Title: Molecular mechanisms of scaffolding proteins and tankyrase in oncogenic Wnt/ß-catenin signalling
Funder: Cancer Research UK

Abstract: Background Dysregulated Wnt/ß-catenin signalling, mostly arising from truncations in the tumour suppressor adenomatous polyposis coli (APC), is a key initiator of colorectal cancer (CRC). The precise molecular consequences of APC truncations are poorly understood, and our limited knowledge of targetable pathway nodes restricts therapeutic opportunities. APC is a core component of the ß-catenin destruction complex, which sequentially phosphorylates and ubiquitinates ß-catenin to trigger its proteasomal degradation. The poly(ADP-ribose)polymerase (PARP) tankyrase minimally PARylates the central destruction complex scaffold AXIN1/2, thereby either limiting destruction complex formation and function or promoting the Wnt-induced inactivation of the destruction complex. Tankyrase self-polymerisation is required for PARP activity, but at high tankyrase levels can drive Wnt/ß-catenin signalling independently of catalysis, via scaffolding. In a subset of APC-truncated CRCs, tankyrase catalytic inhibition substantially restores destruction complex function. We hypothesise that catalytically inhibited tankyrase acts as a scaffold to compensate for the loss of APC function, but that tankyrase accumulation limits the efficacy of catalytic tankyrase inhibitors. Aims We aim to 1) explore the regulation of the intact and APC-mutant destruction complex and AXIN1 by tankyrase, 2) explore the structural basis of tankyrase regulation by controlled polymerisation, and 3) develop inhibitors of the tankyrase-substrate protein-protein interaction to modulate both tankyrase-dependent scaffolding and catalysis. Methods Aim 1: We will reconstitute variants of the ß-catenin destruction complex from purified proteins to analyse complex composition and activity in the context of wild-type and mutant APC. We will next analyse how tankyrase, through catalytic and non-catalytic (scaffolding) activities, modulates the destruction complex. Analyses will include multi-angle light scattering (MALS), electron microscopy (EM), structural proteomics (native, hydrogen-deuterium exchange and cross-linking mass spectrometry), biophysical binding studies, enzyme assays and PARylation site mapping. Complementary functional analyses will be performed using cell models. Aim 2: Using cryo-EM, X-ray crystallography, biochemical, proteomics and cell-based approaches, we will dissect how PARylation-regulated polymerisation controls tankyrase. Aim 3: We aim to develop small molecules to disrupt the recruitment of substrates/binders to tankyrase to inhibit both tankyrase-dependent scaffolding and PARylation. Approaches will include structure-based drug design, biophysical binding assays, in-vitro PARP activity assays and cell-based gene reporter studies. How the results of this research will be used This work will expand our understanding of how oncogenic APC truncations affect the destruction complex, potentially explain why cancer evolution selects for particular APC truncations and reveal how tankyrase modulation can be harnessed to curb oncogenic Wnt/ß-catenin signalling in CRC and other ß-catenin-dependent malignancies.

Lay abstract: Background Dysregulated Wnt/ß-catenin signalling, mostly arising from truncations in the tumour suppressor adenomatous polyposis coli (APC), is a key initiator of colorectal cancer (CRC). The precise molecular consequences of APC truncations are poorly understood, and our limited knowledge of targetable pathway nodes restricts therapeutic opportunities. APC is a core component of the ß-catenin destruction complex, which sequentially phosphorylates and ubiquitinates ß-catenin to trigger its proteasomal degradation. The poly(ADP-ribose)polymerase (PARP) tankyrase minimally PARylates the central destruction complex scaffold AXIN1/2, thereby either limiting destruction complex formation and function or promoting the Wnt-induced inactivation of the destruction complex. Tankyrase self-polymerisation is required for PARP activity, but at high tankyrase levels can drive Wnt/ß-catenin signalling independently of catalysis, via scaffolding. In a subset of APC-truncated CRCs, tankyrase catalytic inhibition substantially restores destruction complex function. We hypothesise that catalytically inhibited tankyrase acts as a scaffold to compensate for the loss of APC function, but that tankyrase accumulation limits the efficacy of catalytic tankyrase inhibitors. Aims We aim to 1) explore the regulation of the intact and APC-mutant destruction complex and AXIN1 by tankyrase, 2) explore the structural basis of tankyrase regulation by controlled polymerisation, and 3) develop inhibitors of the tankyrase-substrate protein-protein interaction to modulate both tankyrase-dependent scaffolding and catalysis. Methods Aim 1: We will reconstitute variants of the ß-catenin destruction complex from purified proteins to analyse complex composition and activity in the context of wild-type and mutant APC. We will next analyse how tankyrase, through catalytic and non-catalytic (scaffolding) activities, modulates the destruction complex. Analyses will include multi-angle light scattering (MALS), electron microscopy (EM), structural proteomics (native, hydrogen-deuterium exchange and cross-linking mass spectrometry), biophysical binding studies, enzyme assays and PARylation site mapping. Complementary functional analyses will be performed using cell models. Aim 2: Using cryo-EM, X-ray crystallography, biochemical, proteomics and cell-based approaches, we will dissect how PARylation-regulated polymerisation controls tankyrase. Aim 3: We aim to develop small molecules to disrupt the recruitment of substrates/binders to tankyrase to inhibit both tankyrase-dependent scaffolding and PARylation. Approaches will include structure-based drug design, biophysical binding assays, in-vitro PARP activity assays and cell-based gene reporter studies. How the results of this research will be used This work will expand our understanding of how oncogenic APC truncations affect the destruction complex, potentially explain why cancer evolution selects for particular APC truncations and reveal how tankyrase modulation can be harnessed to curb oncogenic Wnt/ß-catenin signalling in CRC and other ß-catenin-dependent malignancies.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;5.1 PHARMACEUTICALS
Health categories: CANCER AND NEOPLASMS