Discovery of a potential hematologic malignancies therapy: Selective and potent HDAC7 PROTAC degrader targeting non-enzymatic function

Discovery of a potential hematologic malignancies therapy: Selective and potent HDAC7 PROTAC degrader targeting non-enzymatic function

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Yuheng Jin^a, Xuxin Qi^b, Xiaoli Yu^a, Xirui Cheng^b, Boya Chen^b, Mingfei Wu^a, Jingyu Zhang^a, Hao Yin^b, Yang Lu^a, Yihui Zhou^b, Ao Pang^a, Yushen Lin^b, Li Jiang^b, Qiuqiu Shi^a, Shuangshuang Geng^a, Yubo Zhou^h, Xiaojun Yaoⁱ, Linjie Li^a, Haiting Duan^a, Jinxin Che^a^,^c^,^*, Ji Cao^b^,^d^,^e^,^f^,^g^,^*, Qiaojun He^b^,^d^,^e^,^g^,^*, Xiaowu Dong^a^,^*

Acta Pharmaceutica Sinica B | 2025, 15(3) : 1659 - 1679

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Acta Pharmaceutica Sinica B | 2025, 15(3): 1659-1679

• ORIGINAL ARTICLE •

Discovery of a potential hematologic malignancies therapy: Selective and potent HDAC7 PROTAC degrader targeting non-enzymatic function

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Affiliations

^aCollege of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China

^bInstitute of Pharmacology & Toxicology, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences and Cancer Center, Zhejiang University, Hangzhou 310058, China

^cHangzhou Institute of Innovative Medicine, Zhejiang University, Hangzhou 310058, China

^dInnovation Institute for Artificial Intelligence in Medicine of Zhejiang University, Hangzhou 310018, China

^eEngineering Research Center of Innovative Anticancer Drugs, Ministry of Education, Hangzhou 310000, China

^fCenter for Medical Research and Innovation in Digestive System Tumors, Ministry of Education, Hangzhou 310020, China

^gCancer Center, Zhejiang University, Hangzhou 310058, China

^hZhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Guangdong 528400, China

ⁱCentre for Artificial Intelligence Driven Drug Discovery, Faculty of Applied Sciences, Macao Polytechnic University, Macao 999078, China

About Author:

E-mail addresses: chejx@zju.edu.cn (Jinxin Che)

caoji88@zju.edu.cn (Ji Cao)

qiaojunhe@zju.edu.cn (Qiaojun He)

dongxw@zju.edu.cn (Xiaowu Dong).

^†

These authors made equal contributions to this work.

Author contributions

Yuheng Jin: Writing – original draft, Investigation, Formal analysis, Data curation, Conceptualization. Xuxin Qi: Writing – original draft, Investigation, Formal analysis, Data curation, Conceptualization. Xiaoli Yu: Formal analysis, Data curation. Xirui Cheng: Data curation. Boya Chen: Formal analysis, Data curation. Mingfei Wu: Methodology, Formal analysis. Jingyu Zhang: Methodology. Hao Yin: Formal analysis, Data curation. Yang Lu: Methodology. Yihui Zhou: Formal analysis, Data curation. Ao Pang: Methodology. Yushen Lin: Methodology. Li Jiang: Methodology. Qiuqiu Shi: Methodology, Data curation. Shuangshuang Geng: Methodology, Data curation. Yubo Zhou: Methodology, Conceptualization. Xiaojun Yao: Software. Linjie Li: Methodology. Haiting Duan: Methodology. Jinxin Che: Writing – review & editing, Project administration, Funding acquisition, Formal analysis, Conceptualization. Ji Cao: Writing – review & editing, Resources, Project administration, Funding acquisition, Formal analysis, Conceptualization. Qiaojun He: Supervision, Resources, Project administration, Funding acquisition. Xiaowu Dong: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, Formal analysis, Conceptualization.

