Combining VPS34 inhibitors with STING agonists enhances type I interferon signaling and anti-tumor efficacy.

An immunosuppressive tumor microenvironment promotes tumor growth and is one of the main factors limiting the response to cancer immunotherapy. We have previously reported that inhibition of vacuolar protein sorting 34 (VPS34), a crucial lipid kinase in the autophagy/endosomal trafficking pathway, decreases tumor growth in several cancer models, increases infiltration of immune cells and sensitizes tumors to anti-programmed cell death protein 1/programmed cell death 1 ligand 1 therapy by upregulation of C-C motif chemokine 5 (CCL5) and C-X-C motif chemokine 10 (CXCL10) chemokines. The purpose of this study was to investigate the signaling mechanism leading to the VPS34-dependent chemokine increase. NanoString gene expression analysis was applied to tumors from mice treated with the VPS34 inhibitor SB02024 to identify key pathways involved in the anti-tumor response. We showed that VPS34 inhibitors increased the secretion of T-cell-recruitment chemokines in a cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes protein (STING)-dependent manner in cancer cells. Both pharmacological and small interfering RNA (siRNA)-mediated VPS34 inhibition increased cGAS/STING-mediated expression and secretion of CCL5 and CXCL10. The combination of VPS34 inhibitor and STING agonist further induced cytokine release in both human and murine cancer cells as well as monocytic or dendritic innate immune cells. Finally, the VPS34 inhibitor SB02024 sensitized B16-F10 tumor-bearing mice to STING agonist treatment and significantly improved mice survival. These results show that VPS34 inhibition augments the cGAS/STING pathway, leading to greater tumor control through immune-mediated mechanisms. We propose that pharmacological VPS34 inhibition may synergize with emerging therapies targeting the cGAS/STING pathway.

Cisplatin resistance can be curtailed by blunting Bnip3-mediated mitochondrial autophagy.

Cisplatin (CDDP) is commonly used to treat a multitude of tumors including sarcomas, ovarian and cervical cancers. Despite recent investigations allowed to improve chemotherapy effectiveness, the molecular mechanisms underlying the development of CDDP resistance remain a major goal in cancer research. Here, we show that mitochondrial morphology and autophagy are altered in different CDDP resistant cancer cell lines. In CDDP resistant osteosarcoma and ovarian carcinoma, mitochondria are fragmented and closely juxtaposed to the endoplasmic reticulum; rates of mitophagy are also increased. Specifically, levels of the mitophagy receptor BNIP3 are higher both in resistant cells and in ovarian cancer patient samples resistant to platinum-based treatments. Genetic BNIP3 silencing or pharmacological inhibition of autophagosome formation re-sensitizes these cells to CDDP. Our study identifies inhibition of BNIP3-driven mitophagy as a potential therapeutic strategy to counteract CDDP resistance in ovarian carcinoma and osteosarcoma.

Lighting up the fire in cold tumors to improve cancer immunotherapy by blocking the activity of the autophagy-related protein PIK3C3/VPS34.

Cancer immunotherapy based on Immune checkpoint blockade (ICB) is a promising strategy to treat patients with advanced highly aggressive therapy-resistant tumors. Unfortunately, the clinical reality is that only a small number of patients benefit from the remarkable clinical remissions achieved by ICB. Experimental and clinical evidence claimed that durable clinical benefit observed using ICB depends on the immune status of tumors, notably the presence of cytotoxic effector immune cells. In our paper, we revealed that genetically targeting the autophagy-related protein PIK3C3/VPS34 in melanoma and colorectal tumor cells, or treating tumor-bearing mice with selective inhibitors of the PIK3C3/VPS34 kinase activity, reprograms cold immune desert tumors into hot, inflamed immune infiltrated tumors. Such reprograming results from the establishment of a proinflammatory signature characterized by the release of CCL5 and CXCL10 in the tumor microenvironment, and the subsequent recruitment of natural killer (NK) and CD8+ T cells into the tumor bed. Furthermore, we reported that combining pharmacological inhibitors of PIK3C3/VPS34 improves the therapeutic benefit of anti-PD-1/PD-L1 immunotherapy. Our results provided the proof-of-concept to set-up innovative clinical trials for cold ICB-unresponsive tumors by combining PIK3C3/VPS34 inhibitors with anti-PDCD1/PD-1 and anti-CD274/PD-L1.

