Genetic and immune landscape evolution in MMR-deficient colorectal cancer.

Mismatch repair-deficient (MMRd) colorectal cancers (CRCs) have high mutation burdens, which make these tumours immunogenic and many respond to immune checkpoint inhibitors. The MMRd hypermutator phenotype may also promote intratumour heterogeneity (ITH) and cancer evolution. We applied multiregion sequencing and CD8 and programmed death ligand 1 (PD-L1) immunostaining to systematically investigate ITH and how genetic and immune landscapes coevolve. All cases had high truncal mutation burdens. Despite pervasive ITH, driver aberrations showed a clear hierarchy. Those in WNT/ß-catenin, mitogen-activated protein kinase, and TGF-ß receptor family genes were almost always truncal. Immune evasion (IE) drivers, such as inactivation of genes involved in antigen presentation or IFN-γ signalling, were predominantly subclonal and showed parallel evolution. These IE drivers have been implicated in immune checkpoint inhibitor resistance or sensitivity. Clonality assessments are therefore important for the development of predictive immunotherapy biomarkers in MMRd CRCs. Phylogenetic analysis identified three distinct patterns of IE driver evolution: pan-tumour evolution, subclonal evolution, and evolutionary stasis. These, but neither mutation burdens nor heterogeneity metrics, significantly correlated with T-cell densities, which were used as a surrogate marker of tumour immunogenicity. Furthermore, this revealed that genetic and T-cell infiltrates coevolve in MMRd CRCs. Low T-cell densities in the subgroup without any known IE drivers may indicate an, as yet unknown, IE mechanism. PD-L1 was expressed in the tumour microenvironment in most samples and correlated with T-cell densities. However, PD-L1 expression in cancer cells was independent of T-cell densities but strongly associated with loss of the intestinal homeobox transcription factor CDX2. This explains infrequent PD-L1 expression by cancer cells and may contribute to a higher recurrence risk of MMRd CRCs with impaired CDX2 expression. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

ADP-ribosyltransferases, an update on function and nomenclature.

ADP-ribosylation, a modification of proteins, nucleic acids, and metabolites, confers broad functions, including roles in stress responses elicited, for example, by DNA damage and viral infection and is involved in intra- and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis, and cell death. ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs), which transfer ADP-ribose from NAD+ onto substrates. The modification, which occurs as mono- or poly-ADP-ribosylation, is reversible due to the action of different ADP-ribosylhydrolases. Importantly, inhibitors of ARTs are approved or are being developed for clinical use. Moreover, ADP-ribosylhydrolases are being assessed as therapeutic targets, foremost as antiviral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP-ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP-ribosylation. In addition, characterization of biochemical and structural aspects of the ARTs and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ARTs that will facilitate future discussions when addressing the biochemistry and biology of ADP-ribosylation. This is combined with a brief description of the main functions of mammalian ARTs to illustrate the increasing diversity of mono- and poly-ADP-ribose mediated cellular processes.

Reconstitution of the destruction complex defines roles of AXIN polymers and APC in ß-catenin capture, phosphorylation, and ubiquitylation.

The Wnt/ß-catenin pathway is a highly conserved, frequently mutated developmental and cancer pathway. Its output is defined mainly by ß-catenin's phosphorylation- and ubiquitylation-dependent proteasomal degradation, initiated by the multi-protein ß-catenin destruction complex. The precise mechanisms underlying destruction complex function have remained unknown, largely because of the lack of suitable in vitro systems. Here we describe the in vitro reconstitution of an active human ß-catenin destruction complex from purified components, recapitulating complex assembly, ß-catenin modification, and degradation. We reveal that AXIN1 polymerization and APC promote ß-catenin capture, phosphorylation, and ubiquitylation. APC facilitates ß-catenin's flux through the complex by limiting ubiquitylation processivity and directly interacts with the SCFß-TrCP E3 ligase complex in a ß-TrCP-dependent manner. Oncogenic APC truncation variants, although part of the complex, are functionally impaired. Nonetheless, even the most severely truncated APC variant promotes ß-catenin recruitment. These findings exemplify the power of biochemical reconstitution to interrogate the molecular mechanisms of Wnt/ß-catenin signaling.

Fragment-based screening identifies molecules targeting the substrate-binding ankyrin repeat domains of tankyrase.

