Melanoma Tumor Xenografts Research Articles

A Recombinant Lentiviral Vegfr2-Silencing Vector Attenuates Roxarsone-Promoted Growth of Rat Vascular Endothelial Cells and Angiogenesis in Matrigel Plug and B16F10 Xenograft Models.

Roxarsone (ROX) is widely used as a feed addictive for livestock and poultry. ROX promotes angiogenesis, which can lead to health problems, and it is necessary to identify methods to counter this angiogenic effect of ROX. The VEGF/VEGFR2 signaling pathway is involved in the growth and reconstruction of new blood vessels during angiogenesis. In this study, a recombinant lentiviral vector encoding Vegfr2 shRNA was transfected into rat vascular endothelial cells and used in mouse matrigel plug and melanoma xenograft models to investigate its potential to regulate ROX-induced angiogenesis and tumor growth. Treating endothelial cells with ROX increased cell proliferation, migration, and a tube-like structure of growth relative to the control group. The addition of the lentiviral Vegfr2-silencing vector significantly attenuated the effects of ROX on endothelial cells. The hemoglobin content of mouse matrigel plugs treated with ROX was increased significantly. This effect was dramatically attenuated by the co-administration of shRNA targeting Vegfr2. The volume, weight and CD34 staining of the melanoma xenograft tumors increased by ROX were also attenuated by Vegfr2 silence. These results indicate that the down-regulation of VEGFR2 protein plays an inhibitory role in the ROX-promoted angiogenesis in vivo and in vitro. These data support the targeting of Vegfr2 gene as an effective means to treat ROX-induced angiogenesis and tumor growth.

Tozasertib activates anti-tumor immunity through decreasing regulatory T cells in melanoma

Although immune checkpoint therapy has significantly improved the prognosis of patients with melanoma, urgent attention still needs to be paid to the low patient response rates and the challenges of precisely identifying patients before treatment. Therefore, it is crucial to investigate novel immunosuppressive mechanisms and targets in the tumor microenvironment in order to reverse tumor immune escape. In this study, we found that the cell cycle checkpoint Aurora kinase B (AURKB) suppressed the anti-tumor immune response, and its inhibitor, Tozasertib, effectively activated T lymphocyte cytokine release in vitro and anti-tumor immunity in vivo. Tozasertib significantly inhibited melanoma xenograft tumor growth by decreasing the number of inhibitory CD4+ Treg cells in the tumors, which, in turn, activated CD8+ T cells. Single-cell analysis revealed that AURKB suppressed anti-tumor immunity by increasing MIF-CD74/CXCR4 signaling between tumor cells and lymphocytes. Our study suggests that AURKB is a newly identified anti-tumor immunity suppressor, whose inhibitors may be developed as novel anti-tumor immunity drugs and may have synergistic anti-melanoma effects with immune checkpoint therapies.

Preclinical evaluation of an 18F-labeled Nε-acryloyllysine piperazide for covalent targeting of transglutaminase 2.

Transglutaminase 2 (TGase2) is a multifunctional protein and has a prominent role in various (patho)physiological processes. In particular, its transamidase activity, which is rather latent under physiological conditions, gains importance in malignant cells. Thus, there is a great need of theranostic probes for targeting tumor-associated TGase 2, and targeted covalent inhibitors appear to be particularly attractive as vector molecules. Such an inhibitor, equipped with a radionuclide suitable for noninvasive imaging, would be supportive for answering the general question on the possibility for functional characterization of tumor-associated TGase 2. For this purpose, the recently developed 18F-labeled Nε-acryloyllysine piperazide [18F]7b, which is a potent and selective irreversible inhibitor of TGase2, was subject to a detailed radiopharmacological characterization herein. An alternative radiosynthesis of [18F]7b is presented, which demands less than 300µg of the respective trimethylammonio precursor per synthesis and provides [18F]7b in good radiochemical yields (17 ± 7%) and high (radio)chemical purities (≥ 99%). Ex vivo biodistribution studies in healthy mice at 5 and 60min p.i. revealed no permanent enrichment of 18F-activity in tissues with the exception of the bone tissue. In vivo pretreatment with ketoconazole and in vitro murine liver microsome studies complemented by mass spectrometric analysis demonstrated that bone uptake originates from metabolically released [18F]fluoride. Further metabolic transformations of [18F]7b include mono-hydroxylation and glucuronidation. Based on blood sampling data and liver microsome experiments, pharmacokinetic parameters such as plasma and intrinsic clearance were derived, which substantiated the apparently rapid distribution of [18F]7b in and elimination from the organisms. A TGase2-mediated uptake of [18F]7b in different tumor cell lines could not be proven. Moreover, evaluation of [18F]7b in melanoma tumor xenograft models based on A375-hS100A4 (TGase2 +) and MeWo (TGase 2 -) cells by ex vivo biodistribution and PET imaging studies were not indicative for a specific targeting. [18F]7b is a valuable radiometric tool to study TGase2 in vitro under various conditions. However, its suitability for targeting tumor-associated TGase2 is strongly limited due its unfavorable pharmacokinetic properties as demonstrated in rodents. Consequently, from a radiochemical perspective [18F]7b requires appropriate structural modifications to overcome these limitations.

