Michael Ranes

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: CEA_hi (n = 3), CEA_lo (n = 1) and CEA_mixed PDOs (n = 4), that stably maintained populations of CEA_hi and CEA_lo cells, which has not previously been described in CRC cell lines. CEA_hi PDOs were sensitive whereas CEA_lo PDOs showed resistance to cibisatamab. PDOs with mixed expression showed low sensitivity to cibisatamab, suggesting that CEA_lo cells maintain cancer cell growth. Culture of FACS-sorted CEA_hi and CEA_lo 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/β-catenin pathway activity in CEA_lo cells. Cell surface CEA expression was up-regulated by inhibitors of the WNT/β-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/β-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.

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.

The poly(ADP-ribose)polymerase (PARP) enzyme tankyrase (TNKS/ARTD5, TNKS2/ARTD6) uses its ankyrin repeat clusters (ARCs) to recognize degenerate peptide motifs in a wide range of proteins, thereby recruiting such proteins and their complexes for scaffolding and/or poly(ADP-ribosyl)ation. Here, we provide guidance for predicting putative tankyrase-binding motifs, based on the previously delineated peptide sequence rules and existing structural information. We present a general method for the expression and purification of tankyrase ARCs from Escherichia coli and outline a fluorescence polarization assay to quantitatively assess direct ARC–TBM peptide interactions. We provide a basic protocol for evaluating binding and poly(ADP-ribosyl)ation of full-length candidate interacting proteins by full-length tankyrase in mammalian cells.

Cockayne syndrome B (CSB), best known for its role in transcription-coupled nucleotide excision repair (TC-NER), contains a ubiquitin-binding domain (UBD), but the functional connection between protein ubiquitylation and this UBD remains unclear. Here, we show that CSB is regulated via site-specific ubiquitylation. Mass spectrometry analysis of CSB identified lysine (K) 991 as a ubiquitylation site. Intriguingly, mutation of this residue (K991R) does not affect CSB's catalytic activity or protein stability, but greatly affects genome stability, even in the absence of induced DNA damage. Moreover, cells expressing CSB K991R are sensitive to oxidative DNA damage, but proficient for TC-NER. K991 becomes ubiquitylated upon oxidative DNA damage, and while CSB K991R is recruited normally to such damage, it fails to dissociate in a timely manner, suggesting a requirement for K991 ubiquitylation in CSB activation. Interestingly, deletion of CSB's UBD gives rise to oxidative damage sensitivity as well, while CSB ΔUBD and CSB K991R affects expression of overlapping groups of genes, further indicating a functional connection. Together, these results shed new light on the regulation of CSB, with K991R representing an important separation-of-function-mutation in this multi-functional protein.

Significance: Cockayne syndrome (CS) is an autosomal-recessive, multisystem disorder characterized by neurological disease, growth failure, developmental abnormalities, photosensitivity, and degeneration of organ systems such as the ear and eye, including cataracts. Most patients with CS carry mutations in Cockayne syndrome group B (CSB), best known for its role in transcription-coupled repair. Indeed, because various repair pathways are compromised in patient cells, CS is widely considered a genome instability syndrome. Here, we provide evidence from human and mouse cell models, as well as brain tissue from patients with CS, that the involvement of CSB in regulating gene expression can explain several features of CS. Together, our data suggest that dysregulation of gene regulatory networks rather than DNA repair defects may be the main cause of neurological symptoms in CS.

Formation of a ribonucleoprotein particle (mRNP) competent for export requires the coupling of transcription with mRNA processing and RNA export. A key link between these processes is provided by the THO complex. To progress in our understanding of this coupling, we have performed a search for suppressors of the transcription defect caused by the hpr1Δ mutation. This has permitted us to identify mutations in the genes for the RNA polymerase II mediator component Med10, the Sch9 protein kinase, and the Ypr045c protein. We report a role in transcription elongation for Ypr045c (Thp3) and the Csn12 component of the COP9 signalosome. Thp3 and Csn12 form a complex that is recruited to transcribed genes. Their mutations suppress the gene expression defects of THO complex mutants involved in mRNP biogenesis and export and show defects in mRNA accumulation. Transcription elongation impairment of thp3Δ mutants is shown by in vivo transcript run-on analysis performed in G-less systems. Thp3-Csn12 establishes a novel link between transcription and mRNA processing that opens new perspectives on our understanding of gene expression and reveals novel functions for a component of the COP9 signalosome. Thp3-Csn12 also copurifies with ribosomal proteins, which opens the possibility that it has other functions in addition to transcription.