Edward J. Hartsough

Uveal melanoma (UM) is the most common intraocular malignancy in adults, arising in melanocytes within the uveal tract. Although local disease can be effectively controlled by radiation or enucleation, nearly half of patients ultimately develop fatal liver metastases. Hepatic metastases are strongly associated with loss of the tumor suppressor BAP1, yet the metabolic adaptations that enable BAP1-deficient UM cells to survive and proliferate within the liver remain poorly understood. Transcriptomic analyses revealed that mutant BAP1 UM tumors exhibit elevated expression of genes involved in glutathione (GSH) synthesis. Analysis of human UM cell lines revealed that mutant BAP1 UM cell lines have increased total thiols and an elevated GSH:GSSG ratio, alongside heightened sensitivity to GSH depletion. Given that GSH serves as a cofactor for antioxidant selenoproteins such as GPX4, we investigated the role of selenoproteins in UM liver metastases. We found that mutant BAP1 UM cells exhibited increased dependence on GPX4, as inhibition of GPX4 induced lipid peroxidation and cell death. Because GPX4 requires selenocysteine for its synthesis and activity, we next investigated whether mutant BAP1 UM cells exhibit altered selenium acquisition pathways. Mutant BAP1 UM cells displayed enhanced selenoproteome synthesis and increased utilization of liver-derived selenoprotein P (SEPP) through the receptor LRP8. Moreover, disruption of this BAP1-associated selenoprotein network via LRP8 interference impaired the growth of hepatic lesions and reduced tumor viability in human liver slice models, thereby identifying a potential therapeutic target for metastatic UM. Together, these findings identify a mutant BAP1-associated selenoprotein-dependent vulnerability in metastatic UM.

Uveal melanoma (UM) is an aggressive intraocular malignancy in adults arising from the melanocytes of the uveal tract. While primary UM lesions can be successfully treated, ~ 50% of UM patients develop metastases primarily in the liver. Patients with liver metastatic UM (LMUM) have poor prognosis and few therapeutic options. LMUM lesions are unresponsive to standard chemotherapies, targeted therapies, and immune checkpoint inhibitors — an effect at least in part associated with the detoxification function of the liver and the diverse hepatic immunological landscape. Here, we recount the etiology and molecular mechanisms in the development of LMUM, examine the influences of the hepatic tumor microenvironment (TME) on UM liver tropism, and review how the innate and peripheral immune response contributes to LMUM progression and therapeutic efficacy. The unique immunological properties of the liver coupled with the distinct growth patterns of LMUM lesions present significant challenges for developing effective treatments that can overcome this specialized microenvironment. Improved understanding of the interplay between the liver and LMUM is essential for the development of more effective diagnostic techniques and improved therapeutic outcomes.

Uveal melanoma (UM) is an aggressive intraocular malignancy. Despite effective control of primary tumors, ~50% of UM patients develop metastases, with the liver being the predominant secondary site. BAP1 deficiency, present in ~80% of metastatic UM cases, is strongly associated with increased metastatic risk and poor prognosis. In silico analysis of UM patient samples suggests that reduced BAP1 is linked to enhanced expression of genes involved in fatty acid processing; therefore, we hypothesize that BAP1 deficiency primes UM cells for survival in the hepatic microenvironment by enhancing lipid tolerance and oxidative stress responses. Our findings demonstrate BAP1‐mutant UM resist lipotoxicity, whereas BAP1‐competent UM exhibit sensitivity due to lipid peroxide accumulation—a hallmark of ferroptotic‐like stress, and a response that can be mitigated by ferroptosis inhibition. Using an ex vivo liver slice model, we found that disrupting lipid metabolism with atorvastatin, an HMG‐CoA reductase inhibitor, reduced tumor burden of BAP1‐mutant UM. Moreover, we demonstrate a positive correlation between BAP1 and an epigenetic regulator of lipid homeostasis, ASXL2. Notably, ASXL2 depletion in BAP1‐competent UM phenocopies the lipotoxicity resistance observed in BAP1‐mutant UM—an effect that may be mediated by altered PPAR expression. This study reveals a novel mechanism linking BAP1 expression to lipid sensitivity via ASXL2, providing insights into liver tropism and potential therapeutic avenues for metastatic uveal melanoma.