Abstract A093: Deep Cyclic Inhibition of the MAPK pathway with IMM-6-415, alone and in combination with encorafenib, demonstrates anti-tumor activity and tolerability in RAF mutant tumors in vivo

Abstract Introduction: A significant proportion of cancer patients have tumors addicted to uncontrolled MAPK signaling. Activating mutations in RAS or RAF are often directly responsible and have been observed in over 20% of human tumors1. Because MEK is downstream of RAS and RAF, it is an appealing drug target. However, MEK inhibitors have historically suffered from poor clinical durability, high toxicity and a susceptibility to pathway reactivation that has limited monotherapy activity in the RAS mutant setting. Unlike other MEK inhibitors, IMM-1-104 [NCT05585320] and IMM-6-415 are designed with distinctive features including both (1.) a unique target engagement mechanism that helps resist MAPK pathway reactivation and (2.) a pharmacokinetic (PK) profile that enables fast cadence deep cyclic inhibition (DCI). DCI drives pulsatile targeted inhibition that deprives tumor cells of a critical oncogenic pathway while limiting drug-related toxicities by affording normal cells an adequate PK recovery window between drug doses. To our knowledge, IMM-6-415’s preclinical activity is driven by the shortest drug plasma half-life (0.3-hours in mice) of any MEK inhibitor developed to date. Experimental Procedures: IMM-6-415 has already demonstrated promising activity in RAS-mutant xenograft tumor models (SITC 2022). Here, the antitumor activity of IMM-6-415 was evaluated in over 60 humanized 3D tumor growth assays (3D-TGA), which included 30 BRAF class I-mutant tumor models. Additionally, multiple drug-drug combinations have been explored, including vertical drug combinations with BRAF inhibitors. IMM-6-415, binimetinib and encorafenib were tested head-to-head as single agents and in combination with encorafenib in BRAFV600E melanoma and colorectal subcutaneous tumor xenograft models in female BALB/c nude mice. Summary of New Data: As monotherapy, IMM-6-415 demonstrated antitumor activity in over 50% (34 of 66) of the 3D-TGA models tested, including 30 BRAF-mutant preclinical models in which 19 (63%) showed activity. Therefore, we further explored both monotherapy and combination activity of IMM-6-415 in the A-375 (melanoma) and HT-29 (colorectal) BRAF V600E tumor xenograft models. Monotherapy treatment with encorafenib or IMM-6-415 displayed superior tumor growth inhibition (TGI) when compared to binimetinib. Furthermore, the combination of IMM-6-415 plus encorafenib prompted greater TGI with superior durability of response when tested head-to-head against the combination of binimetinib plus encorafenib in vivo at human equivalent doses for registered drugs. Conclusions: IMM-6-415 demonstrated promising activity and tolerability in preclinical models alone and in combination with encorafenib. In combination with encorafenib, IMM-6-415 achieved a greater TGI in vivo than the combination of encorafenib plus binimetinib in BRAFV600E colorectal cancer and melanoma tumor models, suggesting an opportunity for IMM-6-415 as monotherapy or in combination in BRAF mutant tumors. Citation Format: Anna Travesa, Mai Johnson, Peter King, Praveen Nair, Jason Funt, Sarah Kolitz, Kevin D Fowler, John Brothers II, Amy Axel, Scott Barrett, Benjamin J Zeskind, Brett Hall. Deep Cyclic Inhibition of the MAPK pathway with IMM-6-415, alone and in combination with encorafenib, demonstrates anti-tumor activity and tolerability in RAF mutant tumors in vivo [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A093.

Targeting TREM1 augments antitumor T cell immunity by inhibiting myeloid-derived suppressor cells and restraining anti-PD-1 resistance.

The triggering receptor expressed on myeloid cell 1 (TREM1) plays a critical role in development of chronic inflammatory disorders and the inflamed tumor microenvironment (TME) associated with most solid tumors. We examined whether loss of TREM1 signaling can abrogate the immunosuppressive TME and enhance cancer immunity. To investigate the therapeutic potential of TREM1 in cancer, we used mice deficient in Trem1 and developed a novel small molecule TREM1 inhibitor, VJDT. We demonstrated that genetic or pharmacological TREM1 silencing significantly delayed tumor growth in murine melanoma (B16F10) and fibrosarcoma (MCA205) models. Single-cell RNA-Seq combined with functional assays during TREM1 deficiency revealed decreased immunosuppressive capacity of myeloid-derived suppressor cells (MDSCs) accompanied by expansion in cytotoxic CD8+ T cells and increased PD-1 expression. Furthermore, TREM1 inhibition enhanced the antitumorigenic effect of anti-PD-1 treatment, in part, by limiting MDSC frequency and abrogating T cell exhaustion. In patient-derived melanoma xenograft tumors, treatment with VJDT downregulated key oncogenic signaling pathways involved in cell proliferation, migration, and survival. Our work highlights the role of TREM1 in cancer progression, both intrinsically expressed in cancer cells and extrinsically in the TME. Thus, targeting TREM1 to modify an immunosuppressive TME and improve efficacy of immune checkpoint therapy represents what we believe to be a promising therapeutic approach to cancer.

