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Background: As systemic therapy improves, there has been an increasing number of breast cancer patients who develop brain metastasis. Screening of asymptomatic stage IV breast cancer patients with brain MRIs is not currently recommended by the National Comprehensive Cancer Network (NCCN) Guidelines. Retrospective reports suggest breast cancer patients are more likely to present with more advanced central nervous system disease at the time of brain metastasis diagnosis compared to melanoma and non-small cell lung cancer (NSCLC) patients. This may be in part due to routine screening recommendations in melanoma and NSCLC. Early detection and treatment of brain metastases may improve outcomes for breast cancer patients. Trial Design: The study is designed as a single arm, nonrandomized phase II study, with the goal of investigating the role of screening brain MRIs in neurologically asymptomatic patients with metastatic breast cancer. Breast cancer patients will be allocated based on receptor subtypes into triple negative (TN), HER2+, and hormone receptor (HR)+/HER2- breast cancer. Following study enrollment, patients will undergo a screening brain MRI. Patients will undergo a second brain MRI at first systemic progression or at 6 months whichever event occurs sooner. Eligibility: Asymptomatic, stage IV breast cancer patients that have progressed past first line therapy in the metastatic setting with an ECOG/= 6 months are eligible. Specific Aims: The primary objective is to determine the incidence of asymptomatic brain metastasis in metastatic breast cancer by subtype. Secondary objectives include determining the incidence of asymptomatic leptomeningeal disease, the number and size of brain metastases at diagnosis, the number of patients requiring whole brain radiation therapy vs. stereotactic radiation following diagnosis and overall survival and brain metastasis specific survival following brain metastasis diagnosis in metastatic breast cancer by subtype. Statistical Methods: A total of 30, 30, and 40 TN, HER2+, and HR+/HER2-, breast cancer patients will be enrolled, respectively. Using an incidence rate of 17%, the 95% CI by subtype will be (0.06,0.351), (0.06,0.351), and (0.07,0.322). Patient Accrual: This study is open with 30 patients enrolled at the time of submission. A total of 100 patients will be enrolled. Contact Information: Kamran A. Ahmed MD, Moffitt Cancer Center, email: kamran.ahmed@moffitt.org, Clinical trial information: NCT05115474. Funding: Florida Breast Cancer Foundation. Citation Format: Kamran A. Ahmed, Youngchul Kim, Avan Armaghani, John Arrington, Ricardo Costa, Brian J. Czerniecki, Roberto Diaz, Peter A. Forsyth, Hung Khong, Kimberley Lee, Loretta Loftus, Marilin Rosa, Hatem H. Soliman, Iman Washington, Aixa Soyano, Hyo S. Han. Phase II Study of Screening Brain MRIs in Stage IV Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-19-01.

The overall survival of advanced melanoma has improved dramatically. Immunotherapies, specifically checkpoint inhibitors, have played a large role in this improvement. These agents have also shown benefit in the adjuvant setting, are approved for treatment of resected stage II, III, and IV melanoma, and play an evolving role in the neoadjuvant setting. Although generally well tolerated, immune-related adverse events occur and can be severe. Here we focus on some severe and potentially long term toxicities, including cardiovascular and neurologic toxicities. Our understanding of the acute and long-term toxicities of immune checkpoint inhibitors continues to evolve. Oncologists must continue to balance cancer risk and treatment-related toxicities.

