research Articles

Chloroquine Enhances Rapamycin-induced Apoptosis in MG63 Cells

We previously showed that the use of autophagy inhibitors in combination with chemotherapy can enhance anticancer effects in sarcoma cell lines. In this study, we investigated the combined effect of the autophagy inhibitor chloroquine and the mTOR inhibitor rapamycin on MG63 osteosarcoma cells. Effects of chloroquine and/or rapamycin on cell proliferation were assessed by WST-1 assays. Effects of chloroquine and/or rapamycin on the mTOR pathway components, autophagy, and apoptosis were investigated by western blot, flow cytometry, and fluorescence microscopy using immunocytochemical staining of LC3 and Annexin V-FITC/propidium iodide. Rapamycin suppressed cell growth and inhibited the mTOR pathway. Rapamycin promoted autophagy by blocking the mTOR pathway, and chloroquine enhanced apoptosis by blocking autophagy. Chloroquine enhances the effects of rapamycin in inducing apoptosis via autophagy inhibition in MG63 cells. Thus, the combined therapy of chloroquine and rapamycin may be a potent treatment for osteosarcoma.

Role of Enhanced Autophagy in Resistance of FLT3-ITD AML Stem Cells to FLT3 TKI Treatment

The FLT3-ITD mutation occurs in 25-30% of AML patients, and is associated with increased relapse rate and poor survival. FLT3 tyrosine kinase inhibitors (TKI) have shown encouraging results, but responses are not sustained in most patients. Therefore there is considerable interest in understanding mechanism contributing to resistance and preservation of FLT3-ITD AML stem cells after TKI treatment. Autophagy is an important cellular response that maintains cell survival under stress. Here we investigated the regulation of autophagy in FLT3-ITD+ AML cells, and its role in resistance to FLT3 TKI treatment.Expression of a FLT3-ITD construct in FLT3-ITD negative OCI-AML3 cells using lentiviral vectors resulted in increased autophagic flux compared with wild-type FLT3 or non-transduced controls. However, treatment of FLT3-ITD+ MV4-11 and Molm13 AML cells, or primary FLT3-ITD+ CD34+ cells, with the potent FLT3 TKI AC220 (Quizartinib) did not significantly alter autophagic flux. These results suggest that FLT3-ITD expression increases autophagy but in a kinase-independent manner.MV4-11 and Molm13 cells were transduced with the GFP-LC3-RFP-LC3 ΔG construct (Kaizuka T, et al,2016) to measure autophagic flux. Autophagyhigh AML cells showed lower growth rate compared with Autophagylow cells (0.65, p=0.025), and reduced sensitivity to inhibition by AC220 (0.49 vs 0.71, p=0.003). Treatment of MV4-11 and Molm13 cells with the potent autophagy inhibitor Lys05 in combination with AC220 resulted in significantly enhanced inhibition of AML cell growth (MV4-11: 0.83 vs 0.54, p<0.001; Molm13: 0.58 vs 0.43, p=0.02) and increased induction of apoptosis (MV4-11: 0.81 vs 0.54, p<0.001; Molm13: 0.73 vs 0.49, p=0.024) compared to AC220 alone. We further show that inhibition of autophagy by shRNA-mediated knockdown of the critical autophagy gene ATG5 enhanced sensitivity of AML cells to AC220 treatment (MV4-11: 0.91 vs 0.65, p=0.005; Molm13: 0.88 vs 0.64, p=0.004). Treatment with Lys05 also inhibited autophagic flux in primary FLT3-ITD+ AML CD34+ stem/progenitor cells and resulted in significantly increased inhibition of cell cycle arrest (G0 stage: 40.3% vs 61.8%), increased myeloid differentiation (CD11b: 1 vs 1.24, p=0.009; CD14: 1 vs 1.21, p=0.0024), and increased cell death when combined with AC220 (0.58% vs 0.78%, p=0.0245). These results indicate that increased autophagy in FLT3-ITD+ AML cells is associated with TKI resistance, and that autophagy inhibition enhances sensitivity of FLT3-ITD+ AML cells to TKI treatment.Induction of autophagy by starvation or mTOR inhibitor treatment in MOLM13 and MV4-11 cells was associated with reduced p53 expression, whereas autophagy inhibition by chloroquine or SAR405, a PIK3C3/Vps34 inhibitor, was associated with increased p53 expression. Starvation-mediated autophagy in FLT3-ITD+ AML cells was significantly increased upon p53 knockdown, suggesting that p53 has an inhibitory effect on autophagy. Enhanced inhibition of FLT3-ITD+ AML cell growth by autophagy inhibition in combination with AC220 treatment was prevented by shRNA-mediated knockdown of p53, suggesting that effects of autophagy inhibition on FLT3-ITD+ AML cell growth were p53 dependent.We conclude that FLT3-ITD expression enhances autophagic flux in FLT3-ITD+ AML cells in a kinase-independent manner, and that increased autophagy contributes to TKI resistance of FLT3-ITD+ AML cells. We show that autophagy inhibition can enhance sensitivity of FLT3-ITD+ AML stem cells to TKI treatment. Finally, we demonstrate important roles for autophagy in regulating p53 levels, and conversely for p53 in regulating autophagy and the effects of autophagy inhibition in FLT3-ITD+ AML cells. These studies support further development of strategies for targeting of autophagy and related pathways to enhance efficacy of TKI treatment in eliminating AML stem cells. DisclosuresNo relevant conflicts of interest to declare.

