Adult stem cells maintain tissue homeostasis and regenerative capacity throughout life, but the mechanisms that preserve their integrity during aging and stress remain incompletely understood. Hair follicle pigmentation provides a tractable model to study stem cell maintenance as it relies on melanocyte stem cells (McSCs) whose depletion leads to age-related hair greying. Here, we identify the transcription factor BRN2 ( POU3F2 ) as a key regulator of McSC homeostasis. We show that BRN2 activity is enriched in McSCs and declines with age in both mouse and human hair follicles. Using a melanocyte-specific conditional knockout mouse model, we reveal that loss of Brn2 promotes melanocyte differentiation and accumulation in the hair follicle bulb, accompanied by increased expression of melanocytic genes and a marked downregulation of DNA repair pathways, particularly those involved in nucleotide excision repair and chromosome maintenance. Brn2-deficient cells displayed impaired activation of the DNA damage response following ionizing radiation and entered a p53–p21–associated senescence-like differentiation state rather than undergoing apoptosis. In vivo, Brn2-deficient mice exposed to irradiation-induced stress developed rapid and permanent hair depigmentation associated with depletion of melanocyte lineage cells. Together, these findings identify BRN2 as a suppresor of stress-induced hair greying that links genome maintenance to melanocyte stem cell fate.

Interaction between the tumor microenvironment and cancer cell plasticity drives intratumor phenotypic heterogeneity and underpins disease progression and nongenetic therapy resistance. Phenotype-specific expression of the AXL receptor tyrosine kinase is a pivotal player in dormancy, invasion, and resistance to treatment. However, although the AXL ligand GAS6 is present within tumors, how AXL is activated in metastasizing cells remains unclear. Here, using melanoma as a model, we reveal that AXL is activated by exposure to human adipocytes and to oleic acid, a monounsaturated fatty acid abundant in lymph and in adipocytes. AXL activation triggers SRC-dependent formation and nuclear translocation of a β-catenin-CAV1 complex required for melanoma invasiveness. Remarkably, only undifferentiated AXLHigh melanoma cells engage in symbiosis with human adipocytes, in part by triggering WNT5a-mediated lipolysis, leading to AXL-dependent, but FATP-independent, fatty acid uptake and nuclear localization of the β-catenin-CAV1 complex. Significantly, human melanomas in the vicinity of adipocytes exhibit high levels of nuclear CAV1. The results unveil an AXL- and CAV1-dependent mechanism through which a nutritional input drives phenotype-specific activation of a prometastasis program. Given the key role of AXL in a broad range of cancers, the results offer major insights into the mechanisms of cancer cell dormancy and therapy resistance.

BRCA1-associated protein 1 (BAP1) can function as a tumor suppressor or oncogene depending on context, but its role in colorectal cancer (CRC) is not well understood. Here, we demonstrate that BAP1 suppresses CRC progression primarily by deubiquitinating and stabilizing von Hippel-Lindau tumor suppressor protein (pVHL). BAP1 undergoes covalent modification by ubiquitin-fold modifier 1 (UFM1) at Lys51, Lys61, Lys187, and Lys205, enhancing its interaction with pVHL and promoting pVHL stabilization. Loss of this modification through UFL1 depletion or reconstitution with a UFMylation-defective BAP1 mutant (4KR) impairs pVHL stabilization and promotes tumor progression in CRC cell line-based and patient-derived xenograft models. Clinically, down-regulation of UFL1 and BAP1 correlates with reduced pVHL level and poor prognosis in patients with CRC. These findings identify a previously unrecognized posttranslational mechanism regulating BAP1 activity and highlight UFMylation as essential for maintaining pVHL tumor-suppressive function. Targeting BAP1 UFMylation may represent a potential therapeutic strategy in CRC and other cancers with wild-type BAP1 and VHL.

