USA NIH: EYE & AQUAPORIN PROTEINS

Researchers get close-up view of water pores needed in the eye’s lens NIH-funded study of aquaporins could hold clues to cataract Researchers have achieved dynamic, atomic-scale views of a protein needed to maintain the transparency of the lens in the human eye. The work, funded in part by the National Institutes of Health, could lead to new insights and drugs for treating cataract and a variety of other health conditions. Aquaporin proteins form water channels between cells and are found in many tissues, but aquaporin zero (AQP0) is found only in the mammalian lens, which focuses light onto the retina, at the back of the eye. The lens is primarily made up of unique cells called lens fibers that contain little else besides water and proteins called crystallins. Tight packing of these fibers and of the crystallin proteins within them helps create a uniform medium that allows light to pass through the lens, almost as if it were glass. Views of the open and closed AQP0 channel. This illustration shows two views of a single unit of the aquaporin-0 channel. The left channel is open, allowing the passage of water molecules (red). The right channel is closed because amino acids in its core have flipped inward. Credit: Reichow et al. Nature Structural and Molecular Biology. Abnormal development or age-related changes in the lens can lead to cataract — a clouding of the lens that causes vision loss. Besides age, other risk factors for cataract include smoking, diabetes, and genetic factors. Mutations in the AQP0 gene can cause congenital cataract and may increase the risk of age-related cataract. “The AQP0 channel is believed to play a vital role in maintaining the transparency of the lens and in regulating water volume in the lens fibers, so understanding the molecular details of how water flows through the channel could lead to a better understanding of cataract,” said Dr. Houmam Araj, who oversees programs on lens, cataract and oculomotor systems at NIH’s National Eye Institute (NEI), which helped fund the research. Closing of AQP0 channels is regulated by a calcium-sensitive protein called calmodulin, but the precise mechanism has been unclear. Some models have suggested that calmodulin simply acts as a plug to fill the open channel. The new study, published in Nature Structural and Molecular Biology, reveals a more nuanced process in which calmodulin essentially grasps the open channel and forces it to close. The research was a collaboration between investigators at the University of California, Irvine, and the Janelia Farm Research Campus in Ashburn, Va., a part of the Howard Hughes Medical Institute (HHMI). Drs. James Hall and Douglas Tobias led the effort at UC Irvine. Dr. Tamir Gonen led the effort at Janelia Farm. In prior studies, Dr. Gonen had examined the atomic structure of the AQP0 protein by X-ray crystallography, which involves crystallizing a protein and bombarding it with X-rays. But X-ray crystallography does not work well for large groups of proteins or for proteins in motion. So in the new study, the researchers first used electron microscopy to view AQP0 and calmodulin bound together. Then they combined their microscopy and crystallography data to generate computerized models of how the two proteins interact and to identify the most critical amino acids (the building blocks for proteins) within AQP0. To test their models, they neutralized those amino acids one by one in the actual AQP0 channel. The AQP0 channel is made up of four identical barrel-shaped units, bundled together side by side. The researchers found that in the presence of calcium, calmodulin binds to one unit and then another, as if grabbing a pair of reins. This makes the channel twist slightly, which causes just a few amino acids within each unit to slide into the channel’s core and block the flow of water. “Calmodulin essentially throws a molecular switch that moves in and out of the water pore, like the gate valve of a plumbing fixture,” Dr. Hall said. This new view of AQP0 could help lead to new approaches for treating cataract, Dr. Hall said. Cataracts are the most common cause of blindness worldwide. In the United States, they affect about 1 in 6 people over age 40 and half over age 80. Congenital cataracts (present from birth) affect about 1 in 5,000 American children. Cataracts can be successfully treated with surgery, in which the cloudy lens is removed and replaced with an artificial plastic lens. But the new findings “may be a step toward learning how to prevent or delay cataracts,” said Dr. Hall. The new findings also provide inroads to understanding how calmodulin interacts with a variety of protein channels, and thus could open doors to new drugs for other common health conditions. In addition to aquaporins, our bodies rely on a vast menagerie of channels, many of which are regulated by calmodulin. For example, calmodulin helps control the gating of ion channels, which allow the passage of ions (charged particles) in and out of our cells and are essential for nerve cell firing, muscle contraction, and the rhythmic beating of the heart. This study provides the first structural model of calmodulin bound to any complete protein channel. Drs. Daniel Clemens and Steve Reichow were co-first authors on the study. NIH support for the study came from NEI (grants EY005661, EY018768), the National Institute of General Medical Sciences (NIGMS grant GM079233), a joint program on “Making Sense of Voltage Sensors” co-funded by NIGMS and the National Institute of Neurological Disorders and Stroke (grant GM086685), and the National Library of Medicine (grant LM007443). Additional support came from HHMI, the National Science Foundation, and the German Academy of Sciences. To set up an interview with Dr. Hall, please contact Tom Vasich at UC Irvine (tmvasich@uci.edu, 949-824-6455).