Uveal melanoma (UM) is a rare and aggressive intraocular malignancy arising from melanocytes in the uveal tract. Despite effective local control of primary tumors, approximately 50% of UM patients develop metastases, with the liver being the predominant site of metastatic spread. BAP1 deficiency, present in about 80% of metastatic UM cases, is strongly associated with increased metastatic risk and poor prognosis. Analysis of UM patient samples reveals negative correlations between BAP1 expression and genes involved in long-chain fatty acid metabolism and lipase activity. We hypothesized that BAP1 deficiency primes UM cells for survival in the hepatic microenvironment by enhancing lipid processing and oxidative stress management. Our findings show that BAP1-deficient UM cells tolerate long-chain fatty acid-induced lipotoxicity. Alternatively, BAP1-competent UM is sensitive to lipotoxicity, an effect associated with lipid peroxide accumulation. Notably, this response can be rescued with pharmacological inhibition of ferroptosis. When exposed to liver-secreted factors, only BAP1-competent UM cells respond by upregulating lipid metabolism and oxidative stress management markers, suggesting BAP1-deficient UM possesses inherent stress resistance. In an ex vivo liver MUM model, BAP1-deficient UM cells are highly sensitized to fatty acid metabolism inhibition by atorvastatin, resulting in increased lipid peroxides and reduced tumor burden. Mechanistically, we uncovered a novel relationship between BAP1 and ASXL2, a known regulator of lipid homeostasis. BAP1 loss reduces ASXL2 expression, disrupting their deubiquitinating complex and increasing histone H2A ubiquitination. ASXL2 depletion in BAP1-competent cells recapitulates the lipotoxicity resistance of BAP1-deficient cells, an effect that may be linked to altered PPAR expression patterns. This study reveals a novel mechanism linking BAP1 deficiency to metabolic rewiring in UM through ASXL2 dysregulation, providing insights into liver tropism and opening new possibilities for targeted therapies in metastatic uveal melanoma.

8% of all human cancers are driven by an activating mutation (V600E/K) in the BRAF protein (Mendiratta et al., 2022), a serine-threonine kinase central to the Mitogen-Activated Protein Kinase (MAPK) pathway. Small molecule inhibitors targeting malignant cells carrying a BRAFV600E mutation (BRAFi) have proven to have clinical efficacy. However, patient response varies across different cancer types, and resistance, whether innate or acquired, is a major challenge. Mutant BRAFV600E is predominantly expressed in papillary thyroid carcinomas (PTC) (~60%) and melanomas (~50%) (Owsley et al., 2021). Melanoma patients typically exhibit a significant initial reduction in tumor burden in response to BRAFi treatment. However, resistance is inevitably acquired 6-9 months into treatment (Flaherty et al., 2012). In contrast, patients with PTC respond poorly to BRAFi, indicating an innate resistance. Programmed cell death mechanisms play a role in BRAFi efficacy, and resistance to apoptosis drives relapse in many mutant-BRAF cancers. Therefore, understanding the role of cell death machinery in resistance and relapse could help guide more durable treatment strategies against mutant BRAF cancers. In this study, we investigate the role of programmed cell death mechanisms in providing response to BRAFi in PTC and melanoma models. We first characterize a role for the Bcl-2 family member Mcl-1 in contributing to a failed apoptotic phenotype, which drives innate resistance in BRAFV600E PTC. Additionally, we explore the role of immunogenic cell death in providing anti-tumor immune responses capable of clearing drug-tolerant persisters (DTPs) in a syngeneic mouse model of melanoma brain metastases (MBM). These studies aim to elucidate the mechanisms underlying innate resistance in PTC models and work to combat acquired resistance and minimal residual disease (MRD) in melanoma models. By doing so, we enhance our understanding of therapeutic response to BRAFi, addressing both innate and acquired resistance phenotypes across different mutant BRAFV600E cancers with a goal of contributing to improved treatment outcomes.

We have previously reported that subsets of cancer cells (4-8%) expressing the chemokine receptor CX3CR1 on their surface (CX3CR1Surf+) are endowed with stemness features and behave as metastasis-initiating cells (MICs) when grafted in mice. We show that prostate cancer cells lacking surface CX3CR1 (CX3CR1Surf-) can re-express CX3CR1 on their surface in vitro and re-acquire MIC potential in vivo. We also found that intracellular trafficking is responsible for the early phase of this CX3CR1 switch and is accompanied by a partial Epithelial-Mesenchymal Transition, thus indicating phenotypic plasticity. Our studies also revealed that the changes in CX3CR1 protein expression and its surface localization occur in a cyclical pattern. This unprecedented finding suggests that the CX3CR1 plasticity might be controlled by an autoregulatory feedback loop. Additional experiments showed that disseminated tumors in the skeleton and softtissue organs of mice grafted with human prostate cancer cells harbored higher percentages of CX3CR1Surf+ phenotypes, indicating a key role of the tissue microenvironment in promoting CX3CR1 plasticity. When cancer cells were harvested from metastatic tumors and re-plated in culture, the overall percentage of CX3CR1Surf+ phenotypes rapidly reverted to that of pre-grafting levels, thus suggesting the implication of epigenetic mechanisms. In conclusion, this study identifies novel phenotypic plasticity inducing the emergence of CX3CR1Surf+ cells with MIC properties, which are likely responsible for metastatic recurrence and support the development of CX3CR1 antagonists for therapeutic purposes.

