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The melanoma‐microglia cross‐talk upregulates the expression of aldolase C (ALDOC) in melanoma cells from several patients. The response of melanoma cells from 2 different patients to ALDOC upregulation was diametrically divergent. Whereas several metastasis‐associated functions including brain metastasis formation were augmented in one melanoma cell line (Mel 1), these functions were attenuated in the other melanoma cell line (Mel 2). Previous studies indicated that microglia cells upregulate the expression of aldolase C (ALDOC) in melanoma cells. The present study using brain‐metastasizing variants from three human melanomas explores the functional role of ALDOC in the formation and maintenance of melanoma brain metastasis (MBM). ALDOC overexpression impacted differentially the malignant phenotype of these three variants. In the first variant, ALDOC overexpression promoted cell viability, adhesion to and transmigration through a layer of brain endothelial cells, and amplified brain micrometastasis formation. The cross‐talk between this MBM variant and microglia cells promoted the proliferation and migration of the latter cells. In sharp contrast, ALDOC overexpression in the second brain‐metastasizing melanoma variant reduced or did not affect the same malignancy features. In the third melanoma variant, ALDOC overexpression augmented certain characteristics of malignancy and reduced others. The analysis of biological functions and disease pathways in the ALDOC overexpressing variants clearly indicated that ALDOC induced the expression of tumor progression promoting genes in the first variant and antitumor progression properties in the second variant. Overall, these results accentuate the complex microenvironment interactions between microglia cells and MBM, and the functional impact of intertumor heterogeneity. Since intertumor heterogeneity imposes a challenge in the planning of cancer treatment, we propose to employ the functional response of tumors with an identical histology, to a particular drug or the molecular signature of this response, as a predictive indicator of response/nonresponse to this drug.

Alzheimer’s disease (AD) is the most prevalent cause of dementia in adults. Current available drugs for AD transiently alleviate some of the symptoms, but do not modify the disease mechanism or cure it. Therefore, new drugs are desperately needed. Key contributors to AD are amyloid beta (Aβ)- and reactive oxygen species (ROS)-induced cytotoxicities. Plant-derived substances have been shown to affect various potential targets in various diseases including AD. Therefore, phytochemicals which can protect neuronal cells against these insults might help in preventing and treating this disease. In the following research, we have isolated the sesquiterpene lactone achillolide A from the plant Achillea fragrantissima and, for the first time, characterized its effects on Aβ-treated neuroblastoma cells. Aβ is a peptide derived from the sequential cleavage of amyloid precursor protein, and is part of the pathogenesis of AD. Our current study aimed to determine whether achillolide A can interfere with Aβ-induced processes in Neuro2a cells, and protect them from its toxicity. Our results show that achillolide A decreased Aβ-induced death and enhanced the viability of Neuro2a cells. In addition, achillolide A reduced the accumulation of Aβ-induced ROS and inhibited the phosphorylation of stress-activated protein kinase/c-Jun N-terminal kinase and p44/42 mitogen-activated protein kinase in these cells. We therefore suggest that achillolide A may have therapeutic potential for the treatment of AD.

Achillolide A is a natural sesquiterpene lactone that we have previously shown can inhibit microglial activation. In this study we present evidence for its beneficial effects on astrocytes under oxidative stress, a situation relevant to neurodegenerative diseases and brain injuries. Viability of brain astrocytes (primary cultures) was determined by lactate dehydrogenase (LDH) activity, intracellular ROS levels were detected using 2′,7′-dichlorofluorescein diacetate, in vitro antioxidant activity was measured by differential pulse voltammetry, and protein phosphorylation was determined using specific ELISA kits. We have found that achillolide A prevented the H2O2-induced death of astrocytes, and attenuated the induced intracellular accumulation of reactive oxygen species (ROS). These activities could be attributed to the inhibition of the H2O2-induced phosphorylation of MAP/ERK kinase 1 (MEK1) and p44/42 mitogen-activated protein kinases (MAPK), and to the antioxidant activity of achillolide A, but not to H2O2 scavenging. This is the first study that demonstrates its protective effects on brain astrocytes, and its ability to interfere with MAPK activation. We propose that achillolide A deserves further evaluation for its potential to be developed as a drug for the prevention/treatment of neurodegenerative diseases and brain injuries where oxidative stress is part of the pathophysiology.

