An investigation into the role of the initiator methionine transfer RNA in cell migration and tumour growth

Control of cellular tRNA repertoires can drive specific programmes of translation to favour the maintenance of proliferative or differentiated phenotypes. tRNAiMet is the initiator methionine tRNA, responsible for recognising the start codon and initiating translation. We have investigated how increased expression of tRNAiMet can influence cell behaviour, using both immortalised cell lines in vitro and mouse models in vivo. Levels of tRNAiMet are increased in carcinoma-associated fibroblasts compared to normal fibroblasts. To understand the cellular effects of tRNAiMet overexpression in more detail we generated immortalised mouse embryonic fibroblasts (iMEFs) that overexpressed tRNAiMet (iMEF.tRNAiMet) or an empty vector as control (iMEF.Vector). Full characterisation of iMEF.Vector and iMEF.tRNAiMet cell lines showed that overexpression of tRNAiMet did not affect cell size, energy metabolism, cell spreading, rate of cellular protein synthesis or proliferation. Increased expression of tRNAiMet did, however, have marked effects on cell migration; with iMEF.tRNAiMet cells migrating approximately 1.5 fold faster than iMEF.Vector controls when assessing both directional and random migration. This tRNAiMet-driven increase in cell speed was dependent on the levels of phosphorylated eIF2α, indicating that fibroblast migration might be influenced by tRNAiMet in the ternary complex. Furthermore, the ability of tRNAiMet to increase cell migration depended on the ability of integrin α5β1 to bind its extracellular ligand fibronectin. However, despite the robust and reproducible role of both phospho-eIF2α and integrin α5β1 in this process, the way in which these are mechanistically linked to tRNAiMet levels is yet to be determined. To investigate whether increased stromal tRNAiMet expression may contribute to tumour progression, we utilised a mouse that expressed additional copies of the tRNAiMet gene (2+tRNAiMet mouse), and performed syngeneic allografts into these animals. Subcutaneous allograft tumours of a number of different cancer cell lines became more vascularised and grew significantly more rapidly in 2+tRNAiMet mice by comparison with tumours grown in littermate control animals. The extracellular matrix (ECM) that was deposited by fibroblasts from 2+tRNAiMet mice was found to support enhanced endothelial cell and fibroblast migration. We used SILAC mass spectrometry to compare the secretome of iMEF.Vector and iMEF.tRNAiMet cell lines and found that overexpression of tRNAiMet significantly increased synthesis and secretion of certain types of collagen, in particular collagen II. Moreover, knockdown of collagen II using siRNA and CRISPR approaches opposed the ability of tRNAiMet overexpressing fibroblasts to deposit a pro-migratory ECM. We used the prolyl hydroxylase inhibitor, ethyl-3,4-dihydroxybenzoate (DHB), to determine whether collagen synthesis contributed to the ability of tRNAiMet to drive a pro-tumorigenic stroma in vivo. Administration of DHB had no effect on the growth of syngeneic allografts in wild-type mice, but opposed the ability of 2+tRNAiMet animals to support increased angiogenesis and tumour growth. Collectively these data indicate that increased expression of tRNAiMet contributes to tumour progression by enhancing the ability of stromal fibroblasts to synthesise and secrete a collagen II-rich ECM that supports endothelial cell migration and angiogenesis. Taken together these data provide evidence that the tRNAome, and in particular cellular levels of tRNAiMet, influence both the migration of fibroblasts and the composition of their secretome in a way that promotes the generation of a microenvironment supportive of endothelial cell migration, angiogenesis and tumour growth

The initiator methionine tRNA drives cell migration and invasion leading to increased metastatic potential in melanoma

