Rezafungin acetate is a novel echinocandin in clinical development for prevention and treatment of invasive fungal infections. Rezafungin is differentiated by a pharmacokinetic/pharmacodynamic (PK/PD) profile that includes a long half‐life allowing once‐weekly administration, front‐loaded plasma drug exposures associated with antifungal efficacy, and penetration into deep‐seated infections, such as intra‐abdominal abscesses. In this series of in vivo studies, rezafungin demonstrated efficacy in the treatment of neutropenic mouse models of disseminated candidiasis, including infection caused by azole‐resistant Candida albicans, and aspergillosis. These results contribute to a growing body of evidence demonstrating the antifungal efficacy and potential utility of rezafungin in the treatment of invasive fungal infections.

In order to develop novel κ agonists restricted to the periphery, a diastereo- and enantioselective synthesis of (4aR,5S,8aS)-configured decahydroquinoxalines 5-8 was developed. Physicochemical and pharmacological properties were fine-tuned by structural modifications in the arylacetamide and amine part of the pharmacophore as well as in the amine part outside the pharmacophore. The decahydroquinoxalines 5-8 show single-digit nanomolar to subnanomolar κ-opioid receptor affinity, full κ agonistic activity in the [S]GTPγS assay, and high selectivity over μ, δ, σ, and σ receptors as well as the PCP binding site of the NMDA receptor. Several analogues were selective for the periphery. The anti-inflammatory activity of 5-8 after topical application was investigated in two mouse models of dermatitis. The methanesulfonamide 8a containing the (S)-configured hydroxypyrrolidine ring was identified as a potent (K = 0.63 nM) and highly selective κ agonist (EC = 1.8 nM) selective for the periphery with dose-dependent anti-inflammatory activity in acute and chronic skin inflammation.

Despite advancements of immunotherapy against various cancers, breast cancer still retains poor response to immune checkpoint blockade (ICB) therapy. Therefore, the identification of promising target or new strategy to enhance ICB therapy in breast cancer is crucial. Here, we uncover that the DNA-damaging potential of tamoxifen (TAM) can shape the unfavorable but ready-to-fire tumor immune microenvironment in breast cancer. We discover that a long-term TAM administration unexpectedly induces JAK/STAT signaling and Type I interferon (IFN)-stimulated gene expression to promote T-cell infiltration. Furthermore, TAM also induces the expression of CEACAM1, which acts as an alternative T-cell inhibitory ligand via binding to TIM-3, a “checkpoint” receptor expressed on CD4+ and CD8+ T cells. The chromatin ‘‘reader’’ RACK7/ZMYND8, which functions as a transcriptional repressor of IFN-stimulated gene (ISG) and a critical factor in DNA repair, is found to be downregulated upon tamoxifen exposure and involved in the tamoxifen-mediated cellular modulation. We demonstrate that TAM in conjunction with RACK7-knockdown (KD) triggers robust upregulation of ISGs and CEACAM1 in both estrogen receptor-positive (ER+) and triple negative breast cancer (TNBC) cells. This immunomodulatory effect lead by loss of RACK7 is specific to TAM treatment, and is not observed when combined with other endocrine therapeutics. TAM combined with RACK7-KD promotes mitochondrial DNA damage, which leads to accumulation of cytosolic DNA and subsequent activation of the cGAS/STING pathway. The murine breast orthotopic models with TS/A, EO771 and 4T1 cells further demonstrate that TAM-mediated immunomodulatory in conjunction with RACK7-KD evokes cytokine/chemokine secretion and further induces T-cell infiltration into tumor microenvironment. However, the tumor killing effect is limited due to promotion of T-cell exhaustion from CEACAM1-TIM-3 interaction between tumor and T cells. Thus, our study indicates that targeting CEACAM1-TIM-3 interaction is crucial for TAM-mediated tumor immune response. This brings promising therapeutic approach with TAM by combining RACK7-KD with blockage to CEACAM1-TIM-3 interaction in breast cancer. The resistance of tamoxifen treatment may be overcome, and RACK7 may serve as both a therapeutic target and a biomarker to enable ICB therapy. Citation Format: Marvin A. Aberin, Yu-Ling (Pony) Lee, Xin-Guo Hsu, Chen-Yang Shen, Yao-Ming Chang, Kun-Yuan Lin, Chandan Guha, Shu-Ping Wang. New functional role of tamoxifen in breast cancer immunomodulation: role of RACK7 in activation of type I interferon signaling and CEACAM1/TIM-3-dependent immunosuppression. [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 3646.

