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\begin{document}

\title{PrePrint Journal Club Review: Small-molecule targeting of MUSASHI
RNA-binding activity in acute myeloid~ leukemia~~~~}



\author[1]{David Dilworth}%
\author[1]{Rachel Harding}%
\author[1]{Kirsten Hart}%
\author[1]{Jolene Ho}%
\author[2]{Danton Ivanochko}%
\author[1]{Genna Luciani}%
\author[2]{Mandeep Mann}%
\author[1]{Lea Melin }%
\author[1]{Panagiotis Prinos}%
\affil[1]{Affiliation not available}%
\affil[2]{University of Toronto}%


\vspace{-1em}



  
  \date{May 25, 2024}


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Title: Small-molecule targeting of MUSASHI RNA-binding activity in acute
myeloid leukemia.

Authors: Gerard Minuesa, Steven K Albanese, Arthur Chow, Alexandra
Schurer, Sun-Mi Park, Christina Z. Rotsides, James Taggart, Andrea
Rizzi, Levi N. Naden, Timothy Chou, Saroj Gourkanti, Daniel Cappel,
Maria C Passarelli, Lauren Fairchild, Carolina Adura, Fraser J Glickman,
Jessica Schulman, Christopher Famulare, Minal Patel, Joseph K Eibl,
Gregory M Ross, Derek S Tan, Christina S Leslie, Thijs Beuming, Yehuda
Goldgur, John D Chodera, Michael G Kharas

Date of submission to bioRxiv: May 14, 2018

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Dear Authors,

Thank you for posting your manuscript titled \emph{Small-molecule
targeting of MUSASHI RNA-binding activity in acute myeloid leukemia} as
a preprint on bioRxiv! We reviewed this work at our journal club at the
Structural Genomics Consortium, University of Toronto. Compiled comments
from the attendants are below. To structure the feedback, we used the
quick worksheet guidelines published on PREreview.

\par\null

We hope this feedback will be useful to improve the manuscript.

\par\null

Kind regards,~

\par\null

PreReview Journal Club ~Members, Structural Genomics Consortium,
University of Toronto

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\textbf{What is the main question the study attempts to answer?}

\begin{itemize}
\tightlist
\item
  Can chemical inhibition of Musashi2's RNA-binding function be used to
  selectively target myeloid leukemia cells?
\item
  How does Ro 08-2750 inhibit MSI RNA-binding activity?
\end{itemize}

\textbf{What is (are) the hypothesis?}

Small molecule antagonism of the RNA binding domain of MSI2 has
therapeutic potential in the treatment of acute myeloid leukemia.
Ro-08-2750 binds at an RNA-interacting site and competitively inhibits
RNA binding of MSI2.

\textbf{What techniques/analyses do the researchers adopt to test their
hypothesis(es)?}

The authors use biophysical assays, computational structural biology,
cell biology techniques, and an animal model. These methods are all
common to preclinical drug development and are appropriate to address
the outlined hypothesis and in most cases adequately address the
question being asked.

\textbf{Why is this study relevant?}

Understanding of RNA biology and its importance in human health has
expanded greatly in recent years. While proteins with RNA binding
domains have been implicated in disease, they are typically thought of
as ``undruggable'' as they often lack defined pockets. MSI2
overexpression is common in AML patients with poor clinical prognosis.
This study highlights that (1) RRM domains can be targeted by a small
molecule antagonist and (2) that this approach may have therapeutic
value in the treatment of AML.

\textbf{Write here any general comments you might have about the
research approach.}

\textbf{} \emph{Structural studies}

\begin{itemize}
\tightlist
\item
  The structure-activity-relationship was clearly described considering
  a lack of a co-crystal structure.
\item
  Given that residues F66, F97 and R100 of MSI2 are also conserved in
  MSI1, it would have been interesting to see how Ro 08-2750 compares
  between both proteins.~
\end{itemize}

\emph{Biophysical Assays}

\begin{itemize}
\tightlist
\item
  It would be interesting to see measurements of compound binding to
  MSI2, for example ITC or thermal shift assays.
\end{itemize}

\emph{Cell Biology}

\begin{itemize}
\tightlist
\item
  The assays used appeared to be well thought out and provided good
  evidence for target engagement and therapeutic potential in cellular
  models of AML. However, given the genetic diversity of AML, it would
  be informative to include a sentence to describe why MOLM13 and K562
  cells were selected for these studies.
\end{itemize}

\textbf{Write here any specific comment you might have about
experimental approaches and methods used in the study.}

\begin{itemize}
\tightlist
\item
  In the discussion the authors state that Ro 08-2750 is the first
  ``selective MSI inhibitor''. MSI2 was compared to SYNCRIP for
  selectivity; however, given the extensive repertoire of RNA binding
  domains found in the human genome, we feel that a more extensive
  characterization is warranted to make a definitive statement. This
  could be accomplished by either utilizing a biotinylated compound for
  pulldowns and MS-id or by measuring binding to a panel of RRMs.
\item
  It would be interesting to see KD/KO studies alongside compound
  treatment to evaluate to what degree this compound may phenocopy
  genetic perturbation of MSI2 in AML. In addition, it would also be
  cool to see how the mutants deficient in Ro binding are functional in
  cells, which may hint at potential mechanisms of resistance.
\item
  For the mouse data presented it would be informative to the reader to
  include both survival and tumour volume data. Additionally, have the
  authors done any experiments to test the suitability of this compound
  for in vivo use (ex. What is the PK of Ro?).
\item
  Additional biophysical assay to measure direct binding of the compound
  to MSI2's RRM domain would be informative, ex. ITC or thermal shift
  assay.
\end{itemize}

\emph{Structural studies}

\begin{itemize}
\tightlist
\item
  We found that the molecular dynamics analysis greatly complemented the
  structural data and enabled a thorough description of the potential
  binding mechanism for the Ro compounds. It would be useful to note
  whether co-crystallization of MSI2 and Ro was attempted, though the
  mutagenesis experiments provided evidence of the importance of
  interactions with F66, R100, and F97. It is unclear why YANK was
  selected to perform alchemical analysis given that the program has not
  yet been extensively validated and carries a ``Use at your own risk!''
  warning. It would have been reassuring to see these calculations
  carried out with a more validated software.
\end{itemize}

\textbf{Write here any specific comment/note about figures in the paper
(this could be related to the way data are displayed and your ability to
understand the results just by looking at the figures).}

\begin{itemize}
\tightlist
\item
  Extended figure 7 appears to be mislabeled in text and extended figure
  8 is missing.
\item
  For figure 3d, we felt it would be easier to interpret if annexin V +
  cells were normalized to the DMSO control.
\item
  For the RNA-IP experiments, we would suggest that this data be
  presented as \% input, akin to a ChIP experiment. ~This would account
  for any changes in gene expression that may confound the
  interpretation of this result as well as allow the authors to probe
  RNAs that should not change in response to compound (ie. negative
  control)
\end{itemize}

\textbf{Write here any additional comment you might have (this includes
minor concerns such as typos and structure of the manuscript).}

\begin{itemize}
\tightlist
\item
  On line 216: what does ``proximal'' mean here?
\item
  Classically, the term inhibitor would be reserved for a compound
  disrupting an enzymatic activity. Here\textbf{,} antagonist may be a
  more appropriate term.
\item
  Any supplementary tables listed in the text should be included for
  readers/reviewers. \textbf{}
\end{itemize}

~

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