Paper Review – Selective cell death mediated by small conditional RNAs

Alright then. This is the second paper review I’m writing for the blog. This details a novel method of killing cancer cells selectively. Here, only cancer cells that express a particular gene aberrantly will be killed off through the activation of an innate immune response. This paper details an in vitro study carried out using cell cultures.

You may find it useful to have a copy of the paper handy, and you may find the PDF available from PNAS here

In this paper, the authors elaborate upon a method that uses the physical properties of RNA molecules to drive a chain reaction involving generation of an active molecule of RNA which then goes on to kill cancer cells by apoptosis through the activation of the PKR pathway.


Please note that you will be able to bring up bigger versions of the images you see by clicking on them.

The Actors of the Piece.

PKR (or Protein Kinase R) is a protein that is involved in an immune response to the presence of viral double stranded RNA (dsRNA), it triggers inhibition of protein synthesis and apoptosis when it dimerizes (two molecules of this combine to form one unit) in association with double stranded RNA that is longer than 30 base pairs.

Small Conditional RNA (or scRNA) – These molecules are designed to stay inactive in which a cancer-specific marker is absent, however, when such a marker is present (this shall be referred to as the cognate marker from now on) they will react to form a long double stranded RNA sequence which will go on to activate PKR mediated apoptosis.

The molecules they used in the paper have a 4 base loop region and a 14 base pairs long duplex region, forming a hairpin shaped RNA molecule. These molecules by themselves are incapable of activating PKR signalling. There are two of these molecules (labelled scRNA A and scRNA B from now on) that work in concert. These molecules have a diagnosis domain and a treatment domain. The former part of the molecule binds to the cognate marker and the latter is involved in PKR activating dsRNA polymer formation.

Cognate Marker – This is a sequence of RNA that is expressed only in cancer cells. It is capable of converting normally inactive scRNA to active scRNA. In other words, the activation of the therapeutic pathway in question is selective based on the expression of this marker.

The Script of the Play.

I wish to introduce a diagram from the paper at this juncture.

Graphical Summary of the Process.

I now think we can move on to discuss specifics of the conditionality involved and the mechanics of action.

Case One – Normal Cells where cognate marker is absent.

Both scRNA A and B stay in hairpin configuration through complementary self-pairing. This leaves them unable to trigger PKR mediated apoptosis. This keeps normal cells that do not express the cognate marker safe.

Case Two – Cancer Cells where cognate marker is present.

Here things change rapidly, and this is what happens.

[1] The diagnosis domain on scRNA A binds to a complementary region on the cognate marker. This opens the hairpin structure of scRNA B up into two reactive arms, to one of which the marker binds, the other which is exposed,.

[2] A region complementary to scRNA A on scRNA B then binds complementarily to scRNA A, the other arm is left exposed.

[3] Enzymes cleave the single stranded protruding bit, producing a molecule of dsRNA that is sufficiently long to trigger PKR mediated apoptosis.

[4] PKR dimerizes in complex with the dsRNA that is formed.

[5] Apoptosis is triggered and the cancer cell commits hara-kiri.

Continued expression of the cognate marker triggers continued PKR activation and this can be critical in the chain reaction that is a feature of the work presented.

HCR Design Constraints.

The authors note that currently the design is limited to mutant markers that result from translocation (that is, one sequence finds itself fused with another sequence). This means that the fusion point has to be included in any cognate marker thus defined to ensure that the reaction is triggered specifically in cancer cells.

Constraints on choice of Cognate Marker - An Illustration.

So once a cancer specific marker is picked, scRNA A & B can be designed using knowledge of sequence and base pairing, and once all the actors are in place the script is good to be enacted, and this happens as described above.

The study per se – Highlights.

[1] They created three sets of scRNA corresponding to three markers and then checked if polymerisation would only take place if the markers were present using polyacrylamide electrophoresis (PAGE) which basically separates molecules by size, they found that complexes (large and thus less mobile during PAGE) were formed only when the requisite markers were present.

The markers and the cancer cell lines that they were specific to were

i) Δegfr fusion in the glioblastoma cell line U87MG-ΔEGFR , now this case is interesting because we don’t have the fusion of segments from two different genes, but the fusion of two segments of the same gene which would normally be separated by intervening segments that are deleted in this case.

ii) tpc/hpr fusion in prostrate cancer cell line LNCaP , this translocation involves two genes.

iii) ews/fli1 fusion in TC71, which is an Ewing’s Sarcoma Cell Line.

[2] They mutated scRNA molecules and ensured that them having to hybridize was absolutely essential for HCR and PKR mediated apoptosis to take place. They also ensured that having scRNA A & B in normal cells wasn’t enough to trigger apoptosis, since this is essential for selective killing of cancer cells.

[3] They verified the efficacy of the HCR strategy in cell cultures of the types mentioned in highlight [1] using flow cytometry, both in untreated cells and cells that survived the first round of treatment. It is now time to move on to the results.

Results.

I wish to introduce two graphs from the paper at this juncture.

Results of targeting previously untreated cell lines with scRNA, from the original paper.

Self-explanatory, results from experiment treating cells that survived the first round of treatment.

They found that there is a 20 to 100 fold reduction in populations of the treated cells through PKR mediated apoptosis, they confirmed in the end that this pathway was responsible by blocking it using a chemical inhibitor, which prevented scRNA treatment from having an effect on cancer cells, thus conclusively establishing that PKR mediated apoptosis was involved.

I think this is an elegant method that could be part of therapies in the future. I also happen to think that with the advent of sequencing technology et cetera, personalized medicine using this approach could become reality. At the same time the high rate of mutation in genes of cancer cells may very well make the evolution of resistance possible through the non-complementarity of the cognate marker with the scRNA molecules in question, so that is a challenge that may well crop up.

For more information, I suggest that you read the paper yourself and have fun in the process.

That is all from me about this paper.

– Ankur.

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