Vps34 Inhibitor Research Articles

Abstract C036: Vertical inhibition of the autophagy pathway enhances sensitization to RAS MAPK pathway inhibition in pancreatic ductal adenocarcinoma

Abstract PDAC growth is characterized by dependencies on both mutant KRAS signaling and autophagy. Our group and others previously demonstrated that inhibition of the RAF/MEK/ERK pathway in PDAC induces an increased dependence on autophagy, a metabolic nutrient scavenging process by which cellular components are recycled in times of nutrient stress. Combined inhibition of the ERK MAPK pathway and autophagy inhibited the growth of multiple preclinical models of PDAC. Based on these findings, combined ERK/MEK inhibition and hydroxychloroquine (HCQ) is currently under clinical evaluation (NCT03825289, NCT04386057). However, resistance to this combination has already been described. To identify sensitizers to CQ treatment, our lab performed a CRISPR-Cas9 mediated loss-of- function screen using an sgRNA library targeting genes associated with cancer signaling pathways. Surprisingly, we identified multiple autophagy related genes, indicating that concurrent inhibition of two nodes of the autophagy pathway may be a more effective method of inhibiting autophagy in PDAC. For example, we identified, PIK3C3, the gene that encodes for VPS34, a protein essential for the nucleation of autophagosomes, as a potential sensitizer to CQ treatment. Inhibition of VPS34 with the compound SAR405, resulted in both decreased autophagic flux and impaired proliferation. Additionally, VPS34 inhibition sensitized cells to CQ treatment, leading to further reduced proliferation. These results prompted us to hypothesize that inhibition of ULK1, a serine/threonine kinase critical for the initiation of autophagy, could also sensitize PDAC cells to CQ treatment. We found that ULK1 inhibition further reduced CQ-mediated anti-proliferative effects. Additionally, both ULK1 and VPS34 inhibitor treatment sensitized cells to PIKfyve inhibition with apilimod, a chemically and mechanistically distinct inhibitor of the termination phase of the autophagic pathway. Previous studies have indicated that autophagy inhibition alone is not a viable therapeutic avenue for the treatment of PDAC. Thus, we next determined whether vertical inhibition of the autophagy pathway improves the efficacy of combined inhibition of the RAS and autophagy pathway. We found that both VPS34 inhibition and ULK1 inhibition can decrease RAS inhibitor induced autophagic flux. Finally, vertical inhibition of the autophagic pathway via VPS34 inhibition and CQ sensitized PDAC cells to RAS inhibition to further reduce PDAC proliferation. Ongoing and future studies are aimed at elucidating the mechanism leading to decreased proliferation of combined anti-autophagy and RAS inhibitor therapies, as well as mechanistically understanding the effect of autophagy inhibition at multiple nodes on autophagic flux. Citation Format: Mallory K Roach, Jonathan M DeLiberty, Elyse G Schechter, Runying Yang, Noah L Pieper, Clint A Stalnecker, Kirsten L Bryant. Vertical inhibition of the autophagy pathway enhances sensitization to RAS MAPK pathway inhibition in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C036.

Abemaciclib and Vacuolin-1 decrease aggregate-prone TDP-43 accumulation by accelerating autophagic flux

(Macro)autophagy is a cellular degradation system for unnecessary materials, such as aggregate-prone TDP-43, a central molecule in neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Abemaciclib (Abe) and vacuolin-1 (Vac) treatments are known to induce vacuoles characterized by an autophagosome and a lysosome component, suggesting that they facilitate autophagosome-lysosome fusion. However, it remains unknown whether Abe and Vac suppress the accumulation of aggregate-prone TDP-43 by accelerating autophagic flux. In the present study, the Abe and Vac treatment dose-dependently reduced the GFP/RFP ratio in SH-SY5Y neuroblastoma cells stably expressing the autophagic flux marker GFP-LC3-RFP-LC3ΔG. Abe and Vac also increased the omegasome marker GFP-ATG13 signal and the autophagosome marker mCherry-LC3 localized to the lysosome marker LAMP1-GFP. The Abe and Vac treatment decreased the intracellular level of the lysosome marker LAMP1-GFP in SH-SY5Y cells stably expressing LAMP1-GFP, but did not increase the levels of LAMP1-GFP, the autophagosome marker LC3-II, or the multivesicular body marker TSG101 in the extracellular vesicle-enriched fraction. Moreover, Abe and Vac treatment autophagy-dependently inhibited GFP-tagged aggregate-prone TDP-43 accumulation. The results of a PI(3)P reporter assay using the fluorescent protein tagged-2 × FYVE and LAMP1-GFP indicated that Abe and Vac increased the intensity of the PI(3)P signal on lysosomes. A treatment with the VPS34 inhibitor wortmannin (WM) suppressed Abe-/Vac-facilitated autophagic flux and the degradation of GFP-tagged aggregate-prone TDP-43. Collectively, these results suggest that Abe and Vac degrade aggregate-prone TDP-43 by accelerating autophagosome formation and autophagosome-lysosome fusion through the formation of PI(3)P.

