Targeting a microRNA shows potential to enhance effectiveness of diabetes drugs

Over the past 15 years, University of Alabama at Birmingham endocrinologist Anath Shalev, M.D., has unraveled a crucial biological pathway that malfunctions in diabetes.

Her latest discovery in this beta-cell pathway, published in the journal Diabetes, shows the potential to enhance the effectiveness of existing diabetes drugs, as well as reduce some of the unwelcome side effects of those drugs.

The need for improved treatment is great. Diabetes is a disorder characterized by elevated blood sugar that afflicts one of every 10 U.S. adults and doubles the risk of early death. More than 30 million people in the United States have diabetes, which is the seventh-leading cause of death and also leads to blindness and lower-limb amputations.

In 2013, the UAB researchers found that either diabetes or elevated production of the protein TXNIP induced beta-cell expression of microRNA-204, or miR-204, and this microRNA, in turn, blocked insulin production. The Shalev group has now found another vital role for miR-204 — regulating the cell surface receptor that is the target of many of the newer type 2 diabetes drugs, such as Byetta, Victoza, Trulicity, Januvia, Onglyza and Tradjenta. This drug target is the glucagon-like peptide 1 receptor, or GLP1R. Activation of GLP1R with these drugs helps the beta cell produce and secrete more insulin.

Shalev’s new work was performed in rat beta cells, genetically modified mice, mouse pancreatic islets and human pancreatic islets. Healthy beta cells, which are found in the pancreatic islets, produce insulin to control blood sugar levels; in diabetes the beta cells are impaired and dysfunctional, and have lower GLP1R levels.

In the Diabetes study, Shalev and colleagues found that overexpression of miR-204 decreased expression of GLP1R in rat beta cells and in mouse and human pancreatic islets. Conversely, knock-down of miR-204 increased expression of GLP1R in those cells and pancreatic islets.

Greater GLP1R expression is beneficial because it helps transfer a signal to the beta cell to secrete more insulin, such as after a meal. Also, many of the newer diabetes drugs act as agonists to activate GLP1R. Higher expression can allow use of a lower-drug dose to treat diabetes, thus reducing dose-dependent side effects.

In mice, the UAB researchers found that a deletion of miR-204 caused enhanced GLP1R expression, and also better insulin secretion and glucose control. Furthermore, the knockout mice were more responsive to a GLP1R agonist in glucose tolerance tests. When the GLP1R knockout mice were used in a model of diabetes, where beta cells are damaged by low doses of the toxin streptozotocin, the diabetic mice showed improved glucose control and increased serum insulin levels.

These results suggest that downregulating miR-204, now revealed as an upstream regulator of GLP1R, could lead to better treatment of diabetes.

One key fact about miR-204 may further aid improved treatment. This microRNA is highly expressed in beta cells, but it is not highly expressed in the rest of the pancreas or in cells of the gastrointestinal tract that also express GLP1R and therefore respond to GLP1R agonists. Thus, an inhibitor of miR-204 would be relatively selective for beta cells.

“This novel concept of inhibiting a microRNA in a non-targeted manner, but taking advantage of its restricted tissue distribution and thereby selectively upregulating its target genes in that tissue, may have far reaching implications for microRNA biology and tissue-specific gene targeting in general,” Shalev said.

“Since miR-204 is expressed primarily in pancreatic beta cells, manipulating its levels allows for preferential upregulation of GLP1R in the beta cell, where it helps secrete insulin, rather than in the gastrointestinal system, where it can cause nausea and impaired gastric emptying, or in the pancreas, where it can increase the risk for pancreatitis,” Shalev said. “So by inhibiting miR-204, one could increase the effects of GLP1R agonist drugs on insulin secretion, thereby lowering the necessary dose and avoiding some of the dose-dependent adverse effects.”

The mechanism by which miR-204 downregulates expression of GLP1R is binding of the microRNA to the 3-prime-untranslated region of GLP1R messenger RNA. Such binding is a known method to control gene expression by microRNAs. The UAB researchers discovered this specific binding using microRNA target prediction software. They found two binding sites for miR-204 in the messenger RNA for human GLP1R and one binding site in the messenger RNA for mouse GLP1R. When they mutated those binding sites, it eliminated the regulatory effect of miR-204.

Additionally, the Shalev group showed a novel link between TXNIP and GLP1R signaling. Mice with a beta cell-specific knockout of the protein TXNIP had lower miR-204 levels and higher GLP1R expression, and the mice showed enhanced insulin secretion and glucose control in response to an agonist of GLP1R. Thus, through both control of insulin production and regulation of GLP1R, as well as regulation of the unfolded protein response and beta cell apoptosis, miR-204 appears to play a linchpin role to control the function of beta cells in the pancreas.

