Role of tankyrase scaffolding in the β-catenin destruction complex and WNT signaling

Reviewer #1 (Public review):

Summary:

This manuscript reports the discovery and characterization of the first bifunctional degrader of tankyrase. Notably, the tankyrase degrader exhibits stronger β-catenin inhibition and tumor growth suppression compared to conventional tankyrase inhibitors. Mechanistically, while tankyrase inhibitors stabilize tankyrase and promote Axin puncta formation - thereby impairing β-catenin degradation - the degrader avoids this effect, resulting in deeper suppression of β-catenin signaling. These findings suggest that targeted degradation of tankyrase offers a novel therapeutic strategy for β-catenin-driven cancers. Overall, this is a compelling study with significant translational potential.

Strengths:

(1) The manuscript presents a rigorous and well-executed study on a timely and impactful topic.

(2) The biochemical and cellular characterization of the tankyrase degrader is thorough, and the comparative analysis with tankyrase inhibitors is insightful.

(3) The finding that tankyrase stabilization by inhibitors may interfere with Axin function is novel and significant. It aligns with earlier observations (e.g., Huang 2009) that transient tankyrase overexpression can stabilize β-catenin independently of PAR domain activity.

(4) The use of TNKS1/2 knockout cells expressing catalytically inactive tankyrase to demonstrate β-catenin inhibitory activity of the tankyrase degrader is elegant.

(5) The finding that the tankyrase degrader has superior anti-proliferative effects in colorectal cancer models has important therapeutic implications.

Weaknesses:

(1) A key caveat is that the identified tankyrase degrader also targets GSPT1 for degradation. This raises the possibility that GSPT1 degradation may contribute to the observed β-catenin and tumor growth inhibition.

(2) The authors address this concern reasonably by showing that DLD1 cells resistant to GSPT1 degradation remain sensitive to the tankyrase degraded.

(3) To further strengthen this point, the authors might consider generating TNKS1/2 double knockout cells (e.g., in DLD1 or SW480 backgrounds) and demonstrating that the degrader loses its growth-inhibitory effect in these models. However, given the technical challenges of creating double knockouts in cancer cell lines, such experiments could be considered optional.

Reviewer #2 (Public review):

Summary:

The ADP-ribosyltransferase tankyrase controls many biological processes, many of which are relevant to human disease. This includes Wnt/beta-catenin signalling, which is dysregulated in many cancers, most notably colorectal cancer. Tankyrase is a positive regulator of Wnt/beta-catenin signalling in that it counters the activity of the beta-catenin destruction complex (DC). Catalytic inhibition of tankyrase not only blocks PAR-dependent ubiquitylation and degradation of AXIN1/2, the central scaffolding protein in the DC, but also tankyrase itself. As a result, blocking tankyrase gives rise to tankyrase accumulation, which may accentuate its non-catalytic functions, which have been proposed to drive Wnt/beta-catenin signalling. Most tankyrase catalytic inhibitors have shown limited efficacy and substantial toxicity in vivo. By developing tankyrase-directed PROTACs, the authors aim to block both catalytic and non-catalytic functions of tankyrase, aspiring to achieve a more complete inhibition of Wnt/beta-catenin signalling. The successfully developed PROTAC, based on the existing catalytic inhibitor IWR1, IWR1-POMA, induces the degradation of both TNKS and TNKS2, blocks beta-catenin-dependent transcription without stabilising the DC in puncta/degradasomes, and inhibits cancer cell growth in vitro. Mechanistically, this points to a scaffolding role of tankyrase in the DC, at least under conditions of tankyrase catalytic inhibition, in line with previous proposals.

Strengths:

The study clearly illustrates the incentive for developing a tankyrase degrader, namely, to abolish both catalytic and non-catalytic functions of tankyrase. By and large, the study achieves these ambitions, and the findings support the main conclusions, although the statement that a more complete inhibition of the pathway is achieved requires corroboration. The proteomics studies are powerful. IWR1-POMA constitutes a very useful tool to re-evaluate targeting of tankyrase in oncogenic Wnt/beta-catenin signalling. The paired compounds will benefit investigations of tankyrase scaffolding functions across many different biological systems controlled by tankyrase. The findings are exciting.

