Biology subjects

Kaufman, C. K.

Publications and source records attributed to Kaufman, C. K..

6 recordsLinked to original sources

bioRxiv2025

Mapping of the hSOX10 protein interactome in human melanoma

The transcription factor SOX10 is a central regulator of melanoma biology, influencing tumor initiation, progression, phenotypic plasticity, and therapeutic resistance. However, the molecular mechanisms underlying these diverse functions remain incompletely understood. To elucidate the protein-protein interactions (PPIs) that mediate SOX10 activity in melanoma, we mapped the human SOX10 (hSOX10) interactome for the first time in human melanoma cells (A375 line). Given the challenges of capturing transient and weak transcription factor interactions, we employed miniTurbo (mT) proximity-dependent biotinylation coupled with mass spectrometry (MS). Stable A375 cell lines expressing N- and C-terminal mT-tagged hSOX10 fusion proteins at physiologically relevant levels were generated, enabling unbiased biotinylation and MS-based identification of candidate interacting proteins. This approach revealed 847 melanoma-enriched candidate interactors, including 213 high-confidence hits. These included both known hSOX10 partners and previously unidentified putative interactors. Functional annotation of our hSOX10 interactome highlighted associations with chromatin remodeling, transcriptional regulation, SUMOylation, and DNA damage response pathways. Western blot validation of select interactors supported the robustness of our dataset. This first comprehensive map of the hSOX10 interactome in melanoma provides a critical foundation for future investigations into SOX10-driven transcriptional networks and their potential as therapeutic targets and biomarkers in melanoma.

cancer biology↗

bioRxiv2024

Specific SOX10 enhancer elements modulate phenotype plasticity and drug resistance in melanoma

Recent studies indicate that the development of drug resistance and increased invasiveness in melanoma is largely driven by transcriptional plasticity rather than canonical coding mutations. Understanding the mechanisms behind cell identity shifts in oncogenic transformation and cancer progression is crucial for advancing our understanding of melanoma and other aggressive cancers. While distinct melanoma phenotypic states have been well characterized, the processes and transcriptional controls that enable cells to shift between these states remain largely unknown. In this study, we initially leverage the well-established zebrafish melanoma model as a high-throughput system to dissect and analyze transcriptional control elements that are hijacked by melanoma. We identify key characteristics of these elements, making them translatable to human enhancer identification despite the lack of direct sequence conservation. Building on our identification of a zebrafish sox10 enhancer necessary for melanoma initiation, we extend these findings to human melanoma, identifying two human upstream enhancer elements that are critical for full SOX10 expression. Stable biallelic deletion of these enhancers using CRISPR-Cas9 induces a distinct phenotype shift across multiple human melanoma cell lines from a melanocytic phenotype towards an undifferentiated phenotype and is also characterized by an increase in drug resistance that mirrors clinical data including an upregulation of NTRK1, a tyrosine kinase, and potential therapeutic target. These results provide new insights into the transcriptional regulation of SOX10 in human melanoma and underscore the role of individual enhancer elements and potentially NTRK1 in driving melanoma phenotype plasticity and drug resistance. Our work lays the groundwork for future gene-based and combination kinase-inhibitor therapies targeting SOX10 regulation and NTRK1 as a potential avenue for enhancing the efficacy of current melanoma treatments.

cancer biology↗

bioRxiv2022

Cross-species analysis reveals unique and shared roles of Sox9 and Sox10 (SOXE family) transcription factors in melanoma

SOX9 and SOX10 are two highly similar transcription factors with nearly 100% identity at their DNA binding domains. Both transcription factors play key but distinct roles in neural crest cell fate specification and melanoma formation. High expression of SOX9 and SOX10 appear to be mutually exclusive, with high SOX10 characteristic of proliferative melanoma and high SOX9 characteristic of metastatic melanoma. To further elucidate the role of SOX9 in melanoma, we over-express SOX9 in a zebrafish melanoma model and a human melanoma cell line. Analysis of tumor onset, binding dynamics, and transcriptional identities supports the notion of SOX9 driving a more mesenchymal signature, which is important for metastasis. Additionally, we identified a potential mechanism of SOX9 down-regulation via analysis of a functional and recurrent non-coding variant in human melanoma. Altogether, our results present a dosage-dependent role of SOX9 and, likely, SOX10 in the melanoma lifespan.