Papillary thyroid carcinoma (PTC) is the most frequent form of thyroid cancer. PTC commonly presents with mutations of the serine/threonine kinase BRAF (BRAF^V600E), which drive ERK1/2 pathway activation to support growth and suppress apoptosis. PTC patients often undergo surgical resection; however, since the average age of PTC patients is under 50, adverse effects associated with prolonged maintenance therapy following total thyroidectomy are a concern. The development of mutant-selective BRAF inhibitors (BRAFi), like vemurafenib, has been efficacious in patients with metastatic melanoma, but the response rate is low for mutant BRAF PTC patients. Here, we assay the therapeutic response of BRAFi in a panel of human PTC cell lines and freshly biopsied patient samples. We observed heterogeneous responses to BRAFi, and multi-omic comparisons between susceptible and resistant mutant BRAF PTC revealed overrepresented stress response pathways and the absence of compensatory RTK activation – features that may underpin innate resistance. Importantly, resistant cell lines and patient samples had increased hallmarks of failed apoptosis; a cellular state defined by sublethal caspase activation and DNA damage. Further analysis suggests that the failed apoptotic phenotypes may have features of “minority mitochondrial outer membrane permeabilization (MOMP)” – a stress-related response characterized by fragmented and porous mitochondria known to contribute to cancer aggressiveness. We found that cells presenting with minority MOMP-like phenotypes are dependent on the apoptotic regulator, Mcl-1, as treatment with the Mcl-1 inhibitor, AZD5991, potently induced cell death in resistant cells. Furthermore, PI3K/AKT inhibitors sensitized resistant cells to BRAFi; an effect that was at least in part associated with reduced Mcl-1 levels. Together, these data implicate minority MOMP as a mechanism associated with intrinsic drug resistance and underscore the benefits of targeting Mcl-1 in mutant BRAF PTC.

BRCA1-associated protein 1 (BAP1) is a tumor suppressor gene that is mutated in cancer, including uveal melanoma. Loss-of-function BAP1 mutations are associated with uveal melanoma metastasis and poor prognosis, but the mechanisms underlying these effects remain unclear. Upregulation of cell–cell adhesion proteins is involved with collective migration and metastatic seeding of cancer cells. Here, we show that BAP1 loss in uveal melanoma patient samples is associated with upregulated gene expression of multiple cell adhesion molecules (CAM), including E-cadherin (CDH1), cell adhesion molecule 1 (CADM1), and syndecan-2 (SDC2). Similar findings were observed in uveal melanoma cell lines and single-cell RNA-sequencing data from uveal melanoma patient samples. BAP1 reexpression in uveal melanoma cells reduced E-cadherin and CADM1 levels. Functionally, knockdown of E-cadherin decreased spheroid cluster formation and knockdown of CADM1 decreased growth of BAP1-mutant uveal melanoma cells. Together, our findings demonstrate that BAP1 regulates the expression of CAMs which may regulate metastatic traits.Implications:BAP1 mutations and increased metastasis may be due to upregulation of CAMs.

BRCA1-associated protein 1 (BAP1) is a tumor suppressor gene that is mutated in cancer, including uveal melanoma. Loss-of-function BAP1 mutations are associated with uveal melanoma metastasis and poor prognosis, but the mechanisms underlying these effects remain unclear. Upregulation of cell–cell adhesion proteins is involved with collective migration and metastatic seeding of cancer cells. Here, we show that BAP1 loss in uveal melanoma patient samples is associated with upregulated gene expression of multiple cell adhesion molecules (CAM), including E-cadherin (CDH1), cell adhesion molecule 1 (CADM1), and syndecan-2 (SDC2). Similar findings were observed in uveal melanoma cell lines and single-cell RNA-sequencing data from uveal melanoma patient samples. BAP1 reexpression in uveal melanoma cells reduced E-cadherin and CADM1 levels. Functionally, knockdown of E-cadherin decreased spheroid cluster formation and knockdown of CADM1 decreased growth of BAP1-mutant uveal melanoma cells. Together, our findings demonstrate that BAP1 regulates the expression of CAMs which may regulate metastatic traits.Implications:BAP1 mutations and increased metastasis may be due to upregulation of CAMs.