Autophagy Inhibition In Combination Research Articles

Combined Autophagy Inhibition and Dendritic Cell Recruitment Induces Antitumor Immunity and Enhances Immune Checkpoint Blockade Sensitivity in Pancreatic Cancer.

The effect of immune checkpoint inhibitors is extremely limited in patients with pancreatic ductal adenocarcinoma (PDAC) due to the suppressive tumor immune microenvironment (TIME). Autophagy, which has been shown to play a role in anti-tumor immunity, has been proposed as a therapeutic target for PDAC. Here, single-cell RNA-sequencing of autophagy-deficient murine PDAC tumors revealed that autophagy inhibition in cancer cells induced dendritic cell (DC) activation. Analysis of human PDAC tumors substantiated a negative correlation between autophagy and DC activation signatures. Mechanistically, autophagy inhibition increased intracellular accumulation of tumor antigens, which could activate DCs. Administration of chloroquine (CQ), an autophagy inhibitor, in combination with Flt3 ligand (Flt3L)-induced DC infiltration inhibited tumor growth and increased tumor-infiltrating T lymphocytes. However, autophagy inhibition in cancer cells also induced CD8+ T cell exhaustion with high expression of immune checkpoint LAG3. A triple therapy comprising CQ, Flt3L, and an anti-LAG3 antibody markedly reduced tumor growth in orthotopic syngeneic PDAC mouse models. Thus, targeting autophagy in cancer cells and activating DCs sensitizes PDAC tumors to immune checkpoint inhibitor therapy, warranting further development of this treatment approach to overcome immunosuppression in pancreatic cancer.

Construction of a Multifunctional Upconversion Nanoplatform Based on Autophagy Inhibition and Photodynamic Therapy Combined with Chemotherapy for Antitumor Therapy.

Inhibition of autophagy increases the sensitivity of tumor cells to radiotherapy and chemotherapy and improves the therapeutic effect on tumors. Recently, photodynamic therapy (PDT) combined with chemotherapy has been proven to further improve the efficiency of cancer treatment. As such, combining autophagy inhibition with PDT and chemotherapy may represent a potentially effective new strategy for cancer treatment. However, currently widely studied autophagy inhibitors inevitably produce various toxic side effects due to their inherent pharmacological activity. To overcome this constraint, in this study, we designed an ideal multifunctional upconversion nanoplatform, UCNP-Ce6-EPI@mPPA + NIR (MUCEN). Control, UCNP-EPI@mPPA (MUE), UCNP-EPI@mPPA + NIR (MUEN), Ce6-EPI@mPPA (MCE), Ce6-EPI@mPPA + NIR (MCEN), and UCNP-Ce6-EPI@mPPA (MUCE) groups were set up separately as controls. Based on a combination of autophagy inhibition and PDT, the average particle size of MUCEN was 197 nm, which can simultaneously achieve the double encapsulation of chlorine e6 (Ce6) and epirubicin (EPI). In vitro tests revealed that MUCE was efficiently endocytosed by 4T1 cells under near-infrared light irradiation. Further, in vivo tests revealed that MUCE dramatically inhibited tumor growth. Immunohistochemistry results indicated that MUCE efficiently increased the expression of autophagy inhibitors p62 and LC3 in tumor tissues. The synergistic effect of autophagy inhibition and PDT with MUCE exhibited superior tumor suppression, providing an innovative approach to cancer treatment.

Targeting RPA promotes autophagic flux and the antitumor response to radiation in nasopharyngeal carcinoma

BackgroundAutophagy is involved in nasopharyngeal carcinoma (NPC) radioresistance. Replication protein A 1 (RPA1) and RPA3, substrates of the RPA complex, are potential therapeutic targets for reversing NPC radioresistance. Nevertheless, the role of RPA in autophagy is not adequately understood. This investigation was performed to reveal the cytotoxic mechanism of a pharmacologic RPA inhibitor (RPAi) in NPC cells and the underlying mechanism by which RPAi-mediated autophagy regulates NPC radiosensitivity.Methods and resultsWe characterized a potent RPAi (HAMNO) that was substantially correlated with radiosensitivity enhancement and proliferative inhibition of in vivo and in NPC cell lines in vitro. We show that the RPAi induced autophagy at multiple levels by inducing autophagic flux, AMPK/mTOR pathway activation, and autophagy-related gene transcription by decreasing glycolytic function. We hypothesized that RPA inhibition impaired glycolysis and increased NPC dependence on autophagy. We further demonstrated that combining autophagy inhibition with chloroquine (CQ) treatment or genetic inhibition of the autophagy regulator ATG5 and RPAi treatment was more effective than either approach alone in enhancing the antitumor response of NPC to radiation.ConclusionsOur study suggests that HAMNO is a potent RPAi that enhances radiosensitivity and induces autophagy in NPC cell lines by decreasing glycolytic function and activating autophagy-related genes. We suggest a novel treatment strategy in which pharmacological inhibitors that simultaneously disrupt RPA and autophagic processes improve NPC responsiveness to radiation.

