Development Of Melanoma Brain Metastases Research Articles

Abstract 4127: Combined inhibition of RAF, MEK, and FAK attenuates melanoma brain metastases and prolongs survival in preclinical models

Abstract Despite promising results from recent FDA-approved therapies, many advanced melanoma patients develop resistance to both immunotherapy and targeted therapy. A common resistance mechanism to targeted therapy is upregulation of the PI3K/AKT signaling pathway, which has also been shown to promote the development of melanoma brain metastases. Historically, AKT inhibitors have failed in the clinic due to their limited efficacy or intolerable toxicity. Proteomic analysis comparing non-metastatic vs brain metastatic primary tumors in mice revealed focal adhesion kinase (FAK) as an AKT1 specific effector and a potential alternative therapeutic target. FAK is a non-receptor tyrosine kinase that localizes primarily to focal adhesions to regulate cell migration. To determine whether targeting FAK alone or in combination with the RAF/MEK inhibitor avutometinib reduces brain metastases and prolongs survival, we utilized both autochthonous and syngeneic melanoma mouse models. Mice with either subcutaneous tumors or established brain metastases were treated with FAK inhibitor, RAF/MEK inhibitor, or the combination of FAK and RAF/MEK inhibitors. Each cohort was assessed for tumor onset, growth, metastasis, and survival. Our results show that combined RAF/MEK/FAK inhibition significantly delays tumor onset, causes regression of established tumors, prevents the development of brain metastases, promotes the regression of established brain metastases, and prolongs survival. In addition, patient-derived BRAF V600E melanoma xenograft mouse models resistant to the BRAF inhibitor dabrafenib and the MEK inhibitor trametinib, were sensitive to combined RAF/MEK/FAK blockade. The addition of the BRAF inhibitor encorafenib to these models further enhanced the effect on tumor growth. These results support the initiation of a clinical trial evaluating the efficacy of the RAF/MEK inhibitor avutometinib in combination with the FAK inhibitor defactinib in patients with brain metastases from cutaneous melanoma. Additionally, we are assessing non-canonical roles of FAK in modulating the tumor microenvironment to determine whether avutometinib and defactinib also enhance the efficacy of immune checkpoint inhibition in this disease. Citation Format: Karly A. Stanley, Jared D. Almazan, Tursun Turapov, David A. Kircher, Gennie L. Parkman, MiKaela N. Field, Katie M. Culver, Christopher M. Stehn, Silvia Coma, Jonathan A. Pachter, Howard Colman, Sheri L. Holmen. Combined inhibition of RAF, MEK, and FAK attenuates melanoma brain metastases and prolongs survival in preclinical models [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 4127.

HDAC8-mediated inhibition of EP300 drives a transcriptional state that increases melanoma brain metastasis

Melanomas can adopt multiple transcriptional states. Little is known about the epigenetic drivers of these cell states, limiting our ability to regulate melanoma heterogeneity. Here, we identify stress-induced HDAC8 activity as driving melanoma brain metastasis development. Exposure of melanocytes and melanoma cells to multiple stresses increases HDAC8 activation leading to a neural crest-stem cell transcriptional state and an amoeboid, invasive phenotype that increases seeding to the brain. Using ATAC-Seq and ChIP-Seq we show that increased HDAC8 activity alters chromatin structure by increasing H3K27ac and enhancing accessibility at c-Jun binding sites. Functionally, HDAC8 deacetylates the histone acetyltransferase EP300, causing its enzymatic inactivation. This, in turn, increases binding of EP300 to Jun-transcriptional sites and decreases binding to MITF-transcriptional sites. Inhibition of EP300 increases melanoma cell invasion, resistance to stress and increases melanoma brain metastasis development. HDAC8 is identified as a mediator of transcriptional co-factor inactivation and chromatin accessibility that drives brain metastasis.

