G-quadruplex Nucleic Acids as a target for Pancreatic Cancer Drug Discovery


Pancreatic Cancer

Pancreatic cancer is highly lethal, with a mere 3% survival rate 5 years after diagnosis in England and Wales, and it is the fifth most common cause of cancer death (London School of Hygiene and Tropical Medicine, 2014 & Cancer Research UK, 2014). The reason for the poor prognosis of pancreatic cancer is largely due to the delay in its diagnosis. Symptoms present in a similar manner to other illnesses, and the location of the pancreas, deep inside the body, presents an obstacle for the identification of tumours. Additionally a particular difficulty in drug discovery for pancreatic cancer is that the standard drug used for its treatment, gemcitabine, develops resistance. In the past 40 years there has been no improvement on the 3% survival rate, compared to breast cancer survival rates which have increased from 50 – 80% since the 1970s (Pancreatic Cancer Research Fund, accessed 2016). Accordingly there is a great urgency that the unacceptable prognosis of pancreatic cancer be improved through identifying novel therapeutics for its treatment.

What are G-quadruplexes?

G-quadruplexes are guanine rich secondary structures of DNA. Four guanine bases can associate through Hoogsteen hydrogen bonding to form a square planar structure, known as a guanine tetrad. When 2 or more guanine tetrads stack they form a G-quadruplex, which is further stabilised by the presence of a cation, usually potassium. The cation sits in the central channel between each pair of tetrads. A schematic of the G-quadruplex structure (described above) is shown in Figure 1.

 

Rachael Picture 1 27-01-2016

Figure 1, G-quadruplex structure, taken from Huppert, 2006

Targeting G-quadruplexes

Stabilisation of G-quadruplex structures formed within telomeric DNA with the use of G-quadruplex-selective ligands have the capacity for inhibiting the enzyme telomerase (Haider et al., 2003). Telomerase is a highly attractive target for stop the indefinite division of cancerous cells. It is almost universal in all human cancer cell lines, and is found in over 85% of primary tumours (Kim et al., 1994). Telomerase is responsible for the rebuilding of telomeres, the capping ends of chromosomes (Collins & Mitchell, 2002). During normal cellular replication telomeres shorten. Once the critical limit of telomere length has been met cells are programmed for apoptosis. However, with upregulated telomerase, cancer cells have the ability to replicate indefinitely. Accordingly G-quadruplex stabilising ligands can potentially induce apoptosis in cancerous cells.

Liu et al. (2005) have shown that sub-cytotoxic doses of G-quadruplex targeting ligands TMPyP4 and telomestatin in MIA PaCa-2 cells, pancreatic cancer cells, are associated with an increase in senescent cells by 15 and 10% respectively, as determined by β-galactosidase staining at day 39 and 103 respectively. These results validate that growth inhibition of cells is induced by the progressive loss of telomere DNA. This consequently leads to cellular senescence and cell death.

More recently a great interest has been shown in the vast abundance of G-quadruplexes in the promotor regions of oncogenes. The Neidle group at UCL have designed a compound called MM41, a tetra-substituted naphthalene-diimide derivative, shown in Figure 2, which reduces the growth of pancreatic tumours by 80% in treated mice (Ohnmacht et al., 2015). MM41 was delivered by IV with twice-weekly dosage of 15 mg/kg. Tumour growth prevention by MM41 over a time course is displayed in Figure 3. MM41 binds strongly to G-quadruplexes encoded within the promoter sequences of BCL-2 and k-RAS genes. Blocking these genes, which are prominent in cancer and key to the survival and growth of cancer cells, induces cancer cell apoptosis.

Rachael Picture 2 27-01-2016

Final Remarks

Further refinements of MM41 are underway as it is not yet ready for human trials. A derivative compound of MM41, CM03, has shown to target a gene which is involved in cancer resistance (Neidle, accessed 2016). In gemcitabine-resistant MIA PaCa-2 cells CM03 is still potent, thus showing promise in overcoming the hurdle that drug resistance poses on pancreatic cancer drug discovery (Neidle group, data not published). A key consideration is to ensure that potential drugs for pancreatic cancer which designed to interact with G-quadruplexes are selective in targeting cancerous cells, and do not interfere with normal cellular function. From the ongoing trials undertaken by the Neidle group, progress has been made on the drug discovery front for pancreatic cancer in recent years, and advances to human trials may be on the horizon. Thus there is reason to be optimistic about the future of drug discovery for pancreatic cancer.