Abstract 4711: Evaluation of effect of autophagy inhibition by SAR405, a selective Vps34 inhibitor, on proliferation of pleural mesothelioma cells

Abstract Pleural mesothelioma is a poor prognostic malignancy because the efficacy of therapies remains limited; thus, there is an urgent need to elucidate the pathophysiology leading to newer treatment modalities. Macroautophagy (hereafter referred to as autophagy) is an intracellular degradation system in which cytoplasmic components are degraded by the lysosome. Autophagy plays pivotal roles in various physiological processes, including adaptation to starvation, preimplantation embryonic development, elimination of intracellular pathogens, and regulation of innate and adaptive immunity. In cancer cells, autophagy may have the potential to function protectively for the cells and, alternatively, to work suppressively. It can promote tumor growth in established cancers through autophagy-mediated intracellular recycling, providing substrates for metabolism and maintain that the functional pool of mitochondria. On the other hand, it may lead to a cytotoxic effect called autophagy-dependent cell death. Several reports have demonstrated that autophagy has cytoprotective effects on pleural mesothelioma cell lines. In this study, we assessed effects of inhibition of autophagy using SAR405, which specifically inhibits Vps34 in an early step of autophagy, on pleural mesothelioma cells. Additionally, we investigated whether SAR405 could enhance the growth suppression induced by cisplatin in these cells. Mesothelioma cell lines, including NCI-H28 (H28), NCI-H2452 (H2452), and MSTO-211H (211H), were cultured with 0.1-50 μM SAR405 or 1.0-5.0 μM cisplatin for cell viability analysis using water-soluble tetrazolium salt-8, and cell cycle analysis. For the assessment of autophagy, a plasmid, pMRX-IP-GFP-LC3-RFP-LC3ΔG, was stably transfected into mesothelioma cells. Transfected cells generate GFP-LC3-RFP-LC3ΔG, and it is cleaved into GFP-LC3 and RFP-LC3ΔG by inherent ATG4. GFP-LC3 levels are decreased by autophagy activation, while RFP-LC3ΔG serves as an internal control. SAR405 dose-dependently suppressed cell viability, accompanied by a significantly increase in the proportions of cells in the G2/M phase. The IC50 of SAR405 at 72 hours was 11.5 μM for H28, 16.7 μM for H2452, and 14.9 μM for 211H. SAR405 inhibited autophagy 24 hours after treatment at 2.5 μM for H28 and 211H, and at 5.0 μM for H2452. Furthermore, cisplatin induced autophagy at 5.0 μM for H28, 0.5 μM for H2452, 1.0 μM for 211H. The combination of 2.5-10 µM SAR405 and 0.5-5.0 µM cisplatin exhibited additive or synergistic effects on cell growth inhibition in all plural mesothelioma cell lines. In conclusion, Vps34 inhibition by SAR405 suppressed cell growth in pleural mesothelioma cells, accompanied by autophagy inhibition. The combination of SAR405 and cisplatin resulted in a synergistic inhibition of cell proliferation. Citation Format: Yoshiki Kuwabara, Kosuke Sakai, Shigeru Ishi, Shin Yokosuka, Masatoshi Abe, Tomoyuki Takahashi, Yuichiro Kawano, Hiroaki Nishimura, Maiko Toda-Sasaki, Yumiko Kobayashi-Ogawa, Satoshi Kikuchi, Yusuke Hirata, Hiroyuki Kyoyama, Gaku Moriyama, Nobuyuki Koyama, Kazutsugu Uematsu. Evaluation of effect of autophagy inhibition by SAR405, a selective Vps34 inhibitor, on proliferation of pleural mesothelioma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4711.

Abstract C074: VPS34 inhibition delays activation-induced STING degradation to prolong STING signaling and improve anti-tumor efficacy in preclinical models

Abstract Background: The STING/interferon (IFN) response pathway has been a target of cancer immunotherapy for many years, as a method for reactivating the anti-tumor immune response. Stimulation of the STING pathway leads to activation and infiltration of immune cells into the tumor, and cancer cell death. However, multiple STING agonists have failed in the clinic due to poor efficacy or toxicity1. STING activation leads to its degradation within a few hours through trafficking to the lysosome, as a mechanism to control immune cell hyperactivation2. Although this safety mechanism is important for preventing autoimmunity and chronic inflammation, it diminishes the continuous activation of immune-mediated cancer cell killing, especially in an immunosuppressive tumor microenvironment. VPS34 is a lipid kinase which plays an integral role in endosomal trafficking and autophagy3. Previous publications have demonstrated a role for VPS34 inhibition in increased effector immune cell infiltration, which synergizes with anti-PD1 therapy, demonstrating a link to immune activation4. Herein, we demonstrate that the in vivo combination efficacy with a VPS34 inhibitor and a STING agonist is due to reduced degradation of activated STING, through the inhibition of VPS34-mediated endosomal trafficking. Materials and Methods: Cancer cell lines were cultured using recommended medium. STING activity was measured by Western blot and via a CisBio pTBK1 kit. ADU-S100/MSA-2 were sourced from MedChemExpress. CCL5, CXCL10 and IFNb ELISA’s were purchased from R&D systems. Primers used to measure mRNA expression were obtained from IDT. Results: DP-9244 is a potent and selective VPS34 inhibitor. The combination of DP-9244 with the STING agonist, ADU-S100, enhances IFN-mediated gene expression and cytokine secretion in the murine melanoma cell line, B16F10, and in the human and murine myeloid cell lines THP-1 and DC2.4, demonstrating combination activity in both cancer and immune cells. This combination activity is STING-dependent as demonstrated by the complete loss of IRF and NFκB activity in the THP-1 STING-/- cell line when treated with ADU-S100, DP-9244 or the combination. Mechanistically, VPS34 inhibition delays degradation of activated STING and prolongs downstream signaling of pTBK1 in multiple human and murine cancer cell lines. Importantly, the in vitro effects extend to in vivo efficacy in the B16F10 model, in which the combination of DP-9244 and ADU-S100 results in increased tumor growth control and number of mice responding to treatment. Conclusions: These data provide a strong preclinical rationale to combine a VPS34 inhibitor with a STING agonist as a method of improving STING pathway mediated anti-tumor efficacy.

