oncology – Page 6 – Drug Discovery Opinion

CCR7 Linked to Leukaemia Infiltration of CNS

T-ALL cells T-cell acute lymphoblastic leukaemia (T-ALL) primarily affects children and adolescents, and is characterised by a high rate of infiltration into the central nervous system. Aggressive treatment using cranial irradiation and intrathecal chemotherapy to prevent CNS involvement has improved survival outcomes but at a high cost to patients – side effects can include secondary brain tumours, delayed development and mental retardation. The severe consequences of available treatments to prevent CNS involvement in T-ALL have driven a search for genes and signalling pathways involved in CNS infiltration.

CCR7 was already known as a key player in normal lymphocyte migration and adhesion, and as a binding partner of two chemokines, CCL19 and CCL21. Previous studies had also implicated these receptor-ligand interactions in the metastasis of tumours such as melanomas and breast cancers, and anti-CCR7 antibodies had been shown to block the migration of chronic lymphocytic leukaemia (CLL) cells. Scientists at NYU School of Medicine have now shown that CCR7 is also essential for infiltration of T-ALL cells into the CNS.

The Notch1 signalling pathway is essential in T-cell development and earlier research had linked mutations in Notch1 with the progression of T-ALL. In the new study, the team found that mice expressing overactive forms of Notch1 developed leukaemia and, moreover, that the leukaemia cells efficiently infiltrated the inner layer of the membrane covering the brain. Using gene-expression profiling, CCR7 gene expression was found to be controlled by Notch1 and CCR7 itself to be essential for CNS infiltration. Silencing either CCR7 or one of its cognate ligands, CCL19, in an animal model of T-ALL specifically inhibited CNS infiltration. Mouse brain infiltration by human T-ALL cells was also found to be dependent on CCR7 expression. In four out of five T-ALL cell lines derived from human patients, the gene for CCR7 was found to be upregulated, providing further evidence for its role in the disease. The identification of a single chemokine receptor as necessary and sufficient for CNS infiltration suggests a new possibility for reducing the complications associated with aggressive CNS-targeted therapy for T-ALL using small molecules, antibodies or RNAi directed against CCR7.

The study is in the June 18^th issue of the journal Nature.

Replacement miRNA Treats Liver Cancer in Mice

MicroRNAs (miRNAs) are small (21-23 nucleotides in length) single stranded RNA molecules which are not translated into proteins but whose main function is to regulate gene expression. Almost all types of tumour have abnormal – usually reduced – miRNA expression, and scientists at Johns Hopkins Medical School, Nationwide Children’s Hospital, and Ohio State University have now shown that replacing missing miRNAs in mice with liver cancer can rapidly kill the tumour cells whilst leaving healthy cells untouched. The team found that hepatocellular carcinoma (HCC) cells had reduced levels of miR-26a, a miRNA that is normally expressed at high levels in a variety of tissues. In vitro, expression of miR-26a in liver cancer cells was shown to cause cell-cycle arrest associated with direct targeting of cyclins D2 and E2. Systemic administration of miR-26a to mice with HCC using an adeno-associated virus vector inhibited the proliferation of the cancer cells and protected the animals from disease progression. Normal liver cells were unaffected by the treatment. After three weeks, 8 out of 10 mice treated with the miRNA showed only small tumours or a complete absence of tumours, whereas 6 out of 8 sham-treated mice experienced aggressive disease progression.

The study shows that liver cancer can be successfully treated in an animal model of disease by replacing ‘missing’ miRNA using a delivery vector that is suitable for use in the clinic. Although more work is needed before the technique could be used in patients, it could provide an effective and safe treatment for human HCCs, which account for 80 – 90% of all liver cancers and generally have a poor prognosis. If suitable delivery systems can be developed, the technique could also be used to treat other diseases caused by ‘missing’ miRNA.

The study is published in the June 12^th edition of Cell.

Targeting Cancer Stem Cells

Cancer stem cells are slowly dividing tumourigenic cells that possess characteristics of normal stem cells. It has been proposed that these cells persist in treated tumours and are responsible for tumour re-growth and metastasis. As a consequence, more effective chemotherapy may be achieved by targeting these cells in addition to the rapidly proliferating tumour cells. So far, the difficulty has been the lack of understanding of cancer stem cells and how they might be selectively targeted over normal stem cells.