GREB1 isoform 4 is specifically transcribed by MITF and required for melanoma proliferation

Growth regulation by estrogen in breast cancer 1 (GREB1) is involved in hormone-dependent and -independent tumor development (e.g., hepatoblastoma). In this study, we found that a GREB1 splicing variant, isoform 4 (Is4), which encodes C-terminal half of full-length GREB1, is specifically expressed via microphthalmia-associated transcription factor (MITF) in melanocytic melanoma, and that two MITF-binding E-box CANNTG motifs at the 5’-upstream region of GREB1 exon 19 are necessary for GREB1 Is4 transcription. MITF and GREB1 Is4 were strongly co-expressed in approximately 20% of the melanoma specimens evaluated (17/89 cases) and their expression was associated with tumor thickness. GREB1 Is4 silencing reduced melanoma cell proliferation in association with altered expression of cell proliferation-related genes in vitro. In addition, GREB1 Is4 targeting by antisense oligonucleotide (ASO) decreased melanoma xenograft tumor formation and GREB1 Is4 expression in a BRAFV600E; PTENflox melanoma mouse model promoted melanoma formation, demonstrating the crucial role of GREB1 Is4 for melanoma proliferation in vivo. GREB1 Is4 bound to CAD, the rate-limiting enzyme of pyrimidine metabolism, and metabolic flux analysis revealed that GREBI Is4 is necessary for pyrimidine synthesis. These results suggest that MITF-dependent GREB1 Is4 expression leads to melanoma proliferation and GREB1 Is4 represents a new molecular target in melanoma.

Co-Targeting Nucleus Accumbens Associate 1 and NF-κB Signaling Synergistically Inhibits Melanoma Growth.

Nucleus-accumbens-associated protein-1 (NAC1) is a cancer-related transcriptional factor encoded by the NACC1 gene, which is amplified and overexpressed in various human cancers and has been appreciated as one of the top potential cancer driver genes. NAC1 has therefore been explored as a potential therapeutic target for managing malignant tumors. Here, we show that NAC1 is a negative regulator of NF-κB signaling, and NAC1 depletion enhances the level of the nuclear NF-κB in human melanoma. Furthermore, the inhibition of NF-κB signaling significantly potentiates the antineoplastic activity of the NAC1 inhibition in both the cultured melanoma cells and xenograft tumors. This study identifies a novel NAC1-NF-κB signaling axis in melanoma, offering a promising new therapeutic option to treat melanoma.

Abstract 6639: Preclinical characterization and clinical biomarker studies with RP3, a novel oncolytic immunotherapy expressing a fusogenic protein, a human anti-CTLA-4 antibody, hCD40L and h4-1BBL

Abstract Introduction: RP3, a tumor-directed oncolytic immunotherapy (TDOI), is a selectively replication-competent HSV-1 that contains the codon-optimized sequence for the fusogenic GALV-GP-R- protein, an anti-CTLA-4 antibody-like molecule, CD40L, and 4-1BBL. The expression of GALV-GP-R- potentiates immunogenic tumor cell death through the induction of cell-to-cell fusion. CD40L enhances dendritic cell and macrophage activation and 4-1BBL promotes CD8+ T-cell activation and co-stimulation. The anti-CTLA-4 antibody-like molecule blocks the interaction of CTLA-4 with its ligands B7.1 and B7.2, further enhancing T cell activation. RP3 is currently being assessed in a Phase 1 clinical trial with or without nivolumab in patients with advanced solid tumors (NCT04735978). Methods: CD1 nude mice with A375 melanoma xenograft tumors (Group 1) were treated with a single intratumoral injection of 1 × 107 PFU/mL of RP3. Tumors and blood samples were collected 3 days later, and immunohistochemistry (IHC) was performed to detect CD40L, 4-1BBL and HSV-1. For anti-CTLA-4 detection, two doses of 107 PFU/mL of RP3 were injected intratumorally into C57BL/6 mice bearing 4434 murine BRAFV600E mutant melanoma tumors (Group 2). Tumors and blood samples were collected at 3, 6, 9, 12 and 15-days post-dosing and anti-CTLA-4 ELISA was performed. For clinical biomarker studies, tumor biopsies were collected pre- and post-RP3 treatment. CD8 and PD-L1 were characterized by IHC, and gene expression analysis (GEA) was performed. TCR analysis was also performed using PBMCs. Results: IHC analysis from Group 1 confirmed abundant and widespread expression of CD40-L and 4-1BBL, which were detected alongside with HSV-1 antigen. In the blood samples collected from Group 2, robust levels of anti-CTLA-4 antibody were detected, which stayed in detectable range up to 15 days after the 2nd RP3 injection. IHC analysis of patient tumor samples demonstrated a marked increase in CD8 T cells and PD-L1 expression. GEA of patient tumor biopsies demonstrated increased levels of genes associated with innate and adaptive immune activation and genes previously reported to be associated with responsiveness to anti-PD1 therapy. TCR sequencing of PBMCs and multiplex IHC analysis are ongoing and updated results will be presented. Conclusion: Preclinical experiments from mice models confirmed the robust expression of CD40-L, 4-1BBL and anti-CTLA-4 in RP3-injected tumors. Biomarker studies from patients demonstrate increases in CD8-T cell infiltration and PD-L1 expression post RP3 +/- nivolumab treatment, including increases in the expression of genes associated with response to anti-PD-1 therapy. These data indicate the potential for broad utility of RP3 in a range of tumor types, including in tumors that are not sensitive to PD-1 blockade. Citation Format: Praveen K. Bommareddy, Kevin Harrington, Victoria Roulstone, Joan Kyula, Aaron Clack, Linta Kuncheria, Sylwia Jones, Robert Coffin. Preclinical characterization and clinical biomarker studies with RP3, a novel oncolytic immunotherapy expressing a fusogenic protein, a human anti-CTLA-4 antibody, hCD40L and h4-1BBL. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6639.