Weaknesses:

Although the results are promising and mostly compelling, the claim that the PROTACs provide "a deeper suppression of the WNT/β-catenin pathway activity" requires further corroboration, particularly at endogenous tankyrase levels.

There are also some other points that, if considered, would further improve the manuscript, as detailed below.

(1) Abstract and line 62: Many catalytic tankyrase inhibitors tend to display toxicity, which is likely on-target (e.g., 10.1177/0192623315621192; 10.1158/0008-5472). This constitutes the main limiting factor for these compounds. An incomplete inhibition of Wnt/beta-catenin signalling may contribute to the challenges, but this does not appear to be the dominant problem. A more prominent introduction to this important challenge is probably expected by the field.

(2) The authors do a good job in setting the scene for the need for tankyrase degraders. Their observations relating to the formation of puncta (degradasomes) being tankyrase-dependent are compatible with a previous study by Martino-Echarri et al. 2016 (10.1371/journal.pone.0150484): simultaneous silencing of TNKS and TNKS2 by RNAi abolishes degradasome formation. The paper is cited as reference 17, but only in passing, and deserves more prominence. (It includes an entire paragraph titled "Expression of tankyrases 1 and 2 is required for TNKSi-induced formation of axin puncta").

(3) Moreover, the scaffolding concept has been discussed comprehensively in other studies: 10.1111/bph.14038 and more recently 10.1042/BCJ20230230. There are also a few studies that focus on targeting the ankyrin repeat clusters of tankyrase to disengage substrates (10.1038/s41598-020-69229-y; 10.1038/s41598-019-55240-5) that illustrate the concept of blocking the scaffolding function. In that sense, the hypotheses are mature, and it is interesting to see some of them supported in this study. The authors could improve how they set their work into the context of these other efforts and proposals.

(4) In several places in the manuscript, the DC is referred to as "biomolecular condensate", at times even as a "classic example", implying that it operates through phase separation. This has not been demonstrated. In fact, super-resolution microscopy indicates that the puncta are not droplet-like (10.7554/eLife.08022), which would argue against the condensate hypothesis.

(5) It is beautiful to be able to use IWR1 and IWR1-POMA at identical concentrations for direct comparisons. However, this requires the two compounds to bind to tankyrase similarly well and reach the target to a comparable extent. How sure are authors that target engagement is comparable? Has this been evaluated?

(6) Figure 1F: It is not immediately apparent how IWR1-POMA shows more complete containment of Wnt/beta-catenin signalling. Most Wnt/beta-catenin targets lie close to the perfect diagonal, so I do not see how the statement "that IWR1-POMA controlled WNT/β-catenin signaling more effectively than IWR1" (in the legend of Figure 1F) is supported. Minimally, an expanded explanation would benefit the reader. Providing the colour-coding legend directly in the figure would help improve clarity. Also, the panel is very small and may benefit from a different presentation in the figure.

(7) Figure 2: The conclusion of a "deeper suppression" of signalling relies on overexpression of tankyrase in an otherwise tankyrase-null background. Have the authors attempted to measure reporter activity or endogenous gene expression without tankyrase overexpression, in Wnt3a-stimulated cells (in the context of a normal Wnt/beta-catenin pathway) or CRC cells at the basal level? Non-catalytic activity in a similar assay has previously been observed upon tankyrase overexpression (10.1016/j.molcel.2016.06.019). Whether or not there is a substantial scaffolding effect at endogenous tankyrase levels after tankyrase inhibition remains unconfirmed, and the PROTAC is a valuable tool to address this important question. The findings presented in Figure S7C and D go some way towards answering this question - these data could be presented more prominently, and similar assays could be performed in other cell systems.

(8) Line 237/238: "TNKS accumulation negatively impacts the catalytic activity of the DC (Figure 5D)" - the data do not show this. Beta-catenin levels are a surrogate readout for DC function (phosphorylation and ubiquitylation). Minimally, this requires rewording, with reference to beta-catenin levels.

