Biology subjects

Towler, B. P.

Publications and source records attributed to Towler, B. P..

6 recordsLinked to original sources

bioRxiv2026

RUNX1 aberrations in blast-phase CML induce the RBP SPATS2L which promotes growth, survival and stress granule assembly

The RUNX1 transcription factor is a critical regulator of hematopoiesis and frequently mutated in myeloid malignancies. In the myeloproliferative neoplasm, chronic myeloid leukemia (CML), secondary somatic RUNX1 mutations and RUNX1::MECOM/EVI1, are associated with tyrosine kinase inhibitor (TKI) resistance and progression to the blast-phase (BP-CML). Research has predominantly focussed on transcriptional dysregulation mediated by RUNX1 mutations in myeloid malignancies, whilst post-transcriptional dysregulation remains comparatively unexplored. To address this, we used orthogonal organic phase separation (OOPS), to characterise the RNA-binding proteome of RUNX1 deficient BP-CML cells. RUNX1 depleted BP-CML cells exhibited significant alterations to RBP abundance involved in stress response pathways and translation/ribosome-biogenesis (RiBi). Furthermore, RUNX1 depletion or expression of RUNX1::EVI1 in BP-CML cells induced expression and RNA binding activity of SPATS2L, a component of stress granules (SG); membraneless cytoplasmic condensates protecting mRNAs from degradation, promoting survival under stress. Whilst RUNX1 depletion increased SG-assembly, SPATS2L depletion reduced SG-assembly in BP-CML cells and inhibited the growth and survival of multiple BP-CML cell lines. The translation inhibitor homoharringtonine (HHT), used historically in TKI-resistant CML, ablated SG-assembly in BP-CML cells with RUNX1 depletion, and, primary BP-CML cells with LOF/hypomorphic RUNX1 mutations (characterised by defective DNA-binding/CBF{beta}-interaction) were preferentially sensitised to HHT. Finally, suppressing SPATS2L expression induced by RUNX1 depletion, increased the HHT-sensitivity of RUNX1 depleted BP-CML cells, suggesting SPATS2L contributes to therapeutic resistance in CML with RUNX1 mutations. This study suggests that SPATS2L and SG induction could be critical to RUNX1-mutant leukemias, and, provides preliminary evidence for a mutationally-targeted approach in CML with RUNX1 aberrations.

cancer biology↗

bioRxiv2026

Dual targeting a LIN28B:β-catenin axis in acute myeloid leukaemia

BackgroundWnt/{beta}-catenin signalling is dysregulated in acute myeloid leukaemia (AML), where it lacks effective targeting strategies. Previously, we discovered that {beta}-catenin interacts with several RNA-binding proteins (RBP), indicating post-transcriptional influence which is yet to be therapeutically interrogated in AML. MethodsCo-immunoprecipitation confirmed protein interactions, and TCF/LEF reporters were used to assess Wnt signalling output in leukaemia cells. Regulatory crosstalk was assessed using immunoblotting and RT-qPCR approaches following lentiviral transduction of myeloid cell lines. Targeting of {beta}-catenin and LIN28B was tested through combinations of genetic and pharmacological inhibition in AML cells. ResultsThe most frequent RBP-binding motif amongst {beta}-catenin-bound mRNAs was the GGAG motif targeted by oncofetal miRNA-regulating RBP; LIN28B. {beta}-Catenin:LIN28B interactions were detected in lymphoid and myeloid cell lines, plus primary human CD34 fetal-liver HSCs. LIN28B positively regulated Wnt signalling output through LEF1 regulation involving a post-transcriptional let7 miRNA mechanism. Further miRNA sequencing of {beta}-catenin- and LIN28B-depleted myeloid cells revealed potential cooperative and antagonistic function in miRNA regulation. Finally, dual-targeting both {beta}-catenin and LIN28B through either genetic and/or pharmacological means preferentially reduced AML cell viability. ConclusionThe {beta}-catenin:LIN28B axis could represent a novel synthetically lethal relationship in AML which could be exploited in rare subtypes where LIN28B expression becomes reactivated.

cancer biology↗

bioRxiv2024

A β-catenin:MSI2 axis regulates the expression of LEF1 and subsequent human haematopoietic stem/progenitor cell proliferation

Wnt/{beta}-catenin signaling is important for normal hematopoietic stem/progenitor cell (HSPC) biology and heavily implicated in acute myeloid leukaemia (AML). The central mediator {beta}-catenin is an attractive therapeutic target in AML however its targeting has been hampered by poor characterisation of its molecular interactions in haematopoietic cells. Our previous {beta}-catenin interactome study identified the significant enrichment of RNA-binding proteins (RBP) implying post-transcriptional roles for {beta}-catenin in myeloid cells. To identify {beta}-catenin-associated mRNAs we performed {beta}-catenin RNA-immunoprecipitation coupled to RNA-sequencing (RIP-seq) and identified significantly enriched Wnt signalling pathway transcripts. Using {beta}-catenin cross-linking immunoprecipitation (CLIP) we demonstrated a limited capacity for {beta}-catenin to bind RNA directly implying dependence on other RBPs. {beta}-Catenin was found to interact with MSI2 in both myeloid cell lines and AML patient samples, where expression was significantly correlated. MSI2 knockdown reduced Wnt signalling output (TCF/LEF activity), through suppression of LEF-1 expression and nuclear localization. Through both RIP and CLIP we demonstrate MSI2 binds LEF1 mRNA in a partly {beta}-catenin dependent fashion, and may impact the post-transcriptional control of LEF-1 expression. Finally, we show that MSI2-mediated expansion of human HSPCs could be partly driven through LEF1 regulation. This is the first study to show functional crosstalk between MSI2 and Wnt signalling in human cells, and indicates potential novel post-transcriptional roles for {beta}-catenin in a haematological context.

