Virally programmed extracellular vesicles sensitize cancer cells to oncolytic virus and small molecule therapy

Recent advances in cancer therapeutics clearly demonstrate the need for innovative multiplex therapies that attack the tumour on multiple fronts. Oncolytic or “cancer-killing” viruses (OVs) represent up-and-coming multi-mechanistic immunotherapeutic drugs for the treatment of cancer. In this study, we perform an in-vitro screen based on virus-encoded artificial microRNAs (amiRNAs) and find that a unique amiRNA, herein termed amiR-4, confers a replicative advantage to the VSVΔ51 OV platform. Target validation of amiR-4 reveals ARID1A, a protein involved in chromatin remodelling, as an important player in resistance to OV replication. Virus-directed targeting of ARID1A coupled with small-molecule inhibition of the methyltransferase EZH2 leads to the synthetic lethal killing of both infected and uninfected tumour cells. The bystander killing of uninfected cells is mediated by intercellular transfer of extracellular vesicles carrying amiR-4 cargo. Altogether, our findings establish that OVs can serve as replicating vehicles for amiRNA therapeutics with the potential for combination with small molecule and immune checkpoint inhibitor therapy.

Editorial Note: This manuscript has been previously reviewed at another journal that is not operating a transparent peer review scheme. This document only contains reviewer comments and rebuttal letters for versions considered at Nature Communications. Mentions of prior referee reports have been redacted. Reviewer #2: Remarks to the Author: The authors have done an admirable job of responding to the reviewer concerns and the revised manuscript is much more organized and clear to read. I have no further concerns and believe this work will be of interest to those in the field.
Reviewer #3: Remarks to the Author: I appreciate that the authors addressed majority of the concerns that I raised in the first round of revision. The experimental approach, execution and data presentation are satisfactory. However, while skimming through the figures, I found many typing and grammatical mistakes that need to be addressed prior to publication. Please proof-read the manuscript to make sure that figure legends correspond to right data. 1. Colony formation assay results ( Figure  We thank the reviewer for the time and effort invested into the review of our manuscript, and for the helpful comments and suggestions.
The authors have done an admirable job of responding to the reviewer's concerns and the revised manuscript is much more organized and clear to read. I have no further concerns and believe this work will be of interest to those in the field.
We thank the reviewer for the time and effort invested into the review of our manuscript, and for the helpful comments and suggestions.

Reviewer #3 (Remarks to the Author):
I appreciate that the authors addressed the majority of the concerns that I raised in the first round of revision. The experimental approach, execution and data presentation are satisfactory. However, while skimming through the figures, I found many typing and grammatical mistakes that need to be addressed prior to publication. Please proof-read the manuscript to make sure that figure legends correspond to right data.
We thank the reviewer for the time and effort invested into the review of our manuscript, and for the helpful comments and suggestions. We have extensively edited and proofread the manuscript. We have also carefully verified that the figure legends correspond to the right data. Figure 2C-h): Some of the wells in the figures are highly saturated with blue color. Can we rely on the quantification of those wells where the colonies are not even visible? Software-based analysis of such over-saturated wells is also not accurate.

Colony formation assay results (
The data shown in Figure 2c-h is not derived from colony-forming assays, we apologize if we were not clear in the Figure legend. The data shown in these figures are crystal violet cytotoxicity assays performed as detailed in the Methods section. Crystal violet cytotoxicity assay is one of the methods commonly used to detect virus or drug cytotoxicity. We recognize that we did not make as clear as necessary the type of assay used in Figures 2c-h, we have now edited the associated figure legends to make these links more obvious. Figure 2i