The Combined Inhibition of Autophagy and Diacylglycerol Acyltransferase-Mediated Lipid Droplet Biogenesis Induces Cancer Cell Death during Acute Amino Acid Starvation.

Lipid droplets (LDs) are dynamic organelles involved in the management of fatty acid trafficking and metabolism. Recent studies suggest that autophagy and LDs serve complementary roles in the protection against nutrient stress, but the autophagy-LD interplay in cancer cells is not well understood. Here, we examined the relationship between autophagy and LDs in starving HeLa cervical cancer- and MDA-MB-231 breast cancer cells. We found that acute amino acid depletion induces autophagy and promotes diacylglycerol acyltransferase 1 (DGAT1)-mediated LD accumulation in HeLa cells. Inhibition of autophagy via late-stage autophagy inhibitors, or by knocking down autophagy-related 5 (ATG5), reduced LD accumulation in amino acid-starved cancer cells, suggesting that autophagy contributes to LD biogenesis. On the contrary, knockdown of adipose triglyceride lipase (ATGL) increased LD accumulation, suggesting that LD breakdown is mediated by lipolysis under these conditions. Concurrent inhibition of autophagy by silencing ATG5 and of LD biogenesis using DGAT inhibitors was effective in killing starving HeLa cells, whereas cell survival was not compromised by suppression of ATGL-mediated lipolysis. Autophagy-dependent LD biogenesis was also observed in the aggressive triple-negative MDA-MB-231 breast cancer cells deprived of amino acids, but these cells were not sensitized to starvation by the combined inhibition of LD biogenesis and autophagy. These findings reveal that while targeting autophagy-driven and DGAT-mediated LD biogenesis reduces the resilience of HeLa cervical cancer cells to amino acid deprivation, this strategy may not be successful in other cancer cell types.

Is Autophagy Inhibition in Combination with Temozolomide a Therapeutically Viable Strategy?

Temozolomide is an oral alkylating agent that is used as the first line treatment for glioblastoma multiform, and in recurrent anaplastic astrocytoma, as well as having demonstrable activity in patients with metastatic melanoma. However, as the case with other chemotherapeutic agents, the development of resistance often limits the therapeutic benefit of temozolomide, particularly in the case of glioblastoma. A number of resistance mechanisms have been proposed including the development of cytoprotective autophagy. Cytoprotective autophagy is a survival mechanism that confers upon tumor cells the ability to survive in a nutrient deficient environment as well as under external stresses, such as cancer chemotherapeutic drugs and radiation, in part through the suppression of apoptotic cell death. In this review/commentary, we explore the available literature and provide an overview of the evidence for the promotion of protective autophagy in response to temozolomide, highlighting the possibility of targeting autophagy as an adjuvant therapy to potentially increase the effectiveness of temozolomide and to overcome the development of resistance.

Targeting autophagy increases the efficacy of proteasome inhibitor treatment in multiple myeloma by induction of apoptosis and activation of JNK

BackgroundThe therapeutic armamentarium in multiple myeloma has been significantly broadened by proteasome inhibitors, highly efficient means in controlling of multiple myeloma. Despite the developments of therapeutic regimen in treatment of multiple myeloma, still the complete remission requires a novel therapeutic strategy with significant difference in outcomes. Proteasome inhibitors induce autophagy and ER stress, both pivotal pathways for protein homeostasis. Recent studies showed that the IRE1α-XBP1 axis of the unfolded protein response (UPR) is up-regulated in multiple myeloma patients. In addition, XBP1 is crucial for the maintenance of viability of acute lymphoblastic leukemia (ALL).ResultsWe analyzed the efficacy of targeting IRE1α-XBP1 axis and autophagy in combination with proteasome inhibitor, ixazomib in treatment of multiple myeloma. In this present study, we first show that targeting the IRE1α-XBP1 axis with small molecule inhibitors (STF-083010, A106) together with the ixazomib induces cell cycle arrest with an additive cytotoxic effect in multiple myeloma. Further, we examined the efficacy of autophagy inhibitors (bafilomycin A, BAF and chloroquine, CQ) together with ixazomib in multiple myeloma and observed that this combination treatment synergistically reduced cell viability in multiple myeloma cell lines (viable cells Ixa: 51.8 ± 3.3, Ixa + BAF: 18.3 ± 7.2, Ixa + CQ: 38.4 ± 3.7) and patient-derived multiple myeloma cells (Ixa: 59.6 ± 4.4, Ixa + CQ: 7.0 ± 2.1). We observed, however, that this combined strategy leads to activation of stress-induced c-Jun N-terminal kinase (JNK). Cytotoxicity mediated by combined proteasome and autophagy inhibition was reversed by addition of the specific JNK inhibitor JNK-In-8 (viable cells: Ixa + BAF: 11.6 ± 7.0, Ixa + BAF + JNK-In-8: 30.9 ± 6.1).ConclusionIn this study we showed that combined inhibition of autophagy and the proteasome synergistically induces cell death in multiple myeloma. Hence, we consider the implication of pharmaceutical inhibition of autophagy together with proteasome inhibition and UPR-directed therapy as promising novel in vitro treatment strategy against multiple myeloma.

