Targeting antiapoptotic proteins BCL-xL, BCL-2, and BCL-w has been extensively investigated for cancer treatment. However, robust inhibition of BCL-xL by conventional inhibitors, such as ABT-263, causes thrombocytopenia, a notable drawback that limits the clinical utility of this strategy. To overcome this on-target toxicity, BCL-xL-selective and BCL-xL/BCL-2 dual-targeting proteolysis targeting chimeras (PROTACs) have been developed as alternative therapeutic strategies. In this study, we report a new generation of ABT-263-based PROTACs designed to leverage a novel solvent-exposed region on the bis(sulfonyl)benzene ring of ABT-263, made accessible through regioselective electrophilic aromatic bromination. The lead compounds, 44 and 46, demonstrated effective degradation of BCL-xL and, unexpectedly, degraded BCL-w, while sparing BCL-2. With further optimization, these BCL-xL and BCL-w dual-targeting PROTACs hold great promise as safer, more effective anticancer agents against BCL-xL and BCL-w codependent cancers.

Abstract Critical challenges remain in the clinical management of diffuse large B-cell lymphoma (DLBCL), as up to 40% of patients experience disease relapse or refractoriness. Despite the use of novel therapies, relapsed/refractory DLBCL results in a high risk of mortality, especially in the activated B-cell-like (ABC) subtype. Developing new therapeutic options for those patients becomes a pressing and clinically unmet need. Here, we explore the combined targeting of histone deacetylase (HDAC) 3 and 8 by targeted protein degradation to combat epigenetic deregulation in DLBCL. Through a comprehensive screen using single or dual HDAC3/8-targeting compounds in 12 DLBCL cell lines, we discovered that a highly selective, potent, first-in-class dual HDAC3/8 proteolysis-targeting chimera (PROTAC) degrader YX968 potently suppressed cell survival and proliferation. This effect was particularly more prominent and significantly stronger than single HDAC3 or 8 PROTACs in the ABC-DLBCL cell lines. Further functional evaluations revealed that YX968 specifically induced caspase-3-mediated apoptosis and caused cell cycle arrest. Mechanistically, YX968 profoundly upregulated H3K27 acetylation (H3K27ac), a key epigenetic mark for enhancer regulation, and generated a unique transcriptomic profile with downregulated genes associated with cell cycle regulation and upregulated genes implicated in immune activation. Moreover, we validated the gene expression results using flow cytometry for upregulated surface protein expression of the antigen presentation markers MHC-I and II, and detected enhanced CD8+ T-cell-induced cytotoxicity by YX968 in an in vitro T-cell co-culture assay, indicating its immunomodulating activities to potentiate anti-tumor immunity. Although YX968 showed in vitro efficacy, we found that it had poor metabolic stability, which precluded its potential in vivo application. To enhance the translation value of our approach, we developed a second-generation dual HDAC3/8 PROTAC (YX226) with improved metabolic stability while maintaining potent degradation, cell-killing, and immunomodulating activities. Using global proteomics, we confirmed the on-target activity of YX226 to selectively degrade HDAC3 and 8 but not the other HDAC isoforms. Importantly, we observed significant in vivo HDAC3/8 protein degradation and tumor growth inhibition by YX226 in a xenograft lymphoma model without adverse reduction of body weight, providing proof-of-principle evidence for in vivo efficacy and safety of a dual HDAC3/8 PROTAC. In conclusion, our findings demonstrate that dual HDAC3/8 PROTAC degraders induce significant anti-tumor and immunomodulating effects in our preclinical DLBCL models, supporting further development and evaluation of our second-generation bioavailable compound YX226 as a novel treatment option for DLBCL. Citation Format: Michael Y. He, Yufeng Xiao, Mehran Bakhtiari, Ting Liu, Wenxi Xu, David G. Brooks, Housheng Hansen He, Guangrong Zheng, Robert Kridel. Dual HDAC3/8 PROTAC degraders exert anti-tumor and immunomodulating effects in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4602.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor clinical outcomes and limited therapeutic options. Aberrant activation of the IKKβ-NF-κB pathway occurs in approximately 40% of AML cases and contributes to leukemogenesis. However, pharmacological inhibition of IKKβ has been limited by serious toxicities, including neutrophilia. Here we identify IKKβ and NR4A1 as critical drivers of AML progression in certain models and develop a proteolysis-targeting chimera (PROTAC) capable of degrading the proteins. Although NR4A1 has previously been described as a tumor suppressor in AML, our findings demonstrate that NR4A1 exhibits oncogenic functions in some AMLs of the (pro)monocytic lineage. Notably, elevated expression of IKKβ and NR4A1 in AML is associated with poor clinical outcomes, playing non-redundant oncogenic roles in AML. To therapeutically target IKKβ and NR4A1, we designed and synthesized a series of celastrol-based PROTACs that exploit celastrol's ability to bind both IKKβ and NR4A1. Among these compounds, the lead A9 induces potent cytotoxicity in multiple AML cell lines and primary AML samples through cereblon E3 ligase-dependent degradation of IKKβ and/or NR4A1. In vivo, A9 suppresses leukemia progression in a KMT2A::MLLT3 AML mouse model without inducing neutrophilia, supporting PROTAC-mediated degradation of IKKβ and NR4A1 as a promising therapeutic strategy.

