Tankyrase

Tankyrase 1 and 2 protein (TNKS1/2) both belong to the poly(adenosine diphosphate (ADP)-ribose) polymerase (PARP) family, which are structurally conserved enzymes that participate in both the WNT signaling pathway and Hippo signaling pathway [53]. The TNKS1 isoform differs from TNKS2 by an additional histidine, proline, serine-rich (HPS) domain in the TNKS1 (Figure 7) [53]. The function of the HPS domain is unknown. Both enzymes contain five ankyrin repeat clusters (ARC), a sterile alpha motif (SAM), and a catalytic PARP domain [53, 54]. TNKS1/2 interacts with target proteins through ARC domains. The SAM domain is responsible for homo-oligomerization, while the catalytic domain contains the ADP-ribosyltransferase activity [53, 55].

[53]

1.5.2 Cellular function and enzymatic activity

Tankyrases are enzymes with multiple, overlapping cellular functions, including regulating different signaling pathways, mitosis, telomere maintenance, glucose metabolism, and tumor suppressors[55]. Tankyrases can also function as scaffolding for other proteins through their SAM domains, providing structural functions [56].

The catalytic activity of TNKS1/2 controls different regulations in the cells by poly-Adenosine Diphosphate-ribosylation (PARsylation) [55]. PARsylation results in post-translational

modification, which regulates other cellular processes by stabilizing the target genes [55].

PARsylation involves the hydrolysis of nicotinamide adenine dinucleotide (NAD+) to

ADP-Figure 7. Structure of TNKS1/2 peptide. The HPS domain is one of the structural differences between TNKS1 and 2, while the function of this domain remains unknown at this point. The five ARC domains contribute to the interactions between TNKS1/2 and other target proteins. SAM domain controls self-oligomerization. PARP is the catalytic domain that performs PARsylation. TNKS1/2 domain structures are highly conserved. Figure created with BioRender.com, inspired by [53].

11 ribose and nicotinamide, attaching the ADP-ribose to a target protein which regulates cellular processes including gene transcription, DNA damage repair, and cellular stress (Figure 8) [53].

The growing poly(ADP-ribose) chain is recognized by the E3 ubiquitin ligase RING finger protein 146 (RNF146), which tags the protein for degradation by the proteasome [57].

Figure 8. PARsylation activity of tankyrase. Tankyrase orchestrates the hydrolysis of NAD+ to ADP ribose and nicotinamide. Tankyrase PARsylates the target protein by attaching an ADP-ribose to the target. Figure created with BioRender.com

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1.5.3 Tankyrase inhibition suppress WNT signaling

TNKS1/2 interacts with the TBM domain of AXIN1/2 and PARsylate AXIN1/2, leading to the recognition of PARsylated AXIN1/2 through E3 ligase RNF146 (Figure 9) [34]. RNF146 mediates ubiquitination and degradation of AXIN1/2 through the proteasome. This ultimately destabilizes the destruction complex, resulting in the accumulation of β-catenin, which

translocates to the nucleus [53]. Dysregulated WNT signaling and overexpression of WNT target genes are often found in multiple cancer types [30]. By inhibiting TNKS1/2, AXIN proteins stabilize [34]. Small-molecule tankyrase inhibitors, such as G007-LK, therefore, destabilize β-catenin and decreases WNT signaling. [34]

Figure 9. Tankyrase inhibition suppress WNT signaling. Left panel: Without tankyrase inhibitors, active tankurase binds the TBS domain of AXIN and PARsylates AXIN, leading to phosphorylation, ubiquitination, and degradation by RNF146 and 26S proteasome. Hence, AXIN is not allowed to form the destruction complex, lading to accumulation of β-catenin and gene transcription in the nucleus. Right panel: Tankyrase inhibition stabilizes AXIN, allowing the destruction complex to form and perform ubiquitin-mediated proteosomal degradation of β-catenin, thereby suppressing WNT signaling. Abbreviations: ubiquitin (Ub), Figure created with BioRender.com, inspired by [34].

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1.5.4 Tankyrase inhibition suppress YAP/TAZ activity

AMOTs are interacting partners with YAP and TAZ in the Hippo signaling pathway. AMOT1/2 proteins can regulate YAP signaling directly (Figure 10) [47]. Similar to the WNT signaling pathway, TNKS1/2 PARsylates AMOT proteins lead to their degradation by E3 ligase RNF146, leaving YAP/TAZ free to translocate to the nucleus [48]. Here, YAP/TAZ forms the active transcription complex with TEAD [48]. Multiple efforts have been made to find effective YAP/TAZ activity inhibiting drugs, whereas tankyrase inhibition has shown YAP/TAZ

suppressing functions. By inhibiting TNKS1/2, AMOT proteins can associate with transcription co-factors YAP/TAZ and leave the nucleus. Thus, YAP/TAZ signaling gets suppressed [48]. [48]

Figure 10. Tankyrase inhibition suppresses the Hippo signaling network. Left panel: Without tankyrase inhibitor, active tankyrase PARsylates AMOT proteins, leading to their degradation by RNF146 and 26S proteasome, leaving YAP/TAZ free to translocate into the nucleus. Here, YAP/TAZ forms the active transcription complex with TEAD. Right panel: Tankyrase inhibition leads to an increased level of AMOT proteins, which binds YAP/TAZ and either causes cytoplasmic retention or activation of LATS ½ and thereby ubiquitin-mediated proteasomal degradation. Abbreviations:

phosphate group (P), ubiquitin (Ub), Figure created with BioRender.com, inspired by [48].

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1.5.5 Tankyrase inhibition and tankyrase inhibitors

The druggable effect on the catalytic domains in TNKS1/2 makes the inhibition of the enzymes a topic of interest in cancer treatment, leading to less active pro-oncogenic signaling pathways.

Inhibitors bind to the catalytic domain and can be classified into drugs that attach to the

nicotinamide binding pocket or those that bind to the catalytic domains adenosine binding pocket [58]. The nicotinamide binding pocket is similar for all types of PARPs, and an example of a developed inhibitor that binds there is XAV939. TNKS1/2 are the only proteins in the PARP family that contain adenosine binding pockets, whereas other developed inhibitors such as G007-LK and OM-153 can bind [59]. Hence, G007-G007-LK and OM-153 are more specific TNKS1/2 inhibitors compared to XAV939.

Multiple tankyrase inhibitors have been developed because they inhibit key cancer-promoting signaling pathways [60]. In addition to WNT and Hippo signaling pathway, TNKS1/2 can act on various other signaling pathways such as phosphatidylinositol-4,5-bisphosphate 3-kinase

(PI3K)/AKT serine/threonine kinase 1 (AKT) [48, 60, 61]. Although TNKS1/2 seems like promising target candidates in many of those pathways, there are currently no tankyrase inhibitors available for clinical practice.

Tankyrase inhibitor monotherapy has shown limited responsiveness in malignant melanoma. A synergic effect may be achieved by combining tankyrase inhibition with other therapies, such as chemotherapy, programmed cell death (PD-1), PI3K inhibitor BKM120, and epidermal growth factor receptor (EGFR) inhibitor erlotinib against the different types of cancer [25, 53, 62]. For example, combined inhibition of TNKS1/2 with G007-LK and the breakthrough of immune checkpoint inhibitors in anti-cancer therapy have been reported to sensitize melanoma tumors to anti-PD-1 immune checkpoint therapy [25].