MAPK-mediated PHGDH induction is essential for melanoma formation and represents an actionable vulnerability.

Overexpression of PHGDH, the rate-limiting enzyme in the serine synthesis pathway, promotes melanomagenesis, melanoma cell proliferation, and survival of metastases in serine-low environments such as the brain. While PHGDH amplification explains PHGDH overexpression in a subset of melanomas, we find that PHGDH levels are universally increased in melanoma cells due to oncogenic BRAFV600E promoting PHGDH transcription through mTORC1-mediated translation of ATF4. Importantly, PHGDH expression was critical for melanomagenesis as depletion of PHGDH in genetic mouse models blocked melanoma formation. Despite BRAFV600E-mediated upregulation, PHGDH was further induced by exogenous serine restriction. Surprisingly, BRAFV600E inhibition diminished serine restriction-mediated PHGDH expression by preventing ATF4 induction, creating a potential vulnerability whereby melanoma cells could be specifically starved of serine by combining BRAFV600E inhibition with exogenous serine restriction. Indeed, we show that this combination promoted cell death in vitro and attenuated melanoma growth in vivo. This study identified a melanoma cell-specific PHGDH-dependent vulnerability.

PTEN Lipid Phosphatase Activity Suppresses Melanoma Formation by Opposing an AKT/mTOR/FRA1 Signaling Axis.

Inactivating mutations in PTEN are prevalent in melanoma and are thought to support tumor development by hyperactivating the AKT/mTOR pathway. Conversely, activating mutations in AKT are relatively rare in melanoma, and therapies targeting AKT or mTOR have shown disappointing outcomes in preclinical models and clinical trials of melanoma. This has led to the speculation that PTEN suppresses melanoma by opposing AKT-independent pathways, potentially through noncanonical functions beyond its lipid phosphatase activity. In this study, we examined the mechanisms of PTEN-mediated suppression of melanoma formation through the restoration of various PTEN functions in PTEN-deficient cells or mouse models. PTEN lipid phosphatase activity predominantly inhibited melanoma cell proliferation, invasion, and tumor growth, with minimal contribution from its protein phosphatase and scaffold functions. A drug screen underscored the exquisite dependence of PTEN-deficient melanoma cells on the AKT/mTOR pathway. Furthermore, activation of AKT alone was sufficient to counteract several aspects of PTEN-mediated melanoma suppression, particularly invasion and the growth of allograft tumors. Phosphoproteomics analysis of the lipid phosphatase activity of PTEN validated its potent inhibition of AKT and many of its known targets, while also identifying the AP-1 transcription factor FRA1 as a downstream effector. The restoration of PTEN dampened FRA1 translation by inhibiting AKT/mTOR signaling, and FRA1 overexpression negated aspects of PTEN-mediated melanoma suppression akin to AKT. This study supports AKT as the key mediator of PTEN inactivation in melanoma and identifies an AKT/mTOR/FRA1 axis as a driver of melanomagenesis. SIGNIFICANCE: PTEN suppresses melanoma predominantly through its lipid phosphatase function, which when lost, elevates FRA1 levels through AKT/mTOR signaling to promote several aspects of melanomagenesis.

Genetically engineered mouse models of head and neck cancers.

The head and neck region is one of the anatomic sites commonly afflicted by cancer, with ~1.5 million new diagnoses reported worldwide in 2020 alone. Remarkable progress has been made in understanding the underlying disease mechanisms, personalizing care based on each tumor's individual molecular characteristics, and even therapeutically exploiting the inherent vulnerabilities of these neoplasms. In this regard, genetically engineered mouse models (GEMMs) have played an instrumental role. While progress in the development of GEMMs has been slower than in other major cancer types, several GEMMs are now available that recapitulate most of the heterogeneous characteristics of head and neck cancers such as the tumor microenvironment. Different approaches have been employed in GEMM development and implementation, though each can generally recapitulate only certain disease aspects. As a result, appropriate model selection is essential for addressing specific research questions. In this review, we present an overview of all currently available head and neck cancer GEMMs, encompassing models for head and neck squamous cell carcinoma, nasopharyngeal carcinoma, and salivary and thyroid gland carcinomas.

Distinct Nrf2 Signaling Thresholds Mediate Lung Tumor Initiation and Progression.