Disclosure: A. Chmelnik: None. A. Telerman: None. A. Tirosh: None. Introduction - Pancreatic neuroendocrine tumors (PanNENs) may develop sporadically or as part of an inherited disease, such as von Hippel-Lindau (VHL). VHL disease is caused by a germline pathogenic variant in the VHL gene encoding VHL protein (pVHL). Hypoxia inducible factor (HIF) is responsible for cellular oxygen supply. Its degradation is mediated by pVHL via ubiquitination in normoxic states. In pVHL-deficient state leads to HIF overexpression and pseudohypoxia. Several studies suggested immunomodulatory role for HIF in kidney cancer. Aims- To assess the impact of pseudohypoxia on PanNEN immune tumor microenvironment (iTME). Methods- A total of 16 vPanNEN and 23 sPanNEN were processed. Whole genome DNA methylation was performed by Illumine Infinium EPIC Array and analyzed using ChAMP on RStudio. Immune cells composition was compared using methylation-based deconvolution algorithms (MethylResolver, Robust Partial Correlations, Constrained Projection and Cibersort), and gene set enrichment analysis identified pathways affected in each group based on promoter methylation analysis. Results- Unsupervised hierarchical analysis of immune-related gene promoter methylation demonstrated separation of vPanNEN vs. sPanNEN. In GSEA analysis methylation was altered in immune memory-related genes in vPanNEN, and in T cells receptors-related genes in sPanNEN. Deconvolution algorithms revealed a lower fraction of CD4 and NK cells (p<0.05) and a lower PD-L1 expression in vPanNENs (p=0.037). Conclusion- Pseudohypoxic vPanNEN have distinct iTME than sPanNEN, in immune cells composition and PD-L1 expression. Presentation: Thursday, June 15, 2023

Background Alzheimer’s disease is a neurodegenerative disease, characterized by progressive decline in memory and cognitive functions, that results from loss of neurons in the brain. Amyloid beta (Aβ) protein and oxidative stress are major contributors to Alzheimer’s disease, therefore, protecting neuronal cells against Aβ-induced toxicity and oxidative stress might form an effective approach for treatment of this disease. 3,5,4′-trihydroxy-6,7,3′-trimethoxyflavone (TTF) is a flavonoid we have purified from the plant Achillea fragrantissima; and the present study examined, for the first time, the effects of this compound on Aβ-toxicity to neuronal cells. Methods Various chromatographic techniques were used to isolate TTF from the plant Achillea fragrantissima, and an N2a neuroblastoma cell line was used to study its activities. The cellular levels of total and phosphorylated stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and of total and phosphorylated extracellular signal-regulated kinase (ERK 1/2) were determined by enzyme-linked immunosorbent assay (ELISA). Intracellular reactive oxygen species (ROS) levels were measured by using 2′,7′-dichlorofluorescein diacetate (DCF-DA). Cytotoxicity and cell viability were assessed by using lactate dehydrogenase (LDH) activity in cell-conditioned media, or by crystal violet cell staining, respectively. Results TTF prevented the Aβ-induced death of neurons and attenuated the intracellular accumulation of ROS following treatment of these cells with Aβ. TTF also inhibited the Aβ-induced phosphorylation of the signaling proteins SAPK/JNK and ERK 1/2, which belong to the mitogen-activated protein kinase (MAPK) family. Conclusion TTF should be studied further as a potential therapeutic means for the treatment of Alzheimer’s disease.

