Biology subjects

Makhzami, S.

Publications and source records attributed to Makhzami, S..

9 recordsLinked to original sources

bioRxiv2025

Multiomic profiling of a unique in-transit melanoma cohort identifies melanoma differentiation as predictor of tumor progression and therapy response

Melanoma patients with in-transit metastasis (ITM), a stage of disease where melanoma has metastasized to sites in between the primary lesion and draining lymph node, vary significantly in their clinical outcomes, but the biology driving differential outcomes in ITM is poorly understood. To elucidate the mechanisms of differential outcomes, we utilized multimodal molecular profiling (WES, RNA-seq, highly multiplexed immunofluorescence, spatial transcriptomics) in 1) evolutionary analysis of longitudinal tumor samples and 2) identifying prognostic tumor intrinsic and microenvironmental features in a unique cohort of patients with unresectable ITM. Among other findings, we observed a persistent dedifferentiated AXL/NGFR clonal lineage pre-existing and following immune checkpoint blockade in in-transit and distant metastases. Concordantly, we found that low pigmentation and high T cell exhaustion signatures were independently associated with distant progression. Our findings highlight tumor cell state and immune dysfunction as key predictors and potential biomarkers of metastatic risk in ITM. STATEMENT OF SIGNIFICANCEWhat drives distant progression in melanoma is unclear. Analyzing tumor and immune features in a rare in-transit melanoma patient cohort, we identify biological signals highlighting how immune and tumor states observable in pre-distant metastasis melanomas shape long-term outcomes, and nominate potential prognostic biomarkers.

cancer biology↗

bioRxiv2025

Redirecting resistance evolution in BRAFV600 melanoma by inhibition of the peroxiredoxin-thioredoxin system

Drug-tolerant persister cells (DTPs) exhibit remarkable cell state heterogeneity and phenotypic evolvability. However, the central question of how DTPs epigenetically coordinate their metabolic flexibility to adapt early to therapeutic stress remains unanswered. We have recently shown that the histone demethylase KDM5B, which is intrinsically expressed in differentiated melanoma DTPs, reprograms the metabolic cell landscape. However, the exact mechanism by which KDM5B affects underlying metabolic enzymes, and whether this reveals new druggable vulnerabilities, remain unknown. By transcriptional and epigenetic profiling of BRAFV600 melanoma cells following KDM5B gene silencing, we discovered a direct molecular axis between the epigenetic regulator KDM5B and the PRDX/TXN ROS detoxification system. This metabolic axis is regulated independently of KDM5Bs demethylase activity. Furthermore, RNAi approaches and the pharmacological inhibition of the PRDX/TXN system led to ROS-induced cell death in differentiated melanoma DTPs and a delay of resistance development to MAPK inhibition. This process was independent of lipid-ROS-driven ferroptosis. Additionally, single-cell transcriptome analyses from pre-clinical melanoma models under continuous MAPK inhibitory treatment demonstrated altered cellular differentiation dynamics, with a reduction in the early evolution into the mesenchymal DTP state under concomitant PRDX inhibition. Interestingly, the degree of melanoma cell state differentiation at the onset of treatment was a major determinant for the transition towards the neural crest-like DTP state. Our study identified a high degree of epigenetic-metabolic connectivity and flexibility within the melanoma DTP pool and urges caution with single redox pathway-targeted strategies for tumor elimination in the future. Prospectively, our results point towards a new resistance targeting strategy for BRAFV600 melanoma patients based on pharmacological re-direction of the evolution of melanoma cell states already at therapy onset. HighlightsO_LITranscriptional and epigenetic profiling identified KDM5B as a regulator of the PRDX/TXN-ROS detoxification system C_LIO_LIPRDX inhibition increases the vulnerability of KDM5Bhigh DTPs to ROS, independent of ferroptosis C_LIO_LIPRDX inhibition delays resistance to MAPK inhibition in BRAFV600 melanoma cells C_LIO_LILongitudinal single-cell transcriptome analysis reveals that PRDX inhibition re-directs early DTP evolution C_LIO_LIPRDX/TXN gene expression is predictive for melanoma patient survival C_LI