Inhibiting the Core Autophagy Enzyme ATG4B with Novel Drugs Sensitizes Resistant Leukemic Stem/Progenitor Cells to Standard Targeted Therapy

Leukemic stem cell (LSC) persistence is a major cause of therapy failure and relapse in patients, thus warranting further investigations into the cells' molecular properties to aid in meaningful clinical decisions. Chronic myeloid leukemia (CML) is a clear example and excellent study model with rare, propagating LSCs that are not eradicated by BCR-ABL1-directed tyrosine kinase inhibitor (TKI) monotherapy such as Imatinib Mesylate (IM), since the LSCs do not exclusively rely on BCR-ABL1 for their survival.We and others have shown that the persistence of primitive leukemic cells is mediated by macroautophagy (autophagy), a catabolic cellular recycling process. In particular, we discovered that transcript and protein expressions of all ATG4 family members, including ATG4A, B, C, and D, are significantly increased in CD34+ CML cells compared to CD34+ normal bone marrow (BM) cells. ATG4B expression is also significantly higher in pre-treatment CD34+ CML cells from IM-nonresponders vs. IM-responders, including LSCs. Moreover, we revealed that stable suppression of ATG4B significantly suppressed autophagy, impaired survival of IM-nonresponder stem/progenitor cells and sensitized leukemic cells to TKI treatment. Thus, we identified ATG4B as a critical therapeutic target in CML (Rothe et al., Blood, 2014).To further explore whether targeting of ATG4B would be a viable strategy in the successful treatment of various leukemia, two lead ATG4B inhibitors were recently developed for pre-clinical proof-of-concept studies. Compound 4-28, a styrylquinoline, was identified by in silico screening and high content cell-based screening. Its structure-activity relationship (SAR)-based optimization led to a more stable and potent compound, LV-320. LV-320 was further evaluated by Microscale Thermophoresis and showed consistent Kd values for binding to the ATG4B enzyme (Kd=16±1 μM). LV-320 can inhibit autophagic flux, shows excellent tolerability and a good PK profile. It is also characterised as a non-competitive inhibitor of ATG4B and displays similar potency against ATG4A. Interestingly, inhibition of ATG4B with compound 4-28 decreased viability by 40-60% and increased apoptosis by 30-40% in different BCR-ABL1+ leukemic cell lines upon serum-deprivation as compared to the same cells without treatment of 4-28 (p<0.05). 4-28 treatment also efficiently inhibited autophagic flux in these cells as shown by Western blot analysis of LC3-II/I and p62 accumulation. In addition, compound 4-28 was able to inhibit the clonal growth of patient-derived CML stem/progenitor cells from IM-nonresponders, in particular when combined with various TKIs compared to single agents (20 vs. 50%, p<0.05). However, this combination approach also showed slight toxic effects on healthy BM cells.We then tested the more stable and potent compound LV-320 with superior results. Treatment of several drug-resistant and mutated CML and aggressive BCR-ABL1+ B-ALL cell lines with LV-320 increased apoptosis up to 90% compared to controls and reached almost 100% when combined with various TKIs (p<0.05). Moreover, LV-320 sensitized IM-nonresponder stem and progenitor cells to TKIs in colony-forming short-term and long-term cell assays as compared to single agents (10 % vs. 40 %, p<0.05), but was not toxic to primitive BM cells from healthy donors (up to 10µM). Mechanistically, we found that LV-320 effectively inhibited autophagic flux in leukemic cells: Confocal microscopy demonstrated an increase in LC3-II-positive punctae (autophagosomes) and the presence of yellow punctae (blocked autophagosome-lysosome fusion) when leukemic cells where transduced with a mRFP-GFP-LC3 construct and treated with LV-320 or a combination of LV-320 and TKIs. Drug interaction analysis further indicated synergy between LV-320 plus IM (CI value ≤ 0.9). A novel in vivo model is currently being investigated to validate our proof-of-concept. Together, our results suggest that targeting of ATG4B with novel autophagy inhibitors in combination with TKIs may be able to circumvent drug resistance in CML and possibly other aggressive leukemia. DisclosuresNo relevant conflicts of interest to declare.

Cyclin D1-CDK4 activity drives sensitivity to bortezomib in mantle cell lymphoma by blocking autophagy-mediated proteolysis of NOXA