Disclosure: A. Telerman: None. Y. Yossef: None. A. Chmelnik: None. A. Tirosh: None. Background: Von Hippel-Lindau (VHL) disease is a familial cancer syndrome caused by a germline mutation in the VHL tumor suppressor gene. Although VHL-related pancreatic neuroendocrine neoplasms (vPNEN) have been studied, their molecular pathogenesis is not fully understood. Aims: To generate a mouse model for studying the mechanisms that promote vPNEN development in vivo. Methods: We induced a frameshift mutation in VHL using the CRISPR/Cas9 technique in the BON1 cell line, originating from high-grade PNEN (FS-BON1). We validated the pseudohypoxic nature of the cells by real-time polymerase chain reaction of VEGF and EPO in FS-BON1 compared to VHL wild-type BON1 (WT-BON1). We compared cell line-derived xenografts (CDXs) growth in athymic Nude-Foxn1nu female mice injected with WT-BON1 (n=9), FS-BON1 (n=9) and media (purified bovine serum, n=5). Tumor diameter and calculated volume and mice weight were recorded weekly. Plasma chromogranin A (CgA) levels were measured in mice sera by ELISA. Results: FS-BON1 showed upregulation of VEGF and EPO compared with WT-BON1, confirming their pseudohypoxic microenvironment. At 14 weeks, 0/9 FS-BON1 CDX reached a volume of 1 cm3 compared with 5/9 of the WT-BON1 group (p=0.03). In time-dependent analysis, FS-BON1-derived xenografts grew significantly slower than WT-BON1 xenografts (p=0.02). No significant differences in CgA serum levels were found between the mice groups. Conclusions: We report a new pseudohypoxic in vivo PNEN model. The pVHL-deficient cells show an unexpected indolent course, suggesting it as a new model for low-grade PNEN. This CDX mouse model can be utilized in further studies to evaluate the mechanism responsible for vPNEN development and for evaluating interventions in low-grade PNEN. Presentation: Thursday, June 15, 2023

Cardiovascular complications are increasingly reported with the use of certain tyrosine kinase inhibitors (TKIs) to treat chronic myeloid leukemia (CML). We studied neutrophil extracellular trap (NET) formation in CML and evaluated the effect of TKIs on NET formation. Neutrophils isolated from treatment-naïve patients with CML showed a significant increase in NET formation compared to matched controls at baseline and after stimulation with ionomycin (IO) and phorbol 12-myristate 13-acetate (PMA). Expression of citrullinated histone H3 (H3cit), peptidyl arginine deiminase 4 (PAD4) and reactive oxygen species (ROS) was significantly higher in CML samples compared to controls. Pre-treatment of neutrophils with TKIs was associated with a differential effect on NET formation, and ponatinib significantly augmented NET-associated elastase and ROS levels as compared to controls and other TKIs. BCR-ABL1 retroviral transduced HoxB8-immortalized mouse hematopoietic progenitors, which differentiate into neutrophils in-vitro, demonstrated increased H3cit & myeloperoxidase (MPO) expression consistent with excess NET formation. This was inhibited by Cl-amidine, a PAD4 inhibitor, but not by the NADPH inhibitor diphenyleneiodonium (DPI). Ponatinib pre-exposure significantly increased H3cit expression in HoxB8-BCR-ABL1 cells after stimulation with IO. In summary, CML is associated with increased NET formation, which is augmented by ponatinib, suggesting a possible role for NETs in promoting vascular toxicity in CML.

Glutamate toxicity is a major contributor to the pathophysiology of numerous neurodegenerative diseases including amyotrophic lateral sclerosis and Alzheimer’s disease. Therefore, protecting neuronal cells against glutamate-induced cytotoxicity might be an effective approach for the treatment of these diseases. We have previously purified from the medicinal plant Achillea fragrantissima two bioactive compounds which were not studied before: the sesquiterpene lactone achillolide A and the flavonoid 3,5,4′-trihydroxy-6,7,3′-trimethoxyflavone (TTF). We have shown that these compounds protect astrocytes from oxidative stress-induced cell death and inhibit microglial activation. The current study examined for the first time their effects on differentiated mouse neuroblastoma N2a cells and on glutamate toxicity. We have found that, although these compounds belong to different chemical families, they protect neuronal cells from glutamate toxicity. We further demonstrate that this protective effect might be, at least partially, due to inhibitory effects of these compounds on the levels of reactive oxygen species produced following treatment with glutamate.

