Biology subjects

Giotopoulos, G.

Publications and source records attributed to Giotopoulos, G..

10 recordsLinked to original sources

bioRxiv2025

A Step-wise, Deterministic and Fatal Mouse Model of Myeloid Neoplasm with Spontaneous Acquisition of Patient-relevant RTK-RAS Mutations

Leukaemia arises through the stepwise transformation of healthy haematopoietic cells, yet the asymptomatic premalignant phase and its progression to overt disease remain poorly understood. To model this process, we engineered a patient-derived CEBPA mutation into Hoxb8-FL multipotent murine progenitors and transplanted them into syngeneic mice, capturing a clinically silent premalignant stage. All recipients developed overt disease after [~]12 months with 100% penetrance and all acquired secondary RTK-RAS mutations, often with identical amino acid changes to those in patients. Single-cell transcriptomics and phenotypic profiling showed that premalignant mutant cells adopt a plasmacytoid dendritic progenitor-like state in vitro which generates both myeloid and B-lymphoid lineages during premalignancy in vivo, with individual tumours restricted to one lineage. Specificity for RTK-RAS mutations coupled with ongoing differentiation is reminiscent of Juvenile Myelomonocytic Leukaemia, where cellular models are currently lacking, thus providing a tractable model of myeloid neoplasm for mechanistic studies and drug discovery.

cancer biology↗

bioRxiv2025

Delayed Trp53 activation protects Dnmt3a-mutant hematopoietic stem cells from inflammatory attrition.

Hematopoietic stem cells (HSCs) accumulate somatic mutations over time, some conferring a fitness advantage that can lead to clonal hematopoiesis (CH). Mutations in DNMT3A, particularly at hotspot R882, are the most prevalent in CH and carry an increased risk of acute myeloid leukemia (AML). Although DNMT3A R882 mutations are linked to global DNA hypomethylation, the mechanisms underlying their selective advantage remain unclear. Here, we show that Dnmt3a-R882H mutant HSCs exhibit resilience under inflammatory and genotoxic stress. During IL-1{beta}-induced emergency granulopoiesis, Dnmt3a R882H/+ HSCs uncouple increased proliferation from stem cell exhaustion. In contrast, wild-type HSCs rapidly progress to terminal differentiation. We link this phenotype to a delayed activation of the p53-p21-DREAM axis, that allows mutant HSCs to avoid attrition, despite increased replication stress. Similarly, mutant HSCs exhibit delayed Trp53 activation following irradiation, but eventually recover a physiological Trp53 response. Analysis of patient data reveals shared phenotypic features between DNMT3A and monoallelic TP53 mutations in CH and myeloid neoplasms, highlighting potential functional similarities. Collectively, these findings suggest that the expansion of DNMT3A-mutant clones is affected by impaired TP53 signaling, which confers resilience against stressors. Therapeutic strategies targeting inflammatory pathways or the p53-p21-DREAM axis may reduce DNMT3A-CH expansion and/or progression and its associated risks.

molecular biology↗

bioRxiv2023

Genetic or pharmacological inactivation of CREBBP sensitizes B-cell Acute Lymphoblastic Leukemia to Ferroptotic Cell Death upon BCL2 Inhibition

B-cell acute lymphoblastic leukemia (B-ALL) is a leading cause of death in childhood and outcomes in adults remain dismal. There is therefore an urgent clinical need for therapies that target the highest risk cases. Mutations in the histone acetyltransferase CREBBP associate with high-risk features in B-ALL and have been implicated in chemoresistance. We performed a targeted drug screen in isogenic human cell lines, identifying a number of actionable small molecules that specifically target CREBBP-mutated B-ALL. The most potent was the BCL2 inhibitor Venetoclax, which acts through a non-canonical mechanism resulting in ferroptotic cell death. CREBBP-mutated cell lines showed differences in cell-cycle, metabolism and response to oxidative stress. Lastly, we demonstrate that small-molecule inhibition of CREBBP sensitizes B-ALL cells, regardless of genotype, to Venetoclax-induced ferroptosis in-vitro and in-vivo, providing a potential novel drug combination for broader clinical translation in B-ALL.

cancer biology↗

bioRxiv2022

HOXA9 forms a repressive complex with nuclear matrix-associated protein SAFB to maintain acute myeloid leukemia

