What's cooking?
As the title (wise words of my postdoc supervisor) says, these are snippets of some cool science I am working on!
GFP-MP fusion library to identify and characterize functional microproteins
A new strategy to screen for functional microproteins, where we fuse microproteins from a curated library of short open reading frames to GFP (which could help stabilize them, therefore making them easier to study).
What does this help us achieve? The Elsässer lab has several methods in our toolkit that we have deployed in this study, including GFP pulldowns to elucidate the proteomic interactome of microproteins, and high-throughput imaging to assess microprotein subcellular localization, all of which would help us parse out functional significance of these microproteins. Watch this space for more!
A large-scale CRISPR-Cas9 screen identifies significant microproteins in cancer
Using a high-throughput CRISPR-Cas9 knockout screen targeting a large pool of nearly 12,000 sORFs, we aimed to determine if microproteins could play a role in cancer cell fitness.
Our study revealed that microprotein candidates as small as 13 amino acids could have a significant impact on cancer cell viability when knocked down (how cool is that?!?!).
Our study revealed that microprotein candidates as small as 13 amino acids could have a significant impact on cancer cell viability when knocked down (how cool is that?!?!).
In addition to identifying these microproteins, we further used GFP-microprotein fusion constructs to determine where they localize within the cell and proteins they interact with, to parse out functional significance for our candidates of interest.
While our study highlights a high-throughput pipeline for the screening and functional characterization of putative sORFs, there are several technical limitations in the field that still need addressing, such as the use of GFP fusions to study microproteins rather than untagged versions, which we also discuss in detail. Read more about these microproteins here!
While our study highlights a high-throughput pipeline for the screening and functional characterization of putative sORFs, there are several technical limitations in the field that still need addressing, such as the use of GFP fusions to study microproteins rather than untagged versions, which we also discuss in detail. Read more about these microproteins here!
PIPPI: a microprotein that helps cancer cells overcome ER stress
Could large-scale screens help us identify and characterize functional microproteins (i.e. proteins derived from short open reading frames of less than 100 codons)? At the Elsässer lab, we aimed to find out.
In our study, we used pooled overexpression screens using a curated library of over 11,000 sORFs and then performed phenotypic screens to discover microproteins that confer resistance to cancer cells undergoing selective pressure (in this case, treatment with nucleotide analogue 6-thioguanine). Did we find anything of note? Yes we did! Here, we describe PIPPI, a microprotein from the Morpheus gene cluster that localizes to and interacts with proteins in the endoplasmic reticulum to regulate ER stress response in cancer cells. Read more here!
KMT2D, an epigenetic regulator of 5-fluorouracil drug response in PDAC
Despite decades of research into pancreatic cancer, we have not seen any significant improvements in patient survival. We have identified genetic drivers and mutations associated with PDAC, for which many systemic and targeted therapies have been developed. Yet these therapies fail when taken to the clinic. So, it stands to reason that there is more to PDAC progression and drug response besides genetic drivers, such as epigenetic drivers.
I focused on the MLL/KMT family of histone methyltransferases, in particular, KMT2D, which is mutated in approximately 15% of PDAC patients (thus comprising a decent subset of patients, and highlighting the clinical significance of this mutation).
In my thesis, I was able to demonstrate that even a heterozygous mutation in KMT2D induces resistance to 5-fluorouracil in PDAC cells.
I focused on the MLL/KMT family of histone methyltransferases, in particular, KMT2D, which is mutated in approximately 15% of PDAC patients (thus comprising a decent subset of patients, and highlighting the clinical significance of this mutation).
In my thesis, I was able to demonstrate that even a heterozygous mutation in KMT2D induces resistance to 5-fluorouracil in PDAC cells.
In KPC animal models, we saw poor survival rates and low tissue differentiation, which is directly correlated to worse prognosis and survival. With bulk sequencing techniques like RNA-sequencing and ChIP-sequencing, I determined that this differential chemotherapy response to 5-FU is induced by changes in the expression of genes involved in the 5-FU metabolic pathway.