The development of melanoma brain metastasis is largely dependent on mutual interactions between the melanoma cells and cells in the brain microenvironment. Here, we report that the extracellular cysteine protease inhibitor cystatin C (CysC) is involved in these interactions. Microglia-derived factors upregulated CysC secretion by melanoma. Similarly, melanoma-derived factors upregulated CysC secretion by microglia. Whereas CysC enhanced melanoma cell migration through a layer of brain endothelial cells, it inhibited the migration of microglia cells toward melanoma cells. CysC was also found to promote the formation of melanoma three-dimensional structures in matrigel. IHC analysis revealed increased expression levels of CysC in the brain of immune-deficient mice bearing xenografted human melanoma brain metastasis compared to the brain of control mice. Based on these in vitro and in vivo experiments we hypothesize that CysC promotes melanoma brain metastasis. Increased expression levels of CysC were detected in the regenerating brain of mice after stroke. Post-stroke brain with melanoma brain metastasis showed an even stronger expression of CysC. The in vitro induction of stroke-like conditions in brain microenvironmental cells increased the levels of CysC in the secretome of microglia cells, but not in the secretome of brain endothelial cells. The similarities between melanoma brain metastasis and stroke with respect to CysC expression by and secretion from microglia cells suggest that CysC may be involved in shared pathways between brain metastasis and post-stroke regeneration. This manifests the tendency of tumor cells to highjack physiological molecular pathways in their progression.

Abstract Background: The neuroinflammatory process plays a central role in the initiation and progression of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases, and involves the activation of brain microglial cells. During the neuroinflammatory process, microglial cells release proinflammatory mediators such as cytokines, matrix metalloproteinases (MMP), Reactive oxygen species (ROS) and nitric oxide (NO). In the present study, extracts from 66 different desert plants were tested for their effect on lipopolysaccharide (LPS) - induced production of NO by primary microglial cells. The extract of Achillea fragrantissima (Af ), which is a desert plant that has been used for many years in traditional medicine for the treatment of various diseases, was the most efficient extract, and was further studied for additional anti-neuroinflammatory effects in these cells. Methods: In the present study, the ethanolic extract prepared from Af was tested for its anti-inflammatory effects on lipopolysaccharide (LPS)-activated primary cultures of brain microglial cells. The levels of the proinflammatory cytokines interleukin1β (IL-1β) and tumor necrosis factor-α (TNFα) secreted by the cells were determined by reverse transcriptase-PCR and Enzyme-linked immunosorbent assay (ELISA), respectively. NO levels secreted by the activate cells were measured using Griess reagent, ROS levels were measured by 2'7'-dichlorofluorescein diacetate (DCF-DA), MMP-9 activity was measured using gel zymography, and the protein levels of the proinflammatory enzymes cyclooxygenase-2 (COX-2) and induced nitric oxide synthase (iNOS) were measured by Western blot analysis. Cell viability was assessed using Lactate dehydrogenase (LDH) activity in the media conditioned by the cells or by the crystal violet cell staining. Results: We have found that out of the 66 desert plants tested, the extract of Af was the most efficient extract and inhibited ~70% of the NO produced by the LPS-activated microglial cells, without affecting cell viability. In addition, this extract inhibited the LPS - elicited expression of the proinflammatory mediators IL-1β, TNFα, MMP-9, COX-2 and iNOS in these cells. Conclusions: Thus, phytochemicals present in the Af extract could be beneficial in preventing/treating neurodegenerative diseases in which neuroinflammation is part of the pathophysiology.