HOXA9 is commonly upregulated in acute myeloid leukemia (AML), where it confers poor prognosis. Characterising the protein interactome of endogenous HOXA9 in human AML, we identified a chromatin complex of HOXA9 with the nuclear matrix attachment protein-SAFB. SAFB perturbation phenocopied HOXA9 knockout to decrease AML proliferation, increase differentiation and apoptosis in vitro and prolonged survival in vivo. Integrated genomic, transcriptomic and proteomic analyses further demonstrated that the HOXA9-SAFB-chromatin complex associates with NuRD and HP1{gamma} to repress the expression of factors associated with differentiation and apoptosis, including NOTCH, CEBP{delta}, S100A8, and CDKN1A. Chemical or genetic perturbation of NuRD and HP1{gamma} catalytic activity also triggered differentiation, apoptosis and the induction of these tumor-suppressive genes. Importantly, this mechanism is operative in other HOXA9-dependent AML genotypes. This mechanistic insight demonstrates active HOXA9-dependent differentiation block as a potent mechanism of disease maintenance in AML, that may be amenable to therapeutic intervention via therapies targeting the HOXA9/SAFB interface and/or NuRD and HP1{gamma} activity. Key Points- Identification of the endogenous human HOXA9 protein interactome in AML - HOXA9 forms a repressive complex with S/MAR binding protein (SAFB) that is critical for the maintenance of AML by facilitating proliferation and preventing differentiation and cell death. - The HOXA9/SAFB (H9SB) complex represses gene expression via recruitment of NuRD and HP1{gamma}.

cancer biology↗

bioRxiv2022

Acute resistance to BET inhibitors remodels compensatory transcriptional programs via p300 co-activation

Initial clinical trials with drugs targeting epigenetic modulators - such as bromodomain and extraterminal (BET) inhibitors - demonstrate modest results in acute myeloid leukemia (AML). The main reason for this involves an increased transcriptional plasticity within AML, which allows cells to escape the therapeutic pressure. To study mechanisms of resistance, we investigated immediate epigenetic and transcriptional responses following BET inhibition, and could demonstrate that BET inhibitor-mediated release of BRD4 from chromatin is accompanied by an acute compensatory feedback loop that attenuates inhibition, or even increases expression, of specific transcriptional modules. This adaptation is most marked at key AML maintenance genes and is mediated by p300, suggesting a rational therapeutic opportunity by combining BET- and p300- inhibition. p300 activity is required during all steps of adaptation. However, the transcriptional programs that p300 regulates to induce resistance to BETi differ between AML subtypes. Remarkably, in some AMLs, p300 regulates a series of transitional transcriptional patterns that allow homeostatic adjustments during earlier stages of resistance to BET-inhibitors. In consequence, p300 remains crucial throughout all stages of resistance in sensitive AML-subtypes, although its importance declines following the development of chronic resistance to BET inhibitors in some other AMLs. Altogether, our study elucidates the mechanisms that underlie an "acute" state of resistance to BET inhibition, achieved through p300 activity, and how these mechanisms remodel to become "chronic". Importantly, however, our data also suggest that a sequential treatment with BET- and p300 inhibition may prevent resistance development, thereby improving outcomes. Key pointsO_LIA mechanistic feedback to p300 enables acute tolerance to BET inhibition. C_LIO_LIp300 regulates transcriptional networks that lead to chronic resistance to BET inhibition. C_LIO_LISequential BET-, followed by p300-inhibition, is synthetically lethal in AML, and is optimally deployed during earlier stages of resistance to BET inhibitors. C_LI

cancer biology↗

bioRxiv2022

Systematic functional screening of chromatin factors identifies strong lineage and disease dependencies in normal and malignant haematopoiesis

Interactions between transcription factors (TF) and chromatin factors (CF) regulate gene expression programmes to determine cellular fate. However, unlike for TF, the exact role of CF in this process is poorly understood. Using haematopoiesis as a model system and utilising novel functional CRISPR screens ex vivo and in vivo, coupled with Perturb-Seq, CF binding and genome-wide chromatin accessibility in primary murine cells, we assess the role of 550 chromatin factors in lineage choice in normal haematopoiesis and the maintenance of acute myeloid leukaemia (AML). These studies demonstrate marked specificity for a large number of CFs in lineage determination, highlighting functional diversity within specific families of chromatin regulators, including MLL-H3K4-methyltransferases and different BAF-complexes, that regulate disparate lineage decisions across haematopoiesis. Conversely, we demonstrate that unrelated Repressive complexes function similarly to restrain excessive myeloid differentiation and protect lineage diversity. We identify interactions between CF and TF that, at least in part, explain the regulatory function of CF and link Brd9-loss to a premalignant state. Utilising similar experiments in a relevant murine AML model, we demonstrate opposing effects for CF in normal haematopoiesis and AML, where MLL-H3K4-methyltransferases, c-BAF-remodelers and Repressive complexes prevent differentiation and maintain leukaemic fitness. We show that this alteration relates to differential utilisation of TF by CF complexes between normal and malignant haematopoiesis, highlighting corrupted TF-CF interactions as potential novel avenues for therapeutic intervention in AML. Together, this study provides novel insights on the functional diversity of chromatin factors in governing cell-fate.

genomics↗

bioRxiv2022

Mannose metabolism inhibition sensitizes acute myeloid leukemia cells to cytarabine and FLT3 inhibitor therapy by modulating fatty acid metabolism to drive ferroptotic cell death.