Abstract Proteolysis-targeting chimera (PROTAC) technology has emerged as a powerful therapeutic strategy in drug discovery. Conventional PROACs are heterobifunctional molecules composed of two distinct ligands that independently bind to a protein of interest (POI) and an E3 ligase. However, their inherently large molecular size often leads to suboptimal drug-like properties. In this study, we report a “2-in-1” PROTAC design strategy for developing more compact HDAC8 degraders by integrating the CRBN-recruiting ligand into the capping group of an HDAC8 warhead, thereby enabling a single structural motif to engage both HDAC8 and CRBN. Compared to our first generation HDAC8 degrader YX862, the new degraders exhibit improved selectivity, reduced molecular weight, improved overall drug-like properties, and, importantly, potent HDAC8 degradation in vivo. These findings highlight the therapeutic potential of this new class of HDAC8 degraders, and demonstrate a conceptual framework for integrating E3 ligase recruitment and target engagement into a more efficient and compact PROTAC design.

Proteolysis targeting chimeras (PROTACs) represent an emerging targeted cancer therapy approach. However, their poor cell penetration and instability in vivo pose daunting challenges for wide-spread clinical usage. To enhance the in vivo therapeutic efficacy of PROTACs, we introduced extracellular vesicles (EVs) for in vivo PROTAC delivery, which is leveraged by a novel microfluidic droplet-based EV electro-transfection system (μDES). We previously developed YX968 PROTAC, which can selectively degrade both HDAC3 and HDAC8 in triple negative breast cancer (TNBC) cells and effectively suppress the tumour cell growth without provoking global hyperacetylation. In this manuscript, we demonstrated that YX968 loaded EVs via the μDES system can retain the optimal integrity of drug loaded EVs with improved loading efficiency compared to other transfection approaches, which, in turn, significantly enhances the therapeutic function of PROTAC in vivo in TNBC mouse models. Intraperitoneal injections of YX968 loaded EVs led to significantly enhanced intratumoral degradation of HDAC3 and HDAC8 than YX986 alone, which resulted in advanced TNBC tumour inhibition without noticeable tissue toxicity. Such EV-based delivery strategy, with a scalable EV loading approach, enhanced the in vivo PROTAC drug stability and bioavailability and improved tissue penetration and targeting, filling an important gap in the clinical translation of PROTAC-based cancer therapy.

Acute myeloid leukemia (AML) is a common and aggressive blood cancer with the highest lethality rate among all leukemia subtypes. The cure rate of available therapeutic options is very low, underscoring an urgent need to develop novel and effective AML therapeutics. Here we identify IKKβ and NR4A1 as two closely related drivers of AML progression and develop a proteolysis targeting chimera (PROTAC) drug that has dual degradation activity against IKKβ and NR4A1. IKKβ and its downstream nuclear factor-κB (NF-κB) signaling are aberrantly activated in around 40% AML patients. However, nearly all IKKβ inhibitors have failed prior clinical trials due to their serious side effects such as neutrophilia and systematic inflammation. As opposed to the previously reported tumor suppressive role in AML, we found that NR4A1 promotes AML pathogenesis in a context-dependent manner. Here we designed, synthesized, and validated several celastrol-based PROTACs, with one lead compound A9 that effectively kills several AML cell lines and primary human AML cells via the degradation of IKKβ and NR4A1. At the mechanistic level, A9 degrades both targets through cereblon (CRBN) E3 ligase-mediated proteasomal system by forming ternary complexes with the target proteins and CRBN. More importantly, A9 attenuates AML disease progression in a clinically relevant KMT2A::MLLT3 mouse model and doesn't induce neutrophilia in vivo - a common side effect of IKKβ inhibitors. Our results reveal a potentially novel strategy to treat intractable and aggressive AMLs in the clinic. Key Points:IKKβ and NR4A1 are clinically relevant mediators of AML pathogenesis.A novel celastrol-based PROTAC can effectively degrade both IKKβ and NR4A1 to disrupt AML pathogenesis.

