The lipid droplet protein DHRS3 is a regulator of melanoma cell state

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

Single-cell profiling of MC1R-inhibited melanocytes

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

DNA damage-induced interaction between a lineage addiction oncogenic transcription factor and the MRN complex shapes a tissue-specific DNA Damage Response and cancer predisposition

Since genome instability can drive cancer initiation and progression, cells have evolved highly effective and ubiquitous DNA Damage Response (DDR) programs. However, some cells, in skin for example, 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. Here we show, using melanoma as a model, that the microphthalmia-associated transcription factor MITF, a lineage addition oncogene that coordinates many aspects of melanocyte and melanoma biology, plays a non-transcriptional role in shaping the DDR. On exposure to DNA damaging agents, MITF is phosphorylated by ATM/DNA-PKcs, and unexpectedly its interactome is dramatically remodelled; most transcription (co)factors 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, high MITF levels are associated with increased SNV burden in melanoma. Significantly, the SUMOylation-defective MITF-E318K melanoma predisposition mutation recapitulates the effects of ATM/DNA-PKcs-phosphorylated MITF. Our data suggest that a non-transcriptional function of a lineage-restricted transcription factor contributes to a tissue-specialised modulation of the DDR that can impact cancer initiation

DNA damage remodels the MITF interactome to increase melanoma genomic instability

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

Acetylation reprograms MITF target selectivity and residence time

Abstract The ability of transcription factors to discriminate between different classes of binding sites associated with specific biological functions underpins effective gene regulation in development and homeostasis. How this is achieved is poorly understood. The microphthalmia-associated transcription factor MITF is a lineage-survival oncogene that plays a crucial role in melanocyte development and melanoma. MITF suppresses invasion, reprograms metabolism and promotes both proliferation and differentiation. How MITF distinguishes between differentiation and proliferation-associated targets is unknown. Here we show that compared to many transcription factors MITF exhibits a very long residence time which is reduced by p300/CBP-mediated MITF acetylation at K206. While K206 acetylation also decreases genome-wide MITF DNA-binding affinity, it preferentially directs DNA binding away from differentiation-associated CATGTG motifs toward CACGTG elements. The results reveal an acetylation-mediated switch that suppresses differentiation and provides a mechanistic explanation of why a human K206Q MITF mutation is associated with Waardenburg syndrome

Acetylation reprograms MITF target selectivity and residence time

The ability of transcription factors to discriminate between different classes of binding sites associated with specific biological functions underpins effective gene regulation in development and homeostasis. How this is achieved is poorly understood. The microphthalmia-associated transcription factor MITF is a lineage-survival oncogene that plays a crucial role in melanocyte development and melanoma. MITF suppresses invasion, reprograms metabolism and promotes both proliferation and differentiation. How MITF distinguishes between differentiation and proliferation-associated targets is unknown. Here we show that compared to many transcription factors MITF exhibits a very long residence time which is reduced by p300/CBP-mediated MITF acetylation at K206. While K206 acetylation also decreases genome-wide MITF DNA-binding affinity, it preferentially directs DNA binding away from differentiation-associated CATGTG motifs toward CACGTG elements. The results reveal an acetylation-mediated switch that suppresses differentiation and provides a mechanistic explanation of why a human K206Q MITF mutation is associated with Waardenburg syndrome

ALDH1A3-acetaldehyde metabolism potentiates transcriptional heterogeneity in melanoma

Cancer cellular heterogeneity and therapy resistance arise substantially from metabolic and transcriptional adaptations, but how these are interconnected is poorly understood. Here, we show that, in melanoma, the cancer stem cell marker aldehyde dehydrogenase 1A3 (ALDH1A3) forms an enzymatic partnership with acetyl-coenzyme A (CoA) synthetase 2 (ACSS2) in the nucleus to couple high glucose metabolic flux with acetyl-histone H3 modification of neural crest (NC) lineage and glucose metabolism genes. Importantly, we show that acetaldehyde is a metabolite source for acetyl-histone H3 modification in an ALDH1A3-dependent manner, providing a physiologic function for this highly volatile and toxic metabolite. In a zebrafish melanoma residual disease model, an ALDH1-high subpopulation emerges following BRAF inhibitor treatment, and targeting these with an ALDH1 suicide inhibitor, nifuroxazide, delays or prevents BRAF inhibitor drug-resistant relapse. Our work reveals that the ALDH1A3-ACSS2 couple directly coordinates nuclear acetaldehyde-acetyl-CoA metabolism with specific chromatin-based gene regulation and represents a potential therapeutic vulnerability in melanoma.</p

Deficiency of factor-inhibiting HIF creates a tumor-promoting immune microenvironment

Hypoxia signaling influences tumor development through both cell-intrinsic and -extrinsic pathways. Inhibiting hypoxia-inducible factor (HIF) function has recently been approved as a cancer treatment strategy. Hence, it is important to understand how regulators of HIF may affect tumor growth under physiological conditions. Here we report that in aging mice factor-inhibiting HIF (FIH), one of the most studied negative regulators of HIF, is a haploinsufficient suppressor of spontaneous B cell lymphomas, particular pulmonary B cell lymphomas. FIH deficiency alters immune composition in aged mice and creates a tumor-supportive immune environment demonstrated in syngeneic mouse tumor models. Mechanistically, FIH-defective myeloid cells acquire tumor-supportive properties in response to signals secreted by cancer cells or produced in the tumor microenvironment with enhanced arginase expression and cytokine-directed migration. Together, these data demonstrate that under physiological conditions, FIH plays a key role in maintaining immune homeostasis and can suppress tumorigenesis through a cell-extrinsic pathway

Tuning transcription factor availability through acetylation-mediated genomic redistribution

It is widely assumed that decreasing transcription factor DNA-binding affinity reduces transcription initiation by diminishing occupancy of sequence-specific regulatory elements. However, in vivo transcription factors find their binding sites while confronted with a large excess of low-affinity degenerate motifs. Here, using the melanoma lineage survival oncogene MITF as a model, we show that low-affinity binding sites act as a competitive reservoir in vivo from which transcription factors are released by mitogen-activated protein kinase (MAPK)-stimulated acetylation to promote increased occupancy of their regulatory elements. Consequently, a low-DNA-binding-affinity acetylation-mimetic MITF mutation supports melanocyte development and drives tumorigenesis, whereas a high-affinity non-acetylatable mutant does not. The results reveal a paradoxical acetylation-mediated molecular clutch that tunes transcription factor availability via genome-wide redistribution and couples BRAF to tumorigenesis. Our results further suggest that p300/CREB-binding protein-mediated transcription factor acetylation may represent a common mechanism to control transcription factor availability