BackgroundMantle cell lymphoma (MCL) is an aggressive B-non-Hodgkin lymphoma with generally poor outcome. MCL is characterized by an aberrantly high cyclin D1-driven CDK4 activity. New molecular targeted therapies such as inhibitors of the ubiquitin-proteasome system (UPS) have shown promising results in preclinical studies and MCL patients. Our previous research revealed stabilization of the short-lived pro-apoptotic NOXA as a critical determinant for sensitivity to these inhibitors. It is currently unclear how cyclin D1 overexpression and aberrant CDK4 activity affect NOXA stabilization and treatment efficacy of UPS inhibitors in MCL.MethodsThe effect of cyclin D1-driven CDK4 activity on response of MCL cell lines and primary cells to proteasome inhibitor treatment was investigated using survival assays (Flow cytometry, AnnexinV/PI) and Western blot analysis of NOXA protein. Half-life of NOXA protein was determined by cycloheximide treatment and subsequent Western blot analysis. The role of autophagy was analyzed by LC3-II protein expression and autophagolysosome detection. Furthermore, silencing of autophagy-related genes was performed using siRNA and MCL cells were treated with autophagy inhibitors in combination with proteasome and CDK4 inhibition.ResultsIn this study, we show that proteasome inhibitor-mediated cell death in MCL depends on cyclin D1-driven CDK4 activity. Inhibition of cyclin D1/CDK4 activity significantly reduced proteasome inhibitor-mediated stabilization of NOXA protein, mainly driven by an autophagy-mediated proteolysis. Bortezomib-induced cell death was significantly potentiated by compounds that interfere with autophagosomal function. Combined treatment with bortezomib and autophagy inhibitors enhanced NOXA stability leading to super-induction of NOXA protein. In addition to established autophagy modulators, we identified the fatty acid synthase inhibitor orlistat to be an efficient autophagy inhibitor when used in combination with bortezomib. Accordingly, this combination synergistically induced apoptosis both in MCL cell lines and in patient samples.ConclusionOur data demonstrate that CDK4 activity in MCL is critical for NOXA stabilization upon treatment with UPS inhibitors allowing preferential induction of cell death in cyclin D transformed cells. Under UPS blocked conditions, autophagy appears as the critical regulator of NOXA induction. Therefore, inhibitors of autophagy are promising candidates to increase the activity of proteasome inhibitors in MCL.

Abstract 1335: Withaferin A induces nonprotective autophagy in a STK11-independent manner and mediates breast cancer inhibition via energetic impairment

Abstract Background and Aim: Cancer cells undergo cytoprotective autophagy and evade chemotherapy therefore many clinical trials are investigating the efficacy of autophagy inhibition in combination with chemotherapy. At the functional level, autophagic process can be cytoprotective, cytotoxic, cytostatic or nonprotective. We investigated strategies to convert cellular autophagic response to non-protective autophagy which does not interfere with therapeutic regimens exploiting bioactive molecules. Methods: Utilizing in vivo xenograft models, we established that Withaferin A (WA), a bioactive molecule from Withania Somnifera inhibits breast tumorigenesis. Autophagy studies were conducted utilizing immunoblot, RT-PCR, and immunofluorescence analyses for autophagy markers, transmission electron microscopy and confocal imaging. The fusion of autophagosome and lysosome was examined by using GFP-LC3/LysoTracker-red and GFP-LC3/mCherryRAB7A. Protein degradation activity of lysosomes and ATP levels were examined by DQ-BSA assay, Cathepsin activity and quantitative ATP assay. Results: WA inhibited growth and induced apoptosis in breast cancer cells resulting in inhibition of breast carcinogenesis in vivo. Although WA increased tumor suppressor LKB1 which is known to be involved in autophagy, WA-mediated increased cleavage of Light Chain 3 type II (LC3-II) and punctated LC3-II staining was LKB1-independent. The redistribution of EGFP-LC3 from cytosol to autophagosome indicated increased formation of autophagosomes in WA-treated cells. However, WA-induced increased autophagosome-formation was not mediated by increased activation of autophagy by upstream processes but was due to blockade of lysosomal-degradation as evident by higher level of sequestosome 1 (SQSTM1/p62) and decreased turnover of LC3. WA was found to be a potent lysosomal deacidification agent capable of blocking autophagic flux. Accordingly, inhibiting autophagy by blocking formation of autophagosomes or elevating lysosomal pH did not interfere with WA-mediated growth-inhibition. WA blocked autophagic flux decreasing recycling of cellular fuels leading to reduced energy supply. Investigating this alternative mechanism, we discovered that indeed, WA reduced ATP levels and increased phosphorylation of AMP-activated protein kinase (AMPK). Modulating substrates for tricarboxylic acid (TCA) cycle with methyl pyruvate protected WA-treated cells while 2DG potentiated WA-induced cell death. Conclusion: Our results indicate that WA induces a non-protective autophagy and blocks energy fuels in cancer cells by reducing ATP levels and inhibiting lysosomal acidification hence offering a three-pronged approach to facilitate cancer cell death. WA might be a useful strategic addition to chemotherapy regimens to evade cytoprotective effects of autophagy. Citation Format: Nethaji Muniraj, Arumugam Nagalingam, Neeraj K. Saxena, Dipali Sharma. Withaferin A induces nonprotective autophagy in a STK11-independent manner and mediates breast cancer inhibition via energetic impairment [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 1335.

Targeting Autophagy in the Tumor Microenvironment: New Challenges and Opportunities for Regulating Tumor Immunity.

Cancer cells evolve in the tumor microenvironment, which is now well established as an integral part of the tumor and a determinant player in cancer cell adaptation and resistance to anti-cancer therapies. Despite the remarkable and fairly rapid progress over the past two decades regarding our understanding of the role of the tumor microenvironment in cancer development, its precise contribution to cancer resistance is still fragmented. This is mainly related to the complexity of the “tumor ecosystem” and the diversity of the stromal cell types that constitute the tumor microenvironment. Emerging data indicate that several factors, such as hypoxic stress, activate a plethora of resistance mechanisms, including autophagy, in tumor cells. Hypoxia-induced autophagy in the tumor microenvironment also activates several tumor escape mechanisms, which effectively counteract anti-tumor immune responses mediated by natural killer and cytotoxic T lymphocytes. Therefore, strategies aiming at targeting autophagy in cancer cells in combination with other therapeutic strategies have inspired significant interest to overcome immunological tolerance and promote tumor regression. However, a number of obstacles still hamper the application of autophagy inhibitors in clinics. First, the lack of selectivity of the current pharmacological inhibitors of autophagy makes difficult to draw a clear statement about its effective contribution in cancer. Second, autophagy has been also described as an important mechanism in tumor cells involved in presentation of antigens to T cells. Third, there is a circumstantial evidence that autophagy activation in some innate immune cells may support the maturation of these cells, and it is required for their anti-tumor activity. In this review, we will address these aspects and discuss our current knowledge on the benefits and the drawbacks of targeting autophagy in the context of anti-tumor immunity. We believe that it is important to resolve these issues to predict the use of autophagy inhibitors in combination with immunotherapies in clinical settings.