BRAF and MEK inhibitors are standard treatments for patients with BRAF V600E-mutated melanoma. However, resistance to these therapies poses a significant challenge. Understanding the molecular mechanisms underlying this resistance is critical for the development of more effective treatment strategies. To investigate the role of histone deacetylase 8 (HDAC8) in drug resistance in melanoma and evaluate the potential of HDAC8 degradation as a therapeutic strategy. We utilized overexpression vector to examine the effects of HDAC8 in melanoma cells. The proliferative, migratory, and drug sensitivity phenotypes were assessed in HDAC8-overexpressing melanoma cells. Additionally, we tested YX862, a selective HDAC8 degrader, to explore its effects on HDAC8 activity and downstream cellular processes. Acetylation of SMC3, a cohesin complex protein, was measured, and genome-wide HiC analysis was employed to assess chromatin structural changes. HDAC8 overexpression promoted a more proliferative and migratory phenotype in melanoma cells. Furthermore, HDAC8-overexpressing cells exhibited reduced sensitivity to BRAF and MEK inhibitors. YX862 treatment specifically degraded HDAC8, leading to increased acetylation of SMC3. Genome-wide HiC analysis revealed that changes in SMC3 acetylation altered the three-dimensional structure of chromatin, potentially contributing to drug resistance. These findings highlight the critical role of HDAC8 in melanoma progression and drug resistance. HDAC8 degraders like YX862 may offer a promising therapeutic approach to overcome resistance and inhibit melanoma progression by modulating key epigenetic and cellular pathways. Kanchan Gupta, Richard L. Bennett, Michael F. Emmons, Yufeng Xiao, Guangrong Zheng, Keiran S. Smalley, Jonathan D. Licht. HDAC8 over-expression promotes drug resistance and alters chromatin architecture in melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB419.

Orphan nuclear receptor 4A1 (NR4A1, Nur77) plays a crucial role in regulating immune cell metabolism and function within the tumor microenvironment (TME), thus influencing cancer progression and serving as a potential therapeutic target for cancer immunotherapy. A comprehensive review discussing the multifaceted roles of NR4A1 in immune cells and the exploitation of that knowledge for therapeutic development is lacking in the field. This review explores diverse functions of NR4A1 in tumor-associated immune cells, including T cells, monocytes, natural killer cells, B cells, dendritic cells, macrophages, and neutrophils. NR4A1 contributes to immune regulation by impacting cytokine production, cell differentiation, and immune cell exhaustion. We highlight how NR4A1 in immune cells within the TME may be either a positive (e.g., macrophages in colon cancer) or negative prognostic factor (e.g., T cells in melanoma), depending on the cancer and immune cell context. Additionally, this review also highlights potential therapeutic strategies targeting NR4A1, leading to its inhibition, activation, or degradation to restore immune cell function and enhance anti-tumor immunity. Such therapies could potentially improve patient outcomes by altering immune cell behaviors, blocking intrinsic tumor growth pathways, or via both mechanisms. However, the development of NR4A1-targeted therapies will be dependent on further research to better understand lineage-specific roles of NR4A1 and the underlying mechanisms across different cancer types and immune cells.

Targeted protein degradation (TPD), including the use of proteolysis-targeting chimeras (PROTACs) and molecular glue degraders (MGDs) to degrade proteins, is an emerging strategy to develop novel therapies for cancer and beyond. PROTACs or MGDs function by inducing the proximity between an E3 ligase and a protein of interest (POI), leading to ubiquitination and consequent proteasomal degradation of the POI. Notably, one major issue in TPD is the lack of ligandable E3 ligases, as current studies predominantly use CUL4CRBN and CUL2VHL. The TPD community is seeking to expand the landscape of ligandable E3 ligases, but most discoveries rely on phenotypic screens or serendipity, necessitating systematic target deconvolution. Here, we examine and discuss both current and emerging E3 ligase deconvolution approaches for degraders discovered from phenotypic screens or monovalent glue chemistry campaigns, highlighting future prospects for identifying more ligandable E3 ligases.

