The scientific community has made significant strides in recent years in identifying important genetic contributors to malignancy and developing therapeutic agents that target altered genes and proteins.  A recent approach to treat cancer called synthetic lethality takes advantage of genetic alterations in cancer cells that make them more susceptible to certain drugs. Alan F. List, MD, president and CEO of Moffitt Cancer Center, co-authored an article on synthetic lethality.

“Genetic alterations in cancer in humans may involve gene inactivation, amplification or inactivation,” said List.  These changes are not present in non-malignant cells. Common chemotherapeutic agents aggressively kill tumour cells irrespective of genetic alterations.  They also have a negative impact on normal cells and can cause significant side effects. Synthetic lethality harnesses the genetic differences between tumour cells and normal cells to minimize the effects on normal cells, and maximize a drug’s effects on cancer cells.

Synthetic lethality can target a variety of cellular defects, including alterations in DNA repair, cell-cycle control and metabolism.  This approach can also be used to target interactions between tumour cells and surrounding normal cells that promote tumour survival and oncogenes that drive tumorigenesis that are difficult to target directly.  Many of the synthetic lethal drugs and targets have been identified in large-scale drug screens of the entire human genome.

An example of synthetic lethality is the recent approach being investigated to treat breast cancer patients with BRCA1 and BRCA2 mutations. BRCA1 plays an important role is repairing damaged DNA.  Women who have mutations in BRCA1 or BRCA2 have an increased risk of developing breast and ovarian cancer because their cells cannot properly repair DNA. This suggests that BRCA mutated breast cancer cells may be more susceptible to drugs that target DNA.  Laboratory studies have confirmed this hypothesis by showing that agents that target another DNA repair protein called PARP significantly kill BRCA mutated cells. Several PARP inhibitors are now being investigated in clinical trials in breast cancer patients, and early results are promising.

“The goal of current anticancer approaches is to offer individualized and highly selective therapy. The treatment model for many anticancer approaches has been expanded, with movement away from dose-intensive, non-targeted cytotoxic agents to combination chemoimmunotherapy, new therapeutic combinations and targeted agents,” said List. Synthetic lethality approaches may provide an additional avenue for individualized patient treatment. Moffitt Cancer Center

Analysis of 607 small cell lung cancer (SCLC) lung tumours and neuroendocrine tumours (NET) identified common molecular markers among both groups that could reveal new therapeutic targets for patients with similar types of lung cancer, according to research.

This study examined the clinical specimens of 607 total cases of SCLC tumours (375) and lung NET (232), which included carcinoid, atypical carcinoid and large-cell neuroendocrine tumors. Biomarker testing was achieved through a combination of DNA sequencing (Next-Generation Sequencing (NGS) or Sanger-based); immunohistochemistry (IHC) to identify which proteins are present; and in situ hybridization (ISH) testing, a form of gene amplification, to determine if any of the markers that can cause cancer cells to grow or to become resistant to treatment are present.

Sequencing data were obtained from 201 total specimens (SCLC=115, NET=86). The 115 SCLC tumors harboured a wide spectrum of gene markers. Sequencing revealed mutations in p53 (57 percent), RB1 (11 percent), ATM, cMET (6 percent), PTEN (6 percent), BRAF (3 percent), SMAD4, KRAS (3 percent), ABL1, APB, CTNNB1, EGFR, FBXW7, FGFR2 (2 percent), HNF1A, HRAS, JAK3 (2 percent), MLH1 and PIK3CA (1 percent).

Multiple genes of interest were found in the NET group of 86 tumours, including 66 pulmonary neuroendocrine carcinomas and 20 carcinoid tumours. Among the neuroendocrine tumours, mutations were seen in p53 (44 percent), FGFR2 (9percent), ATM (9 percent), KRAS (6 percent) and PIK3CA (4 percent) as well as EGFR (2 percent) and BRAF (4 percent). Analysis of the carcinoid tumours revealed fewer markers, with notable mutations in p53 (11 percent), HRAS (11 percent), and BRAF (6 percent).

EGFR amplification was verified for 11 percent (5) of the 46 SCLC tumours tested. No SCLC tumours displayed amplification of cMET or HER2. The neuroendocrine tumours exhibited amplification of EGFR (13 percent), cMET (3 percent), and HER2 (4 percent) amplification, while the carcinoid tumours only showed amplification in EGFR (8 percent).