doi: 10.1016/j.apsb.2025.01.021

Outline

Abstract

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HDAC7, a member of class IIa HDACs, plays a pivotal regulatory role in tumor, immune, fibrosis, and angiogenesis, rendering it a potential therapeutic target. Nevertheless, due to the high similarity in the enzyme active sites of class IIa HDACs, inhibitors encounter challenges in discerning differences among them. Furthermore, the substitution of key residue in the active pocket of class IIa HDACs renders them pseudo-enzymes, leading to a limited impact of enzymatic inhibitors on their function. In this study, proteolysis targeting chimera (PROTAC) technology was employed to develop HDAC7 drugs. We developed an exceedingly selective HDAC7 PROTAC degrader B14 which showcased superior inhibitory effects on cell proliferation compared to TMP269 in various diffuse large B cell lymphoma (DLBCL) and acute myeloid leukemia (AML) cells. Subsequent investigations unveiled that B14 disrupts BCL6 forming a transcriptional inhibition complex by degrading HDAC7, thereby exerting proliferative inhibition in DLBCL. Our study broadened the understanding of the non-enzymatic functions of HDAC7 and underscored the importance of HDAC7 in the treatment of hematologic malignancies, particularly in DLBCL and AML.

Key words

HDAC7 / PROTAC / Selectivity / Hematologic malignancies / Non-enzymatic function

Cite this Article

Yuheng Jin, Xuxin Qi, Xiaoli Yu, Xirui Cheng, Boya Chen, Mingfei Wu, Jingyu Zhang, Hao Yin, Yang Lu, Yihui Zhou, Ao Pang, Yushen Lin, Li Jiang, Qiuqiu Shi, Shuangshuang Geng, Yubo Zhou, Xiaojun Yao, Linjie Li, Haiting Duan, Jinxin Che, Ji Cao, Qiaojun He, Xiaowu Dong. Discovery of a potential hematologic malignancies therapy: Selective and potent HDAC7 PROTAC degrader targeting non-enzymatic function[J]. Acta Pharmaceutica Sinica B, 2025 , 15 (3) : 1659 -1679 . DOI: 10.1016/j.apsb.2025.01.021

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1.　Introduction

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Histone Deacetylases (HDACs) constitute a group of epigenetic enzymes pivotal for the regulation of gene expression by specifically deacetylating lysine residues on histones and non-histones^1,2. This deacetylation process intricately modulates the expression of downstream proteins, thereby fulfilling crucial physiological functions³. However, in contrast to class I HDACs, the substitution of tyrosine with histidine in the active pocket of class IIa HDACs (HDAC4, HDAC5, HDAC7, HDAC9) weakens their deacetylating function by 1000-fold rendering them pseudo-enzymes^4,5. The precise mechanism underlying the role of class IIa HDACs as epigenetic enzymes is not yet fully elucidated^6,7.

Among the four class IIa HDACs, HDAC7 has undergone extensive scrutiny, revealing its regulatory roles in gene expression, cell proliferation, differentiation, and survival⁸. HDAC7 has been identified as a potential therapeutic target for various diseases, including autoimmune diseases like Crohn's disease^9,10, solid tumors like non-small-cell lung cancer^11-15, and notably, hematologic malignancies like diffuse large B cell lymphoma (DLBCL) and acute myeloid leukemia (AML)^16-18. Concerning lymphoma, numerous studies have indicated that HDAC7 is significantly expressed in DLBCL, and its high expression is correlated with shorter patient survival¹⁹. In the case of AML, HDAC7 is co-expressed with the oncogene the Src Homology 2-domain containing adapter protein B (SHB)²⁰. These validations were accomplished through methodologies such as gene knockdown or bioinformatics analysis.