BackgroundAlthough the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combinations are effective in advanced melanoma it remains unclear whether their mechanisms of action and resistance overlap.MethodsWe used single cell (sc) RNA-seq, flow cytometry and IHC analysis of responding SM1 and B16 melanoma flank tumors and SM1 brain metastases to explore the mechanism of action of the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combination. CD4+ and CD8+ T cell depletion and ELISPOT assays were used to demonstrate the unique role of CD4+ T cell help in the anti-tumor effects of the anti-PD-1+LAG-3 combination. Tetramer assays confirmed the loss of CD8+ tumor-reactive T cells in brain tumors resistant to the anti-PD-1+LAG-3 combination.ResultsThe anti-PD-1+CTLA-4 combination was associated with the infiltration of FOXP3+ regulatory CD4+ cells (Tregs), fewer activated CD4+ T cells and the accumulation of a subset of IFNγ secreting cytotoxic CD8+ T cells, whereas the anti-PD-1+LAG-3 combination led to the accumulation of CD4+ T helper cells that expressed CXCR4, TNFSF8, IL21R and a subset of CD8+ T cells with reduced expression of cytotoxic markers. T cell depletion studies showed a requirement for CD4+ T cells for the anti-PD-1+LAG-3 combination, but not the PD-1-CTLA-4 combination at both flank and brain tumor sites. In anti-PD-1+LAG-3 treated tumors, CD4+ T cell depletion was associated with fewer activated (CD69+) CD8+ T cells, impaired IFNγ release and increased numbers of myeloid-derived suppressor cells (MDSCs) but, conversely, increased numbers of activated CD8+ T cells and IFNγ release in anti-PD-1+CTLA-4 treated tumors. Analysis of relapsing melanoma brain metastases from anti-PD-1+LAG-3 treated mice showed an increased accumulation of MDSCs and a loss of gp100+ tumor reactive CD8+ T cells. An analysis of the inferred cell-cell interactions from the scRNA-seq data suggested the MDSCs interacted with multiple subsets of T cells in a bi-directional manner.ConclusionsTogether these studies suggest that these two clinically relevant ICI combinations have differential effects upon CD4+ T cell polarization, which in turn, impacted cytotoxic CD8+ T cell function. Further insights into the mechanisms of action/resistance of these clinically-relevant ICI combinations will allow therapy to be further personalized.

Immunotherapy (IT) and targeted therapy (TT) are both effective against melanoma, but their combination is frequently toxic. Here, we investigated whether the sequence of IT (anti–PD-1)→ TT (ceritinib–trametinib or dabrafenib–trametinib) was associated with improved antitumor responses in mouse models of BRAF- and NRAS-mutant melanoma. Mice with NRAS-mutant (SW1) or BRAF-mutant (SM1) mouse melanomas were treated with either IT, TT, or the sequence of IT→TT. Tumor volumes were measured, and samples from the NRAS-mutant melanomas were collected for immune-cell analysis, single-cell RNA sequencing (scRNA-seq), and reverse phase protein analysis (RPPA). scRNA-seq demonstrated that the IT→TT sequence modulated the immune environment, leading to increased infiltration of T cells, monocytes, dendritic cells and natural killer cells, and decreased numbers of tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells. Durable responses to the IT→TT sequence were dependent on T-cell activity, with depletion of CD8+, but not CD4+, T cells abrogating the therapeutic response. An analysis of transcriptional heterogeneity in the melanoma compartment showed the sequence of IT→TT enriched for a population of melanoma cells with increased expression of MHC class I and melanoma antigens. RPPA analysis demonstrated that the sustained immune response induced by IT→TT suppressed tumor-intrinsic signaling pathways required for therapeutic escape. These studies establish that upfront IT improves the responses to TT in BRAF- and NRAS-mutant melanoma models.

S1: IT monotherapy in SW1 model S2: IT->dabrafenib-trametinib therapy in BRAF mutant melanoma. S3: IT->TT sequence in NRAS#5 model S4: Expanded heatmap of all immune cells S5: IT->TT increases accumulation of activated CD8+ T cells S6: The TT->IT sequence on T cells, Tregs and MDSCS S7: Expanded heatmap of T and NK cells S8: IT->TT increases accumulation of activated CD8+ T cells, pathway analysis. S9: Expanded heatmap of myeloid cell data S10: IT->TT sequence increases expression of MHC I mRNAs S11: TT affects melanoma antigens S12: IC50 curves showing resistance to TT S13: Model showing mechanism of IT->TT sequence ST1: tumor weights at the end of the experiment

Supplemental Table 5: Cox proportional hazard model analysis identifies immune cell subpopulations associated with better survival outcomes

Supplementary Figures 1-11. 1. CSF cytology of leptomeningeal patient cohort. 2. Additional clinical timelines of leptomeningeal patient cohort 3. Patient MRIs 4. RNAseq Workflow 5. Differential responses to BRAFi treatment in CSF 6. RNAseq shows CSF from Patient 4 to elicit transcriptional modulation of melanoma cell response to BRAF inhibition 7. CSF from LMM patients elicits transcriptional changes in melanoma cells 8. CSF protects melanoma cells from BRAF inhibitor therapy 9. CSF activates AKT in WM164 10. No-LMM CSF does not induce expression of TGFbeta or activation of AKT 11. ELISA assay showing the levels of TGFβ1 in CSF specimens from extrordi- nary responder (Patient 1) and non-responders.

