Melanoma Brain Metastases Research Articles

Combination of regorafenib with BRAF-/MEK-inhibitors for the treatment of patients with refractory melanoma brain metastases.

e14003 Background: there are no active treatment options available for patients (pts) diagnosed with melanoma brain metastases (MBM) that progress despite treatment with immune checkpoint inhibitors (ICI), and BRAF-/MEK-inhibitors (BRAF/MEKi) in pts with BRAF V600mutant melanoma. Regorafenib (REGO), an oral multi-target kinase inhibitor, including potent inhibition of RAF-dimers, has single-agent activity in pretreated melanoma (AS Vander Mijnsbrugge et al. SMR 2022). Here we report our single-institution experience with REGO in combination with BRAF/MEKi in pts with refractory MBM. Methods: pts were identified prospectively, medical records were reviewed retrospectively. Results: 13 pts with stage IV-M1d melanoma were included (6F, med age 54y [range 33-72], WHO PS: 0/1/2/3 resp. n = 1/4/6/2 pts, BRAF V600mut: n = 9, NRAS Q61mut: n = 4). Pts previously progressed on ICI (anti-PD-1 and -CTLA-4; 13 pts); BRAF-/MEK-inhibitor (all BRAF V600mut pts); chemotherapy (3 pts), T-VEC (2 pts), REGO mono (2 pts), and REGO+ICI (2 pts). At baseline, 11 pts had active MBM (8 pts on corticosteroids); 10 pts had extracranial evaluable disease. Pts were treated with REGO (40-80 mg QD) combined with BRAF/MEKi in 9 BRAF V600mut pts (dabrafenib/trametinib: n = 4; encorafenib/binimetinib: n = 5) or, in 4 NRAS Q61mut pts, with trametinib or binimetinib (+ low-dose BRAF-inhibitor if needed to mitigate skin toxicity). Treatment was stopped in 8 pts because of PD and is ongoing in 5 pts (3-49 wks after initiating therapy). There were no grade 4-5 TRAE. Grade 3 TRAE included: arterial hypertension (n = 4), hepatotoxicity (n = 2), diverticular bleeding (n = 1), and duodenal perforation, treated conservatively (n = 1). None of the 2 pts without active MBM at baseline progressed within the CNS. In 11 response evaluable pts (incl. 10 with evaluable extracranial metastases), best objective intracranial response (according to RANO-BM) was PR in 4 pts (36%; 3 BRAF V600mut pts), and SD in 4 pts (36%; 3 BRAF V600mut pts). Intracranial responses corresponded with extracranial responses in all but 3 pts (1 NRAS Q61mut pt with a PR intra-, had SD extracranially; 1 BRAF V600mut pt and 1 NRAS Q61mut pt with SD intra-, had PD extracranially); considering a potential clinical benefit of therapy beyond first PD, both pts continued treatment and remained clinically stable at the latest FU (resp. 15 and 49 wks after initiating therapy). Median time on REGO+BRAF/MEKi in BRAF V600mut pts was 15.9 wks (range 3-46), and on REGO+MEKi in NRAS Q61mut pts: 7.6 wks (range 3-49). Median PFS and OS: 12.9 and 26.4 wks (resp. 15.3 and 26.4 wks in BRAF V600mut pts; 8.6 and 10.1 wks in NRAS Q61mut pts). Conclusions: in heavily pretreated patients with MBM combination of REGO with BRAF-/MEK-inhibitors demonstrated promising anti-tumor activity. Further investigation of safety and clinical activity in a prospective trial is warranted.

Evaluating novel imaging-based mathematical modeling prediction of immunotherapy response by individual melanoma brain metastasis and patient prognosis.