In recent years, notable progress has been achieved in the research on HDAC inhibitors. Specifically, four pan-HDAC inhibitors—Vorinostat, FK-228, Belinostat, and Chidamide—have obtained approval for the treatment of Peripheral T-cell Lymphoma (PTCL) and/or Cutaneous T-cell Lymphoma (CTCL)^21-23. Givinosta, another pan-HDAC inhibitor, was recently approved by FDA in 2024 for the treatment of Duchenne Muscular Dystrophy (DMD)²⁴. However, the limited selectivity of these drugs has led to the occurrence of adverse side effects, and their clinical use is hindered by the rarity of indications. Despite progress in multi-targeted HDAC inhibitors and combination therapies, the issue of poor selectivity leading to high toxicity with pan-HDAC inhibitors remains unresolved²⁵. Panobinostat, in combination with dexamethasone and bortezomib, was FDA-approved for multiple myeloma in 2015 but was later withdrawn in 2022 due to incomplete post-approval clinical studies, raising concerns about the future development of HDAC inhibitors. Class IIa HDAC inhibitors, such as trifluoromethyl oxadiazole (TFMO) inhibitors^26,27 (TMP269, TMP195, and NVS-HD1) and hydroxamic acid structure inhibitors^28,29 (MC1568, CHDI-390576, and inhibitor F), exhibit enhanced selectivity and distinctive structures (Fig. 1). Nevertheless, due to the high homology of the enzymatic activity domain, achieving selective inhibition among class IIa HDAC isotypes remains a challenge^30,31. Additionally, the inhibitory activity of cell proliferation in hematologic malignancies by class IIa HDAC inhibitors is insufficient^32,33, which may be attributed to the non-enzymatic function of class IIa HDACs^34,35. There is currently no direct way to verify the deeper biological function and target druggability of HDAC7. Therefore, the development of potent and selective HDAC7 drugs is necessary.

Proteolysis Targeting Chimera (PROTAC) is a heterobifunctional molecule that specifically targets protein degradation. Its unique event-driven mechanism offers potential advantages over small molecule inhibitors, including high selectivity and elimination of non-enzymatic functions^36-38. Several HDAC PROTACs have been reported, such as HDAC1/2/3 PROTACs³⁹, HDAC1/2 PROTACs⁴⁰, HDAC3 PROTACs⁴¹, HDAC3/8 PROTACs^42,43, HDAC6 PROTACs^44,45, HDAC8 PROTACs^46,47 and SIRT2 PROTACs⁴⁸. The class I HDAC PROTACs successfully achieved selective degradation of specific isoforms of class I HDAC and broadened their non-enzymatic function in inflammation. Meanwhile, PROTACs targeting HDAC6, HDAC8, and SIRT2 have shown superior efficacy compared to related inhibitors in inflammation, tumors, and other diseases. The discovery of selective HDAC4 PROTAC further supports the feasibility and necessity of developing HDAC7 PROTAC⁴⁹. Consequently, the discovery of HDAC7 PROTAC, which exhibits greater potential therapeutic advantages compared to small molecule inhibitors, is expected to facilitate a deeper understanding of the biological functions of HDAC7.

In this study, we selected TMP269, a class IIa inhibitor, as the ligand for HDAC7. Multiple rounds of structural modifications were conducted to obtain compound B14, which was identified as the selective and potent HDAC7 degrader. B14 demonstrated effectiveness in cells and animal models of DLBCL and AML. This study also highlighted the important regulatory role of HDAC7 in DLBCL and AML, particularly its potential non-enzymatic functions, and provides a solid reference and theoretical basis for further development of HDAC7 PROTAC.

2.　Materials and methods

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2.1.　 Cell line and cell culture

Human cancer cell lines including NB4, HL60, DHL4, DHL6, and DHL2 were purchased from Chinese Academy of Sciences National Collection of Authenticated Cell Cultures (Shanghai, China). The TMD8 cell line was obtained by Dr Lynn Wang (University of Chicago, USA). The OCI-ly10 and Jeko-1 cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, USA). CRBN-knockout and sgCtrl Jeko-1 cells were constructed from the normal Jeko-1 cells. The HEK 293T cell line was purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). The NB4, DHL2, DHL4, DHL6, TMD8, and Jeko-1 (sgCtrl or CRBN-KO) cells were cultured in the RRMI-1640 medium. The HEK 293T cells were cultured in the DMEM medium. Both mediums were supplemented with 10% fetal bovine serum (FBS, Cytiva, SH30396.03, Logan, Massachusetts, USA). The HL60 and OCI-ly10 cells were cultured in the IMDM cell culture medium supplemented with 20% FBS. All cells were cultured in the 37 ℃ constant temperature incubator with 5% CO₂. All cells were authenticated by STR profiling and were monitored for mycoplasma contamination every six months.

