SMYD2 and SMYD3 are lysine methyltransferases. SMYD2 monomethylates the stress response kinase MAPKAPK3 at Lys355. SMYD3 methylates MAP3K2 at K260. Both of these modifications result in increased activity of the respective kinases. In the case of MAPKAPK3 this is thought to be due to decreased protein turnover and therefore higher effective concentration in the cytoplasm. In the case of MAP3K2 the methylation is known to cause the dissociation of the inhibitor protein PP2A [1,2]. SMYD2 and SMYD3 have other protein targets which are relevant to cancer such as p53.
The activation of both kinases promotes pancreatic cancer cell growth. SMYD3 inhibition by small molecule has been shown to inhibit the growth of a wide range of cancers including pancreatic cancer in preclinical animal models . SMYD2 small molecule inhibitors have been developed however they have only been tested in vitro . Combination with MAP kinase inhibitors such as MEK and ERK may prove effective.
- Reynoird, Nicolas, Pawel K. Mazur, Timo Stellfeld, Natasha M. Flores, Shane M. Lofgren, Scott M. Carlson, Elisabeth Brambilla, et al. ‘Coordination of Stress Signals by the Lysine Methyltransferase SMYD2 Promotes Pancreatic Cancer’. Genes & Development, 17 March 2016. doi:10.1101/gad.275529.115.
- Mazur, Pawel K., Nicolas Reynoird, Purvesh Khatri, Pascal W. T. C. Jansen, Alex W. Wilkinson, Shichong Liu, Olena Barbash, et al. ‘SMYD3 Links Lysine Methylation of MAP3K2 to Ras-Driven Cancer’. Nature 510, no. 7504 (21 May 2014): 283–87. doi:10.1038/nature13320.
- Peserico, Alessia, Aldo Germani, Paola Sanese, Armenio Jorge Barbosa, Valeria di Virgilio, Raffaella Fittipaldi, Edoardo Fabini, et al. ‘A SMYD3 Small-Molecule Inhibitor Impairing Cancer Cell Growth’. Journal of Cellular Physiology 230, no. 10 (October 2015): 2447–60. doi:10.1002/jcp.24975.
- Nguyen, Hannah, Abdellah Allali-Hassani, Stephen Antonysamy, Shawn Chang, Lisa Hong Chen, Carmen Curtis, Spencer Emtage, et al. ‘LLY-507, a Cell-Active, Potent, and Selective Inhibitor of Protein-Lysine Methyltransferase SMYD2’. Journal of Biological Chemistry 290, no. 22 (29 May 2015): 13641–53. doi:10.1074/jbc.M114.626861.
|Figure 1: Top panel: Galectin-3 is highly expressed in the 44 pancreatic cancer cell lines in the CCLE. CDKN2A is included for reference. Bottom panel: An analysis of galectin-3 expression from microarray gene-expression profiles of 45 matching pairs of pancreatic tumor and adjacent non-tumor tissues from 45 patients with pancreatic ductal adenocarcinoma (PDAC) (GEO Series GSE28735). RMA = robust multi-array average (mRNA expression).
See also the pancreatic cancer database for papers – here.
GITR (glucocorticoid-induced tumor necrosis factor receptor) is an immune checkpoint cell surface receptor induced within 24-72h on naïve T cells upon engagement with an antigen presenting cell . When GITR engages with its ligand GITR-L effector T cell proliferation is induced. In the pancreatic cancer microenvironment there is a shortage of GITR-L. Therefore one way to improve immune response is with GITR activating antibodies.
In a well known analogy, checkpoint antibodies take the breaks off the immune system, however in the case of pancreatic cancer because it is non-immunogenic, the clinician must also put their foot down so to speak. Mesothelin is a membrane bound glycoprotein that is universally overexpressed in pancreatic cancer. In a preclinical mouse model of pancreatic cancer full length human mesothelin cDNA was used as a vaccine in combination with activating GITR antibodies . In GITR antibody alone treated tumours all remained. In mesothelin alone treated tumours half were eliminated and in GITR antibody and vaccine treated mice 94% had eliminated tumours. This strikingly demonstrates the power of the combination of pancreatic cancer vaccines with immune checkpoint antibodies.
A mesothelin vaccine in combination with GM-CSF immune system stimulation has successfully completed phase II clinical trial . Merck, Bristol-Myers Squibb, and Pfizer are developing GITR antibodies.
- Schaer, David A, Judith T Murphy, and Jedd D Wolchok. ‘Modulation of GITR for Cancer Immunotherapy’. Current Opinion in Immunology 24, no. 2 (April 2012): 217–24. doi:10.1016/j.coi.2011.12.011.
