Anika Hazra

To treat glioblastoma, researchers focus on tumour vulnerabilities

Christopher.Sorensen — Thu, 21 Nov 2024 14:21:00 +0000

To treat glioblastoma, researchers focus on tumour vulnerabilities

Christopher.Sorensen Thu, 11/21/2024 - 09:21

Cutline

From left to right: researchers Graham MacLeod, Fatemeh Molaei and Stéphane Angers, director of U of T’s Donnelly Centre for Cellular and Biomolecular Research (supplied images)

Anika Hazra

Topic

Breaking Research

Temerty Faculty of Medicine

Donnelly Centre for Cellular & Biomolecular Research

Cancer

Research & Innovation

Subheadline

"Our study increases our understanding of this type of cancer and proposes a different approach to treating it that will hopefully improve the prognosis of patients"

A team led by researchers at the ��߲ݴ�ý has uncovered new targets that could be the key to effectively treating glioblastoma, a lethal type of brain cancer.

The targets were identified through a screen for genetic vulnerabilities in patient-derived cancer stem cells that represent the variability found in tumours.

The study was published recently in the journal Cancer Research.

“Glioblastoma tumors have evaded treatment thus far because their composition is highly variable both within and between tumours,” said Graham MacLeod, co-first author on the study and senior research associate at U of T’s Donnelly Centre for Cellular and Biomolecular Research.

“The tumours vary quite a bit from person to person, and even within a single tumour there are multiple cell types that harbour differences at the genetic level.”

Glioblastoma is the most common type of brain cancer in adults. It is also the most challenging to treat due to the resistance of glioblastoma cancer stem cells, from which tumours grow, to therapy. Cancer stem cells that survive after a tumour is treated go on to form new tumours that do not respond to further treatment.

A key finding of the research is that the variability among glioblastoma cancer stem cells can be observed across a gradient between two cell subtypes. On one end is the developmental subtype, which resembles cells in which normal neurodevelopment has gone awry. On the other is the injury-response subtype, which is an inflammatory state. The aim of the study was to identify potential treatment methods to target each subtype, thereby tackling tumours in a more holistic manner.

This study follows earlier research published in Cell Reports that identified vulnerabilities in glioblastoma cancer stem cells that impact their sensitivity to chemotherapy. The next step was to study how vulnerabilities in glioblastoma cancer stem cells vary in a large and diverse set of patient-derived cell lines to identify the most common of these vulnerabilities in each of the subtypes.

The team performed screens in glioblastoma stem cell lines from 30 patients, making this the largest screening study of its kind. The patient-derived cell lines were generated by the lab of Peter Dirks, chief of the division of neurosurgery at SickKids and a U of T professor of surgery and molecular genetics in the Temerty Faculty of Medicine. Within the cancer stem cell samples, the team found genes responsible for the proliferation of the two cell subtypes that could be targeted to prevent tumour growth. Combining drugs to target both cell subtypes simultaneously could potentially make for a more effective glioblastoma treatment.

“A lot of the research on glioblastoma is conducted with a limited number of immortalized cell lines grown in serum,” said Fatemeh Molaei, co-first author on the study and graduate student at the Donnelly Centre and the Leslie Dan Faculty of Pharmacy. “These cells aren’t the best model as they don’t resemble true glioblastoma cells as much as we would like. The findings from our study represent what we see in a patient’s tumour more accurately because our cell lines are derived directly from a large group of patients.

“It’s through our screens of this group of cell lines that we were able to identify the OLIG2 and MEK genes as drug targets for the developmental cell subtype and the FAK and B1-Integrin genes as targets for the injury-response subtype.”

Stéphane Angers, principal investigator on the study and director of the Donnelly Centre, said it had already been established that there are different subtypes of glioblastoma stem cells, but that their differences are not currently being addressed in the clinic.

“In the future, our results will help in designing new treatments that are tailored to patients by targeting the predominant cell subtype, or both subtypes simultaneously,” said Angers, who is also a professor in the Leslie Dan Faculty of Pharmacy and Temerty Faculty of Medicine. “The ability of glioblastoma to adapt to therapeutic treatment is its greatest strength and our biggest challenge. Our study increases our understanding of this type of cancer and proposes a different approach to treating it that will hopefully improve the prognosis of patients.”

This research was supported by the Canadian Institutes of Health Research.

U of T researchers develop AI model to predict 'very dynamic' peptide structures

Christopher.Sorensen — Thu, 15 Aug 2024 12:54:41 +0000

U of T researchers develop AI model to predict 'very dynamic' peptide structures

Christopher.Sorensen Thu, 08/15/2024 - 08:54

Cutline

PhD Graduate Osama Abdin and Professor Philip M. Kim developed a deep-learning model that can predict all possible shapes of peptides, which are are of keen interest to researchers who are developing therapeutics (supplied image)

Anika Hazra

Topic

Breaking Research

Temerty Faculty of Medicine

Donnelly Centre for Cellular & Biomolecular Research

Alumni

Artificial Intelligence

Computer Science

Research & Innovation

Subheadline

The new model expands on the capabilities of Google DeepMind's AlphaFold, the leading AI system for predicting protein structures

Researchers at the ��߲ݴ�ý have developed a deep-learning model that can predict all possible shapes of peptides – chains of amino acids that are shorter than proteins, but perform similar biological functions.