1084 Background: Brain metastasis is the most common malignancy of the central nervous system which causes severe morbidity and mortality in multiple cancer types of patients and represents an unmet medical need. Several critical steps are required for a successful brain metastasis, including local invasion, intravasation, dissemination, extravasation, and colonization. Extensive research has been conducted to elucidate the mechanism of cancer metastasis with limited information toward how cancer cells extravasate and colonize. Methods: To understand the underlying molecular mechanisms and applications of molecular targets for brain metastasis therapy, murine models are employed for investigation of brain extravasation and colonization. To investigate the roles of SQLE in breast-to-brain metastasis in vivo, we silenced SQLE expression directly with lentiviral shRNA in the brain metastatic MDA-MB-231-BR cell line (231-BR/shSQLE) and used 231-BR cells expressing a scramble shRNA (231-BR/shScr) as the control. Brain metastases were induced by intracardiac, orthotopic, or direct intracranial injections of 231-BR/shSQLE or 231-BR/shScr cells into immune deficient mice. The essential roles of SQLE in the specific step(s) of breast-to-brain metastatic process were evaluated by ex vivo immunofluorescence analysis of brain slices from the animals. To verify SQLE as an oncogenic factor that can be selected as a potential therapeutic target in suppressing breast-to-brain metastasis, we evaluated the inhibitory effects of NB-598 (a SQLE inhibitor) in both the 231-BR orthotopic and intracardiac mouse models. Results: Recently, we identified a novel mechanism by which squalene epoxidase (SQLE), the second rate-limiting enzyme in the cholesterol biosynthesis, plays a critical role in the processes of breast cancer metastasized to the brain, especially in brain extravasation and colonization. Interestingly, the pharmacologic inhibition of SQLE has been widely used against fungal infections, and the next-generation SQLE inhibitors have been recently shown to exert an anticancer effect. Our data demonstrated that SQLE is essential for 231-BR cells to extravasate into the parenchyma as well as the formation of micro and macro-metastases in the brain. Interestingly, we found that astrocytes play a key role in supporting 231-BR-developed brain macro-metastasis. In vitro blood-brain barrier (BBB) models further demonstrated the critical roles of SQLE in promoting 231-BR cell invasion and penetration through BBB. Inhibition of SQLE by NB-598 demonstrated anti-tumor proliferation and anti-metastasis. Conclusions: Pharmacologic inhibition of SQLE by NB-598 suppressed 231-BR tumor growth at the mammary fat pad and distal metastases to organs, suggesting that targeting SQLE represents a therapeutic opportunity for breast cancer metastases.

e13101 Background: Brain metastases are serious complications of breast cancer and there is no effective treatment. The major therapeutic issues can be attributed to the unique brain microenvironment as well as the difficulty of drug delivery to the brain due to the blood-brain barrier (BBB). BBB limits access of nutrients from the circulation and thereby makes the brain in a special condition that is hypoxic and composed of depleted metabolites, growth factors, and proteins. Nevertheless, tumor cells that are able to metastasize to the brain can necessitate metabolic adaptations for their nutrient availability and thereby succeed to colonize in the brain. However, it is still poorly understood how breast cancer cells alter their metabolic systems to manipulate brain metastasis by overcoming nutritional limitations. Methods: To probe the molecular mechanism of breast cancer to brain metastasis, we compared gene signatures in primary breast tumors and breast-to-brain metastatic tumors, and identified that SQLE (encodes squalene epoxidase, the second rate-limiting enzyme in the cholesterol biosynthesis) could be a leading-edge gene in breast-to-brain metastasis. Using the brain metastatic MDA-MB-231-BR (231-BR) and its parental MDA-MB-231 (231) cell line expressing SQLE shRNAs, we evaluated the effects of SQLE loss on cancer cell migration, invasion, and stemness by wound-healing, transwell invasion/migration, and tumorsphere formation assays. The in vitro cell-based BBB model and in vivo mouse models that develop 231-BR brain metastases were established to verify our hypothesis. Results: While loss of SQLE greatly attenuated cell invasiveness and stemness in both 231 and 231-BR cells, loss of SQLE could only affect the cell migration activity on 231 cells but not 231-BR cells. Since the ability to invade, migrate, and penetrate is critical for invasion of cancer cells, our results strongly imply the novel function of SQLE in breast cancer cell invasion, penetration, and colonization in the brain. Our RNA-seq data further identified a subset of SQLE-affected genes that is uniquely enriched in 231-BR cells and favors brain extravasation and colonization. The in vitro cell-based BBB model and in vivo mouse models that develop 231-BR brain extravasation and colonization finally verified our hypothesis that SQLE may play a unique function to promote breast cancer metastasis into the brain. Conclusions: Our findings provide new insights into contributions by SQLE in breast-to-brain extravasation and colonization and indicate that targeting SQLE may represent a therapeutic opportunity for breast cancer brain metastases.