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Disseminated cancer cells from primary tumours can seed in distal tissues, but may take several years to form overt metastases, a phenomenon that is termed tumour dormancy. Despite its importance in metastasis and residual disease, few studies have been able to successfully characterize dormancy within melanoma. Here we show that the aged lung microenvironment facilitates a permissive niche for efficient outgrowth of dormant disseminated cancer cells—in contrast to the aged skin, in which age-related changes suppress melanoma growth but drive dissemination. These microenvironmental complexities can be explained by the phenotype switching model, which argues that melanoma cells switch between a proliferative cell state and a slower-cycling, invasive state(1–3). It was previously shown that dermal fibroblasts promote phenotype switching in melanoma during ageing(4–8). We now identify WNT5A as an activator of dormancy in melanoma disseminated cancer cells within the lung, which initially enables the efficient dissemination and seeding of melanoma cells in metastatic niches. Age-induced reprogramming of lung fibroblasts increases their secretion of the soluble WNT antagonist sFRP1, which inhibits WNT5A in melanoma cells and thereby enables efficient metastatic outgrowth. We also identify the tyrosine kinase receptors AXL and MER as promoting a dormancy-to-reactivation axis within melanoma cells. Overall, we find that age-induced changes in distal metastatic microenvironments promote the efficient reactivation of dormant melanoma cells in the lung.

Cellular plasticity describes the ability of cells to transition from one set of phenotypes to another. In melanoma, transient fluctuations in the molecular state of tumor cells mark the formation of rare cells primed to survive BRAF inhibition and reprogram into a stably drug-resistant fate. However, the biological processes governing cellular priming remain unknown. We used CRISPR-Cas9 genetic screens to identify genes that affect cell fate decisions by altering cellular plasticity. We found that many factors can independently affect cellular priming and fate decisions. We discovered a new plasticity-based mode of increasing resistance to BRAF inhibition that pushes cells towards a more differentiated state. Manipulating cellular plasticity through inhibition of DOT1L before the addition of the BRAF inhibitor resulted in more therapy resistance than concurrent administration. Our results indicate that modulating cellular plasticity can alter cell fate decisions and may prove useful for treating drug resistance in other cancers.

Purpose: Angiogenesis is thought to be critical for tumor metastasis. However, inhibiting angiogenesis using antibodies such as bevacizumab (Avastin), has had little impact on melanoma patient survival. We have demonstrated that both angiogenesis and metastasis are increased in older individuals, and therefore sought to investigate whether there was an age-related difference in response to bevacizumab, and if so, what the underlying mechanism could be. Experimental Design: We analyzed data from the AVAST-M trial of 1,343 patients with melanoma treated with bevacizumab to determine whether there is an age-dependent response to bevacizumab. We also examined the age-dependent expression of VEGF and its cognate receptors in patients with melanoma, while using syngeneic melanoma animal models to target VEGF in young versus old mice. We also examined the age-related proangiogenic factor secreted frizzled-related protein 2 (sFRP2) and whether it could modulate response to anti-VEGF therapy. Results: We show that older patients respond poorly to bevacizumab, whereas younger patients show improvement in both disease-free survival and overall survival. We find that targeting VEGF does not ablate angiogenesis in an aged mouse model, while sFRP2 promotes angiogenesis in vitro and in young mice. Targeting sFRP2 in aged mice successfully ablates angiogenesis, while the effects of targeting VEGF in young mice can be overcome by increasing sFRP2. Conclusions: VEGF is decreased during aging, thereby reducing response to bevacizumab. Despite the decrease in VEGF, angiogenesis is increased because of an increase in sFRP2 in the aged tumor microenvironment. These results stress the importance of considering age as a factor for designing targeted therapies.

Older patients with melanoma (>50 years old) have poorer prognoses and response rates to targeted therapy compared with young patients ( Significance: These data show that melanoma cells take up lipids from aged fibroblasts, via FATP2, and use them to resist targeted therapy. The response to targeted therapy is altered in aged individuals because of the influences of the aged microenvironment, and these data suggest FATP2 as a target to overcome resistance. See related commentary by Montal and White, p. 1255.. This article is highlighted in the In This Issue feature, p. 1241

Metastatic melanoma is challenging to clinically address. Although standard-of-care targeted therapy has high response rates in patients with BRAF-mutant melanoma, therapy relapse occurs in most cases. Intrinsically resistant melanoma cells drive therapy resistance and display molecular and biologic properties akin to neural crest-like stem cells (NCLSC) including high invasiveness, plasticity, and self-renewal capacity. The shared transcriptional programs and vulnerabilities between NCLSCs and cancer cells remains poorly understood. Here, we identify a developmental LPAR1-axis critical for NCLSC viability and melanoma cell survival. LPAR1 activity increased during progression and following acquisition of therapeutic resistance. Notably, genetic inhibition of LPAR1 potentiated BRAFi ± MEKi efficacy and ablated melanoma migration and invasion. Our data define LPAR1 as a new therapeutic target in melanoma and highlights the promise of dissecting stem cell–like pathways hijacked by tumor cells. Significance: This study identifies an LPAR1-axis critical for melanoma invasion and intrinsic/acquired therapy resistance.