Hydroxycamptothecin mediates antiproliferative effects through apoptosis and autophagy in A549 cells.

Hydroxycamptothecin (HCPT) represents a new generation of anticancer drugs, with almost no side effects when used for the treatment of a number of types of cancer. Autophagy is becoming recognized as an important biological mechanism in human cancer, including lung cancer. However, the involvement of autophagy in the antiproliferative effects of HCPT on lung cancer remains unclear. In the present study, A549 cells, an accepted model of non-small cell lung cancer (NSCLC) cells, were employed. It was demonstrated that HCPT was able to suppress proliferation and induce apoptosis and autophagy in A549 cells. The molecular mechanism underlying HCPT-induced cell death was attributed to apoptosis and autophagy. Furthermore, it was demonstrated that an autophagy inhibitor, 3-methyladenine, accelerated HCPT-induced cell death in A549 cells. The results of the present study may lead to a deeper understanding of the molecular mechanism by which HCPT regulates NSCLC A549 cells. These results highlight the potential use of autophagy inhibitors in combination with traditional chemotherapy drugs for the treatment of lung cancer.

Combined inhibition of autophagy and Nrf2 signaling augments bortezomib-induced apoptosis by increasing ROS production and ER stress in pancreatic cancer cells.

Pancreatic cancer (PC) is highly resistant to current therapies; thus, there is an urgent need to develop new treatment strategies. The proteasome is crucially important for proteostasis, which is involved in cell proliferation and survival, making it an attractive therapeutic target in cancer. However, recent studies have indicated that bortezomib, a highly selective proteasome inhibitor, has limited effects in solid tumors including PC. Thus, more mechanistic insights into chemo-sensitization strategies for bortezomib are urgently needed. Herein, we demonstrate that bortezomib induced apoptosis and autophagy via a mechanism involving endoplasmic reticulum (ER) stress in PC cells. Additionally, bortezomib treatment led to increased levels of intracellular reactive oxygen species (ROS), which play critical roles in bortezomib-induced ER stress and apoptosis. Moreover, autophagy functions as a compensatory mechanism to eliminate bortezomib-induced ROS and resists ER stress-mediated apoptosis. Additionally, the Nrf2-mediated antioxidative response, which works against with bortezomib-induced autophagy, also protected cells against bortezomib-induced ROS production. Finally, the dual inhibition of autophagy and Nrf2 signaling cooperatively enhanced bortezomib-induced apoptosis by elevating ROS levels and ER stress. Together, these data demonstrate that activation of autophagy and the Nrf2 antioxidant system, which lowers intracellular ROS, are mechanistically how PC cells overcome bortezomib treatment. In summary, combining proteasome inhibitors with drugs targeting autophagy and Nrf2 signaling could be a promising therapeutic approach for PC treatment.

Histone Deacetylase Inhibitor-Induced Autophagy in Tumor Cells: Implications for p53.

Autophagy is an essential process of the eukaryotic cell allowing degradation and recycling of dysfunctional cellular components in response to either physiological or pathological changes. Inhibition of autophagy in combination with chemotherapeutic treatment has emerged as a novel approach in cancer treatment leading to cell cycle arrest, differentiation, and apoptosis. Suberoyl hydroxamic acid (SAHA) is a broad-spectrum histone deacetylase inhibitor (HDACi) suppressing family members in multiple HDAC classes. Increasing evidence indicates that SAHA and other HDACi can, in addition to mitochondria-mediated apoptosis, also promote caspase-independent autophagy. SAHA-induced mTOR inactivation as a major regulator of autophagy activating the remaining autophagic core machinery is by far the most reported pathway in several tumor models. However, the question of which upstream mechanisms regulate SAHA-induced mTOR inactivation that consequently initiate autophagy has been mainly left unexplored. To elucidate this issue, we recently initiated a study clarifying different modes of SAHA-induced cell death in two human uterine sarcoma cell lines which led to the conclusion that the tumor suppressor protein p53 could act as a molecular switch between SAHA-triggered autophagic or apoptotic cell death. In this review, we present current research evidence about HDACi-mediated apoptotic and autophagic pathways, in particular with regard to p53 and its therapeutic implications.