Mutations in the KEAP1-NRF2 (Kelch-like ECH-associated protein 1-nuclear factor-erythroid 2 p45-related factor 2) pathway occur in up to a third of non-small cell lung cancer (NSCLC) cases and often confer resistance to therapy and poor outcomes. Here, we developed murine alleles of the KEAP1 and NRF2 mutations found in human NSCLC and comprehensively interrogated their impact on tumor initiation and progression. Chronic NRF2 stabilization by Keap1 or Nrf2 mutation was not sufficient to induce tumorigenesis, even in the absence of tumor suppressors, p53 or LKB1. When combined with KrasG12D/+, constitutive NRF2 activation promoted lung tumor initiation and early progression of hyperplasia to low-grade tumors but impaired their progression to advanced-grade tumors, which was reversed by NRF2 deletion. Finally, NRF2 overexpression in KEAP1 mutant human NSCLC cell lines was detrimental to cell proliferation, viability, and anchorage-independent colony formation. Collectively, these results establish the context-dependence and activity threshold for NRF2 during the lung tumorigenic process. SIGNIFICANCE: Stabilization of the transcription factor NRF2 promotes oncogene-driven tumor initiation but blocks tumor progression, indicating distinct, threshold-dependent effects of the KEAP1/NRF2 pathway in different stages of lung tumorigenesis.

Gain of Chromosome 1q Perturbs a Competitive Endogenous RNA Network to Promote Melanoma Metastasis.

Somatic copy-number alterations (CNA) promote cancer, but the underlying driver genes may not be comprehensively identified if only the functions of the encoded proteins are considered. mRNAs can act as competitive endogenous RNAs (ceRNA), which sponge miRNAs to posttranscriptionally regulate gene expression in a protein coding-independent manner. We investigated the contribution of ceRNAs to the oncogenic effects of CNAs. Chromosome 1q gains promoted melanoma progression and metastasis at least in part through overexpression of three mRNAs with ceRNA activity: CEP170, NUCKS1, and ZC3H11A. These ceRNAs enhanced melanoma metastasis by sequestering tumor suppressor miRNAs. Orthogonal genetic assays with miRNA inhibitors and target site blockers, along with rescue experiments, demonstrated that miRNA sequestration is critical for the oncogenic effects of CEP170, NUCKS1, and ZC3H11A mRNAs. Furthermore, chromosome 1q ceRNA-mediated miRNA sequestration alleviated the repression of several prometastatic target genes. This regulatory RNA network was evident in other cancer types, suggesting chromosome 1q ceRNA deregulation as a common driver of cancer progression. Taken together, this work demonstrates that ceRNAs mediate the oncogenicity of somatic CNAs. SIGNIFICANCE: The function of CEP170, NUCKS1, and ZC3H11A mRNAs as competitive endogenous RNAs that sequester tumor suppressor microRNAs underlies the oncogenic activity of chromosome 1q gains.

Single-cell Characterization of the Cellular Landscape of Acral Melanoma Identifies Novel Targets for Immunotherapy.

PURPOSE: Acral melanoma is a rare subtype of melanoma that arises on the non-hair-bearing skin of the palms, soles, and nail beds. In this study, we used single-cell RNA sequencing (scRNA-seq) to map the transcriptional landscape of acral melanoma and identify novel immunotherapeutic targets. EXPERIMENTAL DESIGN: We performed scRNA-seq on nine clinical specimens (five primary, four metastases) of acral melanoma. Detailed cell type curation was performed, the immune landscapes were mapped, and key results were validated by analysis of The Cancer Genome Atlas (TCGA) and single-cell datasets. Cell-cell interactions were inferred and compared with those in nonacral cutaneous melanoma. RESULTS: Multiple phenotypic subsets of T cells, natural killer (NK) cells, B cells, macrophages, and dendritic cells with varying levels of activation/exhaustion were identified. A comparison between primary and metastatic acral melanoma identified gene signatures associated with changes in immune responses and metabolism. Acral melanoma was characterized by a lower overall immune infiltrate, fewer effector CD8 T cells and NK cells, and a near-complete absence of γδ T cells compared with nonacral cutaneous melanomas. Immune cells associated with acral melanoma exhibited expression of multiple checkpoints including PD-1, LAG-3, CTLA-4, V-domain immunoglobin suppressor of T cell activation (VISTA), TIGIT, and the Adenosine A2A receptor (ADORA2). VISTA was expressed in 58.3% of myeloid cells and TIGIT was expressed in 22.3% of T/NK cells. CONCLUSIONS: Acral melanoma has a suppressed immune environment compared with that of cutaneous melanoma from nonacral skin. Expression of multiple, therapeutically tractable immune checkpoints were observed, offering new options for clinical translation.

