Potent Autophagy Inhibitor Research Articles

Preimplantation development of in vitro-produced bovine embryos treated with hydroxychloroquine

Hydroxychloroquine (HCQ) is a safe antimalarial drug but its overdosage or inappropriate use, such as during the pandemic, may cause adverse effects once this drug is considered a potent inhibitor of autophagy. Information about HCQ's effects on the reproductive field, including gametes and initial embryos, is limited. In this study, we evaluated the effect of HCQ (1, 6, 12, and 24 μM) on pre-implantation embryo development, autophagy, and apoptosis of bovine embryos produced in vitro. A dose-response experiment showed a reduction (p < 0.05) in cleavage only at the highest concentration. Blastocyst rate was gradually reduced (p < 0.05) with the increase of HCQ dosage starting at 6 μM, with no embryo formation occurring at 24 μM. Further analysis showed that embryos treated with 12 μM of HCQ had a higher (p < 0.05) accumulation of acidic autophagic vesicles on Days 5 and 7 of development and a higher (p < 0.01) apoptotic index on Day 7. To our knowledge, this is the first study to evaluate the effects of HCQ on embryo pre-implantation development in mammals. The results contribute with more information related to the study of autophagy in embryology as well as add some discussion on HCQ toxicology and its effects on reproductive cells.

Abstract C047: Improving the efficacy of dual ERK-MAPK and autophagy inhibition as a therapeutic strategy for pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS- and autophagy-dependent growth. We and others recently demonstrated that inhibition of KRAS signaling through targeting the RAF-MEK-ERK kinase cascade resulted in further reliance on autophagy. We found that targeting this increased reliance on autophagy with the autophagy inhibitor hydroxychloroquine (HCQ)/chloroquine (CQ) together with MEK or ERK inhibition (MEKi, ERKi) synergistically blocked PDAC growth. These findings provided rationale for our initiation of Phase I/II clinical trials evaluating the combination of MEKi (binimetinib; NCT04132505) or ERKi (LY3214996; NCT04386057) with HCQ in PDAC. However, a limitation of this approach is that HCQ/CQ are not specific or potent autophagy inhibitors. Thus, we sought to identify a more efficacious autophagy inhibition strategy. To this end, we performed a CRISPR-Cas9 mediated genetic loss-of-function screen in PDAC cells to identify other targetable mediators of autophagy. We identified PIKfyve, a lipid kinase critical for the recycling dynamics of lysosomes, as an essential autophagy-related gene in PDAC cells. PIKfyve inhibition by the clinical candidate inhibitor apilimod resulted in potent reduction of autophagic flux and growth. Additionally, PIKfyve inhibition induced intracellular vacuolization. These PIKfyve-regulated vacuoles stained positive for the lysosomal marker, LAMP1, and exhibited reduced acidity and impaired cargo degradation. Importantly, PIKfyve inhibition prevented the increased autophagic flux we observed when we inhibited MEK with the clinical stage MEKi, mirdametinib. As a result, co-targeting MEK and PIKfyve led to synergistic impairment of PDAC cell proliferation. Similar results were observed following dual inhibition of KRAS and PIKfyve. We found the synergistic growth inhibition was due in part to a substantial induction of apoptosis unique to combination treatment. We then tested the combination of MEKi and PIKfyvei in a panel of patient derived PDAC organoids and observed a robust synergistic reduction in viability, suggesting this may be an efficacious therapeutic strategy for PDAC treatment. Future work includes in vivo studies, currently underway, to determine the efficacy of single agent PIKfyve inhibition, as well as efficacy studies assessing combined MEKi and PIKfyvei in orthotopic mouse models of PDAC. Taken together, these findings implicate PIKfyve as an effective anti-autophagy target when paired with RAS or ERK-MAPK pathway inhibition in pre-clinical models of pancreatic cancer. Citation Format: Jonathan M. DeLiberty, Mallory K. Roach, Noah L. Pieper, Kristina Drizyte-Miller, Elyse G. Schechter, Runying Yang, Channing J. Der, Adrienne D. Cox, Clint A. Stalnecker, Kirsten L. Bryant. Improving the efficacy of dual ERK-MAPK and autophagy inhibition as a therapeutic strategy for pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C047.

A Nano-Autophagy Inhibitor Triggering Reciprocal Feedback Control of Cholesterol Depletion for Solid Tumor Therapy.