Called PepFlow, the model combines machine learning and physics to model the range of folding patterns that a peptide can assume based on its energy landscape.

Peptides, unlike proteins, are dynamic molecules that can take on a range of conformations. They are involved in many biological processes that are of keen interest to researchers who are developing therapeutics.

“We haven’t been able to model the full range of conformations for peptides until now,” said Osama Abdin, first author on the study and recent PhD graduate of molecular genetics at U of T’s Donnelly Centre for Cellular and Biomolecular Research. “PepFlow leverages deep-learning to capture the precise and accurate conformations of a peptide within minutes.

“There’s potential with this model to inform drug development through the design of peptides that act as binders.”

The study was recently published in the journal Nature Machine Intelligence.

A peptide’s role in the human body is directly linked to how it folds since its 3D structure determines the way it binds and interacts with other molecules.

“Peptides were the focus of the PepFlow model because they are very important biological molecules and they are naturally very dynamic, so we need to model their different conformations to understand their function,” said Philip M. Kim, the study’s principal investigator and a professor at the Donnelly Centre. “They’re also important as therapeutics, as can be seen by the GLP1 analogues, like Ozempic, used to treat diabetes and obesity.”

Peptides are also cheaper to produce than their larger protein counterparts, said Kim, who is also a professor of computer science in U of T’s Faculty of Arts & Science and a professor of molecular genetics in the Temerty Faculty of Medicine.

The new model expands on the capabilities of AlphaFold, the leading Google DeepMind AI system for predicting protein structure. It does this by generating a range of conformations for a given peptide. Taking inspiration from highly advanced physics-based machine learning models, PepFlow can also model peptide structures that take on unusual formations, including the ring-like structure that results from a process called macrocyclization. Peptide macrocycles are currently a highly promising venue for drug development.

“It took two-and-a-half years to develop PepFlow and one month to train it, but it was worthwhile to move to the next frontier beyond models that only predict one structure of a peptide,” Abdin said.

There are, however, limitations given that PepFlow represents the first version of a new model. The study authors noted a number of ways in which PepFlow could be improved, including training the model with explicit data for solvent atoms, which would dissolve the peptides to form a solution, and for constraints on the distance between atoms in ring-like structures.

Yet, even as a first version, the researchers say PepFlow is a comprehensive and efficient model with potential for furthering the development of treatments that depend on peptide binding to activate or inhibit biological processes.

“Modelling with PepFlow offers insight into the real energy landscape of peptides,” said Abdin.

The research was supported by the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada.

U of T researchers develop RNA-targeting technology to precisely manipulate parts of human genes

Christopher.Sorensen — Thu, 08 Aug 2024 18:26:00 +0000

U of T researchers develop RNA-targeting technology to precisely manipulate parts of human genes

Christopher.Sorensen Thu, 08/08/2024 - 14:26

Cutline

From left to right: PhD student Jack Daiyang Li, Professor Benjamin Blencowe and Associate Professor Mikko Taipale (supplied images)

Anika Hazra

Topic

Breaking Research

Temerty Faculty of Medicine

Donnelly Centre for Cellular & Biomolecular Research

Genes

Research & Innovation

Subheadline

“Our new tool makes possible a broad range of applications, from studying gene function and regulation to potentially correcting splicing defects in human disorders and diseases”

Researchers at the ��߲ݴ�ý have harnessed a bacterial immune defence system, known as CRISPR, to efficiently and precisely control the process of RNA splicing.

The technology opens the door to new applications, including systematically interrogating the functions of parts of genes and correcting splicing deficiencies that underlie numerous diseases and disorders.

“Almost all human genes produce RNA transcripts that undergo the process of splicing, whereby coding segments, called exons, are joined together and non-coding segments, called introns, are removed and typically degraded,” said Jack Daiyang Li, first author on the study and PhD student of molecular genetics, working in the labs of U of T researchers Benjamin Blencowe and Mikko Taipale at the Donnelly Centre for Cellular and Biomolecular Research in the Temerty Faculty of Medicine.

Exons from the same gene can be mixed and matched in various combinations to produce different versions of RNA, and consequently, different proteins. This process, called alternative splicing, contributes to the diverse expression of the 20,000 human genes that encode proteins, allowing the development and functional specialization of different types of cells.

However, it is unclear what most exons or introns do and the misregulation of normal alternative splicing patterns is a frequent cause or contributing factor to various diseases, including cancers and brain disorders. In addition, there is a lack of existing methods that allow for the precise and efficient manipulation of splicing.