The PARP enzyme and scaffolding protein tankyrase (TNKS, TNKS2) uses its ankyrin repeat clusters (ARCs) to bind a wide range of proteins and thereby controls diverse cellular functions. A number of these are implicated in cancer-relevant processes, including Wnt/ß-catenin signalling, Hippo signalling and telomere maintenance. The ARCs recognise a conserved tankyrase-binding peptide motif (TBM). All currently available tankyrase inhibitors target the catalytic domain and inhibit tankyrase's poly(ADP-ribosyl)ation function. However, there is emerging evidence that catalysis-independent "scaffolding" mechanisms contribute to tankyrase function. Here we report a fragment-based screening programme against tankyrase ARC domains, using a combination of biophysical assays, including differential scanning fluorimetry (DSF) and nuclear magnetic resonance (NMR) spectroscopy. We identify fragment molecules that will serve as starting points for the development of tankyrase substrate binding antagonists. Such compounds will enable probing the scaffolding functions of tankyrase, and may, in the future, provide potential alternative therapeutic approaches to inhibiting tankyrase activity in cancer and other conditions.

Genomic and Transcriptomic Determinants of Therapy Resistance and Immune Landscape Evolution during Anti-EGFR Treatment in Colorectal Cancer.

Despite biomarker stratification, the anti-EGFR antibody cetuximab is only effective against a subgroup of colorectal cancers (CRCs). This genomic and transcriptomic analysis of the cetuximab resistance landscape in 35 RAS wild-type CRCs identified associations of NF1 and non-canonical RAS/RAF aberrations with primary resistance and validated transcriptomic CRC subtypes as non-genetic predictors of benefit. Sixty-four percent of biopsies with acquired resistance harbored no genetic resistance drivers. Most of these had switched from a cetuximab-sensitive transcriptomic subtype at baseline to a fibroblast- and growth factor-rich subtype at progression. Fibroblast-supernatant conferred cetuximab resistance in vitro, confirming a major role for non-genetic resistance through stromal remodeling. Cetuximab treatment increased cytotoxic immune infiltrates and PD-L1 and LAG3 immune checkpoint expression, potentially providing opportunities to treat cetuximab-resistant CRCs with immunotherapy.

CEA expression heterogeneity and plasticity confer resistance to the CEA-targeting bispecific immunotherapy antibody cibisatamab (CEA-TCB) in patient-derived colorectal cancer organoids.

BACKGROUND: The T cell bispecific antibody cibisatamab (CEA-TCB) binds Carcino-Embryonic Antigen (CEA) on cancer cells and CD3 on T cells, which triggers T cell killing of cancer cell lines expressing moderate to high levels of CEA at the cell surface. Patient derived colorectal cancer organoids (PDOs) may more accurately represent patient tumors than established cell lines which potentially enables more detailed insights into mechanisms of cibisatamab resistance and sensitivity. METHODS: We established PDOs from multidrug-resistant metastatic CRCs. CEA expression of PDOs was determined by FACS and sensitivity to cibisatamab immunotherapy was assessed by co-culture of PDOs and allogeneic CD8 T cells. RESULTS: PDOs could be categorized into 3 groups based on CEA cell-surface expression: CEAhi (n = 3), CEAlo (n = 1) and CEAmixed PDOs (n = 4), that stably maintained populations of CEAhi and CEAlo cells, which has not previously been described in CRC cell lines. CEAhi PDOs were sensitive whereas CEAlo PDOs showed resistance to cibisatamab. PDOs with mixed expression showed low sensitivity to cibisatamab, suggesting that CEAlo cells maintain cancer cell growth. Culture of FACS-sorted CEAhi and CEAlo cells from PDOs with mixed CEA expression demonstrated high plasticity of CEA expression, contributing to resistance acquisition through CEA antigen loss. RNA-sequencing revealed increased WNT/ß-catenin pathway activity in CEAlo cells. Cell surface CEA expression was up-regulated by inhibitors of the WNT/ß-catenin pathway. CONCLUSIONS: Based on these preclinical findings, heterogeneity and plasticity of CEA expression appear to confer low cibisatamab sensitivity in PDOs, supporting further clinical evaluation of their predictive effect in CRC. Pharmacological inhibition of the WNT/ß-catenin pathway may be a rational combination to sensitize CRCs to cibisatamab. Our novel PDO and T cell co-culture immunotherapy models enable pre-clinical discovery of candidate biomarkers and combination therapies that may inform and accelerate the development of immuno-oncology agents in the clinic.

