Biology subjects

Pickett, H. A.

Publications and source records attributed to Pickett, H. A..

11 recordsLinked to original sources

bioRxiv2026

MUTYH activity maintains telomere stability in response to chronic telomeric 8-oxoguanine damage in cancer cells

Telomeres are highly susceptible to oxidative DNA damage, particularly 8-oxoguanine (8-oxoG), which is processed by glycosylase-initiated base excision repair (BER). OGG1 removes 8-oxoG opposite C, and MUTYH removes A misinserted opposite 8-oxoG to prevent mutations. While OGG1 has an established role in telomere protection, the contribution of MUTYH to telomere stability in cancer cells after oxidative DNA damage remains poorly understood. Using a chemoptogenetic system to induce targeted 8-oxoG lesions specifically at telomeres in HeLa cancer cells, we demonstrate that MUTYH is required to prevent telomere shortening, telomere loss, and genomic instability after chronic damage. Yet, telomere damage in MUTYH-deficient cells does not cause sustained DNA damage signaling or reduced cellular proliferation. Whole-genome sequencing further reveals enrichment of G to T transversions within telomeric repeats in MUTYH-deficient cells, consistent with increased mutagenesis due to unrepaired 8-oxoG:A mispairs. Combined loss of MUTYH and OGG1 rescues damage-induced telomere aberrations and genomic instability, implicating BER-generated single-strand break (SSB) intermediates as major contributors to telomere instability. In agreement, exo-FISH and S1-END-seq analyses reveal that repair-proficient cells rapidly accumulate SSB intermediates after damage, which are later resolved, whereas glycosylase-deficient cells exhibit SSBs at later time points. Together, these findings identify MUTYH as a critical guardian of telomere integrity during chronic oxidative stress and provide insight into how defective BER at telomeres contributes to genomic instability in cancer cells, with implications for cancers associated with MUTYH deficiency and mutations.

molecular biology↗

bioRxiv2025

Anillin-dependent actin assembly at PML NBs protects genome stability

Nuclear filamentous actin (F-actin) is emerging as a key regulator of genome stability1-6. During replication stress, transient bursts of nuclear actin assembly in S-phase promote fork remodelling and repair3,4, but how these bursts are initiated, regulated, and whether nuclear F-actin also act on DNA lesions inherited across cell cycles, remains unknown. Here, we show that replication stress induces nuclear actin assembly at liquid-liquid phase-separated (LLPS) promyelocytic leukaemia nuclear bodies (PML NBs), driven by the actin-binding protein Anillin. Although best known as a cytokinesis factor, Anillin localises to PML NBs in interphase, where it concentrates monomeric actin (G-actin) and establishes a favourable environment for spontaneous actin assembly. Filament growth within these condensates remodels PML NB morphology and composition, releasing sequestered AKT into the nucleoplasm. AKT is subsequently phosphorylated and activated, and this nuclear AKT activity is required to regulate 53BP1 nuclear bodies in G1. These protective LLPS compartments shield under-replicated DNA inherited from the stressed S-phase until the following S-phase. This prevents premature engagement by aberrant repair pathways that would otherwise generate toxic intermediates and culminate in mitotic failure. Together, these findings define a condensate-to-filament pathway in which nuclear F-actin dynamically reorganises phase-separated compartments to safeguard genome stability across cell cycles.

cell biology↗

bioRxiv2025

Potent synthetic lethality between PLK1 and EYA-family inhibitors in tumours of the central and peripheral nervous system

The Eyes Absent family of protein phosphatases (EYA1-4) are aberrantly expressed and tumour-promoting across many devastating cancers of neurological origin affecting both children and adults. It has recently been demonstrated that EYA1 and EYA4 promote tumour cell survival by increasing the active pool of Polo-like kinase 1 (PLK1) molecules. This discovery provides a rationale for the therapeutic combination of EYA inhibitors with direct, ATP-competitive, PLK1 inhibitors. Here, we demonstrate potent and synergistic effects of EYA and PLK1 inhibition in cancer cell lines that overexpress EYA1 and/or EYA4, including in neuroblastoma and glioblastoma models. We identify decreases in PLK1 activity and RAD51 foci formation, and increases in mitotic arrest and cell death, as mechanistic contributors to combination sensitivity. Combined EYA and PLK1 inhibition is also effective in glioblastoma stem cell models that overexpress EYA1/EYA4 and specifically targets the cancer stem cell state. Finally, through multi-omic correlational analysis, we identify high levels of the NuRD complex and SOX9 as contributors to combination treatment sensitivity. Overall, this work identifies a novel synthetic lethal combination therapy with potential utility across a wide range of neurological cancers.