e14013 Background: Ipilimumab/Nivolumab (Ipi/Nivo) significantly transformed outcomes for patients with melanoma brain metastases (MBM), however inter- and intra-patient responses can vary widely. The purpose of this study is to develop and calibrate an imaging-based mathematical model to predict lesion-specific immune response to Ipi/Nivo in patients with MBM to guide early local treatments such as radiotherapy and evaluate the prognostic value of identifying treatment resistant lesions. Methods: Baseline and follow-up 3D T1-W contrast MRIs of patients who solely received Ipi/Nivo after diagnosis with MBM were used to segment individual brain metastases and track serial volumetric data for each lesion to develop a mechanistic model (eq 1). After applying appropriate assumptions to our model, tumor burden ( ρ), intrinsic lesion growth rate ( α 0), Ipi/Nivo-induced kill rate ( μ) and immune response strength ( Λ) were quantified for each lesion. Growth rate shortly after the start of treatment ( α 1 ) was calculated using the short-term model solution (eq 2). dρ/dt= (α0- μ+μ.Λ).ρ - μ.Λ.ρ2 (eq 1) ρ(t)≈eα1.t (eq 2) Change in BM volume between baseline and last follow-up was used to classify each lesion as either ‘responder’ or ‘non-responder’; these were compared with the Wilcoxon-rank-sum test. The highest α1 value for any one BM within a patient tumor was evaluated for its potential to sort patient overall survival (OS). Because the median survival was not reached for this cohort, we determined the α1 threshold of 0.005 to sort patients using an optimization routine that generates the maximum AUC to separate the cohort into 2 groups on an ROC curve. Kaplan-Meier (KM) curves were compared between these 2 groups. Results: In 23 patients, we evaluated 61 lesions. At the lesion level, model parameters representing μ and Λ were significantly different between responder and non-responder lesions (p-values <0.0001 and 0.0004, respectively). The highest α1value for any one BM within a patient tumor was used to sort patient overall survival (OS) into prognostically ‘favorable’ or ‘unfavorable’ groups based on patient survival to ±1,200 days. In this limited cohort, OS KM curves by most aggressive lesion for favorable (α1 < 0.005) vs. unfavorable (α1 > 0.005) were not significant by log-rank test (p = 0.3263). Conclusions: In this limited cohort, we demonstrate feasibility to leverage serial conventional imaging and tumor volumetric data to calibrate a mechanistic model to predict per-lesion treatment response of MBM to Ipi/Nivo. Although the p-value between groups was found to be insignificant when predicting survival using α1, this is likely because of the small number of patients included in this study (n=23). Furthermore, when taken together with the notable AUC (=0.7917) observed in the ROC curve, this promising result supports further investigation in a larger cohort.

Combination beta-adrenergic blockade and immunotherapy for the treatment of primary and metastatic brain cancer.

e14017 Background: Brain tumors present a unique challenge to immunotherapeutic success, as immune responses in patients with brain tumors are suppressed both within the tumor microenvironment and peripherally. The systemic immunosuppression observed in brain tumor patients limits the capacity of the immune system to respond to immunotherapy. Brain tumor patients, particularly those with primary brain tumors like glioblastoma, represent a population in dire need of more thoughtful, targeted treatment approaches. Through this work we present an immunosuppressive axis specifically activated in the context of intracranial malignancies that inhibits immunotherapeutic success in these patients. We demonstrate that overactive adrenergic signaling is a major barrier to immunotherapeutic success. More specifically, beta-adrenergic signaling is known to suppress immune responses in various contexts. Our data indicate that combining beta-adrenergic blockade with immunotherapy provides a survival benefit in the setting of brain tumors, where immunotherapy alone has proven ineffective. Methods: Using the SEER Medicaid database, we retrospectively examined outcomes in patients with melanoma and lung adenocarcinoma brain metastasis who received checkpoint blockade therapy alone vs those concurrently on beta-blocker therapy. Results: We found that patients receiving combination therapy showed increased overall survival compared to those receiving checkpoint blockade alone. We then validated these findings in preclinical models of both glioma and melanoma brain metastases, demonstrating that combining immunotherapy and propranolol, a highly-prescribed FDA-approved beta-blocker, extended survival. Conclusions: Moving forward, we suggest that combination therapy with propranolol and immunotherapy, particularly checkpoint blockade, represents a promising translational treatment platform for patients with primary or metastatic intracranial malignancies.

Gene-edited and -engineered stem cell platform drives immunotherapy for brain metastatic melanomas.

Oncolytic virus therapy has shown activity against primary melanomas; however, its efficacy in brain metastases remains challenging, mainly because of the delivery and immunosuppressive nature of tumors in the brain. To address this challenge, we first established PTEN-deficient melanoma brain metastasis mouse models and characterized them to be more immunosuppressive compared with primary melanoma, mimicking the clinical settings. Next, we developed an allogeneic twin stem cell (TSC) system composed of two tumor-targeting stem cell (SC) populations. One SC was loaded with oncolytic herpes simplex virus (oHSV), and the other SC was CRISPR-Cas9 gene-edited to knock out nectin 1 (N1) receptor (N1KO) to acquire resistance to oHSV and release immunomodulators, such as granulocyte-macrophage colony-stimulating factor (GM-CSF). Using mouse models of brain metastatic BRAFV600E/PTEN-/- and BRAFV600E/wt/PTEN-/- mutant melanomas, we show that locoregional delivery of TSCs releasing oHSV and GM-CSF (TSC-G) activated dendritic cell- and T cell-mediated immune responses. In addition, our strategy exhibited greater therapeutic efficacy when compared with the existing oncolytic viral therapeutic approaches. Moreover, the TSCs composed of SC-oHSV and SCN1KO-releasing GM-CSF and single-chain variable fragment anti-PD-1 (TSC-G/P) had therapeutic efficacy in both syngeneic and patient-derived humanized mouse models of leptomeningeal metastasis. Our findings provide a promising allogeneic SC-based immunotherapeutic strategy against melanomas in the CNS and a road map toward clinical translation.

