Biology subjects

Johnstone, R. W.

Publications and source records attributed to Johnstone, R. W..

14 recordsLinked to original sources

bioRxiv2026

Termination dynamics set RNAPII elongation rate and gate the response to CDK12 inactivation

The transcriptional fidelity of RNA polymerase II (RNAPII) is governed by a tight equilibrium between elongation and termination activities, a balance frequently disrupted in human diseases such as cancer. The transcriptional cyclin-dependent kinase 12 (CDK12) maintains RNAPII elongation rate and processivity throughout the gene body. Inactivation of CDK12 disrupts this homeostatic balance and causes elongation stress, slowing RNAPII and triggering premature termination at intronic polyadenylation sites (IPAs). Despite this, the precise executors of intronic premature termination under CDK12-inactivation-induced elongation stress remain poorly understood. Using genome-wide CRISPR screening combined with chemical-genetic approach, we identified a pro-termination mechanism at intronic checkpoints; upon CDK12 inactivation, SCAF4 recruits the cleavage and polyadenylation (CPA) complex through its catalytic endonuclease CPSF3 to execute premature cleavage at IPAs. Disruption of SCAF4-CPA axis prevents early termination, restores full length transcription and confers resistance to CDK12/cyclin K inhibition. Genetic loss of SCAF4 restores the RNAPII elongation rate under CDK12 inhibition, revealing that termination dynamics actively shape the rate of transcription. Supporting this model, we uncovered an anti-termination mechanism driven by KHDRBS1/SAM68, whose depletion promotes proximal termination and sensitises cells to CDK12 targeting. Our findings mechanistically couple elongation and termination activities at intronic checkpoints as joint contributors to both the processivity and elongation rate of RNAPII. This establishes termination dynamics as an active and tractable axis of the cellular response to elongation stress.

molecular biology↗

bioRxiv2026

Target RNA abundance controls the collateral activity of RfxCas13d in human cells and zebrafish embryos

Collateral RNA cleavage by CRISPR-Cas13 effectors presents a critical obstacle to their application in biological research and therapeutics, yet the molecular determinants of this activity remain poorly understood. Here, we systematically investigate the collateral activity of various Cas13 variants in human cells in vitro and zebrafish embryos in vivo. Among these nucleases, RfxCas13d displays robust collateral activity that is highly dependent on target abundance. Targeting moderately expressed RNAs activates only a limited subset of RfxCas13d molecules, resulting in selective degradation of ectopically expressed transcripts, while endogenous RNAs remain largely protected. This selectivity indicates higher accessibility to exogenous RNAs through spatial proximity and temporal colocalization with activated RfxCas13d nuclease domains. In contrast, the recognition of highly abundant RNA targets drives simultaneous activation of a large fraction of cellular RfxCas13d, leading to widespread collateral cleavage of cellular RNAs, disruption of proteome homeostasis, and consequent cell toxicity and developmental defects in zebrafish embryos. Notably, transgenic zebrafish with target RNA expression restricted to endothelial or neuronal cell lineages exhibit localized collateral activity, leading to tissue-specific developmental abnormalities and motility deficits. These findings reveal that RfxCas13ds collateral activity is threshold-dependent, with abundant target RNA acting as a molecular switch for widespread collateral RNA degradation. This work underscores the need for careful consideration of target abundance when deploying RfxCas13d, and highlights PspCas13b as an alternative for RNA silencing free of collateral activity.

molecular biology↗

bioRxiv2025

Enhanced efficacy of a specific HDAC3 inhibitor in combination with 5-Azacitidine against diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) refers to an aggressive lymphoma that arises from germinal center (GC) B-cells, which differentiate into plasma cells (PC) to produce high affinity antibodies. 40% of DLBCL patients relapse or are refractory to the conventional immunochemotherapy treatment, usually with fatal consequences. Therefore, there is an unmet critical need to find more targeted therapies for DLBCL. DLBCL are characterized by profound alterations in the epigenome, which are correlated with poor survival. While epigenetic therapies are used as anti-cancer treatments, their full potential has not been achieved, mainly because of their limited efficacy when used as monotherapies and recurrent side effects associated with their low specificity. The abnormal epigenetic landscape of DLBCL tumors is associated with a blockade in GC exit and differentiation programs, which are regulated by the transcription factor BCL6. This aberrant repression of BCL6-target genes is mediated at least by two epigenetic mechanisms: 1) increased DNA methylation and 2) loss of acetylation of the lysine 27 of histone 3 (H3K27ac)-through recruitment of histone deacetylase 3 (HDAC3). Therefore, we investigated the efficacy against DLBCL of a novel combinatorial epigenetic therapy using the hypomethylating agent (HMA) 5-Azacitidine (5-Aza) and a specific HDAC3 inhibitor (HDAC3i). We found that treatment with 5-Aza and HDAC3i had a potent synergistic anti-tumor activity in vitro and in vivo, which was superior to the effect of each single drug or 5-Aza combined with non-specific HDACi and, importantly, was not associated with toxicity in normal cells. We also demonstrated that the combined 5-Aza and HDAC3i treatment induced the epigenetic remodeling of DLBCL cells, which resulted in a more potent re-expression of PC differentiation genes, including XBP1 and ATF4, compared to each drug used as single agents. Our results highlight the importance of targeting multiple layers of the epigenome to maximize the efficacy of epigenetic-based therapies.