cancer biology↗

bioRxiv2023

Rescuing early Parkinson-induced hyposmia prevents dopaminergic system failure

The classical diagnosis of Parkinsonism is based on motor symptoms that are the consequence of nigrostriatal pathway dysfunction and reduced dopaminergic output. However, a decade prior to the emergence of motor issues, patients frequently experience non-motor symptoms, such as a reduced sense of smell (hyposmia). The cellular and molecular bases for these early defects remain enigmatic. To explore this, we developed a new collection of five fruit fly models of familial Parkinsonism and conducted single-cell RNA sequencing on young brains of these models. Interestingly, cholinergic projection neurons are the most vulnerable cells and genes associated with presynaptic function are the most deregulated. Additional single nucleus sequencing of three specific brain regions of Parkinsons disease patients confirms these findings. Indeed, the disturbances lead to early synaptic dysfunction, notably affecting cholinergic olfactory projection neurons crucial for olfactory function in flies. Correcting these defects specifically in olfactory cholinergic interneurons in flies or inducing cholinergic signaling in Parkinson mutant human induced dopaminergic neurons in vitro using nicotine, both rescue age-dependent dopaminergic neuron decline. Hence, our research uncovers that one of the earliest indicators of disease in 5 different models of familial Parkinsonism is synaptic dysfunction in higher-order cholinergic projection neurons and this contributes to the development of hyposmia. Furthermore, the shared pathways of synaptic failure in these cholinergic neurons ultimately contribute to dopaminergic dysfunction later in life.

neuroscience↗

bioRxiv2022

A TCF4/BRD4-dependent regulatory network confers cross-resistance to targeted and immune checkpoint therapy in melanoma

Primary resistance drastically limits the clinical success of immune checkpoint blockade (ICB) in melanoma. Resistance to ICB may also develop when tumours relapse after targeted therapy. To identify cancer cell-intrinsic mechanisms driving resistance to ICB, we generated single-cell RNA-sequencing (scRNA-seq) data from a prospective longitudinal cohort of patients on ICB therapy, including an early time point obtained after only one cycle of treatment. Comparing these data with murine scRNA-seq datasets, we established a comprehensive view of the cellular architecture of the treatment-naive melanoma ecosystem, and defined 6 evolutionarily conserved melanoma transcriptional metaprograms (Melanocytic or MEL, Mesenchymal-like or MES, Neural Crest-like, Antigen Presentation, Stress (hypoxia response) and Stress (p53 response)). Spatial multi-omics revealed a non-random geographic distribution of cell states that is, at least partly, driven by the tumour microenvironment. The single-cell data allowed unambiguous discrimination between melanoma MES cells and cancer-associated fibroblasts both in silico and in situ, a long-standing challenge in the field. Importantly, two of the melanoma transcriptional metaprograms were associated with divergent clinical responses to ICB. While the Antigen Presentation cell population was more abundant in tumours from patients who exhibited a clinical response to ICB, MES cells were significantly enriched in early on-treatment biopsies from non-responders, and their presence significantly predicted lack of response. Critically, we identified TCF4 (E2-2) as a master regulator of the MES program and suppressor of both MEL and Antigen Presentation programs. Targeting TCF4 expression in MES cells either genetically or pharmacologically using a bromodomain inhibitor increased immunogenicity and sensitivity to targeted therapy. This study describes an increasingly complex melanoma transcriptional landscape and its rapid evolution under ICB. It also identifies a putative biomarker of early response to ICB and an epigenetic therapeutic strategy that increases both immunogenicity of ICB-refractory melanoma and their sensitivity to targeted therapy.

cancer biology↗

bioRxiv2021

Decoding gene regulation in the fly brain

The Drosophila brain is a work horse in neuroscience. Single-cell transcriptome analysis 1-5, 3D morphological classification 6, and detailed EM mapping of the connectome 7-10 have revealed an immense diversity of neuronal and glial cell types that underlie the wide array of functional and behavioral traits in the fruit fly. The identities of these cell types are controlled by - still unknown - gene regulatory networks (GRNs), involving combinations of transcription factors that bind to genomic enhancers to regulate their target genes. To characterize the GRN for each cell type in the Drosophila brain, we profiled chromatin accessibility of 240,919 single cells spanning nine developmental timepoints, and integrated this data with single-cell transcriptomes. We identify more than 95,000 regulatory regions that are used in different neuronal cell types, of which around 70,000 are linked to specific developmental trajectories, involving neurogenesis, reprogramming and maturation. For 40 cell types, their uniquely accessible regions could be associated with their expressed transcription factors and downstream target genes, through a combination of motif discovery, network inference techniques, and deep learning. We illustrate how these "enhancer-GRNs" can be used to reveal enhancer architectures leading to a better understanding of neuronal regulatory diversity. Finally, our atlas of regulatory elements can be used to design genetic driver lines for specific cell types at specific timepoints, facilitating the characterization of brain cell types and the manipulation of brain function.