The Vicious Cycle of Melanoma-Microglia Crosstalk: Inter-Melanoma Variations in the Brain-Metastasis-Promoting IL-6/JAK/STAT3 Signaling Pathway.

Previous studies from our lab demonstrated that the crosstalk between brain-metastasizing melanoma cells and microglia, the macrophage-like cells of the central nervous system, fuels progression to metastasis. In the present study, an in-depth investigation of melanoma-microglia interactions elucidated a pro-metastatic molecular mechanism that drives a vicious melanoma-brain-metastasis cycle. We employed RNA-Sequencing, HTG miRNA whole transcriptome assay, and reverse phase protein arrays (RPPA) to analyze the impact of melanoma-microglia interactions on sustainability and progression of four different human brain-metastasizing melanoma cell lines. Microglia cells exposed to melanoma-derived IL-6 exhibited upregulated levels of STAT3 phosphorylation and SOCS3 expression, which, in turn, promoted melanoma cell viability and metastatic potential. IL-6/STAT3 pathway inhibitors diminished the pro-metastatic functions of microglia and reduced melanoma progression. SOCS3 overexpression in microglia cells evoked microglial support in melanoma brain metastasis by increasing melanoma cell migration and proliferation. Different melanomas exhibited heterogeneity in their microglia-activating capacity as well as in their response to microglia-derived signals. In spite of this reality and based on the results of the present study, we concluded that the activation of the IL-6/STAT3/SOCS3 pathway in microglia is a major mechanism by which reciprocal melanoma-microglia signaling engineers the interacting microglia to reinforce the progression of melanoma brain metastasis. This mechanism may operate differently in different melanomas.

A Survival Tree of Advanced Melanoma Patients with Brain Metastases Treated with Immune Checkpoint Inhibitors.

The efficacy of immune checkpoint inhibitors (ICIs) in patients with advanced melanoma that develop brain metastases (BM) remains unpredictable. In this study, we aimed to identify prognostic factors in patients with melanoma BM who are treated with ICIs. Data from advanced melanoma patients with BM treated with ICIs in any line between 2013 and 2020 were obtained from the Dutch Melanoma Treatment Registry. Patients were included from the time of the treatment of BM with ICIs. Survival tree analysis was performed with clinicopathological parameters as potential classifiers and overall survival (OS) as the response variable. In total, 1278 patients were included. Most patients were treated with ipilimumab-nivolumab combination therapy (45%). The survival tree analysis resulted in 31 subgroups. The median OS ranged from 2.7 months to 35.7 months. The strongest clinical parameter associated with survival in advanced melanoma patients with BM was the serum lactate dehydrogenase (LDH) level. Patients with elevated LDH levels and symptomatic BM had the worst prognosis. The clinicopathological classifiers identified in this study can contribute to optimizing clinical studies and can aid doctors in giving an indication of the patients' survival based on their baseline and disease characteristics.

Health-related quality of life in patients with melanoma brain metastases treated with immunotherapy.

Aims: To describe the health-related quality of life (HRQoL) of melanoma brain metastasis (MBM) patients throughout the first 18weeks of ipilimumab-nivolumab or nivolumab treatment. Materials & methods: HRQoL data (European Organisation for Research and Treatment of Cancer'sCore Quality of Life Questionnaire, additional Brain Neoplasm Module, and EuroQol 5-Dimension 5-Level Questionnaire) were collected as a secondary outcome of the Anti-PD1 Brain Collaboration phase II trial. Mixed linear modeling assessed changes over time, whereas the Kaplan-Meier method was used to determine median time to first deterioration. Results: Asymptomatic MBM patients treated with ipilimumab-nivolumab (n=33) or nivolumab (n=24) maintained baseline HRQoL. MBM patients with symptoms or leptomeningeal/progressive disease treated with nivolumab (n=14) reported a statistically significant trend toward improvement. Conclusion: MBM patients treated with either ipilimumab-nivolumab or nivolumab did not report a significant deterioration in HRQoL within 18weeks of treatment initiation. Clinical trial registration: NCT02374242 (ClinicalTrials.gov).