The new study, published in the journal Molecular Cell, describes how a catalytically deactivated version of an RNA-targeting CRISPR protein, referred to as dCasRx, was joined to more than 300 splicing factors to discover a fusion protein called dCasRx-RBM25. This protein is capable of activating or repressing alternative exons in an efficient and targeted manner.

“Our new effector protein activated alternative splicing of around 90 per cent of tested target exons,” said Li. “Importantly, it is capable of simultaneously activating and repressing different exons to examine their combined functions.”

This multi-level manipulation will facilitate the experimental testing of functional interactions between alternatively spliced variants from genes to determine their combined roles in critical developmental and disease processes.

“Our new tool makes possible a broad range of applications, from studying gene function and regulation, to potentially correcting splicing defects in human disorders and diseases,” said Blencowe, principal investigator on the study, Canada Research Chair in RNA Biology and Genomics, Banbury Chair in Medical Research and a professor of molecular genetics at the Donnelly Centre and Temerty Medicine.

“We have developed a versatile engineered splicing factor that outperforms other available tools in the targeted control of alternative exons,” said Taipale, also principal investigator on the study, Canada Research Chair in Functional Proteomics and Proteostasis, Anne and Max Tanenbaum Chair in Molecular Medicine and associate professor of molecular genetics at the Donnelly Centre and Temerty Medicine. “It is also important to note that target exons are perturbed with remarkably high specificity by this splicing factor, which alleviates concerns about possible off-target effects.”

The researchers now have a tool in hand to systematically screen alternative exons to determine their roles in cell survival, cell-type specification and gene expression.

When it comes to the clinic, the splicing tool has potential to be used to treat numerous human disorders and diseases, such as cancers, in which splicing is often disrupted.

The research was supported by the Canadian Institutes of Health Research and the Simons Foundation.

U of T researchers lead discovery of natural compounds that selectively kill parasites

rahul.kalvapalle — Tue, 21 May 2024 17:46:46 +0000

U of T researchers lead discovery of natural compounds that selectively kill parasites

rahul.kalvapalle Tue, 05/21/2024 - 13:46

Cutline

An international study led by PhD student Taylor Davie (L) and Professor Andrew Fraser (R) of the Donnelly Centre for Cellular and Biomolecular Research could have important implications for treatment of lethal parasitic worms (supplied images)

Anika Hazra

Topic

Breaking Research

Temerty Faculty of Medicine

Donnelly Centre for Cellular & Biomolecular Research

Molecular Genetics

Subheadline

The compounds stall a unique metabolic process that parasitic worms use to survive in the human gut

An international team led by researchers at the ��߲ݴ�ý has found a family of natural compounds that could potentially be harnessed as treatments for parasitic worms, which wreak havoc in developing countries in the tropics.

Infection by these parasites, which are transmitted through soil, leads to malaise, weakness, malnutrition and other debilitating symptoms, and can cause developmental defects and growth impairments in children.

The newly discovered compounds stall the unique metabolic process that the worms use to survive in the human gut, according to the study, which was published in Nature Communications.

“Soil-transmitted parasitic worms infect over one billion people around the world, typically in low-income communities of developing countries without comprehensive health care and infrastructure for sanitation,” said Taylor Davie, first author on the study and a PhD student at U of T’s Donnelly Centre for Cellular and Biomolecular Research. “Parasites are becoming less susceptible to the few anthelmintic drugs available, so there’s an urgent need to find new compounds.”

Many parasitic worm species spend a large portion of their life cycle inside a human host. To adapt to the environmental conditions of the gut, particularly a lack of oxygen, the parasite switches to a type of metabolism that depends on a molecule called rhodoquinone (RQ).

The parasite can survive inside its human host for many months using RQ-dependent metabolism.

The research team chose to target the adaptive metabolic process of the parasitic worm because RQ is only present in the parasite’s system – humans do not produce or use RQ. Therefore, compounds that can regulate the molecule’s production or activity would selectively kill the parasite, with no harm done to the human host.

The researchers conducted a screen of natural compounds isolated from plants, fungi and bacteria on the model organism C. elegans. Although C. elegans is not a parasite, this worm also depends on RQ for metabolism when oxygen is not available.

“This is the first time that we have been able to screen for drugs that specifically target the unusual metabolism of these parasites,” said Andrew Fraser, principal investigator on the study and professor of molecular genetics at the Donnelly Centre and the Temerty Faculty of Medicine.

“The screen was only possible because of recent progress made by our group and others in using C. elegans to study RQ-dependent metabolism, and our collaboration with RIKEN, one of Japan’s biggest research agencies.

“We screened their world-class collection of 25,000 natural compounds, resulting in our discovery of a family of benzimidazole compounds that kills worms relying on this type of metabolism.”

The researchers suggest a multi-dose regimen using the newly discovered family of compounds to treat parasitic worms. While a single-dose treatment is easier to facilitate in mass drug administration programs, a longer treatment program would eliminate the parasite more effectively.