(9) Line 303-304: Beta-catenin is thought to exchange at beta-catenin degradasomes; this is clear from previous FRAP assays and the observation that phospho-beta-catenin accumulates in degradasomes upon proteasome inhibition (10.1158/1541-7786.MCR-15-0125). However, degradasome size hasn't, to my knowledge, been related to activity. Can this be clarified, please?

(10) There are previous hypotheses/proposals that the sensitivity of CRC cells to tankyrase inhibition correlates with APC truncation or PIK3CA status (10.1158/1535-7163.MCT-16-0578; 10.1038/s41416-023-02484-8). Have the authors considered expanding their cell line panel (Figure S7) to sample a wider range of cell lines, including some that are wild-type with regard to APC or Wnt/beta-catenin signalling in general? This would be a valuable addition to the work. Quantitated colony formation data could be moved to the main body of the manuscript.

(11) The manuscript only mentions toxicity (i.e., therapeutic window) in the last sentence of the Discussion section. As this is THE main challenge with tankyrase inhibitors (as mentioned above), can the authors expand their discussion of this aspect? Is there an expectation that PROTACs may be less toxic?

(12) Figures 3, 4, 5A: For fluorescence microscopy experiments, can these be quantified, and can repeat data be included?

(13) Figure 4, S6: An additional channel illustrating the distribution of cells (e.g., nuclei, cytoskeleton, or membrane) would be helpful for orientation and context for the AXIN1 signal.

(14) How were cytosolic fractions of cells prepared to assess cytosolic beta-catenin levels? This detail is missing from the methods.

Reviewer #3 (Public review):

In this manuscript, Wang et al employ a chemical biology approach to investigate the differences between the enzymatic and scaffolding roles of tankyrase during Wnt β-catenin signalling. It was previously established that, in addition to its enzymatic activity, tankyrase 1/2 also plays a scaffolding function within the destruction complex, a property conferred by SAM-domain-dependent polymerization (PMID: 27494558). It is also known that TNKS1/2 is an autoregulated protein and that its enzymatic inhibition leads to accumulation of total TNKS proteins and stabilization of Axin punctae (through the scaffolding function of TNKS1/2), leading to rigidification of the DC and decreased β-catenin turnover. The authors surmised that this could, in part, explain the limited efficacy of TNKS1/2 catalytic inhibition for the treatment of colorectal cancers. To test this hypothesis, they evaluated a series of PROTAC molecules promoting the degradation of TNKS1/2 to block both the catalytic and scaffolding activities. They show that IWR1-POMA (their most active molecule) promotes more efficient suppression of beta-catenin-mediated transcription and is more active in inhibiting colorectal cancer cell and CRC patient-derived organoids growth. Mechanistically, the authors used FRAP to demonstrate that catalytic inhibitors of TNKS led to a reduced dynamic assembly of the DC (rigidification), whereas IWR1-POMA did not affect the dynamics.

Overall, this is an interesting study describing the design and development of a PROTAC for TNKS1/2 that could have increased efficacy where catalytic inhibitors have displayed limited activity. Knowing the importance of the scaffolding role of TNKS1/2 within the destruction complex, targeting both the catalytic and scaffolding roles certainly makes sense. The manuscript contains convincing evidence of the different mechanisms of the PROTAC vs catalytic inhibitors. Some additional efforts to quantify several of the experiments and to indicate the reproducibility and statistical analysis would strengthen the manuscript. Ultimately, it would have been great to evaluate the in vivo efficacy of IWR1-POMA in an in vivo CRC assay (APCmin mice or using PDX models); however, I realize that this is likely beyond the scope of this manuscript.

I have some recommendations listed below for consideration by the authors to strengthen their study:

(1) The title is slightly misleading, as it is already known that the scaffolding function of TNKS is important within the DC. The authors should consider incorporating the PROTAC targeting aspect in the title (e.g., PROTAC-mediated targeting of tankyrase leads to increased inhibition of betacat signaling and CRC growth inhibition).

