Cancer Genome Landscapes

Vogelstein et al have published a very informative review on the genomic landscapes of cancer.

As the cost of genome sequencing has fallen 100 fold in the last ten years it is becoming commonplace to sequence the exomes of sets of 100+ tumours, which is allowing us to study the genomic make up of tumours.

The number of mutations a tumour has is dependent on its type:  solid adult tumours have between 33-66, where as childhood cancers far fewer.

FP1Exceptions to this are lung cancers in smokers and melanomas that have far more mutations due to the mutagenic impact of the carcinogen (smoke/sunlight) that initiates them. Tumours with defects in DNA Damage Response also accrue disproportionally large number of mutations.

Interesting, the number of mutations observed in solid adult tumours in self-renewing tissues (e.g. colon) are proportional to the age of the patient implying that these mutations may be present at the pre-neoplastic stage.

It is estimated that between two to eight sequential alterations that develop over the course of 20 to 30 years are actually causative of the cancer.  These are termed  “driver mutations” and occur in driver genes.  Each alteration causes a selective growth advantage to the cell in which it resides.  The other mutations occur because the cancer is genetically unstable, are termed passenger mutations, and confer no selective advantage to the cell in which they reside,

Driver genes are genes that contain driver mutations and there are two types:

Tumour suppressors that confer a selective advantage to the cell when the are “broken”

Oncogenes who confer a selective advantage to the cell, if they are “activated”.

Vogelstein et al. estimate how many driver genes exist using the 20:20 rule. In tumour suppressors at least 20% of the mutations cause truncation of the gene product, where in oncogenes at least 20% of the missense mutations occur in a single position along the polypeptide chain. (see figure 2)


PIK3CA and IDH1 are oncogenes, where as RB1 and VHL are tumour suppressors.

Using the 20:20 rule Vogelstein et al.identify ~140 genes whose intragenic mutations contribute to cancer (so-called Mut-driver genes).

Interesting this is far fewer than the ~500 genes identified in the Cancer Gene Census as being causative of cancer.  They suggest that other genes (Epi-driver genes) that are altered by epigenetic mechanisms and cause a selective growth advantage, but the definitive identification of these genes has been challenging.

Although every individual tumor, even of the same histopathologic subtype as another tumor, is distinct with respect to its genetic alterations, but the pathways affected in different tumors are similar.  These driver genes function through a dozen signaling pathways that regulate three core cellular processes: cell fate determination, cell survival, and genome maintenance.


They also briefly discuss that currently most anti-cancer drugs available inhibit the activity of an enzyme. However, of ~140 driver genes identified, only 31 could be targeted in this manner.

Indeed the majority of Mut-driver genes encode tumor suppressors, not oncogenes. Drugs generally interfere in the function of a protein – conceptually very difficult to produce a drug that will restore the function of a protein.

They conclude that in the future, the most appropriate management plan for a patient with cancer will be informed by an assessment of the components of the patient’s germline genome and the genome of his or her tumor.  That the inherent heterogeneity of tumours and their metastases makes resistance to targeted therapies ‘inevitable’ and that it is important to research the efficacy of combination therapies. They also suggest that the information from cancer genome studies should be exploited to improve methods for prevention and early detection of cancer, which will be essential to reduce cancer morbidity and mortality.

Targeting leukaemias with a therapeutic human antibody

Currently marketed anticancer monoclonal antibodies (mAbs) recognise extracellular proteins or those expressed on the cell surface. Generally these are not tumour-specific, as oncogenic proteins tend to be nuclear or cytoplasmic. These intracellular proteins can however, be presented on the cell surface as T cell epitopes by the major histocompatibility complex (MHC). These epitopes are recognised by T cell receptors (TCRs). Therefore, the generation of ‘TCR-like’ mAbs that can recognise cell surface epitopes that are derived from tumours are an exciting potential cancer therapy.

Dao et al., recently published a paper in Science Translational Medicine, in which they have exploited phage display technology to produce a specific mAb (ESK1) against Wilms tumor 1 (WT1), an oncoprotein that is overexpressed in both leukaemia and a range of solid tumours such as ovarian cancer and mesothelioma, but is rare in normal tissues. It was also recently ranked as a top cancer target for immunotherapy by the NIH. ESK1 targets a 9mer WT1 derived peptide (RMF) that is processed and presented by HLA-A0201. RMF induces cytotoxic T cells that are able to kill WT1+ tumour cells in vitro.