Purpose: Rhabdoid tumors are highly aggressive pediatric tumors that are usually refractory to available treatments. The purpose of this study was to evaluate the therapeutic potential of two oncolytic viruses, myxoma virus (MV) and an attenuated vesicular stomatitis virus (VSVΔM51), in experimental models of human rhabdoid tumor.Experimental Design: Four human rhabdoid tumor cell lines were cultured in vitro and treated with live or inactivated control virus. Cytopathic effect, viral gene expression, infectious viral titers, and cell viability were examined at various time points after infection. To study viral oncolysis in vivo, human rhabdoid tumor cells were implanted s.c. in the hind flank or intracranially in CD-1 nude mice and treated with intratumoral (i.t.) or i.v. injections of live or UV-inactivated virus. Viral distribution and effects on tumor size and survival were assessed.Results: All rhabdoid tumor cell lines tested in vitro were susceptible to productive lethal infections by MV and VSVΔM51. I.t. injection of live MV or VSVΔM51 dramatically reduced the size of s.c. rhabdoid tumor xenografts compared with control animals. I.v. administration of VSVΔM51 or i.t. injection of MV prolonged the median survival of mice with brain xenografts compared with controls (VSVΔM51: 25 days versus 21 days, log-rank test, P = 0.0036; MV: median survival not reached versus 21 days, log-rank test, P = 0.0007). Most of the MV-treated animals (4 of 6; 66.7%) were alive and apparently “cured” when the experiment was arbitrarily ended (>180 days).Conclusions: These results suggest that VSVΔM51 and MV could be novel effective therapies against human rhabdoid tumor.

The fold changes in gene expression between the vemurafenib treated and vehicle treated cells for each cell culture condition

Supplemental Figure 1. Expression of T, NK, B and Plasma cell markers by metastatic site. Supplemental Figure 2. Expression of myeloid cell markers by metastatic site and gene expression associated with classical and alternative macrophage activation. Supplemental Figure 3. Baseline and therapy induced changes in the LMM immune landscape. Supplemental Figure 4. Baseline and therapy-mediated changes to the MBM immune landscape. Supplemental Figure 5. Expression of MHC class I and II in melanoma cells from samples with and without DC3s. Supplemental Figure 6. Cell-cell interactions between DC3s and T cells defined by SingleCellR. Supplemental Figure 7. Detection of DC3 cells in clinical samples using multiplexed IHC. Supplemental Figure 8. Validation analysis of samples from melanoma metastases in a publicly available validation dataset. Supplemental Figure 9. Expression of key markers that distinguish the major subsets of myeloid cells. Supplemental Figure 10. Correlation of DC3 gene signature and PC loading for DC3 gene signature in TCGA. Supplemental Figure 11. Overall survival for melanoma patients with LMM metastases whose CSF samples contained high DC3 cells vs. low DC3 cells. Supplemental Figure 12. Correlation of individual genes from the DC3 gene signature to overall survival and their univariate cox proportional-hazards models in TCGA dataset.

Purpose:Melanoma brain metastases (MBM) and leptomeningeal melanoma metastases (LMM) are two different manifestations of melanoma CNS metastasis. Here, we used single-cell RNA sequencing (scRNA-seq) to define the immune landscape of MBM, LMM, and melanoma skin metastases.Experimental Design:scRNA-seq was undertaken on 43 patient specimens, including 8 skin metastases, 14 MBM, and 19 serial LMM specimens. Detailed cell type curation was performed, the immune landscapes were mapped, and key results were validated by IHC and flow cytometry. Association analyses were undertaken to identify immune cell subsets correlated with overall survival.Results:The LMM microenvironment was characterized by an immune-suppressed T-cell landscape distinct from that of brain and skin metastases. An LMM patient with long-term survival demonstrated an immune repertoire distinct from that of poor survivors and more similar to normal cerebrospinal fluid (CSF). Upon response to PD-1 therapy, this extreme responder showed increased levels of T cells and dendritic cells in their CSF, whereas poor survivors showed little improvement in their T-cell responses. In MBM patients, therapy led to increased immune infiltrate, with similar T-cell transcriptional diversity noted between skin metastases and MBM. A correlation analysis across the entire immune landscape identified the presence of a rare population of dendritic cells (DC3) that was associated with increased overall survival and positively regulated the immune environment through modulation of activated T cells and MHC expression.Conclusions:Our study provides the first atlas of two distinct sites of melanoma CNS metastases and defines the immune cell landscape that underlies the biology of this devastating disease.