- Gaffney, Mary C., Peter Goedegebuure, Hiroyuki Kashiwagi, John R. Hornick, Reuben I. Thaker, Timothy Eberlein, and William G. Hawkins. ‘DNA Vaccination Targeting Mesothelin Combined with Anti-GITR Antibody Induces Rejection of Pancreatic Adenocarcinoma’. Cancer Research 66, no. 8 Supplement (15 April 2006): 329–329. http://cancerres.aacrjournals.org/content/66/8_Supplement/329.2.
- ‘Clinical Trials | Aduro Biotech’. Accessed 22 March 2016. http://www.aduro.com/pipeline/clinical-trials/.
The GI-4000 vaccine is built from the tarmogen platform . Tarmogens are heat inactivated yeast which contain overexpressed proteins. In the case of GI-4000 they contain peptides of mutated RAS. The KRAS gene is mutated in 90% of pancreatic cancers. In a phase II trial GI-4000 showed a trend towards improved survival in a subset of pancreatic cancer patients, those with post-operative residual disease .
From biopsies of this patient subset a biomarker companion diagnostic was developed retrospectively by MALDI-TOF mass spectrometry that can identify pancreatic cancer patients as likely to respond to GI-4000 . In theory this could be converted to a next generation sequence based diagnostic and this may become a cheaper option as sequencing costs continue to fall. A phase III trial to confirm or refute the effectiveness of GI-4000 in patients identified as eligible by the companion diagnostic remains to be carried out. The potential for combination with checkpoint inhibitors which theoretically should be highly synergistic with the mechanism of action of GI-4000 are no doubt also under consideration.
- ‘GlobeImmune’s $69M IPO Designed to Build Immunotherapy Pipeline – FierceBiotech’. Accessed 21 March 2016. http://www.fiercebiotech.com/story/globeimmunes-69m-ipo-designed-build-immunotherapy-pipeline/2012-07-03.
- Richards, Donald A., Peter Muscarella, Tanios Bekaii-Saab, Lalan S. Wilfong, Vic Velanovich, Julian Raynov, Patrick J. Flynn, et al. ‘Abstract 5314: A Proteomic Signature Predicts Response to a Therapeutic Vaccine in Pancreas Cancer; Analysis from the GI-4000-02 Trial’. Cancer Research 74, no. 19 Supplement (10 January 2014): 5314–5314. doi:10.1158/1538-7445.AM2014-5314.
The glycosaminoglycan hyaluronan (HA) is a major component of the extracellular matrix (ECM). Data from a recent phase II trial suggest that 25-36% of tumours from stage IV previously untreated pancreatic cancer patients have high levels of HA . It is thought that HA can act as a barrier preventing drug penetrance. Hyaluronidase is an enzyme that degrades HA and preliminary data suggests that a systemically delivered formulation of the enzyme can increase overall survival time of late stage pancreatic cancer patients expressing high levels of HA (HAhigh) when combined with nab-paclitaxel and gemcitabine compared with nab-paclitaxel and gemcitabine alone .
It is important to note that due to unavoidable low patient numbers this is only a trend, however subsequent phase III clinical trials will confirm or refute the trend. Increased progression-free survival of HAhigh patients was statistically significant. The stratification of HA status was performed retrospectively and suggests that for future trials eligibility should be determined based on HA status. This is an important strategy that is relevant to all pancreatic cancer therapeutics in development. In the era of next generation sequencing it is important to gather as much patient tumour data as possible with the goal of uncovering biomarkers of efficaciousness which can be used to select patients for future clinical trials.
The importance of HA status necessitated the development of a companion diagnostic. Hyaluronidase can potentially increase drug penetrance and efficaciousness in a subset of pancreatic cancer patients.
- ‘High Response Rate and PFS with PEGPH20 Added to Nab-Paclitaxel/gemcitabine in Stage IV Previously Untreated Pancreatic Cancer Patients with High-HA Tumors: Interim Results of a Randomized Phase II Study.’ Accessed 17 March 2016. http://meetinglibrary.asco.org/print/1991286.
- ‘Halozyme Phase 2 Clinical Study Of Investigational Drug PEGPH20 Shows Doubling Of Progression-Free Survival And Improvement Trend In Overall Survival In High HA Metastatic Pancreatic Cancer Patients’. Accessed 17 March 2016. http://www.streetinsider.com/Press+Releases/Halozyme+Phase+2+Clinical+Study+Of+Investigational+Drug+PEGPH20+Shows+Doubling+Of+Progression-free+Survival+And+Improvement+Trend+In+Overall+Survival+In+High+HA+Metastatic+Pancreatic+Cancer+Patients/10609719.html.
