GNS561, a new lysosomotropic small molecule, for the treatment of intrahepatic cholangiocarcinoma
Among the acquired modifications in cancer cells, changes in lysosomal phenotype and functions are well described, making lysosomes a potential target for novel therapies. Some weak base lipophilic drugs have a particular affinity towards lysosomes, taking benefits from lysosomal trapping to exert anticancer activity. Here, we have developed a new lysosomotropic small molecule, GNS561, and assessed its activity in multiple in vitro intrahepatic cholangiocarcinoma models (HuCCT1 and RBE cell lines and patient-derived cells) and in a chicken chorioallantoic membrane xenograft model. GNS561 significantly reduced cell viability in two intrahepatic cholangiocarcinoma cell lines (IC50 of 1.5 ± 0.2 μM in HuCCT1 and IC50 of 1.7 ± 0.1 μM in RBE cells) and induced apoptosis as measured by caspases activation. We confirmed that GNS561-mediated cell death was related to its lysosomotropic properties. GNS561 induced lysosomal dysregulation as proven by inhibition of late-stage autophagy and induction of a dose-dependent build-up of enlarged lysosomes. In patient-derived cells, GNS561 was more potent than cisplatin and gemcitabine in 2/5 and 1/5 of the patient-derived cells models, respectively. Moreover, in these models, GNS561 was potent in models with low sensitivity to gemcitabine. GNS561 was also efficient in vivo against a human intrahepatic cholangiocarcinoma cell line in a chicken chorioallantoic membrane xenograft model, with a good tolerance at doses high enough to induce an antitumor effect in this model. In summary, GNS561 is a new lysosomotropic agent, with an anticancer activity against intrahepatic cholangiocarcinoma. Further investigations are currently ongoing to fully elucidate its mechanism of action.
KeywordsGNS561 Cholangiocarcinoma Anticancer Lysosome Apoptosis
The authors are very grateful to Dr. Emilien Dosda, Dr. Xavier Rousset and Sylvain Roveda from Inovotion for their work on the CAM study.
This study was supported by private funding.
Compliance with ethical standards
Conflict of interest
All authors declare that they have no conflict of interest and consent to the submission of this manuscript.
According to the French legislation, no ethical approval is needed for scientific experimentations using oviparous embryos (decree n° 2013–118, February 1, 2013; art. R-214–88).
For this type of study, formal consent is not required. This article does not contain any studies with human participants or animals performed by any of the authors.
- 1.World Health Organization (2018) Cancer. https://www.who.int/news-room/fact-sheets/detail/cancer. Accessed December 18, 2018
- 3.Valle J, Wasan H, Palmer DH, Cunningham D, Anthoney A, Maraveyas A, Madhusudan S, Iveson T, Hughes S, Pereira SP, Roughton M, Bridgewater J, Investigators ABCT (2010) Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer. N Engl J Med 362(14):1273–1281. https://doi.org/10.1056/NEJMoa0908721 Google Scholar
- 9.Walter T, Horgan AM, McNamara M, McKeever L, Min T, Hedley D, Serra S, Krzyzanowska MK, Chen E, Mackay H, Feld R, Moore M, Knox JJ (2013) Feasibility and benefits of second-line chemotherapy in advanced biliary tract cancer: a large retrospective study. Eur J Cancer 49(2):329–335. https://doi.org/10.1016/j.ejca.2012.08.003 Google Scholar
- 13.Perera RM, Stoykova S, Nicolay BN, Ross KN, Fitamant J, Boukhali M, Lengrand J, Deshpande V, Selig MK, Ferrone CR, Settleman J, Stephanopoulos G, Dyson NJ, Zoncu R, Ramaswamy S, Haas W, Bardeesy N (2015) Transcriptional control of autophagy-lysosome function drives pancreatic cancer metabolism. Nature 524(7565):361–365. https://doi.org/10.1038/nature14587 Google Scholar
