Parvovirus H-1 an oncolytic virus undergoing clinical trials for pancreatic cancer

Parvovirus H-1 (H-1PV) was originally isolated (reported in 1960) from a number of transplantable human tumour cell lines including HEp 1 and is able to enter normal human cells [1] (figure 1). It is known to infect rats in the field. Its suitability as an oncolytic therapy is under investigation. The H-1PV cognate cellular receptor is unknown however sialic acid is important for cell membrane binding by the virus [2]. Due to its dependence on S phase factors only present during active cell replication and the activation of normal cell innate antiviral programs it is non-pathogenic [3]. Unlike viral vectors such as adenovirus the average person’s immune system has not encountered H-1PV before meaning that it could be suitable for systemic delivery [3]. In proliferative cancer cells H-1PV can replicate and is oncolytic primarily due to the viral protein NS1 [4]. One potential drawback of H-1PV is that due to its dependence on S phase factors it is reasonable to suspect it would not be effective against non-proliferative cancer stem cells unlike other oncolytic vectors such as adenovirus. However it has been shown in in vitro neurosphere cultures that it can infect and lyse brain tumour stem cells [5]. It remains to be seen whether it is as effective against cancer stem cells in vivo. New effective therapies for pancreatic cancer are required due to the dismal effectiveness of current drugs. Parvovirus H-1 is a potential next generation therapeutic currently under investigation for pancreatic cancer.

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Figure 1: 3-D molecular model of parvovirus. Colour scheme is arbitrary. Credit: AJC PHOTOGRAPHY. No changes were made. Creative Commons Attribution-ShareAlike 2.0.

It has been shown in mouse and rat xenograft models of pancreatic cancer that H-1PV synergises with gemcitabine treatment to improve overall survival time [6]. The H-1PV virus however has a dependence upon active SMAD4 expression for replication in pancreatic cancer cell lines [7]. SMAD4 tends to be lost in late stage metastatic pancreatic cancer which also tends to be the group of patients first tested with an investigational new drug. H-1PV is undergoing phase I and II clinical trials in metastatic pancreatic cancer [8]. It would be wise to confirm the SMAD4 status of the patients in these trials so that efficacy can be correlated with SMAD4 status. It would also be wise for future trials to select patients with a confirmed active SMAD4 tumour status.

Refs

  1. Toolan HW, Dalldore G, Barclay M, Chandra S, Moore AE. AN UNIDENTIFIED, FILTRABLE AGENT ISOLATED FROM TRANSPLANTED HUMAN TUMORS. Proceedings of the National Academy of Sciences of the United States of America. 1960;46(9):1256-1258. PMCID: PMC223034
  2. Marchini, Antonio, Serena Bonifati, Eleanor M Scott, Assia L Angelova, and Jean Rommelaere. ‘Oncolytic Parvoviruses: From Basic Virology to Clinical Applications’. Virology Journal 12, no. 1 (2015): 6. doi:10.1186/s12985-014-0223-y.
  3. Allaume, X., N. El-Andaloussi, B. Leuchs, S. Bonifati, A. Kulkarni, T. Marttila, J. K. Kaufmann, et al. ‘Retargeting of Rat Parvovirus H-1PV to Cancer Cells through Genetic Engineering of the Viral Capsid’. Journal of Virology 86, no. 7 (1 April 2012): 3452–65. doi:10.1128/JVI.06208-11.
  4. Hristov, G., M. Kramer, J. Li, N. El-Andaloussi, R. Mora, L. Daeffler, H. Zentgraf, J. Rommelaere, and A. Marchini. ‘Through Its Nonstructural Protein NS1, Parvovirus H-1 Induces Apoptosis via Accumulation of Reactive Oxygen Species’. Journal of Virology 84, no. 12 (15 June 2010): 5909–22. doi:10.1128/JVI.01797-09.
  5. Lacroix, J, R Josupeit, S Kern, C Herold-Mende, F Schlund, H Witt, T Milde, et al. ‘Parvovirus H-1 (H-1PV) exerts oncolytic effects in cell culture models of human brain tumor-initiating cells’. Klin Padiatr 224, no. 06 (9 November 2012): A9. doi:10.1055/s-0032-1320177.
  6. Angelova, A. L., M. Aprahamian, S. P. Grekova, A. Hajri, B. Leuchs, N. A. Giese, C. Dinsart, et al. ‘Improvement of Gemcitabine-Based Therapy of Pancreatic Carcinoma by Means of Oncolytic Parvovirus H-1PV’. Clinical Cancer Research 15, no. 2 (15 January 2009): 511–19. doi:10.1158/1078-0432.CCR-08-1088.
  7. Dempe, Sebastian, Alexandra Y. Stroh-Dege, Elisabeth Schwarz, Jean Rommelaere, and Christiane Dinsart. ‘SMAD4: A Predictive Marker of PDAC Cell Permissiveness for Oncolytic Infection with Parvovirus H-1PV’. International Journal of Cancer, 2010, NA – NA. doi:10.1002/ijc.24992.
  8. ‘Parvovirus H-1 (ParvOryx) in Patients With Metastatic Inoperable Pancreatic Cancer – Full Text View – ClinicalTrials.gov’. Accessed 29 February 2016. https://clinicaltrials.gov/ct2/show/NCT02653313?term=H%E2%80%901PV&rank=2.
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The opportunistic pathogen Pseudomonas aeruginosa

