Figure - available from: Nature
This content is subject to copyright. Terms and conditions apply.
Fin melanocytes have a limb positional identity Related to Fig. 3. (a–c) Validation of successful isolation of melanocytes for RNA-seq. (a) Volcano plots comparing melanocytes (GFP+ sample) to their microenvironment (GFP- sample) across locations and transgenic models. Melanocyte markers are labeled. Genes with FDR-adjusted p-value < 0.05 indicated in blue. P-values calculated using DESeq2. (b) GSEA showing the list of the top pathways enriched in melanocytes (GFP+ sample) compared to their microenvironment (GFP- sample). Colors indicate p-value adjusted for FDR = 0.05. (c) Log normalized counts for the expression of transgenes across all samples. EGFP and mitfa expression are high in all melanocyte samples and CRKL, GAB2, TERT, and Cas9-mCherry expression is high only in acral melanoma model melanocytes. N = 3 biologically independent replicates. Box minima = 25th percentile, centre = 50th percentile, maxima = 75th percentile. Whiskers extends to the largest/smallest value no further than 1.5 * IQR (interquartile range) from the box maxima/minima. Data beyond the end of the whiskers are plotted individually as outliers. (d) Principal component analysis (PCA) for all samples showing principal components 1 (PC1) and 2 (PC2). The percent of transcriptional variation captured by each principal component is indicated. (e) GSEA pathway analysis comparing fin vs body melanocytes from the WT melanocyte model listing the top enriched pathways in fin melanocytes. Limb development and positional identity-related pathways are highlighted. See Supplementary Table 4 for a full list of pathways. (f) GSEA barcode plot showing enrichment of genes in the GO: Appendage Development pathway, generated with weighted kolmogorov smirnov (WKS) testing. NES and FDR = 0.05 adjusted p-values are indicated. (g− h) Volcano plots comparing fin melanocytes vs body melanocytes from the (g) WT melanocyte model and (h) acral melanoma model. Genes with FDR-adjusted p-value < 0.05 indicated in blue. P-values calculated using DESeq2.
Source publication
Oncogenic alterations to DNA are not transforming in all cellular contexts1,2. This may be due to pre-existing transcriptional programmes in the cell of origin. Here we define anatomic position as a major determinant of why cells respond to specific oncogenes. Cutaneous melanoma arises throughout the body, whereas the acral subtype arises on the pa...
Citations
... We performed CRISPR-seq on DNA from both populations to directly measure Cas9 efficiency and specificity, quantifying the proportion of indels at the albino locus. Although we have previously shown that FACS sorting melanocytic cells is possible, it is important to note that we often observe high levels of contaminating keratinocytes in FACS sorted melanocyte populations, which may lead to an underestimation of the true allelic frequency in our CRISPR-seq (43). Despite this, robust inactivation of the albino gene was observed in GFP+ (mitfa-expressing) cells, confirming somatic inactivation (Fig. 2M). ...
... Other plasmids used in this study that were previously developed in the lab include zU6:gRNA-ptena/394, zU6:gRNA-ptenb/394, zU6:gRNA-p53/394, mitfa-BRAF V600E ;cmlc2:eGFP. All guide RNAs used in this study have previously been validated in zebrafish (17,35,43,71). ...
... The epidermal and dermal layers of the skin as well as the fins were separated from the rest of the tissues and diced into 1-3mm pieces. Cells were dissociated with Liberase TL (Millipore Sigma 05401020001) and filtered for single cell suspensions as previously described (43). Samples were then FACS sorted (BD FACSAria) for GFP+ and GFP-cells. ...
The advent of large-scale sequencing in both development and disease has identified large numbers of candidate genes that may be linked to important phenotypes. Validating the function of these candidates in vivo is challenging, due to low efficiency and low throughput of most model systems. This is especially the case in skin cells such as melanocytes, where the background mutation rate is high. We have developed a rapid and scalable system for assessing the role of candidate genes in a melanocyte specific manner using zebrafish. We generated transgenic zebrafish in which Cas9 was knocked-in to the endogenous mitfa locus, a master transcription factor of the melanocyte lineage. By introducing single guide RNA expression cassettes into mitfaCas9 embryos, we were able to achieve highly efficient melanocyte-specific mutation of genes important for melanocyte patterning and survival. These animals can be used to screen for dominant or recessive pigment defects in both the F0 generation (3 days) and F1 generation (3 months). We also utilized the mitfaCas9 line to study the role of melanoma genetic dependencies such as SOX10, demonstrating that loss of SOX10 reduces melanoma initiation yet promotes tumor progression by a switch to a SOX9hi state. This SOX10 to SOX9 switch has previously been observed in human patients, indicating that our system can be used to rapidly uncover biological states with relevance to human disease. Our high efficiency genetic approach can be readily applied to other cell lineages, with relevance to both development and disease.