Loss of AMPKα2 promotes melanoma tumor growth and brain metastasis

AMP-activated protein kinase (AMPK) is a critical cellular energy sensor at the interface of metabolism and cancer. However, the role of AMPK in carcinogenesis remains unclear. Here, through analysis of the TCGA melanoma dataset, we found that PRKAA2 gene that encodes the α2 subunit of AMPK is mutated in ∼9% of cutaneous melanomas, and these mutations tend to co-occur with NF1 mutations. Knockout of AMPKα2 promoted anchorage-independent growth of NF1-mutant melanoma cells, whereas ectopic expression of AMPKα2 inhibited their growth in soft agar assays. Moreover, loss of AMPKα2 accelerated tumor growth of NF1-mutant melanoma and enhanced their brain metastasis in immune-deficient mice. Our findings support that AMPKα2 serves as a tumor suppressor in NF1-mutant melanoma and suggest that AMPK could be a therapeutic target for treating melanoma brain metastasis.

Ipilimumab plus nivolumab in patients with symptomatic melanoma brain metastasis requiring corticosteroids

Study aimTo investigate the efficacy of PD-1-directed antibody-based therapy in patients with symptomatic melanoma brain metastases (MBM) and concurrent treatment with corticosteroids. MethodsThis retrospective cohort study included patients with cutaneous melanoma with symptomatic MBM and concurrent treatment with corticosteroids who received PD-1-directed antibody-based treatment at the Royal Marsden Hospital London between 2016 and 2021. The primary outcome was overall survival (OS), secondary outcomes were intracranial response rate (ORR) and duration of response (DOR). We used the Kaplan-Meier method to describe survival. ResultsBetween 2016 and 2021, 256 patients presented with metastatic melanoma, of whom 29 were eligible with symptomatic MBM requiring corticosteroids and receiving ipilimumab plus nivolumab. Median age was 54 (interquartile range 44, 66). Median OS was 5.45months (95% confidence interval (CI) 2.89, 29.40), with 21% of patients (95% CI 9%, 47%) alive after 3years. ORR was 28% (8/29) and DOR was 7.85months (95% CI 7.85, not estimably [NE]). Responding patients had a median OS of 56.4months (95% CI 46.03, NE). Elevated lactate dehydrogenase and Eastern Cooperative Oncology Group PS> 2 were associated with poorer outcomes (median OS 29.4 versus 3.12months and 6.44 versus 5.13months), no such association was observed for corticosteroid dose, number of lesions, or line of treatment. ConclusionPatients with symptomatic MBM derive only modest benefit from combination immunotherapy treatment. Nevertheless, those with disease response have the potential to derive long-term benefit, justifying ipilimumab plus nivolumab in this group in the absence of other more effective treatment options.

Neuronal Cannabinoid CB1 Receptors Suppress the Growth of Melanoma Brain Metastases by Inhibiting Glutamatergic Signalling.

Melanoma is one of the deadliest forms of cancer. Most melanoma deaths are caused by distant metastases in several organs, especially the brain, the so-called melanoma brain metastases (MBMs). However, the precise mechanisms that sustain the growth of MBMs remain elusive. Recently, the excitatory neurotransmitter glutamate has been proposed as a brain-specific, pro-tumorigenic signal for various types of cancers, but how neuronal glutamate shuttling onto metastases is regulated remains unknown. Here, we show that the cannabinoid CB1 receptor (CB1R), a master regulator of glutamate output from nerve terminals, controls MBM proliferation. First, in silico transcriptomic analysis of cancer-genome atlases indicated an aberrant expression of glutamate receptors in human metastatic melanoma samples. Second, in vitro experiments conducted on three different melanoma cell lines showed that the selective blockade of glutamatergic NMDA receptors, but not AMPA or metabotropic receptors, reduces cell proliferation. Third, in vivo grafting of melanoma cells in the brain of mice selectively devoid of CB1Rs in glutamatergic neurons increased tumour cell proliferation in concert with NMDA receptor activation, whereas melanoma cell growth in other tissue locations was not affected. Taken together, our findings demonstrate an unprecedented regulatory role of neuronal CB1Rs in the MBM tumour microenvironment.

The abscopal effect: inducing immunogenicity in the treatment of brain metastases secondary to lung cancer and melanoma.

The phenomenon of radiation therapy (RT) causing regression of targeted lesions as well as lesions outside of the radiation field is known as the abscopal effect and is thought to be mediated by immunologic causes. This phenomena has been described following whole brain radiation (WBRT) and stereotactic radiosurgery (SRS) of brain metastasis (BM) in advanced melanoma and non-small-cell lung cancer (NSCLC). We systematically reviewed the available literature to identify which radiation modality and immunotherapy (IT) combination may elicit the abscopal effect, the optimal timing of RT and IT, and potential adverse effects inherent to the combination of RT and IT. Using PRISMA guidelines, a search of PubMed, Medline, and Web of Science was conducted to identify studies demonstrating the abscopal effect during treatment of NSCLC or melanoma with BM. 598 cases of irradiated BM of melanoma or NSCLC in 18 studies met inclusion criteria. The most commonly administered ITsincluded PD-1 or CTLA-4 immune checkpoint inhibitors (ICI), with RT most commonly administered within 3 months of ICI. Synergy between ICI and RT was described in 16 studies including evidence of higher tumor response within and outside of the irradiated field. In the 12 papers (n = 232 patients) that reported objective response rate (ORR) in patients with BM treated with RT and concurrent systemic IT, the non-weighted mean ORR was 49.4%; in the 5 papers (n = 110 patients) that reported ORR for treatment with RT or IT alone, the non-weighted mean ORR was 27.8%. No studies found evidence ofsignificantly increased toxicity in patients receiving RT and ICI. The combination of RT and ICIs may enhance ICI efficacy and induce more durable responses via the abscopal effect in patients with brain metastases of melanoma or NSCLC.

