Biology subjects

Bond, J.

Publications and source records attributed to Bond, J..

12 recordsLinked to original sources

bioRxiv2025

Nonsense, but not frameshift, truncating mutations result in Cyclin D2 stabilisation in an induced pluripotent stem cell model of MPPH

Stabilisation of Cyclin D2 is the underlying cause of a range of neurodevelopmental disorders, characterised by megalencephaly, cortical migration defects and overgrowth. Intracellular CCND2 is vital for mTOR pathway signalling, with inhibition of mTOR resulting in CCND2 phosphorylation and degradation by the ubiquitin proteasome system. Mutations at the regulatory c-terminus of CCND2, and in proteins that regulate mTOR such as PTEN, PIK3CA, AKT3 and TSC1/2, result in CCND2-stabilisation and overgrowth. To determine the molecular and cellular mechanisms underpinning the neurodevelopmental defects observed in Megalencephaly-Polymicrogyria-Polydactyly-Hydrocephalus (MPPH) syndrome, we generated human induced pluripotent stem cell (iPSC) derived models of CCND2-associated disease. Using CRISPR-Cas9 we generated lines containing either a pathogenic CCND2 variant (c.814G>T, p.Glu272Ter) or frameshift variants in the final exon of CCND2, all of which truncate CCND2 before the critical Thr-280 residue, required for its phosphorylation and degradation. We observed truncating frameshift variants do not result in CCND2 stabilization, whereas the single nucleotide c.814G>T, p.Glu272Ter substitution does, mimicking the effect seen in MPPH patients. Differentiation into human cortical spheroids (hCS) revealed all CCND2-truncating lines continued to express PAX6 beyond the neural progenitor (NP) expansion phase. Furthermore, both the homozygous and heterozygous p.Glu272Ter hCS failed to produce mature Tbr-1 expressing neurons, while some expression was observed in the frameshift hCS, highlighting differences in neurogenesis between frameshift and nonsense lines. Despite all lines truncating CCND2 and removing Thr-280, our data implies that frameshift truncations do not stabilise CCND2. In comparison, truncation of CCND2 through introduction of a single nucleotide nonsense variant results in CCND2 stabilisation, mimicking MPPH.

developmental biology↗

bioRxiv2025

Diverse epigenomic mechanisms underpin transcriptional dysregulation in Polycomb-altered acute myeloid leukemia

BackgroundPolycomb Repressive Complex 2 (PRC2) modulates chromatin accessibility and architecture to direct tissue-specific gene expression. PRC2 function is frequently altered in cancer by loss-of-function mutation or deletion, but the downstream effects on transcriptional regulation are incompletely understood. ResultsTo gain insights into these mechanisms, we performed a holistic analysis of epigenomic and transcriptional changes in an isogenic model of acute myeloid leukemia (AML) with heterozygous EZH2 deletion that mimics reduced PRC2 function in patient leukemias. PRC2-depleted cells had diverse gene expression changes, including a bias towards more immature monocyte-lineage transcriptional signatures. PRC2 depletion also correlated with marked increases in chromatin accessibility genome-wide, with 10-45% increases in ATAC-seq peaks in EZH2+/- clones. These changes were accompanied by decreased H3K27me3 and increased H3K27ac levels in CUT+RUN assays that were incompletely linked to transcriptional activity. Despite these generalised changes, 3D chromatin architecture assessed by Hi-C was largely maintained, with H3K27me3 preferentially lost in regions with low DNA-DNA contact frequency. Surprisingly, some regions gained broad H3K27me3 domains at heavily compacted chromatin. We notably saw compartmentalisation changes upstream of the transcriptionally upregulated fetal hematopoiesis gene LIN28B in EZH2+/- cells, with corresponding activation of a LIN28B-specific transcriptional program, including upregulation of the CDK6 oncogene. These results correlated with EZH2+/- cell phenotype, including decreased cellular proliferation and increased resistance to CDK6 inhibitor palbociclib. ConclusionsOur findings suggest that PRC2 depletion pleiotropically affects AML transcriptional regulation to directly impact cell phenotype and treatment responsiveness, which may partially explain the aggressive biology seen in these cases.

cancer biology↗

bioRxiv2025

A structure-based modelling approach identifies effective drug combinations for RAS-mutant acute myeloid leukemia