“We are very pleased with the results of the study, which made use of our library,” said Hiroyuki Osada, professor of pharmacy at the University of Shizuoka and group director of the Chemical Biology Research Group at the RIKEN Center for Sustainable Resource Science.

“The study shows the power of the screening approach, allowing researchers in this case to search through a very large number of molecules within a focused collection of natural products. Screens are very efficient, which is key for addressing urgent research questions of global relevance like this one.”

Next steps for the research team are to refine the new class of inhibitors through additional in-vivo testing with parasitic worms, which will be performed by the Keiser lab at the University of Basel in Switzerland, and to continue screening for compounds that inhibit RQ.

“This study is just the beginning,” said Fraser. “We have found several other very powerful compounds that affect this metabolism including, for the first time, a compound that blocks the ability of the worms to make RQ.

“We hope our screens will deliver drugs to treat major pathogens around the world.”

This research was supported by the Canadian Institutes of Health Research and the European Molecular Biology Organization.

U of T researchers' approach to producing neural cells could yield new treatments for Parkinson’s

rahul.kalvapalle — Mon, 13 May 2024 13:03:19 +0000

U of T researchers' approach to producing neural cells could yield new treatments for Parkinson’s

rahul.kalvapalle Mon, 05/13/2024 - 09:03

Cutline

PhD Student Andy Yang, left, and Professor Stephane Angers, right, at the Donnelly Centre for Cellular and Molecular Biology are advancing a novel approach to developing dopaminergic neurons (supplied images)

Anika Hazra

Topic

Breaking Research

Institutional Strategic Initiatives

Temerty Faculty of Medicine

Donnelly Centre for Cellular & Biomolecular Research

Graduate Students

Leslie Dan Faculty of Pharmacy

Medicine by Design

Parkinson's

Research & Innovation

Subheadline

An antibody was used to selectively activate a receptor in a molecular signalling pathway to develop dopaminergic neurons

Researchers at the ��߲ݴ�ý believe they’ve found a way to better control the generation of key neurons depleted in Parkinson’s disease – suggesting a potentially new approach to addressing a disease with no cure and few effective treatments.

In preclinical studies, the researchers used an antibody to selectively activate a receptor in a molecular signalling pathway to develop dopaminergic neurons. These neurons produce dopamine, a neurotransmitter critical to brain health.

While researchers around the world have been working to coax stem cells to differentiate into dopaminergic neurons to replace those lost in patients living with Parkinson’s disease, the efforts have so far been hindered in part by an inability to target specific receptors and areas of the brain.

“We used synthetic antibodies that we had previously developed to target the Wnt signaling pathway,” said principal investigator Stephane Angers, who is director of U of T’s Donnelly Centre for Cellular and Molecular Biology and a professor in the Leslie Dan Faculty of Pharmacy and the Temerty Faculty of Medicine.

“We can selectively activate this pathway to direct stem cells in the midbrain to develop into neurons by targeting specific receptors in the pathway. This activation method has not been explored before.”

Parkinson’s disease is the second-most common neurological disorder after Alzheimer’s, affecting over 100,000 Canadians. It particularly impacts older men, progressively impairing movement and causing pain as well as sleep and mental health issues.

Most previous research efforts to activate the Wnt signaling pathway relied on a GSK3 enzyme inhibitor. This method involves multiple signaling pathways for stem cell proliferation and differentiation, which can have an unintended effect on the newly produced neurons and activate off-target cells.

“We developed an efficient method for stimulating stem cell differentiation to produce neural cells in the midbrain,” said Andy Yang, first author on the study and a PhD student at the Donnelly Centre. “Moreover, cells activated via the FZD5 receptor closely resemble dopaminergic neurons of natural origin.”

Another promising finding of the study, published recently in the journal Development, is that implanting the artificially-produced neurons in a rodent model with Parkinson’s disease led to improvement of the rodent’s locomotive impairment.

“Our next step would be to continue using rodent or other suitable models to compare the outcomes of activating the FZD5 receptor and inhibiting GSK3,” said Yang. “These experiments will confirm which method is more effective in improving symptoms of Parkinson’s disease ahead of clinical trials.”

The research was supported by U of T’s Medicine by Design program, an institutional strategic initiative that receives funding from the Canada First Research Excellence Fund and the Canadian Institutes of Health Research.

Researchers devise new method to find proteins for targeted treatment of disease

Christopher.Sorensen — Tue, 26 Mar 2024 18:12:33 +0000

Researchers devise new method to find proteins for targeted treatment of disease

Christopher.Sorensen Tue, 03/26/2024 - 14:12

Cutline

Visiting Scientist Juline Poirson and Associate Professor Mikko Taipale worked with researchers at Sinai Health to develop a method to interrogate the entire human proteome for “effector” proteins, which can influence the stability of other proteins (supplied images)

Anika Hazra

Topic

Breaking Research

Sinai Health

Temerty Faculty of Medicine

Donnelly Centre for Cellular & Biomolecular Research

Research & Innovation

Subheadline

“Targeting proteins through induced proximity is a new and promising area of biomedical research”

Researchers at Sinai Health and the ��߲ݴ�ý have created a new platform to identify proteins that can be used to control the stability of other proteins – a new and largely unrealized approach to treating diseases.