Abstract LB159: Deregulation of CTC-associated RPL/RPS protein expression inhibits melanoma brain metastasis

Abstract Melanoma brain metastasis (MBM) is associated with poor prognosis and low survival rates. MBM is diagnosed clinically in up to 60% of melanoma patients and in up to 80% of patients at autopsy. Circulating Tumor Cells (CTCs) are “seeds” of fatal metastasis and the smallest functional units of cancer. Our previous studies have employed a multilevel approach to identify a common CTC signature for ribosomal protein large/small subunits (RPL/RPS) which is linked to MBM development and progression1. Here, we hypothesized that targeting ribosomal biogenesis prevents MBM onset in CTC-derived xenografts. First, we treated CTC-injected MBM mice with RPL15 inhibitor omacetaxine to monitor metastatic progression. Second, parallel cohorts of mice were treated with CDK4/CDK6 inhibitor palbociclib with or without omacetaxine to assess effects of cell proliferation. Total flux of MBM signal was measured by IVIS and found to be higher in untreated mice. Mouse necropsies revealed decreased levels of MBM, along with metastatic dissemination to other organs. Third, collected mouse blood was analyzed by the FDA-approved ParsortixTM platform to enumerate CTC/CTC clusters (IHC), and evaluate their gene expression (RNA-seq). Lastly, metabolic profiling of inhibitor-treated versus untreated MBM CTC-derived clones was performed in vitro. Collectively, our findings indicate that the deregulation of ribosomal proteins inhibits MBM progression. This implicates a specific vulnerability of aggressive CTC subsets with high RPL/RPS content in MBM patients. RPL/RPS genetic screening could be a prognostic indicator of MBM severity and clinical outcomes. Our work promotes the concept that therapies targeting RPL/RPS proteins merit exploration as potential suppressors of metastatic progression in general, MBM in particular. 1. Bowley T, Lagutina I, Francis C, Sivakumar S, Selwyn R, Taylor E, et al. The RPL/RPS gene signature of melanoma updates CTCs associates with brain metastasis. Cancer Research Communications 2022;2: 1445-57. Citation Format: Tetiana Yuriyivna Bowley, Seth Merkley, Irina Lagutina, Dario Marchetti. Deregulation of CTC-associated RPL/RPS protein expression inhibits melanoma brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB159.

Metastatic Melanoma: A Preclinical Model Standardization and Development of a Chitosan-Coated Nanoemulsion Containing Temozolomide to Treat Brain Metastasis.

Melanoma is the most aggressive type of skin cancer. Brain metastasis is the worst scenario in metastatic melanoma and the treatment options for these patients are limited. Temozolomide (TMZ) is a chemotherapy agent used to treat primary central nervous system tumors. Our objective was to develop chitosan-coated nanoemulsion containing temozolomide (CNE-TMZ) for nasal route administration to melanoma brain metastasis treatment. A preclinical model of metastatic brain melanoma was standardized, and the efficiency of the developed formulation was further determined in vitro and in vivo. The nanoemulsion was done by spontaneous emulsification method and the formulation was characterized by size, pH, polydispersity index, and zeta potential. Culture assessments to determine cell viability were done in the A375 human melanoma cell line. To determine the safety of formulation, healthy C57/BL6 mice were treated with a nanoemulsion without TMZ. The model in vivo used B16-F10 cells implanted by stereotaxic surgery in C57/BL6 mice brains. The results demonstrate that the preclinical model used showed to be useful to analyze the efficiency of new candidate drugs to treat melanoma brain metastasis. The chitosan-coated nanoemulsions with TMZ showed the expected physicochemical characteristics and demonstrated safety and efficacy, reducing around 70% the tumor size compared to control mice, and presenting a tendency in mitotic index reduction, becoming an interesting approach to treat melanoma brain metastasis.