Despite selective HDAC3 inhibition showing promise in a subset of lymphomas with CREBBP mutations, wild-type tumors generally exhibit resistance. Here, using unbiased genome-wide CRISPR screening, we identify GNAS knockout (KO) as a sensitizer of resistant lymphoma cells to HDAC3 inhibition. Mechanistically, GNAS KO-induced sensitization is independent of the canonical G-protein activities but unexpectedly mediated by viral mimicry-related interferon (IFN) responses, characterized by TBK1 and IRF3 activation, double-stranded RNA formation, and transposable element (TE) expression. GNAS KO additionally synergizes with HDAC3 inhibition to enhance CD8+ T cell-induced cytotoxicity. Moreover, we observe in human lymphoma patients that low GNAS expression is associated with high baseline TE expression and upregulated IFN signaling and shares common disrupted biological activities with GNAS KO in histone modification, mRNA processing, and transcriptional regulation. Collectively, our findings establish an unprecedented link between HDAC3 inhibition and viral mimicry in lymphoma. We suggest low GNAS expression as a potential biomarker that reflects viral mimicry priming for enhanced response to HDAC3 inhibition in the clinical treatment of lymphoma, especially the CREBBP wild-type cases.

Abstract An effective cancer therapy requires both killing cancer cells and targeting tumor-promoting pathways or cell populations within the tumor microenvironment (TME). We purposely search for molecules that are critical for multiple cell types in the TME and identified nuclear receptor subfamily 4 group A member 1 (NR4A1) as one such molecule. NR4A1 has been shown to promote the aggressiveness of cancer cells and maintain the immune suppressive TME. Using genetic and pharmacological approaches, we establish NR4A1 as a valid therapeutic target for cancer therapy. Importantly, we have developed the first-of-its kind proteolysis-targeting chimera (PROTAC, named NR-V04) against NR4A1. NR-V04 effectively degrades NR4A1 within hours of treatment in vitro and sustains for at least 4 days in vivo, exhibiting long-lasting NR4A1-degradation in tumors and an excellent safety profile. NR-V04 leads to robust tumor inhibition and sometimes eradication of established melanoma tumors. At the mechanistic level, we have identified an unexpected novel mechanism via significant induction of tumor-infiltrating (TI) B cells as well as an inhibition of monocytic myeloid derived suppressor cells (m-MDSC), two clinically relevant immune cell populations in human melanomas. Overall, NR-V04-mediated NR4A1 degradation holds promise for enhancing anti-cancer immune responses and offers a new avenue for treating various types of cancer such as melanoma. Citation Format: Lei Wang, Yufeng Xiao, Yuewan Luo, Rohan Master, Jiao Mo, Myung-Chul Kim, Yi Liu, Chandra Maharjan, Urvi Patel, Xiangming Li, Donald Shaffer, Guertin Kevin, Haoyang Zhuang, Emily Moser, Keiran Smalley, Daohong Zhou, Guangrong Zheng, Weizhou Zhang. PROTAC mediated NR4A1 degradation as a novel strategy for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2469.

An effective cancer therapy requires killing cancer cells and targeting the tumor microenvironment (TME). Searching for molecules critical for multiple cell types in the TME, we identified NR4A1 as one such molecule that can maintain the immune suppressive TME. Here, we establish NR4A1 as a valid target for cancer immunotherapy and describe a first-of-its-kind proteolysis-targeting chimera (PROTAC, named NR-V04) against NR4A1. NR-V04 degrades NR4A1 within hours in vitro and exhibits long-lasting NR4A1 degradation in tumors with an excellent safety profile. NR-V04 inhibits and frequently eradicates established tumors. At the mechanistic level, NR-V04 induces the tumor-infiltrating (TI) B cells and effector memory CD8(+) T (Tem) cells and reduces monocytic myeloid-derived suppressor cells (m-MDSC), all of which are known to be clinically relevant immune cell populations in human melanomas. Overall, NR-V04-mediated NR4A1 degradation holds promise for enhancing anticancer immune responses and offers a new avenue for treating various types of cancers such as melanoma.

