Anti-melanoma Activity Research Articles

Anti-melanoma effect of ruthenium(II)-diphosphine complexes containing naphthoquinone ligand

The use of natural products as potential ligands has been explored as a strategy in the development of metal-based chemotherapy. Since ruthenium complexes are promising alternatives to traditional antitumor agents, this study evaluated the anti-melanoma potential of two ruthenium(II) complexes containing the naphthoquinone ligands lapachol (lap), [Ru(lap)(dppm)2]PF6, and lawsone (law), [Ru(law)(dppm)2]PF6, in addition to the bis(diphenylphosphino)methane (dppm) ligand, referred to as complexes (1) and (2), respectively, using a syngeneic murine melanoma model. Activation of the apoptotic pathway by the treatments was assessed by immunohistochemistry in tumor tissue. Additionally, toxicity of the treatments was evaluated by variation in body and organ weight, quantification of biochemical indicators of renal damage, and genotoxicity in bone marrow and hepatocytes. First, the antiproliferative activity of (1) and (2) was observed in B16F10 cells, with IC50 values of 2.78 and 1.68 μM, respectively. The results obtained in mice showed that, unlike complex (1), (2) possesses significant anti-melanoma activity demonstrated by a reduction in tumor volume and mass (88.42%), as well as in mitosis frequency (83.86%). Additionally, complex (2) increased the levels of cleaved caspase-3, inducing tumor cell apoptosis. When compared to the metallodrug cisplatin, complex (2) exhibited similar anti-melanoma activity and lower toxicity considering all parameters evaluated. In silico studies demonstrated no difference in the binding energy of the naphthoquinone complex between complexes (1) and (2). However, the complex containing the lawsone ligand has a lower molar volume, which may be important for interactions with minor DNA grooves. The present results demonstrate the antitumor efficiency of complex (2) and a significantly lower systemic toxicity compared to cisplatin.

Stereoselective Access to Antimelanoma Agents by Hybridization and Dimerization of Dihydroartemisinin and Artesunic acid.

A library of five hybrids and six dimers of dihydroartemisinin and artesunic acid has been synthetized in a stereo‐controlled manner and evaluated for the anticancer activity against metastatic melanoma cell line (RPMI7951). Among novel derivatives, three artesunic acid dimers showed antimelanoma activity and cancer selectivity, being not toxic on normal human fibroblast (C3PV) cell line. Among the three dimers, the one bearing 4‐hydroxybenzyl alcohol as a spacer showed no cytotoxic effect (CC50>300 μM) and high antimelanoma activity (IC50=0.05 μM), which was two orders of magnitude higher than that of parent artesunic acid, and of the same order of commercial drug paclitaxel. In addition, this dimer showed cancer‐type selectivity towards melanoma compared to prostate (PC3) and breast (MDA‐MB‐231) tumors. The occurrence of a radical mechanism was hypothesized by DFO and EPR analyses. Qualitative structure activity relationships highlighted the role of artesunic acid scaffold in the control of toxicity and antimelanoma activity.

Structures and anti-melanoma activities of two polysaccharides from Angelica sinensis (Oliv.) Diels