Solid tumors are characterized by enhanced metabolism of lipid, particularly cholesterol, inspiring the exploration of metabolic therapy through cholesterol oxidase (COD)-mediated cholesterol deprivation. However, the therapeutic efficacy of COD is limited due to the hypoxic tumor microenvironment and the protective autophagy triggered by cholesterol deprivation. Herein, a combination therapy for metabolically treating solid tumors through COD in conjunction with molybdenum oxide nanodots (MONDs), which serve as both potent oxygen generators and autophagy inhibitors, is reported. MONDs convert H2 O2 (arising from COD-mediated cholesterol oxidation) into O2 , which is then recycled by COD to form reciprocal feedback for cholesterol depletion. Concurrently, MONDs can overcome autophagy-induced therapeutic resistance frequently occurring in conventional nutrient deprivation therapy by activating AKT/mTOR pathway phosphorylation. Combination therapy in the xenograft model results in an ≈5-fold increase in therapeutic efficiency as compared with COD treatment alone. This functionally cooperative metabolic coupling strategy holds great promise as a novel polytherapy approach that will benefit patients with solid tumors.

Identification of Fangchinoline as a novel autophagy inhibitor with an adjuvant of chemotherapy against lung cancer

Autophagy is a fundamental recycling pathway that enhances cellular resilience, promoting survival. However, this survival mechanism can impede anti-cancer treatment strategies designed to induce cell death. In this study, we identified a novel autophagy inhibitor, Fangchinoline (Fan) isolated from the traditional Chinese medicine Stephania tetrandra. We speculated that when Fan blocks autophagy, cancer cells lose substantial self-preservation abilities during treatment. Firstly, we examined in detail the mechanism through which Fan inhibits autophagy. Specifically, Fan induced a significant increase in autophagosomes, as indicated by GFP-LC3 labeling, confirmed by the up-regulation of LC3-II. The autophagy receptor protein p62 was also up-regulated, suggesting a potential inhibition of autophagy flux. We further ruled out the possibility of fusion barriers between lysosomes and autophagosomes, as confirmed by their co-localization in double fluorescence staining. However, the lysosomal acid environment might be compromised, as suggested by the diminished fluorescence of acidity-sensitive dyes in the lysosomes and the corresponding decrease in mature forms of lysosomal cathepsin. To test the anti-cancer potential of Fan, we combined it with Cisplatin (Cis) or Paclitaxel (PTX) for lung cancer cell treatment. This combined treatment demonstrated a synergistically enhanced killing effect. These promising anti-tumor results were also replicated in a xenografted tumor model. The significance of this research lies in the identification of Fan as a potent autophagy inhibitor and its potential to enhance the efficacy of existing anti-cancer drugs. By unraveling the mechanisms of Fan's action on autophagy and demonstrating its synergistic effect in combination therapies, our study provides valuable insights for developing novel strategies to overcome autophagy-mediated resistance in cancer treatment.

Linderanidins A–F: Rare oligomeric flavonoids with an unusual C-3–C-4 linkage from the roots of Lindera erythrocarpa and their inhibitory activities on autophagy

Autophagy is a crucial process for maintaining cellular homeostasis by degrading and recycling unnecessary or damaged cellular components. In the process of exploring autophagy regulators in plants, unique nine oligomeric flavonoids linked by the bonding of C-3 and C-4, consisting of three pairs of biflavonoids, linderanidins A–C [(+)-1/(−)-1, (+)-2/(−)-2, and (+)-3/(−)-3], and three trimeric A-type proanthocyanidins, linderanidins D–F (4–6), were isolated from the roots of Lindera erythrocarpa. The structures and absolute configurations of these compounds were determined using various techniques, such as 1D and 2D NMR, mass spectrometry, X-ray crystallography, and electronic circular dichroism. All isolates were evaluated for their ability to regulate autophagy, and compounds (±)-1–(±)-3, (−)-1–(−)-3, (+)-1–(+)-3 and 4 were found to inhibit autophagy by blocking the fusion process between autophagosome and lysosome in HEK293 cells. This study suggests that unique oligomeric flavonoids possessing a C-3–C-4 linkage derived from the roots of L. erythrocarpa are potent autophagy inhibitors.

Discovery of a potent inhibitor of chaperone-mediated autophagy that targets the HSC70-LAMP2A interaction in non-small cell lung cancer cells.

