Two studies led by investigators at Weill Cornell Medical College shed light on the molecular biology of three blood disorders, leading to novel strategies to treat these diseases.
The two new studies propose two new treatments for beta-thalassaemia, a blood disorder which affects thousands of people globally every year. In addition, they suggest a new strategy to treat thousands of Caucasians of Northern European ancestry diagnosed with HFE-related hemochromatosis and a novel approach to the treatment of the rare blood disorder polycythaemia vera.
These research insights were only possible because two teams that included 24 investigators at six American and European institutions decoded the body’s exquisite regulation of iron, as well as its factory-like production of red blood cells.
‘When you tease apart the mechanisms leading to these serious disorders, you find elegant ways to manipulate the system,’ says Dr. Stefano Rivella, associate professor of genetic medicine in pediatrics at Weill Cornell Medical College.
For example, Dr. Rivella says, two different gene mutations lead to different outcomes. In beta-thalassemia, patients suffer from anaemia — the lack of healthy red blood cells — and, as a consequence, iron overload. In HFE-related haemochromatosis, patients suffer of iron overload. However, he adds, one treatment strategy that regulates the body’s use of iron may work for both disorders.
Additionally, investigators found another strategy, based on manipulating red blood cell production, could also potentially treat beta-thalassaemia as well as a very different disorder, polycythaemia vera.
Dr. Rivella and his colleagues tackled erythropoiesis — the process by which red blood cells (erythrocytes) are produced — as a way to decipher and decode the two blood disorders beta-thalassaemia and polycythaemia vera.
Beta-thalassaemia, a group of inherited blood disorders, is caused by a defect in the beta globin gene. This results in production of red blood cells that have too much iron, which can be toxic, resulting in the death of many of the blood cells. What are left are too few blood cells, which leads to anaemia. At the same time, the excess iron from destroyed blood cells builds up in the body, leading to organ damage. In polycythaemia vera, a patient’s bone marrow makes too many red blood cells due to a genetic mutation that doesn’t shut down erythropoiesis — the production of the cells.
The researchers studied both normal erythropoiesis, in which a person makes enough red blood cells to replace those that are old, and a mechanism called stress erythropoiesis, which flips on when a person requires extra blood cells — such as loss of blood from an accident. The hormone erythropoietin (EPO) controls red blood cell production, and can also induce stress erythropoiesis. Iron is also essential, says Dr. Rivella. ‘The two well-known elements needed to switch between normal and stress erythropoiesis are EPO and iron,’ he says.
But Dr. Rivella and his team found that a third player is essential: macrophages, the immune cells that engulf cellular garbage and pathogens. Macrophages had been known to digest the iron left when old blood cells are targeted for destruction, but Dr. Rivella discovered that they also are necessary for stress erythropoiesis. He found macrophages need to physically touch erythroblasts, the factories that make red blood cells, in order for more factories to be created so that they can churn out red blood cells.
‘No one knew macrophages were a part of emergency red blood cell production. We now know they provide fuel to push red blood cell factories to work faster,’ says the study’s lead author Dr. Pedro Ramos, a former postdoctoral researcher at Weill Cornell.
The research team then looked at diseases in which there are too many red blood cell factories. Polycythemia vera was one of the conditions examined. The researchers disabled macrophage functioning in mice with polycythemia vera and found that red blood cell production returned to normal.
In beta-thalassemia, the body increases the number of red blood cell factories to make up for the lack of viable blood cells — a strategy that doesn’t work. As a result, patients develop enlarged spleens and livers due to the overload of erythroblasts in those organs.
The researchers found in mouse models that if they suppress the function of macrophages, the number of blood cell factories revert back to normal levels. However, there was also an additional benefit discovered. One of the functions of macrophages is to put excess recycled iron into erythroblasts. Researchers report if you suppress that function, less iron goes into the red blood cells. ‘So you then make red blood cells that have less iron, and they are now closer in structure to what they should be,’ says Dr. Rivella.
In animal studies, the researchers saw that decoupling macrophages from the erythroblasts not only reduced the number of blood cell factories, but also improved anaemia.
The discovery could be translated into an experimental therapy by finding the molecule that physically binds a macrophage to an erythroblast, and then targeting and inhibiting it. ‘We need macrophages for good health, but it may be possible to decouple the macrophages that contribute to blood disorders,’ Dr. Rivella says. ‘I estimate that up 30 to 40 percent of the beta-thalassaemia population could benefit from this treatment strategy.’
Dr. Rivella also made another connection. He says polycythaemia vera ‘is sort of a tumour of the red cells, because you make too many of them.’ And he notes that previous research on macrophages found that they are very important in cancer metastasis. ‘I see a parallel between the activity of macrophages in supporting the proliferation of cells that are under stress conditions — growing tumors and red blood cells that need to grow,’ he says. ‘It seems to us that macrophages are important in supporting a switch between normal growth and increased growth.’ Weill Cornell Medical College