Inhibition of Vps34 reprograms cold into hot inflamed tumors and improves anti-PD-1/PD-L1 immunotherapy.

One of the major challenges limiting the efficacy of anti-PD-1/PD-L1 therapy in nonresponding patients is the failure of T cells to penetrate the tumor microenvironment. We showed that genetic or pharmacological inhibition of Vps34 kinase activity using SB02024 or SAR405 (Vps34i) decreased the tumor growth and improved mice survival in multiple tumor models by inducing an infiltration of NK, CD8+, and CD4+ T effector cells in melanoma and CRC tumors. Such infiltration resulted in the establishment of a T cell-inflamed tumor microenvironment, characterized by the up-regulation of pro-inflammatory chemokines and cytokines, CCL5, CXCL10, and IFNγ. Vps34i treatment induced STAT1 and IRF7, involved in the up-regulation of CCL5 and CXCL10. Combining Vps34i improved the therapeutic benefit of anti-PD-L1/PD-1 in melanoma and CRC and prolonged mice survival. Our study revealed that targeting Vps34 turns cold into hot inflamed tumors, thus enhancing the efficacy of anti-PD-L1/PD-1 blockade.

STAT3 is activated in multicellular spheroids of colon carcinoma cells and mediates expression of IRF9 and interferon stimulated genes.

Three-dimensional cell cultures, such as multicellular spheroids (MCS), reflect the in vivo architecture of solid tumours and multicellular drug resistance. We previously identified interferon regulatory factor 9 (IRF9) to be responsible for the up-regulation of a subset of interferon (IFN)-stimulated genes (ISGs) in MCS of colon carcinoma cells. This set of ISGs closely resembled a previously identified IFN-related DNA-damage resistance signature (IRDS) that was correlated to resistance to chemo- and radiotherapy. In this study we found that transcription factor STAT3 is activated upstream of IRF9 and binds to the IRF9 promoter in MCS of HCT116 colorectal carcinoma cells. Transferring conditioned media (CM) from high cell density conditions to non-confluent cells resulted in STAT3 activation and increased expression of IRF9 and a panel of IRDS genes, also observed in MCS, suggesting the involvement of a soluble factor. Furthermore, we identified gp130/JAK signalling to be responsible for STAT3 activation, IRF9, and IRDS gene expression in MCS and by CM. Our data suggests a novel mechanism where STAT3 is activated in high cell density conditions resulting in increased expression of IRF9 and, in turn, IRDS genes, underlining a mechanism by which drug resistance is regulated.

Annual Meeting of the International Society of Cancer Metabolism (ISCaM): Cancer Metabolism.

Tumors are metabolic entities wherein cancer cells adapt their metabolism to their oncogenic agenda and microenvironmental influences. Metabolically different cancer cell subpopulations collaborate to optimize nutrient delivery with respect to immediate bioenergetic and biosynthetic needs. They can also metabolically exploit host cells. These metabolic networks are directly linked with cancer progression, treatment, resistance, and relapse. Conversely, metabolic alterations in cancer are exploited for anticancer therapy, imaging, and stratification for personalized treatments. These topics were addressed at the 4th annual meeting of the International Society of Cancer Metabolism (ISCaM) in Bertinoro, Italy, on 19-21 October 2017.

A drug screening assay on cancer cells chronically adapted to acidosis.