cancer biology↗

bioRxiv2022

Functional analysis of recurrent non-coding variants in human melanoma

Small nucleotide variants in non-coding regions of the genome can alter transcriptional regulation, leading to changes in gene expression which can activate oncogenic gene regulatory networks. Melanoma is heavily burdened by non-coding variants, representing over 99% of total genetic variation, including the well-characterized TERT promoter mutation. However, the compendium of regulatory non-coding variants is likely still functionally under-characterized. We developed a pipeline to identify hotspots, i.e. recurrently mutated regions, in melanoma containing putatively functional non-coding somatic variants that are located within predicted melanoma-specific regulatory regions. We identified hundreds of statistically significant hotspots, including the hotspot containing the TERT promoter variants, and focused in on a hotspot in the promoter of CDC20. We found that variants in the promoter of CDC20, which putatively disrupt an ETS motif, lead to lower transcriptional activity in reporter assays. Using CRISPR/Cas9, we generated an indel in the CDC20 promoter in a human A375 melanoma cell line and observed decreased expression of CDC20, changes in migration capabilities, and an altered transcriptional state previously associated with neural crest transcriptional programs and melanoma initiation. Overall, our analysis prioritized several recurrent functional non-coding variants that, through downregulation of CDC20, led to perturbation of key melanoma phenotypes.

genomics↗

bioRxiv2020

SATB2 induction of a neural crest mesenchyme-like program drives invasion and drug resistance in melanoma

Recent genomic and scRNA-seq analyses of melanoma identified common transcriptional states correlating with invasion or drug resistance, but failed to find recurrent drivers of metastasis. To test whether transcriptional adaptation can drive melanoma progression, we made use of a zebrafish mitfa:BRAFV600E;tp53-/- model, in which malignant progression is characterized by minimal genetic evolution. We undertook an overexpression-screen of 80 epigenetic/transcriptional regulators and found neural crest-mesenchyme developmental regulator SATB2 to accelerate aggressive melanoma development. Its overexpression induces invadopodia formation and invasion in zebrafish tumors and human melanoma cell lines. SATB2 binds and activates neural crest-regulators, including pdgfab and snai2. The transcriptional program induced by SATB2 overlaps with known MITFlowAXLhlgh and AQP1+NGFR1high drug resistant states and functionally drives enhanced tumor propagation and resistance to Vemurafenib in vivo. Here we show that melanoma transcriptional rewiring by SATB2 to a neural crest mesenchyme-like program can drive invasion and drug resistance in endogenous tumors.

cancer biology↗

bioRxiv2020

Functional in vivo characterization of zebrafish sox10 enhancers in melanoma and neural crest

The re-emergence of a neural crest transcriptional program, including Sox10 upregulation, is a key step in melanoma initiation in humans and zebrafish. We hypothesize that epigenetic regulation of sox10 modulates melanoma initiation. ATAC-Seq analysis of zebrafish melanoma tumors identifies recurrently open chromatin domains near sox10. Reporter constructs for each putative sox10 enhancer were examined in zebrafish embryos for neural crest activity and in stable transgenic lines for melanoma activity. One element, peak5 (23 kilobases upstream of sox10), drives EGFP reporter expression in a subset of neural crest cells, Kolmer-Agduhr neurons, and early melanoma patches and tumors with high specificity. A [~]200 bp region, conserved across the Cyprinidae family (fish), is required for peak5 activity in neural crest and melanoma, and contains dimeric SoxE binding sites essential for neural crest activity. Our work identifies a novel melanoma transcriptional enhancer, expanding our knowledge of epigenetic regulation of neural crest identity in melanoma.

cancer biology↗