TMIC-47. INHIBITION OF MIR-146A-5P DECREASES PROGRESSION OF MELANOMA BRAIN METASTASIS VIA NOTCH PATHWAY DYSREGULATION IN ASTROCYTES

Abstract Melanoma has a high propensity for brain metastasis, occurring in 40% of late-stage patients. The progression of secondary tumors in the brain depends on the support of the surrounding microenvironment, emphasizing the importance of modulating neighboring cells for successful metastasis. Tumor-derived extracellular vesicles (EVs) play a crucial role in creating a pre-metastatic niche for tumor growth by transferring proteins, RNA, and DNA. This study aims to explore the involvement of melanoma brain metastasis (MBM)-derived EVs in MBM development and identify a target miRNA for potential therapeutic intervention. In an in vivo metastatic model, MBM-EV pre-treated mice exhibited significantly increased MBM tumor burden compared to non-primed mice. In vitro co-cultures of MBM-derived EVs with astrocytes led to astrocyte activation characterized by enhanced proliferation, invasion, cytokine production, and upregulation of GFAP. Microarray analysis revealed significant upregulation of miR-146a-5p in MBM-EVs compared to healthy astrocyte and melanocyte EVs. In situ hybridization of clinical MBM samples showed significantly elevated miR-146a-5p expression compared to healthy brain controls. Sequencing of NHA after co-culture with miR-146a-5p mimic revealed NUMB, a Notch signaling pathway inhibitor, as a miR-146a-5p target. Expression levels of Numb and other downstream Notch proteins were significantly altered in NHA in the presence of MBM-derived EVs and miR-146a. Deserpidine was identified as a potential inhibitor of miR-146a-5p through a drug docking study, and qPCR analysis confirmed reduced miR-146a-5p expression in deserpidine-treated MBM cells and EVs in vitro. In an MBM in vivo model, deserpidine-treated mice exhibited significantly increased survival, reduced tumor burden, and decreased astrocyte activation in both tumor and non-tumor brain tissues. In conclusion, EVs are important regulators of MBM and establish tumor-supporting reactive astrocytes by delivery of miR-146a. Elevated miR-146a levels in patients suggests a potential clinical intervention, and our results indicate deserpidine as a promising candidate for adjuvant therapy.

The RPL/RPS gene signature of melanoma CTCs associates with brain metastasis.

Melanoma brain metastasis (MBM) is linked to poor prognosis and low overall survival. We hypothesized that melanoma circulating tumor cells (CTCs) possess a gene signature significantly expressed and associated with MBM. Employing a multi-pronged approach, we provide first-time evidence identifying a common CTC gene signature for ribosomal protein large/small subunits (RPL/RPS) which associate with MBM onset and progression. Experimental strategies involved capturing, transcriptional profiling and interrogating CTCs, either directly isolated from blood of melanoma patients at distinct stages of MBM progression or from CTC-driven MBM in experimental animals. Second, we developed the first Magnetic Resonance Imaging (MRI) CTC-derived MBM xenograft model (MRI-MBM CDX) to discriminate MBM spatial and temporal growth, recreating MBM clinical presentation and progression. Third, we performed the comprehensive transcriptional profiling of MRI-MBM CDXs, along with longitudinal monitoring of CTCs from CDXs possessing/not possessing MBM. Our findings suggest that enhanced ribosomal protein content/ribogenesis may contribute to MBM onset. Since ribosome modifications drive tumor progression and metastatic development by remodeling CTC translational events, overexpression of the CTC RPL/RPS gene signature could be implicated in MBM development. Collectively, this study provides important insights for relevance of the CTC RPL/RPS gene signature in MBM, and identify potential targets for therapeutic intervention to improve patient care for melanoma patients diagnosed with or at high-risk of developing MBM.

Melanoma brain metastases: Biological basis and novel therapeutic strategies.

The development of brain metastases is the deadliest complication of advanced melanoma and has long been associated with a dismal prognosis. The recent years have seen incredible progress in the development of therapies for melanoma brain metastases (MBM), with both targeted therapies (the BRAF-MEK inhibitor combination) and immune checkpoint inhibitors (the anti-CTLA-4, anti-PD-1 combination) showing impressive levels of activity. Despite this, durations of response for these therapies remain lower at intracranial sites of metastasis compared to extracranial metastases and it has been suggested that there are unique features of the brain microenvironment that contribute to therapeutic escape. In this review, we outline the latest research into the biology and pathophysiology of melanoma brain metastasis development and progression. We then discuss the current status of clinical trial that are open to patients with MBM and end by describing the ongoing challenges for the field.

Upfront Surgical Resection of Melanoma Brain Metastases Provides a Bridge Toward Immunotherapy-Mediated Systemic Control.