Autophagy Contributes to Metabolic Reprogramming and Therapeutic Resistance in Pancreatic Tumors.

Metabolic reprogramming is a feature of cancers for which recent research has been particularly active, providing numerous insights into the mechanisms involved. It occurs across the entire cancer process, from development to resistance to therapies. Established tumors exhibit dependencies for metabolic pathways, constituting vulnerabilities that can be targeted in the clinic. This knowledge is of particular importance for cancers that are refractory to any therapeutic approach, such as Pancreatic Ductal Adenocarcinoma (PDAC). One of the metabolic pathways dysregulated in PDAC is autophagy, a survival process that feeds the tumor with recycled intracellular components, through both cell-autonomous (in tumor cells) and nonautonomous (from the local and distant environment) mechanisms. Autophagy is elevated in established PDAC tumors, contributing to aberrant proliferation and growth even in a nutrient-poor context. Critical elements link autophagy to PDAC including genetic alterations, mitochondrial metabolism, the tumor microenvironment (TME), and the immune system. Moreover, high autophagic activity in PDAC is markedly related to resistance to current therapies. In this context, combining autophagy inhibition with standard chemotherapy, and/or drugs targeting other vulnerabilities such as metabolic pathways or the immune response, is an ongoing clinical strategy for which there is still much to do through translational and multidisciplinary research.

Metabolic Rewiring Is Essential for AML Cell Survival to Overcome Autophagy Inhibition by Loss of ATG3.

Simple SummaryThe importance of autophagy in leukemia progression and survival has been studied previously. However, little is known about the development of resistance mechanisms to autophagy inhibition in leukemia. Here, we present data on the mechanisms by which leukemia cells maintain their cell survival after inhibition of autophagy by the loss of ATG3. After the loss of ATG3, leukemia cells upregulated their energy metabolism by increasing glycolysis and mitochondrial metabolism, in particular oxidative phosphorylation, which resulted in higher ATP levels. Moreover, inhibition of mitochondrial function strongly impaired cell survival in ATG3 deficiency, thus demonstrating the importance of ATG3 in the regulation of metabolism and survival of leukemic cells. Therefore, our data provide a rationale for combining autophagy inhibitors with inhibitors targeting mitochondrial metabolism for the development of leukemia therapy to overcome the potential obstacle of emerging resistance to autophagy inhibition.Autophagy is an important survival mechanism that allows recycling of nutrients and removal of damaged organelles and has been shown to contribute to the proliferation of acute myeloid leukemia (AML) cells. However, little is known about the mechanism by which autophagy- dependent AML cells can overcome dysfunctional autophagy. In our study we identified autophagy related protein 3 (ATG3) as a crucial autophagy gene for AML cell proliferation by conducting a CRISPR/Cas9 dropout screen with a library targeting around 200 autophagy-related genes. shRNA-mediated loss of ATG3 impaired autophagy function in AML cells and increased their mitochondrial activity and energy metabolism, as shown by elevated mitochondrial ROS generation and mitochondrial respiration. Using tracer-based NMR metabolomics analysis we further demonstrate that the loss of ATG3 resulted in an upregulation of glycolysis, lactate production, and oxidative phosphorylation. Additionally, loss of ATG3 strongly sensitized AML cells to the inhibition of mitochondrial metabolism. These findings highlight the metabolic vulnerabilities that AML cells acquire from autophagy inhibition and support further exploration of combination therapies targeting autophagy and mitochondrial metabolism in AML.

RARE-18. NF1-MUTATED TUMORS EXHIBIT INCREASED SENSITIVITY TO AUTOPHAGY INHIBITION ALONE AND IN COMBINATION WITH MEK INHIBITION

BackgroundAutophagy inhibition is a potential treatment for central nervous system (CNS) tumors. Autophagy, a heavily regulated process by which cellular waste is transferred to lysosomes for degradation and processing, is an integral part of tumor cell survival under stressful conditions including nutrient deprivation and chemotherapy. While the efficacy of autophagy inhibition has been demonstrated in CNS tumors with BRAFV600e mutations, it has yet to be explored in other CNS tumor types with MAPK pathway dysregulation including NF1-mutated tumors. Many tumors associated with the NF1 phenotype can be difficult to treat surgically thus development of further pharmacologic interventions is necessary. MethodsA CRISPR/Cas9 mediated NF1 KO was derived from human immortalized Schwann cells and utilized as a tumor model. Autophagy inhibition was achieved pharmacologically by chloroquine (CQ) and genetically via shRNAi of ATG5 and ATG7. Trametinib was used for MEK inhibition. Cell growth and viability were determined by Incucyte, Cell Titer-Glo luminescent assay, and colony-formation assays. Protein expression was measured by western blot. ResultsWe demonstrate increased autophagic activity in NF1 KO cell as compared to control lines both at baseline and in response to cellular stress. Furthermore, we describe that NF1 KO cells exhibit increased sensitivity to CQ alone, CQ in combination with trametinib, and shRNAi-mediated autophagy inhibition in combination with trametinib. ConclusionHere, we describe increased autophagic dependence of NF1 mutated tumors and demonstrate increased tumor sensitivity to autophagy inhibition both alone and in combination with MEK inhibition. These findings indicate that autophagy inhibition via CQ may be an effective adjunctive treatment for NF1 mutated tumors and suggests that diverse CNS tumor types with MAPK pathway dysregulation are susceptible to autophagy inhibition. Clinical investigation of combined MEK and autophagy inhibition has the potential to improve outcomes for NF1 patients with CNS tumors.