cancer biology↗

bioRxiv2024

Mechanism of structure-specific DNA binding by the FANCM branchpoint translocase

FANCM is a DNA repair protein that recognizes stalled replication forks, and recruits downstream repair factors. FANCM activity is also essential for the survival of cancer cells that utilize the Alternative Lengthening of Telomeres (ALT) mechanism. FANCM efficiently recognizes stalled replication forks in the genome or at telomeres through its strong affinity for branched DNA structures. In this study, we demonstrate that the N-terminal translocase domain drives this specific branched DNA recognition. The Hel2i subdomain within the translocase is crucial for effective substrate engagement and couples DNA binding to catalytic ATP-dependent branch migration. Removal of Hel2i or mutation of key DNA-binding residues within this domain diminished FANCMs affinity for junction DNA and abolished branch migration activity. Importantly, these mutant FANCM variants failed to rescue the cell cycle arrest, telomere-associated replication stress, or lethality of ALT-positive cancer cells depleted of endogenous FANCM. Our results reveal the Hel2i domain is key for FANCM to properly engage DNA substrates, and therefore plays an essential role in its tumour-suppressive functions by restraining the hyperactivation of the ALT pathway.

biochemistry↗

bioRxiv2024

Homologous recombination promotes mitotic death to suppress the innate immune response

Double strand breaks (DSBs) can initiate mitotic catastrophe, a complex oncosuppressive phenomenon characterized by cell death during or after cell division. Through single-cell analysis of extended live imaging, we unveiled how cell cycle-regulated DSB repair guides disparate mitotic catastrophe outcomes. Our data reveal that toxic double Holliday junctions (dHjs) generated during homologous recombination (HR) promote non-immunogenic intrinsic apoptosis in the immediate mitosis after S or G2-phase DSB induction. Conversely, the combined activity of non-homologous end joining (NHEJ), microhomology mediated end joining (MMEJ), and single strand annealing (SSA) enable G1 phase cells to tolerate high DSB loads at the cost of aberrant cell division, innate immune response activation and delayed extrinsic lethality. Targeting NHEJ, MMEJ, or SSA promotes HR-dependent mitotic death, while suppressing mitotic death fosters a robust immunogenic response. Together the data indicate that a temporal repair hierarchy, coupled with cumulative DSB load, serves as a reliable predictor of mitotic catastrophe outcomes. In this pathway, HR suppress the innate immune response by promoting mitotic lethality.

cell biology↗

bioRxiv2023

Imetelstat-Mediated Alterations in Fatty Acid Metabolism To Induce Ferroptosis As Therapeutic Strategy for Acute Myeloid Leukemia

Telomerase enables replicative immortality in most cancers including acute myeloid leukemia (AML). Imetelstat is a first-in-class telomerase inhibitor with clinical efficacy in myelofibrosis and myelodysplastic syndromes. Here, we develop an AML patient-derived xenograft (PDX) resource, and perform integrated genomics, transcriptomics, and lipidomics analyses combined with functional genetics to identify key mediators of imetelstat efficacy. In a randomized Phase II-like preclinical trial in PDX, imetelstat effectively diminishes AML burden, and preferentially targets subgroups containing mutant NRAS and oxidative stress-associated gene expression signatures. Unbiased, genome-wide CRISPR/Cas9 editing identifies ferroptosis regulators as key mediators of imetelstat efficacy. Imetelstat promotes the formation of polyunsaturated fatty acid-containing phospholipids, causing excessive levels of lipid peroxidation and oxidative stress. Pharmacological inhibition of ferroptosis diminishes imetelstat efficacy. We leverage these mechanistic insights to develop an optimized therapeutic strategy using oxidative stress-inducing chemotherapy to sensitize patient samples to imetelstat causing significant disease control in AML.

cancer biology↗

bioRxiv2023

Irreversible inhibition of TRF2TRFH recruiting functions: a strategy to induce telomeric replication stress in cancer cells.

The shelterin component telomeric repeat-binding factor 2 (TRF2) is an essential regulator of telomere homeostasis and genomic stability. Mutations in the TRF2TRFH domain physically impair t-loop formation and prevent the recruitment of several factors that promote efficient telomere replication, resulting in a telomeric DNA damage response. Here, we design, synthesize, and biologically test covalent cyclic peptides that irreversibly target the TRF2TRFH domain. We identify APOD53 as our most promising compound. APOD53 forms a covalent adduct with a reactive cysteine residue present in the TRF2TRFH domain and induces phenotypes consistent with TRF2TRFH domain mutants. These include induction of a telomeric DNA damage response in the absence of fusions, increased telomeric replication stress, and impaired recruitment of regulator of telomere elongation helicase 1 (RTEL1) and structure-specific endonuclease subunit (SLX4) to telomeres. We demonstrate that APOD53 impairs cell growth in both a telomerase-positive and an ALT cell line, while sparing the viability of non-cancerous cells. Finally, we find that co-treatment with APOD53 and the G4 stabilizer RHPS4 further exacerbates telomere replication stress.

cancer biology↗

bioRxiv2022

The Eyes Absent family members EYA4 and EYA1 promote PLK1 activation and successful mitosis through tyrosine dephosphorylation.