BACKGROUND: Drug screening for the identification of compounds with anticancer activity is commonly performed using cell lines cultured under normal oxygen pressure and physiological pH. However, solid tumors are characterized by a microenvironment with limited access to nutrients, reduced oxygen supply and acidosis. Tumor hypoxia and acidosis have been identified as important drivers of malignant progression and contribute to multicellular resistance to different forms of therapy. Tumor acidosis represents an important mechanism mediating drug resistance thus the identification of drugs active on acid-adapted cells may improve the efficacy of cancer therapy. METHODS: Here, we characterized human colon carcinoma cells (HCT116) chronically adapted to grow at pH 6.8 and used them to screen the Prestwick drug library for cytotoxic compounds. Analysis of gene expression profiles in parental and low pH-adapted cells showed several differences relating to cell cycle, metabolism and autophagy. RESULTS: The screen led to the identification of several compounds which were further selected for their preferential cytotoxicity towards acid-adapted cells. Amongst 11 confirmed hits, we primarily focused our investigation on the benzoporphyrin derivative Verteporfin (VP). VP significantly reduced viability in low pH-adapted HCT116 cells as compared to parental HCT116 cells and normal immortalized epithelial cells. The cytotoxic activity of VP was enhanced by light activation and acidic pH culture conditions, likely via increased acid-dependent drug uptake. VP displayed the unique property to cause light-dependent cross-linking of proteins and resulted in accumulation of polyubiquitinated proteins without inducing inhibition of the proteasome. CONCLUSIONS: Our study provides an example and a tool to identify anticancer drugs targeting acid-adapted cancer cells.

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.

Spheroid-based 3D cell cultures identify salinomycin as a promising drug for the treatment of chondrosarcoma.

Chondrosarcoma (CS) is a cartilage malignancy of adulthood that is treated by surgery alone, since chemotherapy is considered ineffective. Unfortunately, a large proportion of patients with CS develop lung metastases, and several die of the disease. In this study, we compared 3D-spheroid cultures and conventional cell monolayer models in order to identify the best way to select anticancer agents that could be effective for the systemic control of CS. Using SW1353 cells, we developed a three-dimensional (3D) in vitro culture model to mimic in vivo features of CS microenvironment and evaluated the efficacy of different drugs to modulate CS cell proliferation and survival in 2D versus 3D-cultures. Doxorubicin (DXR) and cisplatin, that are widely employed in sarcomas, were less effective on 3D-CS spheroids when compared to standard monolayer models, whereas treatment with the ionophore salinomycin (SAL) had a strong cytotoxic effect both on 2D and 3D-cultures. Furthermore, as demonstrated by the reduced viability and the enhanced DXR nuclear localization, SAL enhanced DXR cytotoxicity in 3D-CS spheroids also at sub-lethal doses. SAL activity on 3D-CS spheroids was mediated by a significant induction of apoptosis via caspase activation. This study demonstrates that preclinical tests significantly differ in monolayer and 3D cultures of CS cells. Using this approach, SAL, alone or, at sub-lethal concentrations, in combination with DXR, represents a promising agent for the systemic treatment of CS. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Annual Meeting of the International Society of Cancer Metabolism (ISCaM): Metabolic Networks in Cancer.

Cancers are metabolic entities wherein cancer cells adapt their metabolism to their oncogenic agenda and microenvironmental influences. Metabolically different cancer cell subpopulations collaborate to optimize nutrient delivery with respect to immediate bioenergetic and biosynthetic needs. They can also metabolically exploit host cells. These metabolic networks are directly linked with cancer progression, treatment resistance and relapse. Conversely, metabolic alterations in cancer are exploited for anticancer therapy, imaging and personalized medicine. These topics were addressed at the 3rd annual meeting of the International Society of Cancer Metabolism (ISCaM) in Brussels, Belgium, on 26-29 October 2016.

Therapeutic implications of tumor interstitial acidification.

Interstitial acidification is a hallmark of solid tumor tissues resulting from the combination of different factors, including cellular buffering systems, defective tissue perfusion and high rates of cellular metabolism. Besides contributing to tumor pathogenesis and promoting tumor progression, tumor acidosis constitutes an important intrinsic and extrinsic mechanism modulating therapy sensitivity and drug resistance. In fact, pharmacological properties of anticancer drugs can be affected not only by tissue structure and organization but also by the distribution of the interstitial tumor pH. The acidic tumor environment is believed to create a chemical barrier that limits the effects and activity of many anticancer drugs. In this review article we will discuss the general protumorigenic effects of acidosis, the role of tumor acidosis in the modulation of therapeutic efficacy and potential strategies to overcome pH-dependent therapy-resistance.

Pathobiology and Therapeutic Implications of Tumor Acidosis.