2.2.　 Lentivirus infection and cell proliferation

ShRNAs targeting HDAC7 were subcloned into pLKO.1-TRC cloning vectors (Addgene, #10878, Watertown, Massachusetts, USA) using the primers listed in Supporting Information Table S1. Lentivirus was produced by HEK 293T cells with pCMV-dR8.91 (packaging vector), pMD2.G-VSVG (envelope vector), and targeted HDAC7 plasmids co-transfected using Lipofectamine 2000 (Invitrogen, #11668019, Carlsbad, California, USA). The viral medium was harvested 48 h after transfection and filtered by a 0.45 μm Millipore filter. The cells were seeded in 6-well plates with a density of 1 × 10⁶ cells/mL and 1 mL of each virus was added with 2 μL polybrene (6 mg/mL). The cells were centrifuged for 90 min at 2000 rpm (Eppendorf, Centrifuge 5810R, Hamburg, Germany) at 32 ℃ after adding the virus. After the cells were infected for 12–16 h, the medium was changed to a fresh medium. The cells were counted 36 h after infection with the virus at 24 h intervals.

2.3.　 Western blot analysis

Cells with a density of 5 × 10⁵ cells/mL were seeded in 6-well plates and then the cells were treated with compounds for 12 or 24 h. The cells were first washed with cold phosphate-buffered saline (PBS, 0.01 mol/L Na₂HPO₄, 0.0018 mol/L KH₂PO₄, 0.8% (w/v) NaCl, 0.0002% (w/v) KCl) and then lysed with 50 μL loading buffer (20 mmol/L Tris-base, 4% (w/v) SDS, 16% (v/v) glycerol, 3% (w/v) DTT, 0.02% (w/v) bromophenol blue, pH 6.8). After incubation at 95 ℃ for 30 min, the cell lysate was subjected to 10% SDS-PAGE gel, and the protein was transferred to the polyvinylidene difluoride membranes (Millipore, #IPVH00010, Darmstadt, Germany). Then the membrane was blocked using 5% skim milk and incubated with primary antibody overnight at 4 ℃ and secondary antibody at room temperature for 1 h. The immunoreactive bands were then visualized using ECL (Shanghai Pufei Bio-Technology, #36222-A/36222-B, Shanghai, China) and analyzed using the Amersham ImageQuant 800 system (Cytiva, 29399481, Logan, Massachusetts, USA). The primary antibody and second antibody used in this study are shown in Supporting Information Table S2.

2.4.　 Global proteomics analysis

The DHL6 and NB4 cells were individually treated with DMSO or B14 before being rapidly frozen by liquid nitrogen. Quantitative proteomics analysis was performed by Jingjie PTM-Biolab company (Hangzhou, China).

2.5.　 Generation of CRBN-knockout cell lines based on CRISPR-cas9 gene editing technology

CRBN-knockout HEK 293T lines were generated by CRISPR-cas9 technology. CRISPR gRNAs were designed by http://crispor.tefor.net/. The sgRNA targeting CRBN were inserted into Bbs1-digested pSpCas9 (BB)-2A-GFP (PX458) plasmid (Addgene, #48138, Watertown, Massachusetts, USA) to generate PX458-CRBN gRNAs. The sgRNA sequences can be found in Table S1. Wild-type cell lines were transfected using JetPRIME transfection reagents (Polyplus, 101000046, Headquarters – Illkirch, France) carrying sgRNA for 48 h to get the CRBN-knockout cell pools. The single cell was sorted out by flow cytometry (BD BioSciences, FACSAria III, Franklin Lake, New Jersey, USA) and developed in 96 well-plate units. The genomic DNA of individual cell clones was extracted using TIANamp Genomic DNA kit (TIANGEN Biotech, DP304-02, Beijing, China) and was identified by gene sequencing.