Abstract 5590: Inhibition of the autophagy protein Vps34 induces tumor cell secretion of proinflammatory chemokines

Abstract Autophagy is the cellular process by which cytoplasmic contents are degraded and recycled through a lysosomal pathway. In cancer, autophagy can contribute to both tumor promotion and tumor suppression, and emerging evidence supports a role in resistance to immunotherapy. We investigated whether inhibition of autophagy with a small molecule inhibitor of the class III phosphoinositide 3-kinase Vps34 could stimulate anti-tumor immune responses and potentiate the efficacy of immune checkpoint blockade (ICB). Treatment with the Vps34 inhibitor (Vps34i) inhibited autophagy and was weakly cytotoxic to B16F10, MC38, EMT6 and CT26 mouse syngeneic tumor cell lines. In certain cancers, autophagy has been linked to lysosomal degradation of MHC I proteins, thereby downregulating expression and contributing to immune evasion. Notably, we observed minimal upregulation of MHC I surface expression on a subset of mouse and human cell lines. We further explored whether autophagy inhibition would result in release of any soluble immune stimulatory factors from mouse tumor cell lines. Inhibition of Vps34 markedly increased the secretion of several proinflammatory chemokines, including CCL5 and CXCL10. Increased chemokine secretion was also observed in a panel of human cancer cell lines, including NCI-H2009 lung cancer and PC-3 prostate cancer, confirming that the response is observed in human cancer cell lines derived from different tissue origins. As the chemokines CCL5 and CXCL10 are critical to the development of robust anti-tumor immunity, we next tested whether Vps34i treatment could potentiate the anti-tumor efficacy of ICB. We explored the activity of Vps34i as a single agent and in combination with anti-PD-1 antibody in three different syngeneic tumor models, including EMT6, CT26 and MC38. No single agent activity was observed in these models, however, combination of anti-PD-1 and Vps34i significantly inhibited the growth of CT26 tumors compared to control. Anti-tumor efficacy of combination treatment was not significantly different compared to single agents, and no complete tumor regressions were observed. No significant increases in CCL5 and CXCL10 were observed in the serum of combination treated mice, potentially explaining the lack of robust efficacy. Our results suggest that inhibition of autophagy promotes the secretion of proinflammatory cytokines from mouse and human cancer cells in vitro. As the autophagy pathway exhibits cross talk with other major cell signaling pathways, the tumor contexture may be a critical determinant of the role of autophagy in anti-tumor immune responses. Future experiments will explore which tumor contexts are sensitive to combination of autophagy inhibition and ICB. Citation Format: Marie Bernardo, Yu-an Zhang, Martin Graf, Jane Cheng, Fangxian Sun, Virna Cortez-Retamozo, Sukhvinder Sidhu, Eladio Marquez, Donald Jackson, Jack Pollard, Timothy R. Wagenaar, Donald Shaffer. Inhibition of the autophagy protein Vps34 induces tumor cell secretion of proinflammatory chemokines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5590.

Abstract 2116: VPS34 inhibitor SB02024 activates cGAS-STING signaling and sensitizes tumors to STING agonist

Abstract Novel approaches to reduce tumor immunosuppression and improve responses to anti-cancer immunotherapies based on immune-checkpoint inhibitors are needed. Emerging evidence demonstrates that autophagy inhibition enhances anti-tumor immunity by tumor cell-intrinsic and extrinsic mechanisms. Recently, we reported that pharmacological inhibition of VPS34 (PIK3C3), a lipid kinase regulating autophagy initiation, decreases tumor growth and improve the efficacy of anti-PD-1/PD-L1 therapy in melanoma and colorectal cancer mouse models. This effect was dependent on increasing T and NK cell infiltration as well as the expression of CCL5 and CXCL10 chemokines in the tumor microenvironment. Here, we explored the signaling mechanisms underlying the chemokine release following treatment with VPS34 inhibitor SB02024. We found that both pharmacological and RNAi-mediated inhibition of VPS34 activated a cGAS-STING-dependent type I interferon response in renal cell carcinoma (RCC) and melanoma cell lines. Furthermore, combination treatment of VPS34 inhibitor SB02024 with STING agonist ADU-S100 or cGAMP increased the mRNA expression and release of proinflammatory cytokines in human and murine RCC and melanoma cancer cell lines. Oral administration of SB02024 in combination with intratumoral injections of ADU-S100 significantly decreased tumor growth and weight and improved mice survival of B16-F10 tumor-bearing mice. Taken together, our data demonstrates that targeting of VPS34 results in a cGAS/STING-mediated increase of pro-inflammatory cytokine secretion and synergizes with a STING agonist. We believe that systemic VPS34 inhibition using SB02024 would be of major interest in combination or as an alternative to STING agonists to improve anti-tumor immune responses. Citation Format: Yasmin Yu, Muhammad Z. Noman, Santiago Parpal, Simone C. Kleinendorst, Kristine B. Saether, Andrey Alexeyenko, Jenny Viklund, Martin Andersson, Jessica Martinsson, Katja P. Tamm, Angelo De Milito, Bassam Janji. VPS34 inhibitor SB02024 activates cGAS-STING signaling and sensitizes tumors to STING agonist [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2116.