The cremaphor formulation of paclitaxel has dose limiting toxicity which prevent its use for pancreatic cancer. Paclitaxel’s toxicity like the majority of chemotherapeutics stems from its systemic delivery and toxicity to normal cells. However recently an albumin-bound formulation (nab-paclitaxel) has demonstrated increased survival times in combination with gemcitabine compared to gemcitabine alone . And now a new biodegradable device has been developed which can deliver chemotherapeutics including paclitaxel directly to the pancreas limiting systemic toxicities .
The device has been tested with paclitaxel and shown favourable results in mouse xenograft models over systemically delivered paclitaxel. The device is flexible and can be surgically placed over the pancreatic tumour where it rests delivering a steady flow of paclitaxel for the duration of the treatment. The one time insertion is an attractive aspect compared with repeated intravenous deliveries.
Improving delivery and dosing of existing pancreatic cancer chemotherapeutics is a growing area of commercial research. Devices under development include an implantable electroporation device (see here) and encapsulated 293 cells that activate prodrug-chemotherapeutics (see here). Another approach uses low dose chemotherapy to take advantage of pancreatic cancer heterogeneity to maintain stable disease (see here).
- Ma, W. W., and M. Hidalgo. ‘The Winning Formulation: The Development of Paclitaxel in Pancreatic Cancer’. Clinical Cancer Research 19, no. 20 (15 October 2013): 5572–79. doi:10.1158/1078-0432.CCR-13-1356.
- Ligorio, Matteo, Laura Indolfi, David T. Ting, Kristina Xega, Nicola Aceto, Francesca Bersani, Cristina R. Ferrone, et al. ‘Abstract 4584: A Novel Drug-Eluting Platform for Localized Treatment of Pancreatic Cancer’. Cancer Research 74, no. 19 Supplement (10 January 2014): 4584–4584. doi:10.1158/1538-7445.AM2014-4584.
Renalase (RNLS) is a secreted flavoprotein. It is over-expressed by pancreatic cancer and acts as a survival factor blocking apoptosis. Importantly RNLS activates STAT3 and establishes a positive feedback loop whereby STAT3 promotes greater RNLS expression .
STAT3 plays a major role in pancreatic cancer . It will be very important to determine through further research the extent to which STAT3 mediates renalase’s pro-cancer effects. A combination of renalase and STAT3 inhibition may prove effective against pancreatic cancer. A STAT3 inhibitor is already in clinical trials .
As renalase is secreted it may also serve as a useful diagnostic blood biomarker of pancreatic cancer (See here for pancreatic cancer biomarkers under development). This is certainly worth pursuing as late detection is a major reason for the dismal outcomes of pancreatic cancer patients.
- Guo, Xiaojia, Lindsay Hollander, Douglas MacPherson, Ling Wang, Heino Velazquez, John Chang, Robert Safirstein, Charles Cha, Fred Gorelick, and Gary V. Desir. ‘Inhibition of Renalase Expression and Signaling Has Antitumor Activity in Pancreatic Cancer’. Scientific Reports 6 (14 March 2016): 22996. doi:10.1038/srep22996.
- Corcoran, R. B., G. Contino, V. Deshpande, A. Tzatsos, C. Conrad, C. H. Benes, D. E. Levy, J. Settleman, J. A. Engelman, and N. Bardeesy. ‘STAT3 Plays a Critical Role in KRAS-Induced Pancreatic Tumorigenesis’. Cancer Research 71, no. 14 (15 July 2011): 5020–29. doi:10.1158/0008-5472.CAN-11-0908.
- Hong, David, Razelle Kurzrock, Youngsoo Kim, Richard Woessner, Anas Younes, John Nemunaitis, Nathan Fowler, et al. ‘AZD9150, a next-Generation Antisense Oligonucleotide Inhibitor of STAT3 with Early Evidence of Clinical Activity in Lymphoma and Lung Cancer’. Science Translational Medicine 7, no. 314 (18 November 2015): 314ra185. doi:10.1126/scitranslmed.aac5272.
Pancreatic tumours are typically non-immunogenic and those containing so called M2, CD68, CD163 or CD204 positive tumour associated macrophages (TAMs) have a poor prognosis . M1 TAMs are considered to function immunologically against the tumour whereas M2 actively suppress the immune system in the tumour environment. There is a continuous spectrum of macrophage phenotypes between the polar opposites of M1 and M2. Promoting an M1 macrophage phenotype would be highly desirable in pancreatic cancer.
Two secreted cytokines play a role in maintaining the balance: GM-CSF can drive macrophage polarisation to M1 whereas CSF-1 can drive M2 polarisation. Antibodies targeting the macrophage receptor for CSF-1 (CSF-1R) can block the M2 phenotype and tip the balance towards M1 (figure 1) . Several companies including Bristol-Myers Squibb, Roche, and Lilly have anti-CSF-1R antibodies in phase I clinical trial for solid tumours including pancreatic.
|Figure 1: In the balance. Credit: synx508. No changes were made. Creative Commons Attribution-NonCommercial 2.0 Generic (CC BY-NC 2.0).