For the first time, scientists from the German Cancer Research Center (DKFZ) and the National Center for Tumor Diseases (NCT) Heidelberg have characterised cancer cells that can initiate metastasis in the blood of breast cancer patients. These cells have properties of cancer stem cells and are characterised by three surface proteins. Patients with large numbers of these cells found in their blood show a rather unfavourable disease progression. The pattern of the three molecules may therefore be used as a biomarker for disease progression. The scientists plan to investigate whether the characteristic surface molecules may be used as targets for specific therapies for patients with advanced breast cancer.
Individual cancer cells that break away from the original tumour and circulate through the blood stream are considered responsible for the development of metastases. These dreaded secondary tumours are the main cause of cancer-related deaths. Circulating tumour cells (CTCs) detectable in a patient’s blood are associated with a poorer prognosis. However, up until now, experimental evidence was lacking as to whether ‘stem cells’ that lead to metastases can be found among CTCs.
‘We were convinced that only a very few of the various circulating tumour cells are capable of forming a secondary tumour in a different organ,’ says Prof. Andreas Trumpp, a stem cell expert. ‘Many patients do not develop metastases even though they have cancer cells circulating through their blood.’ Trumpp is head of DKFZ’s Division of Stem Cells and Cancer and director of the Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM) at DKFZ. ‘Metastasis is a complex process and cancer cells need to have very specific properties for it,’ he says. ‘Our hypothesis was that the characteristics of cancer stem cells, which are resistant to therapy and very mobile, are best suited.’

Irène Baccelli from Trumpp’s team developed a transplantation test for the experimental detection of metastasis-initiating cells. In collaboration with Prof. Andreas Schneeweiss from the National Center for Tumor Diseases (NCT) Heidelberg, along with colleagues from the Institute of Tumor Biology in Hamburg and the Institute of Pathology of Heidelberg University Hospitals, the researchers analysed the blood of more than 350 breast cancer patients. Using specific surface molecules, Baccelli isolated circulating tumour cells from the blood and directly transplanted them into the bone marrow of mice with defective immune systems. ‘Bone marrow is a perfect niche for tumour sells to colonise,’ Trumpp explains. After more than one hundred transplantations, metastases started forming in the bones, lungs and livers of some of the animals.

This proved that CTCs do contain metastasis stem cells – even though their frequency is apparently low. What characterises these cells? To define their molecular properties, the researchers analysed the surface molecules of the CTCs that had led to metastases after transplantation.

Three molecules characterise the metastasis stem cell
In a systematic screening process, Baccelli first isolated cells carrying a typical breast cancer stem cell protein (CD44) on their surface from the CTCs. This protein helps the cell to settle in bone marrow. Next, the researchers screened this cell population for specific surface markers which help the cells to survive in foreign tissue. These include, for example, a signalling molecule (CD47) that protects them from attacks by the immune system, and a surface receptor that enhances the cells’ migratory and invasive capabilities (MET).

Using a cell sorter, the researchers were then able to isolate CTCs which simultaneously exhibit all three characteristic molecules (CD44, CD47, MET). Another round of transplantation tests showed that these were in fact the cells from which the metastases originated.

Depending on the patient, cells exhibiting all three surface molecules (‘triple-positive’ cells) made up between 0.6 and 33 percent of all CTCs. ‘It is interesting that only cells with the stem cell marker CD44 carry the combination of the other two surface molecules,’ said Irène Baccelli. ‘It looks like the triple-positive cells are a specialized subtype of breast cancer stem cells circulating in the blood.’

Triple-positive cells as prognostic biomarkers

Are the triple-positive cells a more precise biomarker of breast cancer progression than the number of CTCs alone? In a small patient group, the researchers observed that as the disease advances, the number of triple-positive cells increases, but the total number of CTCs does not. In addition, patients with very high numbers of triple-positive cells had particularly high numbers of metastases and a much poorer prognosis than women in whom only a few metastasis-inducing cells were detected. ‘On the whole, triple-positive cells seem to have a substantially higher biological relevance for disease progression than previously studied CTCs,’ Andreas Schneeweiss explains. The researchers plan to confirm these new results in a large study.