Hypoxia Induces Autophagy in Human Dendritic Cells: Involvement of Class III PI3K/Vps34.

Hypoxia is a component of both physiological and pathological conditions, including inflammation, solid tumors, and lymphoid tissues, where O2 demand is not balanced by O2 supply. During their lifespan, dendritic cells (DCs) are exposed to different pO2 and activate different adaptive responses, including autophagy, to preserve their viability and functions. Autophagy plays multiple roles in DC physiology. Very recently, we demonstrated that hypoxia shapes autophagy in DCs upon their differentiation state. Here, we proposed a role for PI3Ks, and especially class III PI3K/Vps34, that could be relevant in hypoxia-induced autophagy, in either immature or mature DCs. Hypoxia inhibited mTOR phosphorylation and activated a pro-autophagic program. By using different pharmacological inhibitors, we demonstrated that hypoxia-induced autophagy was mediated by PI3Ks, especially by Vps34. Furthermore, Vps34 expression was enhanced by LPS, a TLR4 ligand, along with the promotion of autophagy under hypoxia. The Vps34 inhibitor, SAR405, abolished hypoxia-induced autophagy, inhibited pro-survival signaling and viability, and increased the expression of proinflammatory cytokines. Our results underlined the impact of autophagy in the maintenance of DC homeostasis at both cell survival and inflammatory response levels, therefore, contributing to a better understanding of the significance of autophagy in DC physiology and pathology.

Tailored Parameterization of the LIE Method for Calculating the Binding Free Energy of Vps34-Inhibitor Complexes.

Vps34 is the only isoform of the PI3K family in fungi, making this protein an attractive target to develop new treatments against pathogenic fungi. The high structural similarity between the active sites of the human and fungal Vps34 makes repurposing of human Vps34 inhibitors an appealing strategy. Nonetheless, while some of the cross-reactive inhibitors might have the potential to treat fungal infections, a safer approach to prevent undesired side effects would be to identify molecules that specifically inhibit the fungal Vps34. This study presents the parameterization of four LIE models for estimating the binding free energy of Vps34–inhibitor complexes. Two models are parameterized using a multiparametric linear regression leaving one or more free parameters, while the other two are based on the LIE-D model. All of the models show good predictive capacity (R2 > 0.7, r > 0.85) and a low mean absolute error (MAE < 0.71 kcal/mol). The current study highlights the advantages of LIE-D-derived models when predicting the weight of the different contributions to the binding free energy. It is expected that this study will provide researchers with a valuable tool to identify new Vps34 inhibitors for relevant applications such as cancer treatment and the development of new antimicrobial agents.

Mitophagy Induction by ROS-PINK1 Signaling Protects Head and Neck Cancer From Radiotherapy

Approximate 25% of patients with local advanced head and neck cell carcinoma (HNSCC) treated with radiation still suffer from local relapse and are considered radiation resistant. We previously determined that radiation induces autophagy, a pro-survival cellular stress response, in HNSCC. In this study, we examine the consequence of autophagy inhibition and investigate the molecular mechanism underlying RT-induced autophagy.Autophagy was assessed using a nano-Luc LC3 reporter assay (Promega), immunoblotting and immunofluorescence for LC3 and acridine orange in multiple HNSCC cell lines. RNAi knockdown of EGFR, LAPTM4B and PINK1 were used to test the involved signaling molecules. CM-H2DCFDA was used as a measure of reactive oxygen species (ROS). Hydrogen peroxide was used to stimulate ROS production. Trolox, a ROS scavenger, was used to determine the correlation between ROS and RT-induced autophagy. Mitophagy determination was completed by using MitoTracker Red (Invitrogen) combined with immunofluorescence staining for LC3 and immunoblotting. Radiation was delivered using a RS225 cabinet irradiator at a dose rate of approximately 3 Gy/min with dose validation by TLD using custom, geometry specific phantoms. SAR405, a VPS34 inhibitor, was used to inhibit autophagy. A flank xenograft model using A253 cells was used to test the combination of autophagy inhibitors and radiotherapy in vivo.RT caused a two-fold increase in autophagy as assessed using the reporter assay and immunoblotting. Knockdown of EGFR and LAPTM4B, two proteins important in growth-factor deprivation induced autophagy, did not influence autophagy in RT-treated cells. RT did increase the accumulation of ROS (∼50%) and the dephosphorylation of mTOR (∼25%). Addition of Trolox to RT contributed to a considerable decrease in both ROS (∼50%) and autophagy (∼50%). RT also resulted in a mRNA elevation of PINK1 (∼1.6 fold), a mitochondrial modulator. Immunoblotting for LC3 in both the cytoplasmic and mitochondrial fraction indicated RT mostly increases mitophagy rather than bulk autophagy, which was confirmed using immunofluorescent staining for LC3 and MitoTracker red. Using a clonogenic survival assay, the combination of SAR405 and RT resulted in complete loss of cell survival suggesting a radiosensitizing effect. In vivo, SAR405 treatment combined with RT improved tumor control when compared to RT or SAR405 alone.RT-induced mitophagy involves ROS-PINK1 pathway and the regulation of mTOR. Inhibition of autophagy resulted in decreased cell survival in vitro and decreased in vivo tumor growth. These results suggest that targeting mitophagy may be a viable approach to sensitize HNSCC to radiation treatment.

