Peer review of: Enhancing chemotherapy response through augmented synthetic lethality by co-targeting nucleo (2020)

Reviewers' comments: 
Reviewer #1 (Remarks to the Author): 

 
The paper by Kong et al describes a type of synthetic lethality (SL) they term “augmented 
synthetic lethality” (ASL) in which the differential sensitivity of a p53 mutant lung adenocarcinoma 
cancer cell line to cis-platin is facilitated by the simultaneous co-targeting of MK2 and XPA. 
Previous work from the Yaffe laboratory had demonstrated a weaker but significant SL relationship 
between p53 and MK2 (a digenic negative interaction) in the context of DNA damaging 
chemotherapeutic agents. The current work purportedly extends these findings to a potent trigenic 
negative interaction between p53, MK2, and XPA by demonstrating that simultaneous loss of MK2 
and XPA causes increased killing by cis-platin in p53-defective lung adenocarcinoma cells relative 
to targeting either pathway alone. The authors suggest this is due to the hyperactivation of MK2 
signaling in XPA-deficient cells. They go on to utilize an RNAi delivery system developed in their 
laboratory, involving RNAi-peptide nanocomplexes, to simultaneously co-target MK2 and PKA in 
p53-defective tumors in vivo, and observe enhanced tumor control and improved long term 
survival in a fully immunocompetent mouse model of lung adenocarcinoma. The tumor cell killing 
effects of simultaneous targeting of MK2 and PKA and treating with cis-platin in this in vivo test 
system are dramatic. 
 
The work clearly demonstrates a strong synthetic lethal relationship between MK2 and XPA in 
presence of cis-platin in the adenocarcinoma cell line. The authors claim that the lethality is 
dependent on the p53 status of the line, however they do not directly demonstrate that p53 
mutation is necessary to observe the SL effect. The authors claim that the trigenic SL interaction 
between MK2, PKA, and p53, that they call ASL, is novel, and stress that p53 mutations are 
prevalent in many cancer types suggesting broad applicability for differential killing relative to p53-
WT cells. Trigenic negative genetic interactions are not a novel concept, so I am not convinced 
that a conditional trigenic interaction (causing drug sensitivity to a sub-lethal dose of a DNA 
damaging agent) warrants creating a new nomenclature term (ASL), although this is not a major 
concern. Nevertheless, the authors do not formally demonstrate the requirement of p53 loss of 
function in the MK2/PKA co targeting experiments, so the ASL concept has not been demonstrated. 
The claim that the genetic context of p53-mutant vs p53-WT is critical to demonstrating ASL, but it 
is not tested. Would introduction of p53 back into KP7B or H1299 cancer cell lines rescue the 
lethality of simultaneous knockdown of XPA and MK2? 
 
The second half of the paper uses RNA-peptide nanoplexes to downregulate genes in vivo. The 
authors convincingly show that a combination of siRNAs targeting XPA and MK2 shrinks p53-
deficient xenografts in the presence of cisplatin. However, the effect in the absence of cisplatin or 
in p53-proficient tumors is not investigated. It would also be beneficial to know how the treatment 
might affect normal mice (by eg measuring their weight gain/loss over treatment or other 
phenotypes) as the treatment involves a combination of knockdown of two genes and a potent 
genotoxic agent. The authors claim that: 
 
MK2 has a well-established role in innate immunity and inflammatory signaling (26), similar to 
DNA-PK (35), raising the possibility of off-target side effects from systemic inhibition. To overcome 
these limitations, we developed a novel tumor-targeting nanoparticle that delivers siRNAs to the 
site of pre-exisiting MK2-containing tumors and efficiently depletes both its target RNA and 
protein. (page 12). 
 
It is not easy to determine from the text why nanoparticles would target tumors specifically and 
not also affect immune cells and no data for this appears to be given. If the authors are using 
nanoparticles to avoid side effects by specifically targeting tumor cells, this should be clarified. 
 
Additional points: 
 
-Many of the methods seem incomplete. Eg for survival assays, the authors write: 
Cell viability was measured using CellTiter-Glo luminescent cell viability assay as per 
manufacturer’s instructions (Promega) (pg16) 
It would also be useful to know how long the cells were grown and when/how siRNAs and drugs 
were added. 
 
Materials and methods lack explanations about how “expected” values are calculated. 
 