Chaperone-mediated autophagy (CMA) is a selective type of autophagy targeting protein degradation and maintains high activity in many malignancies. Inhibition of the combination of HSC70 and LAMP2A can potently block CMA. At present, knockdown of LAMP2A remains the most specific method for inhibiting CMA and chemical inhibitors against CMA have not yet been discovered. Levels of CMA in non-small cell lung cancer (NSCLC) tissue samples were confirmed by tyramide signal amplification dual immunofluorescence assay. High-content screening was performed based on CMA activity, to identify potential inhibitors of CMA. Inhibitor targets were determined by drug affinity responsive target stability-mass spectrum and confirmed by protein mass spectrometry. CMA was inhibited and activated to elucidate the molecular mechanism of the CMA inhibitor. Suppression of interactions between HSC70 and LAMP2A blocked CMA in NSCLC, restraining tumour growth. Polyphyllin D (PPD) was identified as a targeted CMA small-molecule inhibitor through disrupting HSC70-LAMP2A interactions. The binding sites for PPD were E129 and T278 at the nucleotide-binding domain of HSC70 and C-terminal of LAMP2A, respectively. PPD accelerated unfolded protein generation to induce reactive oxygen species (ROS) accumulation by inhibiting HSC70-LAMP2A-eIF2α signalling axis. Also, PPD prevented regulatory compensation of macroautophagy induced by CMA inhibition via blocking the STX17-SNAP29-VAMP8 signalling axis. PPD is a targeted CMA inhibitor that blocked both HSC70-LAMP2A interactions and LAMP2A homo-multimerization. CMA suppression without increasing the regulatory compensation from macroautophagy is a good strategy for NSCLC therapy.

Targeting PP2A-dependent autophagy enhances sensitivity to ruxolitinib in JAK2V617F myeloproliferative neoplasms

The Janus kinase 2 (JAK2)-driven myeloproliferative neoplasms (MPNs) are chronic malignancies associated with high-risk complications and suboptimal responses to JAK inhibitors such as ruxolitinib. A better understanding of cellular changes induced by ruxolitinib is required to develop new combinatory therapies to improve treatment efficacy. Here, we demonstrate that ruxolitinib induced autophagy in JAK2V617F cell lines and primary MPN patient cells through the activation of protein phosphatase 2A (PP2A). Inhibition of autophagy or PP2A activity along with ruxolitinib treatment reduced proliferation and increased the death of JAK2V617F cells. Accordingly, proliferation and clonogenic potential of JAK2V617F-driven primary MPN patient cells, but not of normal hematopoietic cells, were markedly impaired by ruxolitinib treatment with autophagy or PP2A inhibitor. Finally, preventing ruxolitinib-induced autophagy with a novel potent autophagy inhibitor Lys05 improved leukemia burden reduction and significantly prolonged the mice’s overall survival compared with ruxolitinib alone. This study demonstrates that PP2A-dependent autophagy mediated by JAK2 activity inhibition contributes to resistance to ruxolitinib. Altogether, our data support that targeting autophagy or its identified regulator PP2A could enhance sensitivity to ruxolitinib of JAK2V617F MPN cells and improve MPN patient care.

Neuroprotective Effects of Oxymatrine via Triggering Autophagy and Inhibiting Apoptosis Following Spinal Cord Injury in Rats.

Spinal cord injury (SCI) is a devastating neurological disorder characterized by high morbidity and disability. However, there is still a lack of effective treatments for it. The identification of drugs that promote autophagy and inhibit apoptosis in neurons is critical for improving patient outcomes following SCI. Previous studies have shown that increasing the activity of silent information regulator 1 (SIRT1) and downstream protein AMP-activated protein kinase (AMPK) in rat models of SCI is highly neuroprotective. Oxymatrine (OMT), a quinolizidine alkaloid, has exhibited neuroprotective effects in various central nervous system (CNS) diseases. However, its explicit effect and molecular mechanism in SCI are still unclear. Herein, we aimed to investigate the therapeutic effects of OMT and explore the potential role of autophagy regulation following SCI in rats. A modified compressive device (weight 35 g, time 5 min) was applied to induce moderate SCI in all groups except the sham group. After treatment with drugs or vehicle (saline), our results indicated that OMT treatment significantly reduced the lesion size, promoted survival of motor neurons, and subsequently attenuated motor dysfunction following SCI in rats. OMT significantly enhanced autophagy activity, inhibited apoptosis in neurons, and increased SIRT1 and p-AMPK expression levels. Interestingly, these effects of OMT on SCI were partially prevented by co-treatment with SIRT1 inhibitor EX527. Furthermore, combining OMT with the potent autophagy inhibitor chloroquine (CQ) could effectively abolish its promotion of autophagic flux. Taken together, these data revealed that OMT exerts a neuroprotective role in functional recovery against SCI in rats, and these effects are potentially associated with OMT-induced activation of autophagy via the SIRT1/AMPK signaling pathway.

