Cabozantinib Is Effective in Melanoma Brain Metastasis Cell Lines and Affects Key Signaling Pathways

Melanomas have a high potential to metastasize to the brain. Recent advances in targeted therapies and immunotherapies have changed the therapeutical landscape of extracranial melanomas. However, few patients with melanoma brain metastasis (MBM) respond effectively to these treatments and new therapeutic strategies are needed. Cabozantinib is a receptor tyrosine kinase (RTK) inhibitor, already approved for the treatment of non-skin-related cancers. The drug targets several of the proteins that are known to be dysregulated in melanomas. The anti-tumor activity of cabozantinib was investigated using three human MBM cell lines. Cabozantinib treatment decreased the viability of all cell lines both when grown in monolayer cultures and as tumor spheroids. The in vitro cell migration was also inhibited and apoptosis was induced by cabozantinib. The phosphorylated RTKs p-PDGF-Rα, p-IGF-1R, p-MERTK and p-DDR1 were found to be downregulated in the p-RTK array of the MBM cells after cabozantinib treatment. Western blot validated these results and showed that cabozantinib treatment inhibited p-Akt and p-MEK 1/2. Further investigations are warranted to elucidate the therapeutic potential of cabozantinib for patients with MBM.

Arginine deprivation therapy induces apoptotic cell death in melanoma brain metastasis

Aims The development of melanoma brain metastasis (MBM) occurs in ~50% of metastatic melanoma cases, and significantly worsens prognosis to a median survival of 12.8 months. Melanoma is often reported as an arginine auxotroph due to transcriptional silencing of argininosuccinate synthase 1 (ASS1). Arginine deiminase (ADI) is a non-mammalian enzyme which depletes blood arginine by converting it to citrulline and ammonia, and in its pegylated form ADI shows clinical efficacy in the treatment of a number of cancers via exploiting tumour arginine auxotrophy, resulting in targeted arginine deprivation of tumour cells. While cutaneous melanoma is the prototype cancer for this therapy, studies to date have excluded central nervous system metastasis. We have demonstrated that patient derived primary MBM models are sensitive to arginine deprivation in vitro, confirmed suitable clinical biomarkers of sensitivity, and established the mechanism of tumour cell specific cytotoxicity. Method Patient derived primary cultures of MBM were established and subject to treatment with arginine deprivation. Gene expression and methylation analysis was examined by RT-qPCR, western blot, Illumina mRNA sequencing and Illumina methylated DNA immunoprecipitation-sequencing (MeDIP-seq) on ADI treated and untreated samples. Cell death, cytotoxicity induction and caspase-3 and-7 recruitment was analysed using an Incucyte S3 live-cell imager, by fluorescently labelling cells with Incucyte Cytolight Red Rapid dye, Cytotox Green dye and Caspase-3/7 Green dye, and imaging cells every 2 hours over the course of 2 weeks. 3D spheroid growth and invasion was measured by culturing cells as tumour spheroids before treating with ADI, and imaging spheroids every 2 hours for 2 weeks using an Incucyte S3 live-cell imager. Nuclear leakage and mitochondrial morphology was observed by fluorescently staining treated and untreated cells with DAPI and MitoTracker Red, and imaging on a Leica DMi8 confocal microscope. Results Primary MBMs differentially express ASS1 at substantially lower levels than non-cancerous melanocytes, however some models are capable of upregulating ASS1 following confrontation with arginine deprivation. Despite this, long-term sensitivity of primary MBMs to arginine deprivation was observed in both 2D and 3D models. In addition, arginine deprivation was seen to inhibit MBM invasion in a 3D model – an important feature in MBM pathogenesis. Initially, autophagy was induced in arginine deprived MBM, however in all models the induction of cytotoxicity correlated with recruitment of caspase-3 and -7, and intrinsic apoptotic cell death confirmed. Nuclear leakage, and eventually complete nuclear destruction was observed, in addition to mitochondrial fragmentation. Conclusion Arginine deprivation is highly effective in reducing 2D and 3D MBM growth, as well as limiting invasion. While apoptotic cell death was observed in all models, the initial induction of autophagy could pose threat of resistance development in a clinical setting, and so combinational therapies with autophagic inhibitors and/or additional apoptotic inducers should be investigated. It is unclear whether nuclear leakage and mitochondrial degradation are the cause or product of apoptosis. Considering the strong clinical evidence for the use of arginine deprivation in non-CNS metastatic melanoma and the results of this study, arginine deprivation is a highly suitable treatment for pre-surgical MBM to limit invasion and increase resection, and for post-surgical continuation.

