Biology subjects

Milne, T.

Publications and source records attributed to Milne, T..

9 recordsLinked to original sources

bioRxiv2026

A Unique Promoter Architecture Defines Menin-KMT2A Complex Selectivity

Selective occupancy of chromatin-regulatory complexes is central to developmental gene regulation, yet the sequence determinants of this selectivity remain poorly understood. Using interpretable machine learning, we identify a composite promoter architecture comprising CpG islands flanked by several-kilobase A/T-rich domains (AT-flanks) that denotes high occupancy of the Menin-KMT2A histone-modifying complex at developmental and leukemia-associated genes. Genetic deletion and chromatin-contact analyses show that AT-flanks support KMT2A retention at linked CpG islands. We identify LEDGF as an A/T-rich DNA-associated protein required for Menin engagement at AT-flanks and sustained KMT2A promoter occupancy. AT-flank extent is associated with differential KMT2A loss following pharmacologic disruption of the Menin-KMT2A interaction, providing a genomic basis for heterogeneous target sensitivity to recently approved Menin inhibitors in acute leukemia. Beyond Menin-KMT2A, the same architecture distinguishes promoters with high canonical PRC1 occupancy across diverse cell types, revealing a shared sequence organization associated with distinct chromatin-regulatory states.

molecular biology↗

bioRxiv2025

Zone Equalisation Normalisation For Improved Alignment of Epigenetic Signal

High-throughput genomic technologies have transformed our understanding of biological systems, but analysing these complex datasets remains challenging. Existing normalisation methods often struggle to accurately correct for technical variations across different experimental conditions, potentially leading to misleading biological interpretations. We introduce Zone Equalisation Normalisation (ZEN), a novel approach designed to improve signal normalisation across genomic datasets. By developing a flexible method that reduces technical noise whilst preserving biological signals, ZEN addresses critical limitations in current normalisation techniques. By applying ZEN to diverse genomic techniques, we reveal its robust performance in managing technical variations across different experimental platforms. ZEN provides researchers with a robust tool for more accurate and reliable genomic data analysis, potentially revealing biological insights that were previously obscured by technical variations.

bioinformatics↗

bioRxiv2025

Bi-specific CAR-iNKT cell immunotherapy for high-risk KMT2A-rearranged leukemia outperforms CAR-T in an NKG2D-dependent manner and eradicates leptomeningeal disease

Current therapies, including autologous CAR-T immunotherapy, fail to cure half of infants with KMT2A-rearranged acute lymphoblastic leukemia (KMT2Ar-ALL). Here we deploy allogeneic iNKT cells, innately more powerful effectors than T cells, and equip them with CD19- and/or CD133-targeting CARs. Compared to mono-specific counterparts and bi-specific CAR-T, CD19-CD133 bi-specific CAR-iNKT have more potent anti-leukemia activity, they effectively target CAR antigen-low leukemia, eradicate medullary and leptomeningeal leukemia and induce sustained remissions without discernible hematologic toxicity. Mechanistically, dynamic CAR- and CAR antigen-dependent upregulation of the activating innate receptor NKG2D and its engagement by corresponding ligands on KMT2Ar-ALL cells lead to more potent anti-leukemia effect of CAR-iNKT over CAR-T cells, including against CAR antigen-negative leukemia. Thus, by engaging with two different types of leukemia-associated targets, CAR-iNKT provide a powerful platform for the treatment of KMT2Ar-ALL. This approach can be readily adapted for other high-risk malignancies, including those with otherwise difficult to target leptomeningeal involvement.

cancer biology↗

bioRxiv2023

Dysregulation of chromatin via H3K27 methylation underpins differentiation arrest in Isocitrate dehydrogenase-mutant Acute Myeloid Leukaemia.

Dysregulation of cellular differentiation is a hallmark of cancer. Isocitrate dehydrogenase (IDH) is commonly mutated multiple cancers including glioma, cholangiocarcinoma, lymphoma and Acute Myeloid Leukaemia (AML). Mutant IDH generates d-2-hydroxyglutarate that inhibits enzymes including Jumonji histone demethylases and TET2. Using primary human IDH2-mutant AML cells as a model, single cell RNA-seq and ATAC-seq, we demonstrated the continuum of cell states during restoration of neutrophilic differentiation to leukaemic progenitors by Enasidenib, a mutant IDH2 inhibitor. In cells which ultimately differentiate, there is co-expression of competing GATA2/RUNX3/SOX4-driven stem-progenitor and pro-differentiation EGR1/JUN/FOS programmes, followed by expression of cell cycle and terminal neutrophil programmes involving CEBP family and SPI1/PU.1. Genes upregulated during differentiation display loss of H3K27me3 in bivalent chromatin but not of H3K4me3, while downregulated genes are enriched for PRC2/EZH2 targets. In contrast to previous reports of a TET2-dependent mechanism for IDH-mutations, we observed only a modest link between promoter DNA CpG methylation and gene expression. For the first time in primary AML, we describe the lifting of differentiation block by de-repression of pro-differentiation genes through modulation of H3K27 demethylation in bivalent chromatin, and thus highlight a novel and important mechanism in how IDH mutations disrupt cell fates in cancer.

cancer biology↗

bioRxiv2022

Pharmacologic Inhibition of DYRK1A Results in MYC Hyperactivation and ERK Hyperphosphorylation rendering KMT2A-R ALL Cells Sensitive to BCL2 Inhibition