Immune checkpoint blockade has systemic efficacy in patients with metastatic melanoma, including those with brain metastases (MBMs). However, immunotherapy-induced intracranial tumoral inflammation can lead to neurologic compromise, requiring steroids, which abrogate the systemic efficacy of this approach. We investigated whether upfront neurosurgical resection of MBM is associated with a therapeutic advantage when performed prior to initiation of immunotherapy. An institutional review board-approved, retrospective study identified 142 patients with MBM treated with immune checkpoint blockade between 2010 and 2016 at Massachusetts General Hospital, of whom 79 received surgery. Patients were classified based on the temporal relationship between immunotherapy, surgery, and development of central nervous system metastases. Overall survival (OS) was calculated from the date of diagnosis of MBM until death from any cause. Multivariate model building included a prognostic Cox model of OS, the effect of immunotherapy and surgical sequencing on OS, and the effect of immunotherapy and radiation sequencing on OS. The 2-year overall survival for patients treated with cytotoxic T-lymphocyte antigen 4, programmed death 1, or combinatorial blockade was 19%, 54%, and 57%, respectively. Among immunotherapy-naïve melanoma brain metastases, surgery followed by immunotherapy had a median survival of 22.7 months (95% confidence interval [CI], 12.6-39.2) compared with 10.8 months for patients treated with immunotherapy alone (95% CI, 7.8-16.3) and 9.4 months for patients treated with immunotherapy followed by surgery (95% CI, 4.1 to ∞; p = .12). On multivariate analysis, immunotherapy-naïve brain metastases treated with immunotherapy alone were associated with increased risk of death (hazard ratio, 1.72; 95% CI, 1.00-2.99) compared with immunotherapy-naïve brain metastases treated with surgery followed by immunotherapy. In treatment-naïve patients, early surgical resection for local control should be considered prior to commencing immunotherapy. A prospective, randomized trial comparing the sequence of surgery and immunotherapy for treatment-naïve melanoma brain metastases is warranted. In this retrospective study of 142 patients with melanoma brain metastases treated with immune checkpoint blockade, the development of melanoma brain metastases following immunotherapy was associated with decreased survival compared with diagnosis of immunotherapy-naïve brain metastases. The benefit of surgical intervention was seen in immunotherapy-naïve brain metastases in contrast to brain metastases that developed on immunotherapy. These results suggest that upfront local control with surgery for immunotherapy-naïve melanoma brain metastasis may provide a bridge toward immunotherapy-mediated systemic control.

Melanoma central nervous system metastases: An update to approaches, challenges, and opportunities.

In February 2018, the Melanoma Research Foundation and the Moffitt Cancer Center hosted the Second Summit on Melanoma Central Nervous System (CNS) Metastases in Tampa, Florida. In this white paper, we outline the current status of basic science, translational, and clinical research into melanoma brain metastasis development and therapeutic management. We further outline the important challenges that remain for the field and the critical barriers that need to be overcome for continued progress to be made in this clinically difficult area.

Abstract 3025: Ligand-independent EphA2 signaling drives an amoeboid phenotype that promotes melanoma brain metastasis development

Abstract The majority of melanoma patients treated with BRAF inhibitors (BRAFi) ultimately develop resistance and fail on therapy. It has previously been shown that BRAFi resistance is highly correlated with the adoption of a strongly invasive phenotype through ligand-independent EphA2 signaling. In this study, we used comprehensive mass spectrometry-based proteomic approaches to delineate the global signaling changes associated with the aggressive phenotype driven by ligand-independent EphA2 signaling. Preliminary data have identified new signaling adaptations associated with a mesenchymal-to-amoeboid transition (MAT) phenotype, which was confirmed in 3D collagen cultures. Functional experiments demonstrated that the amoeboid phenotype promoted cell migration and invasion, which was mediated through the interaction of EphA2 and CDC42. These findings were confirmed by expressing a constitutively activated form of CDC42. BRAFi resistant cell lines also exhibited the MAT phenotype and were more invasive compared to their treatment-naïve counterparts, in line with their dependence upon ligand-independent EphA2 signaling following BRAFi selection pressure. To further demonstrate the metastatic potential of these amoeboid cells driven by ligand-independent EpHA2 signaling in vivo, we performed intracardiac injections in mice with cell lines expressing either EpHA2 S897A (inactive) or S897E (constitutively active phosphomimetic). Interestingly, we found a preferential homing of EphA2 S897E cells to the brain, but no difference in metastasis to other organs including the lung and liver. An analysis of brain metastasis specimens from patients failing BRAF and BRAF/MEK inhibitor therapy showed strong staining for the amoeboid phenotype marker EphA2. To investigate whether the amoeboid phenotype may confer a survival advantage in circulation, we carried out shear stress assays. These experiments demonstrated that the amoeboid phenotype driven by ligand-independent EphA2 signaling promoted the survival of melanoma cells under shear stress. Cell attachment assays, trans-endothelial invasion assays and vascular permeability assays show that these cells are better suited to attach to and permeate an endothelial monolayer. We further show that inhibiting PI3K reversed the amoeboid phenotype and limited the EphA2-driven invasive capacity. In summary, we show for the first time that BRAFi resistance is associated with the adoption of an MAT phenotype that increases the metastatic seeding of melanoma cells to the brain, which can be reversed through inhibition of PI3K signaling. Citation Format: Chao Zhang, Inna Smalley, Ritin Sharma, Michael Emmons, Jane Messina, John Koomen, Keiran Smalley. Ligand-independent EphA2 signaling drives an amoeboid phenotype that promotes melanoma brain metastasis development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3025.