Genetic tools for the stable overexpression of circular RNAs.

Circular RNAs (circRNAs) are a class of non-coding RNAs featuring a covalently closed ring structure formed through backsplicing. circRNAs are broadly expressed and contribute to biological processes through a variety of functions. Standard gain-of-function and loss-of-function approaches to study gene functions have significant limitations when studying circRNAs. Overexpression studies in particular suffer from the lack of efficient genetic tools. While mammalian expression plasmids enable transient circRNA overexpression in cultured cells, most cell biological studies require long-term ectopic expression. Here we report the development and characterization of genetic tools enabling stable circRNA overexpression in vitro and in vivo. We demonstrated that circRNA expression constructs can be delivered to cultured cells via transposons, whereas lentiviral vectors have limited utility for the delivery of circRNA constructs due to viral RNA splicing in virus-producing cells. We further demonstrated ectopic circRNA expression in a hepatocellular carcinoma mouse model upon circRNA transposon delivery via hydrodynamic tail vein injection. Furthermore, we generated genetically engineered mice harbouring circRNA expression constructs. We demonstrated that this approach enables constitutive, global circRNA overexpression as well as inducible circRNA expression directed specifically to melanocytes in a melanoma mouse model. These tools expand the genetic toolkit available for the functional characterization of circRNAs.

A Series of BRAF- and NRAS-Driven Murine Melanoma Cell Lines with Inducible Gene Modulation Capabilities.

Murine cancer cell lines are powerful research tools to complement studies in genetically engineered mouse models. We have established 21 melanoma cell lines from embryonic stem cell-genetically engineered mouse models driven by alleles that model the most frequent genetic alterations in human melanoma. In addition, these cell lines harbor regulatory alleles for the genomic integration of transgenes and the regulation of expression of such transgenes. In this study, we report a comprehensive characterization of these cell lines. Specifically, we validated melanocytic origin, driver allele recombination and expression, and activation of the oncogenic MAPK and protein kinase B pathways. We further tested tumor formation in syngeneic immunocompetent recipients as well as the functionality of the integrated Tet-ON system and recombination-mediated cassette exchange homing cassette. Finally, by deleting the transcription factor MAFG with an inducible CRISPR/Cas9 approach, we show the utility of the regulatory alleles for candidate gene modulation. These cell lines will be a valuable resource for studying melanoma biology and therapy.

Functional analysis of the 1p34.3 risk locus implicates GNL2 in high-grade serous ovarian cancer.

The high-grade serous ovarian cancer (HGSOC) risk locus at chromosome 1p34.3 resides within a frequently amplified genomic region signifying the presence of an oncogene. Here, we integrate in silico variant-to-function analysis with functional studies to characterize the oncogenic potential of candidate genes in the 1p34.3 locus. Fine mapping of genome-wide association statistics identified candidate causal SNPs local to H3K27ac-demarcated enhancer regions that exhibit allele-specific binding for CTCF in HGSOC and normal fallopian tube secretory epithelium cells (FTSECs). SNP risk associations colocalized with eQTL for six genes (DNALI1, GNL2, RSPO1, SNIP1, MEAF6, and LINC01137) that are more highly expressed in carriers of the risk allele, and three (DNALI1, GNL2, and RSPO1) were upregulated in HGSOC compared to normal ovarian surface epithelium cells and/or FTSECs. Increased expression of GNL2 and MEAF6 was associated with shorter survival in HGSOC with 1p34.3 amplifications. Despite its activation of ß-catenin signaling, RSPO1 overexpression exerted no effects on proliferation or colony formation in our study of ovarian cancer and FTSECs. Instead, GNL2, MEAF6, and SNIP1 silencing impaired in vitro ovarian cancer cell growth. Additionally, GNL2 silencing diminished xenograft tumor formation, whereas overexpression stimulated proliferation and colony formation in FTSECs. GNL2 influences 60S ribosomal subunit maturation and global protein synthesis in ovarian cancer and FTSECs, providing a potential mechanism of how GNL2 upregulation might promote ovarian cancer development and mediate genetic susceptibility of HGSOC.

Squaring the circle: circRNAs in melanoma.