(2) The authors should comment in the manuscript on the bell-shaped curve obtained with treatment of cells with the PROTACs (Figure S2C). This likely indicates tittering of the targets within a bifunctional molecule with increasing concentration (and likely reveals the auto-inhibition conferred by the catalytic inhibition alone).

(3) The authors comment that using G007-LK as warehead was unsuccessful, but they do not show data. Do the authors know why this was the case?

(4) Throughout the manuscript, the authors need to do a better job at quantifying their results (i.e., the western blots and the IF). For example, the degradation of TNKS1/2 in Figure 1D is not overly convincing. Similarly, the IF data in Figure 3 needs to be quantified in some ways. Along the same lines, the effect of IWR1-POMA treatments on the proliferation of cells and organoids should be quantified using viability assays... There is also no indication of how many times these experiments were performed and whether the blots shown are representative experiments. The quantification should include all experiments.

Author response:

Reviewer #1 (Public Review):

We thank the Reviewer for the favorable feedback. The major concern is the collateral degradation of GSPT1. As the Reviewer noted, IWR1-POMA was able to suppress colony formation in DLD-1 cells resistant to GSPT1/2 degrader, suggesting that TNKS but not GSPT degradation is responsible for growth inhibition.

We also appreciate that the Reviewer brought it to our attention an important early observation of the TNKS scaffolding effects. Cong reported in 2009 that overexpression of TNKS induced AXIN puncta formation in a SAM but not PARP domain-dependent manner (PMID 19759537). We will include this information in the revised manuscript.

Reviewer #2 (Public Review):

We thank the Reviewer for the encouraging and insightful comments. The major critique concerns whether TNKS degraders can suppress WNT/β-catenin signaling more effectively than TNKS inhibitors at endogenous TNKS levels. Fig. 1D shows that IWR1-POMA reduced the level of cytosolic β-catenin more effectively than IWR1 in Wnt3A-stimulated HEK293 cells without protein overexpression, and Fig. S7B shows that IWR1-POMA reduced STF signals more effectively than IWR1 in DLD-1 and SW480 cells with endogenous TNKS expression. We will corroborate these findings with additional cell lines during the revision.

(1) We agree with the Reviewer that on-target toxicities pose challenges to the development of WNT inhibitors. For example, LGK974 that inhibits PORCN to prevent the secretion of all WNT proteins showed significant on-target toxicity in human (PMC10020809), and G007-LK that inhibits TNKS to block canonical WNT signaling selectively exhibited weak efficacy and dose-limiting toxicity at 5‒30 mg/kg BID or 10‒60 mg/kg QD in various mouse xenograft models (PMID: 23539443). Similarly, G-631, another TNKS inhibitor, also showed dose-limiting toxicity without significant efficacy at 25‒100 mg/kg QD in mice (PMID: 26692561). However, G007-LK was well-tolerated at 200 mg/kg QD over 3 weeks in mice in another study (PMC5759193). Treating mice with G007-LK at 10 mg/kg QD over 6 months also improved glucose tolerance without notable toxicity (PMID 26631215). Importantly, constitutive silencing of both TNKS for 150 days in APC-null mice prevented tumorigenesis without damaging the intestines (PMC6774804). Furthermore, basroparib, a selective TNKS inhibitor, was well tolerated in a recent clinical trial (PMC12498271). We are therefore cautiously optimistic that TNKS degraders will have an improved therapeutic index compared with TNKS inhibitors.

(2) We agree with the Reviewer that Henderson's 2016 paper (PMC4773256) shed important light on the role of TNKS scaffolding in the DC. However, whereas this study demonstrated that knocking down both TNKS by siRNA prevented G007-LK to induce AXIN puncta, the function role of TNKS scaffolding in the DC remained unaddressed. We will include a more detailed description on Henderson's discovery.

(3) Indeed, Guettler demonstrated that TNKS scaffolding could promote WNT/β-catenin signaling in 2016, which forms the basis of the current work. Meanwhile, whereas there have been efforts to target the SAM or ARC domain to address TNKS scaffolding, our approach of targeting TNKS for degradation is complementary. We will provide a more detailed discussion of these studies.

