Drug Repurposing Study Sheds Light on Heart Disease Risk

Research team develops computer program for investigating unknown drug side effects and novel uses

A team led by researchers at Brigham and Women’s Hospital has developed a computational technique to reveal the unknown side effects – both good and bad – of hundreds of drugs. That knowledge could help pharmacologists discover new indications for drugs already on the market and repurpose them for other disorders. Using their unique method, the researchers discovered that two drugs commonly prescribed for non-coronary disorders may affect heart disease risk. Their findings were recently published in Nature Communications.

Most medications work by binding to a diseased protein target. However, previous research has shown that some can also interfere with unknown and unintended targets and biological processes. That helps explain why many drugs succeed in single-cell and animal experiments but fail in clinical trials when introduced to the more complex human body.

“The great majority of drugs are not unique to the single target for which they’ve been developed,” said Joseph Loscalzo, MD, PhD, Chair of the Department of Medicine Chair at BWH, who led the study. “They may have many off-target effects – some potentially beneficial, and some adverse.”

The team used archival data to examine how 700 FDA-approved medications interact with various proteins. To fully characterize off-target effects, they built maps of human protein interactomes –networks of protein-protein interactions contained in cells – using existing datasets. They located the subnetworks containing diseased proteins and drug targets unique to heart disease. They identified drugs used for non-cardiovascular disorders whose targets were very near or within the cardiovascular disease subnetworks. Then they explored health care databases containing clinical information from more than 220 million patients and identified the incidence of cardiovascular events in individuals treated with these drugs.

They found that hydroxychloroquine, an anti-rheumatic drug, was associated with lower rates of coronary artery disease. This finding raises the possibility that hydroxychloroquine could be repurposed for treating this common artery-obstructing disorder.

Conversely, they found that carbamazepine, a drug mainly prescribed for epilepsy and neuropathic pain, may increase the risk of coronary artery disease. This could mean that patients who are older, diabetic or otherwise at higher risk for coronary artery disease should consider alternative medications for such neurological conditions.

The research team investigated possible mechanisms for the relationship between these two medications and coronary artery disease risk using lab studies of human vascular cells. They next plan to fully characterize the off-target effects for more drugs and diseases. Their interactome maps and network prediction methods, along with the FDA drug target dataset they used, are open-access for other researchers to utilize.

“Until recently, there hasn’t been a way to look at all possible drug targets,” said Loscalzo. “Now we have the tools to do so, which can facilitate drug repurposing.”

This study was funded by grants from the National Institutes of Health (P50-HG004233, U01-HG001715, and U01-HG007690 from NHGRI, P50-GM107618 from NIGMS and PO1-HL083069, R37-HL061795, RC2-HL101543, U01-HL108630, RC4-HL106373, and K99HL138272 from NHLBI, and ME-1303-5638 from PCORI).

Paper cited: Cheng, F et al. (2018). “Network-based approach to prediction and population-based validation of in silico drug repurposing.” Nature Communications. DOI: 10.1038/s41467-018-05116-5.

Novel Blood Test Predicts Kidney Cancer Risk and Survival Five Years Prior to Diagnosis

Concentrations of KIM-1 in the blood predicted kidney cancer diagnosis and were associated with poorer survival

A critical biomarker of kidney disease may help predict clear cell kidney cancer – the most common form of kidney cancer – years before clinical diagnosis. Kidney-injury-molecule-1 (KIM-1) can be detected in the urine and blood and is generally present at low levels in healthy individuals. Prior research by leaders at Brigham and Women’s Hospital has shown that KIM-1 is an important and highly predictive marker for kidney injury. In a new study published in Clinical Cancer Research, BWH investigators, along with colleagues from Beth-Israel Deaconess Medical Center, explore whether a blood test can detect higher concentrations of KIM-1 in patients who will go on to develop kidney cancer up to five years later. Their results show that KIM-1 substantially helped distinguish between those who went on to develop kidney cancer from those who did not.