Andreas Trumpp considers it good news that metastasis-initiating cells are characterised by the two proteins CD47 and MET. Therapeutic antibodies that target CD47 and inhibit its functions are already being developed. A substance inhibiting the activity of the MET receptor has already been approved and shows good effectiveness in treating a type of lung cancer. The substance may also help breast cancer patients with detectable metastasis-inducing cells. ‘The triple-positive cells we have found turn out to be not only a promising biomarker of disease progression in breast cancer but also a prospect for potential new therapeutic approaches for treating advanced breast cancer,’ says Andreas Trumpp. German Cancer Research Center

Scientists from the University of Iowa and Brigham Young University (BYU) have identified a gene that may be a target for overcoming drug resistance in cancer. The finding could not only improve prognostic and diagnostic tools for evaluating cancer and monitoring patients’ response to treatment but also could lead to new therapies directed at eradicating drug-resistant cancer cells.

Drug resistance is a common problem in many metastatic cancers. It leads to failure of chemotherapy treatments and is associated with poor patient outcomes, including rapid relapse and death.

Fenghuang Zhan, Ph.D.The research team, including Fenghuang (Frank) Zhan, M.D., Ph.D., and Guido Tricot, M.D., Ph.D., from the UI, and David Bearss, Ph.D., from BYU, initially focused on identifying genes linked to the development of drug resistance in multiple myeloma, a bone marrow cancer that affects more than 20,000 Americans and causes almost 11,000 deaths annually.

Working with serial biopsied cells from 19 myeloma patients, the researchers analysed genetic changes in the cells that occurred over the course of treatment with very intensive chemotherapy drugs. This approach identified a gene called NEK2 that is strongly associated with increased drug-resistance, faster cancer growth, and poorer survival for patients.

Guido Tricot, M.D., Ph.D.Having established the relationship between high expression of the NEK2 gene and poor patient outcome in myeloma, the team then examined the relationship in other common cancers—including breast, lung, and bladder cancer—by analysing gene expression profiles from 2,500 patients’ cells with eight different cancers in Zhan’s lab.

Taking the findings back to the lab, the team then examined the effect on cancer cells of either enhancing or blocking the expression of the NEK2 gene. "In all cases, an increase in the NEK2 gene was associated with rapid death of the patient," says Tricot, who is director of Holden Comprehensive Cancer Center’s Bone Marrow Transplant and Myeloma Program at UI Hospitals and Clinics. "So this finding was not unique to myeloma; this is basically seen in every single cancer we looked at."

The research team is now developing compounds to inhibit NEK2 by collaborating with David Bearss, Ph.D., associate professor of physiology and developmental biology at BYU, in the hope that these compounds may overcome drug resistance in cancer cells. "Our studies show that over-expression of NEK2 in cancer cells significantly enhances the activity of drug efflux proteins to pump chemotherapy drugs out of cells, resulting in drug resistance. Furthermore, silencing NEK2 in cancer cells potently decreased drug resistance, induced cell-cycle arrest, cell death, and inhibited cancer cell growth in vitro and in vivo," says Zhan, UI professor of internal medicine.

“We were able to show that if we inhibit NEK2, then we can actually restore sensitivity to drugs that we use right now,” Bearss says.

Although development and clinical testing of such drugs for use in patients is not imminent, Tricot notes that the findings may have clinical use within the next several years.

"NEK2 expression may be a diagnostic or prognostic marker for drug-resistant cancer," he says. "If NEK2 is high, that would suggest that the prognosis is poorer and the patient might benefit from more aggressive treatment. The other potential use is for monitoring the cancer’s response to therapy. If NEK2 levels increase, that would suggest development of increased drug resistance and might indicate that a change of treatment would be helpful."University of Iowa Health Care

Research from Western University and Lawson Health Research Institute sheds new light on a gene called ATRX and its function in the brain and pituitary. Children born with ATRX syndrome have cognitive defects and developmental abnormalities. ATRX mutations have also been linked to brain tumours.
Dr. Nathalie Bérubé, PhD, and her colleagues found mice developed without the ATRX gene had problems in the forebrain, the part of the brain associated with learning and memory, and in the anterior pituitary which has a direct effect on body growth and metabolism. The mice, unexpectedly, also displayed shortened lifespan, cataracts, heart enlargement, reduced bone density, hypoglycemia; in short, many of the symptoms associated with ageing.