Palmitic acid-induced ferroptosis via CD36 activates ER stress to break calcium-iron balance in colon cancer cells.

Ferroptosis, featuring an iron-dependent peroxidation of lipids, is a novel form of programmed cell death that may hold great potential in cancer therapy. Our study found that palmitic acid (PA) inhibited colon cancer cell viability in vitro and in vivo, in conjunction with an accumulation of reactive oxygen species (ROS) and lipid peroxidation. The ferroptosis inhibitor Ferrostatin-1 but not Z-VAD-FMK (a pan-caspase inhibitor), Necrostatin-1 (a potent necroptosis inhibitor), or CQ (a potent inhibitor of autophagy), rescued the cell death phenotype induced by PA. Subsequently, we verified that PA induces ferroptotic cell death through excess iron as cell death was inhibited by iron chelator deferiprone (DFP), while it was exacerbated by a supplement of ferric ammonium citrate (FAC). Mechanistically, PA affects intracellular iron content by inducing endoplasmic reticulum (ER) stress leading to ER calcium release and regulating transferrin transport through increasing cytosolic calcium levels. Furthermore, we observed that cells with high expression of CD36 were more vulnerable to PA-induced ferroptosis. Altogether, our findings reveal that PA engages in anticancer properties by activating ER stress/ER calcium release/transferrin-dependent ferroptosis, and PA might serve as a compound to activate ferroptosis in colon cancer cells with high CD36 expression.

Abstract 4851: Improving the efficacy of dual ERK-MAPK and autophagy inhibition as a therapeutic strategy for pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS- and autophagy-dependent growth. We and others recently demonstrated that inhibition of the RAF-MEK-ERK pathway resulted in upregulated autophagy, and that dual treatment with the autophagy inhibitor hydroxychloroquine (HCQ)/chloroquine (CQ) and MEK or ERK inhibitors (MEKi, ERKi) synergistically blocked PDAC growth. These findings provided rationale for our initiation of Phase I/II clinical trials evaluating the combination of MEKi (binimetinib; NCT04132505) or ERKi (LY3214996; NCT04386057) with HCQ in PDAC. However, HCQ/CQ are not specific or potent autophagy inhibitors. Thus, we sought to identify a more efficacious autophagy inhibition strategy. To this end, we performed a CRISPR-Cas9 mediated genetic loss-of-function screen with a library encompassing cancer signaling pathways in the presence or absence of CQ. In the absence of CQ, we identified PIKfyve, a lipid kinase critical for the recycling dynamics of lysosomes, as an essential autophagy-related gene in PDAC cells. PIKfyve inhibition, by the clinical candidate inhibitor apilimod, resulted in potent reduction of autophagic flux and growth. Additionally, PIKfyve inhibition resulted in robust intracellular vacuolization. Vacuoles stained positive for the lysosomal marker, LAMP1, and exhibited reduced acidity and impaired cargo degradation. Importantly, when we inhibited MEK, with the clinical stage MEKi mirdametinib, and PIKfyve (with apilimod) together, we observed decreased MEKi-induced autophagic flux and synergistic impairment of PDAC cell proliferation. Growth inhibition was due in part to an induction of apoptosis unique to combination treatment. Synergistic growth suppression was maintained in a panel of patient derived PDAC organoids, suggesting this may be an efficacious therapeutic strategy for PDAC treatment. In the presence of CQ, our CRISPR-Cas9 screen showed that loss of upstream autophagy-related genes sensitized PDAC cells to CQ treatment. Validation indicated that vertical inhibition (dual inhibition of distinct nodes of a linear pathway) of the autophagy pathway further reduced autophagic flux relative to inhibition of any single node and synergistically reduced PDAC cell growth. Furthermore, combining ERK-MAPK inhibition with vertical inhibition of autophagy improves the in vitro efficacy of this treatment strategy. Ongoing studies are aimed at delineating the mechanism underlying the synergy observed with anti-autophagy inhibitor combinations and further validation in more advanced preclinical models of PDAC. Citation Format: Jonathan M. DeLiberty, Clint A. Stalnecker, Kristina Drizyte-Miller, Elyse G. Schechter, Noah L. Pieper, Runying Yang, Channing J. Der, Adrienne D. Cox, Kirsten L. Bryant. Improving the efficacy of dual ERK-MAPK and autophagy inhibition as a therapeutic strategy for pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4851.