Resistance to standard and novel therapies remains the main obstacle to cure in acute myeloid leukemia (AML) and is often driven by metabolic adaptations which are therapeutically actionable. Here we identify inhibition of mannose-6-phosphate isomerase (MPI), the first enzyme in the mannose metabolism pathway, as a sensitizer to both cytarabine and FLT3 inhibitors across multiple AML models. Mechanistically, we identify a connection between mannose metabolism and fatty acid metabolism, that is mediated via preferential activation of the ATF6 arm of the unfolded protein response (UPR). This in turn leads to cellular accumulation of polyunsaturated fatty acids, lipid peroxidation and ferroptotic cell death in AML cells. Our findings provide further support to the role of rewired metabolism in AML therapy resistance, unveil a novel connection between two apparently independent metabolic pathways and support further efforts to achieve eradication of therapy-resistant AML cells by sensitizing them to ferroptotic cell death.

cancer biology↗

bioRxiv2021

Transcriptional variability accelerates pre-leukemia by cell diversification and perturbation of protein synthesis

Transcriptional variability facilitates stochastic cell diversification and can in turn underpin adaptation to stress or injury. We hypothesize that it may analogously facilitate progression of pre-malignancy to cancer. To investigate this, we initiated pre-leukemia in mouse cells with enhanced transcriptional variability due to conditional disruption of the histone lysine acetyltransferase gene Kat2a. By combining single-cell RNA-sequencing of pre-leukemia with functional analysis of transformation, we show that Kat2a loss results in global variegation of cell identity and accumulation of pre-leukemic cells. Leukemia progression is subsequently facilitated by destabilization of ribosome biogenesis and protein synthesis, which confer a transient transformation advantage. The contribution of transcriptional variability to early cancer evolution reflects a generic role in promoting cell fate transitions, which, in the case of well-adapted malignancies, contrastingly differentiates and depletes cancer stem cells. In other words, transcriptional variability confers forward momentum to cell fate systems, with differential multi-stage impact throughout cancer evolution. One-sentence summaryLoss of Kat2a enhances transcriptional variability of ribosome biosynthetic programs and transiently accelerates pre-leukemia

cancer biology↗

bioRxiv2020

Mutational synergy coordinately remodels chromatin accessibility, enhancer landscape and 3-Dimensional DNA topology to alter gene expression during leukemia induction

Altered transcription is a cardinal feature of acute myeloid leukemia (AML), however, exactly how mutations synergize to remodel the epigenetic landscape and rewire 3-Dimensional (3-D) DNA topology is unknown. Here we apply an integrated genomic approach to a murine allelic series that models the two most common mutations in AML, Flt3-ITD and Npm1c. We then deconvolute the contribution of each mutation to alterations of the epigenetic landscape and genome organization, and infer how mutations synergize in the induction of AML. These analyses allow the identification of long-range cis-regulatory circuits, including a novel super-enhancer of the Hoxa locus, as well as larger and more detailed gene-regulatory networks, whose importance we demonstrate through perturbation of network members.

cancer biology↗

bioRxiv2018

Loss Of Kat2a Enhances Transcriptional Noise And Depletes Acute Myeloid Leukemia Stem-Like Cells

Acute Myeloid Leukemia (AML) is an aggressive hematological malignancy with abnormal progenitor self-renewal and defective myelo-monocytic differentiation. Its pathogenesis comprises subversion of transcriptional regulation, through mutation and by hijacking normal chromatin regulation. Kat2a is a histone acetyltransferase central to promoter activity that we recently associated with stability of pluripotency networks, and identified as a genetic vulnerability in AML. Through combined chromatin profiling and single-cell transcriptomics, we demonstrate that Kat2a contributes to leukemia propagation through homogeneity of transcriptional programs and preservation of leukemia stem-like cells. Kat2a loss reduces transcriptional bursting frequency in a subset of gene promoters, generating enhanced variability of transcript levels but minimal effects on mean gene expression. Destabilization of target programs shifts cellular equilibrium out of self-renewal towards differentiation. We propose that control of transcriptional variability is central to leukemia stem-like cell propagation, and establish a paradigm exploitable in different tumors and at distinct stages of cancer evolution.

cancer biology↗