Mutations activating RAS/RAF/MEK/ERK signaling are associated with poor outcome in acute myeloid leukemia (AML), but therapeutic targeting of this pathway is challenging. Here, we employ a structure-based, dynamic RAS pathway model to successfully predict RAF inhibitor (RAFi) combinations which synergistically suppress ERK signaling in RAS-mutant AML. Our in silico models predicted therapeutic synergy of two iterations of conformation-specific RAF inhibitors: Type I[1/2] + Type II and Type I + Type II. Predictions were validated in vitro in AML cell lines and patient samples, with synergy verified by the Loewe Additivity model. Lifirafenib (Type II) + encorafenib (Type I[1/2]) was highly synergistic against both NRAS- and KRAS-mutant lines, while synergy of lifirafenib + SB590885 (Type I) was specific to NRAS-mutants. Immunoblotting confirmed that combination efficacy correlated strongly with decreased RAS pathway activation. Leveraging the pharmacokinetic predictions of our in silico model, both combinations were then assessed in a pre-clinical NRAS-mutant AML patient-derived xenograft (PDX) model, showing significantly improved leukaemia growth delay and event-free survival compared with single agent approaches. Assessment of leukemia burden in bone marrow and spleen during treatment further showed site-specific efficacy against circulating and spleen-resident blasts for both combinations. In summary, we report that our structure based-modelling approach can effectively identify novel, non-obvious, and well-tolerated RAFi combinations that are highly effective against in vitro and in vivo models, thereby suggesting alternative potential therapeutic strategies for high-risk RAS-mutant AML.

cancer biology↗

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↗

bioRxiv2024

Enhancer heterogeneity in acute lymphoblastic leukemia drives differential gene expression between patients

Genetic alterations alone cannot account for the diverse phenotypes of cancer cells. Even cancers with the same driver mutation show significant transcriptional heterogeneity and varied responses to therapy. However, the mechanisms underpinning this heterogeneity remain under-explored. Here, we find that novel enhancer usage is a common feature in acute lymphoblastic leukemia (ALL). In particular, KMT2A::AFF1 ALL, an aggressive leukemia with a poor prognosis and a low mutational burden, exhibits substantial transcriptional heterogeneity between individuals. Using single cell multiome analysis and extensive chromatin profiling, we reveal that much transcriptional heterogeneity in KMT2A::AFF1 ALL is driven by novel enhancer usage. Using high resolution Micro-Capture-C in primary patient samples, we also identify patient-specific enhancer activity at key oncogenes such as MEIS1 and RUNX2, driving high levels of expression of both oncogenes in a patient-specific manner. Overall, our data show that enhancer heterogeneity is highly prevalent in KMT2A::AFF1 ALL and may also be a mechanism that drives transcriptional heterogeneity in cancer more generally. Key PointsO_LILeukemia patients with the same driver mutations often display gene expression differences C_LIO_LIUsing chromatin profiling and high resolution 3C methods we show that enhancer heterogeneity drives gene expression differences C_LI

cancer biology↗

bioRxiv2023

Loss of Polycomb Repressive Complex 2 function causes asparaginase resistance in T-acute lymphoblastic leukemia through decreased WNT pathway activity

Loss-of-function mutations and deletions in core components of the epigenetic Polycomb Repressive Complex 2 (PRC2) are associated with poor prognosis and treatment resistance in T-acute lymphoblastic leukemia (T-ALL). We leveraged clinical mutational and transcriptional data to identify a functional link between PRC2 alterations and changes in WNT signaling pathway activity in leukemia cells. Computational integration of transcriptomic, proteomic and phosphoproteomic data from an isogenic T-ALL cellular model revealed reduced activity of the WNT-dependent stabilization of proteins (WNT/STOP) pathway in cells lacking core PRC2 factor EZH2. We discovered that PRC2 loss significantly reduced sensitivity to key T-ALL treatment asparaginase, and that this was mechanistically linked to increased cellular ubiquitination levels that bolstered leukemia cell asparagine reserves. We further found that asparaginase resistance in PRC2-depleted leukemic blasts could be mitigated by pharmaceutical proteasome inhibition, thereby providing a novel and clinically tractable means to tackle induction treatment failure in high-risk T-ALL.

cancer biology↗

bioRxiv2023

Gene essentiality in cancer is better predicted by mRNA abundance than by gene regulatory network-inferred activity