Proteolysis targeting chimeras (PROTACs) are an emerging targeted cancer therapy approach, but wide-spread clinical use of PROTAC is limited due to poor cell targeting and penetration, and instability in vivo. To overcome such issues and enhance the in vivo efficacy of PROTAC drugs, microfluidic droplet-based electroporation (µDES) was developed as a novel extracellular vesicle (EVs) transfection system, which enables the high-efficient PROTAC loading and effective delivery in vivo. Our previously developed YX968 PROTAC drug had shown the selectively degradation of HDAC3 and 8, which effectively suppresses the growth of breast tumor cell lines, including MDA-MB-231 triple negative breast cancer (TNBC) line, via dual degradation without provoking a global histone hyperacetylation. In this study, we demonstrated that µDES-based PROTAC loading in EVs significantly enhanced therapeutic function of PROTAC drug in vivo in the TNBC breast tumor mouse model. NSG mice with pre-established MDA-MB-231 tumors and treated with intraperitoneal injection of EVs for tumor inhibition study, which showed significantly higher HDAC 3 and 8 degradation efficiency and tumor inhibition than PROTAC only group. The liver, spleen, kidney, lung, heart, and brain were collected for safety testing, which exhibited improved toxicity. The EV delivery of PROTAC drug enhances drug stability and bioavailability in vivo, transportability, and drug targeting ability, which fills an important gap in current development of PROTAC therapeutic functionality in vivo and clinical translation. This novel EV-based drug transfection and delivery strategy could be applicable to various therapeutics for enhancing in vivo delivery, efficacy, and safety.

Abstract HDAC8 plays crucial roles in biological processes and is a highly desirable target for therapeutic interventions. However, due to the conserved catalytic domain among HDACs, developing specific inhibitors for these isozymes has proven challenging. HDAC8 also has deacetylase-independent activity which cannot be blocked by an inhibitor. Previously we reported the discoveries of a potent HDAC3 degrader XZ9002 and an HDAC3/8 dual degrader YX968. In this study, we carried out further optimizations based on the warhead of YX968 through rational design, which led to the discovery of YX862 and YL246, novel hydrazide-based HDAC8 selective PROTAC degraders with single-digit nanomolar DC50 and excellent selectivity. We demonstrated that the degradation of HDAC8 affects its non-histone substrates; however, it does not trigger profound histone hyper-acetylation and gene expression alteration, highlighting the unique role of HDAC8. The PROTACs developed in this study are well-characterized HDAC8 degraders without triggering pan-HDAC inhibition which represent valuable tool compounds for exploring the biological and therapeutic potential of HDAC8 in cancers and beyond. Citation Format: Yufeng Xiao, Yi Liu, Nikee Awasthee, Chengcheng Meng, Michael He, Seth Hale, Rashmi Karki, Zongtao Lin, Robert Kridel, Daiqing Liao, Guangrong Zheng. Discovery of hydrazide-based HDAC8 selective PROTACs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3236.

HDAC8 plays crucial roles in biological processes, from gene regulation to cell motility, making it a highly desirable target for therapeutic intervention. HDAC8 also has deacetylase-independent activity which cannot be blocked by a conventional inhibitor. In this study, we report the discovery of YX862, a highly potent and selective hydrazide-based HDAC8-proteolysis targeting chimera (PROTAC) degrader. The selectivity is achieved through rational design of the warhead to spare HDAC3 activity from the previous HDAC3/8 dual degrader YX968. We demonstrate that the degradation of HDAC8 by YX862 increases acetylation levels of its nonhistone substrates such as SMC3 without significantly triggering histone PTM, supporting HDAC8's major role in nonhistone PTM regulation. YX862 exhibits promising on-target antiproliferative activity against DLBCL cells with higher potency than the HDAC8 selective inhibitor PCI-34051. As a selective HDAC8 degrader that avoids pan-HDAC inhibition, YX862 represents a valuable tool for exploring the biological and therapeutic potential of HDAC8.