Supplemental Table 2: Sample and Patient Clinical Correlative Information

Supplemental Table 4: top genes that differentiate each cluster of myeloid cells

Purpose:The development of leptomeningeal melanoma metastases (LMM) is a rare and devastating complication of the late-stage disease, for which no effective treatments exist. Here, we performed a multi-omics analysis of the cerebrospinal fluid (CSF) from patients with LMM to determine how the leptomeningeal microenvironment shapes the biology and therapeutic responses of melanoma cells.Experimental Design:A total of 45 serial CSF samples were collected from 16 patients, 8 of these with confirmed LMM. Of those with LMM, 7 had poor survival (35 months). CSF samples were analyzed by mass spectrometry and incubated with melanoma cells that were subjected to RNA sequencing (RNA-seq) analysis. Functional assays were performed to validate the pathways identified.Results:Mass spectrometry analyses showed the CSF of most patients with LMM to be enriched for pathways involved in innate immunity, protease-mediated damage, and IGF-related signaling. All of these were anticorrelated in the extraordinary responder. RNA-seq analysis showed CSF to induce PI3K/AKT, integrin, B-cell activation, S-phase entry, TNFR2, TGFβ, and oxidative stress responses in the melanoma cells. ELISA assays confirmed that TGFβ expression increased in the CSF of patients progressing with LMM. CSF from poorly responding patients conferred tolerance to BRAF inhibitor therapy in apoptosis assays.Conclusions:These analyses identified proteomic/transcriptional signatures in the CSF of patients who succumbed to LMM. We further showed that the CSF from patients with LMM has the potential to modulate BRAF inhibitor responses and may contribute to drug resistance.See related commentary by Glitza Oliva and Tawbi, p. 2083

Supplemental Table 1: Flow Cytometry Antibodies

Supplemental Table 3: top genes that differentiate each cluster of T/NK cells

Supplementary Figure S1 from Oncolytic Efficacy of Recombinant Vesicular Stomatitis Virus and Myxoma Virus in Experimental Models of Rhabdoid Tumors

Legend for Supplementary Figures S1-S10.

Immunophenotyping of the K1492 glioma microenvironment in response to sterile injury (S1); Immunophenotyping of the K1492 glioma microenvironment after Myxoma virus treatment (S2); Virus infected areas of K1492 tumours are accompanied by polymorphonuclear cell infiltration (S3); K1861 and Spontaneous NPcis gliomas in C57Bl/6 mice have significant microglia and/or GIM recruitment (S4); Non-tumour bearing CCR2-deficient animals are only slightly impaired in clearing MYXV (S5); CCR2-deficient mice have an exaggerated recruitment of NK and T cells to the K1492 glioma in response to Myxoma treatment (S6); Minocycline administration mimics CCR2-null viral clearance kinetics but does not result in a survival advantage (S7); K1492 tumours in IL2Rγ mice are significantly depleted of NK and T cell populations (S8); Non-tumour bearing IL2Rγ-deficient animals are impaired in clearing MYXV (S9); Single low-dose cyclophosphamide combined with Myxoma treatment did result in improved treatment efficacy or viral infection (S10).

