Eric O Neill

Altered nucleoli are a well-established hallmark of cancer, but how oncogenic signalling remodels the nucleolus remains poorly understood. Here we used an inducible mouse model of pancreatic ductal adenocarcinoma (PDAC) to generate spatially resolved proteomic and phosphoproteomic maps of the nucleolus upon RAS oncogene activation. We identify a phosphorylation programme initiated by translocation of the Casein Kinase 2 (CK2) holoenzyme to the nucleolus. This programme amplifies rRNA synthesis and malignant ribosome biogenesis by phosphorylating factors that control RNA polymerase I transcription and early ribosomal RNA (rRNA) processing. Preventing the nucleolar activity of CK2 inhibits oncogene-induced rRNA production, while constitutive nucleolar trapping of CK2 is sufficient to activate rRNA synthesis in the absence of RAS oncogene. Mechanistically, CK2 accumulation in the nucleolus is mediated by direct binding to the 3 prime External Transcribed Sequence (3-ETS) of nascent precursor rRNA, creating an RNA-dependent self-amplifying feedback loop. Nucleolar CK2 accumulation is conserved across diverse human cancers, and its disruption synergises with inhibition of oncogenic RAS signalling to suppress anchorage-independent growth and tumourigenesis. Our study reveals 3-ETS as a CK2 signalling scaffold that amplifies oncogenic ribosome biogenesis, and defines a druggable nucleolar vulnerability that can be exploited by targeting this process.

Apical-basal polarity (ABP) establishment and maintenance is necessary for proper brain development, yet how it is controlled is unclear. Galectin-3 (Gal-3) has been previously implicated in ABP of epithelial cells, and, here, we find that it is apically expressed in human embryonic stem cells (hESCs) during neural induction. Gal-3 blockade disrupts ABP and alters the distribution of junctional proteins in hESC-derived neural rosettes and is rescued by addition of recombinant Gal-3. Transcriptomics analysis shows that blocking Gal-3 regulates expression of genes responsible for nervous system development and cell junction assembly, among others. Last, Gal-3 blockade during embryonic development in vivo reduces horizontal cell divisions, disturbs cortical layering of neural progenitors, and induces gyrification. These data uncover a regulatory mechanism for ABP in the brain and warrant caution in modulating Gal-3 during pregnancy.

Platelets are anucleate blood cells vital for hemostasis and immunity. During cell death and aberrant mitosis, nucleated cells release DNA, resulting in “cell-free” DNA in plasma (cfDNA). An excess of cfDNA is deleterious. Given their ability to internalize pathogen-derived nucleic acids, we hypothesized that platelets may also clear endogenous cfDNA. We found that, despite lacking a nucleus, platelets contained a repertoire of DNA fragments mapping across the nuclear genome. We detected fetal DNA in maternal platelets and cancer-derived DNA in platelets from patients with premalignant and cancerous lesions. As current liquid biopsy approaches utilize platelet-depleted plasma, important genetic information contained within platelets is being missed. This study establishes a physiological role for platelets that has not previously been highlighted, with broad translational relevance.

The mechanical environment is sensed through cell-matrix contacts with the cytoskeleton, but how signals transit the nuclear envelope to affect cell fate decisions remains unknown. Nuclear actin coordinates chromatin motility during differentiation and genome maintenance, yet it remains unclear how nuclear actin responds to mechanical force. The DNA-damage kinase ataxia telangiectasia and Rad3-related protein (ATR) translocates to the nuclear envelope to protect the nucleus during cell motility or compression. Here, we show that ATR drives nuclear actin assembly via recruitment of Filamin-A to the inner nuclear membrane through binding of the hippo pathway scaffold and ATR substrate, RASSF1A. Moreover, we demonstrate how germline RASSF1 mutation disables nuclear mechanotransduction resulting in cerebral cortex thinning and associates with common psychological traits. Thus, defective mechanical-regulated pathways may contribute to complex neurological disorders.

The mechanism by which DNA-damage affects self-renewal and pluripotency remains unclear. DNA damage and repair mechanisms have been largely elucidated in mutated cancer cells or simple eukaryotes, making valid interpretations on early development difficult. Here we show the impact of ionizing irradiation on the maintenance and early differentiation of mouse embryonic stem cells (ESCs). Our findings demonstrate that irradiation induces the upregulation of the p53 family genes, including p53, p63, and p73, resulting in elevated expression of the E3 ubiquitin ligases Itch and Trim32. Consequently, this impairs ESC maintenance by reducing the protein levels of key pluripotency transcription factors in both mouse ESCs and early embryos. Notably, our study reveals that irradiation-induced DNA damage leads to the recruitment of the BAF complex, causing it to dissociate from its binding sites on the target genes associated with the Yap, Wnt, and TGF-β pathways, thereby increasing signaling and promoting differentiation of ESCs into all three lineages. Importantly, pathway inhibition demonstrates that DNA damage accelerated ESC differentiation relies on Wnt and TGF-β, and is selectively dependent on p53 or p63/ p73 for mesoderm and endoderm respectively. Finally, our study reveals that p53 family proteins form complexes with effector proteins of key signaling pathways which actively contribute to ESC differentiation. In summary, this study uncovered a mechanism by which multiple differentiation signaling pathways converge on the p53 family genes to promote ESC differentiation and are impacted by exposure to ionizing radiation.