PURPOSE:To identify the specific intratumoral and microenvironmental heterogeneity of acral melanoma (AM) and mucosal melanoma (MM), we aimed to delineate their distinct cellular compositions, evolutionary trajectories, and subtype-specific therapeutic strategies. EXPERIMENTAL DESIGN:Single-cell transcriptomic and genomic landscapes were analyzed across 42 melanoma (28 AM, 11 MM, and 3 nonacral cutaneous melanoma) samples, supplemented by in vitro and in vivo validation. Tumor and stromal cells were profiled using single-cell RNA sequencing, whole-exome sequencing, and functional assays, including transwell migration, co-culture systems, and xenograft models. RESULTS:Tumor cells exhibited divergent evolutionary routes, with MM dominated by MGP+/PCOLCE+ subpopulations showing high epithelial-to-mesenchymal transition potential. MM displayed elevated neutrophil infiltration and CXCL3+ tumor-associated macrophages, whereas AM was enriched with PI16+ cancer-associated fibroblasts promoting tumor proliferation. Molecular classification revealed MM subtypes: an antigen-presenting subtype linked to favorable outcomes and a proliferative subtype associated with recurrence. TIGIT+ regulatory T cells were enriched in AM, suggesting targeted inhibition potential. Genomic analysis connected BRAF/NRAS mutations to ALDOA+ stem-like tumor cells and identified prostaglandin D2 synthetase as a therapeutic target in triple-wild-type/melanomas. CONCLUSIONS:Our study provides a comprehensive comparison of AM and MM, uncovering subtype-specific stromal-immune interactions and molecular programs. The findings highlight actionable targets (e.g., TIGIT in AM and CXCL3+ macrophages in MM) and propose a framework for precision therapies, biomarker-driven trials, and risk stratification to improve outcomes in these aggressive melanomas.

The impact of the microenvironment on epigenetically plastic cancer cells underpins phenotypic heterogeneity, a major cause of metastatic dissemination and therapy resistance that together represent the primary cause of cancer-related death. Nutrient limitation is a key microenvironmental stress that can cause a phenotypic transition from proliferation to invasion via activation of the integrated stress response. However, whether and how the capacity to store and mobilize nutrients impacts phenotype-switching through metabolic buffering remains unknown. Here, using melanoma as a model, we reveal that the ability to accumulate and mobilize glycogen, that buffers glucose availability, plays a key role in phenotypic transitions in melanoma. While proliferative phenotype cells exhibit high levels of glycogen, invasion is marked by low glycogen levels. Significantly, an inability to store and metabolize glycogen leads to phenotype instability and a switch to invasion. Accordingly, glycogen levels inversely correlate with Clark levels in primary melanomas, with low expression of the glycogen phosphorylases PYGB/L and phosphoglucomutase 1 (PGM1) being associated with worse overall survival. The importance of metabolic buffering in suppressing phenotypic transitions likely extrapolates to other cancer types. Highlights:Melanoma phenotypes are distinguished by their ability to store and mobilize glycogen. Proliferative MITF High melanoma cells store glycogen to improve survival under stressful conditions. Inhibition of glycogen degradation impairs proliferation in MITF High melanoma cells. Lack of PGM1 drives invasion and metastatic dissemination.

Aberrant transcriptional programs mediate malignant transformation of melanoma, the most aggressive form of skin cancer. The lysine methyltransferase SETD6 has been implicated in regulating transcription, cell adhesion, migration, and other processes in various cancers; however its role in melanoma remains unexplored. We recently reported that SETD6 monomethylates the BRD4 at K99 to selectively regulate transcription of genes involved in mRNA (messenger RNA) translation. Here, we observed that BRD4 methylation at K99 by SETD6 occurs in melanoma cells. Knockout of SETD6 or a point mutation at BRD4-K99 disrupts BRD4 genomic occupancy. In addition, we show that SETD6 interacts with MITF, a master transcription factor in melanocytes and melanoma, and influences the genomic distribution of MITF. Mechanistically, we uncover a novel chromatin-localized interaction between BRD4 and MITF in melanoma. Our data suggest that BRD4 binds MITF in melanoma cells and that this interaction is dependent on both SETD6-mediated methylation of BRD4 and MITF acetylation. This chromatin complex plays a pivotal role in selective recruitment of BRD4 and MITF to different genomic loci in melanoma cells.