“Early detection of kidney cancer can be lifesaving. We can cure kidney cancer when we detect it at an early stage, but patients with advanced kidney cancer have a very high death rate,” said Venkata Sabbisetti, PhD, a research faculty member in the BWH Renal Division. However, kidney cancer is asymptomatic and many patients present with advanced kidney cancer at the time of diagnosis. Our results suggest that with further refinement, KIM-1 has the potential to identify patients with early, curable kidney cancer.”

Sabbisetti and colleagues measured KIM-1 concentrations in samples from patients enrolled in the European Prospective Investigation into Cancer and nutrition (EPIC). The team compared KIM-1 levels from 190 participants who went on to develop RCC within the next five years to 190 matched participants (same age, body mass index, smoking status, etc.) who remained healthy. In samples with detectable levels of KIM-1, the average concentration was double in those who would develop kidney cancer.

The team reported that adding KIM-1 to a model for predicting kidney cancer risk approximately doubled the accuracy of that model. KIM-1 was substantially more sensitive for kidney cancer detection than prostate specific antigen is for prostate cancer. However, given how much rarer RCC is, the researchers noted that KIM-1 should be measured along with another kidney disease specific markers to be useful for early detection in the general population.

“We envision that KIM-1 will be useful in settings where the risk of kidney cancer is higher, such as patients undergoing abdominal CT scanning, where KIM-1 could be used to stratify risk of RCC,” the authors wrote. “This will be particularly important given the rise of routine CT scans and the strong association between number of CT scans and number of nephrectomies performed at the regional level in the U.S., indicating a substantial burden of overdiagnosis.”

Funding for this work was provided by the National Institutes of Health (R01 CA196996 and P50 CA101942-12) and a Cancer Research UK Population Research Fellowship.

Paper cited: Scelo G et al. “KIM-1 as a blood-based marker for early detection of kidney cancer: a prospective nested case-control study.” Clinical Cancer Research DOI: 10.1158/1078-0432.CCR-18-1496

Cellular Escape Artists May Help Explain Why Some Women Present with Advanced Ovarian Cancer

Exhaustive study of fallopian tubes reveals evidence of ‘precursor escape’ – a novel concept in high-grade serous carcinoma

More than two-thirds of women with ovarian cancer have high-grade serous carcinoma (HGSC). Up to 90 percent of HGSCs are not detected until they are beyond the ovaries or fallopian tubes, at which point it may not be possible to fully remove the tumor. Many of these cancers are now known to originate in the fallopian tube, which is the site where almost all early HGSCs are discovered in healthy women undergoing resection of fallopian tubes. In contrast, among women who first come into the clinic with symptoms of advanced HGSC that has already spread beyond the gynecological region, clinicians can often find little or no evidence of malignancy in the fallopian tubes. Christopher Crum, MD, in the BWH Division of Women’s and Perinatal Pathology, describes this as the great mystery of HGSC.

“I’ve always been perplexed by the cases where women present with advanced HGSC out of the blue, and we don’t detect anything in the fallopian tube,” said Crum. “This is a fundamental mystery: Why do we catch the cancer so late even when we thought we were catching it early?”

In a new study published recently in The Journal of Pathology, Crum and colleagues conducted an exhaustive analysis of “normal” fallopian tubes from patients with HGSC. Their analysis indicates that normal appearing tubes can contain pre-cancerous cells may escape the tubes, later progressing to cancer in the pelvic or abdominal cavity. This concept – novel in HGSC – may help explain the origins of the disease and supports a potentially important ovarian cancer prevention surgery known as opportunistic salpingectomy (removal of the fallopian tubes).

Fifteen years ago, when clinicians began screening women who were at high-risk for ovarian cancer, such as those with BRCA1 or BRCA2 mutations, Crum and others began seeing evidence of early tubal cancers in addition to cancer in the ovaries. In 2005, Crum began the Sectioning and Extensively Examining the Fimbriated End Protocol (SEE-FIM) at the Brigham in which pathologists thoroughly sectioned the distal fallopian tubes of patients, looking for microscopic lesions. They discovered that a very high percentage of early cancers could be found in the fallopian tubes, changing the thinking around HGSC’s origins. SEE-FIM is now used in clinical laboratories at hospitals across the U.S.

