Breast cancer stem cells (BCSCs) constitute a small population of cells within breast cancer and are characterized by their ability to self-renew, differentiate, and recapitulate the heterogeneity of the tumor. Clinically, BCSCs have been correlated with cancer progression, metastasis, relapse, and drug resistance. The tumorigenic roles of BCSCs have been extensively reviewed and will not be the major focus of the current review. Here, we aim to highlight how the crucial intrinsic signaling pathways regulate the fate of BCSCs, including the Wnt, Notch, Hedgehog, and NF-κB signaling pathways, as well as how different cell populations crosstalk with BCSCs within the TME, including adipocytes, endothelial cells, fibroblasts, and immune cells. Based on the molecular and cellular activities of BCSCs, we will also summarize the targeting strategies for BCSCs and related clinical trials. This review will highlight that BCSC development in breast cancer is impacted by both BCSC endogenous signaling and external factors in the TME, which provides an insight into how to establish a comprehensively therapeutic strategy to target BCSCs for breast cancer treatments.
Immune checkpoint inhibitors (ICIs) use antibodies that block cell surface immune checkpoint proteins with great efficacy in treating immunogenic or “immune hot” tumors such as melanoma, kidney, and lung adenocarcinoma. ICIs have limited response rates to other non-immunogenic cancers. The tumor microenvironment (TME) consists of many cell types that collectively promote tumor progression. Cancer therapeutics are commonly designed to target one molecule in one defined cell type. There is growing evidence that long-term therapeutic responses require the targeting of cancer cells and tumor-promoting populations within the TME. The question remains whether we can identify targetable molecules/pathways that are critical for multiple cell types. Here, we will discuss several molecular targets that may fit a “two or multiple birds, one stone” model, including the B-cell lymphoma-2 (BCL-2) family pro-survival factors, transcriptional factors including signal transducer and activator of transcription 3 (STAT3), the nuclear receptor 4A family (NR4A1, NR4A2, NR4A3), as well as epigenetic regulators such as bromodomain and extra-terminal (BET) family proteins, histone deacetylase (HDAC) family, SET domain bifurcated histone lysine methyltransferase 1 (SETDB1) and lysine-specific demethylase 1 (LSD1/KDM1A). We will focus on the rationale of these targets in immune modulation, as well as the strategies for targeting these important proteins for cancer therapy.
Introduction: Modulating tumor-infiltrating immune cells is the key mission for the development of cancer immunotherapy. However, current immunotherapies including immune-checkpoint inhibitors (ICI) have limited patient response rates and some with adverse effects, which drives us to seek novel targets to improve immunotherapeutic efficiency. Based on recent literature and our bioinformatic analysis, we believe that NR4A1 is an ideal target for cancer immunotherapy due to its important role in T cell exhaustion, Treg cell function as well as neoangiogenesis. Here we developed an immunotherapeutic strategy that targets nuclear receptor subfamily 4 group A member 1 (NR4A1) via proteolysis-targeting chimeric (PROTAC) technology, with an expectation that NR4A1 degradation leads to immune activation and cancer clearance via the inhibition of Treg cell function as well as the activation of effector T cells. Results: We designed, synthesized, and screened over 80 NR4A1 PROTACs based on several reported NR4A1 ligands, including celastrol, cytosporine B, and diindolylmethane analogs. We found that several celastrol-based PROTACs achieved effective degradation of target protein NR4A1. Our current focus is to understand the mechanism of action of the lead NR-V04, a celastrol- and von Hippel-Lindau (VHL)-based PROTAC. We confirmed that the NR-V04-induced NR4A1 degradation is through the proteasome- and VHL-dependent mechanisms as expected and further demonstrated the NR-V04-induced ternary complex formation between NR4A1, VHL, and NR-V04. The lead NR-V04 exhibits outstanding pharmacokinetics and effectively inhibits tumor growth at a very low dose, 1.8mg/kg twice a week, in several tumor models including B16F10, Yummer 1.7, and MC38. Interestingly, we found that NR-V04 significantly enhances anti-tumor immunity in previously mentioned mouse tumor models with seemingly distinct mechanisms of immune activation in a tumor-dependent manner. For example, NR-V04 induces the expansion of B cells and the reduction in myeloid-derived suppressor cells (MDSC) in B16F10; the reduction of Tregs, and the induction of CD8 effector population in Yummer 1.7 and MC38 tumors. we did not find obvious toxicity at these effective doses for up to 2 months. Conclusion: The lead NR-V04 is a very promising agent that induces specific degradation of NR4A1 in tumors, resulting in tumor type-dependent immune activation and tumor reduction. Targeting NR4A1 within the tumor microenvironment is effective and safe. Therefore, the lead NR-V04 or other better ones under test have the potential for translation as a novel immunotherapeutic strategy. Citation Format: Lei Wang, Yufeng Xiao, Rohan P. Master, Zeng Jin, Urvi M. Patel, Yuewan Luo, Daohong Zhou, Guangrong Zheng, Weizhou Zhang. Proteolysis-targeting chimera against NR4A1 for cancer immunotherapy [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 2326.