cancer biology↗

bioRxiv2024

Inhibition of heme biosynthesis triggers cuproptosis in acute myeloid leukaemia.

The ubiquitous metabolite heme has diverse enzymatic and signalling functions in most mammalian cells. Cells can salvage heme from the extracellular environment or synthesise heme de novo from succinyl-CoA and glycine through a series of 8 enzymatic reactions catalysed by heme biosynthesis enzymes (HBEs) localised in the mitochondria and the cytosol1,2. Through integrated analyses of mouse models, human cell lines and primary patient samples, we identify de novo heme biosynthesis as a selective dependency in acute myeloid leukaemia (AML). The dependency is underpinned by a propensity of AML cells, and especially leukaemic stem cells (LSCs) to downregulate HBEs. The resultant low heme state upregulates self-renewal genes via the heme sensing transcription factor BACH1, but also places leukaemia cells on the threshold of heme starvation. Genetic or pharmacological inhibition of HBEs induces cuproptosis, a form of programmed cell death caused by copper accumulation and oligomerisation of lipoylated proteins3. Moreover, we identify pathways that are synthetic lethal with heme biosynthesis, including glycolysis, which can be leveraged for combination strategies. Altogether, our work uncovers a heme rheostat that controls gene expression and drug sensitivity in AML and implicates HBE inhibition as a novel cuproptosis trigger.

cancer biology↗

bioRxiv2024

Long-lived chondroprogenitors are generated by fetal-limb Gli1+ cells and are replenished upon mosaic cell-cycle arrest in the cartilage

The growth-plate cartilage of the developing long bones is a well-known system of spatially segregated stem/progenitor, transient amplifying and terminally differentiated cells. However, the regulation of the number and activity of long-lived cartilage progenitors (LLCPs) is poorly understood, despite its relevance for understanding human-height variation, the evolution of limb size and proportions and the aetiology of skeletal growth disorders. Moreover, whether their behaviour can adapt to developmental perturbations, generating robustness, has not been explored. Here, we show that Gli1+ cells are the fetal precursors of postnatal LLCPs. Moreover, while during normal growth they remain mostly dormant until postnatal stages, in response to cartilage-targeted cell-cycle arrest they increase their cartilage contribution, compensating for the perturbation. We further show that, during the response to challenge, some of the reparative Gli1+ cells originate from Pdgfra+ cells outside the cartilage anlage. Elucidating how stromal cells become Gli1+ LLCPs could shed light on developmental robustness and lead to growth-boosting therapies.

developmental biology↗

bioRxiv2023

Molecular principles of CRISPR-Cas13 mismatch intolerance enable selective silencing of point-mutated oncogenic RNA with single-base precision

Single nucleotide variants (SNVs) are extremely prevalent in human cancers. For instance, KRAS mutations occur in over 90% of pancreatic cancers and [~]40% of colorectal cancers. Virtually all KRAS mutations are SNVs, most of which remain clinically unactionable. The programmable RNA nuclease CRISPR-Cas13 has been deployed to specifically target RNAs such as overexpressed oncogenes and fusion transcripts. However, silencing oncogenic SNVs with single-base precision remains extremely challenging due to the intrinsic mismatch tolerance of Cas13. Here, we developed a comprehensive mutagenesis analysis of target-spacer interactions at single-nucleotide resolution, which revealed key spacer nucleotide positions intolerant to mismatches. We show that introducing synthetic mismatches at these precise positions enables de nova design of CRISPR RNA (crRNA) with strong preferential silencing of SNV transcripts. We demonstrate that our top-performing crRNAs possess prominent SNV-selectivity with dose-dependent silencing activity against all KRAS G12 variants at both the RNA and protein levels with minimal off-target silencing of wildtype KRAS. We applied these design principles to effectively silence oncogenic NRAS G12D and BRAF V600E transcripts, underscoring the adaptability of this platform to silence various SNVs. These findings demonstrate that the CRISPR Cas13 system can be reprogrammed to target mutant transcripts with single-base precision, showcasing the tremendous potential of this tool in personalized transcriptome editing. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/557083v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@20a7b3org.highwire.dtl.DTLVardef@1e1f79corg.highwire.dtl.DTLVardef@4724c1org.highwire.dtl.DTLVardef@be3063_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