Samm50 Promotes Hypertrophy by Regulating Pink1-Dependent Mitophagy Signaling in Neonatal Cardiomyocytes.

Pathological cardiac hypertrophy, the adaptive response of the myocardium to various pathological stimuli, is one of the primary predictors and predisposing factors of heart failure. However, its molecular mechanisms underlying pathogenesis remain poorly understood. Here, we studied the function of Samm50 in mitophagy during Ang II-induced cardiomyocyte hypertrophy via lentiviruses mediated knockdown and overexpression of Samm50 protein. We first found that Samm50 is a key positive regulator of cardiac hypertrophy, for western blot and real-time quantitative PCR detection revealed Samm50 was downregulated both in pressure-overload-induced hypertrophic hearts and Ang II-induced cardiomyocyte hypertrophy. Then, Samm50 overexpression exhibits enhanced induction of cardiac hypertrophy marker genes and cell enlargement in primary mouse cardiomyocytes by qPCR and immunofluorescence analysis, respectively. Meanwhile, Samm50 remarkably reduced Ang II-induced autophagy as indicated by decreased mitophagy protein levels and autophagic flux, whereas the opposite phenotype was observed in Samm50 knockdown cardiomyocytes. However, the protective role of Samm50 deficiency against cardiac hypertrophy was abolished by inhibiting mitophagy through Vps34 inhibitor or Pink1 knockdown. Moreover, we further demonstrated that Samm50 interacted with Pink1 and stimulated the accumulation of Parkin on mitochondria to initiate mitophagy by co-immunoprecipitation analysis and immunofluorescence. Thus, these results suggest that Samm50 regulates Pink1-Parkin-mediated mitophagy to promote cardiac hypertrophy, and targeting mitophagy may provide new insights into the treatment of cardiac hypertrophy.

ALK inhibition activates LC3B-independent, protective autophagy in EML4-ALK positive lung cancer cells

ALK inhibitors effectively target EML4-ALK positive non-small cell lung cancer, but their effects are hampered by treatment resistance. In the present study, we asked whether ALK inhibition affects autophagy, and whether this may influence treatment response. Whereas the impact of targeted therapies on autophagic activity previously have been assessed by surrogate marker proteins such as LC3B, we here thoroughly examined effects on functional autophagic activity, i.e. on the sequestration and degradation of autophagic cargo, in addition to autophagic markers. Interestingly, the ALK inhibitor Ceritinib decreased mTOR activity and increased GFP-WIPI1 dot formation in H3122 and H2228 EML4-ALK+ lung cancer cells, suggesting autophagy activation. Moreover, an mCherry-EGFP-LC3B based assay indicated elevated LC3B carrier flux upon ALK inhibition. In accordance, autophagic cargo sequestration and long-lived protein degradation significantly increased upon ALK inhibition. Intriguingly, autophagic cargo flux was dependent on VPS34 and ULK1, but not LC3B. Co-treating H3122 cells with Ceritinib and a VPS34 inhibitor or Bafilomycin A1 resulted in reduced cell numbers. Moreover, VPS34 inhibition reduced clonogenic recovery of Ceritinib-treated cells. In summary, our results indicate that ALK inhibition triggers LC3B-independent macroautophagic flux in EML4-ALK+ cells to support cancer cell survival and clonogenic growth.

Abstract A01: Vps34 promotes macropinocytosis in Tsc2-deficient cells

Abstract Purpose: The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) is constitutively active in many human cancers and in tuberous sclerosis complex (TSC). mTORC1 hyperactivation drives extensive metabolic reprogramming via several interconnected mechanisms, including glucose and glutamine utilization, nucleic acid synthesis, and lipid synthesis. We recently found that treatment with chloroquine (CQ), a lysosomal/autophagy inhibitor, upregulates the uptake and endosomal processing of cholesterol selectively in TSC2-deficient cells, creating a TSC2-specific dependence on exogenous cholesterol. This dependence on exogenous nutrients and the mechanisms of nutrient uptake may be therapeutically targetable. Our central hypothesis is that Tsc2-deficient cells depend on the uptake of exogenous nutrients via macropinocytosis to maintain cellular metabolic homeostasis and proliferation. Methods and Results: To investigate the role of macropinocytosis-mediated nutrient uptake in Tsc2-deficient cells, we measured the uptake of dextran, a polysaccharide taken up via macropinocytosis. Tsc2-deficient cells showed a striking increase in both dextran (3.6-fold, p&amp;lt;0.001) and exogenous protein uptake (~2-fold, p&amp;lt;0.001) relative to Tsc2-expressing cells. Interestingly, mTOR inhibition using Torin1 decreased dextran uptake (3-fold, p&amp;lt;0.001). Pharmacologic and genetic inhibition of the lipid kinase Vps34 markedly abrogated uptake of dextran in Tsc2-deficient cells (~50%, p&amp;lt;0.0001). Macropinocytosis was further increased in Tsc2-deficient cells that lack autophagic mechanisms (25%, p&amp;lt;0.001), suggesting that autophagy inhibition leads to dependence on exogenous nutrient uptake in Tsc2-deficient cells. Additionally, exogenous protein activates mTORC1 signaling of Tsc2-deficient cells grown in amino acid-free media. Treatment with ethylisopropylamiloride (EIPA) resulted in selective growth inhibition of Atg5-deficient, Tsc2-deficient cells (50%, p&amp;lt;0.0001). Genetic inhibition of autophagy (Atg5-/- MEFs) sensitized cells with Tsc2 downregulation to the Vps34 inhibitor, SAR405, resulting in growth inhibition (75%, p&amp;lt;0.0001). Finally, pharmacologic inhibition of lysosomal function with CQ and macropinocytosis with SAR405 inhibited tumor growth and increased tumor latency in an in vivo xenograft model of TSC. Conclusions: These data suggest that macropinocytosis is upregulated in Tsc2-deficient cells via a Vps34-dependent mechanism to support tumorigenesis. Macropinocytosis-mediated nutrient uptake represents an unexplored opportunity for therapeutic intervention in TSC and may also be relevant to other diseases characterized by mTORC1 hyperactivation. Citation Format: Charilaos Filippakis, Amine Belaid, Brian Siroky, Constance Wu, Nicola Alesi, Thomas Alesi, Julie Nijmeh, Hilaire Colletta Lam, Ellizabeth Henske. Vps34 promotes macropinocytosis in Tsc2-deficient cells [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A01.