The cell's repertoire of transfer RNAs (tRNAs) has been linked to cancer. Recently, levels of the initiator methionine tRNA (tRNAiMet) in stromal fibroblasts have been shown to influence extracellular matrix (ECM) secretion to drive tumour growth and angiogenesis. Here we show that increased tRNAiMet within cancer cells does not influence tumour growth, but drives cell migration and invasion via a mechanism that is independent from ECM synthesis and dependent on α5β1 integrin and levels of the translation initiation ternary complex. In vivo and ex vivo migration (but not proliferation) of melanoblasts is significantly enhanced in transgenic mice which express additional copies of the tRNAiMet gene. We show that increased tRNAiMet in melanoma drives migratory, invasive behaviour and metastatic potential without affecting cell proliferation and primary tumour growth, and that expression of RNA polymerase III-associated genes (which drive tRNA expression) are elevated in metastases by comparison with primary tumours. Thus specific alterations to the cancer cell tRNA repertoire drive a migration/invasion programme that may lead to metastasis

The initiator methionine tRNA drives secretion of type II collagen from stromal fibroblasts to promote tumor growth and angiogenesis

Summary: Expression of the initiator methionine tRNA (tRNAi Met) is deregulated in cancer. Despite this fact, it is not currently known how tRNAi Met expression levels influence tumor progression. We have found that tRNAi Met expression is increased in carcinoma-associated fibroblasts, implicating deregulated expression of tRNAi Met in the tumor stroma as a possible contributor to tumor progression. To investigate how elevated stromal tRNAi Met contributes to tumor progression, we generated a mouse expressing additional copies of the tRNAi Met gene (2+tRNAi Met mouse). Growth and vascularization of subcutaneous tumor allografts was enhanced in 2+tRNAi Met mice compared with wild-type littermate controls. Extracellular matrix (ECM) deposited by fibroblasts from 2+tRNAi Met mice supported enhanced endothelial cell and fibroblast migration. SILAC mass spectrometry indicated that elevated expression of tRNAi Met significantly increased synthesis and secretion of certain types of collagen, in particular type II collagen. Suppression of type II collagen opposed the ability of tRNAi Metoverexpressing fibroblasts to deposit pro-migratory ECM. We used the prolyl hydroxylase inhibitor ethyl- 3,4-dihydroxybenzoate (DHB) to determine whether collagen synthesis contributes to the tRNAi Met-driven pro-tumorigenic stroma in vivo. DHB had no effect on the growth of syngeneic allografts in wild-type mice but opposed the ability of 2+tRNAi Met mice to support increased angiogenesis and tumor growth. Finally, collagen II expression predicts poor prognosis in high-grade serous ovarian carcinoma. Taken together, these data indicate that increased tRNAi Met levels contribute to tumor progression by enhancing the ability of stromal fibroblasts to synthesize and secrete a type II collagen-rich ECM that supports endothelial cell migration and angiogenesis

PATH-38. ROSETTE-FORMING GLIONEURONAL TUMOR IS DEFINED BY FGFR1 ACTIVATING ALTERATIONS WITH FREQUENT ACCOMPANYING PI3K AND MAPK PATHWAY MUTATIONS

Abstract BACKGROUND Rosette-forming glioneuronal tumor (RGNT) is an uncommon CNS tumor originally described in the fourth ventricle characterized by a low-grade glial neoplasm admixed with a rosette-forming neurocytic component. METHODS We reviewed clinicopathologic features of 42 patients with RGNT. Targeted next-generation sequencing was performed, and genome-wide methylation profiling is underway. RESULTS The 20 male and 22 female patients had a mean age of 25 years (range 3–47) at time of diagnosis. Tumors were located within or adjacent to the lateral ventricle (n=16), fourth ventricle (15), third ventricle (9), and spinal cord (2). All 31 tumors assessed to date contained FGFR1 activating alterations, either in-frame gene fusion, kinase domain tandem duplication, or hotspot missense mutation in the kinase domain (p.N546 or p.K656). While 7 of these 31 tumors harbored FGFR1 alterations as the solitary pathogenic event, 24 contained additional pathogenic alterations within PI3-kinase or MAP kinase pathway genes: 5 with additional PIK3CA and NF1 mutations, 4 with PIK3CA mutation, 3 with PIK3R1 mutation (one of which also contained focal RAF1 amplification), 5 with PTPN11 mutation (one with additional PIK3R1 mutation), and 2 with NF1 deletion. The other 5 cases demonstrated anaplastic features including hypercellularity and increased mitotic activity. Among these anaplastic cases, 3 harbored inactivating ATRX mutations and two harbored CDKN2A homozygous deletion, in addition to the FGFR1 alterations plus other PI3-kinase and MAP kinase gene mutations seen in those RGNT without anaplasia. CONCLUSION Independent of ventricular location, RGNT is defined by FGFR1 activating mutations or rearrangements, which are frequently accompanied by mutations involving PIK3CA, PIK3R1, PTPN11, NF1, and KRAS. Whereas pilocytic astrocytoma and ganglioglioma are characterized by solitary activating MAP kinase pathway alterations (e.g. BRAF fusion or mutation), RGNT are genetically more complex with dual PI3K-Akt-mTOR and Ras-Raf-MAPK pathway activation. Rare anaplastic examples may show additional ATRX and/or CDKN2A inactivation

