Enhancer of zeste homolog 2 (EZH2) is a methyltransferase that tri-methylates histone H3K27 as the catalytic subunit of the polycomb repressive complex 2. However, inhibition of EZH2 methyltransferase showed only variable anti-cancer efficacy, suggesting that this approach is insufficient. Here, we demonstrate a methyltransferase-independent mechanism of EZH2 wherein EZH2 interacts with inosine monophosphate dehydrogenase 2 (IMPDH2) in the cytoplasm to promote guanosine-5'-triphosphate (GTP) synthesis. Mass spectrometry identified methyltransferase-independent interactions between the EED-binding domain of EZH2 and the CBS domain of IMPDH2. EZH2 knockdown impeded IMPDH2 and reduced GTP levels, ribosome biogenesis, and cancer progression-effects reversed by guanosine. IMPDH2 knockout antagonized EZH2's tumor-promoting effects in vivo, and increased cytosolic EZH2 and IMPDH2 expression was observed in human melanomas and associated with nucleolar enlargement. EZH2-IMPDH2 complexes were also observed across multiple cancers, wherein Sappanone A (SA), which inhibits EZH2-IMPDH2 interactions, was anti-tumorigenic. These findings reveal a methyltransferase-independent oncogenic mechanism of EZH2.

Intra-tumoral heterogeneity poses a major challenge to treating and managing cancer patients. A characteristic feature of melanoma is its composition of cancer cells with typically heterogeneous content of melanin pigment, the production of which is a hallmark of normal melanocytic differentiation but of poorly understood consequence in melanoma cells, as prospective assessment of pigment heterogeneity in melanoma cells has been experimentally challenging. Here, we describe a novel flow cytometric method for high purity separation of viable melanoma cells based on their melanin content, exploiting the light scattering properties of melanin. By fluorescence-activated cell sorting, we show that cells with low-pigment content (LPCs) in melanoma cell lines and patient tumors are usually far more abundant than high-pigment cells (HPCs) and have substantially increased potentials for colony formation in vitro and tumor formation in vivo. In RNAseq analysis, HPCs showed P53 activation and perturbed cell cycling, whereas LPCs displayed upregulation of MYC-associated transcription and activated ribosome biogenesis. In proof-of-concept studies, the latter was targeted by topoisomerase 2 beta targeting with CX-5461, which induced senescent HPC phenotypes and irreversible loss of clonogenic activity. These data indicate an 'inverted pyramid' hierarchical model of melanoma cell propagation wherein abundant LPCs frequently renew their own malignant potential to propagate disease but also infrequently generate HPCs that spontaneously lose this ability in a manner that might be exploited as an anti-melanoma strategy.

Abstract The biological and molecular mechanisms that underpin the malignant transformation of normal melanocytes to melanomas are largely unknown. In part, this is due to the limited understanding of normal human melanocyte homeostasis and how melanocytes respond to oncogenic insults such as ultraviolet radiation (UVR). This is particularly true for interfollicular epidermal melanocytes, which have the highest levels of UVR exposure and from whence most melanomas are thought to arise. These knowledge gaps impede the development of strategies for active, targeted prevention of melanoma formation. We thus evaluated epidermal melanocytes transcriptionally, phenotypically and functionally after isolating them from human skin. Using single-cell RNA sequencing (scRNA-seq), we identified multiple transcriptionally distinct subpopulations within human epidermal melanocytes. RNA velocity analysis revealed subpopulations in different states of melanocytic differentiation, and immunohistochemistry staining demonstrated their distinct anatomical distribution throughout follicular and interfollicular epidermal compartments. Notably, one melanocyte subgroup, marked by increased expression of neurotrophic receptor tyrosine kinase 2 (NTRK2) and genes associated with ribosome biogenesis, exhibited molecular characteristics of progenitor cells. This subpopulation displayed human embryonic stem cell (hESC)-derived melanoblast markers, and their anatomical localization corresponded to that of intermediate melanocyte progenitors. NTRK2+ melanocytes demonstrated enhanced clonogenicity after UVR exposure in primary cell cultures and in ex vivo whole skin explants. In contrast, NTRK2- melanocytes were suppressed by UVR. Furthermore, scRNA-seq data of ex vivo melanocytes after UVR exposure revealed the upregulation of genes associated with cell proliferation within NTRK2-expressing melanocytes. In mouse back skin, the ratio of Ntrk2+ melanocytes increased within 24 hours of UVB irradiation, suggesting a proliferative response in these cells in vivo following UVR. We thus report the discovery in human epidermis of a putative melanocytic cell hierarchy, and of a candidate melanocyte progenitor subpopulation that responds proliferatively to UVR and is thus a candidate cell of origin of melanoma. Citation Format: Peinan Zhao, Fumihito Noguchi, Christopher Chew, Gamze Kuser Abali, Pacman Szeto, Youfang Zhang, Malaka Ameratunga, Isobel Leece, Jen G. Cheung, Miles Andrews, Nicholas C. Wong, Anthony T. Papenfuss, Mark Shackleton. Molecular and functional characterization of melanocyte subpopulations in human epidermis based on single-cell RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 867.