Supplementary Figure Legends 1-7 from Myxoma Virus Virotherapy for Glioma in Immunocompetent Animal Models: Optimizing Administration Routes and Synergy with Rapamycin

We have shown previously the oncolytic potential of myxoma virus in a murine xenograft model of human glioma. Here, we show that myxoma virus used alone or in combination with rapamycin is effective and safe when used in experimental models of medulloblastoma in vitro and in vivo. Nine of 10 medulloblastoma cell lines tested were susceptible to lethal myxoma virus infection, and pretreatment of cells with rapamycin increased the extent of in vitro oncolysis. Intratumoral injection of live myxoma virus when compared with control inactivated virus prolonged survival in D341 and Daoy orthotopic human medulloblastoma xenograft mouse models [D341 median survival: 21 versus 12.5 days; P = 0.0008; Daoy median survival: not reached (three of five mice apparently “cured” after 223 days) versus 75 days; P = 0.0021]. Rapamycin increased the extent of viral oncolysis, “curing” most Daoy tumor-bearing mice and reducing or eliminating spinal cord and ventricle metastases. Rapamycin enhanced tumor-specific myxoma virus replication in vivo and prolonged survival of D341 tumor-bearing mice (median survival of mice treated with live virus (LV) and rapamycin, versus LV alone, versus rapamycin alone, versus inactivated virus: 25 days versus 19, 13, and 11 days, respectively; P < 0.0001). Rapamycin increased the levels of constitutively activated Akt in Daoy and D341 cells, which may explain its ability to enhance myxoma virus oncolysis. These observations suggest that myxoma virus may be an effective oncolytic agent against medulloblastoma and that combination therapy with signaling inhibitors that modulate activity of the phosphatidylinositol 3-kinase/Akt pathway will further enhance the oncolytic potential of myxoma virus. [Cancer Res 2007;67(18):8818–27]

Supplementary Figures 1-7 from Myxoma Virus Virotherapy for Glioma in Immunocompetent Animal Models: Optimizing Administration Routes and Synergy with Rapamycin

Supplementary Figure 1 from Targeting Human Medulloblastoma: Oncolytic Virotherapy with Myxoma Virus Is Enhanced by Rapamycin

Supplementary Figure 1 Legend from Targeting Human Medulloblastoma: Oncolytic Virotherapy with Myxoma Virus Is Enhanced by Rapamycin

Background: Breast cancer patients with brain metastases have a high unmet clinical need and improved management strategies are needed. There has been interest in studying CDK 4/6 inhibitors in the management of breast cancer brain metastases. A phase II study has shown abemaciclib to have activity in the management of hormone receptor (HR)+/HER2- brain metastases. Pre-clinical data suggests a potential synergy with CDK inhibitors and radiation therapy. Stereotactic radiosurgery (SRS) is a cornerstone in the management of limited brain metastases. We hypothesize treatment with abemaciclib and SRS will be safe and improve intracranial progression free survival (PFS) compared to abemaciclib alone. Trial Design: The study is designed as a prospective, single-arm, nonrandomized, open-label, phase I/II trial of abemaciclib and endocrine therapy with SRS among patients with HR+/HER2- metastatic breast cancer brain metastases. Treatment will be initiated with one week of abemaciclib followed by stereotactic radiation to sites of brain metastases or post-operative cavities with continued abemaciclib. Safety will be monitored initially by a 3+3 design. If unexpected neurologic toxicities are noted, the dose of radiation therapy will be reduced. This will be followed by a phase II study to evaluate intracranial PFS. Eligibility: Eligible patients include those that are HR+/HER2-, ≥18, ECOG ≤2 with ≤15 breast cancer brain metastases with measurable disease per Response Assessment in Neuro-Oncology Brain Metastases (RANO-BM) criteria. Specific Aims: The primary objective of the phase I study is to evaluate the safety and feasibility of abemaciclib and SRS to sites of brain metastases in the management of HR+/HER2- metastatic breast cancer with brain metastases. The primary objective of the phase II portion is to determine PFS intracranially. Secondary objectives include evaluation of extracranial PFS, local and distant intracranial control, and overall survival. Statistical Methods: Safety and feasibility will be monitored in the phase I study using a 3 + 3 design followed by a phase II study to assess intracranial PFS. The phase II study is designed as a single-arm, two-stage trial using the Restricted-Kwak-and-Jung’s method. In the first stage, a total of 21 patients will be enrolled. If pre-specified endpoints are met, an additional 10 patients will be enrolled in the second stage. Patient Accrual: A total of up to 31 patients will be enrolled inclusive of patients in the phase I portion treated at the recommended phase II dose. Clinical trial information: NCT04923542. Citation Format: Kamran A. Ahmed, Youngchul Kim, Michelle DeJesus, Sasha J. Beyer, Nicole O. Williams, Joshua Palmer, Kristina D. Woodhouse, Rashmi K. Murthy, Jing Li, Avan J. Armaghani, John A. Arrington, Ricardo L. Costa, Brian J. Czerniecki, Arnold B. Etame, Peter A. Forsyth, Hung T. Khong, Daniel E. Oliver, Marilin Rosa, Solmaz Sahebjam, Hatem H. Soliman, Aixa E. Soyano, Michael A. Vogelbaum, Michael Yu, Hyo S. Han. Phase I/II study of stereotactic radiation and abemaciclib in the management of hormone receptor positive HER2 negative breast cancer brain metastases [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-09-01.