Brain metastasis, the most lethal form of melanoma and carcinoma, is the consequence of favorable interactions between the invading cancer cells and the brain cells. Peroxisome proliferator–activated receptor γ (PPARγ) has ambiguous functions in cancer development, and its relevance in advanced brain metastasis remains unclear. Here, we demonstrate that astrocytes, the unique brain glial cells, activate PPARγ in brain metastatic cancer cells. PPARγ activation enhances cell proliferation and metastatic outgrowth in the brain. Mechanistically, astrocytes have a high content of polyunsaturated fatty acids that act as “donors” of PPARγ activators to the invading cancer cells. In clinical samples, PPARγ signaling is significantly higher in brain metastatic lesions. Notably, systemic administration of PPARγ antagonists significantly reduces brain metastatic burden in vivo. Our study clarifies a prometastatic role for PPARγ signaling in cancer metastasis in the lipid-rich brain microenvironment and argues for the use of PPARγ blockade to treat brain metastasis. Significance: Brain-tropic cancer cells take advantage of the lipid-rich brain microenvironment to facilitate their proliferation by activating PPARγ signaling. This protumor effect of PPARγ in advanced brain metastases is in contrast to its antitumor function in carcinogenesis and early metastatic steps, indicating that PPARγ has diverse functions at different stages of cancer development. This article is highlighted in the In This Issue feature, p. 1631

Clinical trials repurposing lysosomotropic chloroquine (CQ) derivatives as autophagy inhibitors in cancer demonstrate encouraging results, but the underlying mechanism of action remains unknown. Here, we report a novel dimeric CQ (DC661) capable of deacidifying the lysosome and inhibiting autophagy significantly better than hydroxychloroquine (HCQ). Using an in situ photoaffinity pulldown strategy, we identified palmitoyl-protein thioesterase 1 (PPT1) as a molecular target shared across monomeric and dimeric CQ derivatives. HCQ and Lys05 also bound to and inhibited PPT1 activity, but only DC661 maintained activity in acidic media. Knockout of PPT1 in cancer cells using CRISPR/Cas9 editing abrogates autophagy modulation and cytotoxicity of CQ derivatives, and results in significant impairment of tumor growth similar to that observed with DC661. Elevated expression of PPT1 in tumors correlates with poor survival in patients in a variety of cancers. Thus, PPT1 represents a new target in cancer that can be inhibited with CQ derivatives. Significance: This study identifies PPT1 as the previously unknown lysosomal molecular target of monomeric and dimeric CQ derivatives. Genetic suppression of PPT1 impairs tumor growth, and PPT1 levels are elevated in cancer and associated with poor survival. These findings provide a strong rationale for targeting PPT1 in cancer. This article is highlighted in the In This Issue feature, p. 151

Melanoma is the deadliest form of skin cancer, possessing a diverse landscape of subtypes with distinct molecular signatures and levels of aggressiveness. Although immense progress has been achieved therapeutically for patients with the most common forms of this disease, little is known of how to effectively treat patients with rarer subtypes of melanoma. These subtypes include acral lentiginous (the rarest form of cutaneous melanoma; AL), uveal, and mucosal melanomas, which display variations in distribution across (a) the world, (b) patient age-groups, and (c) anatomic sites. Unfortunately, patients with these relatively rare subtypes of melanoma typically respond worse to therapies approved for the more common, non-AL cutaneous melanoma and do not have effective alternatives, and thus consequently have worse overall survival rates. Achieving durable therapeutic responses in these high-risk melanoma subtypes represents one of the greatest challenges of the field. This review aims to collate and highlight effective preclinical and/or clinical strategies against these rare forms of melanoma.

In senescence, specific genes encoding secreted factors are excluded from senescence-associated heterochromatin foci, but the mechanisms underlying this senescence-associated secretory phenotype (SASP) are unclear. Aird et al. show that the chromatin-bound protein HMGB2 orchestrates the SASP by preventing heterochromatin spreading to these specific loci., Cellular senescence is a stable cell growth arrest that is characterized by the silencing of proliferation-promoting genes through compaction of chromosomes into senescence-associated heterochromatin foci (SAHF). Paradoxically, senescence is also accompanied by increased transcription of certain genes encoding for secreted factors such as cytokines and chemokines, known as the senescence-associated secretory phenotype (SASP). How SASP genes are excluded from SAHF-mediated global gene silencing remains unclear. In this study, we report that high mobility group box 2 (HMGB2) orchestrates the chromatin landscape of SASP gene loci. HMGB2 preferentially localizes to SASP gene loci during senescence. Loss of HMGB2 during senescence blunts SASP gene expression by allowing for spreading of repressive heterochromatin into SASP gene loci. This correlates with incorporation of SASP gene loci into SAHF. Our results establish HMGB2 as a novel master regulator that orchestrates SASP through prevention of heterochromatin spreading to allow for exclusion of SASP gene loci from a global heterochromatin environment during senescence.