Supplementary Figures S1-S8 S1 Heat map of chemokines and receptors S2 Immunohistochemical staining of lymphatic vessels S3 Expression of VEGF-D and chemokines in cancer cell lines S4 Expression of genes in melanoma patient samples S5 Chemokine secretion by tumour cells S6 Chemokine induced migration of LEC S7 Chemokines attract LEC in vivo S8 CCL-293EBNA tumour growth and CCL27 secretion

Expression of YAP, pYAP or TAZ proteins in 10 melanoma cell lines

Cellular heterogeneity in cancer is linked to disease progression and therapy response, although mechanisms regulating distinct cellular states within tumors are not well understood. We identified melanin pigment content as a major source of cellular heterogeneity in melanoma and compared RNAseq data from high-pigmented (HPCs) and low-pigmented melanoma cells (LPCs), suggesting EZH2 as a master regulator of these states. EZH2 protein was found to be upregulated in LPCs and inversely correlated with melanin deposition in pigmented patient melanomas. Surprisingly, conventional EZH2 methyltransferase inhibitors, GSK126 and EPZ6438, had no effect on LPC survival, clonogenicity and pigmentation, despite fully inhibiting methyltransferase activity. In contrast, EZH2 silencing by siRNA or degradation by DZNep or MS1943 inhibited growth of LPCs and induced HPCs. As the proteasomal inhibitor MG132 induced EZH2 protein in HPCs, we evaluated ubiquitin pathway proteins in HPC vs LPCs. Biochemical assays and animal studies demonstrated that in LPCs, the E2-conjugating enzyme UBE2L6 depletes EZH2 protein in cooperation with UBR4, an E3 ligase, via ubiquitination at EZH2’s K381 residue, and is downregulated in LPCs by UHRF1-mediated CpG methylation. Targeting UHRF1/UBE2L6/UBR4-mediated regulation of EZH2 offers potential for modulating the activity of this oncoprotein in contexts in which conventional EZH2 methyltransferase inhibitors are ineffective.

Correlation between mRNA expression and copy number (CN) in the 10 most commonly amplified Hippo pathway genes

Correlation amongst melanoma cell lines of viability following YAP siRNA- and verteporfin-treatment

Correlation between mRNA expression and copy number (CN) in the 10 most commonly deleted Hippo pathway genes

ABSTRACT Functional variation between cancer cells (intra-tumoral heterogeneity) poses a major challenge to treating and managing cancer patients. Melanomas are typically composed of cancer cells with heterogeneous content of melanin pigment. Pigment production is a hallmark of normal melanocytic differentiation, however the functional consequences of melanin production in melanoma cells remains poorly understood owing to a lack of experimental approaches for detection of pigment in unfixed cells. Here, we describe a novel flow cytometric method for high purity separation of viable melanoma cells based on their content of melanin pigment, exploiting the infrared light scattering properties of melanin. By fluorescence-activated cell sorting, we show that melanoma cells with low-pigment content (LPCs) in culture and in patient tumors are far more abundant than high-pigment cells (HPCs), and have substantially increased potentials for colony formation in vitro and for tumor formation in vivo. RNAseq analysis revealed activation of P53 in HPCs associated with perturbed cell cycling, whereas LPCs displayed upregulation of MYC-associated transcription and activated ribosome biogenesis. The latter was reduced by topoisomerase 2 beta targeting with CX-5461, which also induced senescent HPC phenotypes and irreversible loss of clonogenic activity. These data illuminate an ‘inverted pyramid’ hierarchical model of melanoma cell propagation wherein abundant LPCs frequently renew their own malignant potential to propagate disease, but also rarely generate HPCs that lose this ability in a manner that may be promoted as an anti-melanoma strategy.