Transient thermochromic liquid crystal (TLC) experiments can provide high-fidelity spatially resolved heat transfer data for complex geometries, particularly where infrared techniques cannot be applied. One challenge when applying transient methods to internal geometries is the local definition of the driving gas temperature. The transient nature of the streamwise driving gas temperature profile has led to comparisons with steady-state computational fluid dynamics being questioned. This paper explores simulating the temporal behavior of transient TLC experiments directly. A novel technique is developed to account for differences in the gas and solid time scales, where surface temperature is calculated at each spatial location analytically from the surface heat flux, using an impulse response method assuming one dimensional, semi-infinite conduction. Postprocessing of the simulated surface temperature history is performed using the same method as experimentally, allowing for direct comparison. This analytical thermal boundary condition (ATBC) is applied to simulate a transient TLC experiment of a stationary superscaled rib turbulated internal cooling passage in a gas turbine engine. Traditional steady-state simulations were also performed with constant temporal and spatial temperature boundary conditions. Results show that calculations using the new ATBC and traditional steady-state method give very similar Nusselt number distributions and mean values in relation to the experimental data, suggesting the larger discrepancy between simulations and the experiments is not the definition of the driving gas temperature. Analysis of transient variation of Nusselt number indicated brief highly localized maximum variations up to 40%, although this was not found to significantly affect the mean values, and passage-averaged values converged to within 0.5% of the final value within 0.2 s.

The HER3/ERBB3 receptor is an oncogenic receptor tyrosine kinase that forms heterodimers with EGFR family members and is overexpressed in numerous cancers. HER3 overexpression associates with reduced survival and acquired resistance to targeted therapies, making it a potential therapeutic target in multiple cancer types. Here, we report on immunogenic, promiscuous MHC class II–binding HER3 peptides, which can generate HER3-specific CD4+ Th1 antitumor immune responses. Using an overlapping peptide screening methodology, we identified nine MHC class II–binding HER3 epitopes that elicited specific Th1 immune response in both healthy donors and breast cancer patients. Most of these peptides were not identified by current binding algorithms. Homology assessment of amino acid sequence BLAST showed >90% sequence similarity between human and murine HER3/ERBB3 peptide sequences. HER3 peptide–pulsed dendritic cell vaccination resulted in anti-HER3 CD4+ Th1 responses that prevented tumor development, significantly delayed tumor growth in prevention models, and caused regression in multiple therapeutic models of HER3-expressing murine tumors, including mammary carcinoma and melanoma. Tumors were robustly infiltrated with CD4+ T cells, suggesting their key role in tumor rejection. Our data demonstrate that class II HER3 promiscuous peptides are effective at inducing HER3-specific CD4+ Th1 responses and suggest their applicability in immunotherapies for human HER3-overexpressing tumors.

This study assessed the presentation and institutional outcomes treating brain metastases (BM) of breast cancer (BC), non-small cell lung cancer (NSCLC), and melanoma origin. Patients with brain metastases treated between 2014 and 2019 with primary melanoma, NSCLC, and BC were identified. Overall survival (OS) was calculated from dates of initial BM diagnosis using the Kaplan–Meier method. A total of 959 patients were identified including melanoma (31%), NSCLC (51%), and BC (18%). Patients with BC were younger at BM diagnosis (median age: 57) than NSCLC (65) and melanoma patients (62, p

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. scRNA-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.