Intratumoral T cell clonality and survival in a randomized phase II study of preoperative autophagy inhibition in combination with gemcitabine and nab-paclitaxel treatment in patients with resectable pancreatic cancer.

e16001 Background: Autophagy is a cell survival mechanism that is upregulated in pancreatic ductal adenocarcinoma (PDAC). PDAC autophagy results in an altered metabolic phenotype that promotes tumor progression, chemotherapeutic resistance, and immune evasion. Methods: We previously completed a randomized phase II clinical trial of preoperative gemcitabine-nab-paclitaxel with (PGH n = 34) and without (PG, n = 30) autophagy inhibition in patients with resectable and borderline resectable PDAC, which demonstrated increased Evans Grade histopathologic and serum CA 19-9 response with autophagy inhibition (IRB 13-074, NCT01128296 ). Utilizing the resected FFPE tumor specimens from evaluable patients, we completed paired multiplex immunohistochemistry (CD4, CD8, FOXP3, CD20, CD68, Pan-CK) and T & B cell receptor RNA sequencing to assess the intratumoral adaptive immune response and correlates of outcome. Results: Autophagy inhibition increased the number of infiltrating CD8 T cells (1133±490 vs 712±460 average cells per high power field, p = 0.01), CD8:CD20 ratio (2.22±3.1 vs 0.96±1.1, p = 0.02) and reduced the CD4:CD8 ratio (2.04±0.87 vs 3.01±2.09, p = 0.03). No effect was observed on the number of immature or mature germinal center-like tertiary lymphoid structures (TLS), though the number of TLS correlated with increased infiltration of CD4 T cells (r = 0.40, p < 0.001), T-regulatory cells (r = 0.26, p = 0.03) and CD20 B cells (r = 0.65, p < 0.001). Although the total number of productive T and B cell receptors increased with autophagy inhibition (167217±105961 vs 97339±5,1628, p = 0.02), no apparent effects were observed on Vαβ TCR or BCR IgH, Igκ, Igλ clonality. Independent of treatment, intratumoral CD8 counts were associated with an improved CA 19-9 response (r = 0.32, p = 0.04) and in a subset of short term ( < 2 years, n = 17) and long term ( > 4 years, n = 10) survivors (LTS), a lowered CD4:CD8 ratio was identified in LTS (1.83±0.63 vs 2.8±0.90, p = 0.01). Dominance of B cell receptors was a prominent feature of the immune repertoire in all patients (average expression: Vα 0.6%, Vβ 0.8%, IgH 18.9%, Igκ 32.3%, Igλ 47.2%) with an IgA skewed immunoglobulin class switching (mean 63% of all BCRs). Increased αβ T cell receptor clonality above the median level was associated with a CA 19-9 response (r = 0.37, p = 0.06) and greater overall survival (median OS 38.3 vs 19.3 months, p = 0.02), indicative of possible tumor specific clonal expansion. Conclusions: Preoperative autophagy inhibition increased the number of tumor infiltrating CD8 T cells in patients with localized pancreatic cancer. Intratumoral αβ T cell receptor clonality was associated with CA 19-9 response and improved overall survival. Combination treatment regimens increasing PDAC specific CD8 responses are warranted. Clinical trial information: NCT01978184.

NRF2 activates macropinocytosis upon autophagy inhibition.

Su etal. demonstrate that upon inhibiting autophagy, an intracellular nutrient recycling pathway, pancreatic ductal adenocarcinoma cells upregulate NRF2-mediated transcription of macropinocytosis pathway components, thereby triggering an alternate route for tumors to scavenge nutrients from extracellular sources. Accordingly, the combined inhibition of autophagy and macropinocytosis may improve cancer treatment.

LncRNA BCYRN1-induced autophagy enhances asparaginase resistance in extranodal NK/T-cell lymphoma.