Polysaccharide is one of the necessary macromolecules in life activities, and it is also a very promising natural product for tumor prevention and treatment. In this study, two homogeneous polysaccharides (APS-4I and APS-4II) were isolated from Angelica sinensis (Oliv.) Diels. APS-4I was a linear glucan with molecular weight of 16.1 kDa, which was composed of 88.4% α-1,6-Glcp, 4.1% α-1,2-Glcp, 3.9% α-1,3-Glcp, and 2.8% α-T-Glcp. APS-4II was a novel polysaccharide with molecular weight of 11.1 kDa, which consisted of 55.4% α-1,6-Glcp, 10.4% α-1,3,5-Araf, 8.7% α-T-Araf, 9.2% α-1,5-Araf, 4.0% α-1,3-Araf, 3.6% α-1,4-Galp, and 9.1% β-1,3-Galp. NMR results demonstrated that APS-4II has a backbone composed of →6)-α-Glcp-(1 → 6)-α-Glcp-(1 → 5)-α-Araf-.(1 → 5)-α-Araf-(1 → 3,5)-α-Araf-(1 → 3)-β-Galp-(1 → 3)-β-Galp-(1 → 4)-α-Galp-(1 → 3)-α-Araf-(1 → 3,5)-α-Araf-(1→. Both APS-4I and APS-4II inhibited the tumor growth of B16-bearing mice, and the suppressive effect of APS-4II reached 64.7 ± 7.3%. Meanwhile, there were higher lymphocyte numbers and the levels of IL-2, IFN-γ, and TNF-α in peripheral blood of APS-4II-treated mice than those in APS-4I-treated mice. Furthermore, APS-4II showed a higher inhibitory effect on the proliferation of B16 cells and stronger promoting effects on the proliferation of splenocytes, the phagocytosis of peritoneal macrophages, and the cytotoxicity of NK cells. These results demonstrated that APS-4II could be a promising therapeutic agent for melanoma.

Bufadienolides from the Eggs of the Toad Bufo bufo gargarizans and Their Antimelanoma Activities.

Toads produce potent toxins, named bufadienolides, to defend against their predators. Pharmacological research has revealed that bufadienolides are potential anticancer drugs. In this research, we reported nine bufadienolides from the eggs of the toad Bufo bufo gargarizans, including two new compounds (1 and 3). The chemical structures of 1 and 3, as well as of one previously reported semisynthesized compound (2), were elucidated on the basis of extensive spectroscopic data interpretation, chemical methods, and X-ray diffraction analysis. Compound 1 is an unusual 19-norbufadienolide with rearranged A/B rings. A biological test revealed that compounds 2 and 4-8 showed potent cytotoxic activities toward human melanoma cell line SK-MEL-1 with IC50 values less than 1.0 μM. A preliminary mechanism investigation revealed that the most potent compound, 8, could induce apoptosis via PARP cleavage, while 5 and 6 significantly suppressed angiogenesis in zebrafish. Furthermore, an in vivo biological study showed that 5, 6, and 8 inhibit SK-MEL-1 cell growth significantly.

IL-1α Mediates Innate and Acquired Resistance to Immunotherapy in Melanoma.

Inflammation has long been associated with cancer initiation and progression; however, how inflammation causes immune suppression in the tumor microenvironment and resistance to immunotherapy is not well understood. In this study, we show that both innate proinflammatory cytokine IL-1α and immunotherapy-induced IL-1α make melanoma resistant to immunotherapy. In a mouse melanoma model, we found that tumor size was inversely correlated with response to immunotherapy. Large tumors had higher levels of IL-1α, Th2 cytokines, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), and regulatory T cells but lower levels of IL-12, Th1 cytokines, and activated T cells. We found that therapy with adenovirus-encoded CD40L (rAd.CD40L) increased tumor levels of IL-1α and PMN-MDSCs. Blocking the IL-1 signaling pathway significantly decreased rAd.CD40L-induced PMN-MDSCs and their associated PD-L1 expression in the tumor microenvironment and enhanced tumor-specific immunity. Similarly, blocking the IL-1 signaling pathway improved the antimelanoma activity of anti-PD-L1 Ab therapy. Our study suggests that blocking the IL-1α signaling pathway may increase the efficacy of immunotherapies against melanoma.

Ca2+ overload- and ROS-associated mitochondrial dysfunction contributes to \u03b4-tocotrienol-mediated paraptosis in melanoma cells