Histone deacetylases (HDACs) are enzymes that play an essential role in multiple cellular processes such as DNA transcription, translation, replication, recombination, repair, and metabolism, and their dysregulation can be linked to many different diseases. Most commonly, the overexpression of HDACs is found in various cancer types including hematologic cancers, as well as solid malignancies. Of the many HDACs found in the body, class I HDACs, which consist of HDACs 1, 2, 3, and 8, play an essential role in activating oncogenes underlying tumorigenesis, disease progression, and treatment resistance. Several HDAC inhibitors (HDACis) have been approved for cancer treatment, however, they are pan-inhibitors. This lack of specificity poses many disadvantages for HDACis, including toxicity and other off-target effects. Isozyme-selective inhibitors may reduce these off-target effects and thus enhance their safety. Though favorable, selectivity is difficult to achieve from conventional inhibitors due to the highly homologous catalytic domain among HDAC isozymes. In addition, several HDAC isozymes have deacetylase-independent scaffolding functions that cannot be blocked by traditional inhibitors. Recently, there have been advances in Proteolysis Targeting Chimera (PROTAC), an emerging drug discovery technology designed to hijack cell’s existing protein degradation machinery, the ubiquitin-proteasome system (UPS) to selectively degrade target proteins. Selective degradation by PROTAC is a potential solution to many of the concerns associated with current HDACis. Previously, we reported an HDAC3-selective PROTAC, XZ9002, and following that we reported the discovery of PROTAC YX968, which can degrade both HDAC3 and HDAC8 isozymes with single-digit nanomolar DC50, this highly potent dual degrader exhibits distinct effects on modulating gene expression and is much more potent in inhibiting cancer cell proliferation compared to XZ9002. Based on this, we are further modifying the PROTACs to be selective for HDAC8. The HDAC3, HDAC8 selective degrader and HDAC3 and HDAC8 dual degrader we developed could be useful chemical probes to dissect the complex biological function of HDAC3 and HDAC8 and potential therapeutics for treating cancer. Citation Format: Yufeng Xiao, Seth Hale, Nikee Awasthee, Xuan Zhang, Yi Liu, Zhiguang Huo, Dongwen Lyu, Lei Wang, Weizhou Zhang, Megan Mosteiro, Daiqing Liao, Guangrong Zheng. Selective targeting deacetylase 3 (HDAC3) and HDAC8 by PROTACs. [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 5347.

An effective cancer therapy requires both killing cancer cells and targeting tumor-promoting pathways or cell populations within the tumor microenvironment (TME). We purposely search for molecules that are critical for multiple tumor-promoting cell types and identified nuclear receptor subfamily 4 group A member 1 (NR4A1) as one such molecule. NR4A1 has been shown to promote the aggressiveness of cancer cells and maintain the immune suppressive TME. Using genetic and pharmacological approaches, we establish NR4A1 as a valid therapeutic target for cancer therapy. Importantly, we have developed the first-of-its kind proteolysis-targeting chimera (PROTAC, named NR-V04) against NR4A1. NR-V04 effectively degrades NR4A1 within hours of treatment in vitro and sustains for at least 4 days in vivo, exhibiting long-lasting NR4A1-degradation in tumors and an excellent safety profile. NR-V04 leads to robust tumor inhibition and sometimes eradication of established melanoma tumors. At the mechanistic level, we have identified an unexpected novel mechanism via significant induction of tumor-infiltrating (TI) B cells as well as an inhibition of monocytic myeloid derived suppressor cells (m-MDSC), two clinically relevant immune cell populations in human melanomas. Overall, NR-V04-mediated NR4A1 degradation holds promise for enhancing anti-cancer immune responses and offers a new avenue for treating various types of cancer.

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.

Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that degrade target proteins through recruiting E3 ligases. However, their application is limited in part because few E3 ligases can be recruited by known E3 ligase ligands. In this study, we identified piperlongumine (PL), a natural product, as a covalent E3 ligase recruiter, which induces CDK9 degradation when it is conjugated with SNS-032, a CDK9 inhibitor. The lead conjugate 955 can potently degrade CDK9 in a ubiquitin-proteasome-dependent manner and is much more potent than SNS-032 against various tumor cells in vitro. Mechanistically, we identified KEAP1 as the E3 ligase recruited by 955 to degrade CDK9 through a TurboID-based proteomics study, which was further confirmed by KEAP1 knockout and the nanoBRET ternary complex formation assay. In addition, PL-ceritinib conjugate can degrade EML4-ALK fusion oncoprotein, suggesting that PL may have a broader application as a covalent E3 ligase ligand in targeted protein degradation.