Abstract 3301: A three-pronged attack on cancer cells: Induction of non-protective autophagy, inhibition of lysosomal acidification and promotion of energetic impairment

Abstract Background and Aim: Cancer cells undergo cytoprotective autophagy and evade chemotherapy therefore many clinical trials are investigating the efficacy of autophagy inhibition in combination with chemotherapy. At the functional level, autophagic process can be cytoprotective, cytotoxic, cytostatic or nonprotective. We investigated strategies to convert cellular autophagic response to non-protective autophagy which does not interfere with therapeutic regimens exploiting bioactive molecules. Methods: Utilizing in vivo xenograft models, we established that Withaferin A (WA), a bioactive molecule from Withania Somnifera inhibits breast tumorigenesis. Autophagy studies were conducted utilizing immunoblot, RT-PCR, and immunofluorescence analyses for autophagy markers, transmission electron microscopy and confocal imaging. The fusion of autophagosome and lysosome was examined by using GFP-LC3/LysoTracker-red and GFP-LC3/mCherryRAB7A. Protein degradation activity of lysosomes and ATP levels were examined by DQ-BSA assay, Cathepsin activity and quantitative ATP assay. Results: WA inhibited growth and induced apoptosis in breast cancer cells resulting in inhibition of breast carcinogenesis in vivo. Although WA increased tumor suppressor LKB1 which is known to be involved in autophagy, WA-mediated increased cleavage of Light Chain 3 type II (LC3-II) and punctated LC3-II staining was LKB1-independent. The redistribution of EGFP-LC3 from cytosol to autophagosome indicated increased formation of autophagosomes in WA-treated cells. However, WA-induced increased autophagosome-formation was not mediated by increased activation of autophagy by upstream processes but was due to blockade of lysosomal-degradation as evident by higher level of sequestosome 1 (SQSTM1/p62) and decreased turnover of LC3. WA was found to be a potent lysosomal deacidification agent capable of blocking autophagic flux. Accordingly, inhibiting autophagy by blocking formation of autophagosomes or elevating lysosomal pH did not interfere with WA-mediated growth-inhibition. WA blocked autophagic flux decreasing recycling of cellular fuels leading to reduced energy supply. Investigating this alternative mechanism, we discovered that indeed, WA reduced ATP levels and increased phosphorylation of AMP-activated protein kinase (AMPK). Modulating substrates for tricarboxylic acid (TCA) cycle with methyl pyruvate protected WA-treated cells while 2DG potentiated WA-induced cell death. Conclusion: Our results indicate that WA induces a non-protective autophagy and blocks energy fuels in cancer cells by reducing ATP levels and inhibiting lysosomal acidification hence offering a three-pronged approach to facilitate cancer cell death. WA might be a useful strategic addition to chemotherapy regimens to evade cytoprotective effects of autophagy. Citation Format: Nethaji Muniraj, Arumugam Nagalingam, Panjamurthy Kuppusamy, Neeraj K. Saxena, Dipali Sharma. A three-pronged attack on cancer cells: Induction of non-protective autophagy, inhibition of lysosomal acidification and promotion of energetic impairment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3301. doi:10.1158/1538-7445.AM2017-3301

Abstract 3307: Induction of autophagy as a mechanism of therapeutic resistance in head and neck cancer

Abstract Background: In the curative setting for head and neck cancer (HNC) a commonly used treatment is radiation combined with the anti-EGFR monoclonal antibody cetuximab (CTX). However, despite decades of research into improved treatments, therapeutic resistance remains a major challenge for this malignancy with roughly 40% of patients developing recurrent disease. We are studying the molecular mechanisms by which radiation and cetuximab induce autophagy and how this process can be modulated to improve the efficacy of therapy. Methods: We evaluated a panel of both human papillomavirus (HPV) positive and negative HNC cell lines for autophagic response to both CTX treatment and ionizing radiation (XRT). Induction of autophagy was detected by immunoblot flux assays for LC3 and p62 and by immunofluorescent staining of autophagic vesicles. siRNA knockdown of EGFR, LAMPT4B, ULK1, Beclin1 and others was used to probe the involvement of different signaling molecules. The addition of specific autophagy inhibitors, such as the ULK1 inhibitor SBI-0206965, to CTX or XRT treatment was tested to determine whether the reduction of autophagic response reduced cell survival in a clonogenic survival assay. Induction of apoptosis was analyzed by immunoblot against cleaved Caspase and PARP and AnnexinV staining. Results: Flux assays revealed that both CTX and XRT induced autophagy in a time and dose dependent manner. Immunofluorescent staining of LC3 to identify autophagic vesicles showed that a relatively small fraction of the total cell population is able to induce this response. Specific siRNA knockdown of EGFR and LAMPT4B was able to abrogate the induction of autophagy in response to both CTX and XRT. Involvement of alternative downstream pathways involving either ULK1 and Beclin1 or p53 in response to either treatment is under investigation. To determine whether blockade of cytoprotective autophagy can help overcome therapeutic resistance, we tested specific autophagy inhibitors in combination with either CTX or XRT treatment. The addition of the ULK1 inhibitor to CTX or XRT induced apoptosis as shown by caspase activity, AnnexinV staining, and reduced cell survival in clonogenic assays. Conclusions: Autophagy may play a critical and protective role in how head and neck cancers respond to therapeutic stress. The addition of specific autophagy inhibitors to standard treatments may provide a way to overcome resistance to therapy. Citation Format: Jaimee C. Eckers, Adam D. Swick, Tyler Fowler, Justin Skiba, Kwangok P. Nickel, Randall J. Kimple. Induction of autophagy as a mechanism of therapeutic resistance in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3307. doi:10.1158/1538-7445.AM2017-3307

Abstract 1202: Targeting PIK3C3 to overcome drug resistance resulted from prosurvival autophagy in cancer cells