Editing genes one by one throughout colorectal cancer cell genome uncovers new drug target

Cancers driven by mutations in the KRAS gene are among the most deadly. For decades, researchers have tried unsuccessfully to directly target mutant KRAS proteins as a means to treat tumors. Instead of targeting mutant KRAS itself, researchers at University of California San Diego School of Medicine are now looking for other genes or molecules that, when inhibited, kill cancer cells only when KRAS is also mutated.

The team used the CRISPR-Cas9 gene editing technique to systematically inactivate every gene in the genome of human colorectal cancer cells with and without mutant KRAS. They found that growth of KRAS-mutant colorectal cancer cells in mice was reduced by approximately 50 percent when two genes that encode metabolic enzymes — NADK and KHK — were also inactivated.

The study, published September 27 in Cancer Research, provides potential new drug targets for KRAS-driven cancers.

“We did not get these same results with cancer cells grown in the lab — the growth inhibition we saw when the NADK and KHK genes were inactivated only occurs in tumors in a mammalian system, in a more realistic microenvironment where the tumor has to survive,” said senior author Tariq Rana, PhD, professor of pediatrics at UC San Diego School of Medicine and Moores Cancer Center. “That suggests that the metabolic dependencies of tumor cells growing in a laboratory dish may differ dramatically compared to the same cells growing in a living system, underscoring potential limitations of standard laboratory-based cancer cell growth tests.”

Approximately 20 to 30 percent of all human cancers have mutations in the KRAS gene. KRAS mutations occur in many of the most lethal and most difficult to treat cancers, including lung, pancreatic and colorectal cancer. KRAS mutant cancer cells are able to rewire their metabolism in a way that gives them a growth advantage compared to normal cells.

Rana’s approach to treating KRAS-driven cancers — inhibiting other genes or molecules in addition to KRAS — is called “synthetic lethality” because the intervention is only lethal to the mutated cells. In a previous study, Rana’s team used a library of microRNAs, small pieces of genetic material, to systematically block protein production and look for those inhibitions that are synthetic lethal in combination with KRAS mutations.

In their latest study, Rana’s team used CRISPR-Cas9 to systematically inactivate genes in two human colorectal cancer cell lines — one with normal KRAS and one with a mutant KRAS. They then tested the ability of each of these cell lines to grow as tumors in mice. They found that inactivating two metabolic enzymes, NADK and KHK, reduced the growth of KRAS-mutant tumors by approximately 50 percent, but had no effect on normal KRAS tumors. They also blocked these same enzymes with commercially available small molecule inhibitors and saw significant reduction in tumor growth in mice only in tumor cells with mutant KRAS.

Rana and team also identified several new genes that, when inactivated, had the opposite effect — they increased KRAS-mutant tumor growth, but not the growth of normal KRAS tumors. These types of genes are known as “tumor suppressors” because they normally keep cancer cell growth in check.

“One of the most surprising findings from our study is how performing this type of genetic screen directly in a mammalian microenvironment revealed not only new synthetic lethal interactions, but also new tumor suppressor genes that are dependent on KRAS mutations,” said first author Edwin Yau, MD, PhD, a hematology/oncology and Cancer Therapeutics Training Program fellow in Rana’s lab.

One of these new tumor suppressor genes encodes INO80C, a large multi-subunit protein that, among other things, stabilizes the genome. Rana, Yau and colleagues are now taking steps to carry their findings forward, with the ultimate goal of better understanding how KRAS-mutant cancers develop and translating these insights into developing new therapies to stop them.

Bronchial Thermoplasty Helps Asthma Patients Reduce Severe Attacks, Hospitalizations and ER Visits

In a new study presented at the 2017 American Thoracic Society International Conference, adult asthma patients treated with bronchial thermoplasty (BT) had fewer severe exacerbations and were able to reduce their ER visits and hospitalizations in the two years following treatment.  Approved by the FDA in 2010, BT is a new device-based therapy that uses a series of three radio-frequency treatments to open the airways of adults with severe, persistent asthma whose symptoms are not adequately controlled by inhaled corticosteroids or long-acting beta-agonists.

To date, more than 6,800 patients in 33 countries have been treated with BT.

The “Post-FDA Approval Clinical Trial Evaluating BT in Severe Persistent Asthma” (PAS2 study), which involves hundreds of patients at dozens of research centers, looks at the long-term effects and safety of BT.

“The results of the PAS2 study suggest that after a single series of BT procedures, patients experience long-term improvement in their asthma control,” said lead author Geoffrey Chupp, MD, from Yale School of Medicine.  “These results indicate that BT works across the spectrum of severe asthma patients. We believe BT should be more widely considered as a treatment option in patients with poorly controlled severe asthma.”