RNA decay defines the therapeutic response to transcriptional perturbation in cancer

Therapeutic targeting of dysregulated transcriptional programs has arisen as a promising strategy for the treatment of leukaemias. The therapeutic response to small molecule inhibitors of Bromodomain-Containing Proteins (BRD), such as BRD2 and BRD4, P300/cAMP-response element binding protein (CBP) and Cyclin Dependent Kinases (CDKs), is generally attributed to the selective disruption of oncogenic gene expression networks driven by enhancers, super-enhancers (SEs) and lineage-specific transcription factors (TFs), including the c-MYC oncogene. Using technologies such as thiol (SH)-linked alkylation for the metabolic sequencing of RNA sequencing (SLAM-seq) to profile messenger RNA (mRNA) decay and production rates, we demonstrate that gene intrinsic properties largely govern the selectivity associated with transcriptional inhibition, where total mRNA response signatures are dominated with genes that have short transcript half-lives, including those regulated by SEs and oncogenic TFs. Further highlighting that gene sensitivities only occur in the context of short transcript half-lives, stabilisation of the c-MYC transcript through changes in the 3 UTR rendered it insensitive to transcriptional targeting. However, this was not sufficient to rescue c-MYC target gene transcription and anti-leukaemia effects following transcriptional inhibition. Importantly, long-lived mRNAs encoding essential genes that evade transcriptional targeting can be rendered sensitive via modulation of mRNA decay kinetics through inhibition of the RNA Binding Protein (RBP), ELAV Like RNA binding protein 1 (ELAVL1)/ Human Antigen R (HuR). Taken together, these data demonstrate that mRNA decay shapes the therapeutic response to transcriptional perturbation and can be modulated for novel therapeutic outcomes using transcriptional agents in leukaemia.

cancer biology↗

bioRxiv2022

Inhibition of mutant IDH1 promotes cycling of acute myeloid leukemia stem cells

Acute myeloid leukemias (AML) are comprised of multiple cell types with distinct capabilities to propagate the disease and resist therapy. Approximately 20% of AML patients carry gain- of-function mutations in IDH1 or IDH2 that result in over-production of the onco-metabolite 2-HG. Although IDH inhibitors can induce complete morphological remission, almost all patients eventually relapse. Analysis of clinical samples suggests that a population of IDH mutant cells is able to persist during treatment eventually acquiring 2-HG independence and drug resistance. Herein we characterized the molecular and cellular responses to the clinical IDH1 inhibitor AG-120 at high resolution using a novel multi-allelic mouse model of IDH1 mutant AML. We demonstrate that AG-120 exerts cell type-dependent effects on leukemic cells promoting delayed disease regression. Although IDH1 inhibition alone was not able to fully eradicate the disease, we uncovered that it increases cycling of rare leukemic stem cells and triggers transcriptional upregulation of the pyrimidine salvage pathway. Accordingly, AG-120 sensitized IDH1 mutant AML to azacitidine with the combination of AG-120 and azacitidine showing vastly improved efficacy in vivo. Our data highlight the impact of non- genetic heterogeneity on treatment response and provide mechanistic rationale for a drug combination that is being tested in clinical trials. STATEMENT OF SIGNIFICANCEInhibition of mutant IDH1 in AML is insufficient to eliminate the disease but promotes proliferation of quiescent leukemic stem cells. Our data provide a mechanistic explanation for the observed synergy between IDH inhibitors and azacitidine and suggest that IDH inhibitors may also synergize with other drugs that preferentially target actively dividing cells.

cancer biology↗

bioRxiv2021

Stepwise progression of β-selection during T cell development as revealed by histone deacetylation inhibition