(4) Biomolecular condensates are membrane less cellular compartments formed by phase separation of biomolecules, regardless of the physical/material properties (PMID: 28935776 and PMC7434221). Super-resolution microscopy studies by Peifer and Stenmark (PMC4568445 and PMID 26124443) showed that AXIN, APC, TNKS, and β-catenin interacted with each other to assemble into membraneless complexes, wherein AXIN and APC formed filaments throughout the DC. Peifer has also summarized evidence that supports the condensate nature of the DC (PMC6386181). However, we acknowledge that testing the physical properties of reconstituted DC (PMC8403986) will provide a better understanding of the nature, for example liquid vs. gel, of these condensates.

(5) We will evaluate the ability of IWR1 and IWR1-POMA to engage TNKS.

(6) We will modify Fig. 1F to improve clarity and readability.

(7) Fig. S7B shows that IWR1-POMA suppressed WNT/β-catenin signaling more effectively than IWR1 in APC-mut DLD-1 and SW480 CRC cells without TNKS overexpression. Similarly, Fig. S6B shows that IWR1-POMA provided a deeper suppression of STF signals in HeLa cells transfected with AXIN1 and β-catenin while expressing endogenous TNKS. These results provide evidence that inhibitor-induced TNKS scaffolding plays a significant role at endogenous TNKS expression levels. Separately, we will reorganize the figures to better present Fig. 7C and D as suggested by the Reviewer.

(8) We will rephrase "TNKS accumulation negatively impacts the catalytic activity of the DC".

(9) We apologize for confusing β-catenin phosphorylation with β-catenin abundance. Here, we refer the catalytic activity of the DC to as the ability of the DC to promote β-catenin degradation rather than the kinetics of β-catenin phosphorylation and ubiquitination. It is commonly observed that AXIN stabilization by TNKS inhibitors increases the DC size and reduces the β-catenin levels. Peifer has also noted that APC can increase the size and the "effective activity" of the DC (PMC5912785 and PMC4568445). As such, the induction of AXIN puncta by TNKS inhibitors is frequently used as an indicator of WNT/β-catenin pathway inhibition. However, because the DC only primes β-catenin but does not catalyze its degradation, we will revise our manuscript to improve accuracy and clarity.

(10) We will examine the effects of IWR1 and IWR1-POMA in additional cell lines, quantify the colony formation data, and reorganize the figures.

(11) As discussed above, evidence for on-target toxicity of WNT/β-catenin inhibition is mixed. Yet, the observation of no dose-limiting toxicity for basroparib at doses up to 360 mg QD in human (PMC12498271) is encouraging. PROTAC works by catalyzing target degradation, which is different from traditional catalytic inhibitors that require continuous target occupancy at a high level. Because IWR1-POMA has a durable effect on TNKS, we expect that a fully optimized TNKS degrader may allow less frequent dosing than basroparib and consequently an even more favorable therapeutic window.

(12/13) We will include quantification data, replicate information, and nuclei staining or cell outlines for the fluorescence microscopy experiments.

(14) Cytosolic fractions of cells were prepared using a commercial cytoplasmic extraction kit following manufacturer's instructions. We will include detailed information in the revised manuscript.

Reviewer #3 (Public Review):

We thank the Reviewer for the helpful suggestions.

(1) We will modify the title to include the PROTAC aspect.

(2) As the Reviewer suggested, the bell-shaped dose response of the PROTAC originated from the formation of saturated binary complexes. At high PROTAC concentrations, binding of TNKS and CRBN/VHL by separate PROTAC molecules impedes the formation of productive ternary complexes, which results in reduced degradation efficacy and consequently the hook effect.

(3) The structure-activity relationship of PROTACs is often unpredictable, as both the kinetics and thermodynamics of the target and E3 ligase binding play crucial roles. The lack of translation in degradation efficacy from IWR1 to G007-LK derived PROTACs may originate from differences in the binding kinetics or subtle changes in the orientation of the linker exit vector. We will include data on G007-LK in the revised manuscript.

(4) We will quantify the Western blots, immunofluorescence images, colony formation data, and the replicate information.