ABSTRACTThe enhancer of zeste homolog 2 (EZH2) oncoprotein is a histone methyltransferase that functions canonically as a catalytic subunit of the polycomb repressive complex 2 (PRC2) to tri-methylate histone H3 at Lys 27 (H3K27me3). Although targeting EZH2 methyltransferase is a promising therapeutic strategy against cancer, methyltransferase-independent oncogenic functions of EZH2 are described. Moreover, pharmacological EZH2 methyltransferase inhibition was only variably effective in pre-clinical and clinical studies, suggesting that targeting EZH2 methyltransferase alone may be insufficient. Here, we demonstrate a non-canonical mechanism of EZH2’s oncogenic activity characterized by interactions with inosine monophosphate dehydrogenase 2 (IMPDH2) and downstream promotion of guanosine-5’-triphosphate (GTP) production. EZH2-IMPDH2 interactions identified by Liquid Chromatography-Mass Spectrometry (LC-MS) of EZH2 immunoprecipitates from melanoma cells were verified to occur between the N-terminal EED-binding domain of cytosolic EZH2 and the CBS domain of IMPDH2 in a methyltransfersase-independent manner. EZH2 silencing reduced cellular GTP, ribosome biogenesis, RhoA-mediated actomyosin contractility and melanoma cell proliferation and invasion by impeding the activity of IMPDH2. Guanosine, which replenishes GTP, reversed these effects and thereby promoted invasive and clonogenic cell states even in EZH2 silenced cells. IMPDH2 silencing antagonized the proliferative and invasive effects of EZH2, also in a guanosine-reversible manner. In human melanomas, high cytosolic EZH2 and IMPDH2 expression were associated with nucleolar enlargement, a marker of ribosome biogenesis. EZH2-IMPDH2 complexes were also observed in a range of cancers in which Sappanone A (SA), which inhibits EZH2-IMPDH2 interactions, was anti-tumorigenic, although notably non-toxic in normal cells. These findings illuminate a previously unrecognized, non-canonical, methyltransferase-independent, and GTP-dependent mechanism by which EZH2 regulates tumorigenicity in melanoma and other cancers, opening new avenues for development of anti-EZH2 therapeutics.Graphical AbstractHighlightsEZH2 has non-canonical methyltransferase-independent and GTP-dependent tumorigenic and metastatic functions in melanoma.The N-terminal EED-binding domain of EZH2 interacts with the CBS domain of IMPDH2 in a polycomb repressive complex 2- (PRC2-) and methylation-independent manner.EZH2 accumulates with IMPDH2 in the cytoplasm and increases IMPDH2’s tetramerization-mediated activity independently of EZH2 methyltransferase.EZH2 upregulates GTP synthesis by IMPDH2 activation and thereby activates ribosome biogenesis via rRNA synthesis and actomyosin contractility via RhoA GTPase.Sappanone A (SA) inhibits IMPDH2-EZH2 interactions and is anti-proliferative across a range of cancers including melanoma, but not in normal cells.

ABSTRACTCellular heterogeneity in cancer is linked to disease progression and therapy response, although the mechanisms regulating distinct cellular states within tumours are not well understood. To address this, we identified melanin pigment content as a major source of phenotypic and functional heterogeneity in melanoma and compared RNAseq data from high (HPC) and low pigmented melanoma cells (LPC), revealing the polycomb repressor complex protein, EZH2, as a master regulator of these states. EZH2 protein, but not RNA expression, was found to be upregulated in LPCs and inversely correlated with melanin in pigmented patient melanomas. Surprisingly, conventional EZH2 methyltransferase inhibitors, GSK126 and EPZ6438, had no effect on LPC survival, clonogenicity and pigmentation, despite fully inhibiting methyltransferase activity. In contrast, EZH2 silencing by siRNA strategy or DZNep, MS1943 that reduces EZH2 protein levels, significantly inhibited cell growth in LPCs by hampering ribosome biogenesis. In addition, decline in EZH2 protein level induces pigmented cell phenotype by inducing melanin biosynthesis. Proteasomal inhibitor, MG132 treatment induced EZH2 protein levels in HPCs prompted us to look for differentially regulated ubiquitin system proteins in HPC vs LPCs. UBE2L6, E2 conjugating enzyme has been shown to be downregulated significantly in LPCs by UHRF1-mediated CpG methylation. Both biochemical assays and animal studies demonstrated that UBE2L6 expression decline, in turn, promotes EZH2 protein stability due to lack of ubiquitination on K381 residue in LPCs. UBR4 cooperates with UBE2L6 to facilitate this ubiquitination process. Targeting UHRF1/UBE2L6/UBR4 axis can be a better treatment option to trigger HPC state in melanoma in which conventional EZH2 inhibitors are ineffective.