Bromodomain (BRD)-containing proteins are chemically tractable multi-domain scaffolding molecules involved in acetyl lysine (Kac) signaling. BRD inhibitors have shown promise in clinical oncology, including melanomas; however, their narrow therapeutic windows and issues with resistance in pre-clinical models highlight the need to better understand the functions of and interconnection between BRD-containing proteins. Here, we use complementary interaction-mapping techniques (affinity purification and proximity-dependent biotinylation) to map the interactions of 39 of the 42 BRD-containing proteins and 110 additional proteins that physically or functionally associate with them. We uncover 3,892 novel interactions and reveal the intricate connectivity of the Kac machinery. Chemical inhibition of multiple BRD classes revealed that inhibiting BETs—but not mSWI/SNF or CREBBP/EP300 proteins—dramatically rewired the interactome. Finally, we identified MAPKAPK2 activity as a critical determinant of BET inhibitor sensitivity in melanoma through its impact on chromatin composition remodeling. In Brief Kougnassoukou Tchara et al . generate a static protein interaction map of the human acetyl lysine machinery by coupling two complementary functional proteomics approaches (FLAG affinity purification and proximity-dependent biotinylation) to mass spectrometry. They also investigate network changes upon bromodomain inhibition, and describe a novel resistance mechanism mediated by the p38 stress signaling pathway that causes significant metabolic changes. Highlights ### Competing Interest Statement The authors have declared no competing interest.

Phenotypic diversity of cancer cells within tumors generated through bi-directional interactions with the tumor microenvironment has emerged as a major driver of disease progression and therapy resistance. Nutrient availability plays a critical role in determining phenotype, but whether specific nutrients elicit different responses on distinct phenotypes is poorly understood. Here we show, using melanoma as a model, that only MITF Low undifferentiated cells, but not MITF High cells, are competent to drive lipolysis in human adipocytes. In contrast to MITF High melanomas, adipocyte-derived free fatty acids are taken up by undifferentiated MITF Low cells via a fatty acid transporter (FATP)-independent mechanism. Importantly, oleic acid (OA), a monounsaturated long chain fatty acid abundant in adipose tissue and lymph, reprograms MITF Low undifferentiated melanoma cells to a highly invasive state by ligand-independent activation of AXL, a receptor tyrosine kinase associated with therapy resistance in a wide range of cancers. AXL activation by OA then drives SRC-dependent formation and nuclear translocation of a β-catenin-CAV1 complex. The results highlight how a specific nutritional input drives phenotype-specific activation of a pro-metastasis program with implications for FATP-targeted therapies.

Recent development of new immune checkpoint inhibitors has been particularly successfully in cancer treatment, but still the majority patients fail to benefit. Converting resistant tumors to immunotherapy sensitive will provide a significant improvement in patient outcome. Here we identify Mi-2β as a key melanoma-intrinsic effector regulating the adaptive anti-tumor immune response. Studies in genetically engineered mouse melanoma models indicate that loss of Mi-2β rescues the immune response to immunotherapy in vivo. Mechanistically, ATAC-seq analysis shows that Mi-2β controls the accessibility of IFN-γ-stimulated genes (ISGs). Mi-2β binds to EZH2 and promotes K510 methylation of EZH2, subsequently activating the trimethylation of H3K27 to inhibit the transcription of ISGs. Finally, we develop an Mi-2β-targeted inhibitor, Z36-MP5, which reduces Mi-2β ATPase activity and reactivates ISG transcription. Consequently, Z36-MP5 induces a response to immune checkpoint inhibitors in otherwise resistant melanoma models. Our work provides a potential therapeutic strategy to convert immunotherapy resistant melanomas to sensitive ones.

Lipid droplets are fat storage organelles composed of a protein envelope and lipid rich core. Regulation of this protein envelope underlies differential lipid droplet formation and function. In melanoma, lipid droplet formation has been linked to tumor progression and metastasis, but it is unknown whether lipid droplet proteins play a role. To address this, we performed proteomic analysis of the lipid droplet envelope in melanoma. We found that lipid droplet proteins were differentially enriched in distinct melanoma states; from melanocytic to undifferentiated. DHRS3, which converts all-trans-retinal to all-trans-retinol, is upregulated in the MITFLO/undifferentiated/neural crest-like melanoma cell state and reduced in the MITFHI/melanocytic state. Increased DHRS3 expression is sufficient to drive MITFHI/melanocytic cells to a more undifferentiated/invasive state. These changes are due to retinoic acid mediated regulation of melanocytic genes. Our data demonstrate that melanoma cell state can be regulated by expression of lipid droplet proteins which affect downstream retinoid signaling.