2.6.　 Quantitative real-time PCR (qRT-PCR)

Total mRNA was obtained following the protocol of PureLink RNA extraction kit (Thermo Fisher, 12183018A, Waltham, Massachusetts, USA). 2 μg of mRNA was reversely transcripted into cDNA using cDNA synthesis kit (TransGen, AT311-03, Beijing, China). qRT-PCR was performed with the iTaq Universal SYBR Green qPCR supermixes (Bio-Rad, L001752B, Hercules, California, USA) on the quantitative Real-Time PCR Analysis System (Roche, LightCycler 480 II, Rotkreuz, Switzerland). Relative expression values for each gene of interest were obtained by normalizing to β-Actin mRNA expression using the ΔΔCt method. The gene-specific primer pairs are shown in Table S1 in the supporting information.

2.7.　 Pulldown of total ubiquitinylated proteins with Ni-NTA-Beads

HEK 293T cells were co-transfected with the indicated plasmids (HDAC7-Flag and His-ubiquitin) using Jetprime transfection reagents when grown to 50% confluency in a 100 mm dish. After 12 h, the medium was replaced with fresh culture medium containing either DMSO or PROTAC, along with 10 μmol/L MG132. Following an additional 12 h incubation, the cells were collected, washed with PBS, and lysed with 8 mol/L urea buffer (10 mmol/L Tris pH 8.0; 100 mmol/L NaH₂PO₄; 8 mol/L urea) containing 10 mmol/L imidazole followed by ultrasonic crushing for 2 min. Then the cell lysate was centrifuged at 12,000 rpm (Eppendorf, Centrifuge 5427R, Hamburg, Germany) at 4 ℃ for 20 min. The protein concentration was determined with Bradford assay, and cell lysate was incubated with Ni-NTA Beads (Smart-Lifesciences, SA004005, Changzhou, China) for 3 h at room temperature or 4 ℃ overnight. Beads were washed five times with washing buffer (10 mmol/L Tris–HCl pH 6.3, 100 mmol/L NaH₂PO₄, 8 mol/L urea) containing 20 mmol/L imidazole and protein was eluted in 2 × loading buffer at 95 ℃ for 10 min. 20 μg protein was analyzed by Western blotting.

2.8.　 RNA-sequencing analysis

Total RNA was isolated and purified using TRIzol reagent (Invitrogen, 108-95-2, Carlsbad, California, USA) following the manufacturer's procedure. RNA-sequencing was completed by Hangzhou Lianchuan Biotechnology Ltd. Bioinformatic analysis was performed using the OmicStudio tools (https://www.omicstudio.cn). The volcano plot (or other graphics) was drawn based on the R version 4.1.3 (2022-03-10) on the OmicStudio platform (https://www.omicstudio.cn).

2.9.　 Immunoprecipitation

pCMV-BCL6-HA plasmid was transfected into HEK 293T cells using Jetprime transfection reagents. After 12 h transfection, the cells were treated with PROTAC or DMSO for 12 h. The cells were lysed using RIPA lysis buffer containing (50 mmol/L Tris-base, 150 mmol/L NaCl, 5 mmol/L EDTA, 0.1% (w/v) SDS, 1% (v/v) TritonX-100, 0.25% (w/v) Sodium deoxycholate, pH 7.4), then centrifuged at 12,000 rpm (Eppendorf) for 30 min. Immunoprecipitation was performed by Anti-FLAG beads (Smart-Lifesciences, #SA042005, Changzhou, China) overnight at 4 ℃. The beads were washed five times with washing buffer [25 mmol/L Tris-base, 500 mmol/L NaCl, 0.2% (v/v) NP40, pH 7.4]. The protein was eluted in 2 × loading buffer at 95 ℃ for 10 min and followed by Western blot analysis.

2.10.　 HDAC7 in vitro and cells kinase assay

Full-length human HDAC7 fused with C-terminal His tag were recombinantly expressed in Sf9 insect cells and then were purified by His Trap™ HP and using ӒKTA pure™ system. The in vitro kinase assay was performed in assay buffer (1 mol/L Tris–HCl, 4 mol/L NaCl, pH 7.4) at 30 ℃ using 0.1 μmol/L purified HDAC7 enzyme and 50 μmol/L Ac-Leu-Gly-Lys (TFAc)-AMC substrate. The IC₅₀ values of in vitro enzymatic activity for representative PROTACs were performed by Bioduro-sundia company (Shanghai, China).