Advantages of FBPA PET in evaluating early response of anti-PD-1 immunotherapy in B16F10 melanoma-bearing mice: Comparison to FDG PET.

PET with L-4-borono-2-[18F] fluoro-phenylalanine (FBPA) was reported to be useful to differentiate malignant tumors and inflammation. Although immunotherapy with immune checkpoint inhibitors (ICIs) has been applied to cancer treatment recently, FDG PET may not be suitable to determine the effect of ICIs because of false-positive findings caused by treatment-related inflammation. In this study, we aimed to demonstrate that FBPA PET allowed detection of the early response of anti-PD-1 immunotherapy in tumor-bearing mice, comparing the results with those of FDG PET. Mice with B16F10 melanoma tumor xenografts were prepared. Anti-mouse PD-1 antibody or PBS was administered twice intraperitoneally to the tumor-bearing mice on Day 0 (3 days after inoculation) and Day 5 (treatment or control group <TrG or CoG>). PET/CT imaging was performed twice for each mouse on Day 0 before the anti-PD-1 antibody/PBS administration and on Day 7 using a micro-PET/CT scanner. FBPA and FDG PET/CT studies were conducted separately. SUVmax and the tumor to liver ratio (T/L ratio) were used as parameters exhibiting tumor activity. Tumor uptake volume (TUV) and metabolic tumor volume (MTV) were also calculated for FBPA and FDG, respectively. Changes between pre- and posttreatment SUVmax or T/L ratio were observed using the formula as follows: [(posttreatment parameter values/pretreatment values - 1) × 100] (%). Tumors in TrG were smaller than those in CoG on Day 7. SUVmax and T/L ratio represented no differences between TrG and CoG in FBPA and FDG PET before treatment. FBPA PET on Day 7 demonstrated that SUVmax, T/L ratio, and TUV in TrG were statistically smaller than those in CoG. %T/L ratio and %SUVmax exhibited the same trend in FBPA PET. However, FDG PET on Day 7 revealed no differences in all parameters between TrG and CoG. T/L ratio and %SUVmax in TrG represented larger values than those in CoG without statistical significances. This study demonstrated that FBPA PET allowed detection of the early response of anti-PD-1 immunotherapy in B16F10 melanoma-bearing mice. FDG PET did not detect the response. Further studies are required to determine whether FBPA PET is useful in evaluating the treatment effect of ICIs in humans.

Newly synthesized methionine aminopeptidase 2 inhibitor hinders tumor growth.

Methionine aminopeptidase 2 (MetAp2) inhibition has been recognized as a promising approach for suppressing angiogenesis and cancer progression. Small molecule fumagillol derivatives with adamantane side groups were synthesized and evaluated for MetAp2 inhibition activity, and a lead molecule with superior abilities to inhibit the enzymatic activity of MetAp2 was identified. The compound, referred to as AD-3281, effectively suppressed proliferation of cancer and endothelial cells and impaired tube formation of endothelial cells in vitro. When administered systemically, AD-3281 was well tolerated and led to a significant suppression of human melanoma and mammary tumor xenografts grown in mice. The activity in vivo was associated with reduced angiogenesis and tumor proliferation as detected histologically. In order to develop a formulation that can solubilize AD-3281 with a minimal content of organic solvents, biodegradable nanoparticles comprised of poly-lactic-co-glycolic acid (PLGA) were fabricated and characterized. Compared with the free compound, AD-3281-loaded nanoparticles showed an advantageous cellular availability and uptake, leading to higher activity in cells and better transport in three-dimensional (3D) cultures. Taken together, we introduce a novel MetAp2 inhibitor with high anti-cancer activity and a stable nano-formulation with a high potential for future clinical translation.