- 15.De Duve C, Pressman BC, Gianetto R, Wattiaux R, Appelmans F (1955) Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat-liver tissue. Biochem J 60(4):604–617Google Scholar
- 16.Xu H, Ren D (2015) Lysosomal physiology. Annu Rev Physiol 77:57–80. https://doi.org/10.1146/annurev-physiol-021014-071649 Google Scholar
- 17.de Duve C (1983) Lysosomes revisited. Eur J Biochem 137(3):391–397Google Scholar
- 18.Boyer MJ, Tannock IF (1993) Lysosomes, lysosomal enzymes,. Adv Cancer Res 60:269–291Google Scholar
- 22.Davidson SM, Vander Heiden MG (2017) Critical functions of the lysosome in Cancer biology. Annu Rev Pharmacol Toxicol 57:481–507. https://doi.org/10.1146/annurev-pharmtox-010715-103101 Google Scholar
- 27.Glunde K, Guggino SE, Solaiyappan M, Pathak AP, Ichikawa Y, Bhujwalla ZM (2003) Extracellular acidification alters lysosomal trafficking in human breast cancer cells. Neoplasia 5(6):533–545Google Scholar
- 30.Domagala A, Fidyt K, Bobrowicz M, Stachura J, Szczygiel K, Firczuk M (2018) Typical and atypical inducers of lysosomal cell death: a promising anticancer strategy. Int J Mol Sci 19(8). https://doi.org/10.3390/ijms19082256
- 33.Ono K, Kim SO, Han J (2003) Susceptibility of lysosomes to rupture is a determinant for plasma membrane disruption in tumor necrosis factor alpha-induced cell death. Mol Cell Biol 23(2):665–676Google Scholar
- 34.Petersen NH, Olsen OD, Groth-Pedersen L, Ellegaard AM, Bilgin M, Redmer S, Ostenfeld MS, Ulanet D, Dovmark TH, Lonborg A, Vindelov SD, Hanahan D, Arenz C, Ejsing CS, Kirkegaard T, Rohde M, Nylandsted J, Jaattela M (2013) Transformation-associated changes in sphingolipid metabolism sensitize cells to lysosomal cell death induced by inhibitors of acid sphingomyelinase. Cancer Cell 24(3):379–393. https://doi.org/10.1016/j.ccr.2013.08.003 Google Scholar
- 40.Nitta T, Sato Y, Ren XS, Harada K, Sasaki M, Hirano S, Nakanuma Y (2014) Autophagy may promote carcinoma cell invasion and correlate with poor prognosis in cholangiocarcinoma. Int J Clin Exp Pathol 7(8):4913–4921Google Scholar
- 41.Thongchot S, Yongvanit P, Loilome W, Seubwai W, Phunicom K, Tassaneeyakul W, Pairojkul C, Promkotra W, Techasen A, Namwat N (2014) High expression of HIF-1alpha, BNIP3 and PI3KC3: hypoxia-induced autophagy predicts cholangiocarcinoma survival and metastasis. Asian Pac J Cancer Prev 15(14):5873–5878Google Scholar
- 43.Boya P, Gonzalez-Polo RA, Poncet D, Andreau K, Vieira HL, Roumier T, Perfettini JL, Kroemer G (2003) Mitochondrial membrane permeabilization is a critical step of lysosome-initiated apoptosis induced by hydroxychloroquine. Oncogene 22(25):3927–3936. https://doi.org/10.1038/sj.onc.1206622 Google Scholar
- 52.Bernstein HN (1991) Ocular safety of hydroxychloroquine. Ann Ophthalmol 23(8):292–296Google Scholar
- 53.Prudent R, Vassal-Stermann E, Nguyen CH, Mollaret M, Viallet J, Desroches-Castan A, Martinez A, Barette C, Pillet C, Valdameri G, Soleilhac E, Di Pietro A, Feige JJ, Billaud M, Florent JC, Lafanechere L (2013) Azaindole derivatives are inhibitors of microtubule dynamics, with anti-cancer and anti-angiogenic activities. Br J Pharmacol 168(3):673–685. https://doi.org/10.1111/j.1476-5381.2012.02230.x Google Scholar