Summary Metastatic melanoma is an aggressive disease, despite recent improvements in therapy. Eradicating all melanoma cells even in drug-sensitive tumors is unsuccessful in patients because a subset of cells can transition to a slow-cycling state, rendering them resistant to most targeted therapy. It is still unclear what pathways define these subpopulations and promote this resistant phenotype. In the current study, we show that Wnt5A, a non-canonical Wnt ligand that drives a metastatic, therapy-resistant phenotype, stabilizes the half-life of p53 and uses p53 to initiate a slow-cycling state following stress (DNA damage, targeted therapy, and aging). Inhibiting p53 blocks the slow-cycling phenotype and sensitizes melanoma cells to BRAF/MEK inhibition. In vivo, this can be accomplished with a single dose of p53 inhibitor at the commencement of BRAF/MEK inhibitor therapy. These data suggest that taking the paradoxical approach of inhibiting rather than activating wild-type p53 may sensitize previously resistant metastatic melanoma cells to therapy.

Purpose: We have shown that the aged microenvironment increases melanoma metastasis, and decreases response to targeted therapy, and here we queried response to anti-PD1. Experimental Design: We analyzed the relationship between age, response to anti-PD1, and prior therapy in 538 patients. We used mouse models of melanoma, to analyze the intratumoral immune microenvironment in young versus aged mice and confirmed our findings in human melanoma biopsies. Results: Patients over the age of 60 responded more efficiently to anti-PD-1, and likelihood of response to anti-PD-1 increased with age, even when we controlled for prior MAPKi therapy. Placing genetically identical tumors in aged mice (52 weeks) significantly increased their response to anti-PD1 as compared with the same tumors in young mice (8 weeks). These data suggest that this increased response in aged patients occurs even in the absence of a more complex mutational landscape. Next, we found that young mice had a significantly higher population of regulatory T cells (Tregs), skewing the CD8+:Treg ratio. FOXP3 staining of human melanoma biopsies revealed similar increases in Tregs in young patients. Depletion of Tregs using anti-CD25 increased the response to anti-PD1 in young mice. Conclusions: While there are obvious limitations to our study, including our inability to conduct a meta-analysis due to a lack of available data, and our inability to control for mutational burden, there is a remarkable consistency in these data from over 500 patients across 8 different institutes worldwide. These results stress the importance of considering age as a factor for immunotherapy response. Clin Cancer Res; 24(21); 5347–56. ©2018 AACR. See related commentary by Pawelec, p. 5193

Purpose: Aging is a poor prognostic factor for melanoma. We have shown that melanoma cells in an aged microenvironment are more resistant to targeted therapy than identical cells in a young microenvironment. This is dependent on age-related secreted factors. Klotho is an age-related protein whose serum levels decrease dramatically by age 40. Most studies on klotho in cancer have focused on the expression of klotho in the tumor cell. We have shown that exogenous klotho inhibits internalization and signaling of Wnt5A, which drives melanoma metastasis and resistance to targeted therapy. We investigate here whether increasing klotho in the aged microenvironment could be an effective strategy for the treatment of melanoma. Experimental Design: PPARγ increases klotho levels and is increased by glitazones. Using rosiglitazone, we queried the effects of rosiglitazone on Klotho/Wnt5A cross-talk, in vitro and in vivo, and the implications of that for targeted therapy in young versus aged animals. Results: We show that rosiglitazone increases klotho and decreases Wnt5A in tumor cells, reducing the burden of both BRAF inhibitor–sensitive and BRAF inhibitor–resistant tumors in aged, but not young mice. However, when used in combination with PLX4720, tumor burden was reduced in both young and aged mice, even in resistant tumors. Conclusions: Using glitazones as adjuvant therapy for melanoma may provide a new treatment strategy for older melanoma patients who have developed resistance to vemurafenib. As klotho has been shown to play a role in other cancers too, our results may have wide relevance for multiple tumor types. Clin Cancer Res; 23(12); 3181–90. ©2017 AACR.

Summary The MEK inhibitor MEK162 is the first targeted therapy agent with clinical activity in patients whose melanomas harbor NRAS mutations; however, median PFS is 3.7 months, suggesting the rapid onset of resistance in the majority of patients. Here, we show that treatment of NRAS-mutant melanoma cell lines with the MEK inhibitors AZD6244 or trametinib resulted in a rebound activation of phospho-ERK (pERK). Functionally, the recovery of signaling was associated with the maintenance of cyclin-D1 expression and therapeutic escape. The combination of a MEK inhibitor with an ERK inhibitor suppressed the recovery of cyclin-D1 expression and was associated with a significant enhancement of apoptosis and the abrogation of clonal outgrowth. The MEK/ERK combination strategy induced greater levels of apoptosis compared with dual MEK/CDK4 or MEK/PI3K inhibition across a panel of cell lines. These data provide the rationale for further investigation of vertically co-targeting the MAPK pathway as a potential treatment option for NRAS-mutant melanoma patients.