Background: The highly homologous T-box transcription factors TBX2 and TBX3 are critical for embryonic development, and their overexpression in postnatal tissues contributes to a wide range of malignancies, including melanoma and rhabdomyosarcoma. Importantly, when TBX2 and TBX3 are depleted in cancers where they are overexpressed, the malignant phenotype is inhibited, and they have therefore been regarded as druggable targets. However, the time and costs associated with de novo drug development are challenging and result in drugs that are costly, especially for patients in low- and middle-income countries. In the current study, we therefore combined a targeted and drug repurposing approach to identify drugs that are expected to be more efficacious and cost-effective with significantly reduced side effects. Methods: A high-throughput cell-based immunofluorescence screen was performed to identify drugs in the Pharmakon 1600 drug library that can negatively regulate TBX2 and/or TBX3 levels. "Hit" drugs were validated for their effect on TBX2/TBX3 levels and cytotoxicity in TBX2/TBX3-dependent melanoma and rhabdomyosarcoma cells. To this end, immunofluorescence, western blotting, quantitative real-time PCR, and MTT cell viability assays were performed. Results: Niclosamide, piroctone olamine, and pyrvinium pamoate, were identified as TBX2 and/or TBX3-targeting drugs, and they exhibited cytotoxicity in a TBX2/TBX3-dependent manner. Furthermore, these "Hit" drugs were shown to induce senescence and/or apoptosis. Conclusions: Niclosamide, piroctone olamine, and pyrvinium pamoate are promising, cost-effective therapeutic agents for the treatment of TBX2/TBX3-dependent cancers.

The human red hair color (RHC) trait is caused by increased pheomelanin (red-yellow) and reduced eumelanin (black-brown) pigment in skin and hair due to diminished melanocortin 1 receptor (MC1R) function. In addition, individuals harboring the RHC trait are predisposed to melanoma development. While MC1R variants have been established as causative of RHC and are a well-defined risk factor for melanoma, it remains unclear mechanistically why decreased MC1R signaling alters pigmentation and increases melanoma susceptibility. Here, we use single-cell RNA sequencing (scRNA-seq) of melanocytes isolated from RHC mouse models to define a MC1R-inhibited Gene Signature (MiGS) comprising a large set of previously unidentified genes which may be implicated in melanogenesis and oncogenic transformation. We show that one of the candidate MiGS genes, TBX3, a well-known anti-senescence transcription factor implicated in melanoma progression, binds both E-box and T-box elements to regulate genes associated with melanogenesis and senescence bypass. Our results provide key insights into further mechanisms by which melanocytes with reduced MC1R signaling may regulate pigmentation and offer new candidates of study toward understanding how individuals with the RHC phenotype are predisposed to melanoma.

Since genome instability can drive cancer initiation and progression, cells have evolved highly effective and ubiquitous DNA damage response (DDR) programs. However, some cells (for example, in skin) are normally exposed to high levels of DNA-damaging agents. Whether such high-risk cells possess lineage-specific mechanisms that tailor DNA repair to the tissue remains largely unknown. Using melanoma as a model, we show here that the microphthalmia-associated transcription factor MITF, a lineage addition oncogene that coordinates many aspects of melanocyte and melanoma biology, plays a nontranscriptional role in shaping the DDR. On exposure to DNA-damaging agents, MITF is phosphorylated at S325, and its interactome is dramatically remodeled; most transcription cofactors dissociate, and instead MITF interacts with the MRE11–RAD50–NBS1 (MRN) complex. Consequently, cells with high MITF levels accumulate stalled replication forks and display defects in homologous recombination-mediated repair associated with impaired MRN recruitment to DNA damage. In agreement with this, high MITF levels are associated with increased single-nucleotide and copy number variant burdens in melanoma. Significantly, the SUMOylation-defective MITF-E318K melanoma predisposition mutation recapitulates the effects of DNA-PKcs-phosphorylated MITF. Our data suggest that a nontranscriptional function of a lineage-restricted transcription factor contributes to a tissue-specialized modulation of the DDR that can impact cancer initiation.