Azithromycin, a potent autophagy inhibitor for cancer therapy, perturbs cytoskeletal protein dynamics

BackgroundAutophagy plays an important role in tumour cell growth and survival and also promotes resistance to chemotherapy. Hence, autophagy has been targeted for cancer therapy. We previously reported that macrolide antibiotics including azithromycin (AZM) inhibit autophagy in various types of cancer cells in vitro. However, the underlying molecular mechanism for autophagy inhibition remains unclear. Here, we aimed to identify the molecular target of AZM for inhibiting autophagy.MethodsWe identified the AZM-binding proteins using AZM-conjugated magnetic nanobeads for high-throughput affinity purification. Autophagy inhibitory mechanism of AZM was analysed by confocal microscopic and transmission electron microscopic observation. The anti-tumour effect with autophagy inhibition by oral AZM administration was assessed in the xenografted mice model.ResultsWe elucidated that keratin-18 (KRT18) and α/β-tubulin specifically bind to AZM. Treatment of the cells with AZM disrupts intracellular KRT18 dynamics, and KRT18 knockdown resulted in autophagy inhibition. Additionally, AZM treatment suppresses intracellular lysosomal trafficking along the microtubules for blocking autophagic flux. Oral AZM administration suppressed tumour growth while inhibiting autophagy in tumour tissue.ConclusionsAs drug-repurposing, our results indicate that AZM is a potent autophagy inhibitor for cancer treatment, which acts by directly interacting with cytoskeletal proteins and perturbing their dynamics.

Blockage of autophagosome-lysosome fusion through SNAP29 O-GlcNAcylation promotes apoptosis via ROS production

ABSTRACT Macroautophagy/autophagy has been shown to exert a dual role in cancer i.e., promoting cell survival or cell death depending on the cellular context and the cancer stage. Therefore, development of potent autophagy modulators, with a clear mechanistic understanding of their target action, has paramount importance in both mechanistic and clinical studies. In the process of exploring the mechanism of action of a previously identified cytotoxic small molecule (SM15) designed to target microtubules and the interaction domain of microtubules and the kinetochore component NDC80/HEC1, we discovered that the molecule acts as a potent autophagy inhibitor. By using several biochemical and cell biology assays we demonstrated that SM15 blocks basal autophagic flux by inhibiting the fusion of correctly formed autophagosomes with lysosomes. SM15-induced autophagic flux blockage promoted apoptosis-mediated cell death associated with ROS production. Interestingly, autophagic flux blockage, apoptosis induction and ROS production were rescued by genetic or pharmacological inhibition of OGT (O-linked N-acetylglucosamine (GlcNAc) transferase) or by expressing an O-GlcNAcylation-defective mutant of the SNARE fusion complex component SNAP29, pointing to SNAP29 as the molecular target of SM15 in autophagy. Accordingly, SM15 was found to enhance SNAP29 O-GlcNAcylation and, thereby, inhibit the formation of the SNARE fusion complex. In conclusion, these findings identify a new pathway in autophagy connecting O-GlcNAcylated SNAP29 to autophagic flux blockage and autophagosome accumulation, that, in turn, drives ROS production and apoptotic cell death. Consequently, modulation of SNAP29 activity may represent a new opportunity for therapeutic intervention in cancer and other autophagy-associated diseases.

Inhibition of autophagy rescues HT22 hippocampal neurons from erastin-induced ferroptosis.

Ferroptosis is a regulated form of cell death which is considered an oxidative iron-dependent process. The lipid hydroperoxidase glutathione peroxidase 4 prevents the iron (Fe2+)-dependent formation of toxic lipid reactive oxygen species. While emerging evidence indicates that inhibition of glutathione peroxidase 4 as a hallmark of ferroptosis in many cancer cell lines, the involvement of this biochemical pathway in neuronal death remains largely unclear. Here, we investigate, first whether the ferroptosis key players are involved in the neuronal cell death induced by erastin. The second objective was to examine whether there is a cross talk between ferroptosis and autophagy. The third main was to address neuron response to erastin, with a special focus on ferritin and nuclear receptor coactivator 4-mediated ferritinophagy. To test this in neurons, erastin (0.5-8 µM) was applied to hippocampal HT22 neurons for 16 hours. In addition, cells were cultured with the autophagy inhibitor, 3-methyladenin (10 mM) and/or ferroptosis inhibitors, ferrostatin 1 (10-20 µM) or deferoxamine (10-200 µM) before exposure to erastin. In this study, we demonstrated by immunofluorescence and western blot analysis, that erastin downregulates dramatically the expression of glutathione peroxidase 4, the sodium-independent cystine-glutamate antiporter and nuclear receptor coactivator 4. The protein levels of ferritin and mitochondrial ferritin in HT22 hippocampal neurons did not remarkably change following erastin treatment. In addition, we demonstrated that not only the ferroptosis inhibitor, ferrostatin1/deferoxamine abrogated the ferroptotic cell death induced by erastin in hippocampal HT22 neurons, but also the potent autophagy inhibitor, 3-methyladenin. We conclude that (1) erastin-induced ferroptosis in hippocampal HT22 neurons, despite reduced nuclear receptor coactivator 4 levels, (2) that either nuclear receptor coactivator 4-mediated ferritinophagy does not occur or is of secondary importance in this model, (3) that ferroptosis seems to share some features of the autophagic cell death process.