Non-coding RNAs are emerging as critical molecules in the genesis, progression, and therapy resistance of cutaneous melanoma. This includes circular RNAs (circRNAs), a class of non-coding RNAs with distinct characteristics that forms through non-canonical back-splicing. In this review, we summarize the features and functions of circRNAs and introduce the current knowledge of the roles of circRNAs in melanoma. We also highlight the various mechanisms of action of the well-studied circRNA CDR1as and describe how it acts as a melanoma tumor suppressor. We further discuss the utility of circRNAs as biomarkers, therapeutic targets, and therapeutic agents in melanoma and outline challenges that must be overcome to comprehensively characterize circRNA functions.

Pseudogenes as Competitive Endogenous RNAs: Target Prediction and Validation.

Pseudogenes may regulate expression of their parental genes as well as other protein-coding genes through various mechanisms. One such mechanism is the ability to act as competitive endogenous RNA (ceRNA) and participate in microRNA-mediated cross-regulation. Here, we outline how to predict the targets of pseudogene ceRNAs bioinformatically and how to validate them experimentally.

Pseudogenes as Competitive Endogenous RNAs: Testing miRNA Dependency.

Pseudogenes may function as competitive endogenous RNAs (ceRNAs), where they regulate the expression of genes by sequestering shared miRNAs. ceRNAs are becoming more extensively identified and studied, and demonstrating the dependence of their effects on miRNA sequestration is critical to establish them as ceRNAs. Here, we outline an experimental approach to assess the miRNA dependency of a candidate pseudogene ceRNA.

Strategies to Study the Functions of Pseudogenes in Mouse Models of Cancer.

Aberrant expression of pseudogenes has been observed in many cancer types. Deregulated pseudogenes engage in a multitude of biological processes at the DNA, RNA, and protein levels and eventually facilitate disease progression. To investigate pseudogene functions in cancer, cell lines and cell line transplantation models have been widely used. However, cancer biology is best studied in the context of an intact organism. Here, we present various strategies to investigate pseudogenes in genetically engineered mouse models and discuss advantages and disadvantages of the different approaches.

A Mutational Survey of Acral Nevi.

IMPORTANCE: Acral skin may develop nevi, but their mutational status and association with acral melanoma is unclear. OBJECTIVE: To perform targeted next-generation sequencing on a cohort of acral nevi to determine their mutational spectrum. DESIGN, SETTING, AND PARTICIPANTS: Acral nevi specimens (n = 50) that had been obtained for diagnostic purposes were identified from the pathology archives of a tertiary care academic cancer center and a university dermatology clinic. Next-generation sequencing was performed on DNA extracted from the specimens, and mutations called. A subset of samples was stained immunohistochemically for the BRAF V600E mutation. RESULTS: A total of 50 nevi from 49 patients (19 males and 30 females; median [range] age, 48 [13-85] years) were examined. Analysis of the sequencing data revealed a high prevalence of BRAF mutations (n = 43), with a lower frequency of NRAS mutations (n = 5). Mutations in BRAF and NRAS were mutually exclusive. CONCLUSIONS AND RELEVANCE: In this cohort study, nevi arising on mostly sun-protected acral skin showed a rate of BRAF mutation similar to that of acquired nevi on sun-exposed skin but far higher than that of acral melanoma. These findings are in contrast to the well-characterized mutational landscape of acral melanoma.

A MAPK/miR-29 Axis Suppresses Melanoma by Targeting MAFG and MYBL2.

The miR-29 family of microRNAs is encoded by two clusters, miR-29b1~a and miR-29b2~c, and is regulated by several oncogenic and tumor suppressive stimuli. While in vitro evidence suggests a tumor suppressor role for miR-29 in melanoma, the mechanisms underlying its deregulation and contribution to melanomagenesis have remained elusive. Using various in vitro systems, we show that oncogenic MAPK signaling paradoxically stimulates transcription of pri-miR-29b1~a and pri-miR-29b2~c, the latter in a p53-dependent manner. Expression analyses in melanocytes, melanoma cells, nevi, and primary melanoma revealed that pri-miR-29b2~c levels decrease during melanoma progression. Inactivation of miR-29 in vivo with a miRNA sponge in a rapid melanoma mouse model resulted in accelerated tumor development and decreased overall survival, verifying tumor suppressive potential of miR-29 in melanoma. Through integrated RNA sequencing, target prediction, and functional assays, we identified the transcription factors MAFG and MYBL2 as bona fide miR-29 targets in melanoma. Our findings suggest that attenuation of miR-29b2~c expression promotes melanoma development, at least in part, by derepressing MAFG and MYBL2.

Melanoma models for the next generation of therapies.