Within cells multiple related transcription factors targeting the same sequences may co-exist, leading to potential regulatory cooperativity, redundancy or competition. Yet the differential roles and biological functions of co-targeting transcription factors is poorly understood. In melanoma, three highly-related transcription factors are co-expressed: The mTORC1-regulated TFEB and TFE3, that are key effectors of a wide range of metabolic and microenvironmental cues; and MITF, that controls melanoma phenotypic identity. Here we reveal the functional specialization of MITF, TFE3 and TFEB and their impact on cancer progression. Notably, although all bind the same sequences, each regulates radically different and frequently opposing gene expression programs to coordinate differentiation, metabolic reprogramming, protein synthesis, and expression of immune modulators. The results uncover a hierarchical cascade in which microenvironmental stresses, including glucose limitation, lead MITF, TFEB and TFE3 to drive distinct biologically important transcription programs that underpin phenotypic transitions in cancer.

Melanoma is characterized by high degrees of phenotypic heterogeneity originating from the impact of the microenvironment on epigenetically plastic cancer cells, with different phenotypes exhibiting different sensitivities to both targeted and immunotherapies. For example, MITFLow invasive melanoma cells are more resistant to BRAFi but exhibit an increased sensitivity to ferroptosis. What determines sensitivity to ferroptosis is not well understood. Here, we reveal that TFE3, an MITF-related transcription factor expressed in melanomas, plays a key role in sensitizing cells to ferroptotic cell death. The results highlight a novel mechanism underpinning a potential therapeutic vulnerability in melanoma. Melanoma is a highly heterogeneous malignancy that can present through multiple states of dedifferentiation, each with its own set of phenotypic and metabolic adaptations and vulnerabilities. Phenotypic identity is in part regulated by the lineage-specific microphthalmia-associated transcription factor (MITF), a master regulator of melanocyte development that can control multiple facets of melanoma biology (Goding & Arnheiter, 2019). MITF is also a useful marker of distinct phenotypic states: high levels of MITF are associated with differentiation, while intermediate activity promotes proliferation and low levels are linked to invasion. Under stressful conditions, such as nutrient limitation, melanoma cells shutdown MITF expression and undergo a switch from a proliferative to invasive state (Falletta et al., 2017). Efforts have been focused on understanding the key metabolic, and potentially therapeutic, vulnerabilities that cancer cells acquire while undergoing phenotypic transitions that enable cells to cope with and adapt to microenvironmental stresses. Invasive melanoma cells have been linked to a higher susceptibility to oxidative stress and ferroptosis (Ubellacker et al., 2020), a form of nonapoptotic cell death. In ferroptosis, Fenton chemistry via iron causes reactive oxygen species to target membrane phospholipids leading to lipid peroxidation (Dixon et al., 2012). This cell death pathway highlights an essential role of iron metabolism in cell survival that might represent a potential targetable vulnerability of invasive cells. Lysosomes are key drivers of iron homeostasis and lysosomal dysfunction can contribute to ferroptosis by sequestering and accumulating iron, leading to oxidative stress and ultimately lysosomal membrane permeabilization (Mai et al., 2017). Interestingly, the master regulators of lysosomal biogenesis are TFEB and TFE3, two members of the MITF family that are retained in the cytoplasm when phosphorylated by mTORC1 under nutrient-replete conditions. Upon mTORC1 inhibition, via amino acid depletion for example, TFEB/3 translocate to the nucleus and drive adaptation to acute cellular stress through the upregulation of autophagic and lysosomal target genes (Settembre et al., 2011). Deprivation of the amino acid cyst(e)ine impacts mTORC1 function and activates ferroptosis (Zhang et al., 2021). In cases where stress is prolonged and unresolved, TFEB/3 can also control cell death via the induction of ATF4 and other genes that comprise part of the Integrated Stress Response (ISR), a pathway that can also regulate cyst(e)ine metabolism (Kreß et al., 2023; Martina et al., 2016). While MITFHigh melanoma cells can achieve resistance to the induction of lipid peroxidation through the lineage-restricted expression of enzymes like the long chain fatty acid desaturase SCD (Kreß et al., 2023; Vivas-García et al., 2019), it is currently not well understood how MITFLow invasive cells might transcriptionally regulate ferroptosis. Therefore, we sought to understand the potential role of MIT/TFE family members in driving an adaptive response to ferroptosis in melanoma. As previous work from others (Tsoi et al., 2018) showed that melanoma cells can undergo ferroptotic cell death, we first assessed the proliferation of melanoma cells under treatment with ferroptosis inducers (FINs) via crystal violet staining (Materials and Methods are provided in the supporting information). We observed that MITFLow IGR39 and A375M cells were highly sensitive to drugs directly inhibiting the main lipid peroxide detoxifying enzyme GPX4, such as RSL-3 (Figure 1a) and ML162 (Figure 1b) while growth of MITFHigh 501mel and IGR37 cells was much less affected with much higher levels of drug required to