Abstract Melanoma is usually driven by mutations in BRAF or NRAS that trigger hyperactivation of mitogen-activated protein kinase (MAPK) signaling. However, MAPK targeted therapies are not sustainably effective in most patients. Accordingly, characterizing mechanisms that cooperatively drive melanoma progression is key to improving patient outcomes. One possible mechanism is the Hippo signaling pathway, which regulates cancer progression via its central oncoproteins YAP and TAZ, although it is relatively rarely affected by direct mutation. As YAP hyperactivation occurs in uveal melanoma, we investigated this oncogene in cutaneous melanoma. YAP protein expression was elevated in most benign nevi and primary cutaneous melanomas but present at only very low levels in normal melanocytes. In patient-derived xenografts and melanoma cell lines, we observed variable reliance of cell viability on Hippo pathway signaling that was independent of classical melanoma driver mutations such as BRAF and NRAS. YAP activity was enriched in invasive melanoma cell lines and was necessary for their invasive properties in culture. In in vivo studies, we found that expression of hyperactive YAP induced spontaneous melanoma metastasis in mice. Finally, in genotyping studies of melanoma, we observed the first ever hyperactivating YAP mutations in a human cancer, manifest as seven distinct missense point mutations that caused serine to alanine transpositions. Strikingly, these mutate four of five known serine residues targeted by the Hippo pathway, and we show that they lead to hyperactivation of YAP function. Our studies highlight the YAP oncoprotein as a potential therapeutic target in select subgroups of melanoma patients, although successful treatment with anti-YAP therapies will depend on identification of biomarkers other than YAP protein expression. Citation Format: Xiaomeng Zhang, Lie Yang, Ismael Vergara, Pacman Szeto, Youfang Zhang, Anthony Papenfuss, Mark Shackleton, Kieran Harvey. The Hippo pathway in melanoma [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr IA03.

Melanoma is a deadly form of skin cancer that accounts for a disproportionally large proportion of cancer-related deaths in younger people. Compared with most other skin cancers, a feature of melanoma is its high metastatic capacity, although the mechanisms that confer this are not well understood. The Hippo pathway is a key regulator of organ growth and cell fate that is deregulated in many cancers. To analyse the Hippo pathway in cutaneous melanoma, we generated a transcriptional signature of melanoma cells that overexpressed YAP, the key downstream Hippo pathway oncoprotein. YAP-mediated transcriptional activity varied in melanoma cell lines but did not cluster with known genetic drivers of melanomagenesis such asBRAFandNRASmutations. Instead, it correlated strongly with published gene expression profiles linked to melanoma cell invasiveness and varied throughout the metastatic cascade in melanoma patient tumours. Consistent with this, YAP was both necessary and sufficient for melanoma cell invasion in vitro. In vivo, YAP promoted spontaneous melanoma metastasis, whilst the growth of YAP-expressing primary tumours was impeded. Finally, we identified the YAP target genesAXL,THBS1andCYR61as key mediators of YAP-induced melanoma cell invasion. These data suggest that YAP is a critical regulator of melanoma metastasis.

Abstract Melanoma is usually driven by mutations in BRAF or NRAS, which trigger hyperactivation of MAPK signaling. However, MAPK-targeted therapies are not sustainably effective in most patients. Accordingly, characterizing mechanisms that co-operatively drive melanoma progression is key to improving patient outcomes. One possible mechanism is the Hippo signaling pathway, which regulates cancer progression via its central oncoproteins YAP and TAZ, although is thought to be only rarely affected by direct mutation. As YAP hyperactivation occurs in uveal melanoma, we investigated this oncogene in cutaneous melanoma. YAP protein expression was elevated in most benign nevi and primary cutaneous melanomas but present at only very low levels in normal melanocytes. In patient-derived xenografts and melanoma cell lines, we observed variable reliance of cell viability on Hippo pathway signaling that was independent of TAZ activity and also of classical melanoma driver mutations such as BRAF and NRAS. Finally, in genotyping studies of melanoma, we observed the first ever hyperactivating YAP mutations in a human cancer, manifest as seven distinct missense point mutations that caused serine to alanine transpositions. Strikingly, these mutate four serine residues known to be targeted by the Hippo pathway and we show that they lead to hyperactivation of YAP. Implications: Our studies highlight the YAP oncoprotein as a potential therapeutic target in select subgroups of melanoma patients, although successful treatment with anti-YAP therapies will depend on identification of biomarkers additional to YAP protein expression.