Albendazole-induced autophagy blockade contributes to elevated apoptosis in cholangiocarcinoma cells through AMPK/mTOR activation

Albendazole (ABZ) is a broad-spectrum anti-parasitic drug that exhibits antitumor effects against several carcinomas. The effects of ABZ on cholangiocarcinoma (CCA) and its underlying mechanisms are still unclear. Our study aims to investigate the role of ABZ in inducing autophagy-mediated apoptosis of cholangiocarcinoma cells. The antitumor effects of ABZ were evaluated against CCA cells and HIBEC intrahepatic biliary epithelial cells. Furthermore, the apoptosis rates, and autophagy flux in RBE and FRH-0201 cells treated with ABZ were investigated. ABZ inhibited proliferation, induced cell death and apoptosis in CCA cells in vitro. In vivo, tumors from ABZ- treated BALB/c nude mice were significantly smaller than untreated mice. ABZ also induced the initiation of autophagy via AMPK/mTOR pathways, resulting in the formation of autophagosome. In addition, ABZ blocked autophagic flux by inhibiting the fusion of autophagosome-lysosome, which increased the apoptotic death of CCA cells. However, the apoptotic death of CCA cells induced by ABZ was reversed by 3-methyladenine (3-MA), an autophagosome formation inhibitor, but increased by chloroquine (CQ), an autophagosome-lysosome fusion inhibitor.Our work provides novel mechanisms for anti-tumor effects of ABZ on CCA, suggesting that ABZ may be used as a potent autophagy inhibitor in the treatment of CCA.

Abstract 3600: DCC-3116, a first-in-class selective inhibitor of ULK1/2 kinases and autophagy, synergizes with the KRASG12C inhibitor sotorasib resulting in tumor regression in KRAS mutant NSCLC xenograft models

Abstract Mutationally-activated RAS oncoproteins are detected in approximately 19% of newly diagnosed human cancers and have been targets for drug discovery for over 40 years. Recently, the FDA approved sotorasib, a covalent inhibitor of KRASG12C, for the treatment of non-small cell lung cancer (NSCLC). Although sotorasib demonstrated clear clinical benefits, the emergence of drug resistance has led to drug combination approaches with the goal of deepening and sustaining the durability of patient responses. One potential mechanism of drug resistance is the induction of autophagy, which cancer cells use to survive during periods of stress such as treatment with pathway-targeted therapies. Indeed, KRAS-mutated cancer cells have been shown to exhibit constitutive autophagy for survival. Additionally, such cells further increase autophagy when treated with pathway-targeted inhibitors of RAS&amp;gt;RAF&amp;gt;MEK&amp;gt;ERK MAP kinase signaling as a resistance mechanism. Hence, the combination of a KRASG12C inhibitor with a specific and potent autophagy inhibitor could lead to deeper and more sustained clinical responses. DCC-3116 is an investigational, potent and selective pharmacological inhibitor of the protein kinases ULK1 and ULK2, which are critical initiating components of the autophagy pathway. Treatment of KRASG12C non-small cell lung cancer (NSCLC) cell lines with sotorasib induced autophagy by ~2-fold via activation of ULK kinases as measured by an increase in ULK-mediated phosphorylation of ATG13. Sotorasib-mediated ULK kinase activation, and resulting autophagic flux, was inhibited by DCC-3116 in a dose-dependent manner with IC50 values of 81-160 nM in NSCLC cell lines. These effects translated to in vivo efficacy. In the Calu-1 and H358 KRASG12C NSCLC xenograft models, the combination of DCC-3116 and sotorasib resulted in tumor regression whereas single treatment arms afforded only inhibition of tumor growth. A NSCLC KRASG12C patient derived xenograft (PDX) model further supported the DCC-3116 and sotorasib combination with increased tumor growth inhibition compared to sotorasib alone. These data demonstrate a compelling rationale to study DCC-3116 in combination with KRASG12C inhibitors such as sotorasib in NSCLC patients. DCC-3116 is currently in a Phase 1 clinical trial in patients with advanced solid tumors with documented KRAS, NRAS or BRAF mutations (NCT04892017). Citation Format: Martin McMahon, Madhumita Bogdan, Mary J. Timson, Hikmat Al-Hashimi, Phaedra Ghazi, Yu Zhan, Bryan D. Smith, Conan G. Kinsey, Daniel L. Flynn. DCC-3116, a first-in-class selective inhibitor of ULK1/2 kinases and autophagy, synergizes with the KRASG12C inhibitor sotorasib resulting in tumor regression in KRAS mutant NSCLC xenograft models [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 3600.