Melanoma is an aggressive tumor with still poor therapy outcomes. δ-tocotrienol (δ-TT) is a vitamin E derivative displaying potent anti-cancer properties. Previously, we demonstrated that δ-TT triggers apoptosis in human melanoma cells. Here, we investigated whether it might also activate paraptosis, a non-canonical programmed cell death. In accordance with the main paraptotic features, δ-TT was shown to promote cytoplasmic vacuolization, associated with endoplasmic reticulum/mitochondrial dilation and protein synthesis, as well as MAPK activation in A375 and BLM cell lines. Moreover, treated cells exhibited a significant reduced expression of OXPHOS complex I and a marked decrease in oxygen consumption and mitochondrial membrane potential, culminating in decreased ATP synthesis and AMPK phosphorylation. This mitochondrial dysfunction resulted in ROS overproduction, found to be responsible for paraptosis induction. Additionally, δ-TT caused Ca2+ homeostasis disruption, with endoplasmic reticulum-derived ions accumulating in mitochondria and activating the paraptotic signaling. Interestingly, by using both IP3R and VDAC inhibitors, a close cause-effect relationship between mitochondrial Ca2+ overload and ROS generation was evidenced. Collectively, these results provide novel insights into δ-TT anti-melanoma activity, highlighting its ability to induce mitochondrial dysfunction-mediated paraptosis.Graphic δ-tocotrienol induces paraptotic cell death in human melanoma cells, causing endoplasmic reticulum dilation and mitochondrial swelling. These alterations induce an impairment of mitochondrial function, ROS production and calcium overload.

CYP27A1-dependent anti-melanoma activity of limonoid natural products targets mitochondrial metabolism

Three limonoid natural products with selective anti-proliferative activity against BRAF(V600E) and NRAS(Q61K)-mutation-dependent melanoma cell lines were identified. Differential transcriptome analysis revealed dependency of compound activity on expression of the mitochondrial cytochrome P450 oxidase CYP27A1, a transcriptional target of melanogenesis-associated transcription factor (MITF). We determined that CYP27A1 activity is necessary for the generation of a reactive metabolite that proceeds to inhibit cellular proliferation. A genome-wide small interfering RNA screen in combination with chemical proteomics experiments revealed gene-drug functional epistasis, suggesting that these compounds target mitochondrial biogenesis and inhibit tumor bioenergetics through a covalent mechanism. Our work suggests a strategy for melanoma-specific targeting by exploiting the expression of MITF target gene CYP27A1 and inhibiting mitochondrial oxidative phosphorylation in BRAF mutant melanomas.

Identification of Piperidine-3-carboxamide Derivatives Inducing Senescence-like Phenotype with Antimelanoma Activities.

This study evaluated the potential use of senescence-inducing small molecules in the treatment of melanoma. We screened commercially available small-molecule libraries with high-throughput screening and high-content screening image-based technology. Our findings showed an initial hit with the embedded N-arylpiperidine-3-carboxamide scaffold-induced senescence-like phenotypic changes in human melanoma A375 cells without serious cytotoxicity against normal cells. A focused library containing diversely modified analogues were constructed and examined to evaluate the structure-activity relationship of N-arylpiperidine-3-carboxamide derivatives starting from hit 1. This work identified a novel compound with remarkable antiproliferative activity in vitro and demonstrated the key structural moieties within.

The Effect of Nutmeg Seed (M. fragrans) Extracts Induces Apoptosis in Melanoma Maligna Cell’s (B16-F10)

BACKGROUND: Nutmeg (Myristica fragrans H.), one of native plants of Maluku Indonesia, has long been used as traditional medicines especially to treat tumors, externally to treat skin infections. M. fragrans also has important biological activities as anticancer. However, antimelanoma activity of M. fragrans remains unknown. The aim of this study is to compare M. fragrans extracts as anticancer on melanoma B16-F10 cells by inducing apoptosis.METHODS: M. fragrans seed was extracted with ethanol then fractionated with n-hexane, ethyl acetate, and n-butanol. B16-F10 melanoma cells were cultured and treated with various doses and tested using resazurin reduction assay. Apoptosis signalling via caspase-3 was measured by using western blot.RESULTS: The extract and fractions of M. fragrans reduced viability of cells with IC50 value for ethanol extract 21.83 µg/mL, ethyl acetate fraction 21.66 µg/mL, n-hexane fraction 47.53 µg/mL, and n-butanol fraction >1,000 µg/mL. The active fraction of ethyl acetate induced apoptosis via caspase-3 proteins similar with cisplatin as positive control in B16-F10 cells at 10 hours treatment.CONCLUSION: Taken together, M. fragrans ethyl acetate fraction has the highest IC50 than n-hexane and n-butanol fractions that significantly inhibited B16-F10 cell proliferation by inducing apoptosis via caspase-3. It provides the insight that it has the most potential activity as a chemopreventive agent for addressing melanoma skin cancer.KEYWORDS: M. fragrans, apoptosis, fraction, melanoma

The Glutaminase Inhibitor CB-839 (Telaglenastat) Enhances the Antimelanoma Activity of T-Cell-Mediated Immunotherapies.