Phytogalactolipid dLGG Inhibits Mouse Melanoma Brain Metastasis through Regulating Oxylipin Activity and Re-Programming Macrophage Polarity in the Tumor Microenvironment

Current conventional cancer therapies for melanoma brain metastasis (MBM) remain ineffective. In this study, we demonstrated the bioefficacy of a phyto-glyceroglycolipid, 1,2-di-O-α-linolenoyl-3-O-β-galactopyranosyl-sn-glycerol (dLGG) alone, or in combination with liposomal doxorubicin (Lip-DOX) or Avastin against MBM in a syngeneic B16BM4COX−2/Luc brain-seeking melanoma mouse model. Treatment with dLGG–10, dLGG–25, dLGG–10 + Avastin–5, Lipo-DOX–2, dLGG–10 + Lipo-DOX–2 or Lipo-DOX–2 + Avastin–5 suppressed, respectively, 17.9%, 59.1%, 55.7%, 16.2%, 44.5% and 72.4% of MBM in mice relative to the untreated tumor control. Metastatic PD-L1+ melanoma cells, infiltration of M2-like macrophages and CD31+ endothelial cells, and high expression levels of 15-LOX/CYP450 4A enzymes in the brain tumor microenvironment of the tumor control mice were significantly attenuated in dLGG-treated mice; conversely, M1-like resident microglia and cytotoxic T cells were increased. A lipidomics study showed that dLGG promoted B16BM4 cells to secrete oxylipins 9,10-/12,13-EpOMEs into the culture medium. Furthermore, the conditioned medium of B16BM4 cells pretreated with dLGG or 9,10-EpOMEs + 12,13-EpOMEs drove M2-like macrophages to polarize into M1-like macrophages in vitro. An ex vivo 3D-culture assay further demonstrated that dLGG, 9,10-EpOME or 9,10-EpOME + 12,13-EpOME pretreatment attenuated B16BM4 cells invading brain tissue, and prevented microglia/macrophages infiltrating into the interface of melanoma plug and brain organ/tissue. In summary, this report provides a novel therapeutic strategy and mechanistic insights into phytogalactolipid dLGG for combating MBM.

Is radiation necrosis in radiated melanoma brain metastasis increasing because immunotherapy is contributing to this or are patients just living longer?

e21518 Background: The use of immune checkpoint inhibitors, particularly combination ipilimumab and nivolumab, has drastically changed the management of patients with melanoma brain metastasis. Select patients also benefit from brain-directed stereotactic radiation. Radiation necrosis is a risk associated with stereotactic radiation that oncologists have been observing more frequently in the era of immunotherapy. Methods: Patients were identified who had a history of metastatic melanoma treated with stereotactic brain radiation who subsequently developed radiation necrosis. Brain tissue from those patients with a subsequent resection of their radiation necrosis was obtained and examined for immune infiltrate and other factors. The tissue obtained was evaluated by blinded pathologists who graded % viable tissue, % necrosis, % tumor and % fibrosis. In addition, they graded inflammation on a scale of 1-3. Results: Seven patients were identified who had surgery for radiation necrosis following radiation to melanoma brain metastasis. Tissue was available for five patients. Two patients had received no prior immunotherapy, one patient had received ipilimumab and two patients received combination ipilimumab and nivolumab. The samples obtained consisted of almost entirely viable brain tissue or necrosis. There was minimal inflammation seen in all patients’ samples including those who had not received immunotherapy and those who had. Conclusions: Radiation necrosis in patients on immunotherapy who receive brain-directed stereotactic radiation is a rising problem. On pathologic evaluation increased immune infiltrate is not observed in patients on immunotherapy with radiation necrosis compared to those who never received immunotherapy. This suggests that the increased rates of radiation necrosis may be more likely associated with longer survival as opposed to a direct causative effect from the immunotherapy although with our limited sample size this will need further exploration.[Table: see text]