The researchers developed a method to interrogate the entire human proteome for “effector” proteins, which can influence the stability of other proteins via induced proximity. The approach effectively attempts to hijack cellular processes for therapeutic purposes.

The study marks the first time researchers have searched for effector proteins on this scale, and has identified many new effectors that could potentially be used to develop new drugs.

“We found more than 600 new effector proteins in 14,000 genes,” said Juline Poirson, first author on the study and visiting scientist at U of T’s Donnelly Centre for Cellular and Biomolecular Research. “Over 200 of the new effectors can efficiently degrade their target proteins, while about 400 effectors were capable of stabilizing, and thereby increasing the abundance of, an artificial target protein.”

The study, which involved researchers at Sinai Health’s Lunenfeld-Tanenbaum Research Institute, was published in the journal Nature.

“Targeting proteins through induced proximity is a new and promising area of biomedical research,” said Mikko Taipale, principal investigator on the study and an associate professor of molecular genetics at the Donnelly Centre and in the Temerty Faculty of Medicine. “Not only did we find new effectors worth further investigation for drug discovery, we developed a synthetic platform that can be used to conduct unbiased, proteome-wide, induced-proximity screens to continue expanding the library of effector proteins.”

The effectors currently in use for targeted protein degradation and stabilization are E3 ubiquitin ligases (E3s) and deubiquitinases (DUBs), respectively. E3 is an enzyme that transfers the ubiquitin molecule to the target protein, which essentially flags the protein for a proteosome to digest it. On the other hand, a DUB enzyme removes the ubiquitin tag from a protein, thereby preventing the protein from being recognized and degraded by a proteosome.

The results of the study demonstrate that E3s are quite varied in the degree to which they can degrade target proteins. The research team also discovered four of what they call “angry E3s,” which consistently degrade targets regardless of other factors, such as the location of the target within the cell.

One surprising finding was that some of the strongest effectors for targeted protein degradation were E2 conjugating enzymes, instead of E3s. These differ from E3s in that they are involved at an earlier step of protein degradation and do not directly engage the target protein. Because E2s were not considered to be easily targeted with drugs, they had not been harnessed for protein degradation until recently. They represent, however, the untapped potential of stronger effectors than ones currently in use.

The study demonstrates that exploring the whole proteome for induced proximity offers enormous opportunities for therapeutic interventions.

KLHL40, one of the identified effectors, could potentially be hijacked for targeted protein stabilization to treat skeletal muscle disorders. The research team also found that targeted protein degradation with FBXL12 and FBXL15 effectors could be particularly useful in treating chronic myeloid leukemia.

Targeted protein degradation and stabilization are innovative methods of drug discovery that have thus far been plagued with the “protein pair problem,” where the best effector for a target protein cannot be predicted accurately. Matching a target protein with the right effector is essential to successfully and safely facilitate degradation and stabilization processes in tissues.

“The synthetic screening platform developed by our team solves the protein matching issue through rapid, large-scale testing of effector and target protein interactions,” said Poirson. “We’re confident that an unbiased induced-proximity approach can be used to find effectors for almost any target.”

The research was supported by the David Dime and Elisa Nuyten Catalyst Fund, the Mark Foundation for Cancer Research, the Charles H. Best Postdoctoral Fellowship and the Canadian Institutes of Health Research (CIHR) Fellowship.

Researchers pinpoint issue that could be hampering common chemotherapy drug

Christopher.Sorensen — Mon, 18 Mar 2024 15:00:03 +0000

Researchers pinpoint issue that could be hampering common chemotherapy drug

Christopher.Sorensen Mon, 03/18/2024 - 11:00

Cutline

(photo by Glasshouse Images/Getty Images)

Anika Hazra

Topic

Breaking Research

Temerty Faculty of Medicine

Donnelly Centre for Cellular & Biomolecular Research

Biochemistry

Cancer

Research & Innovation

Subheadline

Study finds two enzymes that work against the chemotherapy drug gemcitabine, preventing it from effectively treating pancreatic cancer

Researchers at the ��߲ݴ�ý’s Donnelly Centre for Cellular and Biomolecular Research have found two enzymes that work against the chemotherapy drug gemcitabine, preventing it from effectively treating pancreatic cancer.

The enzymes – APOBEC3C and APOBEC3D – increase during gemcitabine treatment and promote resistance to DNA replication stress in pancreatic cancer cells.

This, in turn, counteracts the effects of gemcitabine and allows for the growth of cancer cells.

Tajinder Ubhi and Grant Brown (supplied images)

“Pancreatic cancer has proven to be very challenging to treat, as it is usually diagnosed at stage 3 or 4,” said Tajinder Ubhi, first author on the study and a former PhD student in biochemistry in U of T’s Temerty Faculty of Medicine.