NEJM: REVLIMID + DEXAMETHASONE in plasma cell cancer. Dr.M.M. MATEOS; SALAMANCA

Revlimid Plus Dexamethasone Delays Progression And Extends Survival In High-Risk Smoldering Myeloma Results from a Spanish Phase 3 study published late Wednesday in the New England Journal of Medicine indicate that treatment of high-risk smol­dering multiple myeloma with Revlimid plus dexamethasone delays pro­gression to symptomatic multiple myeloma and extends overall sur­viv­al. In addition, the Revlimid (lenalidomide)-dexamethasone (Decadron) regi­men had an acceptable safety profile, according to the investigators. Despite these findings – which would tend to support active treatment of high-risk smoldering myeloma – myeloma experts believe it is too early to begin treating all high-risk smoldering myeloma patients with anti-myeloma therapies. Dr. María-Victoria Mateos of the University Hospital in Sala­manca, Spain is one of the lead investigators of the study. She told The Beacon that “This is the first randomized trial showing a significant benefit to early treatment of high-risk smoldering myeloma patients. However, Revlimid-dexamethasone is not an approved combination for newly diagnosed myeloma patients, and we therefore have to wait to obtain this approval and to obtain additional results of other trials that are currently ongoing in high-risk smoldering myeloma patients.” Whether or not Revlimid is officially approved as a treatment for smoldering myeloma, however, is likely to be a more important consideration outside of the United States. U.S. physicians typically have more flexi­bil­i­ty to prescribe drugs “off label” than their international counterparts. “These results are very promising and strongly support further studies of novel agents in this group of patients with early stage disease,” said Dr. Paul Richardson from the Dana-Farber Cancer Institute in Boston. Dr. Richardson, who was not involved with the study, added, however, that “to recommend that all high-risk smoldering myeloma pa­tients now receive combination therapy as a standard of care is premature in my view.” “Moreover, the need to re-evaluate definitions of high-risk smoldering myeloma is an important next step, especially given the striking differences seen in outcome between patients who were treated, and those who were only observed,” noted Dr. Richardson. Dr. Richardson stated that he currently recommends high-risk smoldering myeloma patients participate in clinical trials, and that he also considers treatment with bisphosphonates for patients with evidence of early bone disease, such as osteopenia. Common bisphosphonates include Aredia (pamidronate) and Zometa (zoledronic acid). Dr. Mateos added that other treatment regimens should also be studied in high-risk smoldering myeloma patients. “I think that high-risk smoldering multiple myeloma constitutes an attractive population in which to evaluate novel agents,” stated Dr. Mateos. She also said that there are several additional ongoing trials “evaluating the role of other agents – such as proteasome inhibitors, monoclonal antibodies, Revlimid alone without dexamethasone, etc. – in this patient population.” Background Smoldering, or asymptomatic, multiple myeloma is a precursor to multiple myeloma in which the patient experiences none of the symptoms typically associated with active (symptomatic) multiple myeloma, such as ele­vated calcium levels, kidney failure, anemia, or bone lesions. Smoldering myeloma is characterized by an excess of monoclonal protein in the blood and urine. A diag­nosis of smoldering multiple myeloma is made when a patient’s monoclonal protein level is at least 30 g/L or the proportion of plasma cells in the bone marrow is at least 10 percent, but the patient does not experi­ence any of the typical myeloma symptoms. The risk of progression from smoldering myeloma to symptomatic disease is around 10 percent during each of the first five years after diagnosis, and decreases to 3 percent per year for the following five years, and to 1 percent per year thereafter. This means that the median time to progression to active disease for a newly diagnosed smoldering myeloma patient is about five years. Although smoldering myeloma patients are at a higher risk of developing active myeloma than the general public, the current standard of care