- Kurahara, Hiroshi, Hiroyuki Shinchi, Yuko Mataki, Kousei Maemura, Hidetoshi Noma, Fumitake Kubo, Masahiko Sakoda, Shinichi Ueno, Shoji Natsugoe, and Sonshin Takao. ‘Significance of M2-Polarized Tumor-Associated Macrophage in Pancreatic Cancer’. The Journal of Surgical Research 167, no. 2 (15 May 2011): e211–19. doi:10.1016/j.jss.2009.05.026.
- Ries, Carola H., Michael A. Cannarile, Sabine Hoves, Jörg Benz, Katharina Wartha, Valeria Runza, Flora Rey-Giraud, et al. ‘Targeting Tumor-Associated Macrophages with Anti-CSF-1R Antibody Reveals a Strategy for Cancer Therapy’. Cancer Cell 25, no. 6 (June 2014): 846–59. doi:10.1016/j.ccr.2014.05.016.
The chemotherapeutic ifosfamide is activated in the liver by cytochrome P450 (P450) enzymes and must travel systemically to the tumour site. The greater the distance of the tumour from the liver the greater the dose requirement for effective delivery. The toxicities induced by an effective dose for pancreatic cancer are too great. Activated ifosfamide can however be targeted to the pancreatic cancer reducing the dose requirement.
A new technology for encapsulating (figure 1) cells in polymers of cellulose sulphate has been developed . This technology protects the enclosed cells from the immune system but allows the free flow of soluble proteins and chemicals. Clinical studies have shown that it is possible to encapsulate 293 cells overexpressing P450 and deliver the capsules to the pancreas via the blood vessels without adverse effects. Lower doses of ifosfamide can then be systemically delivered and yet have a high active local concentration at the pancreas. A phase II trial is planned to confirm effectiveness in pancreatic cancer patients refractory to gemcitabine and abraxane or FOLFIRINOX .
- Gunzburg, W. H., and Brian Salmons. ‘Use of Cell Therapy as a Means of Targeting Chemotherapy to Inoperable Pancreatic Cancer’. ACTA BIOCHIMICA POLONICA-ENGLISH EDITION- 52, no. 3 (2005): 601. https://www.researchgate.net/profile/Brian_Salmons/publication/236627164_Cell_and_gene_therapy_to_improve_cancer_treatment/links/0deec52d89a7e213a2000000.pdf.
- ‘PharmaCyte Biotech Issues Update on Preparations for Its Pancreatic Cancer Clinical Trial Other OTC:PMCB’. Accessed 10 March 2016. https://globenewswire.com/news-release/2016/02/22/812825/0/en/PharmaCyte-Biotech-Issues-Update-on-Preparations-for-Its-Pancreatic-Cancer-Clinical-Trial.html.
Mucins such as MUC1, MUC4, MUC5AC, and MUC16 are highly expressed in pancreatic cancer and have diverse consequences including reducing the penetrance of chemotherapeutics. Mucins are secreted or membrane bound highly glycosylated proteins that make up what is recognised as mucus. Cell surface mucins are a major constituent of what is known as the apical glycocalyx of all mucosal epithelia . Most studies have focused on modulating the protein expression of individual mucins, however a new study has identified an enzyme GCNT3 which catalyses the core glycosylation of all mucins . By disrupting GCNT3 one can effectively eliminate the mucus layer generated by pancreatic cancer.
By targeting GCNT3 with CRISPR and the anti-inflammatory drug talniflumate the authors demonstrated reduced viability of pancreatic cancer cell lines. In a genetically engineered mouse model (GEM) the number of precursor lesions to pancreatic cancer were reduced when the mice were treated with talniflumate. This is an interesting strategy to knock out the mucus layer of pancreatic cancer that could increase the permeability of tumours to chemotherapeutics and thus further drug combination studies will be important.
- Linden SK, Sutton P, Karlsson NG, Korolik V, McGuckin MA. Mucins in the mucosal barrier to infection. Mucosal Immunol. 2008 May;1(3):183-97. doi: 10.1038/mi.2008.5. Review. PubMed PMID: 19079178.
- Rao, C. V., N. B. Janakiram, V. Madka, G. Kumar, S. Edgar, G. Pathuri, T. Bryant, et al. ‘Small Molecule Inhibition of GCNT3 Disrupts Mucin Biosynthesis and Malignant Cellular Behaviors in Pancreatic Cancer’. Cancer Research, 15 February 2016. doi:10.1158/0008-5472.CAN-15-2820.