Abstract Autophagy is an important cellular recycling mechanism in which portions of cytosol or organelles are sequestered into a double-membrane structure and delivered to lysosome for degradation. PIK3C3 is the only class III PI3K, which specifically generates phosphatidylinositol 3-phosphate (PtdIns3P). It in turn recruits proteins containing FYVE or PX domains, thereby initiating various complexes at the membranes of endosomes, phagosomes and autophagosomes. Recently, several studies have shown that autophagy is upregulated in cancer cells when exposing to chemotherapy and radiotherapy. Therefore, the rationale of targeting autophagy addiction in cancer was proposed by combining autophagy inhibition with agents that induce autophagy as a pro-survival response to increase their therapeutic efficacy. We stably knocked down PIK3C3 in cancer cell lines and combined with clinical therapeutic agents reportedly induce pro-survival autophagy. Cell viability was examined by MTT assay and combination index was determined by Compusyn software. The autophagy flux was evaluated by detecting protein levels of LC-3II and p62 through western blot analysis. The results showed PIK3C3 knockdown synergistically sensitize the killing effects of gefitinib and gemcitabine in A549 cells and Panc1 cells, respectively. Both of gefitinib and gemcitabine increased autophagy flux, as evidence by the increase of LC3-II and decrease of autophagy substrate, p62. However, the combination of PIK3C3 knockdown and therapeutic agents increased both of the protein levels of LC3-II and p62. These data suggest incomplete autophagy when the function of PIK3C3 was inhibited. The phenomenon was confirmed by bombing autophagy inducer and PIK3C3 inhibitors. Moreover, no appreciable apoptotic cell death was observed. Taken together, our study revealed a potential strategy by targeting PIK3C3 to overcome drug resistance associated with the pro-survival autophagy in cancer. Citation Format: CHUN-HAN CHEN. Targeting PIK3C3 to overcome drug resistance resulted from prosurvival autophagy in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1202. doi:10.1158/1538-7445.AM2017-1202

Combined inhibition of autophagy with mTOR inhibitor to enhance cell death in renal cell carcinoma.

450 Background: mTOR (mammalian target of rapamycin) and autophagy are increasingly recognized as being a central cellular and pathological process for numerous human diseases, including renal cell carcinoma (RCC). Depending on the cellular context, autophagy may promote cancer cell survival or cell death. However, little is known about the mechanisms of regulating mTOR activity and autophagic function in RCC. We hypothesize that autophagy promotes cell survival via mTOR mediated-phosphatidylinositol 3-kinase (PI3K)/AKT pathway and is regulated by the von Hippel-Lindau (VHL) tumor suppressor. Methods: RCC cells were stably lentiviral transduced with expression of VHL or mCherry-EGFP tandemfluorescent-tagged LC3B for studying autophagic flux. Cell viability was evaluated by cytotoxic XTT and clonogenic assays and flow cytometry. The efficacy of PI3K/AKT/mTOR pathway inhibition by RAD001, PI-103, MK2206, AZD8055, and/or lysosomotropic inhibitors were evaluated by immunoblots and immunofluorescence for autophagy process of autophagosome and lysosome. Results: We show that mTOR is hyperactive in VHL-deficient cells compared to cells with wild-type VHL or VHL-expressing cells. AZD8055-induced toxicity occurs in a VHL-independent manner via cell cycle arrest and clonogenic senescent cell death, but results in significantly increased expression of autophagic marker LC3-II and the formation of autophagic vacuoles. Pharmacologic inhibition or siRNA silencing of autophagy pathway components promotes AZD8055-induced cell death in VHL-deficient cells. Interestingly, defective autophagy marked by the presence of sustained p62 expression in VHL-deficient cells appears to contribute to cell survival via mTOR signaling, which in turn influences autophagosome-lysosome fusion, and thus controls autophagic flux by acting at the termination stage of the process. Conclusions: These results support mTOR and autophagy pathways as potential targets of anticancer drugs and reveal VHL in control of the autophagic program in RCC. Further, this work suggests that combined inhibition of autophagy along with mTOR inhibitors could be a novel therapeutic strategy for the treatment of RCC.

Antitumor adaptive immunity remains intact following inhibition of autophagy and antimalarial treatment.

The rising success of cancer immunotherapy has produced immense interest in defining the clinical contexts that may benefit from this therapeutic approach. To this end, there is a need to ascertain how the therapeutic modulation of intrinsic cancer cell programs influences the anticancer immune response. For example, the role of autophagy as a tumor cell survival and metabolic fitness pathway is being therapeutically targeted in ongoing clinical trials that combine cancer therapies with antimalarial drugs for the treatment of a broad spectrum of cancers, many of which will likely benefit from immunotherapy. However, our current understanding of the interplay between autophagy and the immune response remains incomplete. Here, we have evaluated how autophagy inhibition impacts the antitumor immune response in immune-competent mouse models of melanoma and mammary cancer. We observed equivalent levels of T cell infiltration and function within autophagy-competent and -deficient tumors, even upon treatment with the anthracycline chemotherapeutic doxorubicin. Similarly, we found equivalent T cell responses upon systemic treatment of tumor-bearing mice with antimalarial drugs. Our findings demonstrate that antitumor adaptive immunity is not adversely impaired by autophagy inhibition in these models, allowing for the future possibility of combining autophagy inhibitors with immunotherapy in certain clinical contexts.