There is a lack of appropriate melanoma models that can be used to evaluate the efficacy of novel therapeutic modalities. Here, we discuss the current state of the art of melanoma models including genetically engineered mouse, patient-derived xenograft, zebrafish, and ex vivo and in vitro models. We also identify five major challenges that can be addressed using such models, including metastasis and tumor dormancy, drug resistance, the melanoma immune response, and the impact of aging and environmental exposures on melanoma progression and drug resistance. Additionally, we discuss the opportunity for building models for rare subtypes of melanomas, which represent an unmet critical need. Finally, we identify key recommendations for melanoma models that may improve accuracy of preclinical testing and predict efficacy in clinical trials, to help usher in the next generation of melanoma therapies.

PHGDH supports liver ceramide synthesis and sustains lipid homeostasis.

BACKGROUND: d-3-phosphoglycerate dehydrogenase (PHGDH), which encodes the first enzyme in serine biosynthesis, is overexpressed in human cancers and has been proposed as a drug target. However, whether PHGDH is critical for the proliferation or homeostasis of tissues following the postnatal period is unknown. METHODS: To study PHGDH inhibition in adult animals, we developed a knock-in mouse model harboring a PHGDH shRNA under the control of a doxycycline-inducible promoter. With this model, PHGDH depletion can be globally induced in adult animals, while sparing the brain due to poor doxycycline delivery. RESULTS: We found that PHGDH depletion is well tolerated, and no overt phenotypes were observed in multiple highly proliferative cell compartments. Further, despite detectable knockdown and impaired serine synthesis, liver and pancreatic functions were normal. Interestingly, diminished PHGDH expression reduced liver serine and ceramide levels without increasing the levels of deoxysphingolipids. Further, liver triacylglycerol profiles were altered, with an accumulation of longer chain, polyunsaturated tails upon PHGDH knockdown. CONCLUSIONS: These results suggest that dietary serine is adequate to support the function of healthy, adult murine tissues, but PHGDH-derived serine supports liver ceramide synthesis and sustains general lipid homeostasis.

Translational pathology, genomics and the development of systemic therapies for acral melanoma.

Acral melanomas arise on the non-hair bearing skin of the palms, soles and in the nail beds. These rare tumors comprise 2-3 % of all melanomas, are not linked to UV-exposure, and represent the most frequent subtype of melanomas in patients of Asian, African and Hispanic origin. Although recent work has revealed candidate molecular events that underlie acral melanoma development, this knowledge is not yet been translated into efficacious local, regional, or systemic therapies. In the current review, we describe the clinical characteristics of acral melanoma and outline the genetic basis of acral melanoma development. Further discussion is given to the current status of systemic therapy for acral melanoma with a focus on ongoing developments in both immunotherapy and targeted therapy for the treatment of advanced disease.

A Versatile ES Cell-Based Melanoma Mouse Modeling Platform.

The cumbersome and time-consuming process of generating new mouse strains and multiallelic experimental animals often hinders the use of genetically engineered mouse models (GEMM) in cancer research. Here, we describe the development and validation of an embryonic stem cell (ESC)-GEMM platform for rapid modeling of melanoma in mice. The platform incorporates 12 clinically relevant genotypes composed of combinations of four driver alleles (LSL-BrafV600E, LSL-NrasQ61R, PtenFlox, and Cdkn2aFlox) and regulatory alleles to spatiotemporally control the perturbation of genes of interest. The ESCs produce high-contribution chimeras, which recapitulate the melanoma phenotypes of conventionally bred mice. Using the ESC-GEMM platform to modulate Pten expression in melanocytes in vivo, we highlighted the utility and advantages of gene depletion by CRISPR-Cas9, RNAi, or conditional knockout for melanoma modeling. Moreover, complementary genetic methods demonstrated the impact of Pten restoration on the prevention and maintenance of Pten-deficient melanomas. Finally, we showed that chimera-derived melanoma cell lines retain regulatory allele competency and are a powerful resource to complement ESC-GEMM chimera experiments in vitro and in syngeneic grafts in vivo Thus, when combined with sophisticated genetic tools, the ESC-GEMM platform enables rapid, high-throughput, and versatile studies aimed at addressing outstanding questions in melanoma biology.Significance: This study presents a high-throughput and versatile ES cell-based mouse modeling platform that can be combined with state-of-the-art genetic tools to address unanswered questions in melanoma in vivo See related commentary by Thorkelsson et al., p. 655.