An iron-dependent metabolic vulnerability underlies VPS34-dependence in RKO cancer cells.

VPS34 is a key regulator of endomembrane dynamics and cargo trafficking, and is essential in cultured cell lines and in mice. To better characterize the role of VPS34 in cell growth, we performed unbiased cell line profiling studies with the selective VPS34 inhibitor PIK-III and identified RKO as a VPS34-dependent cellular model. Pooled CRISPR screen in the presence of PIK-III revealed endolysosomal genes as genetic suppressors. Dissecting VPS34-dependent alterations with transcriptional profiling, we found the induction of hypoxia response and cholesterol biosynthesis as key signatures. Mechanistically, acute VPS34 inhibition enhanced lysosomal degradation of transferrin and low-density lipoprotein receptors leading to impaired iron and cholesterol uptake. Excess soluble iron, but not cholesterol, was sufficient to partially rescue the effects of VPS34 inhibition on mitochondrial respiration and cell growth, indicating that iron limitation is the primary driver of VPS34-dependency in RKO cells. Loss of RAB7A, an endolysosomal marker and top suppressor in our genetic screen, blocked transferrin receptor degradation, restored iron homeostasis and reversed the growth defect as well as metabolic alterations due to VPS34 inhibition. Altogether, our findings suggest that impaired iron mobilization via the VPS34-RAB7A axis drive VPS34-dependence in certain cancer cells.

Neuroprotective effects of memantine via enhancement of autophagy

IntroductionChemical intervention of autophagy has been investigated in clinical trials for various age-related conditions such as sarcopenia and neurodegeneration. However, at present, no autophagy inducer has been established as a disease-modifying agent against neurodegenerative diseases. MethodsWe screened a library consisting of 796 medicines clinically approved (in Japan) for autophagy enhancers as potential neurodegeneration therapeutics using HeLa cells stably expressing green fluorescent protein-microtubule-associated protein light chain 3 (GFP-LC3) followed by an analysis of the molecular mechanisms using various neuronal models. ResultsThe primary screening identified 152 hits in a static cellular state. A widely available Alzheimer's disease drug, memantine, which antagonizes N-Methyl-d-aspartate receptor (NMDAR), was one of the hits. Memantine increased the levels of LC3-II in a dose-dependent and time-dependent manner, and upregulated autophagic flux. In addition, the pharmacological effects of memantine on autophagy were independent of mTORC1 activity and NMDAR activation. Furthermore, a VPS34 inhibitor suppressed the memantine-induced LC3-II upregulation, suggesting that memantine may affect VPS34 complex activity. Notably, intracellular Huntington's disease-specific aggregates of elongated huntingtin, a well-established autophagy substrate, were significantly decreased by memantine. In addition, memantine enhanced elimination of degraded mitochondrial in neurons derived from induced pluripotent stem cells of PARK2 or PARK6 patients, who exhibited defective PINK1/parkin-mediated mitophagy, suggests that memantine accelerated the clearance of damaged mitochondria. ConclusionThese findings indicate that memantine may be beneficial for the treatment of neurodegeneration characterized by the abnormal accumulation of autophagy or mitophagy substrates.

Abstract 4273: Inhibition of autophagy increases HNSCC sensitivity to cancer therapies

Abstract Background: Radiation and EGFR-targeted therapies are commonly used in the treatment of head and neck squamous cell carcinoma (HNSCC). These treatments fail to control a significant number of cancers resulting in a 5-year survival rate that remains around 40-50%. We have shown that both cetuximab and radiation induce autophagy, a pro-survival cellular stress response, in head and neck cancer. In this study, we examine the consequence of autophagy inhibition and investigate the molecular mechanism underlying therapy-induced autophagy. Methods: Autophagy was assessed using a nano-Luc LC3 reporter (Promega), immunofluorescence for LC3, p62, and acridine orange in HNSCC cell lines. RNAi knockdown of EGFR and LAPTM4B were used to test the involved signaling molecules. Radiation was delivered using a RS225 cabinet irradiator at a dose rate of approximately 3 Gy/min with dose validation by TLD using custom phantoms. Cetuximab was delivered via intraperitoneal injection. Vps34 inhibitor SAR405 and ULK1 inhibitor SBI-0206965 were used to determine whether inhibition of autophagy reduces cell survival or represses cancer cell growth in the clonogenic assay. A flank xenograft model using A253 cells was used to test the combination of autophagy inhibitors and current therapies in vivo. Results: As previously shown, both cetuximab and radiation induced autophagy by two times. Knockdown of EGFR and LAPTM4B decreased autophagy (62.5% and 65%, respectively) when assessed using the nano-Luc reporter assay. Similar results were seen using IF. Using a clonogenic survival assay, the combination of SAR405 and radiation resulted in complete loss of cell survival suggesting a radiosensitizing effect. In vivo, SAR405 treatment improved tumor control when combined with radiation or cetuximab when compared to either treatment alone. Conclusions: Therapy induced autophagy is dependent upon expression of both EGFR and LAPTM4B. Inhibition of autophagy resulted decreased cell survival in vitro and resulted in decreased in vivo tumor growth. These results suggest that inhibition of autophagy may be a viable approach to sensitize HNSCC to anti-cancer treatments. Citation Format: Yong-Syu Lee, Justin Skiba, Jaimee Kubatzke, Kwangok P. Nickel, Randall Kimple. Inhibition of autophagy increases HNSCC sensitivity to cancer therapies [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 4273.