Background: Asparaginase (ASP) is the cornerstone drug in the treatment of extranodal NK/T-cell lymphoma (ENKTCL), and the mechanisms of resistance to ASP remain largely unknown. Long non-coding RNAs play important roles in chemotherapy resistance in various cancers. However, the expression of BCYRN1 and its role in ENKTCL still remain unidentified.Methods: Lentivirus-mediated BCYRN1 overexpression and knockdown were performed in SNK-6 cells. Cell autophagy was analyzed by adenovirus expressing GFP-LC3B fusion protein. RNA pull-down and RNA Binding Protein Immunoprecipitation Assay were performed to investigate the relationship between BCYRN1 and p53. Western blot analysis was performed to assess the effect of BCYRN1 on different autophagy pathways. Finally, in vivo xenograft tumor model was constructed to analyze the effect of BCYRN1 on tumor growth and ASP resistance.Results: BCYRN1 was overexpressed in ENKTCL than normal NK cells, and patients with higher expression had significantly inferior progression-free survival (PFS). The IC50 value of ASP was significantly increased in BCYRN1-overexpressed SNK-6 cells and BCYRN1 overexpression could resist the inhibitory effect of ASP on proliferation. ASP could induce concurrent apoptosis and autophagy in ENKTCL, and the latter process was enhanced by overexpression of BCYRN1, mainly through affecting both PI3K/AKT/mTOR and p53/mTOR pathways. BCYRN1 could induce the degradation of p53 via ubiquitination, thus resulting in enhancement of autophagy and ASP resistance, which could be reversed by drug-induced autophagy inhibition. The effect of BCYRN1 on tumor growth and autophagy were confirmed in vivo xenograft model.Conclusions: It was found that BCYRN1 was a valuable prognostic biomarker in ENKTCL. BCYRN1 could promote resistance to ASP by inducing autophagy, which could be reversed by inhibition of autophagy. Our findings highlight the feasibility of combining autophagy inhibition and ASP in the treatment of ENKTCL.

Abstract 1225: Co-inhibition of autophagy and MAPK signaling in RAS-driven cancers

Abstract RASis mutated in a number of cancers, including KRAS-driven colorectal and pancreatic cancer, HRAS-driven bladder cancer, and NRAS-driven melanoma, all of which result in downstream activation of the RAF>MEK>ERK (MAPK) and PI3K>AKT signaling pathways. Most RAS GTPases cannot be targeted directly, and strategies blocking both MAPK and PI3K signaling simultaneously are limited by high toxicity and compensatory signaling mechanisms. Interestingly, oncogene-activated MAPK or PI3K signaling pathways are reasonably well described orchestrators of metabolic transformation through multiple pathways. Our lab has recently shown that autophagy,a conserved metabolic process of self-digestion that recycles intracellular components, is increased in KRAS-driven pancreatic ductal adenocarcinoma (PDA) upon MAPK pathway inhibition. We further showed that co-inhibition of autophagy and MEK1/2, a MAPK component, leads to tumor regression of patient-derived PDA xenografts in mice. Our proposed combination therapy has recently been translated into a phase I/II clinical trial for PDA patients with advanced disease. Our most recent data show that inhibition of RAS>RAF>MEK>ERK signaling also results in induction of autophagy in other RAS-driven cancers, including KRAS-driven colorectal cancer, HRAS-driven bladder cancer and NRAS-driven melanoma. Furthermore, ourin vivodata demonstrate a robust regression of tumors upon combined inhibition of autophagy and MEK1/2 in engrafted KRAS-driven colorectal cancer cells xenografted in mice. Patient data from two KRAS-driven colorectal cancer patients, who were recently treated off-label, indicated clinical responses to the combination treatment co-targeting autophagy and MEK1/2. Altogether, these data suggest that co-inhibition of autophagy and oncogenic signaling may represent a potential new treatment strategy for multiple RAS-driven cancer types. Future experiments aim for a better mechanistic understanding of the combination treatment co-targeting autophagy and oncogenic signaling, with the objective to propose novel therapeutic strategies for cancer patients with RASmutations. Citation Format: Mona Foth, Conan Kinsey, Sophia Schuman, Benjamin Battistone, Emilio Cortes Sanchez, David Kircher, Bryan Welm, Sheri Holmen, Martin McMahon. Co-inhibition of autophagy and MAPK signaling in RAS-driven cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1225.

Clarithromycin effectively enhances doxorubicin-induced cytotoxicity and apoptosis in MCF7 cells through dysregulation of autophagy

PurposeUse of autophagy inhibitors in combination with chemotherapy has become a novel chemotherapeutic strategy. In this study, we aimed to determine whether the effectiveness of doxorubicin (DOX) is augmented by clarithromycin (CAM) in MCF7 cells and the molecular mechanisms involved. Materials and methodsCombined cytotoxicity of CAM and DOX was assessed by MTT assay and was analyzed using the Chou-Talalay's method. To clarify the underlying mechanisms, several factors, including apoptosis (Annexin V/propidium iodide staining), intracellular level of DOX (spectrofluorimetry) and P-glycoprotein activity (Rhodamin 123 efflux assay) were measured. In addition, autophagy was evaluated by intracellular labeling with anti-LC3II and LysoTrackerGreen (LTG) staining and analyzed by flowcytometry. ResultsThe anti-proliferation effect of DOX was synergistically enhanced by CAM in MCF7 cells and was associated with an increase in the apoptotic cell death. However, the intracellular level of DOX remained unchanged in the presence of CAM. Based on the findings, 100 μM of CAM did not exhibit any inhibitory effects on P-glycoprotein activity. Flow cytometric analysis indicated that DOX at IC20 concentration induced the autophagy flux, as confirmed by the increased level of LC3II and LTG signals. Moreover, combined treatment with DOX and CAM resulted in more pronounced LTG signals, but no change in LC3II. These results indicate that CAM blocks the autophagy flux induced by DOX. ConclusionsThese findings suggest that suppression of autophagy by CAM may promote chemotherapeutic outcome in breast cancer. However, further investigations are needed to evaluate the application of CAM in adjuvant breast cancer therapy.