The immunosuppressive microenvironment in pancreatic ductal adenocarcinoma (PDAC) prevents tumor control and strategies to restore anti-cancer immunity (i.e. by increasing CD8 T-cell activity) have had limited success. Here, we demonstrate how inducing localized physical damage using ionizing radiation (IR) unmasks the benefit of immunotherapy by increasing tissue-resident natural killer (trNK) cells that support CD8 T activity. Our data confirms that targeting mouse orthotopic PDAC tumors with IR together with CCR5 inhibition and PD1 blockade reduces E-cadherin positive tumor cells by recruiting a hypoactive NKG2D^-ve NK population, phenotypically reminiscent of trNK cells, that supports CD8 T-cell involvement. We show an equivalent population in human single-cell RNA sequencing (scRNA-seq) PDAC cohorts that represents immunomodulatory trNK cells that could similarly support CD8 T-cell levels in a cDC1-dependent manner. Importantly, a trNK signature associates with survival in PDAC and other solid malignancies revealing a potential beneficial role for trNK in improving adaptive anti-tumor responses and supporting CCR5 inhibitor (CCR5i)/αPD1 and IR-induced damage as a novel therapeutic approach.

Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer, a disease with dismal overall survival. Advances in treatment are hindered by a lack of preclinical models. Here, we show how a personalized organotypic “avatar” created from resected tissue allows spatial and temporal reporting on a complete in situ tumor microenvironment and mirrors clinical responses. Our perfusion culture method extends tumor slice viability, maintaining stable tumor content, metabolism, stromal composition, and immune cell populations for 12 days. Using multiplexed immunofluorescence and spatial transcriptomics, we identify immune neighborhoods and potential for immunotherapy. We used avatars to assess the impact of a preclinically validated metabolic therapy and show recovery of stromal and immune phenotypes and tumor redifferentiation. To determine clinical relevance, we monitored avatar response to gemcitabine treatment and identify a patient avatar-predictable response from clinical follow-up. Thus, avatars provide valuable information for syngeneic testing of therapeutics and a truly personalized therapeutic assessment platform for patients.

The immunosuppressive microenvironment in pancreatic ductal adenocarcinoma (PDAC) prevents tumor control and strategies to restore anti-cancer immunity (i.e. by increasing CD8 T cell activity) have had limited success. Here we demonstrate how inducing localized physical damage using ionizing radiation (IR) unmasks the benefit of immunotherapy by increasing tissue-resident NK (trNK) cells that support CD8 T activity. Our data confirms that targeting mouse orthotopic PDAC tumors with IR together with CCR5 inhibition and PD1 blockade reduces E-cadherin positive tumor cells by recruiting a hypoactive NKG2D-ve NK population, phenotypically reminiscent of trNK cells, that supports CD8 T cell involvement. We show an equivalent population in human PDAC cohorts that represents immunomodulatory trNK cells that could similarly support CD8 T cell levels in a cDC1-dependent manner. Importantly, a trNK signature associates with survival in PDAC and solid malignancies revealing a potential beneficial role for trNK in improving adaptive anti-tumor responses and supporting CCR5i/αPD1 and IR-induced damage as a novel therapeutic approach.

The immunosuppressive microenvironment in pancreatic ductal adenocarcinoma (PDAC) prevents tumor control and strategies to restore anti-cancer immunity (i.e. by increasing CD8 T cell activity) have had limited success. Here we demonstrate how inducing localized physical damage using ionizing radiation (IR) unmasks the benefit of immunotherapy by increasing tissue-resident NK (trNK) cells that support CD8 T activity. Our data confirms that targeting mouse orthotopic PDAC tumors with IR together with CCR5 inhibition and PD1 blockade reduces E-cadherin positive tumor cells by recruiting a hypoactive NKG2D ^-ve NK population, phenotypically reminiscent of trNK cells, that supports CD8 T cell involvement. We show an equivalent population in human PDAC cohorts that represents immunomodulatory trNK cells that could similarly support CD8 T cell levels in a cDC1-dependent manner. Importantly, a trNK signature associates with survival in PDAC and solid malignancies revealing a potential beneficial role for trNK in improving adaptive anti-tumor responses and supporting CCR5i/αPD1 and IR-induced damage as a novel therapeutic approach.

DNA lesions occur across the genome and constitute a threat to cell viability; however, damage at specific genomic loci has a relatively greater impact on overall genome stability. The ribosomal RNA gene repeats (rDNA) are emerging fragile sites. Recent progress in understanding how the rDNA damage response is organized has highlighted a key role of adaptor proteins. Here, we show that the scaffold tumor suppressor RASSF1A is recruited to rDNA breaks. RASSF1A recruitment to double‐strand breaks is mediated by 53BP1 and depends on RASSF1A phosphorylation at Serine 131 by ATM kinase. Employing targeted rDNA damage, we uncover that RASSF1A recruitment promotes local ATM signaling. RASSF1A silencing, a common epigenetic event during malignant transformation, results in persistent breaks, rDNA copy number alterations and decreased cell viability. Overall, we identify a novel role for RASSF1A at rDNA break sites, provide mechanistic insight into how the DNA damage response is organized in a chromatin context, and provide further evidence for how silencing of the RASSF1A tumor suppressor contributes to genome instability.