... (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Our results also add to the growing appreciation that tissue context can influence the output of 282 gene mutations in cancer, for example, as illustrated by the differential susceptibility of cells from 283 particular tissues or tissue locations to certain oncogenic events [54][55][56][57] . Our findings extend this 284 concept to metastasis by showing how organ site can have a profound impact on a single driver 285 mutation in a tumor from the same tissue of origin. ...
Driver gene mutations can increase the metastatic potential of the primary tumor, but their role in sustaining tumor growth at metastatic sites is poorly understood. A paradigm of such mutations is inactivation of SMAD4 - a transcriptional effector of TGFβ signaling - which is a hallmark of multiple gastrointestinal malignancies. SMAD4 inactivation mediates TGFβ's remarkable anti- to pro-tumorigenic switch during cancer progression and can thus influence both tumor initiation and metastasis. To determine whether metastatic tumors remain dependent on SMAD4 inactivation, we developed a mouse model of pancreatic ductal adenocarcinoma (PDAC) that enables Smad4 depletion in the pre-malignant pancreas and subsequent Smad4 reactivation in established metastases. As expected, Smad4 inactivation facilitated the formation of primary tumors that eventually colonized the liver and lungs. By contrast, Smad4 reactivation in metastatic disease had strikingly opposite effects depending on the tumor's organ of residence: suppression of liver metastases and promotion of lung metastases. Integrative multiomic analysis revealed organ-specific differences in the tumor cells' epigenomic state, whereby the liver and lungs harbored chromatin programs respectively dominated by the KLF and RUNX developmental transcription factors, with Klf4 depletion being sufficient to reverse Smad4's tumor-suppressive activity in liver metastases. Our results show how epigenetic states favored by the organ of residence can influence the function of driver genes in metastatic tumors. This organ-specific gene-chromatin interplay invites consideration of anatomical site in the interpretation of tumor genetics, with implications for the therapeutic targeting of metastatic disease.
... We know that onco genic alterations to DNA are not transforming in all cellular contexts (7). It has been shown that anatomic position determines oncogenic responses due to preexisting transcriptional pro grammes of the cell of origin (45). ...
Our understanding of how human skin cells differ according to anatomical site and tumour formation is limited. To address this, we have created a multiscale spatial atlas of healthy skin and basal cell carcinoma (BCC), incorporating in vivo optical coherence tomography, single-cell RNA sequencing, spatial global transcriptional profiling, and in situ sequencing. Computational spatial deconvolution and projection revealed the localisation of distinct cell populations to specific tissue contexts. Although cell populations were conserved between healthy anatomical sites and in BCC, mesenchymal cell populations including fibroblasts and pericytes retained signatures of developmental origin. Spatial profiling and in silico lineage tracing support a hair follicle origin for BCC and demonstrate that cancer-associated fibroblasts are an expansion of a POSTN + subpopulation associated with hair follicles in healthy skin. RGS5+ pericytes are also expanded in BCC suggesting a role in vascular remodelling. We propose that the identity of mesenchymal cell populations is regulated by signals emanating from adjacent structures and that these signals are repurposed to promote the expansion of skin cancer stroma. The resource we have created is publicly available in an interactive format for the research community.
... No significant relationship emerged between LZTR1 expression and CDK4i/6i sensitivity in our data (Supplementary Fig. 2D). Further, CRKL, a signaling adaptor protein in pathways including the IGF1R-PI3K axis, was proposed to serve as an oncogenic driver of ALM [14]. We also did not observe a significant correlation between CRKL expression and CDK4i/6i sensitivity ( Supplementary Fig. 2E). ...