During T cell development, the first step in creating a unique T Cell Receptor (TCR) is the genetic recombination of the TCR{beta} chain. The quality of the newly recombined TCR{beta} is assessed at the {beta}-selection checkpoint. Most cells fail this checkpoint and die, but the coordination of the complex events that control fate at the {beta}-selection checkpoint is not yet understood. We shed new light on fate determination during {beta}-selection using a selective inhibitor of histone deacetylase 6, ACY1215. ACY1215 disrupted the {beta}-selection checkpoint. Characterising the basis for this disruption revealed a new, pivotal stage in {beta}-selection, bookended by upregulation of the TCR co-receptors, CD28 and CD2 respectively. Within this DN3bPre stage, CD5 and Lef1 are upregulated to reflect pre-TCR signalling and their expression correlates with proliferation. During this phase, ACY1215-mediated disruption of the organisation of the {beta}-selection immunological synapse is associated with a breakdown in connectivity of expression of pre-TCR, CD5 and Lef1. Subsequent deregulation of pre-TCR-induced proliferation leads to bypass of the {beta}-selection checkpoint and subsequent failure to progress. We propose that the progressive expression of CD28, CD5 and Lef1, then CD2 reports and modulates the pre-TCR signal to orchestrate passage through the {beta}-selection checkpoint. These findings suggest a refined model of {beta}-selection in which a coordinated increase in expression of pre-TCR, CD28, CD5 and Lef1 allows for modulating TCR signalling strength, and culminates in the expression of CD2 to enable exit from the {beta}-selection checkpoint.

developmental biology↗

bioRxiv2021

Inhibition of pyrimidine biosynthesis targets protein translation in AML.

The mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) catalyzes one of the rate-limiting steps in de novo pyrimidine biosynthesis, a pathway that provides essential metabolic precursors for nucleic acids, glycoproteins and phospholipids. DHODH inhibitors (DHODHi) are clinically used for autoimmune diseases and are emerging as a novel class of anti-cancer agents, especially in acute myeloid leukemia (AML) where pyrimidine starvation was recently shown to reverse the characteristic differentiation block in AML cells. Herein we show that DHODH blockade rapidly shuts down protein translation in leukemic stem cells (LSCs) by down-regulation of the multi-functional transcription factor YY1, has potent activity against AML in vivo and is well tolerated with minimal impact on normal blood development. Moreover, we find that ablation of CDK5, a gene that is recurrently deleted in AML and related disorders, increases the sensitivity of AML cells to DHODHi. Our studies provide important molecular insights and identify a potential biomarker for an emerging strategy to target AML.

cancer biology↗

bioRxiv2021

BET Inhibition Enhances TNF Mediated Anti-Tumor Immunity

Targeting chromatin binding proteins and modifying enzymes can concomitantly affect tumor cell proliferation and survival, as well as enhance anti-tumor immunity and augment cancer immunotherapies. By screening a small molecule library of epigenetics-based therapeutics, BET bromodomain inhibitors (BETi) were identified as agents that promote the anti-tumor activity of CD8+ T-cells. BETi sensitized diverse tumor types to the cytotoxic effects of the pro-inflammatory cytokine TNF. By preventing the recruitment of BRD4 to p65-bound cis-regulatory elements, BETi suppressed the induction of inflammatory gene expression, including the key NF-{kappa}B target genes BIRC2 (cIAP1) and BIRC3 (cIAP2). Disruption of pro-survival NF-{kappa}B signaling by BETi led to unrestrained TNF-mediated activation of the extrinsic apoptotic cascade and tumor cell death. Administration of BETi in combination with T-cell bispecific (TCB) antibodies increased bystander killing of tumor cells and enhanced tumor growth inhibition in vivo in a TNF-dependent manner. This novel epigenetic mechanism of immunomodulation may guide future use of BETi as adjuvants for immune oncology agents. STATEMENT OF SIGNIFICANCEManipulating the epigenome is an evolving strategy to enhance anti-tumor immunity. We demonstrate that BET bromodomain inhibitors potently sensitize solid tumors to CD8+ T-cell killing in a TNF-dependent manner. This immunomodulatory mechanism can be therapeutically leveraged to augment immuno-oncology therapies, including TCB antibodies and immune checkpoint blockade.

cancer biology↗

bioRxiv2020

Epigenetic reprogramming of plasmacytoid dendritic cells drives type I interferon-dependent differentiation of acute myeloid leukemias for therapeutic benefit