Drug resistance and therapeutic failure are important causes of disease relapse and progression and may be considered as major obstacles preventing cure of cancer patients. Tumors use a large number of molecular, biochemical and cellular mechanisms to evade chemotherapy and targeted therapy. Important determinants of drug efficacy are the intrinsic pharmacological characteristics of drugs which may be largely affected by the tumor physiology. One feature of solid tumors is the acidic extracellular pH, resulting from metabolic shift and increased metabolic rates combined with low tissue perfusion due to defective vasculature. Besides its role in tumor pathobiology promoting tumor growth and metastasis, the acidic tumor environment creates a chemical barrier for many anticancer drugs, thus limiting their activity. The content of this review will be focused on the pathobiology of tumor acidosis and on its role in therapeutic resistance.

The Role of Autophagy in the Maintenance of Stemness and Differentiation of Mesenchymal Stem Cells.

Regulated self-consumption, also known as autophagy, is an evolutionary conserved process that degrades cellular components by directing them to the lysosomal compartment of eukaryotic cells. As a major intracellular degradation and recycling pathway, autophagy is crucial for maintaining and remodeling cellular homeostasis during normal cellular and tissue development. Recent studies have demonstrated that autophagy is necessary for the maintenance of cellular stemness and for a number of differentiation processes, including the lineage determination of mesenchymal stem cells. These are multipotent progenitor cells with self-renewal capacities that can give rise to a subset of tissues and thus hold a consistent potential in regenerative medicine. Here, we review the current literature on the complex liaison between autophagy induced by various extra- or intracellular stimuli and the molecular targets that affect mesenchymal stem cells proliferation and differentiation.

Altered pH gradient at the plasma membrane of osteosarcoma cells is a key mechanism of drug resistance.

Current therapy of osteosarcoma (OS), the most common primary bone malignancy, is based on a combination of surgery and chemotherapy. Multidrug resistance mediated by P-glycoprotein (P-gp) overexpression has been previously associated with treatment failure and progression of OS, although other mechanisms may also play a role. We considered the typical acidic extracellular pH (pHe) of sarcomas, and found that doxorubicin (DXR) cytotoxicity is reduced in P-gp negative OS cells cultured at pHe 6.5 compared to standard 7.4. Short-time (24-48 hours) exposure to low pHe significantly increased the number and acidity of lysosomes, and the combination of DXR with omeprazole, a proton pump inhibitor targeting lysosomal acidity, significantly enhanced DXR cytotoxicity. In OS xenografts, the combination treatment of DXR and omeprazole significantly reduced tumor volume and body weight loss. The impaired toxicity of DXR at low pHe was not associated with increased autophagy or lysosomal acidification, but rather, as shown by SNARF staining, with a reversal of the pH gradient at the plasma membrane (ΔpHcm), eventually leading to a reduced DXR intracellular accumulation. Finally, the reversal of ΔpHcm in OS cells promoted resistance not only to DXR, but also to cisplatin and methotrexate, and, to a lesser extent, to vincristine. Altogether, our findings show that, in OS cells, short-term acidosis induces resistance to different chemotherapeutic drugs by a reversal of ΔpHcm, suggesting that buffer therapies or regimens including proton pump inhibitors in combination to low concentrations of conventional anticancer agents may offer novel solutions to overcome drug resistance.

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

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

Guidance Molecule SEMA3A Restricts Tumor Growth by Differentially Regulating the Proliferation of Tumor-Associated Macrophages.

Accumulation of tumor-associated macrophages (TAM) correlates with malignant progression, immune suppression, and poor prognosis. In this study, we defined a critical role for the cell-surface guidance molecule SEMA3A in differential proliferative control of TAMs. Tumor cell-derived SEMA3A restricted the proliferation of protumoral M2 macrophages but increased the proliferation of antitumoral M1, acting through the SEMA3A receptor neuropilin 1. Expansion of M1 macrophages in vivo enhanced the recruitment and activation of natural killer (NK) cells and cytotoxic CD8(+) T cells to tumors, inhibiting their growth. In human breast cancer specimens, we found that immunohistochemical levels of SEMA3A correlated with the expression of genes characteristic of M1 macrophages, CD8(+) T cells, and NK cells, while inversely correlating with established characters of malignancy. In summary, our results illuminate a mechanism whereby the TAM phenotype is controlled and identify the cell-surface molecule SEMA3A as a candidate for therapeutic targeting. Cancer Res; 76(11); 3166-78. ©2016 AACR.