Abstract 2129: Induction of autophagy as a mechanism of therapeutic resistance in head and neck cancer

Abstract Background and purpose: Despite decades of research into improved treatments for head and neck cancer, therapeutic resistance remains a major challenge for this malignancy, with roughly 40% of patients developing recurrent disease. Among the plethora of intrinsic and acquired mechanisms of resistance, recent evidence has suggested the cellular process of autophagy may be an additional contributor. This stress response, which normally functions during periods of nutrient deprivation to maintain cellular homeostasis, may have the unintended consequence of protecting tumor cells from the cytotoxic effects of chemotherapy and radiotherapy. We show that autophagy mediates resistance to chemotherapies targeting the EGFR and mTORC pathways in addition to radiotherapy in several head and neck cancer models, begin to elucidate the mechanism of this resistance, and demonstrate that the addition of a specific autophagy inhibitor can block cytoprotective autophagy. Methods and results: Building on well established evidence that mTORC inhibition can directly induce autophagy, recent work has established a novel mechanism by which kinase inactive EGFR resulting from either growth factor starvation or anti-EGFR antibody (cetuximab) can upregulate this response via an interaction with LAMPT4B. Treatment of a panel of HNC cell lines with either serum starvation or cetuximab produced increased formation of LC3-B puncta, a marker for autophagasome formation, by IF staining, and was confirmed by Western blot for increased LC3-B flux and decreased p62. SiRNA knockdown of either EGFR or LAMPT4B in HNC cells abrogated the induction of autophagy in response to either serum starvation or cetuximab treatment. We have also shown that radiation induces autophagy in a time and dose dependent manner. The mechanism of radiation-induced autophagy remains a subject of study; preliminary data suggest that both EGFR and LAMPT4B play a role as siRNA knockdown of those mRNAs reduces the autophagic response. To determine whether blockade of cytoprotective autophagy can help overcome therapeutic resistance, we have tested specific autophagy inhibitors in combination with either chemotherapy or radiation. The novel ULK1 inhibitor SBI-0206965 successfully blocks the induction of autophagy in a panel of HNC lines. More compellingly, while neither SBI-0206965 nor the NEDD4 inhibitor Heclin, impacted cell growth in untreated cells, both were effective in reducing cell survival following treatment with radiation, cetuximab, or mTORC inhibitor (AZD8055). Summary: These pre-clinical studies have established the proof of concept for the cytoprotective effect of autophagy in response to anti-cancer treatments including EGFR or mTORC inhibition and radiotherapy. Further we have identified the addition of specific autophagy inhibitors to standard treatments as a potential strategy to overcome this mechanism of resistance. Citation Format: Adam D. Swick, Tan Xiaojun, Tyler Fowler, Kwangok Nickel, Richard A. Anderson, Randall J. Kimple. Induction of autophagy as a mechanism of therapeutic resistance in head and neck cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2129.

Blockage of autophagy pathway enhances Salmonella tumor-targeting.

Previous studies have shown that strains of Salmonella typhimurium specifically target tumors in mouse models of cancer. In this study, we report that tumor-targeting Salmonella typhimurium A1-R (A1-R) or VNP20009 induced autophagy in human cancer cells, which serves as a defense response. Functionally, by knockdown of essential autophagy genes Atg5 or Beclin1 in bacteria-infected cancer cells, the autophagy pathway was blocked, which led to a significant increase of intracellular bacteria multiplication in cancer cells. Genetic inactivation of the autophagy pathway enhanced A1-R or VNP20009-mediated cancer cell killing by increasing apoptotic activity. We also demonstrate that the combination of pharmacological autophagy inhibitors chloroquine (CQ) or bafilomycin A1 (Baf A1) with tumor-targeting A1-R or VNP20009 significantly enhanced cancer-cell killing compared with Salmonella infection alone. These findings provide a proof-of-concept of combining autophagy inhibitors and tumor-targeting Salmonella to enhance cancer-cell killing.

Targeting autophagy in cancer management - strategies and developments.

Autophagy is a highly regulated catabolic process involving lysosomal degradation of intracellular components, damaged organelles, misfolded proteins, and toxic aggregates, reducing oxidative stress and protecting cells from damage. The process is also induced in response to various conditions, including nutrient deprivation, metabolic stress, hypoxia, anticancer therapeutics, and radiation therapy to adapt cellular conditions for survival. Autophagy can function as a tumor suppressor mechanism in normal cells and dysregulation of this process (ie, monoallelic Beclin-1 deletion) may lead to malignant transformation and carcinogenesis. In tumors, autophagy is thought to promote tumor growth and progression by helping cells to adapt and survive in metabolically-challenged and harsh tumor microenvironments (ie, hypoxia and acidity). Recent in vitro and in vivo studies in preclinical models suggested that modulation of autophagy can be used as a therapeutic modality to enhance the efficacy of conventional therapies, including chemo and radiation therapy. Currently, more than 30 clinical trials are investigating the effects of autophagy inhibition in combination with cytotoxic chemotherapies and targeted agents in various cancers. In this review, we will discuss the role, molecular mechanism, and regulation of autophagy, while targeting this process as a novel therapeutic modality, in various cancers.

Autophagy inhibition re-sensitizes pulse stimulation-selected paclitaxel-resistant triple negative breast cancer cells to chemotherapy-induced apoptosis.