Patients with metastatic acral lentiginous melanoma (ALM) suffer worse outcomes relative to patients with other forms of cutaneous melanoma (CM), and do not benefit as well to approved melanoma therapies. Identification of cyclin-dependent kinase 4 and 6 (CDK4/6) pathway gene alterations in >60% of ALMs has led to clinical trials of the CDK4/6 inhibitor (CDK4i/6i) palbociclib for ALM; however, median progression free survival with CDK4i/6i treatment was only 2.2 months, suggesting existence of resistance mechanisms. Therapy resistance in ALM remains poorly understood; here we report hyperactivation of MAPK signaling and elevated cyclin D1 expression serve as a mechanism of intrinsic early/adaptive CDK4i/6i resistance. ALM cells that have acquired CDK4i/6i resistance following chronic treatment exposure also exhibit hyperactivation of the MAPK pathway. MEK and/or ERK inhibition increases CDK4i/6i efficacy against therapy naïve and CDK4i/6i-resistant AM cells in xenograft and patient-derived xenograft (PDX) models and promotes a defective DNA repair, cell cycle arrested and apoptotic program. Notably, gene alterations poorly correlate with protein expression of cell cycle proteins in ALM or efficacy of CDK4i/6i, urging additional strategies when stratifying patients for CDK4i/6i trial inclusion. Concurrent targeting of the MAPK pathway and CDK4/6 represents a new approach for patients with metastatic ALM to improve outcomes.
... For example, the transcription factor SOX10 specifies a developmental stage in the neural crest-melanoblast-melanocyte trajectory in which BRAF, an oncogene mutated in 50% of cutaneous melanomas, is capable of cellular transformation and tumor initiation (34). Interestingly, acral melanomas, which develop on the palms and feet, depend on a different set of lineage transcription factors and mutations (35). Lineage factors can thus establish a permissive cellular state that facilitates oncogenesis by some genetically activated pathways but not others. ...
Cancer cells adapt and survive through the acquisition and selection of molecular modifications. This process defines cancer evolution. Building on a theoretical framework based on heritable genetic changes has provided insights into the mechanisms supporting cancer evolution. However, cancer hallmarks also emerge via heritable nongenetic mechanisms, including epigenetic and chromatin topological changes, and interactions between tumor cells and the tumor microenvironment. Recent findings on tumor evolutionary mechanisms draw a multifaceted picture where heterogeneous forces interact and influence each other while shaping tumor progression. A comprehensive characterization of the cancer evolutionary toolkit is required to improve personalized medicine and biomarker discovery.
Significance
Tumor evolution is fueled by multiple enabling mechanisms. Importantly, genetic instability, epigenetic reprogramming, and interactions with the tumor microenvironment are neither alternative nor independent evolutionary mechanisms. As demonstrated by findings highlighted in this perspective, experimental and theoretical approaches must account for multiple evolutionary mechanisms and their interactions to ultimately understand, predict, and steer tumor evolution.
... Transposase-accessible chromatin sites are enriched in promotor and enhancer regions in melanoma, as well as several other cancers (Gupta et al, 2020). Finally, histone modifications at HOX gene clusters are important regulators of this gene family (Afzal and Krumlauf, 2022), an important regulator of cell tissueespecific function linked with melanoma (Weiss et al, 2022). Sequence variants in several genes, including HOX genes themselves, can play a role in gene silencing and posttranslational chromatin modification, including by regulating expression of DNA methyltransferases and chromatin remodeling complexes (Cust et al., 2020;Feng et al, 2021;Gupta et al, 2020). ...
... CM are related to the anatomical site. A recent integrated DNA sequencing/RNA-seq study identified amplification of GAB2 and CRKL, two adaptor proteins that amplify receptor tyrosine kinase (RTK) signaling, in AM vs CM [40]. The introduction/silencing of the identified AM genes in a zebrafish model led to the development of melanomas in the fins, whereas expression of CM-associated genes led to skin melanomas [40]. ...
... A recent integrated DNA sequencing/RNA-seq study identified amplification of GAB2 and CRKL, two adaptor proteins that amplify receptor tyrosine kinase (RTK) signaling, in AM vs CM [40]. The introduction/silencing of the identified AM genes in a zebrafish model led to the development of melanomas in the fins, whereas expression of CM-associated genes led to skin melanomas [40]. Mechanistic studies showed that HOX13 was instrumental in regulating IGF signaling in fin melanocytes and that CRKL amplification served to amplify both HOX13 and IGFR signaling through the PI3K pathway [40]. ...
... The introduction/silencing of the identified AM genes in a zebrafish model led to the development of melanomas in the fins, whereas expression of CM-associated genes led to skin melanomas [40]. Mechanistic studies showed that HOX13 was instrumental in regulating IGF signaling in fin melanocytes and that CRKL amplification served to amplify both HOX13 and IGFR signaling through the PI3K pathway [40]. This work demonstrated for the first time that microenvironment of the anatomical site dictated the underlying transcriptional state of the melanocytes, conferring susceptibility to distinct oncogenes. ...
