What's cooking?
As the title goes, these are snippets of some cool science I am working on!
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 (criminally underrated, I mean, under-investigated locus despite evolutionary conservation) that localizes to and interacts with proteins in the endoplasmic reticulum to regulate ER stress response in cancer cells. Read more here!
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 (criminally underrated, I mean, under-investigated locus despite evolutionary conservation) that localizes to and interacts with proteins in the endoplasmic reticulum to regulate ER stress response in cancer cells. Read more here!
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!
A novel role for KMT2D in pancreatic adenocarcinoma
PDAC is a severe form of pancreatic cancer with a low five-year survival rate. PDAC has well-established highly recurrent mutations in four driver genes: oncogene KRAS, and tumor suppressors TP53, CDKN2A and SMAD4. However, barring KRAS, these genetic drivers are not currently therapeutically actionable. Decades of research into therapeutic targeting of these genetic drivers as well as other targets in PDAC have often been unsuccessful in clinical settings. So we aimed to shift our focus on non-genetic drivers of the disease, in particular, epigenetic players like histone methyltransferases.
KMT2D belongs to the 6-member histone-lysine N-methyltransferase 2 (KMT2) family that catalyzes the methylation of H3K4 to promote genome accessibility and subsequent transcriptional activation. KMT2D carries the highest incidence of pathogenic mutations in PDAC cases.
In our study, we assessed the biological role of KMT2D in two independent genetically engineered mouse models driven by mutant Kras in the presence and absence of the tumor suppressor Tp53.
The loss of Kmt2d increased tumor incidence with a poorly differentiated phenotype, reducing survival in both models even in absence of Tp53. Similarly, in human PDAC, the loss of TP53 does not affect the survival of KMT2D low cases. Kmt2d depletion alone was not sufficient to impact pancreatic development and survival. Of note, inactivation of Kmt2d resulted in increased levels of Ki67 positive cells in vivo.
This study highlights a novel role for KMT2D in Kras-driven PDAC as a candidate tumour suppressor. Read more about the study here!
KMT2D belongs to the 6-member histone-lysine N-methyltransferase 2 (KMT2) family that catalyzes the methylation of H3K4 to promote genome accessibility and subsequent transcriptional activation. KMT2D carries the highest incidence of pathogenic mutations in PDAC cases.
In our study, we assessed the biological role of KMT2D in two independent genetically engineered mouse models driven by mutant Kras in the presence and absence of the tumor suppressor Tp53.
The loss of Kmt2d increased tumor incidence with a poorly differentiated phenotype, reducing survival in both models even in absence of Tp53. Similarly, in human PDAC, the loss of TP53 does not affect the survival of KMT2D low cases. Kmt2d depletion alone was not sufficient to impact pancreatic development and survival. Of note, inactivation of Kmt2d resulted in increased levels of Ki67 positive cells in vivo.
This study highlights a novel role for KMT2D in Kras-driven PDAC as a candidate tumour suppressor. Read more about the study here!
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 our subsequent study, 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 our subsequent study, 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.