- 54.Al Dhaheri Y, Attoub S, Arafat K, Abuqamar S, Viallet J, Saleh A, Al Agha H, Eid A, Iratni R (2013) Anti-metastatic and anti-tumor growth effects of Origanum majorana on highly metastatic human breast cancer cells: inhibition of NFkappaB signaling and reduction of nitric oxide production. PLoS One 8(7):e68808. https://doi.org/10.1371/journal.pone.0068808 Google Scholar
- 55.El Hasasna H, Saleh A, Al Samri H, Athamneh K, Attoub S, Arafat K, Benhalilou N, Alyan S, Viallet J, Al Dhaheri Y, Eid A, Iratni R (2016) Rhus coriaria suppresses angiogenesis, metastasis and tumor growth of breast cancer through inhibition of STAT3, NFkappaB and nitric oxide pathways. Sci Rep 6:21144. https://doi.org/10.1038/srep21144 Google Scholar
- 56.Gilson P, Josa-Prado F, Beauvineau C, Naud-Martin D, Vanwonterghem L, Mahuteau-Betzer F, Moreno A, Falson P, Lafanechere L, Frachet V, Coll JL, Fernando Diaz J, Hurbin A, Busser B (2017) Identification of pyrrolopyrimidine derivative PP-13 as a novel microtubule-destabilizing agent with promising anticancer properties. Sci Rep 7(1):10209. https://doi.org/10.1038/s41598-017-09491-9 Google Scholar
- 57.de Duve C, de Barsy T, Poole B, Trouet A, Tulkens P, Van Hoof F (1974) Commentary. Lysosomotropic agents. Biochem Pharmacol 23(18):2495–2531Google Scholar
- 59.Yoshimori T, Yamamoto A, Moriyama Y, Futai M, Tashiro Y (1991) Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J Biol Chem 266(26):17707–17712Google Scholar
- 60.Klionsky DJ, Abdelmohsen K, Abe A et al (2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12 (1):1–222. https://doi.org/10.1080/15548627.2015.1100356
- 64.Ventola CL (2017) Cancer immunotherapy, part 3: challenges and future trends. P T 42(8):514–521Google Scholar
- 65.DeBord LC, Pathak RR, Villaneuva M, Liu HC, Harrington DA, Yu W, Lewis MT, Sikora AG (2018) The chick chorioallantoic membrane (CAM) as a versatile patient-derived xenograft (PDX) platform for precision medicine and preclinical research. Am J Cancer Res 8(8):1642–1660Google Scholar
- 66.Repnik U, Borg Distefano M, Speth MT, Ng MYW, Progida C, Hoflack B, Gruenberg J, Griffiths G (2017) L-leucyl-L-leucine methyl ester does not release cysteine cathepsins to the cytosol but inactivates them in transiently permeabilized lysosomes. J Cell Sci 130(18):3124–3140. https://doi.org/10.1242/jcs.204529 Google Scholar
- 68.Hsu SPC, Kuo JS, Chiang HC, Wang HE, Wang YS, Huang CC, Huang YC, Chi MS, Mehta MP, Chi KH (2018) Temozolomide, sirolimus and chloroquine is a new therapeutic combination that synergizes to disrupt lysosomal function and cholesterol homeostasis in GBM cells. Oncotarget 9(6):6883–6896. https://doi.org/10.18632/oncotarget.23855 Google Scholar
- 72.Uchimoto T, Nohara H, Kamehara R, Iwamura M, Watanabe N, Kobayashi Y (1999) Mechanism of apoptosis induced by a lysosomotropic agent, L-Leucyl-L-Leucine methyl ester. Apoptosis 4(5):357–362Google Scholar
- 73.Thiele DL, Lipsky PE (1990) Mechanism of L-leucyl-L-leucine methyl ester-mediated killing of cytotoxic lymphocytes: dependence on a lysosomal thiol protease, dipeptidyl peptidase I, that is enriched in these cells. Proc Natl Acad Sci U S A 87(1):83–87Google Scholar
- 76.Fehrenbacher N, Bastholm L, Kirkegaard-Sorensen T, Rafn B, Bottzauw T, Nielsen C, Weber E, Shirasawa S, Kallunki T, Jaattela M (2008) Sensitization to the lysosomal cell death pathway by oncogene-induced down-regulation of lysosome-associated membrane proteins 1 and 2. Cancer Res 68(16):6623–6633. https://doi.org/10.1158/0008-5472.CAN-08-0463 Google Scholar
- 77.ClinicalTrials.gov (2018) Study of GNS561 in Patients with liver cancer - full text view - ClinicalTrials.gov. https://clinicaltrials.gov/ct2/show/NCT03316222. Accessed December 18, 2018