Molecular insights into melanoma brain metastases.

Substantial proportions of patients with metastatic melanoma develop brain metastases during the course of their disease, often resulting in significant morbidity and death. Despite recent advances with BRAF/MEK and immune-checkpoint inhibitors in the treatment of patients who have melanoma with extracerebral metastases, patients who have melanoma brain metastases still have poor overall survival, highlighting the need for further therapy options. A deeper understanding of the molecular pathways involved in the development of melanoma brain metastases is required to develop more brain-specific therapies. Here, the authors summarize the currently known preclinical data and describe steps involved in the development of melanoma brain metastases. Only by knowing the molecular background is it possible to design new therapeutic agents that can be used to improve the outcome of patients with melanoma brain metastases. Cancer 2017;123:2163-75. © 2017 American Cancer Society.

The metastatic microenvironment: Claudin-1 suppresses the malignant phenotype of melanoma brain metastasis.

Brain metastases occur frequently in melanoma patients with advanced disease whereby the prognosis is dismal. The underlying mechanisms of melanoma brain metastasis development are not well understood. Identification of molecular determinants regulating melanoma brain metastasis would advance the development of prevention and therapy strategies for this disease. Gene expression profiles of cutaneous and brain-metastasizing melanoma variants from three xenograft tumor models established in our laboratory revealed that expression of tight junction component CLDN1 was lower in the brain-metastasizing variants than in cutaneous variants from the same melanoma. The objective of our study was to determine the significance of CLDN1 downregulation/loss in metastatic melanoma and its role in melanoma brain metastasis. An immunohistochemical analysis of human cells of the melanocyte lineage indicated a significant CLDN1 downregulation in metastatic melanomas. Transduction of melanoma brain metastatic cells expressing low levels of CLDN1 with a CLDN1 retrovirus suppressed their metastatic phenotype. CLDN1-overexpressing melanoma cells expressed a lower ability to migrate and adhere to extracellular matrix, reduced tumor aggressiveness in nude mice and, most importantly, eliminated the formation of micrometastases in the brain. In sharp contrast, the ability of the CLDN1-overexpressing cells to form lung micrometastases was not impaired. CLDN1-mediated interactions between these cells and brain endothelial cells constitute the mechanism underlying these results. Taken together, we demonstrated that downregulation or loss of CLDN1 supports the formation of melanoma brain metastasis, and that CLDN1 expression could be a useful prognostic predictor for melanoma patients with a high risk of brain metastasis.

Melanoma brain colonization involves the emergence of a brain-adaptive phenotype.

The brain offers a unique microenvironment that plays an important role in the establishment and progression of metastasis. However, the molecular determinants that promote development of melanoma brain metastases are largely unknown. Utilizing two species of immune-compromised animals, with in vivo cultivated metastatic tissues along with their corresponding host tissues in a metastasis model, we here identify molecular events associated with melanoma brain metastases. We find that the transcriptional changes in the melanoma cells, as induced by the brain-microenvironment in both host species, reveal the opportunistic nature of melanoma in this biological context in rewiring the molecular framework of key molecular players with their associated biological processes. Specifically, we identify the existence of a neuron-like melanoma phenotype, which includes synaptic characteristics and a neurotransmission-like circuit involving glutamate. Regulation of gene transcription and neuron-like plasticity by Ca(2+)-dependent signaling appear to occur through glutamate receptor activation. The brain-adaptive phenotype was found as more prominent in the early metastatic growth phases compared to a later phase, emphasizing a temporal requirement of critical events in the successful colonization of the brain. Analysis of the host tissue uncovered a cooperative inflammatory microenvironment formed by activated host cells that permitted melanoma growth at the expense of the host organism. Combined experimental and computational approaches clearly highlighted genes and signaling pathways being shared with neurodegenerative diseases. Importantly, the identification of essential molecular networks that operate to promote the brain-adaptive phenotype is of clinical relevance, as they represent leads to urgently needed therapeutic targets.