Acral melanoma is a rare subtype of melanoma that arises on the non-hair bearing skin of the nail bed, palms of the hand and soles of the feet. It is unique among melanomas in not being linked to ultraviolet radiation (UVR) exposure from the sun, and, as such, its incidence is similar across populations who are of Asian, Hispanic, African and European origin. Although research into acral melanoma has lagged behind that of sun-exposed cutaneous melanoma, recent studies have begun to address the unique genetics and immune features of acral melanoma. In this review we will discuss the latest progress in understanding the biology of acral melanoma across different ethnic populations and will outline how these new discoveries can help to guide the therapeutic management of this rare tumor.
... Previous studies have highlighted the distinct genomic landscape of MMs and acral melanoma compared to CMs (6). To investigate whether there are distinct expression profiles in MMs, we integrated our transcriptome data with published RNA-seq data of CMs and acral melanoma (see Materials and Methods) (31). Given the relatively small number of samples for comparison, we performed multiple comparisons of canonical pathways between any two of the three melanoma subtypes. ...
... RNA-seq data of acral and CM were downloaded from Weiss et al. (31). The provided counts value was used for comparison in our analyses. ...
Mucosal melanoma (MM), an aggressive rare subtype of melanoma, is distinct from cutaneous melanoma and has poor prognoses. We addressed the lack of cell models for MM by establishing 30 organoids of human oral MM (OMM), which retained major histopathological and functional features of parental tumors. Organoid groups derived from chronologically or intratumorally distinct lesions within the same individual displayed heterogeneous genetics, expression profiles, and drug responses, indicating rapid tumor evolution and poor clinical response. Furthermore, transcriptome analysis revealed receptor tyrosine kinases (RTKs) signaling, particularly NGFR , a nerve growth factor receptor, was significantly up-regulated in OMMs and organoids from patients resistant to anti–programmed cell death protein 1 (anti–PD-1) therapy. Combining anti–PD-1 with anlotinib (a phase 2 multitarget RTK inhibitor for OMM) or NGFR knockdown enhanced the effective activity of immune cells in organoid–immune cell coculture systems. Together, our study suggested that OMM organoids serve as faithful models for exploring tumor evolution and immunotherapy combination strategies.
... Weiss et al. [18] discovered that acral melanocytes have distinct positional identity programs that make them more likely to develop acral melanoma than their counterparts in other parts of the body. They discovered, using transgenic zebrafish animal models, that CRKL amplifications, in conjunction with the "pro-acral melanoma" transcriptional state provided by the HOX13 genes, favor the progression of acral melanoma at glabrous sites of the extremities [18]. ...
... Weiss et al. [18] discovered that acral melanocytes have distinct positional identity programs that make them more likely to develop acral melanoma than their counterparts in other parts of the body. They discovered, using transgenic zebrafish animal models, that CRKL amplifications, in conjunction with the "pro-acral melanoma" transcriptional state provided by the HOX13 genes, favor the progression of acral melanoma at glabrous sites of the extremities [18]. This means that, when compared to other anatomical sites, volar surfaces are genetically predisposed to develop a specific subtype of melanoma. ...
Melanoma affecting glabrous skin is a challenging entity that needs to be managed by an interdisciplinary team of dermatologists, oncologists, and surgeons. The thin subcutaneous layer of glabrous skin, which speeds up its metastatic spread, is one of the key elements that contributes to the aggressiveness of this form of cutaneous cancer when identified in this anatomical region. Acral lentiginous melanoma is a rare melanocytic malignancy that is usually associated with ominous outcomes, especially in those with dark skin. Moreover, more extensive research is needed to elucidate the puzzle of molecular drivers and their relationship with thermal injury. We reported our experience in order to highlight the value of timely diagnosis and treatment.
... Recent study focused on the positional identity linked to the anatomical location is a determining factor for transformation potential for oncogenes in Melanoma. Compared to melanocytes from other anatomic regions, acral melanocytes were more vulnerbale to CRKL transformation [23]. ...
Skin cancer is a prevalent and heterogenous disease with several subtypes, such as melanoma, basal cell carcinoma, and squamous cell carcinoma. Among them, melanoma is the most aggressive subtype, with a higher propensity to spread compared to most solid tumors. The application of OMICS approaches has revolutionized the field of melanoma research by providing comprehensive insights into the molecular alterations and biological processes underlying melanoma development and progression. This review aims to offer an overview of melanoma biology, covering its transition from primary to malignant melanoma, as well as the key genes and pathways involved in the initiation and progression of this disease. Utilizing online databases, we extensively explored the general expression profile of genes, identified the most frequently altered genes, and gene mutations, and examined genetic alterations responsible for drug resistance. Additionally, we studied the mechanisms responsible for immune checkpoint inhibitors resistance in melanoma.