Guided self-help cognitive-behaviour Intervention for VoicEs (GiVE): results from a pilot randomised controlled trial in a transdiagnostic sample

Background: Few patients have access to cognitive behaviour therapy for psychosis (CBTp) even though at least 16 sessions of CBTp is recommended in treatment guidelines. Briefer CBTp could improve access as the same number of therapists could see more patients. In addition, focusing on single psychotic symptoms, such as auditory hallucinations (‘voices’), rather than on psychosis more broadly, may yield greater benefits. Method: This pilot RCT recruited 28 participants (with a range of diagnoses) from NHS mental health services who were distressed by hearing voices. The study compared an 8-session guided self-help CBT intervention for distressing voiceswith a wait-list control. Data were collected at baseline and at 12 weekswith post-therapy assessments conducted blind to allocation. Voice-impact was the pre-determined primary outcome. Secondary outcomes were depression, anxiety, wellbeing and recovery. Mechanism measures were self-esteem, beliefs about self, beliefs about voices and voice-relating. Results: Recruitment and retention was feasible with low study (3.6%) and therapy (14.3%) dropout. There were large, statistically significant between-group effects on the primary outcome of voice-impact (d=1.78; 95% CIs: 0.86–2.70), which exceeded the minimum clinically important difference. Large, statistically significant effects were found on a number of secondary and mechanism measures. Conclusions: Large effects on the pre-determined primary outcome of voice-impact are encouraging, and criteria for progressing to a definitive trial are met. Significant between-group effects on measures of self-esteem, negative beliefs about self and beliefs about voiceomnipotence are consistentwith these beingmechanisms of change and this requires testing in a future trial

Activating p53 abolishes self-renewal of quiescent leukaemic stem cells in residual CML disease

Whilst it is recognised that targeting self-renewal is an effective way to functionally impair the quiescent leukaemic stem cells (LSC) that persist as residual disease in chronic myeloid leukaemia (CML), developing therapeutic strategies to achieve this have proved challenging. We demonstrate that the regulatory programmes of quiescent LSC in chronic phase CML are similar to that of embryonic stem cells, pointing to a role for wild type p53 in LSC self-renewal. In support of this, increasing p53 activity in primitive CML cells using an MDM2 inhibitor in combination with a tyrosine kinase inhibitor resulted in reduced CFC outputs and engraftment potential, followed by loss of multilineage priming potential and LSC exhaustion when combination treatment was discontinued. Our work provides evidence that targeting LSC self-renewal is exploitable in the clinic to irreversibly impair quiescent LSC function in CML residual disease – with the potential to enable more CML patients to discontinue therapy and remain in therapy-free remission

Hsa-mir183/EGR1-mediated regulation of E2F1 is required for CML stem/progenitor cell survival