ESK1 was shown to bind to acute myeloid leukaemia (AML) CD34+/CD33+ cells expressing HLA-A02 and WT1 but not to normal peripheral blood mononuclear cells. The specificity of the binding was confirmed with other cell types with no general cross-reactivity to healthy or leukemic cells that do not express WT1. Radioimmunoassay experiments then demonstrated that there was adequate RMF expressed on the surface of many cancer and leukaemia cells, whilst the levels of epitope on WT1 negative healthy cells are low.

mAbs can cause cytotoxicity in four ways: antibody-dependent cell-mediated cytotoxicity (ADCC), complement-mediated cytotoxicity (CMC), antibody-dependent cellular phagocytosis (ADCP) and inducing apoptosis. ESK1 was shown to be active in ADCC assays against the following cell types: JMN mesothelioma, BV173 leukaemia, ovarian carcinoma, colon carcinoma cell lines and AML cells. No other form of mAb-mediated cytotoxicity was observed.

The efficacy of ESK1 was then tested in vivo in mice that had been xenografted with BV173 acute lymphoblastic leukaemia (ALL) cells or BA25 acute lymphocytic leukaemia. Two intravenous doses of 100ug of ESK1, when administered in conjunction with human effector cells, suppressed the growth of  leukemic cells in both animal models. Prolonged or, in some cases, leukaemia-free survival were observed. Two control studies confirmed that the RMF/A2 epitope was required for the therapeutic effect observed. Further, no evidence of toxicity was observed in transgenic mice when given therapeutic doses of ESK1.


Survival of NSG mice with BV173 leukaemia. **P<0.01 for all treatment groups compared to untreated control animals or animals treated with isotype control hIgG (log-rank Mantel-Cox test).

The ESK1 antibody could therefore be promising as a new cancer drug, with a large clinical impact for those patients with WT1+ tumours or leukaemia with HLA-A02 expression.


Assessment of Cancer Genes for Drug Discovery

The Cancer Gene Census documents a list of genes which when genetically altered are known to contribute directly to cancer.

A recent paper by Patel et al describes a systematic, computational protocol, that they have used to identify which of these genes code for proteins that would be possible candidate targets, suitable for therapeutic modulation in the treatment of cancer.  A suite of analyses were undertaken to explore the biological and chemical space of these proteins (shown below).


Following the computational analysis, the authors prioritize these proteins for drug development.  First they identified twenty-five proteins already known to be drug targets, with compounds with full FDA approval.  They suggest that some of the compounds may be useful for repurposing in different types of cancer.  For instance Smoothened SMO is the target of Vismodegib was recently approved for the treatment of basal cell carcinoma.  By mining multi-omic data from The Cancer Genome Atlas the authors suggest that Vismodegib might also be of use in treating Multiforme Glioblastoma (GB), as SMO was over-expressed in 95% of the GB samples analysed.

A  further eight-six proteins had active chemical compounds with submicromolar activity in biochemical or binding assays reported in the Chembl database.

They also explored which proteins had a known structure and predicted potential druggable pockets. Figure 2 illustrates the three-dimensional structure of GNAS with the druggable cavity displayed as a surface.  GNAS has an activating dominant mutation in pituitary adenoma, and further activating mutations have also been identified in kidney, thyroid, adenocortical, colorectal and Leydig tumours.  The authors suggest that small-molecule inhibitors of this enzyme regulator may have potential therapeutic applications.


Of the 488 cancer gene census proteins, the authors identify 103 with good evidence of chemical tractability and group them by  “drug development” risk. They identify 46 proteins, whose genes are known to be genetically altered in cancer, whose structures are predicted to be druggable, with few or no know active small molecule modulators, that may be potential therapeutic targets. They suggest that these targets indicate new biological areas for chemical exploration in the treatment of cancer, but they also represent a high potential drug development risk.