Abstract Metastasis via the lymphatic vasculature is an important step in cancer progression. The formation of new lymphatic vessels (lymphangiogenesis), or remodeling of existing lymphatics, is thought to facilitate the entry and transport of tumor cells into lymphatic vessels and on to distant organs. The migration of lymphatic endothelial cells (LEC) toward guidance cues is critical for lymphangiogenesis. While chemokines are known to provide directional navigation for migrating immune cells, their role in mediating LEC migration during tumor-associated lymphangiogenesis is not well defined. Here, we undertook gene profiling studies to identify chemokine–chemokine receptor pairs that are involved in tumor lymphangiogenesis associated with lymph node metastasis. CCL27 and CCL28 were expressed in tumor cells with metastatic potential, while their cognate receptor, CCR10, was expressed by LECs and upregulated by the lymphangiogenic growth factor VEGFD and the proinflammatory cytokine TNFα. Migration assays demonstrated that LECs are attracted to both CCL27 and CCL28 in a CCR10-dependent manner, while abnormal lymphatic vessel patterning in CCR10-deficient mice confirmed the significant role of CCR10 in lymphatic patterning. In vivo analyses showed that LECs are recruited to a CCL27 or CCL28 source, while VEGFD was required in combination with these chemokines to enable formation of coherent lymphatic vessels. Moreover, tumor xenograft experiments demonstrated that even though CCL27 expression by tumors enhanced LEC recruitment, the ability to metastasize was dependent on the expression of VEGFD. These studies demonstrate that CCL27 and CCL28 signaling through CCR10 may cooperate with inflammatory mediators and VEGFD during tumor lymphangiogenesis. Significance: The study shows that the remodeling of lymphatic vessels in cancer is influenced by CCL27 and CCL28 chemokines, which may provide a future target to modulate metastatic spread.

Ryk is a member of the receptor tyrosine kinase (RTK) family of proteins that control and regulate cellular processes. It is distinguished by binding Wnt ligands and having no detectable intrinsic protein tyrosine kinase activity suggesting Ryk is a pseudokinase. Here, we show an essential role for Ryk in directing morphogenetic events required for normal cardiac development through the examination of Ryk-deficient mice. We employed vascular corrosion casting, vascular perfusion with contrast dye, and immunohistochemistry to characterize cardiovascular and pharyngeal defects in Ryk(-/-) embryos. Ryk(-/-) mice exhibit a variety of malformations of the heart and outflow tract that resemble human congenital heart defects. This included stenosis and interruption of the aortic arch, ventriculoarterial malalignment, ventricular septal defects and abnormal pharyngeal arch artery remodelling. This study therefore defines a key intersection between a subset of growth factor receptors involved in planar cell polarity signalling, the Wnt family and mammalian cardiovascular development.

Remodelling of lymphatic vessels in tumours facilitates metastasis to lymph nodes. The growth factors VEGF-C and VEGF-D are well known inducers of lymphatic remodelling and metastasis in cancer. They are initially produced as full-length proteins requiring proteolytic processing in order to bind VEGF receptors with high affinity and thereby promote lymphatic remodelling. The fibrinolytic protease plasmin promotes processing of VEGF-C and VEGF-D in vitro, but its role in processing them in cancer was unknown. Here we explore plasmin's role in proteolytically activating VEGF-D in vivo, and promoting lymphatic remodelling and metastasis in cancer, by co-expressing the plasmin inhibitor α2-antiplasmin with VEGF-D in a mouse tumour model. We show that α2-antiplasmin restricts activation of VEGF-D, enlargement of intra-tumoural lymphatics and occurrence of lymph node metastasis. Our findings indicate that the fibrinolytic system influences lymphatic remodelling in tumours which is consistent with previous clinicopathological observations correlating fibrinolytic components with cancer metastasis.