Immune-checkpoint inhibitors and adoptive tumor-infiltrating lymphocyte (TIL) therapies have profoundly improved the survival of patients with melanoma. However, a majority of patients do not respond to these agents, and many responders experience disease relapse. Although numerous innovative treatments are being explored to offset the limitations of these agents, novel therapeutic combinations with immunotherapies have the potential to improve patient responses. In this study, we evaluated the antimelanoma activity of immunotherapy combinations with Telaglenastat (CB-839), a potent glutaminase inhibitor (GLSi) that has favorable systemic tolerance. In in vitro TIL:tumor coculture studies, CB-839 treatment improved the cytotoxic activity of autologous TILs on patient-derived melanoma cells. CB-839 treatment decreased the conversion of glutamine to alpha-ketoglutarate (αKGA) more potently in tumor cells versus TILs in these cocultures. These results suggest that CB-839 may improve immune function in a tumor microenvironment by differentially altering tumor and immune cell metabolism. In vivo CB-839 treatment activated melanoma antigen-specific T cells and improved their tumor killing activity in an immune-competent mouse model of adoptive T-cell therapy. Additionally, the combination of CB-839 with anti-PD1 or anti-CTLA4 antibodies increased tumor infiltration by effector T cells and improved the antitumor activity of these checkpoint inhibitors in a high mutation burden mouse melanoma model. Responsiveness to these treatments was also accompanied by an increase of interferon gamma (IFNγ)-associated gene expression in the tumors. Together, these results provide a strong rationale for combining CB-839 with immune therapies to improve efficacy of these treatments against melanoma.

Allyl Dibenzoylmethane Derivative: Antimelanoma Activity and Study of Its Molecular Mechanism of Interaction with DNA

In this study, we assessed the cytotoxic effect of 1,3-diphenyl-2-allyl-1,3-propanedione (DPAP) on B16F10 and Tm5 melanoma cells and investigated its interaction with DNA (deoxyribonucleic acid) using optical tweezers. The compound showed to be effective against B16F10 cells with selectivity index (SI) of 7.92 and interaction to the outer surface of the double helix, probably at the minor groove of DNA. This likely induced a bending deformation of the polymer chain, decreasing persistence length, and indicated that each DPAP molecule occupies a single base pair upon binding to the double helix.

Juniperus indica Bertol. extract synergized with cisplatin against melanoma cells via the suppression of AKT/mTOR and MAPK signaling and induction of cell apoptosis.

Juniperus indica Bertol. is an herbal plant that belongs to the genus Juniperus, which is commonly used in traditional medicine to refresh the mind and for diuretic use. However, few studies have reported the function of J. indica Bertol. Hence, this study aimed to investigate the anti-tumor and synergistic potential of J. indica Bertol. extract (JIB extract) for melanoma cells. Our results indicated the anti-melanoma activity of JIB extract. JIB extract induced cell cycle arrest at the G0/G1 phase and decreased cyclin and cdk protein expressions. In addition, AKT/mTOR signaling and MAPK signaling were inhibited by JIB extract to suppress melanoma cell growth and proliferation. Additionally, JIB extract induced B16/F10 cell apoptosis via the caspase cascade. According to the JIB extract's anti-melanoma capacity, to assess the synergistic effects of cisplatin and JIB extract. The results demonstrated that JIB extract combined with cisplatin enhanced the inhibition of cell growth, proliferation, and survival through the obstruction of cell cycle progression and AKT/mTOR and MAPK signaling as well as the induction of cell apoptosis. Collectively, our results indicate that JIB extract showed anti-tumor effects and synergized with cisplatin against B16/F10 cells, indicating the possibility of JIB extract to be developed as adjuvant therapy for melanoma.

