Peer review of: AXL confers cell migration and invasion by hijacking a PEAK1-regulated focal adhesion (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

This is an involved study that implicates receptor tyrosine kinase organized by the enzyme AXL as 
a potential mechanism to enhance metastasis and acquisition of resistance to cancer drugs. This is 
a very complicated study with many parts that is compounded by the frequent reference to 
supplemental data. Some of which should be omitted and some of which should be included in the 
body of the text. Using a variety of indirect methods, the authors propose that AXL signaling 
contributes to FA dynamics. This work could benefit from more mechanistic experiments that 
clearly define the role of AXL and the pseudokinase PEAK1 in tumor growth and metastasis. More 
specific criticisms are listed below. 

On page 4 it states “We identified an overrepresentation of MAPK, CDK, PKA and PKC kinase motifs 
in most of the clusters, suggesting that AXL may signal 5via regulation of these kinases 
(Supplementary Information, Fig. S2d). This data is waek and extremely speculative. Since it 
covers several of the predominant kinases it is hard to know what this means. I recommend 
removal of this data. 

The proximity ligation data presented in fig 2a is poor and hard to evaluated. Likewise, The data in 
figures 2b and c is not very convincing. First, quantitation of the PLA data should and Quantitation 
of the number of focal adhesions as dot scatter plots is a more compelling way to show the relative 
effect of changing AXL expression or action. 

Evidence for changes in the number of focal adhesions presented in figure 2c and the 
supplementary information is far from convincing. 

The Bio ID proteomic data in figures 3f and g is poorly connected to the rest of the paper. This is a 
rather speculative element of the work that needs some more experimental evidence to warrant its 
inclusion in the study. 

Reviewer #2 (Remarks to the Author): 

This manuscript offers a fascinating and important study of AXL signaling, which is timely because 
of the growing appreciation of the key role AXL plays in tumor resistance to targeted and 
chemotherapeutic drugs. The kind of understanding being pursued here, of nodes downstream of 
AXL activation could be very useful in suggesting effective combination treatments as well as in 
gaining insights into novel receptor tyrosine kinase signaling biochemistry. The authors bring 
together multiple approaches, experimental and computational, to ascertain relevant pathway 
elements including NEDD9, PEAK1, CSK, and Paxillin, toward enhancing invasive tumor cell 
migration via focal adhesion turnover. The resulting story is appealing and appears to be generally 
sound. 

My main point of concern relates to the technical details of Gas6 treatment for AXL stimulation. 
The authors seem to be not familiar with certain complications inherent in the Gas6/AXL system, 
such that mere treatment with soluble Gas6 is well-known to have variable effects depending on 
availability and characteristics of lipid vesicle-mediated interactions (for instance, as studied by 
Meyer et al in Cell Systems [2015]), such that AXL activation effects will vary with factors that are 
not explicitly considered in the experimental protocols outlined here. I believe that the authors 
need to provide more detailed characterization of their Gas6 protocols, including whether the cells 
being treated express Gas6 themselves in autocrine manner and to what degree apoptotic cell 
debris might be present due to low levels of nonviability in the culture conditions. These details will 

be vital for other laboratories in any attempts to reproduce and build upon the findings here. 

Reviewer #3 (Remarks to the Author): 

In this paper the authors try to elucidate the major signaling pathways activated by the receptor 
tyrosine kinase, AXL. 
The authors have used systems biology and proteomic approaches and find that AXL modulates 
the phosphorylation of a network of focal adhesion (FA) proteins that culminates in faster FA 
disassembly. They go on to deliniate a rather convoluted pathway whereby AXL phosphorylates the 
FA protein NEDD9, which then promotes NEDD9/CRKII-mediated phosphorylation of PEAK1. This 
pseudokinase then complexes wilt CSK kinase to phosphorylate paxillin, resulting in FA turnover. 

The strengths of this work lie in the biochemistry and systems biology, which are carried out very 
well. The finding that a major network of AXL-mediated phosphorylation events occur in FA 
proteins is novel, and this work does suggest that AXL can modulate FA turnover. 

The weaknesses lie in the biology and physiological relevance of the findings to tumor progression, 
especially the relevance of focal adhesion turnover-per se to AXL-mediated metastasis. 
Thus while the final data in Figs 5 and S7 using PEAK KO MDA-MB-231 cells suggest that PEAK 
may contribute to metastasis, The authors have not connected the pathway back to AXL. Thus, 
PEAK may have completely AXL-independent role in regulating metastasis. To make the case that 
it is the FA turnover pathway 
mediated by AXL that promotes metastasis, the authors need to demonstrate that some of the key 
phosphorylation and protein-protein interactions leading PEAK-mediated to paxillin phosphorylation 
and FA turnover also promote metastasis. 
Otherwise the paper does not hang together very well as it represents some excellent biochemistry 
and a separate finding that PEAK KO modulates metastasis. 

Specific Points that should be addressed: 

1. Introduction and Discussion sections are missing. These need to be added to provide a context 
to this work and to demonstrate originality of the work 

2. All the phospho-blots lack blots of the total proteins. This is essential to show that it is the 
phosporylation of the protein and not the total expression of the proteins that is changing. 

3.In Fig 2 the analyses of the FAs should also include effects of GAS6 induced effects in addition to 
the constitutive 
situation, i.e. effects of GAS6 + R428. 

4. p12. Does inhibition of paxillin phosphorylation ( directly or by using phospho-mutants) have 
the same effect on metastasis as PEAK KO? 

5. The effect of AXL KO or the effect of AXL inhibitor on metastasis has to be carried out. Is this 
effect rescued by PEAK? 

6. The metastasis data ( PEAK KO) need to be carried out in another TNBC cell line in addition to 
MDA-MB-231 cells 

7 Fig s7j, why were the lungs probed ex vivo. Could in vivo signal be detected? 

8. Fig s7d/e: Tumor volumes should be measured , not only IVIS signal. 

9. The pathway model should be depicted in the main figures.