The overexpression of cKIT (64 percent vs. 37 percent), RRM1 (54 percent vs. 28 percent), TOP2A (91 percent vs. 48 percent), TOP01 (63 percent vs. 43 percent), and TS (46 percent vs. 25 percent) was found more frequently in SCLC tumours compared to lung NET, respectively (p=0.0001 for all). Low expression of PTEN was more often identified in SCLC tumours compared to lung NET (56 percent vs. 36 percent; p=0.001).

Molecular profiling of these lung cancer subtypes is not routinely performed, however, numerous mutations were found to be in common with non-small cell lung cancer tumours. Specifically, an EGFR mutation was noted in one small cell lung cancer specimen and one neuroendocrine specimen, an ALK rearrangement was detected in a neuroendocrine tumor, and HER2 amplification was seen in a neuroendocrine specimen.

“Even cancers that appear to be very similar can be dramatically different at the molecular level, and these differences may reflect unique vulnerabilities that could positively impact therapeutic options and decisions,” said Stephen V. Liu, MD, senior study author and Assistant Professor of Medicine in the Division of Hematology/Oncology at Georgetown University’s Lombardi Comprehensive Cancer Center in Washington, DC. “We are pleased that this research confirms these rarer subtypes; it calls for additional investigation on a larger scale. Once confirmed, molecular profiling of small cell tumours and NET could become standard, as it is currently for non-small cell lung cancers, which will be especially important as more molecularly targeted chemotherapy agents are developed.” ASTRO

By combing through the DNA of more than 100,000 people, researchers at Broad Institute, Massachusetts General Hospital, and elsewhere have identified rare, protective genetic mutations that lower the levels of LDL cholesterol — the so-called “bad” cholesterol — in the blood. The researchers’ findings reveal that these naturally occurring mutations also reduce a person’s risk of coronary heart disease by about 50 percent. Remarkably, the mutations disrupt a gene called Niemann-Pick C1-Like 1 (NPC1L1) — the molecular target of the FDA-approved drug ezetimibe, often used as a treatment for high LDL.

“Protective mutations like the one we’ve just identified for heart disease are a treasure trove for understanding human biology,” said Sekar Kathiresan, a senior author of the study, Broad associate member, and director of preventive cardiology at Massachusetts General Hospital. “They can teach us about the underlying causes of disease and point to important drug targets.”

Over the past several years, evidence has been mounting that certain loss-of-function mutations — mutations that reduce or completely eliminate a gene’s ability to work — can, at the same time, protect against disease. With this latest discovery, the list now stands at four genes that have been found to offer protective effects against either heart or metabolic disease. (The genes PCSK9, AP0C3, and now NPC1L1 have been found to protect against heart disease, and SLC30A8 has been shown to protect against type 2 diabetes.)

The scientific community is interested in these protective mutations not only because of what they can reveal about the biological basis of disease, but also for their ability to suggest potential paths toward new therapeutics. From a pharmaceutical perspective, it is much more feasible to develop a drug that disables, rather than activates, a gene.

Kathiresan’s long-standing interest in the genetics of blood cholesterol and heart disease first led him to uncover rare mutations in the NPC1L1 gene in just a handful of patients. He wondered if other patients carried similar mutations, so he set off on a massive hunt.

With the combined expertise of Broad Institute’s Genomics Platform, led by Stacey Gabriel, and major support from the National Human Genome Research Institute, Kathiresan and his colleagues sequenced the exomes (the protein-coding portions of the genome) of over 20,000 people of European, African, or South Asian ancestry. They discovered 15 distinct mutations in NPC1L1, all of which serve to inactivate or dampen gene activity. Roughly 1 in 650 people carries one of these inactivating NPC1L1 mutations.

“When it comes to rare variant studies, there is simply no substitute for extremely large sample sizes,” said co-author Gabriel, director of Broad Institute’s Genomics Platform. “This has become crystal clear through our work on NPC1L1 as well as several other similar projects here at the Broad. We now know the right path to get statistically robust results, and that’s the path we are on.”