2.11.　 Cell apoptosis analysis

Cells were centrifuged at about 1000×g (Eppendorf) for 3–5 min to precipitate the cells, after being treated with the B14. The cells were re-suspended with 1 mL pre-cooled PBS and transferred to a 1.5 mL centrifuge tube. Centrifuged again and carefully removed the supernatant. Then cells were immobilized by 1 mL pre-cool 70% ethanol at 4 ℃ for 30 min. 0.5 mL of propyl iodide staining solution (Beyotime, C1052, Shanghai, China) was added to each tube of cell samples, and the red fluorescence was detected at the excitation wavelength of 488 nm by flow cytometry (BD FACSCanto II).

2.12.　 Cell viability assay by cell counting Kit-8 (CCK8)

Cancer cells in a complete cell culture medium were seeded in 96-well plates (100 μL/well) at the optimized densities (500–10000 suspension cells). Compound treatments were prepared in complete cell culture media and 100 μL of 2 × treatment-containing media were added to each well. Complete cell culture media without treatment was added in control wells. The outer wells of the 96-well plate were not used for treatment and were filled with 200 μL of medium to reduce the evaporation of media from the inner wells. Each compound/combination was tested at eight different concentrations with three replicates unless otherwise specified. The cell viability was determined by CCK8 assay. 20 μL of CCK8 (TargetMol, C0005, Shanghai, China) was added to each control and treatment well. The cells were incubated for 1 h at 37 ℃ and 5% CO₂, and then the absorbance was recorded at 450 nm using Biotek's Synergy Neo2 multi-mode plate reader (TECAN, SPARK, Grödig, Austria).

2.13.　 Bioinformatic analysis

Tissue expression analysis and correlation analysis of HDAC7 were obtained from Gene Expression Profiling Interactive Analysis (GEPIA) at http://gepia.cancer-pku.cn/ (Peking University, China). The data for survival curve analysis were obtained from The Cancer Genome Atlas Program (TCGA) database and survival curve graphs were analyzed and exported from http://www.oncolnc.org/ (National Cancer Institute, USA).

2.14.　 PK evaluation

Three ICR mice were selected at each time point. Blood samples were collected from the orbit at 0.25, 0.5, 1, 2, 4, 8, and 24 h after administering 30 mg/kg B14 through gavage or intraperitoneal injection. Protein precipitation was performed by adding an acetonitrile solution to the plasma samples. After vortexing for 3 min and centrifugation at 10,000 r/min (Yooning Instrument, YN-H06-2320, Hangzhou, China) for 5 min, the supernatant was used for LC‒MS analysis using the positive ion method. The experimental data were analyzed using DAS 3.0 pharmacokinetic software, which enabled the determination of key absorption kinetic parameters such as C_max, T_max, and AUC_0‒t.

2.15.　 Xenograft tumor model

2.15.1.　 HL60 xenograft tumor model

Tumors were established by subcutaneously injecting HL60 cells (1 × 10⁷ cells) into 5-week-old BALB/c female athymic nude mice sourced from National Rodent Laboratory Animal Resource (Shanghai, China). The mice were individually housed and provided with sterilized food, water, and bedding. Environmental conditions were maintained at 25 ℃ and the humidity was between 50% and 70%. After 12 days, intratumoral injection of B14 or vehicle solvent every other day. The maintenance and experimental procedures for the mice studies were approved by The Innovation Institute for Artificial Intelligence in Medicine, Zhejiang University's Institutional Animal Care and Use Committee (DW202305101640). At the end of the experiment, the tumors were dissected and weighed. Tumor measurements were taken every other day, and volumes were calculated with Eq. (1):

Tumor volume= （ Width 2 × Length ） /2

(1)

2.15.2.　 DHL6 xenograft tumor model

Tumors were established by subcutaneously injecting DHL6 cells (1 × 10⁷ cells) into 4 or 5-week-old NSG female mice sourced from Shanghai Model Organisms Center, Inc. (Shanghai, China). The mice were individually housed and provided with sterilized food, water, and bedding. Environmental conditions were maintained at 25 ℃ and the humidity was between 50% and 70%. After 19 days, intraperitoneal injection of B14 or vehicle solvent every day. The maintenance and experimental procedures for the mice studies were approved by The Innovation Institute for Artificial Intelligence in Medicine, Zhejiang University's Institutional Animal Care and Use Committee (DW202305101640). Tumor measurements were taken randomly one or two times every three days, and volumes were calculated with Eq. (1).