Insights into the Molecular Mechanisms Mediating Extravasation in Brain Metastasis of Breast Cancer, Melanoma, and Lung Cancer

Brain metastasis is an incurable end-stage of systemic cancer associated with poor prognosis, and its incidence is increasing. Brain metastasis occurs through a multi-step cascade where cancer cells spread from the primary tumor site to the brain. The extravasation of tumor cells through the blood-brain barrier (BBB) is a critical step in brain metastasis. During extravasation, circulating cancer cells roll along the brain endothelium (BE), adhere to it, then induce alterations in the endothelial barrier to transmigrate through the BBB and enter the brain. Rolling and adhesion are generally mediated by selectins and adhesion molecules induced by inflammatory mediators, while alterations in the endothelial barrier are mediated by proteolytic enzymes, including matrix metalloproteinase, and the transmigration step mediated by factors, including chemokines. However, the molecular mechanisms mediating extravasation are not yet fully understood. A better understanding of these mechanisms is essential as it may serve as the basis for the development of therapeutic strategies for the prevention or treatment of brain metastases. In this review, we summarize the molecular events that occur during the extravasation of cancer cells through the blood-brain barrier in three types of cancer most likely to develop brain metastasis: breast cancer, melanoma, and lung cancer. Common molecular mechanisms driving extravasation in these different tumors are discussed.

Abstract 1301: Evolution of heterogenous BRAFi/MEKi+CDK4/6i resistance mechanisms and targeting site-specific resistant populations in metastatic cutaneous melanoma

Abstract Intrapatient heterogeneity among different metastatic sites contributes to targeted therapy resistance. Determining the proteomic profile of metastatic melanoma lesions that develop within the same patient could help elucidate adaptive mechanisms that drive tumor progression during therapy. Tumors that persist through treatment and colonize distant organs adapt to and rely on organ-specific microenvironments. To assess how protein expression is altered in cutaneous melanoma derived from different metastatic sites in a single patient, we utilized patient-derived BRAFi/MEKi and BRAFi/MEKi+CDK4/6i-treated samples from the LOGIC2 clinical trial. Eight cell lines were established from patient biopsies at chronological time points throughout treatment and subsequent post-mortem resection of the bone, ovary, and brain metastatic sites. While the initial metastatic tumor in the breast was BRAFV600E mutant, NRASQ61 mutations were acquired after treatment with CDK4/6i in the bone and ovary tumors. RPPA was performed on six of the cell lines to characterize site-specific differences in protein expression. We detected upregulation of ErbB3 in the brain metastatic tumor. Since neuregulin 1, a ligand for ErbB3, is abundant in brain tissue, we tested its ability to promote resistance to BRAFi/MEKi in melanoma brain metastases. Neuregulin 1 rescued the growth of brain metastasis cells and restored phospho-AKT and phospho-ERK1/2 levels in the presence of BRAFi/MEKi. Therefore, the ErbB3-Neuregulin axis may be targeted, for example by using ErbB3 inhibitors to improve response to MAPK inhibitors in melanoma brain metastases. Further studies aim to investigate the evolution of metastatic cutaneous melanoma longitudinally in response to targeted therapies. Site-specific signaling mechanisms will be functionally examined as possible avenues for metastasis-specific targeted therapies to improve patient outcomes. Citation Format: Haley P. Wilson, Timothy J. Purwin, Claudia Capparelli, Phil F. Cheng, Mitch P. Levesque, Reinhard Dummer, Jessica Teh, Andrew E. Aplin. Evolution of heterogenous BRAFi/MEKi+CDK4/6i resistance mechanisms and targeting site-specific resistant populations in metastatic cutaneous melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1301.

Abstract 632: Exploiting pyroptosis in melanoma brain metastasis

Abstract Melanoma is the deadliest form of skin cancer due to its frequent metastasis to the brain. Melanoma brain metastases (MBM) are a significant clinical challenge, and current model systems and therapeutic strategies are poor. Inhibitors targeting the most common oncogenic driver of melanoma, BRAFV600E (BRAFi), disrupt mitogenic signaling and promote caspase-3 associated cell death. Though caspase-3 is a well-known apoptotic driver, it can also cleave Gasdermin E (GSDME) to create pore-forming N-terminal fragments that oligomerize at plasma and mitochondrial membranes and trigger pyroptosis - a form of immunogenic cell death (ICD). Our recent work illustrates the importance of GSDME mediated pyroptosis and associated immunogenicity in providing a durable therapeutic response against melanoma. Since pyroptosis can be activated by GSDME cleavage via caspase-3, and is prominent in various neuropathologies, a better understanding of the interplay between central nervous system (CNS) immune cells and BRAFi-mediated ICD is critical for designing more effective therapies against MBM. Here, we engineered a panel of inducible pyroptotic melanoma cells to model BRAFi-mediated pyroptosis. Using ex vivo CNS slice co-cultures, we found that inducible N-GSDME expression in human and murine melanoma cells drives propidium iodide uptake and release of inflammatory activators like HMGB1 and IL-1 - hallmarks of pyroptosis. Importantly, conditioned media from these pyroptotic cells stimulates a more robust phagocytic response from resident macrophages of the CNS and pyroptotic melanoma elicit stronger cytotoxicity from syngeneic CD8+ T cells, further highlighting the benefits of BRAFi-mediated ICD. Our findings provide evidence that resident CNS immune cells and peripheral T cells respond to pyroptotic melanoma, suggesting that induction of ICD should be a consideration in the development of anti-MBM therapies. Citation Format: Maria R. Cavallo. Exploiting pyroptosis in melanoma brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 632.

