Peer review of: ABL1-dependent OTULIN phosphorylation promotes genotoxic Wnt/β-catenin activation to (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
In the manuscript submitted by Wang et al., the authors provide evidence for the DNA damage-
induced activation of Wnt/-catenin signaling through a mechanism which is independent of 
canonical signaling through FZD/LRP heterodimeric receptors and Wnt ligands. They show that 
genotoxic stress induces the phosphorylation of OTULIN at Tyr56, which in turn allows for its 
engagement with-catenin. This inhibits linear ubiquitination of -catenin by LUBAC and 
subsequent Lys48-linked polyubiquitination by -TRCP, thereby attenuating the proteasomal 
degradation of -catenin upon DNA damage. Furthermore, through a limited siRNA screen with a 
tyrosine kinase sub-library, they identified ABL1 as the kinase responsible for phosphorylating 
OTULIN upon genotoxic stress. OTULIN-mediated Wnt/-catenin activation upon genotoxic stress 
promotes drug resistance in TNBC tumors, thus suggesting that targeting OTULIN may be an 
appropriate therapeutic strategy to reduce metastasis and drug resistance in TNBC patients. While 
most experiments are thoroughly executed, the following additional experiments should be 
considered to warrant publication: 
 
Major points: 
 
1. The finding that Dox treatment induces the association between endogenous OTULIN and 
catenin is very subtle (i.e. Fig 4A), although this is a major part of the mechanism for OTULIN-
mediated activation of Wnt signaling. It would be helpful to have a single figure comparing relative 
binding of -catenin to OTULIN, HOIP, and -TRCP with and without Dox treatment to better 
appreciate how these differences in binding contribute to the mechanism for -catenin 
stabilization. For example, in the model in Figure 6J, the authors suggest that LUBAC is bound to 
-catenin regardless of whether Wnt signaling is turned on and OTULIN is bound. Would increasing 
the concentration of LUBAC in a dose-dependent manner outcompete OUTLIN for access to -
catenin for linear ubiquitination? Also, the model indicates that -TRCP is bound to -catenin in 
both the ON and OFF states, yet the data in Figure 3 showed that -TrCP binding was reduced in 
response to genotoxic stress. The model should be re-drawn to better emphasize this point. 
2. The model in Figure 6J also indicates that c-Abl is translocated from the nucleus to 
phosphorylate OTULIN in the cytoplasm, although no direct evidence was provided for this. The 
authors should include immunofluorescence data showing the nuclear localization of c-Abl and 
OTULIN with or without Dox treatment to better clarify this point. 
3. In addition to c-Abl, ITK was also found in the tyrosine kinase screen (Fig. 5A) to induce the 
phosphorylation of OTULIN, yet it was not followed up on. The authors should provide a 
supplemental figure to demonstrate that ITK phosphorylates OTULIN but does not impact Wnt-
signaling in combination with c-Abl, as they suggest. 
4. In Figure 6, the authors show a correlation between OTULIN levels and drug resistance in TNBC 
cells. It would also be interesting to observe whether there is also any correlation with LUBAC 
expression levels in the TCGA dataset. Does modulation of LUBAC attenuate the effects of OTULIN 
on drug resistance in the TNBC cells? 
 
 
Minor points: 
 
1. Figure S3E has a misplaced K48-Ub data label. 
2. The following sentence should reference Figure S5B instead of S5C: “In contrast, inhibiting c-Abl 
did not affect Wnt3a-induced b-catenin stabilization.” (pg. 13) 
3. Methods section is not complete, no info on where the pY56-Otulin antibody came from and no 
info on sources of chemicals and antibodies, cell lines, etc 
 
 
 

Reviewer #2: 
Remarks to the Author: 
This manuscript describes the underlying mechanism that Wnt/β-catenin signaling is activated by 
genotoxic agents in breast cancer cells. The authors showed that OTULIN, a linear linkage-specific 
deubiquitinase, is required for the DNA damage-induced β-catenin activation. OTULIN was 
phosphorylated at Tyr56 by ABL1 and activated, resulting in mediating deubiquitination of the M1 
ubiquitin chain of β-catenin at lysine 133. M1-linked linear ubiquitination of β-catenin by LUBAC 
promoted the interaction of β-TrCP with β-catenin, thereby inducing K48-linked polyubiquitination 
and degradation of β-catenin. In addition, the authors showed that OTULIN depletion decreased 
TNBC cells resistance to chemotherapeutics in vivo. 
 
From these results, the authors proposed that OTULIN activates Wnt/β-catenin signaling by 
inhibiting linear ubiquitination, K48-linked polyubiquitination, and proteasomal degradation of β-
catenin, upon DNA damage. Although this paper is potentially interesting, there are several issues 
that the authors need to address. 
 