blunt their growth. We confirmed that treatment with GPX4 inhibitors led to an increase in lipid peroxidation by BODIPY C11 staining (Figure 1c). To assess the specificity of ferroptosis induction under more physiologically relevant conditions, melanoma cells grown overnight in cystine-free media also exhibited elevated lipid peroxidation (Figure 1d). To rule out any contribution of other cell death pathways like apoptosis or necroptosis, 501mel cells were deprived of cystine and treated at the same time with ZVAD or Nec1s, which inhibit apoptosis and necroptosis respectively, or Ferrostatin1 and Deferoxamine (DFO) that block ferroptosis. Cell death was then measured by Propidium Iodide (PI) staining (Figure 1e). The results confirmed that cell death was inhibited using a ferroptosis inhibitor, but not those blocking apoptosis or necroptosis. We then starved the MITFLow IGR39 cells of cystine and showed using the PI staining assay that cell death was again prevented using the ferroptosis inhibitors (Figure 1f). Gene expression analysis by qRT-qPCR confirmed that cystine deprivation led to the upregulation of targets associated with a ferroptotic response, especially CHAC1, PTGS2, ATF4, and the cystine transporter SLC7A11 (Figure 2a) (Upadhyayula et al., 2023). Next, we sought to understand whether the behavior of MIT/TFE factors in melanoma was altered upon induction of ferroptosis and whether their canonical target genes involved in lysosomal biogenesis would be affected. Firstly, we determined the protein levels of MIT/TFE members in both MITFHigh and MITFLow melanoma cells and observed that while TFEB is not detected in the A375M cells, TFE3 was expressed in all four melanoma cell lines examined, with the highest expression in the MITFLow IGR39 cell line (Figure 2b). Second, quantification by qRT-PCR showed upregulation of lysosomal genes after 12 h of cystine starvation, with TFE3 mRNA, but not TFEB mRNA, showing an increase close to 4-fold (Figure 2c). Surprisingly, data from the Cancer Therapeutics Response Portal which analyses correlations between sensitivity to 481 drugs and gene expression revealed a strong positive correlation between sensitivity to FINs and expression of TFE3 in melanoma, meaning that high expression of TFE3 in melanoma correlates with increased sensitivity to FINs (Figure 2d). Note that of the 481 drugs tested, only those lying in the outlier 1.5× interquartile range are shown. We therefore focused on the role of TFE3 as a potential mediator of ferroptosis in melanoma. Treatment with FINs (Figure 2e, left panel) or cystine starvation (Figure 2e, right panel) revealed a shift in mobility of TFE3, consistent with its dephosphorylation and nuclear translocation. The moderately elevated TFE3 protein levels also observed following cystine starvation may be explained by the almost 4-fold increase in TFE3 mRNA levels (Figure 2c). Nuclear localization was confirmed by immunofluorescence of cells stably expressing physiological levels of HA-tagged TFE3 (Figure 2f). To test whether the induction of TFE3 mRNA following cystine starvation might be mediated by ATF4, a key effector of the integrated stress response whose translation is increased in response to cystine starvation (Kreß et al., 2023), we used RNA-seq from previously published 501mel cells engineered to induce ATF4 expression using doxycycline (Falletta et al., 2017). The results (Figure 2g) revealed after induction with doxycycline, ATF4 was able to induce TFE3 mRNA levels around 2-fold. No significant effect was observed for TFEB mRNA, consistent with the lack of induction of TFEB mRNA by cystine deprivation (Figure 2c), while VEGF, a known ATF4 target, was also induced. After establishing that melanoma cells can drive a transcriptional response to ferroptosis by upregulating lysosomal gene expression, potentially via regulating TFE3 levels and activity, we next asked how these cells might behave in the absence of TFE3. Using CRISPR we generated TFE3 knockout (KO) cells in both 501mel and IGR39 melanoma lines (Figure 2h). qRT-PCR revealed a dramatic reduction in the expression of CHAC1 mRNA, encoding a critical regulator of ferroptosis, in both 501mel and IGR39 TFE3 KO cells (Figure 2i). ChIP-seq data revealed that TFE3 directly binds upstream from the CHAC1 gene and can therefore potentially directly influence its transcriptional expression (Figure 2j), although we cannot be certain that the binding of TFE3 detected at the CHAC1 locus is productive. Lastly, PI staining revealed the high levels of ferroptotic cell death induced by overnight cystine deprivation were significantly suppressed in both the 501mel and IGR39 TFE3 KO cell lines (Figure 2k). No effect on cell survival of the TFE3 KO was seen under standard culture conditions. In summary, we have shown that in melanoma TFE3 is required for the expression of genes such as CHAC1 that promote ferroptosis. Consequently, loss or inactivation of TFE3, for example under nutrient-rich conditions may render cells less susceptible to ferroptosis. These findings shine a light on the regulation of ferroptosis by TFE3 and provide a new target to treat metastatic melanoma by inducing TFE3 activity and ferroptotic cell death in invasive cells. This work was funded by FCT PD/BD/114127/2015 as part of the Graduate Program in Areas of Basic and Applied Biology (DD) and Ludwig Cancer Research (CRG, PL and DD). The authors declare no conflict of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request. Appendix S1 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