Gene regulatory networks (GRNs) are often deregulated in tumor cells, resulting in altered transcriptional programs that facilitate tumor growth. These altered networks may make tumor cells vulnerable to the inhibition of specific regulatory proteins. Consequently, the reconstruction of GRNs in tumors is often proposed as a means to identify therapeutic targets. While there are examples of individual targets identified using GRNs, the extent to which GRNs can be used to predict sensitivity to targeted intervention in general remains unknown. Here we use the results of genome-wide CRISPR screens to systematically assess the ability of GRNs to predict sensitivity to gene inhibition in cancer cell lines. Using GRNs derived from multiple sources, including GRNs reconstructed from tumor transcriptomes and from curated databases, we infer regulatory gene activity in cancer cell lines from ten cancer types. We then ask, in each cancer type, if the inferred regulatory activity of each gene is predictive of sensitivity to CRISPR perturbation of that gene. We observe slight variation in the correlation between gene regulatory activity and gene sensitivity depending on the source of the GRN and the activity estimation method used. However, we find that there is consistently a stronger relationship between mRNA abundance and gene sensitivity than there is between regulatory gene activity and gene sensitivity. This is true both when gene sensitivity is treated as a binary and a quantitative property. Overall, our results suggest that gene sensitivity is better predicted by measured expression than by GRN-inferred activity.

bioinformatics↗

bioRxiv2022

Oxytocin receptor is not required for social attachment in prairie voles

Prairie voles are among a small group of mammals that display long-term social attachment between mating partners. Many pharmacological studies show that signaling via the oxytocin receptor (OxtR) is critical for the display of social monogamy in these animals. We used CRISPR-mutagenesis to independently generate three different OxtR null mutant prairie vole lines. OxtR mutants displayed social attachment such that males and females showed a behavioral preference for their mating partners over a stranger of the opposite sex when assayed using different paradigms. Mothers lacking OxtR delivered viable pups, and parents displayed care of their young and raised them to the weanling stage. Together, our studies unexpectedly reveal that OxtR-mediated signaling is genetically dispensable for social attachment, parturition, and parental behavior.

animal behavior and cognition↗

bioRxiv2020

Drug and siRNA screens identify ROCK2 as a therapeutic target for ciliopathies.

Primary cilia are microtubule-based organelles that act as cellular antennae to mediate vertebrate development and growth factor signalling. Defects in primary cilia result in a group of inherited developmental conditions known as ciliopathies. Ciliopathies often present with cystic kidney disease, a major cause of early renal failure that requires renal replacement therapies. Currently, only one drug, Tolvaptan, is licensed to slow the decline of renal function for the ciliopathy polycystic kidney disease. Novel therapeutic interventions for these conditions remain a pressing clinical need. We screened clinical development compounds for positive effects on cilia formation and function and identified fasudil hydrochloride as the top hit. Fasudil is a generic, off-patent drug that is a potent but broadly selective Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor. In a parallel whole genome siRNA-based reverse genetics phenotypic screen of positive modulators of cilia formation, we identified ROCK2 as the target molecule. We demonstrate that ROCK2 is a key mediator of cilium formation and function through effects on actin cytoskeleton remodelling. Our results indicate that specific ROCK2 inhibitors such as belumosudil (KD-025) could be repurposed for pharmacological intervention in cystic kidney disease. We propose that ROCK2 inhibition represents a novel, disease-modifying therapeutic approach for heterogeneous ciliopathies.

cell biology↗

bioRxiv2019

A transcriptomic continuum of differentiation arrest identifies myeloid interface acute leukemias with poor prognosis

Classification of acute lymphoblastic and myeloid leukemias (ALL and AML) remains heavily based on phenotypic resemblance to normal hematopoietic precursors. This framework can provide diagnostic challenges for immunophenotypically heterogeneous immature leukemias, and ignores recent advances in understanding of developmental multipotency of diverse normal hematopoietic progenitor populations that are identified by transcriptional signatures. We performed transcriptional analyses of a large series of acute myeloid and lymphoid leukemias and detected significant overlap in gene expression between cases in different diagnostic categories. Bioinformatic classification of leukemias along a continuum of hematopoietic differentiation identified leukemias at the myeloid/T-lymphoid interface, which shared gene expression programs with a series of multi or oligopotent hematopoietic progenitor populations, including the most immature CD34+CD1a-CD7- subset of early thymic precursors. Within these interface acute leukemias (IALs), transcriptional resemblance to early lymphoid progenitor populations and biphenotypic leukemias was more evident in cases originally diagnosed as AML, rather than T-ALL. Further prognostic analyses revealed that expression of IAL transcriptional programs significantly correlated with poor outcome in independent AML patient cohorts. Our results suggest that traditional binary approaches to acute leukemia categorization are reductive, and that identification of IALs could allow better treatment allocation and evaluation of therapeutic options.