After defining the mutational landscape of NPC1L1 in the initial study group of 20,000 people, Kathiresan and his colleagues correlated those mutations with LDL levels. The researchers examined the genomes of another 91,000 people and found that those with inactivating mutations in NPC1L1 tended to have lower LDL levels than those without such mutations. The reductions averaged about 12mg/dL, a 10 percent drop that is similar to what is seen in patients receiving ezetimibe therapy.

Individuals who carry inactivating NPC1L1 mutations also have a lower risk of coronary heart disease — roughly half the risk compared to those individuals without those mutations. Broad Institute

Mice bred to carry a gene variant found in a third of ALS patients have a faster disease progression and die sooner than mice with the standard genetic model of the disease, according to Penn State College of Medicine researchers. Understanding the molecular pathway of this accelerated model could lead to more successful drug trials for all ALS patients.

Amyotrophic lateral sclerosis, commonly known as Lou Gehrig’s disease, is a degeneration of lower and upper motor neurons in the brainstem, spinal cord and the motor cortex. The disease, which affects 12,000 Americans, leads to loss of muscle control. People with ALS typically die of respiratory failure when the muscles that control breathing fail.

Penn State researchers were the first to discover increased iron levels in the brains of some patients with the late-onset neurodegenerative disorders Parkinson’s disease and Alzheimer’s disease. A decade ago, they also identified a relationship between ALS and excess iron accumulation when they found that 30 percent of ALS patients in their clinic carried a variant of a gene known as HFE that is associated with iron overload disease.

For this study, the researchers crossbred mice with the HFE gene variant with the standard mice used in ALS research.

‘When we followed the disease progression and the behaviour of our crossbred mice compared to the standard mice, we saw significant differences,’ said James Connor, vice chair of neurosurgery research and director of the Center for Aging and Neurodegenerative Diseases. The crossbred mice performed significantly worse on tests of forelimb and hindlimb grip strength and had a 4 percent shorter life span.

‘The disease progression was much faster in the crossbred mice than in the standard mice,’ Connor said. ‘What we found is that when ALS happens in the presence of the HFE gene variant, things go downhill more quickly.’

The lead investigator on this project, graduate student Wint Nandar, noticed that the HFE gene variant sped up disease progression and death in females but not males. Males with ALS die faster, on average, than females.

Connor said the variant may not have had time to accelerate the pace of the disease in male mice. An accelerated progression may show up in clinical trials in human males, who live longer with the disease than mice.

The researchers also studied how the HFE gene modified the pace of the disease in mice. The crossbred mice showed increased oxidative stress and microglial activation. Microglial cells normally help with repair in the body, but when over-activated they can promote unhealthy inflammation.

‘They can make things worse instead of better,’ Connor said.

The mice were also found to have disruption of the neurofilaments, the tiny cables that transport nutrients through nerve cells.

 ‘It’s a much worse environment when the gene variant is present,’ Connor said. ‘This makes it much easier for the disease to take off.’

The findings could help direct more successful clinical testing of new drug treatments, which have traditionally had disappointing results. Because patients with H63D HFE have an accelerated form of the disease, their results could skew study findings.

‘There might be drugs out there that work for 70 percent of the ALS population even though the studies don’t show that when all of the data are looked at without consideration of the genetic background,’ Connor said.

Separating the data out could help find effective treatments for both those with the gene variant and the rest of the ALS population.

‘How a drug is going to work on a carrier of the gene variant could be worse or it could be better, but it’s likely going to be different,’ Connor said. Penn State

Damage to DNA is an issue for all cells, particularly in cancer, where the mechanisms that repair damage typically fail. The same agents that damage DNA also damage its sister molecule messenger RNA (mRNA), which ferries transcripts of the genes to the tens of thousands of ribosomes in each cell. But little attention has been paid to this damage.

 “There may be cases where messenger RNA is just as important as DNA,” said Carrie Simms, PhD, a postdoctoral associate in Zaher’s lab. “Clearly oxidative damage to RNA is somehow involved in neurodegenerative diseases, such as Alzheimer’s and ALS. It’s not necessarily causing the disease; it may just be some sort of by-product; but it’s in the mix.”

“Under normal conditions only about 1 percent of the cellular mRNAs are oxidized,” Zaher said, “but if you have oxidative stress, for whatever reason, a higher percentage can be damaged.

One of the hallmarks of Alzheimer’s is oxidative stress, and studies have shown that in people with advanced Alzheimer’s, half of the RNA molecules in the neurons may be oxidized.