The Switch between Protective and Nonprotective Autophagy; Implications for Autophagy Inhibition as a Therapeutic Strategy in Cancer

Autophagy, a process of cellular self-degradation and cell survival whereby the cell generates energy and metabolic intermediates under conditions of stress (i.e., nutrient deprivation), is also commonly induced in tumor cells in response to chemotherapy and radiation. While chemotherapy-induced autophagy and radiation-induced autophagy are generally considered to have cytoprotective functions, thereby reducing tumor cell sensitivity (and potentially conferring resistance) to various treatment modalities, autophagy can also be nonprotective; furthermore, the nature of the autophagy can be altered via the “autophagic switch” depending on such factors as the p53 status of the tumor cells. Defective or compromised autophagy has also been associated with neurodegenerative diseases, raising concerns as to the impact of autophagy inhibition on normal tissue function. Furthermore, the impact of autophagy inhibition on the immune system response to therapy as well as the influence of autophagy inhibition in combination with chemotherapy or radiation on critical tissue sites such as the bone marrow remain uncertain. These are factors requiring serious consideration within the context of current clinical efforts to exploit autophagy inhibition as a therapeutic strategy in cancer.

Characterization of Bypassing Kinases Inferred By Phosphoproteomics in a Midostaurin Resistant Myeloid Cell Line Model

Background: Chronic myeloid neoplasms are heterogeneous malignancies caused by sequential accumulation of genetic lesions in hematopoietic stem cells with a tendency to evolve towards acute myeloid leukemia. Genomic approaches can stratify patients according to their mutational landscape but are limited in predicting response to therapeutic agents. Reliable biomarkers to identify patients' chances for response to targeting compounds remain a crucial necessity in the current post-genomic era of precision medicine. We hypothesize that differential phosphoproteomic (PP) profiles might represent a suitable functional biological layer that allows to identify relevant determinants of oncogenic phenotypes. Aim: The aim of the project was to build a bioinformatics pipeline using PP data to i) identify differentially phosphorylated sites, ii) infer targetable kinases and iii) characterize involved oncogenic pathways. Here, we present results from further exploratory experiments of a previously established PP analysis pipeline that enables to infer sensitive/bypassing kinase activities in a Midostaurin/PKC412 (MIDO) resistant myeloid cell line model. Methods: For the validation of our analysis pipeline, we have previously used the human myeloid cell lines K562 and MOLM13, driven by the oncogenic BCR-ABL1 and FLT3 kinases, and exposed to the kinase inhibitors Nilotinib (NILO) and MIDO, respectively. For the current biological study, we applied our pipeline to explore MIDO resistance mechanisms in published MIDO-resistant (rMOLM13) and sensitive (sMOLM13) cell lines (kindly provided by E. Weisberg, Dana Farber Cancer Institute, USA). r/sMOLM13 were cultured in triplicates for 24hrs and subsequently exposed for 1h to 25nM MIDO or DMSO (CTRL). PPs were enriched with titanium-dioxide and analyzed by mass spectrometry (nanoLC-MS2). Our previously validated Kinase Activity Enrichment Analysis (KAEA) bioinformatics pipeline was further applied to infer kinase activities based on the SetRank enrichment algorithm. KAEA integrates substrate-kinase datasets from five experimentally validated databases complemented with NetworKIN in-silico predictions. The pipeline is supported by a Shiny web-app interface to allow interactive visualization and interrogation of the data. Results: In our previous validation experiments, K562 treated with NILO showed expected inhibition of ABL1 and KIT, whereas MOLM13 treated with MIDO showed expected inhibition of PRKC and downstream kinases of FLT3 (AKT1 and MAPK). In the current biological exploration, we compared rMOLM13 vs sMOLM13 exposed to MIDO and CTRL. rMOLM13 showed slower proliferation and vacuolization compared to sMOLM13. 13,682 phosphorylation sites were identified and the three replicates clustered adequately in the corresponding four conditions sMOLM13_CTRL, sMOLM13_MIDO, rMOLM13_CTRL and rMOLM13_MIDO (fig. 1A). Clusters of over- or underexpressed sites associated with distinct conditions emerged in the heatmap. We performed a KAEA comparison of rMOLM13_MIDO vs sMOLM13_MIDO (fig. 1B) and rMOLM13_CTRL vs sMOLM13_CTRL (fig. 1C). CDK1 and other CDKs enriched as underactive in rMOLM13, in line with the observed reduced proliferation. Most interestingly, kinases PDHK1, PDHK2 and PKM, all pyruvate kinases involved in regulation of mitochondrial metabolism, enriched as overactive, as well as MAPK1/3 and CK2. Reduced proliferation, vacuolization and patterns of enriched kinases pointed towards autophagy as potential resistance mechanism in rMOLM13. We performed preliminary experiments with autophagy inhibitors in combination with MIDO, which supported a synergistic activity (fig. 1D). More results of combined inhibition of the identified kinases will be shown at the meeting. Conclusions: With our PP analysis pipeline, we were able to characterize, in unanticipated detail, dynamic changes of PP profiles, kinase activities and potentially involved oncogenic pathways in a MIDO resistant myeloid cell line model. We identified biologically relevant mechanisms of resistance that will be explored as potential targets for combination therapy. Our experiments in myeloid cell lines provide a proof of concept for our analysis pipeline. They allow us to move forward to the analysis of primary patient samples and promote precision medicine based on functional biomarkers identified by PP in patients with myeloid neoplasms. Disclosures Jankovic: Celgene: Other: financial support for travel. Bonadies:Roche: Other: financial support for travel, Research Funding; Novartis: Other: financial support for travel, Research Funding; Celgene: Other: financial support for travel, Research Funding; Janssen: Other: financial support for travel; Amgen: Other: financial support for travel; Sanofi Genzyme: Other: financial support for travel.