“It is the most lethal type of cancer in Canada, with an average survival time of less than two years. While chemotherapy with gemcitabine has increased survival by a few months in clinical trials, options for treatment of pancreatic cancer remain limited.”

The findings were published in the journal Nature Cancer.

Replication stress is the key process by which gemcitabine stops cancer cells from continuing to multiply. It involves the dysregulation of DNA replication, which occurs when cells divide. Replication stress can transform a healthy cell into a cancerous one, but can also be activated within cancer cells to eliminate them.

Gemcitabine has been used for nearly three decades to treat a wide variety of cancers, including pancreatic, breast and bladder cancer. However, a downside of using gemcitabine to target dividing cells is that it can produce toxic side effects in tissues that aren’t being targeted for treatment.

Ubhi and other members of Professor Grant Brown’s lab at the Donnelly Centre have been trying to understand the possible causes of replication stress and its impacts. One way to do this is by studying the stress response mechanisms in cancer cells treated with gemcitabine.

“We conducted a genome-wide CRISPR screen to find genes that could increase the sensitivity of pancreatic cancer cells to gemcitabine,” said Brown, professor of biochemistry at the Donnelly Centre and in the Temerty Faculty of Medicine who is the principal investigator on the study.

“We were excited to identify APOBEC3C and APOBEC3D because other enzymes in the APOBEC3 family can cause cancers to eventually become resistant to treatment. We discovered a more direct role for the enzymes, where they actually protect pancreatic cancer cells from gemcitabine therapy.”

Neither enzyme is naturally found in high concentrations within healthy or cancerous cells. The catch is that the replication stress the drug causes in pancreatic cancer cells in turn triggers an increase in both enzymes. The research team found that removing either APOBEC3C or APOBEC3D kills pancreatic cells by stymieing DNA repair and destabilizing the cell genome.

“What is most exciting is that the removal of just APOBEC3C or APOBEC3D is enough to stop the replication of gemcitabine-treated pancreatic cancer cells,” said Ubhi. “This indicates that the enzymes could be effective new targets for treating this form of cancer.”

The research received support from the Canada Foundation for Innovation, the Canadian Cancer Society, Canadian Friends of the Hebrew University, the Canadian Institutes of Health Research, Cold Spring Harbor Laboratory, the Government of Ontario, the Lustgarten Foundation, the Ministry for Culture and Innovation of Hungary, the U.S. National Institutes of Health, the Northwell Health Affiliation, the Ontario Institute for Cancer Research, Pancreatic Cancer Canada, the Princess Margaret Cancer Foundation, the Simons Foundation, the Terry Fox Research Institute and the Thompson Foundation.

Researchers discover one million new components of the human genome

Christopher.Sorensen — Thu, 08 Feb 2024 19:16:32 +0000

Researchers discover one million new components of the human genome

Christopher.Sorensen Thu, 02/08/2024 - 14:16

Cutline

Timothy Hughes, professor and chair of U of T’s department of molecular genetics in the Temerty Faculty of Medicine, is the principal researcher on a study that found nearly one million new exons, or stretches of DNA that are expressed in mature RNA (photo courtesy of the Donnelly Centre)

Anika Hazra

Topic

Breaking Research

Temerty Faculty of Medicine

Donnelly Centre for Cellular & Biomolecular Research

Genome

Molecular Genetics

Research & Innovation

Subheadline

“We’ve started to chip away at the dark genome by finding nearly one million previously unknown exons through a method called exon trapping”

Researchers at the ��߲ݴ�ý’s Donnelly Centre for Cellular and Biomolecular Research have found nearly one million new exons – stretches of DNA that are expressed in mature RNA – in the human genome.

There are around 20,000 protein-coding genes in humans that contain approximately 180,000 known internal exons. These protein-coding regions account for only one per cent of the entire human genome. The vast majority of what remains is a mystery – aptly referred to as the “dark genome.“

“We’ve started to chip away at the dark genome by finding nearly one million previously unknown exons through a method called exon trapping,” said Timothy Hughes, principal investigator on the study and professor and chair of the department of molecular genetics in U of T’s Temerty Faculty of Medicine.

“The technique involves an assay with plasmids to find exons in DNA fragments of unknown composition,” said Hughes, who holds the Canada Research Chair in decoding gene regulation and the John W. Billes Chair of Medical Research at U of T. “While exon trapping is not widely used anymore, it proved to be effective when used in combination with high-throughput sequencing to scan the entire human genome.”

The findings were published recently in the journal Genome Research.

Exons are segments of the genome that can encode proteins to direct tissue development and biological processes within the body. They are considered to be autonomous if they don’t require external assistance to splice into a mature RNA transcript, which is then translated into a protein.

The team behind the study was driven to test the exon definition model that guides research in molecular genetics after questioning one of its assumptions – that the accurate removal of non-protein-coding intron regions of the genome is aided by clear and consistent indicators of where exons begin and end. This assumption does not seem to hold in all cases as the splicing of exons does not always go smoothly, sometimes resulting in mature RNA transcripts that contain non-functional components.