How is this relevant to PDAC though? Understanding drug resistance mechanisms, particularly for drugs like 5-FU that’s commonly used in the clinic, helps us stratify patients as either responders or non-responders depending on their KMT2D mutational status, or expression of 5-FU metabolic genes, thereby personalizing PDAC treatment and improving patient survival outcomes, which is crucial for a disease in which the 5-year survival rate has not exceeded 10% in four decades, making it crucial that we administer the right drugs to patients.
Read more about my findings on KMT2D and 5-FU response in my thesis, and more on KMTs in my review article here.
How is this relevant to PDAC though? Understanding drug resistance mechanisms, particularly for drugs like 5-FU that’s commonly used in the clinic, helps us stratify patients as either responders or non-responders depending on their KMT2D mutational status, or expression of 5-FU metabolic genes, thereby personalizing PDAC treatment and improving patient survival outcomes, which is crucial for a disease in which the 5-year survival rate has not exceeded 10% in four decades, making it crucial that we administer the right drugs to patients.
Read more about my findings on KMT2D and 5-FU response in my thesis, and more on KMTs in my review article here.
Modeling adaptive therapy in PDAC and CRC
Cancer through the lens of evolution is essentially assessing cancer through time and space. You heard that right folks! When this concept was pitched by a clinical fellow in my PhD lab, I was unsure about what that entailed.
However, the more I dove into the field, the more obvious this approach became, particularly for treatment recalcitrant diseases like PDAC and CRC. Despite significant research in the field and countless drugs, we are nowhere close to improving patient survival.
GI cancer (PDAC, CRC), like many other cancers, is an eco-evolutionary process. Cancer cells evolve over time in response to their microenvironment, other competing cell populations, space, nutrient availability, applied stress like chemotherapy, to name a few factors. In understanding these principles and how they apply to cancer, we can optimize treatment for patients. I developed PDAC and CRC spheroids with which I could model cellular dynamics within the tumour (drug sensitive and resistant cells). With this model, I could alter cell:cell ratios, apply different chemotherapeutic agents, and monitor cell growth over time. To dive into the eco-evolutionary landscape of PDAC, read our review and abstract.
GI cancer (PDAC, CRC), like many other cancers, is an eco-evolutionary process. Cancer cells evolve over time in response to their microenvironment, other competing cell populations, space, nutrient availability, applied stress like chemotherapy, to name a few factors. In understanding these principles and how they apply to cancer, we can optimize treatment for patients. I developed PDAC and CRC spheroids with which I could model cellular dynamics within the tumour (drug sensitive and resistant cells). With this model, I could alter cell:cell ratios, apply different chemotherapeutic agents, and monitor cell growth over time. To dive into the eco-evolutionary landscape of PDAC, read our review and abstract.
Sex-specific neurobehavioural toxicity of endocrine disruptors
Endocrine disruptors have recently become a buzzword on social media. Everyone knows to look out for these chemicals, which are persistent in our daily life. While most regulatory agencies set limits and controls on usage of these chemicals, we theorized that EDCs could have a significant impact on our biology even at sub-lethal doses, especially when the exposure occurs during gestation.
We studied the effect of EDCs like bisphenol-A and methylparaben using zebrafish larvae and rat pups. In our first study, using zebrafish larvae, we were able to easily visualize early developmental deformities such as heart rate (which we counted using a microscope!) and morphological defects. We also developed customized tests to assess behavioural outcomes in F1 offspring, more of which you can read about here.
In another study right after, we graduated to animal models (specifically, rats) and assessed in utero exposure to sub-lethal doses of bisphenol-A, with the hypothesis that any potential behavioural and biochemical alterations we observe would be sex-specific. We noted an increase in anxiety-like behaviour that was specific to male offspring (so it does look like these effects are sex-specific!!). We also saw differential expression in BDNF and CYP19A1 in male and female offspring, further validating our hypothesis that EDC exposure could have sex-specific outcomes on offspring. More about this exciting study here.
In another study right after, we graduated to animal models (specifically, rats) and assessed in utero exposure to sub-lethal doses of bisphenol-A, with the hypothesis that any potential behavioural and biochemical alterations we observe would be sex-specific. We noted an increase in anxiety-like behaviour that was specific to male offspring (so it does look like these effects are sex-specific!!). We also saw differential expression in BDNF and CYP19A1 in male and female offspring, further validating our hypothesis that EDC exposure could have sex-specific outcomes on offspring. More about this exciting study here.