Toosendanin, a novel potent vacuolar-type H+-translocating ATPase inhibitor, sensitizes cancer cells to chemotherapy by blocking protective autophagy.

Macroautophagy/autophagy is the process of self-digestion through the lysosomes; it disassembles unnecessary or dysfunctional long-lived proteins and damaged organelles for the recycling of biomacromolecules. Unfortunately, cancer cells can hijack this mechanism to survive under metabolic stress or develop drug resistance during chemotherapy. Increasing evidence indicates that the combination of autophagy inhibition and chemotherapy is a promising cancer treatment strategy. However, effective autophagy inhibitors with satisfied potency, bioavailability, and clearly-defined drug targets are still rare. Here, we report the identification of a potent autophagy inhibitor toosendanin which can effectively block autophagosome maturation, causing the accumulation of autophagy substrates in multiple cancer cells. Toosendanin did not inhibit the fusion process between autophagosome and lysosome but elevated lysosomal pH and impaired lysosomal enzymes activity. Using rat liver lysosome fraction and purified yeast V-ATPase, we found that toosendanin directly inhibited V-ATPase activity. By applying cellular thermal shift assay (CETSA), immunoprecipitation-coupled LC-MS/MS analysis, and biotin-toosendanin pull-down assay, we confirmed the direct binding between toosendanin and V-ATPase. Furthermore, toosendanin blocked chemotherapy-induced protective autophagy in cultured cancer cells and xenograft tumor tissues to significantly enhance anti-cancer activity. These results suggest that toosendanin has the potential to be developed into an anti-cancer drug by blocking chemotherapy-induced protective autophagy.

Multiplex Gene Tagging with CRISPR-Cas9 for Live-Cell Microscopy and Application to Study the Role of SARS-CoV-2 Proteins in Autophagy, Mitochondrial Dynamics, and Cell Growth.

The lack of efficient tools to label multiple endogenous targets in cell lines without staining or fixation has limited our ability to track physiological and pathological changes in cells over time via live-cell studies. Here, we outline the FAST-HDR vector system to be used in combination with CRISPR-Cas9 to allow visual live-cell studies of up to three endogenous proteins within the same cell line. Our approach utilizes a novel set of advanced donor plasmids for homology-directed repair and a streamlined workflow optimized for microscopy-based cell screening to create genetically modified cell lines that do not require staining or fixation to accommodate microscopy-based studies. We validated this new methodology by developing two advanced cell lines with three fluorescent-labeled endogenous proteins that support high-content imaging without using antibodies or exogenous staining. We applied this technology to study seven severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/COVID-19) viral proteins to understand better their effects on autophagy, mitochondrial dynamics, and cell growth. Using these two cell lines, we were able to identify the protein ORF3a successfully as a potent inhibitor of autophagy, inducer of mitochondrial relocalization, and a growth inhibitor, which highlights the effectiveness of live-cell studies using this technology.

PIK3C3 Inhibition Promotes Sensitivity to Colon Cancer Therapy by Inhibiting Cancer Stem Cells.