As a population of homogeneous cells with both self-renewal and differentiation potential, stem cell pools are highly compartmentalized and contain distinct subsets that exhibit stable but limited heterogeneity during homeostasis. However, their striking plasticity is showcased under natural or artificial stress, such as injury, transplantation, cancer, and aging, leading to changes in their phenotype, constitution, metabolism, and function. The complex and diverse network of cell-extrinsic niches and signaling pathways, together with cell-intrinsic genetic and epigenetic regulators, tightly regulate both the heterogeneity during homeostasis and the plasticity under perturbation. Manipulating these factors offers better control of stem cell behavior and a potential revolution in the current state of regenerative medicine. However, disruptions of normal regulation by genetic mutation or excessive plasticity acquisition may contribute to the formation of tumors. By harnessing innovative techniques that enhance our understanding of stem cell heterogeneity and employing novel approaches to maximize the utilization of stem cell plasticity, stem cell therapy holds immense promise for revolutionizing the future of medicine.

Supplementary Movie 3 from Intravital Imaging Reveals Transient Changes in Pigment Production and Brn2 Expression during Metastatic Melanoma Dissemination

Supplementary Figure, Table, and Movie Legends from Intravital Imaging Reveals Transient Changes in Pigment Production and Brn2 Expression during Metastatic Melanoma Dissemination

The redox regulator NRF2 becomes activated upon oxidative and electrophilic stress and orchestrates a response program associated with redox regulation, metabolism, tumor therapy resistance, and immune suppression. Here, we describe an unrecognized link between the integrated stress response (ISR) and NRF2 mediated by the ISR effector ATF4. The ISR is commonly activated after starvation or ER stress and plays a central role in tissue homeostasis and cancer plasticity. ATF4 increases NRF2 transcription and induces the glutathione-degrading enzyme CHAC1, which we now show to be critically important for maintaining NRF2 activation. In-depth analyses reveal that NRF2 supports ATF4-induced cells by increasing cystine uptake via the glutamate-cystine antiporter xCT. In addition, NRF2 upregulates genes mediating thioredoxin usage and regeneration, thus balancing the glutathione decrease. In conclusion, we demonstrate that the NRF2 response serves as second layer of the ISR, an observation highly relevant for the understanding of cellular resilience in health and disease.