Structural Basis for Auto-Inhibition of the NDR1 Kinase Domain by an Atypically Long Activation Segment.

The human NDR family kinases control diverse aspects of cell growth, and are regulated through phosphorylation and association with scaffolds such as MOB1. Here, we report the crystal structure of the human NDR1 kinase domain in its non-phosphorylated state, revealing a fully resolved atypically long activation segment that blocks substrate binding and stabilizes a non-productive position of helix αC. Consistent with an auto-inhibitory function, mutations within the activation segment of NDR1 dramatically enhance in vitro kinase activity. Interestingly, NDR1 catalytic activity is further potentiated by MOB1 binding, suggesting that regulation through modulation of the activation segment and by MOB1 binding are mechanistically distinct. Lastly, deleting the auto-inhibitory activation segment of NDR1 causes a marked increase in the association with upstream Hippo pathway components and the Furry scaffold. These findings provide a point of departure for future efforts to explore the cellular functions and the mechanism of NDR1.

Genome-wide and high-density CRISPR-Cas9 screens identify point mutations in PARP1 causing PARP inhibitor resistance.

Although PARP inhibitors (PARPi) target homologous recombination defective tumours, drug resistance frequently emerges, often via poorly understood mechanisms. Here, using genome-wide and high-density CRISPR-Cas9 "tag-mutate-enrich" mutagenesis screens, we identify close to full-length mutant forms of PARP1 that cause in vitro and in vivo PARPi resistance. Mutations both within and outside of the PARP1 DNA-binding zinc-finger domains cause PARPi resistance and alter PARP1 trapping, as does a PARP1 mutation found in a clinical case of PARPi resistance. This reinforces the importance of trapped PARP1 as a cytotoxic DNA lesion and suggests that PARP1 intramolecular interactions might influence PARPi-mediated cytotoxicity. PARP1 mutations are also tolerated in cells with a pathogenic BRCA1 mutation where they result in distinct sensitivities to chemotherapeutic drugs compared to other mechanisms of PARPi resistance (BRCA1 reversion, 53BP1, REV7 (MAD2L2) mutation), suggesting that the underlying mechanism of PARPi resistance that emerges could influence the success of subsequent therapies.

Regulation of Wnt/ß-catenin signalling by tankyrase-dependent poly(ADP-ribosyl)ation and scaffolding.

The Wnt/ß-catenin signalling pathway is pivotal for stem cell function and the control of cellular differentiation, both during embryonic development and tissue homeostasis in adults. Its activity is carefully controlled through the concerted interactions of concentration-limited pathway components and a wide range of post-translational modifications, including phosphorylation, ubiquitylation, sumoylation, poly(ADP-ribosyl)ation (PARylation) and acetylation. Regulation of Wnt/ß-catenin signalling by PARylation was discovered relatively recently. The PARP tankyrase PARylates AXIN1/2, an essential central scaffolding protein in the ß-catenin destruction complex, and targets it for degradation, thereby fine-tuning the responsiveness of cells to the Wnt signal. The past few years have not only seen much progress in our understanding of the molecular mechanisms by which PARylation controls the pathway but also witnessed the successful development of tankyrase inhibitors as tool compounds and promising agents for the therapy of Wnt-dependent dysfunctions, including colorectal cancer. Recent work has hinted at more complex roles of tankyrase in Wnt/ß-catenin signalling as well as challenges and opportunities in the development of tankyrase inhibitors. Here we review some of the latest advances in our understanding of tankyrase function in the pathway and efforts to modulate tankyrase activity to re-tune Wnt/ß-catenin signalling in colorectal cancer cells. LINKED ARTICLES: This article is part of a themed section on WNT Signalling: Mechanisms and Therapeutic Opportunities. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.24/issuetoc.

MOB1 Mediated Phospho-recognition in the Core Mammalian Hippo Pathway.