Pharmacological inhibition of epigenetic enzymes can have therapeutic benefit, particularly against hematological malignancies. While these agents can affect tumor cell growth and proliferation, recent studies have demonstrated that pharmacological de-regulation of epigenetic modifiers may additionally mediate anti-tumor immune responses. Here we discovered a novel mechanism of immune regulation through the inhibition of histone deacetylases (HDACs). In a genetically engineered model of t(8;21) AML, leukemia cell differentiation and therapeutic benefit mediated by the HDAC inhibitor panobinostat required activation of the type I interferon (IFN) signaling pathway. Plasmacytoid dendritic cells (pDCs) were identified as the cells producing type I IFN in response to panobinostat, through transcriptional activation of IFN genes concomitant with increased H3K27 acetylation at these loci. Depletion of pDCs abrogated panobinostat-mediated activation of type I IFN signaling in leukemia cells and impaired therapeutic efficacy, while combined treatment of panobinostat and recombinant IFN improved therapeutic outcomes. These discoveries offer a new therapeutic approach for t(8;21) AML and demonstrate that epigenetic rewiring of pDCs enhances anti-tumor immunity, opening the possibility of exploiting this cell type as a new target for immunotherapy.

cancer biology↗

bioRxiv2020

Reprogramming of serine metabolism is an actionable vulnerability in FLT3-ITD driven acute myeloid leukaemia

Activating FMS-like tyrosine kinase 3 (FLT3) mutations occur in approximately 30% of all acute myeloid leukaemias (AMLs) and are associated with poor prognosis. The limited clinical efficacy of FLT3 inhibitor monotherapy has highlighted the need for alternative therapeutic targets and treatments for FLT3-mutant AML. Using human and murine models of MLL-rearranged AML harbouring FLT3 internal tandem duplication (FLT3-ITD) and primary patient samples, we have demonstrated that FLT3-ITD promotes serine uptake and serine synthesis via transcriptional regulation of neutral amino acid transporters (SLC1A4 and SLC1A5) and genes in the de novo serine synthesis pathway (PHGDH and PSAT1). Mechanistically, dysregulation of serine metabolism in FLT3-mutant AML is dependent on the mTORC1-ATF4 axis, that drives RNA-Pol II occupancy at PHGDH, PSAT1, SLC1A4 and SLC1A5. Genetic or pharmacological inhibition of the de novo serine synthesis pathway selectively inhibited the proliferation of FLT3-ITD AML cells, and this was potentiated by withdrawal of exogenous serine. Purine supplementation effectively rescued the antiproliferative effect of inhibiting de novo serine synthesis, consistent with the idea that serine fuels purine nucleotide synthesis in FLT3-mutant AML. Pharmacological inhibition of the de novo serine synthesis pathway, using the PHGDH inhibitor WQ-2101, sensitises FLT3-mutant AML cells to the standard of care chemotherapy agent cytarabine via exacerbation of DNA damage. Collectively, these data reveal new insights as to how FLT3 mutations reprogram metabolism in AML, and reveal a combination therapy strategy to improve the treatment of FLT3-mutant AML. Statement of SignificanceFLT3 mutations are common in AML and are associated with poor prognosis. We show that FLT3-ITD stimulates serine metabolism, thereby rendering FLT3-ITD leukemias dependent on serine for proliferation and survival. This metabolic dependency can be exploited pharmacologically to sensitize FLT3-mutant AML to chemotherapy.

cancer biology↗

bioRxiv2019

Efficient CRISPR/Cas9 gene ablation in uncultured naive mouse T cells for in vivo studies

CRISPR/Cas9 technologies have revolutionised our understanding of gene function in complex biological settings, including T cell immunology. Current CRISPR-mediated gene deletion strategies in T cells require in vitro stimulation or culture that can both preclude studies of gene function within unmanipulated naive T cells and can alter subsequent differentiation. Here we demonstrate highly efficient gene deletion within uncultured primary naive murine CD8+ T cells by electroporation of recombinant Cas9/sgRNA ribonucleoprotein immediately prior to in vivo adoptive transfer. Using this approach, we generated single and double gene knock-out cells within multiple mouse infection models. Strikingly, gene deletion occurred even when the transferred cells were left in a naive state, suggesting that gene deletion occurs independent of T cell activation. This protocol thus expands CRISPR-based probing of gene function beyond models of robust T cell activation, to encompass both naive T cell homeostasis and models of weak activation, such as tolerance and tumour models.

immunology↗