Pseudomonas aeruginosa is a gram negative, aerobic, polarly flagellated bacillus [1]. It is a common inhabitant of soil, water, plants, animals and is an opportunistic pathogen, causing wound, burn, and urinary tract infections in humans. Infections by P. aeruginosa are a great risk for burn victims, where it’s the single greatest cause of death [1]. Cancer, cystic fibrosis, and chemotherapy patients are also at risk. P. aeruginosa is particularly dangerous in hospitals as it is unusually resistant to antibiotics [1].

In 2000 the sequencing of the large 6.3Mbp genome was completed with gene analysis predicting 5570 genes [2]. This approaches the genetic complexity of the simple eukaryote Saccharomyces cerevisiae, whose genome encodes about 6,200 proteins [2]. However, only 54.2% of the genes could be assigned a function [2]. Of the unidentified genes 38% appeared to be unique to P. aeruginosa as they are not homologous with any reported gene sequence. As only genes of known function can be discussed only half the genetics of P. aeruginosa can be described at present. P. aeruginosa is predicted to have all the housekeeping genes for protein synthesis, DNA replication, amino acid synthesis, etc as do other bacteria. However it does have a disproportionately high number of outer membrane protein genes compared with other bacteria, a very large number of genes involved in the uptake of nutrients, a large number of genes involved in β oxidative metabolism, four multi-drug efflux systems, three protein secretion systems, and four chemosensing and chemotaxis systems [2]. The huge array of genetic systems available to P. aeruginosa goes a long way to explaining its ubiquitous and opportunistic pathogenic nature.

Refs

  1. Lim, D. (1998) Procaryotes: The bacteria and the archaea. In: Microbiology. 2nd ed. pp. 319-320. WBC / McGraw-Hill.
  2. Stover, C.K., Pham, X.Q., Erwin, A.L., Mizoguchi, S.D., Warrener, P., Hickey, M. J., Brinkman, F.S.L., Hufnagle, W.O.D., Kowalik, J., Lagrou, M., Garber, R.L., Goltry, L., Tolentino, E., Westbrock-Wadman, S., Yuan, Y., Brody, L.L., Coulter, S.N., Folger, K.R., Kas, A., Larbig, K., Lim, R., Smith, K., Spencer, D., Wong, G.K.-S., Wu, Z., Paulsenk, I.T., Reizer, J., Saier, M.H., Hancock, R.E.W., Lory, S. & Olson, M.V. (2000) Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature. 406:959-964.

The green revolution

In 1798 Thomas Malthus noted in his “Essay on the principle of population”, that the population was increasing geometrically while food production was only increasing arithmetically. He concluded that mass starvation would occur especially among the poor majority of the population. In 1798 the world population was 900 million, today it is greater than 6 billion. The U.N. predicts that by 2050 the world population will reach 9.3 billion. However so far Malthus’ prediction of mass starvation has proved incorrect apart from isolated situations where war and poor governance have led to starvation rather than a world shortage of food.