Cancer stem cells- a new target to inhibit tumour growth

One of the keys for drug discovery is to be able to target the diseased pathways/cells without affecting the healthy cell/pathways. This is particularly evident in cancer, where the challenge is to target the cancerous cells without affecting healthy cells. Three recent publications have raised the bar of this for drug discovery. These studies have identified stem cells at the heart of the tumour which appear to be the drivers for certain types of cancer that are resistant to current chemotherapy. Simultaneously providing potentially revolutionary novel targets for cancer, whilst also increasing the challenge to hit these cells without targeting the healthy cells.

To put these findings in context, for some time there has been debate as to whether stem cells sit at the heart of cancers and the role that these cells may have. Up until this point, the evidence for stem cells has mainly been from immunohistochemical staining/FACs sorting and assaying them in vitro. The problem with using these methods is whether the in vivo phenotype is being altered by in vitro culturing. However, by use of lineage tracing, three separate groups (Luis Parada at the South Western Medical School, University of Texas; Cedric Blanpain of the Free University of Brussels and Hans Clevers at the Hubrecht Institute in Utrecht) in different tumours, in the brain, gut and skin, have demonstrated in each, a subpopulation of stem cells that may propagate and spread the cancer.

Glioblastoma multiforme (GMB) is an aggressive tumour that initially responds to chemotherapy however, the cancer nearly always returns. As such GBM is considered incurable and has a median survival of 15 months. In the first of these studies Chen et al., (1) used mice bred to develop GBM, in which they labelled healthy adult neural stem cells, but not their descendants, with a genetic marker. They found all the tumours again contained at least a few labelled cells, along with the unlabelled cells. Chemotherapy with temozolomide killed the unlabelled cells, but the tumours returned. When the animals were tested again, the tumours contained unlabelled cells that came from the labelled stem cells. When they used the chemotherapeutic treatment alongside a technique to supress the labelled stem cells they found the tumours shrank back to “residual vestiges” that bore no resemblance to GBM. Hence, they identified a chemotherapeutically resistant tumour cell that behaved more like stem cells. These cells themselves do not rapidly divide; however, they give rise to rapidly dividing progeny that are susceptible to current chemotherapy.

In a similar way Schepers et al., (3) used genetically engineered mice to label healthy gut cells and stem cells in benign intestinal tumours, a precursors of cancer. These labels carried a drug-inducible marker, that, when activated, fluoresce one of four colours. They found that even though the tumours consist of many different cell types, each tumour fluoresced the same colour, suggesting they arose from one single stem cell. To double check this, the researchers added a lower dose of the drug that caused the stem cells to fluoresce a different colour. In doing so, they demonstrated the stem cells were consistently producing progeny of different cell types.

In the final study Driessens et al., (2) also used linage tracing and identified distinct proliferative cell compartments within a benign papilloma. The majority of the cells had only limited proliferative potential, however a fraction had the capacity to persist long term. The former population gave rise to a terminally differentiated cell population; however, the more persistent population had stem cell-like characteristics and produced many progeny. In addition, as the tumours became more aggressive they were also more likely to produce new stem cells and less likely to produce terminally differentiated cells.

These studies present clear evidence of the existence of cancer stem cells, explaining the re-occurrence of some cancers after successful chemotherapy, but also critically providing new lines of research for drug targeting. Clearly research will now be focussed on killing these cells to eradicate the cancer, however, targeting the stem cell’s proliferative capacity, or encouraging them to differentiate into non-dividing cells may also be as effective.  Since the numbers of stem cells within the tumours appear to be so small and readily able to differentiate, isolating them to study in vitro is unlikely to be fruitful. However, the labelling and tracking the stem cells and their progeny in vivo demonstrated by these groups enable these cells to be studied in vivo and will be vital for drug discovery programmes. This will enable micro-dissection; genomic sequencing and micro-array analysis to be more easily performed on a pure population of cancer stem cells to be used for target identification/validation. In addition by enabling the tracking of these cells and their progeny in vivo the efficacy of any compounds targeted to either kill the stem cells, or prevent the production of progeny could be assed relatively easily.

The challenge of targeting a small cell population that relatively little is known about, without damaging healthy tissue-resident stem cells will be great. However, the research provides great tools to aid this process together with a new way in to provide novel therapeutic agents for cancer therapy.

1.           Chen J, Li Y, Yu T-S, McKay RM, Burns DK, Kernie SG, Parada LF. A restricted cell population propagates glioblastoma growth after chemotherapy. Nature (August 1, 2012). doi: 10.1038/nature11287.