“There was a sudden paradigm shift in the field,” said Crum. “There’s now a push in the field to do prophylactic salpingectomy – removal of the fallopian tubes – for women who are coming in for routine surgery. The hope is that this will reduce risk of HGSC.”

Salpingectomy removes the fallopian tubes while allowing women to keep the ovaries and, therefore, maintain estrogen production and not face early menopause. Data on the protective effects of the surgery are forthcoming.

But there is a paradox: Among women who enter the clinic with symptomatic cancer, only about 30 to 40 percent show evidence of early cancer in the fallopian tubes. This inconsistency troubled Crum, who was concerned that he could not rule out that the cancer might be beginning somewhere else.

To investigate, Crum and colleagues analyzed the fallopian tubes of women with widespread cancer but without obvious abnormalities in the tubes. This analysis, led by first author Thing Rinda Soong, MD, PhD, MPH, formerly of the BWH Department of Pathology, revealed that 13 of the 32 women who had at first appeared to have normal tubes had very early signs of precancerous cells – which the researchers termed early serous proliferations (ESPs). ESPs are relatively common in the general population, but most women with these proliferations never develop ovarian cancer. On a hunch that some of these proliferations could still be important contributors to the development of HGSC, the investigators sequenced TP53 – a gene that is mutated in all HGSCs. In 12 of the 13 women with ESPs, the team detected a mutation in these precancerous cells that matched the mutation in the cancer found in the pelvis.

“This opens up the prospect that these little cells can escape off the fallopian tube, into the pelvis and, at some point in time, become malignant,” said Crum. “This could tie a very high percentage of HGSCs to the fallopian tube – the tumor could begin either as a malignancy in the tube or in the peritoneal cavity from precursor cells that had previously escaped from the tube. If validated, this will be an important advance in our understanding of the pathogenesis of HGSC, with obvious implications for prevention of this disease.”

This study was supported by the Department of Defense (Pilot award OC130500; Ovarian Cancer Academy Grant OC160444).

Paper cited: Soong, TR et al. “Evidence for lineage continuity between early serous proliferations (ESPs) in the fallopian tube and disseminated high-grade serous carcinomas” Journal of Pathology DOI: 10.1002/path.5145

A Deeper Look at Severe Asthma Yields NET Results

Study identifies key inflammatory and immunological underpinnings of disease with implications for future treatment

Of the more than 24 million people in the U.S. who have asthma, 10 percent have severe asthma – a form of the disease that does not respond to treatment. The immunological mechanisms underlying severe asthma and asthmatic lung inflammation are not well understood. A new study by investigators from Brigham and Women’s Hospital published this week in Science Immunology models allergic lung inflammation and provides new insights into how asthma develops and progresses, with important implications for the most clinically advanced drugs designed to treat severe asthma.

“Asthmatic patients are not identical. Asthma genetics and clinical characterization of patients have provided evidence for differences that have profoundly affected our approach to clinical care,” said corresponding author Bruce Levy, MD, chief of the Division of Pulmonary and Critical Care Medicine. “But we still have an incomplete understanding of the origins of asthma, and only limited information is available on long-term asthma prognosis. No therapies are available to cure asthma, and there remains much work to be done.”

To model allergic lung inflammation in a dirty indoor environment, the team exposed a mouse model to a common environmental indoor allergen – house dust mite – as well as to endotoxin, a toxin released by bacterial cells. Exposure to both stimuli triggered complex lung inflammation, including a phenomenon known as lung NETosis.

In response to inflammatory triggers, white blood cells known as neutrophils form “neutrophil extracellular traps” (NETs). NETosis is the process by which NETs get activated and released. NETs can play a significant role in helping defend a host from invaders, but they can also cause organ injury and inflammation. Vital NETosis is a process in which neutrophils extrude their nuclear material, including DNA, to form NETs and then reseal their membranes to create cytoplasts – cells that lack a nucleus. Levy and colleagues found that in their model, NETosis and cytoplasts appeared to play a key role in triggering and amplifying an allergen-initiated neutrophilic immune response in lung inflammation.