The green revolution has transformed the productivity of the land. In Malthus’ time ~ 20,000 m2 was required to feed one person per year, now only 2000 m2 of land is needed. Until 1960 there was only a very gradual increase in yield of crops such as wheat and rice due to improved varieties achieved through breeding, development of agricultural methods such as crop rotation, the deployment of fertilisers especially natural, and mechanisation with the introduction of the plough. During the decade between 1960 and 1970 the green revolution dramatically increased crop yields.

The green revolution was a culmination of a number of factors including the development of high yielding varieties of wheat, rice, and maize. Advancements were made in the field of fertilisers, pesticides, herbicides, insecticides, and fungicides. Mechanisation was taken to another level with the development of new tractors and mechanisms for harvesting and planting on an industrial scale. Improvements in irrigation allowed two crops per season. All this led to a huge increase in food production worldwide.

The Neuromuscular Junction

A neuromuscular junction (NMJ) is the synapse or junction of the axon terminal of a motor neuron with muscle fibre plasma membrane [1]. Motor neurons are required for the coordinated contraction of skeletal muscles. In mature muscle, motor axon terminals are located in deep and regular invaginations of the muscle plasma membrane termed postjunctional folds [2]. Acetylcholine receptors (AChRs) are concentrated at the crests of these postjunctional folds. The nerve terminal contains synaptic vesicles (SVs), each of which contains 5000 to 10000 molecules of acetylcholine (ACh) [1]. An action potential at the nerve terminal results in the opening of voltage-gated calcium channels. The ensuing increase in cytosolic Ca2+ initiates a train of events which result in the fusing of the SVs with the nerve terminal plasma membrane and the release of ACh into the synaptic cleft [1]. AChR is a transmembrane ligand gated cation channel consisting of five subunits arranged as a pentameric unit. The channel pore opens in response to the binding of two ACh molecules. This allows sodium to enter and depolarize the muscle cell membrane, which initiates the propagation of action potentials across the surface of the muscle, which through a number of steps results in muscle contraction [1]. In vertebrates the site of junction formation is not predetermined [3]. Functional synapses form within minutes to hours after contact between developing motor neurons and myotubes. However, in mammals, fully differentiated NMJs require several weeks after the first contacts are made to mature [3].

Refs

  1. Hirsch, N.P. 2007. Neuromuscular junction in health and disease. British Journal of Anaesthesia. 99:132-138.
  2. Burden, S.J. 2002. Building the Vertebrate Neuromuscular Synapse. J Neurobiol. 53:501–511.
  3. Burden, S.J. 1998. The formation of neuromuscular synapses. Genes & Dev. 12:133-148.

Turning live cancer cells into drugs

Cancer drugs come in many forms: small molecules, proteins such as cytokines and antibodies, bacteria, and viruses to name a few. Some cancer vaccines utilise irradiated and therefore non-proliferative cancer cell lines. However an emerging concept is the use of a patient’s live proliferating cancer cells as vectors to activate the immune system or deliver drugs to the tumour mass.

In a recent study of a mouse xenograft model of cancer the injection of live cancer cells expressing tumour necrosis factor alpha (TNF) reduced the tumour volume of primary tumours and metastases composed of the same cells as the TNF vector cells [1]. This is thought to be due to the phenomenon of “tumour cell seeding” – circulating tumour cells returning to their site of origin [2]. The clinical extension of this study would be to extract circulating tumour cells from a patient’s blood (not simple for solid tumours) or take a biopsy if possible from the primary tumour, transfect/ transduce with TNF and return the cells to the blood stream.

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Figure 1: Immunofluorescence microscopy image of a group of killer T cells (green and red) surrounding a cancer cell (blue, center). Credit: Alex Ritter, Jennifer Lippincott Schwartz and Gillian Griffiths, National Institutes of Health. No changes were made. Creative Commons Attribution 2.0 Generic.