-Figure 1- the authors switch between calling pMK2 levels a measure of MK2 activity and/or MK2 
activation which is confusing 
 
-Figure 1D- the pMK2 protein levels should be normalized to the levels of MK2 in the blot; it’s not 
clear if this was done 
 
-Figure 2A- the symbols in the graph legend do not match the ones in the figure 
 
-Figure 2C- the DAPI staining in the nuclei appears abnormally punctate bringing into question the 
viability of these cells; additionally, gamma-H2AX foci were counted for Figure 2D and it is unclear 
how a cell like the one at the bottom of the siMK2/XPA picture could even have their foci 
individualized to be counted. H2AX foci numbers do not correlate well with the quantification graph 
in Fig 2D. 
 
-Figure 2D- for siM/X, please write out in full (also Figure 4D) 
 
-Although the authors show dual knockdown by western blot in Figure 4, it might also be nice to 
see that dual knockdown occurs at a single cell level (using eg immunofluorescence) 
-Figure 4F-the authors claim in the text that the dual siRNA-containing nanoplexes are “more than 
doubling the survival of longest lived animals in any of the other cohorts” (pg 11), and perhaps the 
x-axis should be changed to “Time (post-treatment)” to better reflect what I think this statement 
means 
-Supplemental Figure 1D and E- it is not explained where the expected line on the graph came 
from and it seems like data is missing (eg where are the data for the effect of each single siRNA 
alone); should cisplatin be relabeled siControl? 
 
-Supplemental Figure 2C- could use a better explanation for what is increasing, maybe the 
concentration of the nanoplexes? 
 
- Sup fig 1B & C are not mentioned in the text. Why the DOX experiments 
 
 

Reviewer #2 (Remarks to the Author): 
 
In this study, Kong et al., expand on data indicating that loss of MK2 promotes sensitivity to DNA 
damaging agents only in the absence of p53. Augmented synthetic lethality shows that depletion 
of a third gene, XPA, enhances the synthetic lethality. 
Specific comments: 
1. Figures S1B and C are not cited in the text. 
2. Text page 10 says “allow the tumors to grow larger and the cancer even to be more 
advanced…” What are the data that indicate the larger tumors are more advanced? 3. The MK2 and pMK2 blots differ in figures 1B and C (one, two or three bands are shown). Please 
explain these differences. Also in figure 1B, is the upper MK2 band representative of active MK2? 
The description is not clear. 
4. Also, the western blots in figures 1B and C should have actin controls for each blot that was 
probed unless all of the westerns represent a single blot. 
5. While the experiments are well done with appropriate controls, only one lung cancer cell line, 
KP7B was used. As thus, the generality of this augmented synthetic relationship is limited. 
6. In figure 2A, the symbols do not match. For example, the red line has triangles but triangles are 
blue and the red line has squares. Thus, I am not sure which line represents which experiment. 
7. In figure 2D, it appears that there might be statistical differences that were not noted. Also 
please indicate what columns are being compared for the asterisks indicated. 
8. It is surprising that in this synthetic lethal interaction, not all cells die. Do nanoplexes target all 
cells? If not, what percent of cells are targeted? 
 
 
 
Reviewer #3 (Remarks to the Author): 
 
The authors have developed a new approach for improving synthetic lethality induction in NSCLC 
using polymeric siRNA nanocomplexes. The study design is systematic and nicely articulated. 
However, there are several concerns with experimental controls and analysis as mentioned below. 
These needs to be addressed: 
1. In line 264 the model is described in Figure 3D and not 3A as written in the text. 
2. The authors should conduct a more precise quantification of in vivo siRNA delivery to the tumors 
via siRNA specific qPCR or any other quantitative method (but not fluorescently based method) in 
order to really know how much of the siRNAs are reaching the tumors and how much is really 
functional. 
3. The authors should use a MK2 small molecule inhibitor as a control in all in vivo experiments to 
demonstrate the superiority of the siRNA. Furthermore, the toxicity of this control should also be 
evaluated. 
4. The authors should evaluate the in vivo silencing of MK2 and XPA by the nanoplexes as they 
conducted in Figure 3F-H. Furthermore, the authors should further validate the mechanism of the 
combination of siMK2 and siXPA to induce synthetic lethality in vivo as they have conducted in 
Figure 3S.