Abstract 1361: Astrocytes activate neuronal programming in melanoma brain metastases

Abstract Melanoma Brain Metastases (MBM) pose a major challenge for treating metastatic melanoma. Up to 70% of advanced melanoma patients develop MBM, resulting in significant morbidity and mortality. Melanocytes, the originating cell type of melanomas, are derived from neural crest cells in development. Melanoma’s proclivity to metastasize to the brain has been anecdotally connected to its neural crest origins. Recently, transcriptomic analyses have demonstrated that MBM express a neuronal-like phenotype, upregulating neural crest factors such as NCAM (Neural cell adhesion molecule), L1CAM (L1 cell adhesion molecule protein), while expressing melanocytic markers like MITF (melanocyte induction transcription factor). The activation of neuronal development programming may be a key mediator in brain-metastatic growth, and thus necessitates a better understanding of how melanoma cells adapt to the brain. Astrocytes have emerged as the major host cell type that cancer cells encounter and interact with during brain metastasis formation. Typically, astrocytes regulate brain homeostasis and support neuronal function. Their response to injury, termed reactive astrocytosis, is a process defined by increased secretion of growth factors and inflammatory cytokine production. Post-mortem analyses of MBM patients reveal perilesional reactive astrocytosis. Here we demonstrate that astrocyte reactivity promotes MBM growth, using in vitro and in vivo model systems. We identify that astrocytes secrete factors that increase tumor growth and expression of neural development factors. Moreover, pharmacological inhibition of neural development pathways, like Wnt signaling, attenuate the effects of astrocyte secreted factors. We developed a brain adapted melanoma line, D4M3a BrJ, and demonstrate the time course of astrocyte reactivity in vivo in its relationship with tumor growth. Our findings provide evidence that astrocyte reactivity promotes MBM growth and activation of neural development programming, unveiling a new therapeutic avenue for targeting the brain metastatic niche. Citation Format: Julia K. Farnan, Maria R. Cavallo, Edward J. Hartsough, Joshua G. Jackson. Astrocytes activate neuronal programming in melanoma brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1361.

Abstract 3606: HDAC8-mediated inhibition of EP300 drives a neural crest-like transcriptional state that increases melanoma brain metastasis

Abstract Melanomas are heterogeneous and adopt multiple transcriptional states that can confer an invasive phenotype and resistance to therapy. Little is known about the epigenetic drivers of these cell states, limiting our ability to regulate melanoma heterogeneity and tumor progression. Here we identify stress-induced HDAC8 activity as the driver of a neural crest stem cell (NCSC)-like transcriptional state that increased the formation of melanoma brain metastases (MBM). Exposure of melanocytes and melanoma cells to multiple different stresses led to HDAC8 activation, a switch to a NCSC gene expression signature and the adoption of an amoeboid, invasive phenotype. This cell state enhanced the survival of melanoma cells under shear stress conditions and increased the formation of metastases in the brain. Single cell RNA-seq analyses showed that HDAC8 expression was correlated with the NCSC cell state in clinical MBM specimens. ATAC-Seq and ChIP-Seq analysis showed HDAC8 to alter chromatin structure by increasing H3K27ac and accessibility at c-Jun binding sites without changing global histone acetylation. The increased accessibility of Jun binding sites was paralleled by decreased H3K27ac and accessibility at MITF binding sites and loss of melanoma-lineage gene expression. Mass spectrometry-based acetylomics demonstrated that HDAC8 deacetylated the histone acetyltransferase (HAT) EP300 leading to its enzymatic inactivation. This, in turn, led to an increased binding of EP300 to Jun-transcriptional sites and decreased binding to MITF-transcriptional sites. Increased expression of EP300 decreased invasion and increased the sensitivity of melanoma cells to multiple stresses while inhibition of EP300 function increased invasion and resistance to stress. We identified HDAC8 as a novel mediator of transcriptional co-factor inactivation and chromatin accessibility that increases MBM development. Citation Format: Michael Emmons, Richard Bennett, Alberto Riva, Chao Zhang, Robert Macaulay, Daphne Dupéré-Richér, Bin Fang, John Koomen, Jiannong Li, Ann Chen, Edward Seto, Jonathan Licht, Keiran Smalley. HDAC8-mediated inhibition of EP300 drives a neural crest-like transcriptional state that increases melanoma brain metastasis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3606.