Major comments; 
(1) Is OTULIN-mediated deubiquitination of β-catenin involved in Wnt ligand-dependent activation 
of β-catenin signaling? Wnt3a did not induce OTULIN phosphorylation (sFig. 4G) and ABL1 or DNA-
PK inhibitor that attenuates OTULIN phosphorylation and functions did not affect β-catenin 
stabilization by Wnt3a (sFig. 5B, J). In contrast, OTULIN KO inhibited Wnt3a-induced stabilization 
of β-catenin and target gene expression (Fig. 2A-C). These results are controversial, therefore, the 
authors should interpret carefully them. 
 
(2) CYLD, a USP family deubiquitinase, binds the PUB domain of HOIP and cleaves M1- and K63-
ubiquitin linkage. It is crucial to test whether CYLD regulates β-catenin levels in the presence of 
genotoxic agents and Wnt3a. 
 
(3) In Fig. 3B, polyubiquitination of β-catenin seems to be greatly reduced by OTULIN or OTUB1, 
whereas K48-linked and M1-linked polyubiquitin chains, respectively, should not be affected by 
these deubiquitinases. The authors need to confirm by immunoblotting using chain type specific 
antibodies. 
 
(4) In Fig. 3E, the authors should show the effect of genotoxic agents and Wnt3a on the enhanced 
M1-ubiquitination of β-catenin in OTULIN-KO cells. The authors suggested that linear ubiquitination 
of β-catenin may occur prior to K48 ubiquitination. Then, it is necessary to examine K48-linked 
ubiquitination and proteasomal degradation of β-catenin in HOIP-KO cells. 
 
(5) The authors identified that K133 is a linear ubiquitination site in β-catenin. This reviewer is 
wondering what is the physiological roles of M1-linked linear ubiquitination of β-catenin at steady 
state? It has been reported that β-catenin Lys133 is methylated by SMYD2 and this modification is 
critical for the binding to FOXM1, nuclear translocation, and activation of Wnt signaling 
[PMID:28915556]. The authors need to test the relationship or interdependence between M1-
linked linear ubiquitination and methylation at Lys133 
 
(6) In sFig. 3E, some mutants of β-catenin other than K133R reduced M1-ubiquitination. The 
authors should examine the ubiquitination sites of β-catenin by mass spectrometry. Otherwise, in 
vitro ubiquitination assay is necessary to confirm that K113 is the M1-ubiquitination site. 
 
(7) In related to Fig. 3K, the authors should show the stability of β-catenin K133R that has defect 
of binding to β-TrCP compared with β-catenin K19/49R and β-catenin SA mutant 
(S33A/S37A/T41A/S45A, which is known to be constitutively activated). In addition, does OTULIN 
affect β-catenin stabilization by GSK3 inhibitors such as CHIR99021 that inhibits β-catenin 
phosphorylation? 
 

(8) In Fig. 3A and I, the authors showed that LUBAC expression induced M1-linked linear 
ubiquitination and degradation of β-catenin. To clarify that K19/K49 are K48-ubiqitination sites of 
β-catenin, whether LUBAC degrades β-catenin K19/K49R mutant should be tested. 
 
(9) As the molecular anatomy, the ABL1-binding region of OTULIN should be determined. 
Furthermore, does the region overlap with β-catenin-binding region of OUTLIN? 
 
(10) The authors should clarify the relationship between phosphorylation and M1-linked linear 
ubiquitination of β-catenin. In Fig. 3K, M1-ubiqitination defect (K133R) did not affect β-catenin 
phosphorylation at T41/S45 which is required for β-TrCP binding. Does the state of β-catenin 
phosphorylation at S33/S37 affect M1-ubiquitination of β-catenin? M1-ubiquitination state of β-
catenin SA mutant (S33A/S37A/T41A/S45A) with or without DOX should be analyzed. 
 
(11) The authors identified that ABL1 is responsible for phosphorylation of OTULIN, which 
regulates the Wnt/β-catenin pathway in Fig. 5. However, many studies reported that OUTLIN is 
involved in NF-κB signaling and cell death. Therefore, the authors need to test whether the NF-κB, 
MAPK, and cell death pathways are activated by genotoxic agents and inflammatory cytokines in 
ABL1-KO cells. 
 
(12) In Fig. 5E, ABL1 KO phenotypes should be rescued by expression of WT ABL1 but not ABL1 
kinase dead mutant. 
 
(13) In Fig. 5I, the total β-catenin level was unchanged by DOX treatment. The result is not 
consistent with other results such as Fig. 1A. 
 
(14) In Fig. 6, direct evidence that in vivo phenotypes induced by OTULIN KO cells are mediated 
by Wnt/β-catenin signaling was not shown The authors should show that phenotypes induced by 
OTULIN KO are rescued by Wnt signaling activation such as the expression of β-catenin SA 
mutant.