cell biology↗

bioRxiv2023

Characterisation of the in-vivo miRNA landscape in Drosophila ribonuclease mutants reveals Pacman mediated regulation of the highly conserved let-7 cluster during apoptotic processes

The control of gene expression is a fundamental process essential for correct development and to maintain homeostasis. Many post-transcriptional mechanisms exist to maintain the correct levels of each RNA transcript within the cell. Controlled and targeted cytoplasmic RNA degradation is one such mechanism with the 5-3 exoribonuclease Pacman (XRN1) and the 3-5 exoribonuclease Dis3L2 playing crucial roles. Loss of function mutations in either Pacman or Dis3L2 have been demonstrated to result in distinct phenotypes, and both have been implicated in human disease. One mechanism by which gene expression is controlled is through the function of miRNAs which have been shown to be crucial for the control of almost all cellular processes. Although the biogenesis and mechanisms of action of miRNAs have been comprehensively studied, the mechanisms regulating their own turnover are not well understood. Here we characterise the miRNA landscape in a natural developing tissue, the Drosophila melanogaster wing imaginal disc, and assess the importance of Pacman and Dis3L2 on the abundance of miRNAs. We reveal a complex landscape of miRNA expression and show that whilst a null mutation in dis3L2 has a minimal effect on the miRNA expression profile, loss of Pacman has a profound effect with a third of all detected miRNAs demonstrating Pacman-sensitivity. We also reveal a role of Pacman in regulating the highly conserved let-7 cluster (containing miR-100, let-7 and miR-125) and present a genetic model outlining a positive feedback loop regulated by Pacman which begins to explain the apoptotic phenotype observed in Pacman mutants.

developmental biology↗

bioRxiv2020

Genome-wide analyses of XRN1-sensitive targets in osteosarcoma cells identifies disease-relevant transcripts containing G-rich motifs.

XRN1 is a highly conserved exoribonuclease which degrades uncapped RNAs in a 5-3 direction. Degradation of RNAs by XRN1 is important in many cellular and developmental processes and is relevant to human disease. Studies in D. melanogaster demonstrate that XRN1 can target specific RNAs, which have important consequences for developmental pathways. Osteosarcoma is a malignancy of the bone and accounts for 2% of all paediatric cancers worldwide. 5 year survival of patients has remained static since the 1970s and therefore furthering our molecular understanding of this disease is crucial. Previous work has shown a downregulation of XRN1 in osteosarcoma cells, however the transcripts regulated by XRN1 which might promote osteosarcoma remain elusive. Here, we confirm reduced levels of XRN1 in osteosarcoma cell lines and patient samples and identify XRN1-sensitive transcripts in human osteosarcoma cells. Using RNA-seq in XRN1-knockdown SAOS-2 cells, we show that 1178 genes are differentially regulated. Using a novel bioinformatic approach, we demonstrate that 134 transcripts show characteristics of direct post-transcriptional regulation by XRN1. Long non-coding RNAs (lncRNAs) are enriched in this group suggesting that XRN1 normally plays an important role in controlling lncRNA expression in these cells. Among potential lncRNAs targeted by XRN1 is HOTAIR, which is known to be upregulated in osteosarcoma and contribute to disease progression. We have also identified G-rich and GU motifs in post-transcriptionally regulated transcripts which appear to sensitise them to XRN1 degradation. Our results therefore provide significant insights into the specificity of XRN1 in human cells which is relevant to disease.

molecular biology↗

bioRxiv2019

Dis3L2 regulates cellular proliferation through a PI3-Kinase dependent signalling pathway

Dis3L2 is a highly conserved 3-5 exoribonuclease which is mutated in the human overgrowth disorders Perlman syndrome and Wilms tumour. Here, we have generated a new dis3L2 null mutant and a UAS-nuclease-dead line in Drosophila to demonstrate that the catalytic activity of Dis3L2 is required to control proliferation in imaginal discs. Using RNA-seq on dis3L2 mutant wing discs, we show that the imaginal disc growth factor Idgf2 is responsible for driving the wing overgrowth observed in dis3L2 null mutants. IDGFs are conserved proteins homologous to human chitinase-like proteins such as CHI3L1/YKL-40 which are implicated in tissue regeneration as well as cancers including colon cancer and non-small cell lung cancer. We also show that loss of DIS3L2 in human HEK-293T cells results in cell proliferation, illustrating the conservation of this important cell proliferation pathway. Using these human cells we show that loss of DIS3L2 results in an increase in the activity of the PI3-Kinase/AKT signalling pathway, which we subsequently show to also have a role in driving the proliferation phenotype in Drosophila. Our work therefore provides the first mechanistic explanation for DIS3L2-induced overgrowth in humans and flies and identifies an ancient proliferation pathway controlled by Dis3L2 to regulate cell proliferation and tissue growth.

molecular biology↗