Ashley Watson, a PhD candidate working in the Bérubé lab and the first author on the paper, discovered the loss of ATRX caused DNA damage especially at the ends of chromosomes which are called telomeres. She investigated further and discovered the damage is due to problems during DNA replication, which is required before the onset of cell division. Basically, the ATRX protein was needed to help replicate the telomere.
Working with Frank Beier of the Department of Physiology and Pharmacology at Western’s Schulich School of Medicine & Dentistry, the researchers made another discovery. ‘Mice that developed without ATRX were small at birth and failed to thrive, and when we looked at the skeleton of these mice, we found very low bone mineralisation. This is another feature found in mouse models of premature ageing,’ says Bérubé, an associate professor in the Departments of Biochemistry and Paediatrics at Schulich Medicine & Dentistry, and a scientist in the Molecular Genetics Program at the Children’s Health Research Institute within Lawson. ‘We found the loss of ATRX increases DNA damage locally in the forebrain and anterior pituitary, resulting in systemic defects similar to those seen in ageing.’
The researchers say the lack of ATRX in the anterior pituitary caused problems with the thyroid, resulting in low levels of a hormone called insulin-like growth factor-one (IGF-1) in the blood. There are theories that low IGF-1 can deplete stores of stem cells in the body, and Bérubé says that’s one of the explanations for the premature ageing. University of Western Ontario

A handheld diagnostic device that Massachusetts General Hospital (MGH) investigators first developed to diagnose cancer has been adapted to rapidly diagnose tuberculosis (TB) and other important infectious bacteria. Two papers describe portable devices that combine microfluidic technology with nuclear magnetic resonance (NMR) to not only diagnose these important infections but also determine the presence of antibiotic-resistant bacterial strains.
‘Rapidly identifying the pathogen responsible for an infection and testing for the presence of resistance are critical not only for diagnosis but also for deciding which antibiotics to give a patient,’ says Ralph Weissleder, MD, PhD, director of the MGH Center for Systems Biology (CSB) and co-senior author of both papers. ‘These described methods allow us to do this in two to three hours, a vast improvement over standard culturing practice, which can take as much as two weeks to provide a diagnosis.’
Investigators at the MGH CSB previously developed portable devices capable of detecting cancer biomarkers in the blood or in very small tissue samples. Target cells or molecules are first labelled with magnetic nanoparticles, and the sample is then passed through a micro NMR system capable of detecting and quantifying levels of the target. But initial efforts to adapt the system to bacterial diagnosis had trouble finding antibodies – the detection method used in the earlier studies – that would accurately detect the specific bacteria. Instead the team switched to targeting specific nucleic acid sequences.
The system detects DNA from the tuberculosis bacteria in small sputum samples. After DNA is extracted from the sample, any of the target sequence that is present is amplified using a standard procedure, then captured by polymer beads containing complementary nucleic acid sequences and labelled with magnetic nanoparticles with sequences that bind to other portions of the target DNA. The miniature NMR coil incorporated into the device – which is about the size of a standard laboratory slide – detects any TB bacterial DNA present in the sample.
Tests of the device on samples from patients known to have TB and from healthy controls identified all positive samples with no false positives in less than three hours. Existing diagnostic procedures can take weeks to provide results and can miss up to 40 percent of infected patients. Results were even stronger for patients infected with both TB and HIV – probably because infection with both pathogens leads to high levels of the TB bacteria – and specialized nucleic acid probes developed by the research team were able to distinguish treatment-resistant bacterial strains.
Another paper describes a similar system using ribosomal RNA (rRNA) – already in use as a bacterial biomarker – as a target for nanoparticle labelling. The investigators developed both a universal nucleic acid probe that detects an rRNA region common to many bacterial species and a set of probes that target sequences specific to 13 clinically important pathogens, including Streptococcus pneumoniae, Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA).
The device was sensitive enough to detect as few as one or two bacteria in a 10 ml blood sample and to accurately estimate bacterial load. Testing the system on blood samples from patients with known infections accurately identified the particular bacterial species in less than two hours and also detected two species that had not been identified with standard culture techniques.
While both systems require further development to incorporate all steps into sealed, stand-alone devices, reducing the risk of contamination, Weissleder notes that the small size and ease of use of these devices make them ideal for use in developing countries. ‘The magnetic interactions that pathogen detection is based on are very reliable, regardless of the quality of the sample, meaning that extensive purification – which would be difficult in resource-limited setting – is not necessary. The ability to diagnose TB in a matter of hours could allow testing and treatment decisions within the same clinic visit, which can be crucial to controlling the spread of TB in developing countries.’ Massachusetts General Hospital