genomics↗

bioRxiv2019

Cross-species analysis of melanoma enhancer logic using deep learning

Genomic enhancers form the central nodes of gene regulatory networks by harbouring combinations of transcription factor binding sites. Deciphering the combinatorial code by which these binding sites are assembled within enhancers is indispensable to understand their regulatory involvement in establishing a cells phenotype, especially within biological systems with dysregulated gene regulatory networks, such as melanoma. In order to unravel the enhancer logic of the two most common melanoma cell states, namely the melanocytic and mesenchymal-like state, we combined comparative epigenomics with machine learning. By profiling chromatin accessibility using ATAC-seq on a cohort of 27 melanoma cell lines across six different species, we demonstrate the conservation of the two main melanoma states and their underlying master regulators. To perform an in-depth analysis of the enhancer architecture, we trained a deep neural network, called DeepMEL, to classify melanoma enhancers not only in the human genome, but also in other species. DeepMEL revealed the presence, organisation and positional specificity of important transcription factor binding sites. Together, this extensive analysis of the melanoma enhancer code allowed us to propose the concept of a core regulatory complex binding to melanocytic enhancers, consisting of SOX10, TFAP2A, MITF and RUNX, and to disentangle their individual roles in regulating enhancer accessibility and activity.

genomics↗

bioRxiv2019

Identification of genomic enhancers through spatial integration of single-cell transcriptomics and epigenomics

Single-cell technologies allow measuring chromatin accessibility and gene expression in each cell, but jointly utilizing both layers to map bona fide gene regulatory networks and enhancers remains challenging. Here, we generate independent single-cell RNA-seq and single-cell ATAC-seq atlases of the Drosophila eye-antennal disc and spatially integrate the data using a virtual latent space that mimics the organization of the 2D tissue. To validate spatially predicted enhancers, we use a large collection of enhancer-reporter lines and identify [~]85% of enhancers in which chromatin accessibility and enhancer activity are coupled. Next, we infer enhancer-to-gene relationships in the virtual space, finding that genes are regulated by multiple redundant enhancers. Exploiting cell-type specific enhancers, we deconvolute cell-type specific effects of bulk-derived chromatin accessibility QTLs. Finally, we discover that Prospero drives neuronal differentiation through the binding of a GGG motif. In summary, we provide a comprehensive spatial characterization of gene regulation in a 2D tissue.

genomics↗

bioRxiv2019

Single-cell gene regulatory network analysis reveals new melanoma cell states and transition trajectories during phenotype switching

Melanoma is notorious for its cellular heterogeneity, which is at least partly due to its ability to transition between alternate cell states. Similarly to EMT, melanoma cells with a melanocytic phenotype can switch to a mesenchymal-like phenotype. However, scattered emerging evidence indicates that additional, intermediate state(s) may exist. In order to search for such new melanoma states and decipher their underlying gene regulatory network (GRN), we extensively studied ten patient-derived melanoma cultures by single-cell RNA-seq of >39,000 cells. Although each culture exhibited a unique transcriptome, we identified shared gene regulatory networks that underlie the extreme melanocytic and mesenchymal cell states, as well as one (stable) intermediate state. The intermediate state was corroborated by a distinct open chromatin landscape and governed by the transcription factors EGR3, NFATC2, and RXRG. Single-cell migration assays established that this \"transition\" state exhibits an intermediate migratory phenotype. Through a dense time-series sampling of single cells and dynamic GRN inference, we unraveled the sequential and recurrent arrangement of transcriptional programs at play during phenotype switching that ultimately lead to the mesenchymal cell state. We provide the scRNA-Seq data with 39,263 melanoma cells on our SCope platform and the ATAC-seq data on a UCSC hub to jointly serve as a resource for the melanoma field. Together, this exhaustive analysis of melanoma cell state diversity indicates that additional states exists between the two extreme melanocytic and mesenchymal-like states. The GRN we identified may serve as a new putative target to prevent the switch to mesenchymal cell state and thereby, acquisition of metastatic and drug resistant potential.

genomics↗

bioRxiv2017

A single-cell catalogue of regulatory states in the ageing Drosophila brain

The diversity of cell types and regulatory states in the brain, and how these change during ageing, remains largely unknown. Here, we present a single-cell transcriptome catalogue of the entire adult Drosophila melanogaster brain sampled across its lifespan. Both neurons and glia age through a process of \"regulatory erosion\", characterized by a strong decline of RNA content, and accompanied by increasing transcriptional and chromatin noise. We identify more than 50 cell types by specific transcription factors and their downstream gene regulatory networks. In addition to neurotransmitter types and neuroblast lineages, we find a novel neuronal cell state driven by datilografo and prospero. This state relates to neuronal birth order, the metabolic profile, and the activity of a neuron. Our single-cell brain catalogue reveals extensive regulatory heterogeneity linked to ageing and brain function and will serve as a reference for future studies of genetic variation and disease mutations.

genomics↗