Abstract P240: Benchmarking the novel dual-MEK inhibitor, IMM-1-104, head-to-head and in combination with sotorasib (AMG-510) in the MIA PaCa-2 (KRAS-G12C) pancreatic cancer xenograft model

Abstract Background: Approximately 9 out of 10 patients diagnosed with pancreatic cancer have tumors driven by a single activating mutation in KRAS. These tumors remain unaddressable by current therapies and collectively represent a high unmet clinical need. KRAS is the most commonly mutated form of RAS and represents approximately 85% of all RAS mutant human tumors with HRAS and NRAS accounting for the remaining 15%. Sotorasib (AMG-510), a covalent KRAS-G12C inhibitor, recently received accelerated regulatory approval for KRAS-G12C lung cancer in the United States. While the G12C mutation has been documented in up to 15% of all KRAS mutant tumors, it is rarely observed (&amp;lt;1%) in human pancreatic cancer, which could severely limit this type of new inhibitor in certain histologies. IMM-1-104 is a novel dual-MEK inhibitor that resists CRAF-bypass (goal: improve activity against RAS mutant tumors), and is designed to have a short plasma drug half-life, which leads to deep, cyclic inhibition of the MAPK pathway (goal: improved tolerability). Materials &amp; Methods: The novel, orally bioavailable dual-MEK inhibitor, IMM-1-104, was evaluated for antitumor activity, as a single agent (50, 100, 150 mg/kg BID p.o.) in a head-to-head format against sotorasib (10, 30, 100 mg/kg QD p.o.) or in combination (50, 100, 150 mg/kg BID p.o. IMM-1-104 with 30 mg/kg QD p.o. sotorasib) in the MIA PaCa-2 KRAS-G12C mutant pancreatic tumor xenograft model. Tumor-bearing animals were treated for 21 days of oral dosing, before drug treatments were terminated and animals monitored for additional 3 weeks. Results: IMM-1-104 and sotorasib were well-tolerated as single agents and in combination with each other across all dose levels tested. Both drugs demonstrated dose-dependent antitumor activity with tumor regressions noted at higher dose levels. IMM-1-104 in combination with sotorasib produced deep regressions that were sustained longer than either drug alone. Conclusions: The dual-MEK mechanism and short half-life of IMM-1-104 has demonstrated broad activity with low toxicity across multiple RAS and RAF mutant preclinical tumor models. Tumor regressions were observed in NRAS mutant melanoma (SK-MEL-2) and KRAS mutant colorectal (Colon-26) rodent tumor models, and tumor stasis was observed in KRAS mutant lung (A549) and BRAF mutant melanoma (A375) tumor xenograft models. Here, we report that IMM-1-104 treatment resulted in tumor regressions similar to that observed for sotorasib in the recently benchmarked KRAS-G12C mutant pancreatic cancer xenograft model (MIA PaCa-2). IMM-1-104 in combination with sotorasib promoted deep, durable tumor regressions, when compared to either drug alone. Therefore, future drug-drug combinations with upstream inhibitors such as sotorasib may afford greater durability in combination for patients with KRAS-G12C and other select tumor types. In totality, these data suggest the potential for broad, single agent activity of IMM-1-104 in tumors with inappropriately elevated MAPK signaling, including a large percentage of KRAS mutant pancreatic cancer. Citation Format: Peter J. King, Amy E. Axel, Kevin D. Fowler, Sarah E. Kolitz, Scott Barrett, Benjamin J. Zeskind, Brett M. Hall. Benchmarking the novel dual-MEK inhibitor, IMM-1-104, head-to-head and in combination with sotorasib (AMG-510) in the MIA PaCa-2 (KRAS-G12C) pancreatic cancer xenograft model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P240.

Abstract 3169: The therapeutically inflamed tumor microenvironment drives melanoma progression