Simple SummaryColorectal cancer (CRC) represents a heterogeneous population of tumor cells and cancer stem cells (CSCs) where CSCs are postulated to resist the chemotherapy, and support cancer malignancy. Eliminating CSC can therefore improve CRC therapy and patient survival; however, such strategies have not yielded the desired outcome. Inhibiting autophagy has shown promise in suppressing therapy resistance; however, current autophagy inhibitors have failed in the clinical trials. In the current study, we provided data supporting the efficacy of 36-077, a potent inhibitor of PIK3C3/VPS34, in inhibiting autophagy to kill the CSC to promote the efficacy of colon cancer therapy.Background: Despite recent advances in therapies, resistance to chemotherapy remains a critical problem in the clinical management of colorectal cancer (CRC). Cancer stem cells (CSCs) play a central role in therapy resistance. Thus, elimination of CSCs is crucial for effective CRC therapy; however, such strategies are limited. Autophagy promotes resistance to cancer therapy; however, whether autophagy protects CSCs to promote resistance to CRC-therapy is not well understood. Moreover, specific and potent autophagy inhibitors are warranted as clinical trials with hydroxychloroquine have not been successful. Methods: Colon cancer cells and tumoroids were used. Fluorescent reporter-based analysis of autophagy flux, spheroid and side population (SP) culture, and qPCR were done. We synthesized 36-077, a potent inhibitor of PIK3C3/VPS34 kinase, to inhibit autophagy. Combination treatments were done using 5-fluorouracil (5-FU) and 36-077. Results: The 5-FU treatment induced autophagy only in a subset of the treated colon cancer. These autophagy-enriched cells also showed increased expression of CSC markers. Co-treatment with 36-077 significantly improved efficacy of the 5-FU treatment. Mechanistic studies revealed that combination therapy inhibited GSK-3β/Wnt/β-catenin signaling to inhibit CSC population. Conclusion: Autophagy promotes resistance to CRC-therapy by specifically promoting GSK-3β/Wnt/β-catenin signaling to promote CSC survival, and 36-077, a PIK3C3/VPS34 inhibitor, helps promote efficacy of CRC therapy.

Cytotoxic Effects of Arsenite in Combination With Gamabufotalin Against Human Glioblastoma Cell Lines.

Glioblastoma is a fatal primary malignant brain tumor, and the 5-year survival rate of treated glioblastoma patients still remains <5%. Considering the sustained development of metastasis, tumor recurrence, and drug resistance, there is an urgent need for the novel therapeutic approaches to combat glioblastoma. Trivalent arsenic derivative (arsenite, AsIII) with remarkable clinical efficacy in leukemia has been shown to exert cytocidal effect against glioblastoma cells. Gamabufotalin, an active bufadienolide compound, also shows selective cytocidal effect against glioblastoma cells, and has been suggested to serve as a promising adjuvant therapeutic agent to potentiate therapeutic effect of conventional anticancer drugs. In order to gain novel insight into therapeutic approaches against glioblastoma, the cytotoxicity of AsIII and gamabufotalin was explored in the human glioblastoma cell lines U-87 and U-251. In comparison with U-251 cells, U-87 cells were highly susceptible to the two drugs, alone or in combination. More importantly, clinically achieved concentrations of AsIII combined with gamabufotalin exhibited synergistic cytotoxicity against U-87 cells, whereas showed much less cytotoxicity to human normal peripheral blood mononuclear cells. G2/M cell cycle arrest was induced by each single drug, and further augmented by their combination in U-87 cells. Downregulation of the expression levels of cdc25C, Cyclin B1, cdc2, and survivin was observed in U-87 cells treated with the combined regimen and occurred in parallel with G2/M arrest. Concomitantly, lactate dehydrogenase leakage was also observed. Intriguingly, SB203580, a specific inhibitor of p38 MAPK, intensified the cytotoxicity of the combined regimen in U-87 cells, whereas wortmannin, a potent autophagy inhibitor, significantly rescued the cells. Collectively, G2/M arrest, necrosis and autophagy appeared to cooperatively contribute to the synergistic cytotoxicity of AsIII and gamabufotalin. Given that p38 MAPK serves an essential role in promoting glioblastoma cell survival, developing a possible strategy composed of AsIII, gamabufotalin, and a p38 MAPK inhibitor may provide novel insight into approaches designed to combat glioblastoma.

Targeting Cytoprotective Autophagy to Enhance Anticancer Therapies.

Autophagy is a highly regulated multi-step process that occurs at the basal level in almost all cells. Although the deregulation of the autophagy process has been described in several pathologies, the role of autophagy in cancer as a cytoprotective mechanism is currently well established and supported by experimental and clinical evidence. Our understanding of the molecular mechanism of the autophagy process has largely contributed to defining how we can harness this process to improve the benefit of cancer therapies. While the role of autophagy in tumor resistance to chemotherapy is extensively documented, emerging data point toward autophagy as a mechanism of cancer resistance to radiotherapy, targeted therapy, and immunotherapy. Therefore, manipulating autophagy has emerged as a promising strategy to overcome tumor resistance to various anti-cancer therapies, and autophagy modulators are currently evaluated in combination therapies in several clinical trials. In this review, we will summarize our current knowledge of the impact of genetically and pharmacologically modulating autophagy genes and proteins, involved in the different steps of the autophagy process, on the therapeutic benefit of various cancer therapies. We will also briefly discuss the challenges and limitations to developing potent and selective autophagy inhibitors that could be used in ongoing clinical trials.