New Delivery Route of Gambogic Acid Via Skin for Topical Targeted Therapy of Cutaneous Melanoma and Reduction of Systemic Toxicity

Cutaneous melanoma is the deadliest form of skin cancer, and gambogic acid (GA) exhibits potent anti-melanoma activity. However, clinical application of GA via intravenous injection and oral administration is limited by systemic toxicity and rapid metabolism in the blood. Here, we developed a new, topical route of GA delivery for anti-melanoma activity and reduction of systemic toxicity. The results indicated that the barrier of the stratum corneum (SC) and low diffusion of GA in the hydrophilic viable skin (epidermis and dermis) limited the GA penetration through intact skin. The combination of azone (AZ) and propylene glycol (PG) showed obvious synergistic effects on skin penetration by GA via improving the permeability of the SC and greatly increasing the skin accumulation of GA, thereby forming a high drug concentration in the skin and achieving a topical targeted treatment of melanoma. In addition, GA (AZ-PG) achieved the same anti-melanoma effect via topical delivery as via intravenous injection. Intravenous injection and oral administration of GA induced remarkable pathological changes in various organs in mice, whereas GA was not toxic to various organs or to the skin via topical delivery. These findings indicated that topical administration of GA is an alternative route for melanoma treatment.

Molecular design and anti-melanoma activity of a novel bullfrog antibacterial peptide RGD-chimera.

Melanoma is a common malignant skin tumor, which is the only fatal skin tumor at present. Melanoma has a high degree of malignancy and metastasis. The activity of modified Temporin-La (T-La) peptides from bullfrog skin were evaluated for antitumor activity and improved targeting in melanoma cells. The amino acid sequence of T-La was modified, resulting in the antitumor peptide, T-La (FS). T-La and T-La (FS) were coupled to the RGD small molecule polypeptide to form the chimeric peptides RGD-T-La and RGD-T-La (FS), respectively. The secondary structures for the peptides, evaluated using circular dichroism, were found to be α-helical. The structure of T-La was evaluated using bioinformatics. In addition, the antitumor effects of the modified peptide and the targeting of RGD chimeric peptide to the tumor in vivo and in vitro were analyzed. Antitumor activity was measured in vitro using the MTT assay. Tumor cells with high integrin αvβ3 expression were detected using flow cytometry, and tumor cells were screened for sensitivity to RGD-T-La (FS) to establish a tumor model in nude mice. The effects of the peptides on tumor cells were measured using laser confocal microscopy in real-time. The mechanism of the peptide antitumor activity in tumor cells was evaluated with scanning electron microscopy. B16 melanoma cells were the most sensitive to the peptides, for which the cell survival rate was 24.65% for 10 µg/ml RGD-T-La (FS). RGD-La (FS) had a rapid effect on tumor cells. RGD chimeric polypeptides exhibited site-targeting cytotoxic effects in tumor cells. In the B16 melanoma mouse model, the peptides exhibited antitumor effects against early melanoma development and induced tumor apoptosis, possibly by inhibiting VEGF and promoting caspase-3 expression. Overall, the present study provides a scientific basis for the application of small molecule antimicrobial peptides as targeted antitumor agents and lays the foundation for the clinical application of these peptides as antitumor drugs.

Revealing the anti-melanoma mechanism of n-BuOH fraction from the red kidney bean coat extract based on network pharmacology and transcriptomic approach