The Hippo tumor suppressor pathway regulates organ size and tissue homoeostasis in response to diverse signaling inputs. The core of the pathway consists of a short kinase cascade: MST1 and MST2 phosphorylate and activate LATS1 and LATS2, which in turn phosphorylate and inactivate key transcriptional coactivators, YAP1 and TAZ (gene WWTR1). The MOB1 adapter protein regulates both phosphorylation reactions firstly by concurrently binding to the upstream MST and downstream LATS kinases to enable the trans phosphorylation reaction, and secondly by allosterically activating the catalytic function of LATS1 and LATS2 to directly stimulate phosphorylation of YAP and TAZ. Studies of yeast Mob1 and human MOB1 revealed that the ability to recognize phosphopeptide sequences in their interactors, Nud1 and MST2 respectively, was critical to their roles in regulating the Mitotic Exit Network in yeast and the Hippo pathway in metazoans. However, the underlying rules of phosphopeptide recognition by human MOB1, the implications of binding specificity for Hippo pathway signaling, and the generality of phosphopeptide binding function to other human MOB family members remained elusive.Employing proteomics, peptide arrays and biochemical analyses, we systematically examine the phosphopeptide binding specificity of MOB1 and find it to be highly complementary to the substrate phosphorylation specificity of MST1 and MST2. We demonstrate that autophosphorylation of MST1 and MST2 on several threonine residues provides multiple MOB1 binding sites with varying binding affinities which in turn contribute to a redundancy of MST1-MOB1 protein interactions in cells. The crystal structures of MOB1A in complex with two favored phosphopeptide sites in MST1 allow for a full description of the MOB1A phosphopeptide-binding consensus. Lastly, we show that the phosphopeptide binding properties of MOB1A are conserved in all but one of the seven MOB family members in humans, thus providing a starting point for uncovering their elusive cellular functions.

AXIN Shapes Tankyrase ARChitecture.

The poly(ADP-ribose)polymerase (PARP) Tankyrase uses ankyrin repeat modules to capture substrates via Tankyrase-binding peptide motifs. In this issue of Structure, Eisemann et al. (2016) describe how the signaling protein AXIN can access and conformationally adapt the multivalent ankyrin repeat region of Tankyrase and discuss potential implications for enzymatic substrate modification.

Tankyrase Requires SAM Domain-Dependent Polymerization to Support Wnt-ß-Catenin Signaling.

The poly(ADP-ribose) polymerase (PARP) Tankyrase (TNKS and TNKS2) is paramount to Wnt-ß-catenin signaling and a promising therapeutic target in Wnt-dependent cancers. The pool of active ß-catenin is normally limited by destruction complexes, whose assembly depends on the polymeric master scaffolding protein AXIN. Tankyrase, which poly(ADP-ribosyl)ates and thereby destabilizes AXIN, also can polymerize, but the relevance of these polymers has remained unclear. We report crystal structures of the polymerizing TNKS and TNKS2 sterile alpha motif (SAM) domains, revealing versatile head-to-tail interactions. Biochemical studies informed by these structures demonstrate that polymerization is required for Tankyrase to drive ß-catenin-dependent transcription. We show that the polymeric state supports PARP activity and allows Tankyrase to effectively access destruction complexes through enabling avidity-dependent AXIN binding. This study provides an example for regulated signal transduction in non-membrane-enclosed compartments (signalosomes), and it points to novel potential strategies to inhibit Tankyrase function in oncogenic Wnt signaling.

The genomic landscape of oesophagogastric junctional adenocarcinoma.

The incidence of oesophagogastric junctional (OGJ) adenocarcinoma is rising rapidly in western countries, in contrast to the declining frequency of distal gastric carcinoma. Treatment options for adenocarcinomas involving the oesophagogastric junction are limited and the overall prognosis is extremely poor. To determine the genomic landscape of OGJ adenocarcinoma, exomes of eight tumours and matched germline DNA were subjected to massively parallel DNA sequencing. Microsatellite instability was observed in three tumours which coincided with an elevated number of somatic mutations. In total, 117 genes were identified that had predicted coding alterations in more than one tumour. Potentially actionable coding mutations were identified in 67 of these genes, including those in CR2, HGF , FGFR4, and ESRRB. Twenty-nine genes harbouring somatic coding mutations and copy number changes in the MSS OGJ dataset are also known to be altered with similar predicted functional consequence in other tumour types. Compared with the published mutational profile of gastric cancers, 49% (57/117) of recurrently mutated genes were unique to OGJ tumours. TP53, SYNE1, and ARID1A were amongst the most frequently mutated genes in a larger OGJ cohort. Our study provides an insight into the mutational landscape of OGJ adenocarcinomas and confirms that this is a highly mutated and heterogeneous disease. Furthermore, we have uncovered somatic mutations in therapeutically relevant genes which may represent candidate drug targets.