Blog written by Rachael Besser

References

Cancer Research UK Website (2014). Pancreatic Cancer. Available at: http://publications.cancerresearchuk.org/downloads/Product/CS_KF_PANCREAS.pdf (accessed 20th January 2016)

Collins, K. & Mitchell, J.R. (2002) Telomerase in the human organism. Oncogene. 21(4) 564 – 579

Haider, S.M., Parkinson, G.N. & Neidle, S. (2003) Structure of a G-guadruplex-ligand complex. Journal of Molecular Biology 326(1) 117-125

Huppert, J Website (2006) Biophysics and Bioinformatics of Nucleic Acids. Available at: http://people.bss.phy.cam.ac.uk/~jlh29/index.html (accessed 21st January 2016)

Kim, N.W., Piatyszek, M.A., Prowse, K.R., Harley, C.B., West, M.D., Ho, P.L., Coviello, G.M., Wright, W.E., Weinrich, S.L. & Shay, J.W. (1994) Specific association of human telomerase activity with immortal cells and cancer. Science. 266(5193) 2011–2015

Liu, W., Sun, D. & Hurley, L.H. (2005) Binding of G-quadrupex-ineractive Agents to Distinct G-Quadruplexes Induces Different Biological Effects in MiaPaCa Cells. Nucleosides, Nucleotides and Nucleic Acids. 24: 1801-1815

London School of Hygiene and Tropical Medicine data accessed via Cancer Research UK Website (2014) Pancreatic cancer survival statistics. Available at: http://www.cancerresearchuk.org/health-professional/cancer-statistics/statistics-by-cancer-type/pancreatic-cancer/survival#ref-0 (accessed 14th January 2016)

Neidle, S. UCL School of Pharmacy Website. Targeting cancer genes – A novel approach to the treatment of pancreatic cancer. Available at: https://www.ucl.ac.uk/pharmacy/research/drug-discovery/drug-discovery-projects/pancreatic-cancer (accessed 21st January 2016)

Ohnmacht, S.A., Marchetti, C., Gunaratnam, M.E., Besser, R.J., Haider, S.M., Di Vita, G., Lowe, H.L., Mellinas-Gomez, M., Diocou, S., Robson, M., Sponer, J., Islam, B., Pedley, R.B., Hartley, J.A. & Neidle, S. (2015) A G-quadruplex-binding compound showing anti-tumour activity in an in vivo model for pancreatic cancer. Scientific Reports. 5(11385)

Pancreatic Cancer Research Fund Website. Why We Exist. Available at: http://www.pcrf.org.uk/pages/why-we-exist.html (accessed 14th January 2016)

Rhodes, D. and Lipps, H.J. (2015) G-Quadruplexes and their regulatory roles in biology. Nucleic Acids Research. 43(18) 8627 – 8637

 

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3rd generation EGFR TKIs


Here is a topical paper (Robert Heald et al; J. Med. Chem. 2015, 58, 8877-8895) by the group at Argenta/Genentech on the discovery of third generation EGFR tyrosine kinase inhibitors. First generation EGFR TKIs like Gefitinib or Erlotinib show convincing early responses in lung cancer, but resistance quickly develops that has limited their overall effectiveness. A recent study published in the British Journal of Cancer (2014, 110, 55-62) that looked at 106 patients with EGFR sensitising mutations showed a 70% response rate to Gefitinib and progression free survival of 9.7 months. Approximately 60% of the acquired resistance is caused by a T790M mutation of the gatekeeper residue that substantially reduces the affinity of first generation inhibitors for the ATP binding site. Several pharma companies are in competition to market a new generation of EGFR TKI’s that also inhibit the mutated kinase. Recent news from the FDA announced the approval of AstraZeneca’s TagrissoTM ahead of the Clovis drug, Rociletinib, for treating patients with EGFR T790M mutation positive metastatic non-small cell lung cancer. Together with the recently approved companion diagnostic, CobasTM, TagrissoTM could see rapid uptake in the clinic with estimated sales potential of $3 billion. (http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm472525.htm)

The T790M mutation reduces the effectiveness of first generation inhibitors by both occluding a portion of the ATP binding site and increasing the enzymes’ affinity for ATP. To overcome the low Km[ATP], both Clovis and AstraZeneca have targeted the mutant tyrosine kinase with covalent modifiers that juxtapose the poorly conserved Cys797 with an acrylamide acting as a Michael acceptor. Unlike the second generation inhibitors, both compounds show good selectivity against the wild-type EGFR. Unfortunately, the EGFR is a moving target with the potential for further mutations always one step away.

The lead optimisation case study published by the Argenta/Genentech group comments on recent reports of a C797S mutation in samples from patients who have developed resistance to third generation therapies. Loss of the cysteine nucleophile would render the third generation covalent inhibitors ineffective, but how quickly this becomes a problem will have to await further clinical study.