Red kidney bean coat (RKBC) extract contains bioactive compounds that are known to exhibit anti-melanoma activity in vitro. However, knowledge on antitumor component and mechanism of RKBC extract has not been fully clarified. Here, RKBC extract was portioned with different solvent sequentially, and based on the cell viability assay, cell migration assay, AO/EB and Hoechst 33342 staining assay, and Annexin V-FITC/PI double staining, n-BuOH (BU) fraction was identified as the most potent antitumor fraction. It exhibited potential anti-melanoma activity via the induction of apoptosis and vacuolization in B16-F10 cells. Transcriptomic and bioprocess-target network analysis revealed that BU fraction triggered apoptosis and vacuolization through regulating PI3K-AKT-FOXO, MDM2-p53 pathway and increasing the expression of Bcl-xl. In addition, quercetin might be served as one of the key anti-melanoma compounds in BU fraction through the similar mechanism. Although the anti-melanoma activity and mechanism of BU fraction have not been elucidated completely, this study effectively expands our understanding for the anti-melanoma activity of RKBC extract and provided the basis for the further functional food research and development using red kidney bean, as well as a new possibility for treating melanoma.

In Vitro Antitumor Potential of Sulfated Polysaccharides from Seaweed Caulerpa cupressoides var. flabellata.

Seaweeds are important source of bioactive compounds, including sulfated polysaccharides (SP). Because of their structural heterogeneity, these compounds are promising sources of anticancer compounds. SP from brown and red seaweeds have shown antimelanoma activity in different in vitro and in vivo models. However, SP from green seaweed are still poorly evaluated. Therefore, SP were extracted from the green alga Caulerpa cupressoides var. flabellata, and their antiproliferative, anti-migratory, and inhibitory effect on melanin production on B16-F10 melanoma cells was evaluated. Cell assays, including flow cytometry, demonstrated that SP (100-1000μgmL-1) are non-cytotoxic, do not induce apoptosis or necrosis, and do not interfere with cell cycle. However, SP (1000μgmL-1) were found to significantly inhibit cell colony formation (80-90%), cell migration (40-75%), and melanin production (~ 20%). In summary, these results showed that SP inhibited important melanoma development events without cytotoxicity effects, suggesting that C. cupressoides may be an important source of SP with antitumor properties.

Nicotinamide inhibits melanoma in vitro and in vivo

BackgroundEven though new therapies are available against melanoma, novel approaches are needed to overcome resistance and high-toxicity issues. In the present study the anti-melanoma activity of Nicotinamide (NAM), the amide form of Niacin, was assessed in vitro and in vivo.MethodsHuman (A375, SK-MEL-28) and mouse (B16-F10) melanoma cell lines were used for in vitro investigations. Viability, cell-death, cell-cycle distribution, apoptosis, Nicotinamide Adenine Dinucleotide+ (NAD+), Adenosine Triphosphate (ATP), and Reactive Oxygen Species (ROS) levels were measured after NAM treatment. NAM anti-SIRT2 activity was tested in vitro; SIRT2 expression level was investigated by in silico transcriptomic analyses. Melanoma growth in vivo was measured in thirty-five C57BL/6 mice injected subcutaneously with B16-F10 melanoma cells and treated intraperitoneally with NAM. Interferon (IFN)-γ-secreting murine cells were counted with ELISPOT assay. Cytokine/chemokine plasmatic levels were measured by xMAP technology. Niacin receptors expression in human melanoma samples was also investigated by in silico transcriptomic analyses.ResultsNAM reduced up to 90% melanoma cell number and induced: i) accumulation in G1-phase (40% increase), ii) reduction in S- and G2-phase (about 50% decrease), iii) a 10-fold increase of cell-death and 2.5-fold increase of apoptosis in sub-G1 phase, iv) a significant increase of NAD+, ATP, and ROS levels, v) a strong inhibition of SIRT2 activity in vitro. NAM significantly delayed tumor growth in vivo (p ≤ 0.0005) and improved survival of melanoma-bearing mice (p ≤ 0.0001). About 3-fold increase (p ≤ 0.05) of Interferon-gamma (IFN-γ) producing cells was observed in NAM treated mice. The plasmatic expression levels of 6 cytokines (namely: Interleukin 5 (IL-5), Eotaxin, Interleukin 12 (p40) (IL12(p40)), Interleukin 3 (IL-3), Interleukin 10 (IL-10) and Regulated on Activation Normal T Expressed and Secreted (RANTES) were significantly changed in the blood of NAM treated mice, suggesting a key role of the immune response. The observed inhibitory effect of NAM on SIRT2 enzymatic activity confirmed previous evidence; we show here that SIRT2 expression is significantly increased in melanoma and inversely related to melanoma-patients survival. Finally, we show for the first time that the expression levels of Niacin receptors HCAR2 and HCAR3 is almost abolished in human melanoma samples.ConclusionNAM shows a relevant anti-melanoma activity in vitro and in vivo and is a suitable candidate for further clinical investigations.