Abstract Even the most therapy responsive melanomas eventually evade immune checkpoint inhibition. Interestingly, initial responses seem to benefit from localized inflammation, but this can become prohibitive when treating advanced disease. Subsequently, there is an urgent need to elucidate how inflammation impacts the tumor microenvironment (TME). Here, inhibition of EGFR and Ras/Raf/MEK is shown to drive the release of a pro-inflammatory secretome that activates the TME to promote progression. This secretome was enriched with immune recruiting cytokines and members of the transforming growth factor beta (TGFβ) superfamily. Both healthy and cancer associated fibroblasts (CAFs) were hyperactivated in response to the inflamed secretome with induction of the inflammatory marker, alpha smooth muscle actin (αSMA). Pharmacologic inhibition of TGFβ receptor type I (TGFβRI) using SB431542 or clinically relevant TEW-7197, diminished CAF αSMA phenotype, disrupted TGFβ and phospo-SMAD2/3 signaling, and blunted CAF expression of tumor promoting extracellular matrix and adhesion molecules. Invasion of melanoma spheroids was enhanced ~2.5-fold in CAF coculture transwells. However, treatment with MEK inhibitor AZD6244 and TEW-7197 only limited invasion of mixed melanoma and CAF multi-cell spheroids in vitro. Prompted by this, the extracellular matrix of patient-derived and melanoma xenograft tumors was analyzed for inflammatory protein expression. Micrometastatic foci were flanked by peri-tumor αSMA+ CAFs and enriched for inflammatory Tenascin-C (TNC) at the invasive front. The analysis of human melanoma tissue microarrays corroborated the experimentally observed expression of αSMA CAFs and TNC which statistically increase with malignant tumor grade. Conversely, the anti-invasive Decorin (DCN), decreased in malignant melanoma while collagen-1 increased during late stage tumor fibrosis. These findings prompted the use of ex vivo human skin organ cultures (SOCs) to define how cell matrix impacted melanoma. Addition of DCN to the SOC eliminated melanoma vertical invasion whereas pathological TNC promoted progression. Thus, a BRAFV600E resistant patient-derived metastatic cell line was used to establish intrasplenic xenografts that were treated after one week using AZD6244 and TEW-7197. Both monotherapy and combination therapy were well tolerated and resulted in reduced splenic tumor burden, αSMA staining, and halted metastasis to the liver. Citation Format: Andrew M. Bradshaw, Erica Kuo, Jelena Grahovac, Kyle Sylakowski, Cindy Sander, Howard Edington, John M. Kirkwood, Alan Wells. The therapeutically inflamed tumor microenvironment drives melanoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3169.

Abstract 1473: Nonclinical development of PF-07284890 (ARRY-461), a potent, brain-penetrant, small molecule inhibitor of BRAF V600-mutation-driven tumors in vitro and in vivo

Abstract Although approved BRAF inhibitors have transformed the treatment of patients with certain BRAF V600 mutant cancers, their long-term efficacy is thought to be limited by poor brain penetration. As a result, disease progression in the brain is a significant cause of morbidity and mortality. PF-07284890 (ARRY-461) is an orally bioavailable, brain penetrant, potent, small molecule inhibitor targeting BRAF V600 mutant tumors and is in clinical development in patients with BRAF V600 metastatic melanoma with progression to the brain. In biochemical in vitro studies, PF-07284890 inhibits BRAF and CRAF with IC50's of 5.8 and 4.1 nM, respectively. Additionally, PF-07284890 inhibits the clinically relevant kinase domain BRAF V600 mutants (V600E and V600K) (IC50 = 24-25 nM). In cell-based systems, PF-07284890 potently inhibits phosphorylation of ERK, a downstream marker of BRAF inhibition, and potently inhibits proliferation of BRAF V600E/K mutant melanoma cell lines (IC50 18-38 nM). PF-07284890 was designed to distribute to the brain and, as such, in vitro experiments indicate that PF-07284890 has high cellular membrane permeability and is not a substrate for human P glycoprotein (P-gp). After oral administration of PF-07284890 to mice and rats, PF-07284890 distributes to the brain where the free fraction adjusted exposure in the brain was approximately proportional to the free fraction adjusted exposure in plasma (i.e., Cbrain,u/Cplasma,u approaches 1.0). In vivo, PF-07284890 inhibits phosphorylation of ERK in A375 BRAF V600E tumors, achieving maximal target inhibitions at a dose of 10 mg/kg. PF-07284890 has been evaluated for its ability to control BRAF V600E cell line and patient-derived melanoma xenograft tumor growth in nude mice when implanted both subcutaneously and intracranially. Dose-related tumor growth inhibition was demonstrated at dose levels ranging from 1 to 30 mg/kg, BID in both subcutaneous and intracranial xenograft models. In all models, regardless of tumor location, maximal efficacy was observed in dose ranges of 10-30 mg/kg BID. In the intracranial A375-luc BRAF V600E melanoma xenograft model, significant and durable tumor regressions were seen at doses of 10 and 30 mg/kg/day which translated to a profound survival benefit in animals receiving PF-07284890 (median survival &amp;gt; 55 days post-implantation) compared to control animals (median survival = 15 days). GLP safety studies demonstrated a good safety profile for PF-07284890. PF-07284890 is fully brain penetrant, with the potential to address this key unmet medical need and thereby defines a new class of brain penetrant, potent and selective BRAF inhibitors. Citation Format: Karyn Bouhana, Deborah Anderson, Walter DeWolf, Suzy Brown, Lance Williams, Li Ren, David Moreno, Ross Wallace, Jay Brad Fell, Dylan Hartley, Patrice Lee. Nonclinical development of PF-07284890 (ARRY-461), a potent, brain-penetrant, small molecule inhibitor of BRAF V600-mutation-driven tumors in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1473.