In a model of murine acute lymphoblastic leukaemia (ALL) it was found that administering mouse ALL cells transduced to express IL-12, an immune system activating cytokine, resulted in an immune response and clearing of the ALL [3]. Importantly in mouse models of squamous cell carcinoma, osteosarcoma, prostate cancer and Lewis lung carcinoma administering cancer cells transduced with IL-12 also resulted in immune system activation and elimination of the tumours [4]. Figure 1 depicts a cancer cell surrounded by activated T-cells. The image is unrelated to study [4]. The effectiveness of live cell vaccines may be related both to their ability to amplify the immune stimulatory signal through initial proliferation and their ability to physically target their tumour of origin. The biotech AvroBio is taking the concept forward into clinical trials for acute myeloid leukemia [5].

Refs

  1. Dondossola, Eleonora, Andrey S. Dobroff, Serena Marchiò, Marina Cardó-Vila, Hitomi Hosoya, Steven K. Libutti, Angelo Corti, Richard L. Sidman, Wadih Arap, and Renata Pasqualini. ‘Self-Targeting of TNF-Releasing Cancer Cells in Preclinical Models of Primary and Metastatic Tumors’. Proceedings of the National Academy of Sciences, 8 February 2016, 201525697. doi:10.1073/pnas.1525697113.
  2. Kim, Mi-Young, Thordur Oskarsson, Swarnali Acharyya, Don X. Nguyen, Xiang H.-F. Zhang, Larry Norton, and Joan Massagué. ‘Tumor Self-Seeding by Circulating Cancer Cells’. Cell 139, no. 7 (December 2009): 1315–26. doi:10.1016/j.cell.2009.11.025.
  3. Labbe, Alain, Megan Nelles, Jagdeep Walia, Lintao Jia, Caren Furlonger, Takahiro Nonaka, Jeffrey A. Medin, and Christopher J. Paige. ‘IL-12 Immunotherapy of Murine Leukaemia: Comparison of Systemic versus Gene Modified Cell Therapy’. Journal of Cellular and Molecular Medicine 13, no. 8b (2 August 2009): 1962–76. doi:10.1111/j.1582-4934.2008.00412.x.
  4. Wei, Louis Z., Yixin Xu, Megan E. Nelles, Caren Furlonger, James C.M. Wang, Marco A. Di Grappa, Rama Khokha, Jeffrey A. Medin, and Christopher J. Paige. ‘Localized Interleukin-12 Delivery for Immunotherapy of Solid Tumours’. Journal of Cellular and Molecular Medicine 17, no. 11 (November 2013): 1465–74. doi:10.1111/jcmm.12121.
  5. ‘Cancer Immunotherapy | AvroBio’. Accessed 22 February 2016. http://www.avrobio.com/technology/cancer-immunotherapy/.

Immunosuppressive enzyme IDO1 – an inhibitor target for pancreatic cancer

The enzyme IDO1 (Indoleamine-pyrrole 2,3-dioxygenase) catalyzes the degradation of the amino acid L-tryptophan to N-formylkynurenine. The activity of IDO1 in cancer cells leads to suppression of the immune system. Catabolites of N-formylkynurenine are directly toxic to T-cells. Furthermore the activity of IDO1 in tumour cells leads to the depletion of L-tryptophan in the microenvironment. Depletion of L-tryptophan in T-cells and dendritic cells present in the tumour microenvironment leads to the build-up of uncharged tryptophan tRNA which triggers a stress response leading to their deactivation [1]. IDO1 mRNA is highly expressed by 14% of the pancreatic cancer cell lines in the CCLE (figure 1) and is up-regulated in cancer tissue (GEO database).

IDO1
Figure 1: Comparison of IDO1 (purple) and for reference CDKN2A (blue) mRNA expression in 44 pancreatic cancer cell lines (data source CCLE). RMA = robust multiarray average (mRNA expression).

Phase I/II metastatic pancreatic cancer clinical trials for IDO1 inhibitors in combination with chemotherapies such as gemcitabine and others are currently recruiting [2]. IDO1 inhibitors are currently in clinical trial in combination with the mesothelin LADD vaccine (see here) in other cancers [3]. In theory this combination should also be effective for pancreatic cancer. IDO1 inhibitors are also in trials in combination with immune checkpoint inhibitors such as PD-1 [4]. IDO1 inhibitor combination with oncolytic viral vectors such as Imlygic (see here) should also be synergistic.