“Almost none of the newly discovered exons are found consistently across genomes of different species,” said Hughes. “They seem to appear in the human genome mainly due to random mutation and are unlikely to play a significant role in our biology. This is evidence that evolution in humans involves a lot of trial and error – most likely enabled by the vast size of our genome.”

It is helpful to document randomly mutated exons within the human genome as their translation could potentially be harmful. Long non-coding RNA exons, which are autonomous but often have no known function, have been connected to the development of cancer. Of the roughly 1.25 million known and unknown exons the team found through exon trapping, almost four per cent were long non-coding RNA exons.

In addition, the exons residing within non-coding introns, called pseudoexons, can mutate to make a weak splice site stronger. This results in the exon being included in a mature RNA transcript, potentially leading to disease.

“This is an interesting study that broadens our knowledge of sequences across the human genome that have the potential to be recognized as exons in transcribed RNA,” said Benjamin Blencowe, professor of molecular genetics in U of T’s Temerty Faculty of Medicine, who was not involved in the study. “While the significance of the majority of the newly detected exons is unclear, some of them may be activated in certain contexts – for example, by disease mutations – and therefore cataloguing them is important. This study will further serve as a valuable resource facilitating ongoing efforts directed at deciphering the splicing code.”

A stronger understanding of the factors impacting exon inclusion in mature RNA can help improve programs like SpliceAI, a widely used tool for predicting splice sites and aberrant splicing. SpliceAI can be trained on new data such as that produced through this study to refine its prediction capabilities.

“SpliceAI often doesn’t provide details on the characteristics of exons and has a poor ability to predict splicing in exons that aren’t already catalogued,” said Hughes. “Our exon trapping data contains biologically meaningful information that can be fed into SpliceAI and other splicing predictors to open up new paths for exploring the dark genome.”

The research was supported by the Canadian Institutes of Health Research and the U.S. National Institutes of Health.

With heart-on-a-chip, researchers study genetic mutation underlying cardiac muscle disease

Christopher.Sorensen — Tue, 09 Jan 2024 16:44:17 +0000

With heart-on-a-chip, researchers study genetic mutation underlying cardiac muscle disease

Christopher.Sorensen Tue, 01/09/2024 - 11:44

Cutline

Professor Milica Radisic and U of T alumna Marianne Wauchop developed a heart-on-a-chip device to study the effects of a genetic mutation that causes dilated cardiomyopathy (supplied images)

Anika Hazra

Topic

Breaking Research

Institute of Biomedical Engineering

Temerty Faculty of Medicine

Donnelly Centre for Cellular & Biomolecular Research

Faculty of Applied Science & Engineering

Research & Innovation

University Health Network

Subheadline

Device was used to observe the effects of a sodium channel mutation that disrupts regular electrical activity in the heart

Researchers at the ��߲ݴ�ý and its partner hospitals have led the development of a heart-on-a-chip device to study the effects of a genetic mutation that causes dilated cardiomyopathy, a heart muscle disease that impairs blood flow throughout the body.

While it’s been difficult to study cardiac muscle cells due to incomplete tissue maturation in the lab, the U of T-led team was able to use the device to mature stem cells into adult cardiac tissue to observe the effects of a sodium channel mutation that disrupts regular electrical activity in the heart.

“Thanks to our heart-on-a-chip, we were able to overcome the challenges associated with the developmental regulation of the sodium channel mutation,” said Marianne Wauchop, first author of the study and former PhD student in the department of physiology in the Temerty Faculty of Medicine and the department of chemical engineering and applied chemistry in the Faculty of Applied Science & Engineering.

“Using the same device, we were also able to determine the effects of the mutation on cardiac function.”

The journal Biomaterials recently published the findings.

The results could help advance personalized treatment for heart diseases such as dilated cardiomyopathy, a heart condition in which the chambers of the heart lose their ability to contract, through a mutation-targeted approach – even in cases where the mutation is not often linked to the disease or associated symptoms. If left untreated, the condition can result in arrhythmia – and eventually heart failure.

“Currently, patients with dilated cardiomyopathy caused by sodium channel mutations are treated for heart failure, such as with beta blockers and diuretics,” said Milica Radisic, a co-principal investigator on the study and professor at U of T’s Donnelly Centre for Cellular and Biomolecular Research and Institute of Biomedical Engineering. “Responses to these treatment methods are varied because they don’t target the specific mutations responsible for dilated cardiomyopathy.”

The researchers found the mutation disrupts interactions between sodium channels and structural proteins that are critical for cardiac muscle cells, leading to tissue dilation and impaired contractile performance.

The cells that carry the mutation were provided for the study by a patient of Kumaraswamy Nanthakumar, senior scientist in the Toronto General Hospital Research Institute at University Health Network and professor at U of T’s Institute of Medical Science in the Temerty Faculty of Medicine.