Tubeimoside-1 induces TFEB-dependent lysosomal degradation of PD-L1 and promotes antitumor immunity by targeting mTOR

Programmed cell death ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) cascade is an effective therapeutic target for immune checkpoint blockade (ICB) therapy. Targeting PD-L1/PD-1 axis by small-molecule drug is an attractive approach to enhance antitumor immunity. Using flow cytometry-based assay, we identify tubeimoside-1 (TBM-1) as a promising antitumor immune modulator that negatively regulates PD-L1 level. TBM-1 disrupts PD-1/PD-L1 interaction and enhances the cytotoxicity of T cells toward cancer cells through decreasing the abundance of PD-L1. Furthermore, TBM-1 exerts its antitumor effect in mice bearing Lewis lung carcinoma (LLC) and B16 melanoma tumor xenograft via activating tumor-infiltrating T-cell immunity. Mechanistically, TBM-1 triggers PD-L1 lysosomal degradation in a TFEB-dependent, autophagy-independent pathway. TBM-1 selectively binds to the mammalian target of rapamycin (mTOR) kinase and suppresses the activation of mTORC1, leading to the nuclear translocation of TFEB and lysosome biogenesis. Moreover, the combination of TBM-1 and anti-CTLA-4 effectively enhances antitumor T-cell immunity and reduces immunosuppressive infiltration of myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells. Our findings reveal a previously unrecognized antitumor mechanism of TBM-1 and represent an alternative ICB therapeutic strategy to enhance the efficacy of cancer immunotherapy.

Modulated Electro-Hyperthermia Facilitates NK-Cell Infiltration and Growth Arrest of Human A2058 Melanoma in a Xenograft Model.

Modulated electro-hyperthermia (mEHT), induced by 13.56 MHz radiofrequency, has been demonstrated both in preclinical and clinical studies to efficiently induce tumor damage and complement other treatment modalities. Here, we used a mouse xenograft model of human melanoma (A2058) to test mEHT (~42°C) both alone and combined with NK-cell immunotherapy. A single 30 min shot of mEHT resulted in significant tumor damage due to induced stress, marked by high hsp70 expression followed by significant upregulation of cleaved/activated caspase-3 and p53. When mEHT was combined with either primary human NK cells or the IL-2 independent NK-92MI cell line injected subcutaneously, the accumulation of NK cells was observed at the mEHT pretreated melanoma nodules but not at the untreated controls. mEHT induced the upregulation of the chemoattractant CXCL11 and increased the expression of the matrix metalloproteinase MMP2 which could account for the NK-cell attraction into the treated melanoma. In conclusion, mEHT monotherapy of melanoma xenograft tumors induced irreversible heat and cell stress leading to caspase dependent apoptosis to be driven by p53. mEHT could support the intratumoral attraction of distantly injected NK-cells, contributed by CXCL11 and MMP2 upregulation, resulting in an additive tumor destruction and growth inhibition. Therefore, mEHT may offer itself as a good partner for immunotherapy.

Redox signals at the ER–mitochondria interface control melanoma progression

Reactive oxygen species (ROS) are emerging as important regulators of cancer growth and metastatic spread. However, how cells integrate redox signals to affect cancer progression is not fully understood. Mitochondria are cellular redox hubs, which are highly regulated by interactions with neighboring organelles. Here, we investigated how ROS at the endoplasmic reticulum (ER)–mitochondria interface are generated and translated to affect melanoma outcome. We show that TMX1 and TMX3 oxidoreductases, which promote ER–mitochondria communication, are upregulated in melanoma cells and patient samples. TMX knockdown altered mitochondrial organization, enhanced bioenergetics, and elevated mitochondrial‐ and NOX4‐derived ROS. The TMX‐knockdown‐induced oxidative stress suppressed melanoma proliferation, migration, and xenograft tumor growth by inhibiting NFAT1. Furthermore, we identified NFAT1‐positive and NFAT1‐negative melanoma subgroups, wherein NFAT1 expression correlates with melanoma stage and metastatic potential. Integrative bioinformatics revealed that genes coding for mitochondrial‐ and redox‐related proteins are under NFAT1 control and indicated that TMX1, TMX3, and NFAT1 are associated with poor disease outcome. Our study unravels a novel redox‐controlled ER–mitochondria–NFAT1 signaling loop that regulates melanoma pathobiology and provides biomarkers indicative of aggressive disease.

Antitumor and Apoptosis-inducing Effects of Piperine on Human Melanoma Cells.

Piperine is a major pungent alkaloid present in black pepper (Piper nigrum L). This study investigated the potential anticancer effects of piperine on human melanoma cells and explored the potential pharmacological mechanisms in vitro and in vivo. Studies were performed using the MTT assay, 4',6-diamidino-2-phenylindole (DAPI) staining, western blotting, a xenograft model, the terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and immunohistochemistry. Piperine inhibited the growth of melanoma cells. Several apoptotic events were observed following treatment, as revealed by DAPI staining. Piperine increased the expression of BCL2-associated X, apoptosis regulator (BAX), cleaved poly(ADP-ribose)polymerase, cleaved caspase-9, phospho-c-Jun N-terminal kinase and phospho-p38, and reduced that of B-cell lymphoma 2 (BCL2), X-chromosome-linked inhibitor of apoptosis, and phospho-extracellular signal-regulated protein kinase (ERK1/2) in a concentration-dependent manner. Treatment of mice for 4 weeks with piperine inhibited tumor growth without apparent toxicity. Piperine increased the expression of apoptotic cells and cleaved-caspase-3 protein and reduced the expression of phospho-ERK1/2 protein in melanoma tumors. Piperine suppressed the growth of human melanoma cells by the induction of apoptosis via the inhibition of tumor growth of human melanoma cells and tumor xenograft models.