Chemotherapy is the mainstay of systemic treatment for triple negative breast cancer (TNBC); however, the development of drug resistance limits its effectiveness. Therefore, we investigated the underlying mechanism for drug resistance and potential approaches to overcome it for a more effective treatment for TNBCs. Using a pulse-stimulated selection strategy to mimic chemotherapy administration in the clinic, we developed a new paclitaxel-resistant MDA-MB-231 cell line and analyzed these cells for changes in autophagy activity, and the role and mechanisms of the increased autophagy in promoting drug resistance were determined. We found that the pulse-stimulated selection strategy with paclitaxel resulted in MDA-MB-231 variant cells with enhanced resistance to paclitaxel. These resistant cells were found to have enhanced basal autophagy activity, which confers a cytoprotective function under paclitaxel treatment stress. Inhibition of autophagy enhanced paclitaxel-induced cell death in these paclitaxel-resistant cells. We further revealed that up-regulated autophagy in resistant cells enhanced the clearance of damaged mitochondria. Last, we showed that the paclitaxel-resistant cancer cells acquired cross resistance to epirubicin and cisplatin. Together, these results suggest that combining autophagy inhibition with chemotherapy may be an effective strategy to improve treatment outcome in paclitaxel-resistant TNBC patients.

Abstract A37: Role of autophagy inhibition in metastatic disease utilizing mouse models

Abstract Modification of autophagy to enhance cancer treatment is being aggressively pursued in the laboratory and clinic; however, it remains to be seen if inhibition or induction of autophagy is going to result in better outcomes. In addition, the advantages of autophagic modification with neoadjuvant chemotherapy vs. surgical adjuvant chemotherapy have yet to be reported. The 4T1-luc metastatic mammary mouse cell line modified to express luciferase was used to induce lung metastases, both from orthotopic growth of primary tumors or experimentally induced via tail vein injection. Both pharmacologic inhibition (chloroquine (CQ) administered 60 mg/kg daily to mice) and genetic knockdown of a key protein necessary for autophagy alone (4T1-luc-shBeclin-1 cells) did not alter time to metastasis (as determined by luciferase positive lungs) when 4T1-luc cells were injected via tail vein. In addition, inhibiting autophagy in 4T1-luc cells with CQ in vitro prior to injection via the tail did not alter time to metastasis. Similar results were observed with pharmacologic inhibition on experimentally induce lung metastases using the metastatic mouse melanoma B16-F10 cell line. In vitro work, by us and others, has lead us to conclude that pharmacologic inhibition plus cisplatin leads to greater growth inhibition and cell kill in vitro than either alone. Effect of autophagy inhibition in combination with cisplatin on metastatic formation in a neoadjuvant setting was tested by treating mice with daily with CQ and cisplatin (q14d) one day post tumor cell injection in the mammary fat pad. Additional experiments were conducted to determine if autophagy inhibition plus cisplatin in a surgical adjuvant setting delays metastasis formation. In each instance, primary tumors were removed at ~100mm3 and mice were followed until luciferase positive lungs were observed. From these results we conclude that autophagy inhibition, using CQ, does not alter metastatic growth alone and appears to slightly enhance the efficacy of cisplatin on treating metastases. It remains to be seen if autophagy inhibition with other compounds would alter results. Citation Format: Rebecca Barnard, Ryan J. Hansen, Paola Maycotte, Andrew Thorburn, Daniel L. Gustafson. Role of autophagy inhibition in metastatic disease utilizing mouse models. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A37.

Abstract 156: Autophagic degradation of granzyme B impairs NK-mediated killing of hypoxic tumor cells

Abstract Natural killer (NK) cells are effectors of the innate immune system, able to kill cancer cells through the release of the cytotoxic protease granzyme B. NK-based therapies have recently emerged as promising anticancer strategies. However, it is well established that hypoxic tumor microenvironment interferes with the antitumor function of immune cells and constitutes a major obstacle for defining cancer immunotherapies. Recent studies demonstrated that autophagy regulates the innate immune response by mechanisms which are not fully understood. In this study, we showed that hypoxia decreases breast cancer cell susceptibility to NK-mediated lysis by a mechanism involving the activation of autophagy in tumor cells. Targeting autophagy was sufficient to restore NK-mediated tumor cell killing. We showed that the resistance of hypoxic tumor cells was neither related to a defect in their recognition by NK cells, nor to a defect in the cytolytic function of NK cells toward hypoxic cells. We provided evidence that autophagy activation degrades NK-derived granzyme B in lysosomes of hypoxic cells making them less sensitive to NK-mediated killing. Genetic and pharmacological inhibition of autophagy restored granzyme B levels and reverted the resistance of hypoxic cells in vitro. Our results highlight autophagy as a critical factor in modulating NK-mediated anti-tumor immune response. We have validated this concept in vivo by showing that targeting autophagy significantly improved NK-mediated tumor shrinking in breast and melanoma models. This study provides a cutting-edge advance in our understanding of how hypoxia-induced autophagy impairs NK-mediated lysis and paves the way for formulating more effective NK-based antitumor therapy by combining autophagy inhibitors. Citation Format: Joanna Baginska, Elodie Viry, Guy Berchem, Aurélie Poli, Muhammad Zaeem Noman, Kris van Moer, Sandrine Medves, Takouhie Mgrditchian, Jacques Zimmer, Anais Oudin, Simone P. Niclou, R. Chris Bleackley, Salem Chouaib, Bassam Janji. Autophagic degradation of granzyme B impairs NK-mediated killing of hypoxic tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 156. doi:10.1158/1538-7445.AM2014-156