The patient went into cardiac arrest while giving birth and has a family history of arrythmia, an irregular heartbeat. Nanthakumar sequenced the patient’s genome to identify the particular sodium channel mutation.

“We were faced with the challenge of culturing stem cells that express the mutation in question, as this only occurs following maturation of the cells,” says Wauchop. “To address this issue, we developed a heart-on-a-chip device, also called a biowire, to mature the heart tissues cultured from stem cells.”

The research team created biowires with either tissues taken directly from Nanthakumar’s patient or tissues in which the mutation was corrected with CRISPR, a gene-editing tool.

While it typically takes a few years for patients to start experiencing symptoms of dilated cardiomyopathy after heart cells carrying the sodium channel mutation mature to their adult form, the biowires allowed the team to study the impacts of the mutation on cardiac tissue after only eight weeks.

Throughout this period, the researchers stimulated maturation of the stem cells through electrical current.

Dilated cardiomyopathy and other heart diseases can be caused by more than one type of mutation, Wauchop notes. But, she adds, “We can give patients a fighting chance by first making the connection between the mutation and the disease, and then developing targeted treatments for that specific mutation.”

This research was supported by the Canadian Institutes of Health Research (CIHR), the Peter Munk Cardiac Centre Innovation Committee and the National Institutes of Health (NIH).

Study reveals genes that set humans apart from other primates in cognitive ability

Christopher.Sorensen — Tue, 05 Dec 2023 16:14:14 +0000

Study reveals genes that set humans apart from other primates in cognitive ability

Christopher.Sorensen Tue, 12/05/2023 - 11:14

Cutline

(Rick Madonik/Toronto Star via Getty Images)

Anika Hazra

Topic

Breaking Research

Temerty Faculty of Medicine

Donnelly Centre for Cellular & Biomolecular Research

Research & Innovation

Subheadline

The researchers found 139 genes that are common across the primate groups but highly divergent in their expression in human brains

An international team led by researchers at the ��߲ݴ�ý has uncovered over 100 genes that are common to primate brains but have undergone evolutionary divergence only in humans – and which could be a source of our unique cognitive ability.

The researchers, led by Associate Professor Jesse Gillis from the Donnelly Centre for Cellular and Biomolecular Research and the department of physiology at U of T’s Temerty Faculty of Medicine, found the genes are expressed differently in the brains of humans compared to four of our relatives – chimpanzees, gorillas, macaques and marmosets.

The findings, published in Nature Ecology & Evolution, suggest that reduced selective pressure, or tolerance to loss-of-function mutations, may have allowed the genes to take on higher-level cognitive capacity. The study is part of the Human Cell Atlas, a global initiative to map all human cells to better understand health and disease.

“This research contributes to our understanding of differences in the brain between humans and other primates at the cellular level, but it has also resulted in a database that can be used to further characterize genetic similarities and differences across primates,” said Gillis.

The team, which includes researchers from Cold Spring Harbour Laboratory and the Allen Institute for Brain Science in the U.S, created a brain map for each primate species based on single-cell analysis, a relatively new technique that enables more specific genetic sequencing than standard methods. They used a BRAIN Initiative Cell Census Network (BICCN) dataset created from samples taken from the middle temporal gyrus of the brain.

In all, the team found 139 genes that are common across the primate groups but highly divergent in their expression in human brains. These genes displayed a stronger ability to withstand mutations without impacting their function, suggesting they may have evolved under more relaxed selective pressure.

“The genes that have diverged in humans must be tolerant to change,” said Hamsini Suresh, first author on the study and a research associate at the Donnelly Centre. “This manifests as tolerance to loss-of-function mutations, and seems to allow for rapid evolutionary change in the human brain.”

Our higher cognitive function may have resulted from the adaptive evolution of human brain cells to a multitude of less threatening mutations over time. It’s also worth noting that around a quarter of the human-divergent genes identified in the study are associated with various brain disorders.

The divergent genes the researchers identified are found in 57 brain cell types, grouped by inhibitory neurons, excitatory neurons and non-neurons. A quarter of the genes were only expressed differently in neuronal cells, also known as grey matter, and half were only expressed differently in glial cells, which are white matter.

Grey matter in the brain consists of neurons, while white matter consists of other cell types, including those responsible for vasculature and immune function.

This study is part of the BICCN initiative to identify and catalogue the diverse cell types in the brains of humans and other species. In 2021, the consortium published a comprehensive census of cell types in the mouse, monkey and human primary motor cortex in the journal Nature. The initiative is shedding light on the evolution of the brain by studying neurotransmission and communication at the finest resolution.

“There are around 570,000 cells in the cross-primate single cell atlas of the middle temporal gyrus,” said Suresh. “Defining a catalogue of shared cell types in this area of the brain provides a framework for exploring the conservation and divergence of cellular architecture across primate evolution. We can use the resulting information to study evolution and disease in a more targeted manner.”

This research was supported by the U.S. National Institutes of Health and the U.S. National Alliance for Research on Schizophrenia and Depression.