In research carried out at The Jackson Laboratory in mice, Alox5 (5-Lipoxygenase) has been shown to be essential for development and maintenance of cancer stem cells in bcr-abl dependent chronic myeloid leukemia (CML). CML did not develop in mice without the Alox5 gene as a result of impaired function of the leukemia stem cells. Importantly, the lack of Alox5 did not affect normal stem cells, indicating distinct pathways in normal and leukemic stem cells for differentiation and self-renewal.

zileuton structure Alox5 is known to be essential for processing fatty acids to leukotrienes, key components of the inflammatory response, and inhibitors of Alox5 have been developed for the treatment of asthma. Using the approved (for asthma) Alox5 inhibitor, Zileuton, the scientists at The Jackson Laboratory were able to demonstrate a greater therapeutic effect in the CML model than the gold standard, Gleevec. Combining the two therapeutics provided an even better response.

The full study is published in the journal Nature Genetics.

Alox5 has also been shown to be over-expressed in certain cancers, for example in colon cancer. The availability of a clinically approved Alox5 inhibitor should enable a rapid progression to clinical studies in cancer.

Targeting ‘Normal’ Proteins to Kill Cancer Cells

For the most part, cancer therapy has been aimed at exploiting pathways that are present in cancer cells and not in normal cells but two studies published in the May 29^th issue of the journal Cell suggest potential for an alternative approach. Blocking the activity of oncogenic protein kinases – either with antibodies or small molecules – has become an important field of interest in cancer research but, despite the prevalence of RAS mutations in human tumours, inhibition of oncogenic RAS has not been realised as a therapeutic strategy. As an alternative to directly targeting RAS, US researchers have now identified ‘normal’ genes that are needed for cell survival in the presence of mutant, but not wild-type, KRAS.

To identify genes that are essential for survival only in the context of mutant KRAS, the researchers used a short hairpin RNA (shRNA) library to carry out high-throughput loss-of-function RNA interference (RNAi) screens in cancer cell lines as well as in normal cells. Dozens of potential drug targets were identified including serine/threonine kinase 33 (STK33) and mitotic polo-like kinase 1 (PLK1).

Patients with KRAS tumours are more likely to survive if they also have reduced expression of genes in the PLK1 pathway, suggesting that PLK1 inhibitors may have the potential to prolong survival. Although not required by normal cells, STK33 was found to be essential for the survival of cancer cells, irrespective of their tissue of origin, again suggesting therapeutic potential for inhibitors.

As well as identifying new targets for which it may be possible to develop therapeutically useful inhibitors, the two studies demonstrate the potential of RNAi screens to discover functional dependencies between oncogenes and normal genes in cancer cells. Targeting proteins which are essential for the survival of cancer cells, but not normal cells, could lead to a substantial therapeutic window, especially if only partial knock-down is needed to kill cancer cells.

AT1 Receptor Linked to Subset of Breast Cancers

pink bridge Increasingly, scientists and doctors are trying to personalise cancer treatment by identifying genetic alterations involved in the disease in individual patients. One example is the development of a monoclonal antibody, trastuzumab (Herceptin™), directed against the ErbB-2 (HER2) protein which is overexpressed in a subset of breast cancers and leads to a highly aggressive form of the disease.

DNA microarrays have been widely used to study gene expression in cancer and a team of US researchers have now used the technique to search for other genes that are overexpressed in breast cancers. Writing in the journal PNAS, the team describe a meta analysis of 31 breast cancer gene profiling studies comprising almost 3,200 microarrays. As well as correctly identifying the known breast cancer-associated gene, ErbB-2, the study found that the angiotensin II receptor type I (AT₁) was markedly overexpressed (up to 100-fold) in 10-20% of tumours across multiple independent cohorts. AT₁ overexpression was found only in oestrogen receptor (ER)-positive tumours and was also restricted to samples that did not overexpress ErbB-2. In primary mammary epithelial cells, ectopic overexpression of AT₁ together with angiotensin II stimulation led to a highly invasive phenotype that was attenuated by the AT₁ antagonist, losartan. In mice, treatment with losartan (90 mg/kg/day) for eight weeks was found to reduce tumour growth by 30% in AT₁-positive breast cancer xenografts.

AT₁ has previously been linked to cancer and cancer-related signalling pathways and the new study suggests that women with AT₁-overexpressing breast cancer may benefit from treatment with an AT₁ antagonist. Earlier studies have linked polymorphisms in the angiotensin pathway with breast cancer incidence and, although studies have not found a significant relationship between antihypertensive therapy and breast cancer incidence, the authors believe that the studies may not have been sufficiently powered to detect the small changes in incidence that might be expected from a subset of only 10-20% of AT₁-positive patients.

First Total Synthesis of (+)-11,11′-Dideoxyverticillin A

shiraia bambusicola Although it is ten years since the anti-cancer properties of (+)-11,11′-dideoxyverticillin A were first described, it is only now that the first total synthesis has been reported. The compound was originally isolated from the mycelium of a marine-derived fungus of the genus Penicillium and has since been found in several other fungal species including Shiraia bambusicola, a parasitic fungus which grows on some species of bamboo.