The Eyes Absent family of proteins (EYA1-4) are a biochemically unique group of tyrosine phosphatases known to be tumour promoting across a range of cancer types. To date, the molecular targets of EYA phosphatase activity remain largely uncharacterised. Here, we identify Polo-like kinase 1 (PLK1) as a direct interactor and phosphatase substrate of both EYA4 and EYA1, with pY445 on PLK1 being the primary target site. EYA-mediated dephosphorylation of PLK1 in the G2 phase of the cell cycle is required for centrosome maturation, PLK1 localization to centrosomes, and polo-box domain (PBD) dependent interactions between PLK1 and the PLK1-activating proteins BORA and CEP192. Molecular dynamics simulations support the rationale that pY445 confers a structural impairment to PBD-substrate interactions that is relieved by EYA-mediated dephosphorylation. Depletion of EYA4 or EYA1, or chemical inhibition of EYA phosphatase activity, dramatically reduces PLK1 activation, causing mitotic defects and cell death. Overall, we have characterized a novel phosphotyrosine signalling network governing PLK1 and mitosis. This work provides a mechanism of cell killing for EYA phosphatase inhibitors with important therapeutic implications.

cell biology↗

bioRxiv2022

Replication stress tolerance and management differs between naive and primed pluripotent cells

Replication stress is an endemic threat to genome stability. For reasons unknown, replication stress response factors become essential during peri-implantation development. This coincides with a stem cell potency switch from the naive to the primed state. Using genetically matched, chimera-derived mouse naive embryonic (mESC) and primed epiblast stem cells (mEpiSC) we found that replication stress management differs between potency states. Primed mEpiSCs rely on Atr activity to prevent replication catastrophe, minimize genomic damage, avoid apoptosis, and re-enter the cell cycle. Conversely, under replications stress, mESCs readily activate Atm regardless of Atr activity, undergo replication catastrophe, and induce apoptosis. Primed pluripotent cells therefore engage Atr to counteract replication difficulties and maintain viability, whereas cells in the naive state are more readily cleared under the same conditions. We anticipate these divergent strategies enable pluripotent cells of different potency states to meet associated proliferative or developmental demands during early development.

developmental biology↗

bioRxiv2018

The Medical Genome Reference Bank: Whole genomes and phenotype of 2,570 healthy elderly

Population health research is increasingly focused on the genetic determinants of healthy ageing, but there is no public resource of whole genome sequences and phenotype data from healthy elderly individuals. Here we describe the Medical Genome Reference Bank (MGRB), comprising whole genome sequence and phenotype of 2,570 elderly Australians depleted for cancer, cardiovascular disease, and dementia. We analysed the MGRB for single-nucleotide, indel and structural variation in the nuclear and mitochondrial genomes. Individuals in the MGRB had fewer disease-associated common and rare germline variants, relative to both cancer cases and the gnomAD and UK BioBank cohorts, consistent with risk depletion. Pervasive age-related somatic changes were correlated with grip strength in men, suggesting blood-derived whole genomes may also provide a biologic measure of age-related functional deterioration. The MGRB provides a broadly applicable reference cohort for clinical genetics and genomic association studies, and for understanding the genetics of healthy ageing. This research has been conducted using the UK Biobank Resource under Application Number 17984.

genomics↗

bioRxiv2018

Telomere-loop dynamics in chromosome end protection

We used super-resolution microscopy to investigate the role of macromolecular telomere structure in chromosome end protection. In murine and human cells with reduced TRF2, we find that ATM-activation at chromosome ends occurs with a structural change from t-loops to linearized chromosome ends through t-loop unfolding. Comparably, we find Aurora B kinase regulates telomere linearity concurrent with ATM activation at telomeres during mitotic arrest. Using a separation of function allele, we find that the TRFH domain of TRF2 regulates t-loop formation while suppressing ATM activity. Notably, we demonstrate that telomere linearity and ATM activation occur separately from telomere fusion via non-homologous end-joining (NHEJ). Further, we show that linear DDR-positive telomeres can remain resistant to fusion, even during an extended G1-arrest when NHEJ is most active. Collectively, these results suggest t-loops act as conformational switches that regulate ATM activation at chromosome ends independent of mechanisms to suppress chromosome end fusion.

molecular biology↗