Abstract 835: Translational nanotechnology-based cancer vaccine to re-educate host immune response against metastatic cancers

Abstract Sixty percent of melanoma patients present brain metastases, having low response rates to current systemic therapies, which do not prolong survival. New therapeutic options to address this clinical need are urgent. Here, we report the development of a polymeric nanoparticle as a cancer nanovaccine capable of selectively co-targeting toll-like receptor function and PD-L1 expression to improve antigen presentation and subsequent effector immune cell function within brain microenvironment. Mannose-poly(lactic-co-glycolic acid)/poly(lactic acid) (man-PLGA/PLA) nanoparticles (NP) were prepared by a modified double emulsion solvent evaporation method, to deliver combinations of melanoma neoantigens, toll-like receptor ligands and regulators of the PD-1/PD-L1 axis. NP physicochemical properties were fully characterized, including size, surface charge and morphology. The amount of melanoma neoantigens and immune regulators entrapped within NP was determined by HPLC. Immature dendritic cells (DC) were used to evaluate the impact of NP on cell viability, and to assess NP uptake kinetics by flow cytometry. NP ability to target and trigger the activation of DC and T cells, was assessed in the lymph nodes and spleen of immunized mice. The NP anti-tumor immune-mediated effect was evaluated in vivo in two primary melanoma-bearing immunocompetent mouse models (B16F10 and B16MO5), and in a melanoma brain metastasis (MBM) mouse model (B16F10), which also included the immune profiling within tumor site by flow cytometry, before and after treatment.NP presented an average diameter of 180 nm, narrow polydispersity index, surface charge close to neutrality, spherical morphology, and high loadings of the neoantigens and immunoregulators. NP did not affect DC viability and were extensively internalized by immature DC. NP were preferentially taken-up in vivo by DC, increasing the expression of activation/maturation markers at DC surface, such as CD80 and CD86. NP elicited antigen-specific T-cell responses, by the significant increase in the expression of TNF-alpha and IFN-gamma (TH1-guided response). In vivo combination of NP with anti-PD-L1 induced a potent immune-mediated anti-tumor response in both primary and preclinical models of MBM, overcoming tumor development with an increased overall survival. This combination re-shaped immune and stroma cell populations in primary tumor and MBM microenvironment, which presented a marked infiltration of cytotoxic T cells that correlated with a decreased expression of PD-1 and PD-L1 within the TME. The synergy between our nanovaccine and anti-PD-L1 provide essential insights to devise alternative combinations regiments to improve the efficacy of immune checkpoint inhibitors in metastatic melanoma, thus opening new line for this unmet medical need. Citation Format: Barbara Carreira, Rita A. Acúrcio, Sabina Pozzi, Ron Kleiner, Liane I. Moura, Ana I. Matos, Daniela vaskovich, Carina Peres, Adelaide Fernandes, Sara Xapelli, Ronit Satchi-Fainaro, Helena F. Florindo. Translational nanotechnology-based cancer vaccine to re-educate host immune response against metastatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 835.

876 Significant Survival Improvements for Patients with Melanoma Brain Metastases: Can We Reach Cure in the Current Era?

INTRODUCTION: New therapies for melanoma have been associated with increasing survival expectations, as opposed to the dismal outcomes of only a decade ago. METHODS: We reviewed 171 melanoma patients with BM receiving stereotactic radiosurgery (SRS) who were followed with point-of-care data collection between 2012-2020. Clinical, molecular and imaging data were collected, including systemic treatment and radiosurgical parameters. RESULTS: Mean age was 63 ± 15 years, 39% were female and 29% had BRAF-mutated tumors. Median overall survival after radiosurgery was 15.7 months (95% Confidence Interval: 11.4-27.7) and 25 months in patients managed since 2015. Thirty-two patients survived 5 years from their initial SRS. BRAF mutation-targeted therapies showed a survival advantage in comparison to chemotherapy (p = 0.009), but not to immunotherapy (p = 0.09). In a multivariable analysis, both immunotherapy and the number of metastases at 1st SRS were predictors of long-term survival (5 years) from initial SRS (p = 0.023 and p = 0.018, respectively). Five patients (16%) of the long-term survivors required no active treatment for 5 years. CONCLUSIONS: Long-term survival in patients with melanoma BM is achievable in the current era of SRS combined with immunotherapies. For those alive 5 years after first SRS, 16% had been also off systemic or local brain therapy for over 5 years. Given late recurrences of melanoma, caution is warranted, however prolonged survival off active treatment in a subset of our patients raises the potential for cure.