To address this concern the Argenta/Genentech medicinal chemists have designed a non-covalent inhibitor. Among the challenges are the low Km[ATP] of the mutant EGFR kinase requiring inhibitors to have high affinity for the ATP binding site to overcome the large intracellular ATP concentrations. The paper builds on their initial publication (J. Med. Chem. 2014, 57, 10176-10191) that identified potent and selective inhibitors based on a 4-imidazopyridine substituted diaminopyrimidine scaffold. Their second paper focuses primarily on the DMPK challenges towards designing compounds with predicted low to moderate in vivo human clearance. However, in doing so it covers many areas of medicinal chemistry with an amalgamation of DMPK, structure based design and lead optimisation metrics. In the authors words; “ …this work highlights a number of aspects of medicinal chemistry doctrine: the structural similarity of leads and optimised compounds, the utility of fluorine in the optimisation of small molecule drugs, and the judicious application of compound quality metrics as an aid to interpretation of SAR”. I would also add to the list, in vivo mouse xenograft PK/PD that clearly shows biomarker modulation consistent with the free drug hypothesis, and some ambitious chemistry with the final compound containing no fewer than 4 chiral centres!

Darren 16-12-2015 Picture 1

Darren 16-12-2015 Picture 2

Compound 42 was administered orally (po) to H1975 tumor-bearing mice once a day for 3 days with either 100 or 300 mg/ kg, respectively. Tumors were collected at indicated times (after last dose), and phosphorylation of EGFR, ERK, and AKT was determined using commercially available MSD assays. Lines on the free concentration plot show the in vitro H1975 p-EGFR IC50 and IC90. Error bars represent the mean ± SD. Plasma concentrations of 42 were measured using a liquid chromatography−tandem mass spectrometry assay, and free concentration was calculated using Fu of 0.207.

The authors suggest follow up studies on highlighted compounds to demonstrate an improved therapeutic index over current third generation treatments and evaluating the potency against C797S mutant cell lines.

I can’t find any mention of them on the current Genentech pipeline. Does anybody know about their progress?

Blog written by Darren Le Grand

 

Targeting the spliceosome to treat MYC-driven cancers


MYC overexpression or hyperactivation is a well known driver of human cancer. Despite being the subject of intense study for many years, attempts to therapeutically inhibit MYC directly have been unsuccessful, making synthetic lethal strategies attractive. MYC is a transcription factor, and indeed its oncogenic activity is attributed to its prominent role in gene expression. However, whilst increased production of RNA, and thereby proteins, may enable cancer cell growth, there is a resultant burden on these cells to process all this RNA. Last month, Hsu et al. reported that the spliceosome, which is required for processing of precursor mRNA to mature mRNA, is a target of oncogenic stress in MYC-driven cancers.1 They identified components of the core spliceosome which are synthetically lethal with MYC activation and showed that genetic or pharmacological inhibition of these spliceosomal factors impaired survival of MYC-dependent breast cancers.

In a genome-wide MYC-synthetic lethal screen performed previously, BUD31 was identified as a MYC-synthetic lethal gene.2 While in yeast BUD31 had been linked to the spliceosome, its function in mammalian systems had yet to be determined. Using co-immunoprecipitation and bimolecular fluorescence complementation experiments it was shown that BUD31 associates with 79 of the 134 core spliceosomal components, which are involved in a variety of the major spliceosomal subcomplexes, inferring that it is present at several stages of spliceosomal assembly. Furthermore, knockdown experiments indicated that loss of BUD31 significantly inhibited pre-mRNA splicing and led to defects in early spliceosome assembly. It appears therefore that mammalian BUD31 functions as a core spliceosomal protein.

Accordingly the authors suggest that cells with oncogenic MYC require BUD31 for survival due to this role in the spliceosome. Indeed it was shown that if BUD31 is unable to associate with the spliceosome, the proliferation of MYC-driven cancer cells is significantly inhibited. Other components of the spliceosome assembly were consequently examined. It was found that partial depletion of all the components studied led to loss of cell viability and increased apoptosis of MYC-hyperactivated cells. One of these components was SF3B1 (splicing factor 3b, subunit 1). To test whether pharmacological inhibition of the spliceosome is synthetically lethal with MYC, SD6 was developed as a bioavailable small molecule inhibitor of SF3B1.3 Low (10-20 nM) concentrations of SD6 were able to selectively suppress colony formation and induce apoptosis of MYC-hyperactivated cells.

By comparing intron retention (IR) after BUD31 knockdown in MYC-normal or MYC-hyperactivated cells it was shown that in combination MYC activation and partial spliceosome inhibition result in increased IR. This indicates that the MYC-induced increase in mRNA synthesis increases cellular dependency on the spliceosome. Moreover, depletion of BUD31 caused a considerably larger decrease in cellular polyadenylated mRNA following inhibition of transcription in MYC-hyperactivated cells than in control cells, indicative of defects in pre-mRNA maturation and stability.