(+)-11,11'-dideoxyverticillin A structure With ten rings and eight stereogenic centres, (+)-11,11′-dideoxyverticillin A is one of the most complex of a family of dimeric epidithiodiketopiperazine natural products. Writing in the journal Science, chemists at the Massachusetts Institute of Technology have now described an enantioselective 11-step synthesis, starting from the commercially available amino acids, tryptophan and alanine. The synthesis was designed to mimic a plausible biosynthetic pathway and, as well as providing ready access to (+)-11,11′-dideoxyverticillin A itself, should provide access to analogues which may have enhanced pharmacological activity. The elegant synthesis used a minimum of protecting groups and, by taking full advantage of the inherent reactivity of intermediates, offers insights into the natural biosynthetic pathways. (+)-11,11′-Dideoxyverticillin A inhibits the tyrosine kinase activity of the epidermal growth factor receptor with an IC₅₀ of 0.14nM, shows anti-angiogenic activity, and prevents the growth of several cancer cell lines.

Blazing a TRAIL

Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is one of several members of the TNF gene superfamily that induce apoptosis through engagement of death receptors. The protein is a potentially attractive treatment for cancer since it induces apoptosis in a variety of cancer cells. However, not all cells of a particular type react uniformly to anti-cancer treatments and, although a promising drug candidate, TRAIL is not 100% successful. Genetic explanations have been put forward to explain the differences in cell responses to treatment but a team of scientists at Harvard University wanted to investigate the mechanisms involved in more detail. They exposed both cancer cells and normal cells to varying concentrations of TRAIL and found that a proportion of them always survived. When the surviving cells were isolated, they and their immediate progeny remained highly resistant to the apoptotic effects of TRAIL for a short time. After reproducing for several days, however, the sensitivity of the cells to TRAIL reverted to that of the original colonies with around 90% of the cells dying and 10% surviving. Using a variety of imaging techniques, the team showed that levels of proteins involved in TRAIL-induced apoptosis were different in the sensitive and resistant cells, despite the fact that the cells were genetically identical. They found that the altered protein levels were initially inherited by progeny cells but that inheritance was transient. The initial differences in protein expression between the cells were completely random – cells do not produce proteins uniformly but rather in bursts, with the timing and level of production varying from cell to cell.

The findings offer an alternative explanation to the cancer stem-cell hypothesis about why some cells are more resistant to chemotherapy or radiation treatment and the team hope that the new insight may contribute to the design of more effective treatments.

The study was published online on April 12^th in Nature.

New Anti-Cancer Bisphosphonates

Etidronic acid structure Bisphosphonates prevent loss of bone mass and are used to treat postmenopausal osteoporosis and also to reduce bone loss in metastatic disease in cancer patients. Bisphosphonates can be divided into two sub-classes, those that do not contain nitrogen such as etidronic acid, and those that do contain nitrogen such as zoledronic acid.

zoeldronic acid structure Bisphosphonates that do not contain nitrogen interfere with ATP-linked energy metabolism in osteoclasts whereas the activity of nitrogenous bisphosphonates is related to their ability to inhibit farnesyl diphosphate synthase (FPPS) a key enzyme in the HMG-CoA reductase (mevalonate) pathway. Farnesyl diphosphate (FPP) is used for the post-translational prenylation of small GTPases such as Ras, and depletion of FPP is thought to be the primary mechanism for inhibition of osteoclast function. The anticancer activity of nitrogenous bisphosphonates has also been attributed to inhibition of FPPS and results from recent clinical trials in oestrogen-receptor positive breast cancer and hormone-refractory prostate cancer have been very encouraging. The transition states involved in FPP biosynthesis by FPPS are expected to be broadly similar to those of other prenyltranferases and inhibition of geranylgeranyl diphosphate synthase (GGPPS) has been suggested to be more important than inhibition of FPPS for the anti-cancer activity of bisphosphonates.

bph-715 structure An international team led by Professor Eric Oldfield at the University of Illinois set out to design compounds that would inhibit both FPPS and GGPPS but have lower affinity for bone tissue and so be more effective in reaching other tissues. Writing in the Journal of the American Chemical Society, they describe potent inhibitors of FPPS and GGPPS that are effective in blocking tumour cell growth and invasiveness, both in vitro and in vivo. One of the compounds, BPH-715, is about 200 times more active in killing tumour cells than zoledronic acid and, since it is more lipophilic, has a lower affinity for bone. Studies showed that although BPH-175 binds to both FPPS and GGPPS, it inhibits GGPPS more strongly.

