Kyle Cottrell

Our second preprint is out today! Post thread to come later.

So, when this gets packaged into an exosome, is it a virus? I'm only being a little sarcastic.

A moment of beauty in the garden provides a brief distraction from the slow death of science in this country.

That image is from the 2022 NCI budget request. Weren't they optimistic?

This isn't even close to the historical norm.

I wonder if tenure committees and university administration will consider this when making tenure/promotion decisions?

This is terrible, and unfortunately, based on my experience sharing this information with others, many in academia (especially more senior faculty) aren't even aware of this change.

A lot of them are non-specific.

I'm team 'immunoblot' - at least in writing.

Cottrell Lab in front of a panda sign at San Diego Zoo.

I treated the lab to a trip to the San Diego Zoo to celebrate our first preprint and for the members attending their first conference, surviving #RNA25. While the zoo is famous for the pandas, the koalas were my favorite!

This evening, the newest Cottrell Lab graduate student, Cassandra Smoak, will be presenting poster 402. Over the last few months she has been evaluating a recently published ADAR1 inhibitor - ZYS-1. I'll let her tell you if it is a good inhibitor or not... #RNA25

Purdue Biochemistry and @cancer-inst-purdue.bsky.social at #RNA25

PACT prevents aberrant activation of PKR by endogenous dsRNA without sequestration - PubMed The innate immune sensor PKR for double-stranded RNA (dsRNA) is critical for antiviral defense, but its aberrant activation by cellular dsRNA is linked to various diseases. The dsRNA-binding protein P...

pubmed.ncbi.nlm.nih.gov/40199855/, pubmed.ncbi.nlm.nih.gov/40185749/, pubmed.ncbi.nlm.nih.gov/40280134/ 15/n

I'd be much more excited about this project if we hadn't gotten scooped by multiple publications in the last month or so (links below). While that is annoying, I'm still proud of this manuscript! Thanks to support from Purdue Biochemistry, @cancer-inst-purdue.bsky.social, and the NIH. 14/n

So, we think PACT could be a good therapeutic target for TNBC. There is one big problem though, PACT isn't an enzyme. However, since PACT dimerization is required, it may be possible to ‘inhibit’ PACT by blocking its dimerization. 13/n

Graph of PACT dependency score in BRCA subtypes

Correlation between PACT dependency score and PKR protein abundance

PKR RNA expression in human tumors

So, we found that PACT suppresses PKR activation through dsRNA binding and dimerization, and is partially redundant with ADAR1 in some cells. Why is this important? PACT is essential in many TNBC cell lines. It is overexpressed in TNBC and so is PKR (which is a good marker for PACT-dependency). 12/n

Multiple figures showing that overexpression of ADAR1 isoforms partially rescued PACT depletion phenotypes.

Interestingly (for the ADAR1 aficionados at least) we found that overexpression of both ADAR1 isoforms partially rescued PKR activation and cell death. This is something we really want to study further. 11/n

Immunoblot indicating activation of PKR in PACT and ADAR1 depleted cells

We tested this by depleting both PACT and ADAR1 - which caused robust activation of PKR. Indicating that PACT and ADAR1 function redundantly (in some cells) to inhibit PKR. To further evaluate this, we asked if overexpression of ADAR1 isoforms could rescue the phenotypes of PACT depletion. 10/n

The requirement of PACT dsRNA binding for PKR inhibition is something that PACT has in common with ADAR1. ADAR1 also suppresses PKR activation by binding to endogenous dsRNAs. With that in mind, we hypothesized that in PACT-independent cells ADAR1 compensates for loss of PACT. 9/n

Multiple panels showing that dsRNA binding is required for inhibition of PKR by PACT.

Multiple panels showing that dimerization is required for inhibition of PKR by PACT

We used a series of knockout-rescue experiments to evaluate how PACT prevents PKR activation. These experiments revealed that dsRNA binding and dimerization of PACT is required to suppress PKR activation. The dimerization requirement is important - keep reading. 8/n

Dot plot of GSEA results for RNA-seq from PACT depleted or control cells

Multiple panels showing that PKR is required for PACT depletion phenotypes

We did, however, observe activation of two pathways known to be activated by PKR: the integrated stress response (ISR) and NF-kB. Through a double-KO experiment we found that activation of both pathways (and cell death) in PACT-KO cells was dependent on PKR. 7/n

Immunoblots indicating PKR activation in PACT depleted cells

Instead, we found the exact opposite. When we depleted PACT in PACT-dependent cells we observed robust activation of PKR and cell death. The same phenotypes were not observed in PACT-independent cell lines. Markers for activation of other dsRNA sensors were unchanged. 6/n

Immunoblot indicating that PACT overexpressing doesn't cause PKR activation.

PACT is a dsRNA binding protein (dsRBP), that like ADAR1 binds with dsRNA via dsRNA binding domains (dsRBD) - and has no enzymatic domains. PACT was originally identified as an activator of PKR, but we found no evidence that PACT overexpression caused PKR activation. 5/n

In addition to studying ADAR1, my lab has also been interested in identifying other proteins that function as suppressors of dsRNA sensing. The correlation between ADAR1 dependency score and PACT dependency scores suggested that they have overlapping functions. 4/n

ADAR1 prevents sensing of endogenous dsRNA by dsRNA sensors MDA5 and PKR. This role is essential in many cancer cell lines, ~half of TNBC. Depleting ADAR1 in those cells induces ‘viral mimicry’, a phenotype that has potential for cancer therapeutics. 3/n

Volcano plot of Pearson correlation statistics for pairwise comparisons of gene dependency scores with ADAR1 dependency score

This project started by analysis of public DepMap data. As a postdoc studying ADAR1, I noticed that cellular sensitivity to ADAR1 loss (ADAR1-dependency) correlates with PACT-dependency. I didn't have the time or resources to study this further then, but that changed when I started my own lab. 2/n

PACT suppresses PKR activation through dsRNA binding and dimerization, and is a therapeutic target for triple-negative breast cancer Triple-negative breast cancer (TNBC), the deadliest breast cancer subtype, lacks broadly applicable targeted therapies. Induction of viral mimicry by activation of viral double-stranded RNA (dsRNA) se...

Just in time for #RNA25, the Cottrell Lab has posted our first independent preprint to bioRxiv. This is the first first-author publication from graduate student Addison Young and the first publication for the four undergraduate co-authors.
www.biorxiv.org/content/10.1... 1/n

I haven't had time to make a post thread for this, but if you are at #RNA25, the first-author Addison Young is presenting a poster on this work tonight - P1-108.

This is our first preprint! The post thread is coming later today.

The Cottrell Lab (minus the undergrads) is on our way to RNA25!