Smart pH-Responsive Nanocube-Controlled Delivery of DNA Vaccine and Chemotherapeutic Drugs for Chemoimmunotherapy.

The combination of chemotherapeutic agents with immune stimulating agents for treating degenerative diseases, called chemoimmunotherapy, has emerged as a promising cancer treatment modality. Despite the tremendous potential, chemoimmunotherapy by the combination of drugs and immune stimulators often suffers because of the lack of controlled delivery nanostructures in the microenvironment. To this end, we show that by using pH-responsive smart nanocubes (NCs), cancer cells and tumor-associated immune cells can be precisely targeted with a chemotherapeutic agent (doxorubicin, DOX) and immune stimulating agent (plasmid ovalbumin, pOVA) for enhanced chemoimmunotherapy. The pH-responsive smart NCs protect payloads from nuclease degradation and avoid renal clearance and undergo supersensitive structural change at the extracellular tumor regions that mediate efficient release. Concurrent release of pOVA vaccines encoding tumor-specific antigen laden with polyplexes were loaded on tumor-associated immune cells and produce antigen-specific humoral immune response, whereas DOX enables effective infiltration into the cancer cells and is involved in the eradication of tumor tissues. The amount of anti-OVA IgG1 antibody produced by the intravenous administration of NC formulation was similar to that of free OVA formulation. Importantly, the combined delivery of pDNA and DOX using NCs showed significantly enhanced antitumor efficacy in B16/OVA melanoma tumor xenografts, which remarkably outperforms the monotherapy counterparts. These results suggest that pH-responsive smart NCs laden with pDNA and DOX provide a promising nanostructure for chemoimmunotherapy that simultaneously involves cancer cell killing and stimulates antigen-specific immune response to prevent cancer recurrence.

MiR-145-5p inhibits tumor occurrence and metastasis through the NF-κB signaling pathway by targeting TLR4 in malignant melanoma.

Compelling evidence shows that deregulated microRNAs (miRNAs) are important regulators in the progression of melanoma. miR-145-5p has been suggested to exhibit antitumorigenic activity in melanoma. However, the molecular mechanism underlying the biological activity of miR-145-5p in melanoma remains to be further understood. Herein, quantitative real-time polymerase chain reaction was used to examine the miR-145-5p expression in malignant melanoma tissues and cells. The interaction between miR-145-5p and toll-like receptor 4 (TLR4) was explored by bioinformatics analyses, luciferase reporter assay, and Western blot. The effects of miR-145-5p or combined with TLR4 on cell proliferation, colony formation, migration, and invasion abilities were investigated by (4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, colony formation, wound healing, and transwell assays, respectively. The melanoma xenograft tumor models were established to determine the biological activity of miR-145-5p in melanoma in vivo. In addition, the changes of the nuclear factor kappa B (NF-κB) pathway were analyzed by detecting the NF-κB activity and the NF-κB p65 protein level. We observed that the miR-145-5p expression was underexpressed in melanoma tissues and cells. miR-145-5p suppressed the TLR4 expression by binding to its 3'untranslated region in melanoma cells. Moreover, TLR4 overexpression abolished the inhibition of cell proliferation, colony formation, migration, and invasion abilities induced by miR-145-5p in melanoma cells. Meanwhile, miR-145-5p was confirmed to restrain melanoma tumor growth in vivo by targeting TLR4. Furthermore, miR-145-5p overexpression inactivated the NF-κB pathway in melanoma in vitro and in vivo, which was reversed by TLR4 overexpression. We concluded that miR-145-5p hindered the occurrence and metastasis of melanoma cells in vitro and in vivo by targeting TLR4 via inactivation of the NF-κB pathway.

Establishment of Melanoma Tumor Xenograft Using Single Cell Line Suspension and Co-injection of Patient-Derived T Cells in Immune-Deficient NSG Mice.

When primary tumor cells are grown in vitro, they are exposed to an environment that is vastly different from the tumor environment they originate from. The in vitro environment can lack the three-dimensional structure of the tumor, other cell types present within the tumor microenvironment, and important growth factors. Humanized mouse models allow researchers to study primary tumor cells in a more natural environment. With further development of several strains of immune-deficient mice, the mouse model allows for observation of the patient-derived tumor xenograft (PDTX) growth alone as well as in the presence of a human immune system. We describe how this can be accomplished with injection of single cell suspension of melanoma tumor cells into immune-deficient NOD-scid IL2Rγnull (NSG) mice. We also describe how tumor cells and immune cells can be co-injected, using Winn assay, and the possibility to use that method to study immune therapies for cancer.