2014P - IGF1R inhibition affects the survival of HT29 cancer cells by alterations of the TLR9- and autophagy signaling

BackgroundIn HT29 cells, an interplay between self-DNA-induced TLR9-signaling and autophagy response was found with significant effects on cell survival and kinetic parameters. The IGF/IGF1R system plays a determinant role in the pathogenesis and progression of colorectal cancer. This pathway is upstream of mTORC1, a negative regulator of autophagy. I mammalian systems chronic IGF1R inhibition was shown to attenuate autophagosome formation. The interrelated action of IGF1R inhibition and TLR9/autophagy signaling in cancer cells has not yet been clarified. The present study was designed to assess this complex network using HT29 colon carcinoma cells. MethodsHT29 cells were incubated for 72h with genomic (g), hypermethylated (m), and fragmented (f) tumor self-DNAs, and with/without inhibitors of IGF1R (picropodophyllin), autophagy (chloroquine) and TLR9 (ODN2088), respectively. Cell viability was measured by MTT assay. Transcriptional changes of TLR9-signaling, IGF1R, mTORC, Akt, and the autophagy process were assayed by Human v3 miRNA Assay (NanoString). Autophagy proteins were detected by immunocytochemistry, while morphology of apoptosis and autophagy by transmission electron microscopy (TEM). ResultsIn case of autophagy g- and f-DNAs caused significant upregulation of Beclin1, Atg16L1, LC3 mRNAs, and downregulation of mTORC, and Akt, verified by immunocytochemistry, as well. IGF1R-inhibition alone altered inversely the autophagy-associated gene- and protein-expressions. Upon combined inhibition of autophagy, TLR9 and/or IGF1R-signaling varying degree of autophagy was detected in all groups of tumor cells according to NanoString and TEM. Incubation with IGF1R inhibitor and with m-DNA no expected suppression of tumor cell survival, induction of apoptotic death, and activation of mitophagy were found. Moreover, IGF1R inhibition affected negatively the cell-protective effect f-DNA on macroautophagy and lipophagy. ConclusionsOur study provided evidence for an interaction between the inhibited IGF1R and TLR9/autophagy signaling with major influences on survival, proliferation and death of HT29 cells subjected to intact/modified self-DNAs. (Funded by StartUp.). Legal entity responsible for the studyFerenc Sipos. FundingStartUp. DisclosureAll authors have declared no conflicts of interest.

2016P - Modulation of TLR9-dependent autophagy response via inhibition of c-Met signaling influences the survival of HT29 cancer cells

BackgroundIn HT29 cells, an interplay between self-DNA-induced TLR9- and autophagy responses was found with remarkable effects on survival and differentiation of tumor cells. c-Met activation is known to drive the progression of colorectal cancer by promoting signaling cascades that mainly result in alterations of cell motility, survival and proliferation. c-Met inhibition was shown to inhibit autophagy. In cancer cells the interrelated role of c-Met inhibition and TLR9/autophagy signaling has not yet been clarified, so we aimed to assess this complex interplay. MethodsHT29 cells were incubated for 72h with genomic (g), hypermethylated (m), and fragmented (f) tumor self-DNAs, and with/without inhibitors of c-Met (diisothiocyanatostilbene), autophagy (chloroquine) and TLR9 (ODN2088), respectively. Cell viability was measured by MTT assay. Transcriptional changes of TLR9-signaling, PI3K, CD95, c-Met, Bcl2, cytochrome-c, and the autophagy process were assayed by Human v3 miRNA Assay (NanoString). Autophagy proteins were detected by immunocytochemistry, while morphology of apoptosis and autophagy by transmission electron microscopy (TEM). ResultsSelf-DNAs g and f resulted in significant upregulation of Beclin1, Atg16L1, LC3 mRNAs, and downregulation of PI3K, Bcl2, CD95, and cytochrome-c, verified by immunocytochemistry, as well. c-Met inhibition alone altered inversely the autophagy-associated gene- and protein-expressions. In each group of tumor cells using combined inhibition of autophagy, TLR9 and/or c-Met-signaling varying degree of autophagy was observed according to NanoString and TEM. Following combined incubation with c-Met inhibitor and m-DNAs no expected suppression of tumor cell survival and induction of apoptosis and mitophagy were detected. Further, c-Met inhibition changed the cell-protective effect f-DNA on macroautophagy. ConclusionsOur study provided evidence for an intense crosstalk between the inhibited c-Met canonical and non-canonical signaling pathways, and the TLR9/autophagy response with profound impacts on survival, proliferation and death of HT29 cells subjected to intact/modified self-DNAs. Legal entity responsible for the studyFerenc Sipos. FundingStartUp. DisclosureAll authors have declared no conflicts of interest.