Chronic myeloid leukemia (CML) stem/progenitor cells (SPC) express a transcriptional program characteristic of proliferation, yet can achieve and maintain quiescence. Understanding the mechanisms by which leukemic SPC maintain quiescence will help to clarify how they persist during long-term targeted treatment. We have identified a novel BCR-ABL1 protein kinase dependent pathway mediated by the up-regulation of hsa-mir183, the down-regulation of its direct target EGR1 and, as a consequence, up-regulation of E2F1. We show here that inhibition of hsa-mir183 reduced proliferation and impaired colony formation of CML SPC. Downstream of this, inhibition of E2F1 also reduced proliferation of CML SPC, leading to p53-mediated apoptosis. In addition, we demonstrate that E2F1 plays a pivotal role in regulating CML SPC proliferation status. Thus, for the first time, we highlight the mechanism of hsa-mir183/EGR1-mediated E2F1 regulation and demonstrate this axis as a novel, critical factor for CML SPC survival, offering new insights into leukemic stem cell eradication

Characterising neutrophil subtypes in cancer using scRNA sequencing demonstrates the importance of IL-1β/CXCR2 axis in generation of metastasis specific neutrophils

Neutrophils are a highly heterogeneous cellular population. However, a thorough examination of the different transcriptional neutrophil states between health and malignancy has not been performed. We utilized single-cell RNA sequencing of human and murine datasets, both publicly available and independently generated, to identify neutrophil transcriptomic subtypes and developmental lineages in health and malignancy. Datasets of lung, breast, and colorectal cancer were integrated to establish and validate neutrophil gene signatures. Pseudotime analysis was used to identify genes driving neutrophil development from health to cancer. Finally, ligand–receptor interactions and signaling pathways between neutrophils and other immune cell populations in primary colorectal cancer and metastatic colorectal cancer were investigated. We define two main neutrophil subtypes in primary tumors: an activated subtype sharing the transcriptomic signatures of healthy neutrophils; and a tumor-specific subtype. This signature is conserved in murine and human cancer, across different tumor types. In colorectal cancer metastases, neutrophils are more heterogeneous, exhibiting additional transcriptomic subtypes. Pseudotime analysis implicates IL1β/CXCL8/CXCR2 axis in the progression of neutrophils from health to cancer and metastasis, with effects on T-cell effector function. Functional analysis of neutrophil-tumoroid cocultures and T-cell proliferation assays using orthotopic metastatic mouse models lacking Cxcr2 in neutrophils support our transcriptional analysis. We propose that the emergence of metastatic-specific neutrophil subtypes is driven by the IL1β/CXCL8/CXCR2 axis, with the evolution of different transcriptomic signals that impair T-cell function at the metastatic site. Thus, a better understanding of neutrophil transcriptomic programming could optimize immunotherapeutic interventions into early and late interventions, targeting different neutrophil states

Investigation of hospital discharge cases and SARS-CoV-2 introduction into Lothian care homes

Background The first epidemic wave of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in Scotland resulted in high case numbers and mortality in care homes. In Lothian, over one-third of care homes reported an outbreak, while there was limited testing of hospital patients discharged to care homes. Aim To investigate patients discharged from hospitals as a source of SARS-CoV-2 introduction into care homes during the first epidemic wave. Methods A clinical review was performed for all patients discharges from hospitals to care homes from 1st March 2020 to 31st May 2020. Episodes were ruled out based on coronavirus disease 2019 (COVID-19) test history, clinical assessment at discharge, whole-genome sequencing (WGS) data and an infectious period of 14 days. Clinical samples were processed for WGS, and consensus genomes generated were used for analysis using Cluster Investigation and Virus Epidemiological Tool software. Patient timelines were obtained using electronic hospital records. Findings In total, 787 patients discharged from hospitals to care homes were identified. Of these, 776 (99%) were ruled out for subsequent introduction of SARS-CoV-2 into care homes. However, for 10 episodes, the results were inconclusive as there was low genomic diversity in consensus genomes or no sequencing data were available. Only one discharge episode had a genomic, time and location link to positive cases during hospital admission, leading to 10 positive cases in their care home. Conclusion The majority of patients discharged from hospitals were ruled out for introduction of SARS-CoV-2 into care homes, highlighting the importance of screening all new admissions when faced with a novel emerging virus and no available vaccine