Two-hundred eighty four patients were enrolled in the study at 27 centers in the U.S. and Canada.  Two-hundred seventy-nine study subjects had at least one BT procedure, and 271 had all three procedures.  In the 12 months prior to the first BT procedure, 78 percent of subjects had at least one severe exacerbation, 16 percent required hospitalization and 29 percent had ER visits.  In the first year follow up, 50.6 percent had severe exacerbations; 45.4 percent had exacerbations in the second year follow up.  Asthma-related hospitalizations and ER visits also saw significant, continuing reductions:  14.4 percent and 12.7 percent of subjects had hospitalizations, respectively, in

Precision Medicine: UAB Study Creates ‘Mini-Lung’ to Study Effect of Pulmonary Fibrosis Drugs

Pulmospheres, three dimensional multicellular spheroids composed of lung cells from individual patients, were shown to be effective in predicting the efficacy of medications for idiopathic pulmonary fibrosis, according to findings from University of Alabama at Birmingham scientists presented today in JCI Insight, a journal of the American Society for Clinical Investigation.

Pulmospheres are tiny spheres — about one millimeter in diameter — which contain all the various cell types found in a human lung and are grown from tissue obtained from a surgical lung biopsy. Pulmospheres give researchers a 3D model to study various aspects of cell biology and disease mechanisms.

“Our results suggest that pulmospheres simulate the microenvironment in the lung and serve as a personalized and predictive model for assessing responsiveness to antifibrotic drugs in patients with IPF,” said Veena Antony, M.D., professor in the Division of Pulmonary, Allergy and Critical Care Medicine within the Department of Medicine, and primary investigator of the study.

The UAB research team grew pulmospheres from 20 patients with idiopathic pulmonary fibrosis — a devastating lung disease — and nine control patients. They then examined whether the pulmospheres reacted to one of two commonly used medications for IPF.

“There is no cure for IPF, but there are two FDA-approved drugs that can help slow the rate of decline caused by the disease and improve quality of life,” said Antony. “Not all patients respond to both drugs, and some don’t respond to either. Having a reliable clinical test that can predict which drug works best for which patient is urgently needed.”

The pulmospheres were grown to useable size in about 24 hours following the biopsy, then exposed to the two medications, pirfenidone and nintedanib. Within about 16 hours, researchers were able to observe if the spheres responded favorably to one, both or neither of the medications.

“This is a wonderful example of precision medicine,” said Victor Thannickal, M.D., professor and director of the Division of Pulmonary, Allergy and Critical Care Medicine and a study co-author. “Using pulmospheres derived from a patient’s own cells may allow clinicians to tailor specific drugs to an individual patient without exposing that patient to potential side-effects or harm from treatments that are unlikely to be effective.”

Of the 20 subjects enrolled in this study, three patient’s pulmospheres responded only to nintedanib and the pulmospheres of four patient’s responded only to pirfenidone. Eleven patient’s pulmospheres responded to both drugs and two patient’s pulmospheres did not respond to either drug.

Researchers confirmed the findings by following the patients over time, establishing that the response predicted by the pulmospheres was clinically observed in the patients.

Antony says there is a critical need for a better predictive model for IPF. Animal models of the disease are disappointing, and more traditional two dimensional cellular models are insufficient.

“The lungs are three dimensional organs and to truly understand the dynamics of IPF medications on the disease we require a 3D model, one that contains all the cell types found in a lung and that is able to function as a microcosm of the lung,” Antony said. “Many drugs have shown promise in pre-clinical studies, only to fail in subsequent clinical trials. Three dimensional modeling might change that.”

Antony says pulmospheres were developed in cancer research as a means of targeting potential drugs. A hallmark of IPF is an aggressive invasion by cells known as myofibroblasts, which mirrors the invasive phenotype of malignant cells, giving researchers reason to think that pulmospheres might work in IPF. The UAB trial was the first to study pulmospheres in IPF.

Antony also hopes that modern drug discovery techniques, using high throughput screening technology to quickly screen numerous compounds for disease-modifying properties, will be enhanced by the use of pulmospheres.

“There are many potential therapeutic agents for IPF in the discovery pipeline now, and this technique might prove to be a very effective way to determining which are the most promising,” she said.

Thannickal serves as the program director of a translational program project grant, sponsored by the National Heart, Lung and Blood Institute, that is focused on identifying novel diagnostic and therapeutic approaches to fibrotic lung disease.

“Without support of the NHLBI, and in particular the tPPG funding mechanism, the tissue biorepository which was critical to developing these innovative screening methods would not have been possible,” he said.

Funding for the study came from the National Institutes of Health. NIH describes IPF as a disease in which tissue deep in the lungs becomes thick and stiff, or scarred, over time. As the lung tissue thickens, the lungs are unable to properly move oxygen into the bloodstream.

IPF is a serious disease that usually affects middle-aged and older adults. IPF has no cure and many people live only about three to five years after diagnosis.