Abstract B29: Systemic antimelanoma immunity induced by oncolytic adenovirus Delta-24-RGDOX

Abstract OX40 ligand (OX40L)-expressing oncolytic adenovirus Delta-24-RGDOX induces efficacious antiglioma immunity in syngeneic intracranial glioma models of immunocompetent mice. It is unknown if the virus is effective to treat metastatic melanomas. To study the abscopal immunity against metastatic melanomas induced by intratumoral injection of the virus in primary melanomas, we established subcutaneous/subcutaneous (s.c./s.c.) and subcutaneous/intracranial (s.c./i.c.) melanoma models with B16-Red-FLuc cells in C57BL/6 mice. In the s.c./s.c. model, compared to treatment with PBS, through monitoring the tumor growth with bioluminescence imaging, we found three doses of intratumoral injection of the virus significantly inhibited the growth of both the injected and the untreated distant tumors, resulting in prolonged survival of the mice with 50% long-term survival (P = 0.001). The surviving mice are resistant to rechallenging with the same tumor cells but are susceptible to lung cancer cells, suggesting the development of immune memory specific to the virus-injected tumor type. In the s.c./i.c. model, viral injection into the s.c. tumor induced antimelanoma activity in the brain, resulting in growth inhibition of both the s.c. and i.c. tumors and an improved survival of the animals (p = 0.005). Through flow cytometry analysis of the tumor-infiltrating lymphocytes, we found the virus injection increased the presence of CD3+ T lymphocytes, CD3+CD4+ helper T cells, CD3+CD8+ cytotoxic T cells, and the frequency of PD-1+ and effector (CD44+ CD62L-) T cells and decreased the amount of exhausted (PD-1+ TIM3+) and regulatory T cells in the injected tumors. Consistently, analysis of T cells in the blood, spleen, and brain hemispheres with untreated tumor revealed the same virus-mediated changes. Interestingly, through monitoring the T cells specific for tumor-associated antigen (TAA) in the mice through bioluminescence imaging, we found injecting Delta-24-RGDOX into the first s.c. B16-OVA melanoma increased the proliferation of OT-I/Luc CD8+ T cells infused in the same tumor and their migration to the distant untreated tumor originated from B16-OVA cells but not to the tumor from B16F10 cells, providing direct evidence that intratumoral viral injection promotes the in situ expansion of TAA-specific T cells. In summary, localized intratumoral injection of Delta-24-RGDOX induced an in situ autovaccination of the treated melanoma, of which the effect changes the immune landscape of the treated mice, resulting in the immunity against the disseminated s.c. or i.c. tumors. Citation Format: Hong Jiang, Dong Ho Shin, Caroline Carrillo, Verlene Henry, Xuejun Fan, Teresa T. Nguyen, Yisel Rivera-Molina, Frederick F. Lang, Candelaria Gomez-Manzano, Juan Fueyo. Systemic antimelanoma immunity induced by oncolytic adenovirus Delta-24-RGDOX [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B29.

Abstract PR06: The glutaminase inhibitor CB-839 potentiates antimelanoma activity of standard-of-care targeted therapies and immunotherapies