Zaher, Simms and their colleagues report that when they fed oxidized mRNA to ribosomes, the nanomachines that convert mRNA to protein, the ribosomes jammed and stopped.

A stuck ribosome could be rescued by factors that released it from the mRNA and chewed up the damaged transcipt. But if the factors involved in this quality-control system were absent, damaged mRNA accumulated in the cell, just as it does in Alzheimer’s.

The three cellular processes essential to life—making copies of DNA, copying DNA into mRNA, and translating the mRNA into protein—have been penalized for billions of years by evolution, are astonishingly accurate, because evolution has heavily penalized any sloppiness.

Errors in DNA copying occur only once every billion events. When DNA is transcribed to mRNA, there is a mistake about once every ten thousand events .and when the mRNA is translated to protein, there might be an error once every thousand events.

To test the robustness of translation, the Zaher lab set out to break it, by giving faulty mRNA transcripts to ribosomes. They damaged one letter in a three-letter mRNA coding unit, oxidizing a G (the base guanine), to create what is called 8-oxo-G.

“We chose this oxidized base,” he said,” because we knew that when DNA is copied, an oxidized G causes a mistake. Instead of pairing with a C, as it normally would, 8-oxo-G will pair with an A.”

He thought the ribosome would read the three-letter codon C[8-oxo-G]C not as CGC but rather as CAC and conseqeuntly put the wrong amino acid in the protein chain it was making.

But when 8-oxo-G was added to a soup that contained all the factors needed to translate mRNA into protein, something surprising happened.

“We expected that we might get aberrant proteins,“ Simms said. “But the ribosome didn’t make mistakes.  It just stopped. It couldn’t deal with the mRNA at all”

The scientsts could tell it was stuck because levels of the protein the faulty mRNA encodes plummeted.

To make sure it was the presence rather than the position of the 8-oxo-G that mattered, Simms made mRNAs with the 8-oxo-G in each of the three positions of the three-letter coding unit. Each time the ribosome stalled.

Knowing they had found something interesting, the scientists upped their game. Simms built a longer 300-nucleotide mRNA to use as a probe. And instead of adding the damaged mRNA to a reconstituted bacterial system, she put it in extracts of plant and animal cells.

“We couldn’t look at ribosomes in the extracts,” Simms said, “but we could look at the proteins they made. They made short proteins, exactly the length you’d expect if the ribosome were stopping at the damaged base. “

A single mRNA typically has several ribosomes traveling along it, all simultaneously translating this transcript into protein. When the first ribosome stops, the others pile up behind it.

“You get this small product that is telling you the ribosome cannot go through the 8-oxo-G and then you get even smaller products that are telling you there are multiple ribosomes stuck behind the first ribosome. So the backed up ribosomes make a ladder of peptides,” Zaher said.

“This is a problem,” he said. “Among other things, the ribosome is an expensive machine that the cell has invested a lot of energy in making, and now it’s stuck on an mRNA. You need those ribosomes back.”

Fortunately ribosomes have three quality-control systems that keep watch for errors in the mRNA and rescue the ribosome if spot serious mistakes. One of these systems is “no-go decay.” When ribosomes are stuck and can’t go forward, they recruit factors that come in to pry open the ribosome, chew up the mRNA and add a tag to the defective peptide  that marks it for degradation.”

But no-go decay was originally discovered by throwing artificial roadblocks in the ribosome’s way: mRNAs with large hairpin turns in them that the ribosome could not unwind or plow through.

“Four billion years of evolution has made sure your genome does not have sequences that make hairpins, so these are clearly not the intended targets for no-go decay,” Zaher said.

To find out, the scientists turned to yeast cells. If the yeast’s ribosomes jammed on the oxidized mRNA but were rescued by no-go decay, very little damaged mRNA would accumulate in the cell. This proved to be the case.

Simms then deleted the gene for a factor that releases the ribosome from the mRNA when it jams. In these knockout yeast the level of oxidized mRNA went up. Then she deleted the gene for a factor that is recruited to degrade the mRNA after the ribosome is released, and again the level of oxidized mRNA rose. Without no-go decay, the cells were clearly in trouble.

“The system that translates mRNA into protein is highly conserved, so what’s true for yeast is probably true for people as well,” said Zaher. Washington University in St. Louis.