Vps34-mediated macropinocytosis in Tuberous Sclerosis Complex 2-deficient cells supports tumorigenesis

Tuberous Sclerosis Complex (TSC), a rare genetic disorder with mechanistic target of rapamycin complex 1 (mTORC1) hyperactivation, is characterized by multi-organ hamartomatous benign tumors including brain, skin, kidney, and lung (Lymphangioleiomyomatosis). mTORC1 hyperactivation drives metabolic reprogramming including glucose and glutamine utilization, protein, nucleic acid and lipid synthesis. To investigate the mechanisms of exogenous nutrients uptake in Tsc2-deficient cells, we measured dextran uptake, a polysaccharide internalized via macropinocytosis. Tsc2-deficient cells showed a striking increase in dextran uptake (3-fold, p < 0.0001) relative to Tsc2-expressing cells, which was decreased (3-fold, p < 0.0001) with mTOR inhibitor, Torin1. Pharmacologic and genetic inhibition of the lipid kinase Vps34 markedly abrogated uptake of Dextran in Tsc2-deficient cells. Macropinocytosis was further increased in Tsc2-deficient cells that lack autophagic mechanisms, suggesting that autophagy inhibition leads to dependence on exogenous nutrient uptake in Tsc2-deficient cells. Treatment with a macropinocytosis inhibitor, ethylisopropylamiloride (EIPA), resulted in selective growth inhibition of Atg5-deficient, Tsc2-deficient cells (50%, p < 0.0001). Genetic inhibition of autophagy (Atg5−/− MEFs) sensitized cells with Tsc2 downregulation to the Vps34 inhibitor, SAR405, resulting in growth inhibition (75%, p < 0.0001). Finally, genetic downregulation of Vps34 inhibited tumor growth and increased tumor latency in an in vivo xenograft model of TSC. Our findings show that macropinocytosis is upregulated with Tsc2-deficiency via a Vps34-dependent mechanism to support their anabolic state. The dependence of Tsc2-deficient cells on exogenous nutrients may provide novel approaches for the treatment of TSC.

SAR405, a Highly Specific VPS34 Inhibitor, Disrupts Auditory Fear Memory Consolidation of Mice via Facilitation of Inhibitory Neurotransmission in Basolateral Amygdala

BackgroundAutophagy has been demonstrated to play an important role in memory deficits as well as the degradation of neurotransmitter receptors. SAR405 is a newly discovered inhibitor that can specifically inhibit vacuolar sorting protein 34 and prevent autophagosome biogenesis. However, the effects of SAR405 on memory processes remain largely unknown. MethodsWestern blotting, immunofluorescence, and transmission electron microscopy were used to assess the level of autophagy after fear conditioning and SAR405 treatment. Behavioral tests, biotinylation assay, electrophysiology, and co-immunoprecipitation were used to unravel the mechanisms of SAR405 in memory consolidation. ResultsSAR405 infusion into the basolateral amygdala impaired long-term memory through autophagy inhibition. Furthermore, the trafficking of gamma-aminobutyric acid type A receptors (GABAARs) following fear conditioning was disrupted by SAR405, and the decreased frequency and amplitude of miniature inhibitory postsynaptic currents induced by fear conditioning were also reversed by SAR405, suggesting that SAR405 disrupted memory consolidation through blockade of the downregulated inhibitory neurotransmission in basolateral amygdala. GABAAR-associated protein (GABARAP) and its interaction with GABAAR γ2 subunit were found to be upregulated after fear conditioning, and SAR405 could suppress this increased interaction. Moreover, disruption of the GABARAP-GABAAR binding by a trans-activating transcriptional activator–GABARAP inhibitory peptide blocked the decrease in surface expression of GABAARs and attenuated long-term memory. ConclusionsThe present study suggests that SAR405 can prevent the memory consolidation via intervening autophagy and GABAAR trafficking and has a potential therapeutic value for disorders characterized by exaggerated fear memories, such as posttraumatic stress disorder.

