Disruption of WSB2-mediated NOXA Degradation Induces Synthetic Lethality to Anti-apoptotic BCL-2 Family Protein Inhibitors

Reviewer #1 (Public Review):

Summary:

In this manuscript, Jiao D et al reported the induction of synthetic lethal by combined inhibition of anti-apoptotic BCL-2 family proteins and WSB2, a substrate receptor in CRL5 ubiquitin ligase complex. Mechanistically, WSB2 interacts with NOXA to promote its ubiquitylation and degradation. Cancer cells deficient in WSB2, as well as heart and liver tissues from Wsb2-/- mice exhibit high susceptibility to apoptosis induced by inhibitors of BCL-2 family proteins. The anti-apoptotic activity of WSB2 is partially dependent on NOXA.

Overall, the finding, that WSB2 disruption triggers synthetic lethality to BCL-2 family protein inhibitors by destabilizing NOXA, is rather novel. The manuscript is largely hypothesis-driven, with experiments that are adequately designed and executed. However, there are quite a few issues for the authors to address, including those listed below.

Specific comments:

(1) At the beginning of the Results section, a clear statement is needed as to why the authors are interested in WSB2 and what brought them to analyze "the genetic co-dependency between WSB2 and other proteins".

(2) In general, the biochemical evidence supporting the role of WSB2 as a SOCS box-containing substrate-binding receptor of CRL5 E3 in promoting NOXA ubiquitylation and degradation is relatively weak. First, since NOXA2 binds to WSB2 on its SOCS box, which consists of a BC box for Elongin B/C binding and a CUL5 box for CUL5 binding, it is crucial to determine whether the binding of NOXA on the SOCS box affects the formation of CRL5WSB2 complex. The authors should demonstrate the endogenous binding between NOXA and the CRL5WSB2 complex. Additionally, the authors may also consider manipulating CUL5, SAG, or ElonginB/C to assess if it would affect NOXA protein turnover in two independent cell lines. Second, in all the experiments designed to detect NOXA ubiquitylation in cells, the authors utilized immunoprecipitation (IP) with FLAG-NOXA/NOXA, followed by immunoblotting (IB) with HA-Ub. However, it is possible that the observed poly-Ub bands could be partly attributed to the ubiquitylation of other NOXA binding proteins. Therefore, the authors need to consider performing IP with HA-Ub and subsequently IB with NOXA. Alternatively, they could use Ni-beads to pull down all His-Ub-tagged proteins under denaturing conditions, followed by the detection of FLAG-tagged NOXA using anti-FLAG Ab. The authors are encouraged to perform one of these suggested experiments to exclude the possibility of this concern. Furthermore, an in vitro ubiquitylation assay is crucial to conclusively demonstrate that the polyubiquitylation of NOXA is indeed mediated by the CRL5WSB2 complex.

(3) In their attempt to map the binding regions between NOXA and WSB2, the authors utilized exogenous proteins of both WSB2 and NOXA. To strengthen their findings, it would be more convincing to perform IP with exogenous wt/mutant WSB2 or NOXA and subsequently perform IB to detect endogenous NOXA or WSB2, respectively. Additionally, an in vitro binding assay using purified proteins would provide further evidence of a direct binding between NOXA and WSB2.

Reviewer #2 (Public Review):

Summary:

Exploring the DEP-MAP database and two drug-screen databases, the authors identify WSB2 as an interactor of several BCL2 proteins. In follow-up experiments, they show that CRL5/WSB2 controls NOXA protein levels via K48 ubiquitination following direct protein-protein interaction, and cell death sensitivity in the context of BH3 mimetic treatment, where WSB2 depletion synergizes with drug treatment.

Strengths:

The authors use a set of orthogonal methods across different model cell lines and a new WSB2 KO mouse model to confirm their findings. They also manage to correlate WSB2 expression with poor prognosis in prostate and liver cancer, supporting the idea that targeting WSB2 may sensitize cancers for treatment with BH3 mimetics.

Weaknesses:

The conclusions drawn based on the findings in cancer patients are very speculative, as regulation of NOXA cannot be the sole function of CRL5/WSB2 and it is hence unclear what causes correlation with patient survival. Moreover, the authors do not provide a clear mechanistic explanation of how exactly higher levels of NOXA promote apoptosis in the absence of WSB2. This would be important knowledge, as usually high NOXA levels correlate with high MCL1, as they are turned over together, but in situations like this, or loss of other E3 ligases, such as MARCH, the buffering capacity of MCL1 is outrun, allowing excess NOXA to kill (likely by neutralizing other BCL2 proteins it usually does not bind to, such as BCLX). Moreover, a necroptosis-inducing role of NOXA has been postulated. Neither of these options is interrogated here.