Structural basis and sequence rules for substrate recognition by Tankyrase explain the basis for cherubism disease.

The poly(ADP-ribose)polymerases Tankyrase 1/2 (TNKS/TNKS2) catalyze the covalent linkage of ADP-ribose polymer chains onto target proteins, regulating their ubiquitylation, stability, and function. Dysregulation of substrate recognition by Tankyrases underlies the human disease cherubism. Tankyrases recruit specific motifs (often called RxxPDG "hexapeptides") in their substrates via an N-terminal region of ankyrin repeats. These ankyrin repeats form five domains termed ankyrin repeat clusters (ARCs), each predicted to bind substrate. Here we report crystal structures of a representative ARC of TNKS2 bound to targeting peptides from six substrates. Using a solution-based peptide library screen, we derive a rule-based consensus for Tankyrase substrates common to four functionally conserved ARCs. This 8-residue consensus allows us to rationalize all known Tankyrase substrates and explains the basis for cherubism-causing mutations in the Tankyrase substrate 3BP2. Structural and sequence information allows us to also predict and validate other Tankyrase targets, including Disc1, Striatin, Fat4, RAD54, BCR, and MERIT40.

Molecular basis for G-actin binding to RPEL motifs from the serum response factor coactivator MAL.

Serum response factor transcriptional activity is controlled through interactions with regulatory cofactors such as the coactivator MAL/MRTF-A (myocardin-related transcription factor A). MAL is itself regulated in vivo by changes in cellular actin dynamics, which alter its interaction with G-actin. The G-actin-sensing mechanism of MAL/MRTF-A resides in its N-terminal domain, which consists of three tandem RPEL repeats. We describe the first molecular insights into RPEL function obtained from structures of two independent RPEL(MAL) peptide:G-actin complexes. Both RPEL peptides bind to the G-actin hydrophobic cleft and to subdomain 3. These RPEL(MAL):G-actin structures explain the sequence conservation defining the RPEL motif, including the invariant arginine. Characterisation of the RPEL(MAL):G-actin interaction by fluorescence anisotropy and cell reporter-based assays validates the significance of actin-binding residues for proper MAL localisation and regulation in vivo. We identify important differences in G-actin engagement between the two RPEL(MAL) structures. Comparison with other actin-binding proteins reveals an unexpected similarity to the vitamin-D-binding protein, extending the G-actin-binding protein repertoire.

Mutant actins that stabilise F-actin use distinct mechanisms to activate the SRF coactivator MAL.

Nuclear accumulation of the serum response factor coactivator MAL/MKL1 is controlled by its interaction with G-actin, which results in its retention in the cytoplasm in cells with low Rho activity. We previously identified actin mutants whose expression promotes MAL nuclear accumulation via an unknown mechanism. Here, we show that actin interacts directly with MAL in vitro with high affinity. We identify a further activating mutation, G15S, which stabilises F-actin, as do the activating actins S14C and V159N. The three mutants share several biochemical properties, but can be distinguished by their ability to bind cofilin, ATP and MAL. MAL interaction with actin S14C is essentially undetectable, and that with actin V159N is weakened. In contrast, actin G15S interacts more strongly with MAL than the wild-type protein. Strikingly, the nuclear accumulation of MAL induced by overexpression of actin S14C is substantially dependent on Rho activity and actin treadmilling, while that induced by actin G15S expression is not. We propose a model in which actin G15S acts directly to promote MAL nuclear entry.

A myristoyl/phosphotyrosine switch regulates c-Abl.

The c-Abl tyrosine kinase is inhibited by mechanisms that are poorly understood. Disruption of these mechanisms in the Bcr-Abl oncoprotein leads to several forms of human leukemia. We found that like Src kinases, c-Abl 1b is activated by phosphotyrosine ligands. Ligand-activated c-Abl is particularly sensitive to the anti-cancer drug STI-571/Gleevec/imatinib (STI-571). The SH2 domain-phosphorylated tail interaction in Src kinases is functionally replaced in c-Abl by an intramolecular engagement of the N-terminal myristoyl modification with the kinase domain. Functional studies coupled with structural analysis define a myristoyl/phosphotyrosine switch in c-Abl that regulates docking and accessibility of the SH2 domain. This mechanism offers an explanation for the observed cellular activation of c-Abl by tyrosine-phosphorylated proteins, the intracellular mobility of c-Abl, and it provides new insights into the mechanism of action of STI-571.