Refs

  1. Soliman, Hatem, Melanie Mediavilla-Varela, and Scott Antonia. ‘Indoleamine 2,3-Dioxygenase’. Cancer Journal (Sudbury, Mass.) 16, no. 4 (2010). doi:10.1097/PPO.0b013e3181eb3343.
  2. ‘Study of IDO Inhibitor in Combination With Gemcitabine and Nab-Paclitaxel in Patients With Metastatic Pancreatic Cancer – Full Text View – ClinicalTrials.gov’. Accessed 22 February 2016. https://clinicaltrials.gov/ct2/show/NCT02077881.
  3. ‘Safety and Efficacy of CRS-207 With Epacadostat in Platinum Resistant Ovarian, Fallopian, or Peritoneal Cancer – Full Text View – ClinicalTrials.gov’. Accessed 22 February 2016. https://clinicaltrials.gov/ct2/show/NCT02575807?term=Epacadostat&rank=1.
  4. Gangadhar, Tara C, Omid Hamid, David C Smith, Todd M Bauer, Jeffrey S Wasser, Jason J Luke, Ani S Balmanoukian, et al. ‘Preliminary Results from a Phase I/II Study of Epacadostat (incb024360) in Combination with Pembrolizumab in Patients with Selected Advanced Cancers’. Journal for ImmunoTherapy of Cancer 3, no. Suppl 2 (2015): O7. doi:10.1186/2051-1426-3-S2-O7.

Reovirus – pancreatic cancer’s natural enemy

Version française

Human reovirus type 3 Dearing (T3D) is a naturally occurring virus that can be used as a tumour specific oncolytic agent (figure 1). Infection by T3D is usually asymptomatic. It does not normally infect healthy cells. However it has been found that cancer cells with activated RAS pathway are readily infected and lysed [1]. KRAS is activated in 90% of the most common form of pancreatic cancer – ductal adenocarcinoma (PDAC). Therefore T3D is naturally selective for pancreatic cancer.

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Figure 1: 3D print of reovirus. Created by NIAID. No changes were made. Creative Commons Attribution 2.0 Generic.

When surgery is possible for pancreatic cancer the five year survival rate is around 22%. This indicates that recurrence is a large problem in pancreatic cancer. This is largely due to cancer stem cells which escape surgery and are highly resistant to chemotherapy. In fact chemotherapeutic agents for pancreatic cancer such a gemcitabine enrich for cancer stem cells [2]. One of the benefits of T3D and other oncolytic viruses is that they can infect slow proliferating cancer stem cells [3]. This a major benefit over small molecule inhibitors which are largely unable to destroy cancer stem cells due to high expression of drug efflux receptors, the cells slow proliferation and resistance to apoptosis. T3D has been shown to replicate in patient pancreatic tumour tissue and clinical trials are ongoing.

Refs

  1. Coffey, Matthew C., James E. Strong, Peter A. Forsyth, and Patrick W. K. Lee. ‘Reovirus Therapy of Tumors with Activated Ras Pathway’. Science 282, no. 5392 (13 November 1998): 1332–34. doi:10.1126/science.282.5392.1332.
  2. Sobrevals, Luciano, Ana Mato-Berciano, Nerea Urtasun, Adela Mazo, and Cristina Fillat. ‘uPAR-Controlled Oncolytic Adenoviruses Eliminate Cancer Stem Cells in Human Pancreatic Tumors’. Stem Cell Research 12, no. 1 (January 2014): 1–10. doi:10.1016/j.scr.2013.09.008.
  3. Marcato, Paola, Cheryl A Dean, Carman A Giacomantonio, and Patrick WK Lee. ‘Oncolytic Reovirus Effectively Targets Breast Cancer Stem Cells’. Molecular Therapy 17, no. 6 (June 2009): 972–79. doi:10.1038/mt.2009.58.