Abstract Nearly all metastatic melanoma patients who respond to targeted therapies will relapse with the disease within a year. Although more durable responses are seen with immune therapies, about half of the melanoma patients do not respond to them, and a significant number of responders eventually relapse. Most relapsed melanomas also exhibit post-treatment resistance to these treatments. Hence, there is a clear and present need to develop therapeutics that counteract resistance associated with relapse. We and others earlier showed that melanomas with elevated mitochondrial activity possess improved cellular rigor and are intrinsically resistant to the antitumor effects of BRAF and MEK inhibitors. In many other instances, melanomas that initially respond to these inhibitors acquire resistance by elevating mitochondrial activity. Mitochondrial activity is elevated in part by increased cellular uptake of glutamine, and its conversion to alpha-ketoglutarate in the TCA cycle, with glutaminase enzyme playing a rate-limiting role. In this study, we show that BRAFV600E-mutant melanomas with intrinsic or acquired resistance to MAPK pathway inhibitors have lower glucose uptake and increased glutamine uptake compared to those that are sensitive. Treatment of these resistant melanomas with single-agent glutaminase inhibitor, CB-839, moderately inhibited their growth. However, a more robust inhibition of their growth was achieved when CB-839 was combined with BRAF and MEK inhibitors. In addition, CB-839 increased the in vivo activity of tumor-infiltrating lymphocytes (TILs) in a mouse vaccine model and also enhanced the proapoptotic effect of human autologous patient-derived TILs on their cognate melanoma cells. Seahorse bioenergetics stress tests showed that CB-839 inhibited mitochondrial OxPhos in tumor cells to a greater extent than in activated TILs. Additional molecular studies are currently in progress. A recent clinical trial in melanoma patients showed that combination treatment with CB-839 and the immune checkpoint blocker, nivolumab, caused an objective response in three melanoma patients who had earlier progressed on treatment with immune checkpoint blockade. Our preclinical results complement this clinical finding and suggest that CB-839 combination could potentiate the efficacy of targeted and immune therapies in refractory melanomas. Citation Format: Sruthy Varghese, Snigdha Pramanik, Rishika Prasad, Hannah Hodges, Leila Williams, Weiyi Peng, Hussein Tawbi, Vashisht Yennu Nanda. The glutaminase inhibitor CB-839 potentiates antimelanoma activity of standard-of-care targeted therapies and immunotherapies [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR06.

Abstract A28: Identification of NGLY1-mediated protein deglycosylation as a vulnerable point in melanoma

Abstract Although NGLY1 is known as a pivotal enzyme that catalyzes the deglycosylation of denatured glycoproteins, information regarding the responses of human cancer and normal cells to NGLY1 suppression is limited. While studying the NGLY1 deficiency-caused neurodevelopmental variations in patients with a congenital deglycosylation disorder, we made an intriguing discovery that the expression of NGLY1 appears to be dysregulated in human melanoma cells. We have examined how NGLY1 expression affects viability, tumor growth, and responses to therapeutic agents in melanoma cells and an animal model. Molecular mechanisms contributing to NGLY1 suppression-induced anticancer responses were revealed by systems biology and chemical biology studies. Using computational and medicinal chemistry-assisted approaches, we established novel NGLY1-inhibitory small molecules. Compared with normal cells, NGLY1 was upregulated in melanoma cell lines and patient tumors. NGLY1 knockdown caused melanoma cell death and tumor growth retardation. Targeting NGLY1 in melanoma cells induced pleiotropic responses, predominantly stress signaling-associated apoptosis and cytokine surges, which synergize with the antimelanoma activity of standard chemotherapy and targeted therapy agents. Pharmacologic and molecular biology tools that inactivate NGLY1 elicited highly similar responses in melanoma cells. Unlike normal cells, melanoma cells presented distinct responses and high vulnerability to NGLY1 suppression. Our work demonstrated the significance of NGLY1 in melanoma cells, provided mechanistic insights into how NGLY1 inactivation leads to eradication of melanoma with limited impact on normal cells, and suggested that targeting NGLY1 represents a novel antimelanoma strategy with the opportunity of a broad therapeutic window. Citation Format: Victor Lin, Ashwini Zolekar, Nigam Mishra, Yin Ying Ho, Hamed Hayatshahi, Abhishek Parab, Rohit Sampat, Xiaoyan Liao, Peter Hoffmann, Jin Liu, Kyle Emmitte, Yu-Chieh Wang. Identification of NGLY1-mediated protein deglycosylation as a vulnerable point in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A28.