In addition to studying animals, the team also examined samples of fluid from the lungs of human severe asthma patients, finding that a subset of patients had high neutrophil counts and detectable NETs and cytoplasts – important implications for how to design more precise clinical trials for severe asthma treatment.

Currently, clinical trials for new drugs to treat moderate and severe asthma do not stratify patients by neutrophil count or other important markers of inflammation. The team notes that markers of NETosis – including NETs and cytoplasts in sputum – may provide an opportunity to better tailor trials and treatments in subsets of asthma patients for future clinical research.

Levy will address questions about his latest research in a Facebook Live event on Aug. 8 at 1 p.m. held by Science Immunology. For more details and to participate, please visit: https://www.facebook.com/ScienceImmunology/  

Funding for this work was provided by the National Heart, Lung, and Blood Institute (U10 HL109172; U10 HL109164; U10 HL109257; U10 HL109250; U10 HL109146; U10 HL109250; U10 HL109168; U10 HL109152; U10 HL109086; R35HL135765; RO1HL122531); NIH National Center for Advancing Translational Sciences awards (UL1 TR001420; UL1 TR000427; UL1 TR001102; UL1 TR000454); a Canadian Institutes of Health Research postdoctoral fellowship (K12 HD047349 and K08 HL130540); and a fellowship from the German Society for Pediatric Pneumology.

Paper cited: Krishnamoorthy, N et al. “Neutrophil cytoplasts induce TH17 differentiation and skew inflammation toward neutrophilia in severe asthma” Science Immunology DOI: 10.1126/sciimmunol. aao4747

Newly Characterized Molecule Offers Possibilities for Novel Alzheimer’s Treatments

The study of neuroprotective microRNAs like miR-132 may lead to new avenues for treating Alzheimer’s disease and other tau pathologies

Alzheimer’s disease is an increasingly prevalent, neurodegenerative condition that erodes memory and other cognitive functions. Treatments for this complex disease have been elusive, although researchers have previously uncovered its main biological features: amyloid-beta plaques and tau tangles.

A study by researchers from Brigham and Women’s Hospital (BWH), recently published in Acta Neuropathologica, investigated lesser-known molecules involved in tauopathies like Alzheimer’s. They focused on microRNAs (miRNAs), gene expression regulators that bind to and destroy protein-encoding messenger RNAs. They discovered that some of these miRNAs showed neuroprotective effects; their supplementation therefore holds potential as a treatment for tauopathies.

The researchers looked at several miRNAs but focused on miR-132, which previous research has shown is downregulated in Alzheimer’s and other tauopathies. They discovered that miR-132 seemed to protect against toxic amyloid-beta and tau in both rodent models and human neurons.

“Our results support the idea that miR-132 is a master regulator of neuronal health with potential as a treatment target,” said lead investigator and BWH scientist, Anna Krichevsky, PhD.

The team first looked at primary cortical and hippocampal neurons taken from both normal and tauopathic mice. To examine the neuroprotective properties of naturally occurring miRNAs, they tested 63 neuronal miRNAs, then inhibited them with miRNA-binding molecules called anti-miRNAs. They found that inhibition of some miRNAs seemed to protect against, and others to exacerbate, amyloid-beta pathology and associated glutamate excitotoxicity. Of these, miR-132 was the most neuroprotective miRNA.

They confirmed the neuroprotective properties of miR-132 by designing miR-132 mimics and introducing them to the mouse cells. They observed reduced levels of toxic forms of tau, glutamate excitotoxicity and cell death. They also examined miR-132 supplementation in live mice models of human neurodegenerative disease by injecting miR-132 by way of a viral vector. Compared to controls, miR-132-injected mice showed reduced tau pathology and enhanced hippocampal long-term potentiation, a process involved in memory formation.

When the researchers next introduced miR-132 mimics to human cells, they saw similar results: reduced toxic forms of tau and less cell death.