2.           Driessens G, Beck B, Caauwe A, Simons BD, Blanpain C. Defining the mode of tumour growth by clonal analysis. Nature (August 1, 2012). doi: 10.1038/nature11344.

3.           Schepers a. G, Snippert HJ, Stange DE, van den Born M, van Es JH, van de Wetering M, Clevers H. Lineage Tracing Reveals Lgr5+ Stem Cell Activity in Mouse Intestinal Adenomas. Science 730, 2012.

Why tumours eat tryptophan?

Sometimes (more often than I care to admit) I read papers because their titles intrigue me rather than I’m interested in the topic. The conjured image of tumours eating tryptophan [i] pulled me in and it turned out to be of more interest than I first thought, leading me into an area of which I was previously completely unaware.

For some time it has long been a matter for conjecture as to how cancers evade immune responses. It now seems the mystery is beginning to be unravelled and the elevated consumption of tryptophan by tumours plays a key role. Tryptophan is metabolised by a well established route (Figure 1) [ii].

Figure 1. Metabolism of L-tryptophan into serotonin and melatonin (left) and niacin (right). Transformed functional groups after each chemical reaction are highlighted in red.

The first enzymes on the pathway to niacin are the indoleamine 2,3-dioxygenase, IDO & IDO2. The publication by Opitz et al [iii] identified tryptophan dioxygenase (TDO) as an enzyme carrying out the same reaction. The downstream product, kynurenine (Kyn), was identified as the endogenous ligand for the human aryl hydrocarbon receptor. The up-regulation of tryptophan dioxygenase (TDO) was key to this discovery. The roles of IDO and IDO2 in cancer have been known for some time. What is new is the identification of TDO as a key enzyme in this process as well.

Kyn suppresses antitumour immune responses and promotes tumour-cell survival and motility through the AHR in an autocrine/paracrine fashion (Figure 2). Binding of Kyn to the AHR leads to translocation of the receptor to the nucleus and upregulation of genes promoting cell growth. Additionally Kyn inhibits the activation of T-cells and dentritic cells and regulatory B-cells. The upshot of this is to down-regulate the immune system and prevent it from attacking the growing tumour.

Figure 2. Autocrine and paracrine effects of TDO-derived Kyn on cancer cells and immune cells through the AHR.The immunomodulating properties of imatinib have already been noted [iv]. These suggest that in gastrointestinal stromal tumour (GIST) patients one of imatinib’s effects is to stimulate an anticancer immune response by alleviating IDO-mediated immunosuppression. Inhibition of cKit with imatinib leads to down regulation of IDO expression and prevents local immunosupression (Figure 3).

Figure 3. Activated c-KIT induces expression of the transcription factor ETV4, which transactivates IDO stimulating Treg cells, resulting in the local inhibition of CD8+ cytotoxic T lymphocytes and NK cellsThis work identifies TDO activation as also promoting cancer-cell migration, something that IDO has not been reported to do. This suggests some divergence in function between TDO and IDOs, despite their shared ability to generate Kyn.TDO is structurally dissimilar to IDO and IDO2, but all three enzymes can consume substrates other than tryptophan. If TDO’s substrate preference differs from that of the IDO enzymes, this might differentiate its biological functions from those of IDO or IDO2 to some extent. Whatever the case, Opitz and colleagues’ work suggests that TDO inhibitors might be important for cancer studies, both because they may treat IDO independent cancers and because TDO activation could be one way for tumours to acquire resistance to IDO inhibitors.To address whether the TDO–Kyn–AHR signalling pathway is activated in cancers, microarray data from a diverse collection of tumour samples were analysed [iii]. Interestingly, TDO expression correlated with the expression of the AHR target gene CYP1B1 in glioma, B-cell lymphoma, Ewing sarcoma, bladder carcinoma, cervix carcinoma, colorectal carcinoma, lung carcinoma and ovarian carcinoma. So, the TDO–Kyn–AHR pathway seems to be a common trait of cancers.Analysis of the Rembrandt database revealed that the overall survival of patients with glioma (WHO grades II–IV) with high expression of TDO, the AHR or the AHR target gene CYP1B1 was reduced in comparison with patients with intermediate or low expression of these genes. Also, in patients with glioblastoma (WHO grade IV), the expression of the AHR targets CYP1B1, IL1B, IL6 and IL8, which are regulated by TDO-derived Kyn in glioma cells, were found to predict survival independently of WHO grade, thus further confirming the importance of AHR activation for the malignant phenotype of gliomas.