Introduction: Melanoma originates from melanocytes within the epidermis and is one of the most common cancers, with nearly 100000, new cases yearly. Despite numerous advancements in therapies to treat melanoma, a prevalent population of patients still do not respond to currently approved therapies. Recent studies have highlighted the role of transcription factor nuclear receptor subfamily 4 group A member 1 (NR4A1) in melanoma for cancer survival, invasion, and metastasis. NR4A1 is also involved in glucose metabolism. Our project aims to identify an effective degrader of NR4A1 using a PROTAC strategy and then validate its effectiveness in reducing the growth of melanoma. PROTACs consists of three domains: a warhead that binds to the protein of interest, a ligand to an E3 ligase, and a linker that brings both domains in proximity to one another. The PROTAC can recruit an E3 ligase to ubiquitinate NR4A1 and degrade it via the ubiquitin-proteasome system (UPS). Results: Our first goal was to identify valid PROTAC candidates that can effectively degrade NR4A1. We identified NR-V04, which demonstrated a dose-dependent degradation of NR4A1 in various melanoma cell lines. We further investigated time-dependent degradation, and NR-V04 was able to achieve in vitro degradation of NR4A1 16 hours after treatment. Additionally, we validated the mechanism of degradation via the UPS through various models. NR-V04 treatment of cells with a VHL knockout, which removes the E3 ligase recruited, experienced no degradation of NR4A1, and treatment of cells treated with MG132, a proteasome inhibitor, also showed no degradation. We observed degradation in vitro of numerous mouse and human cell lines. To investigate the cancer-killing effects of NR-V04, we completed MTS assays on the human melanoma cell lines CHL1 and A375, which showed an EC50 of 0.723 μM and 1.025 μM, respectively. When comparing NR-V04 treated and untreated CHL1 cells, NR-V04 was able to significantly decrease melanoma cell viability. NR4A1 knockout in CHL1 also showed decreased melanoma cell viability, and when comparing NR-V04 treated and untreated in NR4A1 knockout, there was no further decrease in melanoma cell viability. As for in vivo models, NR4A1 knockout in CHL1 and A375 exhibited slower tumor growth compared to the wild type. Furthermore, NR-V04 showed suppression of CHL1 and A375 tumor-bearing NSG mice melanoma growth at low dose concentrations of 1 mg/kg after seven days compared to vehicle and warhead treatment. Western blot analysis of tumor tissue provides support for the ability of NR-V04 to degrade tumor-intrinsic NR4A1. Conclusion: NR-V04 can selectively degrade NR4A1, in vitro and in vivo, to decrease melanoma cancer cell viability via the UPS. NR-V04 holds promising therapeutic potential as a cancer therapy for patients with melanoma. Citation Format: Rohan Master, Yuewan Luo, Yufeng Xiao, Daohong Zhou, Lei Wang, Guangrong Zheng, Weizhou Zhang. Developing a PROTAC-based NR4A1 degrader for melanoma cancer therapy. [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 4451.
Modulating tumor-infiltrating immune cells is the key mission for the development of cancer immunotherapy. Based on recent literature and our bioinformatic analysis, nuclear receptor subfamily 4 group A member 1 (NR4A1) is an ideal target for cancer immunotherapy due to its important role in T cell exhaustion, regulatory T (Treg) cell function. Here we targets NR4A1 via proteolysis-targeting chimeric (PROTAC) technology, expecting to achieve immune activation and cancer clearance. We designed and synthesized over 80 NR4A1 PROTACs based on several reported NR4A1 ligands, such as celastrol. We found that several celastrol-based PROTACs achieved effective degradation of target protein NR4A1. We studied the therapeutic efficiency of the lead NR-V04, a celastrol- and von Hippel-Lindau (VHL)-based PROTAC. As expected, we confirmed that the NR-V04-induced NR4A1 degradation is through the uniquitin proteasome system with a tenery complex formation among NR4A1, VHL and NR-V04. The lead NR-V04 exhibits outstanding pharmacokinetics and effectively inhibits tumor growth at a low dose, 1.8mg/kg twice a week, in B16F10, Yummer 1.7, and MC38 mouse tumor models. We proved that NR-V04 significantly enhances anti-tumor immunity with seemingly distinct mechanisms of immune activation in a tumor-dependent manner. For example, NR-V04 induces the expansion of B cells and the reduction in myeloid-derived suppressor cells (MDSC) in B16F10; the reduction of Tregs, and the induction of CD8 effector population in Yummer 1.7 and MC38 tumors. we did not find obvious toxicity at these effective doses for up to 2 months. Therefore, the lead NR-V04 or other better ones under test have the potential for translation as a novel immunotherapeutic strategy. BC200100 - “Developing a Novel PROTAC-Based NR4A1 Degrader for Breast Cancer Therapy” (DEPARTMENT OF THE ARMY)
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