According to Krichevsky, miRNA research offers a fresh perspective in the search for possible Alzheimer’s treatments. MicroRNAs were discovered more recently than mRNAs and proteins, and their complex roles in multiple biological pathways have caused many to doubt that their manipulation could be a viable therapeutic strategy.

“Now that we have the knowledge and technologies that enable manipulation of miRNA, we can explore new possibilities,” said Krichevsky. “In the last 30 years, research has focused mostly on amyloid. We’re still hopeful about that approach, but we must invest in new strategies as well.”

This study was funded by grants from Alzheimer’s Association (NIRG-09-132844) and Tau Consortium/Rainwater foundation.

Paper cited: Fatimy R et al. “MicroRNA‑132 Provides Neuroprotection for Tauopathies via Multiple Signaling Pathways.” Acta Neuropathologica. DOI: 10.1007/s00401-018-1880-5.

Advancing the Search for Antibodies to Treat Alzheimer’s Disease

Two new studies shed light on the most toxic forms of amyloid-beta and how to find antibodies to target these structures 

Two new studies published by investigators from Brigham and Women’s Hospital illustrate that not all forms of amyloid-beta (Aβ) protein – the protein thought to initiate Alzheimer’s disease – play an equally menacing role in the progress of the disease. Using a new way of preparing and extracting the protein as well as a new technique to search for promising drug candidates, researchers have highlighted the importance of testing and targeting different forms of Aβ. Their work may help advance the search for more precise and effective drugs to prevent or halt the progress of Alzheimer’s disease.

“Many different efforts are currently underway to find treatments for Alzheimer’s disease, and anti-Aβ antibodies are currently the furthest advanced. But the question remains: what are the most important forms of Aβ to target? Our study points to some interesting answers,” said Dominic Walsh, PhD, a principal investigator in the Ann Romney Center.

Aβ protein can take forms ranging from monomers – single molecules – to twisted tangles of plaques that can pollute the brain and are large enough that they can be seen with a traditional microscope. Walsh compares monomers to single Lego bricks, which can start sticking together to form complex structures of varying sizes. The two recently published studies investigate how to find new potential therapeutics that can target the structures most likely to cause harm.

Most Alzheimer’s disease studies use synthetic Aβ to approximate what conditions in the brain of an Alzheimer’s patient might be like. A small number of researchers have used Aβ extracted from human brain, but the extraction process is crude. In a study published in Acta Neuropathologica in April, Walsh and colleagues developed a much gentler extraction protocol to prepare samples from subjects with Alzheimer’s disease. The team found that Aβ was far more abundant in traditional crude extracts, but that the bulk of the extracted Aβ was innocuous. In contrast, much less Aβ was obtained with the gentler protocol, but in this case most of the Aβ was toxic.

In a second study published in Nature Communications in July, Walsh and colleagues developed a screening test to try to find potential drugs to target the toxic forms of Aβ. The new technique uses extracts of brain samples from Alzheimer’s disease patients and live-cell imaging of stem-cell derived brain cells to find promising therapeutics. The team reports on 1C22, an Aβ antibody that they found could protect against toxic forms of amyloid-beta more effectively than the most clinically advanced Alzheimer’s disease therapeutics currently in clinical trials.

“We anticipate that this primary screening technique will be useful in the search to identify more potent anti-Aβ therapeutics in the future,” said Walsh.

Funding for the study appearing in Acta Neuropathologica was provided by the National Institutes of Health (AG046275), Bright Focus, and by the Massachusetts Alzheimer’s Disease Research Center (AG05134). Funding for the study appearing in Nature Communications was provided by the National Institute on Aging (AG046275, AG006173 and AG015379), Health Research Board Ireland (Grant RP/2008/30) and Bright Focus.

Papers cited: Hong W et al. “Diffusible, highly bioactive oligomers represent a critical minority of soluble Aβ in Alzheimer’s disease brain” Acta Neuropathologica DOI: 10.1007/s00401-018-1846-7

Jin M et al. “An in vitro paradigm to assess potential anti-Aβ antibodies for Alzheimer’s disease” Nature Communications DOI: 10.1038/s41467-018-05068-w