cancer biology↗

bioRxiv2019

A high-content screen profiles cytotoxic microRNAs in pediatric and adult glioblastoma cells and identifies miR-1300 as a potent inducer of cytokinesis failure

BackgroundMicroRNAs play an important role in the regulation of mRNA translation, and have therapeutic potential in cancer and other diseases.\n\nMethodsTo profile the landscape of microRNAs with significant cytotoxicity in the context of glioblastoma (GBM), we performed a high-throughput screen using a synthetic oligonucleotide library representing all known human microRNAs in adult and pediatric GBM cells. Bio-informatics analysis were used to refine this list and the top seven microRNAs were validated in a larger panel of cells by flow-cytometry, and RTqPCR. The downstream mechanism of the strongest and most consistent candidate was investigated by siRNAs, 3UTR luciferase assays and Western Blotting.\n\nResultsOur screen identified [~]100 significantly cytotoxic microRNAs with 70% concordance between cell lines. MicroRNA-1300 (miR-1300) was the most potent and robust candidate. We observed a striking binucleated phenotype in miR-1300 expressing cells and characterized the mechanism of action as cytokinesis failure followed by apoptosis, which was observed in an extended GBM cell panel including two stem-like patient-derived cultures. We identified the physiological role of miR-1300 as a regulator of endomitosis in megakaryocyte differentiation where blockade of cytokinesis is an essential step. In glioblastoma cells, the oncogene Epithelial Cell Transforming 2 (ECT2) was validated as a direct key target of miR-1300. ECT2 siRNA phenocopied the effects of miR-1300, and its overexpression led to a significant rescue of miR-1300 induced binucleation.\n\nConclusionMiR-1300 was identified as a novel regulator of endomitosis with translatable potential for therapeutic application. The datatasets will be a resource for the neuro-oncology community.\n\nKey points (2 or 3 key points 85 characters plus spaces each)70% of cytotoxic microRNAs were shared between adult and pediatric glioblastoma cells\n\nMiR-1300 expression is restricted to endomitosis within megakaryocyte differentiation\n\nMiR-1300s ectopic expression is a potent and promising therapeutic tool in cancer\n\nImportance of StudyPrevious functional studies of microRNAs involved in the regulation of glioblastoma cell proliferation and/or survival have focused on adult glioblastoma alone and are restricted to only a few microRNAs at a time. Our study provides the first encompassing landscape of potent cytotoxic microRNAs in pediatric and adult glioblastoma.\n\nNot only, does our data provide an invaluable resource for the research community but it also revealed that 70% of microRNAs with significant cytotoxicity were shared by adult and pediatric cells. Finally, we identified and characterized the previously undescribed role of microRNA-1300 in the tight regulation of megakaryocyte differentiation into platelets and how, when expressed outside of this context, miR-1300 consistently causes cytokinesis failure followed by apoptosis, and thus represents a powerful cytotoxic tool with potential for translation towards therapeutic applications.

cancer biology↗

bioRxiv2018

CBFβ-MYH11 interferes with megakaryocyte differentiation via modulating a gene program that includes GATA2 and KLF1

The inv(16) acute myeloid leukemia associated CBF{beta}-MYH11 fusion is proposed to block normal myeloid differentiation, but whether this subtype of leukemia cells is poised for a unique cell lineage remains unclear. Here, we surveyed the functional consequences of CBF{beta} - MYH11 in primary inv(16) patient blasts, upon expression during hematopoietic differentiation in vitro and upon knockdown in cell lines by multi-omics profiling. Our results reveal that primary inv(16) AML cells share common transcriptomic signatures and epigenetic determiners with megakaryocytes and erythrocytes. Using in vitro differentiation systems, we reveal that CBF{beta}-MYH11 knockdown interferes with normal megakaryocyte maturation. Two pivotal regulators, GATA2 and KLF1, are identified to complementally occupy RUNX1 binding sites upon fusion protein knockdown, and overexpression of GATA2 partly restores megakaryocyte directed differentiation suppressed by CBF{beta}-MYH11. Together, our findings suggest that in inv(16) leukemia, the CBF{beta}-MYH11 fusion inhibits primed megakaryopoiesis by attenuating expression of GATA2/KLF1 and interfering with a balanced transcriptional program involving these two factors.

cancer biology↗