Finally the authors queried whether MYC-driven breast cancers exhibit increased sensitivity to knockdown of spliceosomal genes. A pronounced correlation was observed between MYC-dependency and spliceosome-dependency in basal breast cancer lines. In one example the effects of genetic and pharmacological inhibition of the spliceosome were tested on MYC-dependent metastatic triple-negative breast cancer (TNBC) models. Use of BUD31 or SF3B1 shRNA reduced cell viability and the TNBC cells were significantly more sensitive (IC50 ≈ 4nM) to treatment with inhibitor SD6 than were MYC-normal cell lines (IC50 ≈ 53nM). Overall the results suggested that MYC-driven breast cancers are more highly dependent on the spliceosome.

This study clearly highlights targeting of the core spliceosome as a promising strategy for treatment of MYC-driven cancers and explains the basis for the synthetic lethality of BUD31 and MYC.

  1. Hsu, T. Y.-T. et al. The spliceosome is a therapeutic vulnerability in MYC-driven cancer. Nature 525, 384–388 (2015).
  2. Kessler, J. D. et al. A sumoylation-dependent transcriptional subprogram is required for Myc-driven tumorigenesis. Science 335, 348–353 (2012).
  3. Lagisetti, C. et al. Optimization of antitumor modulators of pre-mRNA splicing. J. Med. Chem. 56, 10033–10044 (2013).

Blog written by: Katie Duffell

Single cell analysis of human metastatic breast cancer cells


Metastasis is still the cause of most deaths from cancer, despite major advances in the fields of molecular and genetic characterisation of tumours. Developing an understanding of how metastatic cells arise and go on to form tumours may uncover more information to aid in the development of new treatments. Current thinking is that tumour cells with stem cell like properties may initiate the formation of metastatic tumours.

Lawson et al., (Nature, 2015) have published an elegant study in which they have succeeded in   isolating early stage metastatic cells from patient-derived xenograft (PDX) models of triple negative breast cancer. The authors then examined the gene expression profiles of metastatic cell populations.

Normal human breast epithelium were used to establish a 49 gene differentiation signature as a reference against which to analyse gene differentiation in metastatic cells. They examined these differentiation gene signatures in populations of both basal lineage cells, containing stem cells, and luminal lineage cells containing progenitor and mature cells.

PDX models of triple negative breast cancer were used due to the aggressive, metastasis forming nature of the tumours formed in these mice. Metastatic cells were isolated from mouse lung, lymph node, bone marrow, liver, brain and peripheral blood using PDX breast cancer specific cell surface marker genes (human CD298). The authors found that expression of this marker correlated with the tumour burden observed in the animal and so could then investigate the gene expression signatures of low-burden (early-stage metastatic) vs high-burden (advanced-stage metastatic) disease.

Sarah's 5-10-15 Pic 1

Identification of human metastatic cells in PDX mice

They found that in low-burden tissues, the metastatic cells found were different from the main tumours that they were derived from and that the differentiation gene signature in these cells was of a basal, stem-cell nature. This stem cell differentiation gene signature was conserved in low-burden metastatic cells across all animals and models tested. These metastatic cells expressed very high levels of the pluripotency genes OCT4 and SOX2 that are found in embryonic stem cells. The authors also observed that genes involved in cellular processes such as DNA damage response, chromatin modification, differentiation, apoptosis and cell cycle control were differentially expressed in low-burden metastatic cells. When these low-burden cells were transplanted into mammary glands they found that an unusually high amount of tumour formation was observed, suggesting that these early-stage metastatic cells can initiate tumour formation. The authors concluded that primary tumours contain a rare sub-population of stem-like cells and that a higher percentage of these cells within the tumour correlates with a higher metastatic potential.

Higher-burden metastatic cells expressed lower levels of quiescence and dormancy genes compared to the low-burden metastatic cells, but higher levels of cell cycle genes such as MYC and CDK2, suggesting that there is a shift to a more proliferative gene expression signature with an increasing ability to metastasise. By using a CDK inhibitor (Dinaciclib) the authors were able to demonstrate a reduction in the number of animals presenting with metastatic cells.

Treatment with Dinaciclib dramatically reduced metastasis in mice

Treatment with Dinaciclib dramatically reduced metastasis in mice

What relevance does this study have for drug discovery? The identification and isolation of metastatic cells to study their particular characteristics may enable the identification of potential new targets against metastatic disease. The methods used here to assess metastatic cells could also be used in the development of novel compounds and potentially translate into the clinic to assess patient response to new therapies.

Blog written by Sarah Walker