KMT2A-rearranged (KMT2A-R) B cell acute lymphoblastic leukemia (ALL) is a high-risk disease in children and adults that is often chemotherapy resistant. To identify non-cytotoxic approaches to therapy, we performed a domain-specific kinome-wide CRISPR screen in KMT2A-R cell lines and patient derived xenograft samples (PDX) and identified dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) as a potential target. Pharmacologic inhibition of the KMT2A-fusion transcriptional co-regulator Menin released the KMT2A-fusion complex from the DYRK1A promoter thereby lowering DYRK1A expression levels confirming DYRK1A as a direct target of the KMT2A fusion oncogene. Direct pharmacologic inhibition of DYRK1A decreased cell proliferation of KMT2A-R ALL, thereby confirming the requirement of DYRK1A in this ALL subtype. To further understand the biologic function of DYRK1A in KMT2A-R ALL, we leveraged pharmacologic DYRK1A inhibitors in KMT2A-R PDX and cell line models. DYRK1A inhibition consistently led to upregulation of MYC protein levels, and hyperphosphorylation of ERK, which we confirmed via in vivo treatment experiments. Furthermore, DYRK1A inhibition decreased ALL burden in mice. Our results further demonstrate that DYRK1A inhibition induces the proapoptotic factor BIM, but ERK hyperphosphorylation is the driving event that induces cell cycle arrest. In contrast, combined treatment of KMT2A-R ALL cells in vitro and in vivo with DYRK1A inhibitors and the BCL2 inhibitor, venetoclax, synergistically decreases cell survival and reduced the leukemic burden in mice. Taken together these results demonstrate a unique function of DYRK1A specially in KMT2A-R ALL. Synergistic inhibition of DRYK1A and BCL2 may provide a low-toxic approach to treat this high risk ALL subtype.

cancer biology↗

bioRxiv2022

PAF1 and FACT cooperate with MLL-AF4 to drive enhancer activity in leukemia

Aberrant enhancer activation has been identified as a key mechanism driving oncogene expression in many cancers. Here we use TOPmentation (Transcription factor-OPtimized ChIPmentation) to probe enhancer usage in primary MLL-rearranged acute lymphoblastic leukemia. We find that MLL-AF4, commonly held to promote transcription by binding to gene promoters, is also present at many active enhancers, where it assembles a complex of transcriptional co-activators normally found in the gene body. This includes DOT1L, ENL, PAF1, and a newly identified interaction with the histone chaperone FACT. By chemical degradation, we demonstrate that PAF1 and FACT are required for enhancer activity, including maintaining histone H3K27 acetylation, enhancer RNA transcription and enhancer-promoter interactions. This work identifies novel roles for PAF1 and FACT in enhancer function, and reveals an enhancer-targeting mechanism by which MLL-AF4 upregulates transcription, recruiting transcription machinery through a network of multivalent interactions to control enhancer activity and gene expression in acute leukemias.

molecular biology↗

bioRxiv2021

A novel tubulin binding molecule drives differentiation of acute myeloid leukaemia cells

Acute Myeloid Leukaemia (AML) continues to have a poor prognosis, especially in the elderly. One reason for this is that many treatment regimens are not well tolerated by elderly patients. Much current focus is on the development of therapies that can target specific vulnerabilities of AML while having fewer toxic side effects. However, despite much recent progress in developing better drugs, many patients with AML still die within a year of diagnosis, partly due to the fact that it is difficult to identify therapeutic targets that are effective across multiple AML subtypes. One common factor across AML subtypes is the presence of a block in differentiation. Thus screening for compounds that can overcome this block in genetically diverse AML models should allow for the identification of agents that are not dependent on a specific mutation for their efficacy. Here, we used a phenotypic screen to identify novel compounds that stimulate differentiation in several AML cell lines. Lead compounds were shown to decrease tumour burden and to increase survival in vivo. Using multiple complementary target deconvolution approaches, these compounds were revealed to be anti-mitotic tubulin disruptors that cause differentiation by inducing a G2-M mitotic arrest. Together, these results reveal a novel function for tubulin disruptors in causing differentiation of AML cells.

cancer biology↗

bioRxiv2020

A phenotypic screen identifies a compound series that induces differentiation of acute myeloid leukemia cells in vitro and shows anti-tumour effects in vivo

Induction of differentiation is a promising therapeutic strategy against acute myeloid leukemia. However, current differentiation therapies are effective only to specific patient populations. To identify novel differentiation agents with wider efficacy, we developed a phenotypic high-throughput screen with a range of genetically diverse cell lines. From the resulting hits, one chemical scaffold was optimised in terms of activity and physicochemical properties to yield OXS007417, which was also able to decrease tumour volume in a murine in vivo xenograft model.

cancer biology↗

bioRxiv2019

BET inhibition disrupts transcription but retains enhancer-promoter contact

In higher eukaryotes, enhancers are DNA sequences that enable complex temporal and tissue-specific regulation of genes. Although it is not entirely clear how enhancer-promoter interactions can increase gene expression, this proximity has been observed in multiple systems at multiple loci and is thought to be essential for the maintenance of gene expression. The formation of phase condensates is thought to be an essential component of enhancer function. Here, we show that pharmacological targeting of cells with inhibitors of BET (Bromodomain and Extra-Terminal domain) proteins can have a strong impact on transcription but very little impact on enhancer-promoter interactions. Treatment with 1,6-hexanediol, which dissolves phase condensate structures and reduces BET and Mediator protein binding at enhancers, can also have a strong effect on gene transcription, without disrupting enhancer-promoter interactions. These results suggest that activation of transcription and maintenance of enhancer-promoter interactions are separable events. Our findings further suggest that enhancer-promoter interactions are not dependent on high levels of BRD4 (Bromodomain-containing protein 4) and Mediator, and are likely maintained by a complex set of factors including additional activator complexes and loop extrusion by CTCF/cohesin.

molecular biology↗