The hypothesis that IDO inhibition might enhance the efficacy of cancer treatments is is being currently evaluated. Results from in vitro and in vivo studies have suggested an improvement of the efficacy of therapeutic vaccination or chemotherapy by concomitant administration of an IDO inhibitor [v].

Inhibitors of both IDO [vi] and TDO[vii] are beginning to appear in the scientific literature, primarily from the Drug Design and Discovery Centre, University of Namur, Belgium. At present these compounds are structurally related to tryptophan and of moderate potency (Figure 5 & 6). The most potent TDO inhibitor (Figure 4) possessed a TDO IC50 2mM, was non-toxic (TD50 >400mM) and was soluble (>300uM). The compound was advanced to an in vivo efficacy study to decipher the exact role of TDO in cancer immunosuppression. Mice were immunized and challenged with compound administered in the drinking water. Systemic treatment of immunized mice with compound at 160 mg/kg/day prevented the growth of TDO-expressing P815 tumour cells with no obvious signs of toxicity.

Figure 4.

Figure 5. IDO inhibitors

Figure 6. TDO inhibitors


 [i] Cancer: Why tumours eat tryptophan, Prendergast George C, Nature (2011), 478(7368), 192-4
[iii] An endogenous tumour-promoting ligand of the human aryl hydrocarbon receptor, Opitz, Christiane A.; Litzenburger, Ulrike M.; Sahm, Felix; Ott, Martina; Tritschler, Isabel; Trump, Saskia; Schumacher, Theresa; Jestaedt, Leonie; Schrenk, Dieter; Weller, Michael; Nature (2011), 478(7368), 197-203
[iv] Imatinib potentiates antitumor T cell responses in gastrointestinal stromal tumor through the inhibition of Ido, Balachandran, V.P. et al. Nat. Med. 17, 1094–1100 (2011).
[v] Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase, Uyttenhove, C.; Pilotte, L.; Theate, I.; Stroobant, V.; Colau, D.; Parmentier, N.; Boon, T.; Van den Eynde, B. J. Nat. Med. 2003, 9, 1269. Inhibition of indoleamine 2,3-dioxygenase, an immunoregulatory target of the cancer suppression gene Bin1, potentiates cancer chemotherapy, Muller, A. J.; DuHadaway, J. B.; Donover, P. S.; Sutanto-Ward, E.; Prendergast, G. C. Nat. Med. 2005, 11, 312. Indoleamine 2,3-dioxygenase in cancer: targeting pathological immune tolerance with small-molecule inhibitors, Muller, A. J.; Malachowski, W. P.; Prendergast, G. C. Expert Opin. Ther. Targets 2005, 9, 831. Marrying immunotherapy with chemotherapy: why say IDO? Muller, A. J.; Prendergast, G. C. Cancer Res. 2005, 65, 8065. Indoleamine 2,3-dioxygenase in immune suppression and cancer, Muller, A. J.; Prendergast, G. C. Curr. Cancer Drug Targets 2007, 7, 31.
[vi] Indol-2-yl ethanones as novel indoleamine 2,3-dioxygenase (IDO) inhibitors, Dolusic, Eduard; Larrieu, Pierre; Blanc, Sebastien; Sapunaric, Frederic; Norberg, Bernadette; Moineaux, Laurence; Colette, Delphine; Stroobant, Vincent; Pilotte, Luc; Colau, Didier; et al, Bioorganic & Medicinal Chemistry (2011), 19(4), 1550-1561
[vii] Tryptophan 2,3-Dioxygenase (TDO) Inhibitors. 3-(2-(Pyridyl)ethenyl)indoles as Potential Anticancer Immunomodulators, Dolusic, Eduard; Larrieu, Pierre; Moineaux, Laurence; Stroobant, Vincent; Pilotte, Luc; Colau, Didier; Pochet, Lionel; Van den Eynde, Benoit; Masereel, Bernard; Wouters, Johan; et al, Journal of Medicinal Chemistry (2011), 54(15), 5320-5334