Abstract 4271: Induction of STK11-dependent cytoprotective autophagy in breast cancer cells upon Honokiol treatment

Abstract Background and Aim: Honokiol, a natural phenolic compound isolated from an extract of seed cones from Magnolia grandiflora, is widely known for its therapeutic potential as an antioxidant, anti-inflammatory, antithrombosis, and anti-depressant agent. Our recent studies show that honokiol impedes breast carcinogenesis. Cancer cells undergo cytoprotective autophagy and evade chemotherapy therefore many clinical trials are investigating the efficacy of autophagy inhibition in combination with chemotherapy. In the present study, we investigated the involvement of autophagic process in honokiol-mediated functional networks in breast cancer and explored the efficacy of combination regimens involving honokiol and chloroquine. Methods: Autophagy studies were conducted utilizing immunoblot, RT-PCR, immunofluorescence analyses, confocal imaging and transmission electron microscopy for autophagy markers. Autophagic flux was analyzed using a plasmid tfLC3B and acridine orange staining. The fusion of autophagosome and lysosome was examined by using GFP-LC3/LysoTracker-red. Functional impact of autophagic process was evaluated using genetic knockout (KO) of AGT7 and BECN1 in MCF7 cells as well as combined treatment with autophagy inhibitors (3-MA, Baf-1 and CQ) and honokiol. Alterations in ATP levels were measured by ATPliteTM luminescence assay. In vivo studies using mammary gland implantation of cancer cells in NOD-SCID mice were conducted to evaluate the efficacy of combination regimen of Honokiol and Chloroquine. Results: We found that Honokiol induces autophagy flux, increases accumulation of autophagosomes and elevates LC3B-II-conversion. Utilizing tandem-mCherry-GFP-LC3B assay and LysoTracker Red-staining, we observed that honokiol increases the autophagosome/lysosome fusion in breast cancer cells. We found that Honokiol induces autophagic response in a STK11-dependent manner as STK11-null breast cancer cells do not exhibit LC3B-II-puncta in response to Honokiol. Next, we explored the functional impact of autophagy in Honokiol mediated breast cancer inhibition and found that inhibiting autophagosome formation, abrogating autophagosome-lysosome fusion or genetic-knockout of BECN1/Beclin1 and ATG7 effectively increases the efficacy of Honokiol. These results clearly showed the cytoprotective nature of Honokiol-mediated autophagy and put forth the notion that a combined strategy of autophagy-inhibition with Honokiol would be more effective. Indeed, our in vivostudies showed that a combined treatment with Honokiol and Chloroquine can effectively inhibit primary tumor growth as well as metastatic progression. Conclusion: Together, these results implicate that honokiol is a potent inducer of cytoprotective autophagy and a combined treatment of honokiol and chloroquine is a promising therapeutic strategy for breast cancer. Citation Format: Nethaji Muniraj, Marey Shriver, Arumugam Nagalingam, Sumit Siddharth, Sheetal Parida, Neeraj K Saxena, Dipali Sharma. Induction of STK11-dependent cytoprotective autophagy in breast cancer cells upon Honokiol treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4271.

Autophagy inhibition specifically promotes epithelial-mesenchymal transition and invasion in RAS-mutated cancer cells

ABSTRACTMacroautophagy/autophagy inhibition is a novel anticancer therapeutic strategy, especially for tumors driven by mutant RAS. Here, we demonstrate that autophagy inhibition in RAS-mutated cells induces epithelial-mesenchymal transition (EMT), which is associated with enhanced tumor invasion. This is at least partially achieved by triggering the NFKB/NF-κB pathway via SQSTM1/p62. Knockdown of ATG3 or ATG5 increases oncogenic RAS-induced expression of ZEB1 and SNAI2/Snail2, and activates NFKB activity. Depletion of SQSTM1 abolishes the activation of the NFKB pathway induced by autophagy inhibition in RAS-mutated cells. NFKB pathway inhibition by depletion of RELA/p65 blocks this EMT induction. Finally, accumulation of SQSTM1 protein correlates with loss of CDH1/E-cadherin expression in pancreatic adenocarcinoma. Together, we suggest that combining autophagy inhibition with NFKB inhibitors may therefore be necessary to treat RAS-mutated cancer.Abbreviations: 4-OHT: 4-hydroxytamoxifen; DIC: differential interference contrast; EMT: epithelial-mesenchymal transition; ESR: estrogen receptor; MAPK/ERK: mitogen-activated protein kinase; iBMK: immortalized baby mouse kidney epithelial cells; MET: mesenchymal-epithelial transition; PI3K: phosphoinositide 3-kinase; RNAi: RNA interference; TGFB/TGF-β: transforming growth factor beta; TNF: tumor necrosis factor; TRAF6: TNF receptor associated factor 6.