is the so-called “watch and wait” approach, in which smoldering mye­lo­ma patients are regularly monitored and treatment only begins once the disease progresses to multiple myeloma. However, certain factors have been shown to be associated with an increased risk of progression to symp­to­matic disease in patients with smoldering multiple myeloma. These include: a monoclonal protein level exceeding 30 g/L; plasma cells exceeding 10 percent in the bone marrow; an elevated level of abnormal plasma cells in the bone marrow; an abnormal ratio of small parts of antibodies (known as an abnormal free light chain ratio); the presence of chromosomal abnormalities in the plasma cells; and lower-than normal levels of one or more types of immunoglobulin. Studies at the Mayo Clinic and in Spain have found that patients with several of these risk factors can be considered to have “high risk” smoldering myeloma, meaning that the patients have a higher risk of pro­gressing to symptomatic myeloma than other smoldering myeloma patients. An earlier Spanish study showed, for example, that smoldering myeloma patients with several high-risk disease factors had a median time to progression of about two years. Several clinical trials during the past 30 years have investigated early treatment of smoldering myeloma patients with melphalan (Alkeran) plus prednisone, thalidomide (Thalomid), and/or bisphosphonates. None of these trials supported early treatment of smoldering myeloma patients, with no benefits seen in terms of significantly longer time to progression or overall survival. These older trials, however, did not distinguish between standard-risk and high-risk smol­dering multiple myeloma patients. The investigators of the current study state that high-risk smoldering myeloma patients are more likely to benefit from early treatment than standard-risk patients. Since these patients do not have any symptoms, the researchers add that the ideal therapy for them should have limited side effects. Dr. Mateos previously presented some of the results from this study and discussed their potential im­pli­ca­tions at the International Myeloma Workshop this spring (see related Beacon news). Additionally, Dr. Ola Landgren, a smoldering myeloma expert from the National Institutes of Health who was not involved with the study, discussed the implications of preliminary results from the study early last year (see related Beacon news). Study Design In this Phase 3 clinical trial, researchers from across Spain investigated whether treatment with Revlimid plus dexa­methasone would prolong the time it took for high-risk smoldering multiple myeloma patients to progress to symptomatic multiple myeloma. A total of 119 high-risk smoldering myeloma patients recruited between 2007 and 2010 were randomly assigned to receive early treatment (57 patients) or to be observed until progression (62 patients). The two groups of patients had median ages of 63 years and 69 years, respectively. The criteria for being considered high-risk were plasma-cell bone marrow infiltration of at least 10 percent and a monoclonal component (defined as an IgG level of at least 30 g/L, an IgA level of at least 20 g/L, or a urinary Bence Jones protein level of more than 1 g per 24 hours). Alternatively, the patients could have only one of the two criteria described above, plus at least 95 percent abnormal plasma cells in the bone marrow, with reductions in one or two uninvolved immunoglobulins of more than 25 percent compared to normal values. Patients in the treatment arm received an initial therapy consisting of nine four-week cycles of Revlimid plus dexamethasone. During each cycle, they received 25 mg of Revlimid daily on days 1 to 21 and 20 mg of dexamethasone daily on days 1 to 4 and 12 to 15. They then received maintenance therapy consisting of 10 mg of Revlimid on days 1 to 21 every four weeks until disease progression or two years of treatment. Patients in the non-treatment arm did not receive any form of treatment until progression to symptomatic multiple myeloma. The median follow-up time was 40 months. Results After initial treatment with Revlimid plus dexamethasone, 79 percent of high-risk smoldering myeloma patients responded, with 7 percent achieving a stringent complete re­sponse, 7 percent a complete re­sponse, 11 percent a