Targeting autophagy by small molecule inhibitors of vacuolar protein sorting 34 (Vps34) improves the sensitivity of breast cancer cells to Sunitinib

Resistance to chemotherapy is a challenging problem for treatment of cancer patients and autophagy has been shown to mediate development of resistance. In this study we systematically screened a library of 306 known anti-cancer drugs for their ability to induce autophagy using a cell-based assay. 114 of the drugs were classified as autophagy inducers; for 16 drugs, the cytotoxicity was potentiated by siRNA-mediated knock-down of Atg7 and Vps34. These drugs were further evaluated in breast cancer cell lines for autophagy induction, and two tyrosine kinase inhibitors, Sunitinib and Erlotinib, were selected for further studies. For the pharmacological inhibition of autophagy, we have characterized here a novel highly potent selective inhibitor of Vps34, SB02024. SB02024 blocked autophagy in vitro and reduced xenograft growth of two breast cancer cell lines, MDA-MB-231 and MCF-7, in vivo. Vps34 inhibitor significantly potentiated cytotoxicity of Sunitinib and Erlotinib in MCF-7 and MDA-MB-231 in vitro in monolayer cultures and when grown as multicellular spheroids. Our data suggests that inhibition of autophagy significantly improves sensitivity to Sunitinib and Erlotinib and that Vps34 is a promising therapeutic target for combination strategies in breast cancer.

Abstract 1350: Inhibiting VPS34 suppresses Wnt/beta-catenin signaling and promotes chemotherapeutic efficacy in colon cancer cells

Abstract A significant obstacle in the successful management of colon cancer (CRC) patients remains intrinsic or acquired drug resistance. Addressing these obstacles remain priority for reducing CRC-associated deaths. Autophagy is a survival-promoting program and upregulated in cancers to sustain metabolism and survive microenvironmental stress. In preclinical cancer models, inhibition of autophagy can restore chemosensitivity and enhance tumor cell death. However, clinical applicability of the currently available autophagy inhibitors is problematic due to potential toxicity as these inhibitors primarily interfere with lysosomal trafficking program, implicated in essential cellular processes. To overcome this limitation, we examined the effects of inhibiting VPS34, a class III PI-3 Kinase implicated in autophagy initiation, to overcome resistance to therapy by inhibiting autophagy. Notably, #36-077, the inhibitor used in our studies was synthesized based on the recently described novel VPS34 inhibitor, SAR405. Using colon cancer cells and colon tumoroids from APCmin mice, we confirmed marked upregulation of autophagy. Tumoroids exposure to #36-077 inhibited autophagy (LC-3I/II and P-62 expression) and induced apoptosis (Cleaved caspase-3 expression), and thus confirmed survival role for autophagy in CRC. We further examined efficacy of using #36-077 for adjuvant therapy. Combinatorial treatment efficacy of #36-077with 5-Fluorouracil (5-FU) and Erlotinib (anti-EGFR therapy) was tested. SW480 (highly tumorigenic) and HCT116 (Highly Metastatic) cells were used. MTT-assay helped determine cell viability. We found synergy between #36-077 and 5-FU in the killing CRC cells, and the EC50 was 4.9µm, 4.7µm and 2.40µm for 5-FU, #36-077 and 5-FU+#36-077, respectively. Combinatorial use of the #36-077 also boosted the cancer cell killing effects of Erlotinib, and the EC50 was 25.05µm ,5.59µm and 4.09 µm for Erlotinib, #36-077 and Erlotinib+#36-077, respectively. In further studies, we focused on the β-catenin regulation due to its role in promoting Wnt-signaling and autophagy. Indeed, in both, colon cancer cells and tumoroids, combinatorial treatment with 5FU+#36-077 resulted in sharp increases in phospho-β-catenin (Ser33) expression. Of note, phosphorylation of β-catenin at Ser33 by GSK-3β leads to its destabilization and degradation. TOP-FLASH reporter analysis (p&amp;lt;0.001 vs control) further confirmed significant inhibition of Wnt-signaling in cells subjected to combinatorial treatment. In accordance, in cells subjected to combinatorial treatment, Survivin expression (a Wnt-signaling target; p&amp;lt;0.001 vs control) was markedly suppressed while cleaved caspse-3 expression (p&amp;lt;0.05 vs control) was upregulated. Taken together, our data provide strong rationale for the therapeutic targeting of VPS34 in improving therapeutic efficacy of anti-CRC treatments. Citation Format: Balawant Kumar, Rizwan Ahmad, Sandeep Rana, Amarnath Natarajan, Punita Dhawan, Amar B. Singh. Inhibiting VPS34 suppresses Wnt/beta-catenin signaling and promotes chemotherapeutic efficacy in colon cancer cells [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 1350.

Vps34 PI 3-kinase inactivation enhances insulin sensitivity through reprogramming of mitochondrial metabolism

Vps34 PI3K is thought to be the main producer of phosphatidylinositol-3-monophosphate, a lipid that controls intracellular vesicular trafficking. The organismal impact of systemic inhibition of Vps34 kinase activity is not completely understood. Here we show that heterozygous Vps34 kinase-dead mice are healthy and display a robustly enhanced insulin sensitivity and glucose tolerance, phenotypes mimicked by a selective Vps34 inhibitor in wild-type mice. The underlying mechanism of insulin sensitization is multifactorial and not through the canonical insulin/Akt pathway. Vps34 inhibition alters cellular energy metabolism, activating the AMPK pathway in liver and muscle. In liver, Vps34 inactivation mildly dampens autophagy, limiting substrate availability for mitochondrial respiration and reducing gluconeogenesis. In muscle, Vps34 inactivation triggers a metabolic switch from oxidative phosphorylation towards glycolysis and enhanced glucose uptake. Our study identifies Vps34 as a new drug target for insulin resistance in Type-2 diabetes, in which the unmet therapeutic need remains substantial.