As well as inhibiting FPPS and GGPPS, bisphosphonates such as zoledronic acid and BPH-715 also stimulate γδ T-cells which help to kill tumour cells.

Liquorice Does Allsorts

Liquorice, which is used as a traditional medicine, a culinary spice and an ingredient of confectionary, is an expectorant and mild laxative, has antiviral properties and also increases blood pressure at high doses. One of the active ingredients of liquorice-root extract is the natural sweetener glycyrrhizin, which is over 50 times sweeter than sucrose. Two recent reports describe additional properties of liquorice.

liquorice Writing in the Journal of Clinical Investigation, researchers at Vanderbilt University Medical Center suggest that glycyrrhizic acid may offer a new approach to preventing colorectal cancer (CRC), the second leading cause of cancer deaths in the United States. Prostaglandin E₂ (PGE₂) has been shown to promote CRC progression and both non-selective cyclooxygenase (COX) inhibitors and selective COX-2 inhibitors, which block production of PGE₂, reduce the number and size of colonic adenomas. Although COX-2 inhibition, which reduces the number and size of colon polyps in both mice and patients with a predisposition to colon cancer, is a promising target for chemoprevention of CRC, both non-selective NSAIDs and selective COX-2 inhibitors have side effects that limit their prophylactic use. The Vanderbilt team had previously shown that inhibiting 11β–hydroxysteroid dehydrogenase type II (11βHSD2) in the kidney suppresses COX-2 expression and, since the colon is one of the only other organs with high expression of 11βHSD2, speculated that this enzyme might play a role in colorectal cancer progression. Expression of 11βHSD2, which converts active glucocorticoids to inactive keto-forms, was found to be increased in polyps found in both mice and humans and correlated with COX-2 expression and activity. Inhibition of 11βHSD2 with glycyrrhizic acid was found to inhibit COX-2–mediated PGE₂ production in tumours, and to prevent polyp formation, tumour growth, and metastasis in mice.

glycyrrhizin structure Inhibition of 11βHSD2 also increases cortisol levels in the kidney and contributes to the increases in blood pressure caused by large doses of glycyrrhizic acid. The team did not see any increases in blood pressure in the treated mice, but believe that this relatively benign side effect could easily be treated by diuretics. The team now want to design more potent and selective inhibitors of 11βHSD2 and explore the role of 11βHSD2 in other cancers.

Chemists in Taiwan have also recently warned that both glycyrrhizin and liquorice extracts can block the absorption of cyclosporine, a drug used to prevent rejection of organ transplants and as a treatment for rheumatoid arthritis. Speaking at the annual meeting of the American Chemical Society in Salt Lake City, Dr Pei-Dawn Lee Chao described how levels of cyclosporine were reduced in laboratory rats when co-administered with liquorice. Although it is not known how much liquorice would have to be eaten to have a similar effect in humans and the mechanism of the effect is not understood, this study adds to a growing number of reports that liquorice can trigger potentially dangerous drug interactions.

New Pathway for Regulation of NF-κB

flames NF-κB is a transcription factor that plays a key role in regulating cellular responses to stimuli such as stress and bacterial and viral infections. In un-stimulated cells, NF-κB dimers are sequestered in the cytoplasm by inhibitors known as IκBs (inhibitors of κB) which mask the nuclear localization signals of NF-κB. When the cell is stimulated, IκB proteins are degraded and the NF-κB complex is free to enter the nucleus where it can activate gene expression. The activation of particular genes by NF-κB then leads to a physiological response such as inflammation, an immune response, cell survival or cell proliferation. Once in the nucleus, NF-κB also turns on expression of IκBs, thus forming a feedback loop which regulates activity. Inappropriately regulated NF-κB has been linked to many different types of tumour as well as to inflammatory diseases.

Recent studies have shown that degradation of NF-κB in the nucleus provides an alternative mechanism for regulating its activity and researchers at the University of Illinois have now shown how this process is controlled. They found that TNF-α stimulates methylation of the RelA subunit of NF-κB by lysine methyltransferase Set9 at lysine residues 314 and 315 both in vitro and in vivo. Methylation of RelA inactivates NF-κB by inducing proteasome-mediated degradation of promoter-associated RelA. Depletion of Set9 by siRNA or mutation of the RelA methylation sites was shown to prolong DNA binding of NF-κB and enhance TNF-α-induced expression of NF-κB target genes.

The study, which is published in the journal EMBO, reveals methylation of the RelA subunit of NF-κB as a novel mechanism regulating the turnover of NF-κB and controlling the NF-κB-mediated inflammatory response. The ability to inhibit NF-κB signalling could have applications in the treatment of both cancer and inflammatory diseases.