very good partial response, and 54 percent a partial response. In total, 88 percent of the treated patients completed initial therapy and started maintenance therapy. Among those patients, the overall response rate was 90 percent. In addition, 24 percent of patients experienced improvements in their quality of response during the maintenance phase of treatment. Treatment of high-risk smoldering myeloma significantly delayed time to progression. Specifically, the medi­an time to progression was not yet reached for patients who received treatment, compared to 21 months for those who were observed. Three years after entering the study, the progression-free survival rate was 77 percent for patients who re­ceived treat­ment and 30 percent for those who did not. Click on image to view a larger version of it. In addition, patients who received treatment had significantly longer overall survival. Five years after di­agnosis, the overall survival rate was 94 percent for those who received treatment compared to 78 percent for those who were not treated. (Click here or on the image to the right to see a larger graph of the key overall survival results.) The investigators found that the time from di­ag­nosis to the time the patient started the clinical trial did not influence their time to pro­gres­sion to multiple myeloma. Severe side effects during the initial Revlimid-dexamethasone therapy phase were more common among the patients who received treatment (12 percent) compared to those who were observed (3 percent). No patients experienced severe side effects during maintenance therapy. The most common severe side effects during the initial treatment phase were infections (6 percent of pa­tients), weakness (6 percent), low white blood cell counts (5 percent), and rash (3 percent). The research­ers noted that infections were equally common among patients who received treatment and those who did not. One patient died from a respiratory infection that was identified as a side effect of treatment. A total of 6 percent of treated patients and 2 percent of those who were not treated reported developing secondary cancers. The researchers stated that 75 percent of these patients had early signs of a second cancer at the time they started the study. For more information, see the study in the New England Journal of Medicine (abstract), the related Celgene press release, and a presentation from the 2013 International Myeloma Workshop which summarizes some of the study results (courtesy of Dr. Mateos).

5% hypertensives have CONN SYNDROME

Quoted in UK DAILY MAIL Somatic mutations in ATP1A1 and CACNA1D underlie a common subtype of adrenal hypertension Elena A B Azizan, Hanne Poulsen, Petronel Tuluc, Junhua Zhou, Michael V Clausen, Andreas Lieb, Carmela Maniero, Sumedha Garg, Elena G Bochukova, Wanfeng Zhao, Lalarukh Haris Shaikh, Cheryl A Brighton, Ada E D Teo, Anthony P Davenport, Tanja Dekkers, Bas Tops, Benno Küsters, Jiri Ceral, Giles S H Yeo, Sudeshna Guha Neogi, Ian McFarlane, Nitzan Rosenfeld, Francesco Marass, James Hadfield, Wojciech Margas et al. Affiliations Contributions Corresponding authors Nature Genetics (2013) doi:10.1038/ng.2716 Received 04 March 2013 Accepted 03 July 2013 Published online 04 August 2013 At least 5% of individuals with hypertension have adrenal aldosterone-producing adenomas (APAs). Gain-of-function mutations in KCNJ5 and apparent loss-of-function mutations in ATP1A1 and ATP2A3 were reported to occur in APAs1, 2. We find that KCNJ5 mutations are common in APAs resembling cortisol-secreting cells of the adrenal zona fasciculata but are absent in a subset of APAs resembling the aldosterone-secreting cells of the adrenal zona glomerulosa3. We performed exome sequencing of ten zona glomerulosa–like APAs and identified nine with somatic mutations in either ATP1A1, encoding the Na+/K+ ATPase α1 subunit, or CACNA1D, encoding Cav1.3. The ATP1A1 mutations all caused inward leak currents under physiological conditions, and the CACNA1D mutations induced a shift of voltage-dependent gating to more negative voltages, suppressed inactivation or increased currents. Many APAs with these mutations were <1 cm in diameter and had been overlooked on conventional adrenal imaging. Recognition of the distinct genotype and phenotype for this subset of APAs could facilitate diagnosis