The metastatic microenvironment: Brain‐residing melanoma metastasis and dormant micrometastasis

Brain metastasis occurs frequently in melanoma patients with advanced disease whereby the prognosis is dismal. The underlying mechanisms of melanoma brain metastasis development are not well understood. We generated a reproducible melanoma brain metastasis model, consisting of brain-metastasizing variants and local, subdermal variants that originate from the same melanomas thus sharing a common genetic background. The brain-metastasizing variants were obtained by intracardiac inoculation. Brain metastasis variants when inoculated subdermally yielded spontaneous brain dormant micrometastasis. Cultured cells from the spontaneous brain micrometastasis grew very well in vitro and generated subdermal tumors after an orthotopic inoculation. Expression analysis assays indicated that the brain metastasis and micrometastasis cells expressed higher levels of angiopoietin-like 4, prostaglandin-synthesizing enzyme cyclooxygenase-2, matrix metalloproteinase-1 and preferentially expressed antigen in melanoma and lower levels of claudin-1 and cysteine-rich protein 61 than the corresponding cutaneous variants. The reproducible models of human melanoma metastasizing experimentally and spontaneously to the brain will facilitate the identification of novel biomarkers and targets for therapy and contribute to the deciphering of mechanisms underlying melanoma metastasis.

Clinical variables and primary tumor characteristics predictive of the development of melanoma brain metastases and post‐brain metastases survival

Melanoma patients who develop brain metastases (B-Met) have limited survival and are excluded from most clinical trials. In the current study, the authors attempted to identify primary tumor characteristics and clinical features predictive of B-Met development and post-B-Met survival. A prospectively accrued cohort of 900 melanoma patients was studied to identify clinicopathologic features of primary melanoma (eg, thickness, ulceration, mitotic index, and lymphovascular invasion) that are predictive of B-Met development and survival after a diagnosis of B-Met. Associations between clinical variables present at the time of B-Met diagnosis (eg, extracranial metastases, B-Met location, and the presence of neurological symptoms) and post-B-Met survival were also assessed. Univariate associations were analyzed using Kaplan-Meier survival analysis, and the effect of independent predictors was assessed using multivariate Cox proportional hazards regression analysis. Of the 900 melanoma patients studied, 89 (10%) developed B-Met. Ulceration and site of the primary tumor on the head and neck were found to be independent predictors of B-Met development on multivariate analysis (P = .001 and P = .003, respectively). Clinical variables found to be predictive of post-B-Met survival on multivariate analysis included the presence of neurological symptoms (P = .008) and extracranial metastases (P = .04). Ulceration was the only primary tumor characteristic that remained a significant predictor of post-B-Met survival on multivariate analysis (P = .04). Primary tumor ulceration was found to be the strongest predictor of B-Met development and remained an independent predictor of decreased post-B-Met survival in a multivariate analysis inclusive of primary tumor characteristics and clinical variables. The results of the current study suggest that patients with ulcerated primary tumors should be prospectively studied to determine whether heightened surveillance for B-Met can improve clinical outcome.

Founder of “Archiv für Protistenkunde”: Fritz Schaudinn – his Unfinished Life

The recent years have seen significant progress in the development of systemic therapies to treat patients with advanced melanoma. Use of these new treatment modalities, which include immune checkpoint inhibitors and small molecule BRAF inhibitors, lead to increased overall survival and better outcomes. Although revolutionary, these therapies are often less effective against melanoma brain metastases, and frequently the CNS is the major site of treatment failure. The development of brain metastases remains a serious complication of advanced melanoma that is associated with significant morbidity and mortality. New approaches to both prevent the development of brain metastases and treat established disease are urgently needed. In this review we will outline the mechanisms underlying the development of melanoma brain metastases and will discuss how new insights into metastasis biology are driving the development of new therapeutic strategies. Finally, we will describe the latest data from the ongoing clinical trials for patients with melanoma brain metastases.