2.16.　 Computational simulation

Crystal structures of HDAC7 protein and CRBN were obtained from the PDB database. Any non-standard residues present in the protein were restored to their original form. Subsequently, all structures were preprocessed using Schrödinger 2019. This preprocessing involved tasks such as adding hydrogen atoms, assigning bond orders, filling in missing side chains, removing water molecules, and minimizing the structure using the OPLS_2005 force field. The warheads and E3 ligands in each PROTAC were also preprocessed using the LigPrep module in Schrödinger. The force field was set to OPLS_2005, allowing up to four stereoisomers per ligand. Other parameters were kept at their default values. Docking of the warheads and E3 ligands in each PROTAC was performed using Glide in standard precision (SP) scoring mode. Molecular dynamics (MD) simulations were then carried out using the Molecular Operating Environment (MOE) to complete the structure of the PROTAC. The generated PROTAC model was constrained to match the conformations of the warhead and E3 ligand in the protein model. Only PROTAC conformations with a root mean square deviation (RMSD) of less than 2.5 Å between the two bound ligands in the computationally generated PROTAC model, after optimal superposition, were retained.

2.17.　 Statistics

Graphical representation and statistical analyses were calculated using GraphPad Prism 9.4.1. Values are presented as the means ± standard error of the mean (SEM) or the means ± standard deviation (SD). Differences between means were determined using unpaired Student's t tests, one-way ANOVA, or two-way ANOVA as appropriate. For all numerical tests, a probable value (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, n.s.: not significant) was considered to be statistically significant.

3.　Results and discussion

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3.1.　 Correlation verification of class IIa HDACs with DLBCL and AML

Given the potential regulatory role of HDAC7 in DLBCL and AML based on literature reports^19,20, we conducted bioinformatic analysis, revealing a marked upregulation of HDAC7 in DLBCL compared to normal tissues (Fig. 2A). Furthermore, we observed a significant correlation between elevated HDAC7 expression and diminished survival rates among AML patients (P = 0.002), while HDAC4, HDAC5, and HDAC9 exhibited no such effect (Supporting Information Fig. S1). These findings not only validate the crucial role of HDAC7 in DLBCL and AML but also emphasize the necessity for the development of drugs that selectively target HDAC7. To delve deeper into this inquiry, we opted to examine the proliferation inhibition of DLBCL and AML cells using the class IIa HDAC inhibitor TMP269. We chose DHL6 and DHL4 as test cells for DLBCL, NB4, and HL60 for AML. Notably, TMP269 demonstrated negligible proliferation inhibitory activity across all four cell lines (IC₅₀ > 10 μmol/L) (Fig. 2B). Meanwhile, significant proliferation inhibition was observed in DLBCL and AML cell lines upon the knockdown of HDAC7 (Fig. 2C and D). This suggests a potential biological role for HDAC7 in DLBCL and AML, possibly mediated through its non-enzymatic function. Consequently, the development of HDAC7 degraders emerges as a promising and imperative avenue for the therapy of DLBCL and AML.

3.2.　 Design of HDAC7 degraders

To design HDAC7 PROTACs, we selected the class IIa HDAC inhibitor TMP269 as the lead compound for the HDAC7-targeting ligand. Molecular docking analysis revealed that TMP269 binds to the metal catalytic pocket of HDAC7 (PDB code: 3ZNR). The trifluoromethyl oxadiazole's oxygen atom coordinated with zinc ions, while the benzene ring connected to the trifluoromethyl oxadiazole exhibited a π‒π stacking effect with Phe 679. Similarly, the thiazole ring demonstrated a π‒π stacking effect with His 843, and the benzene ring linked to the thiazole ring also showed a π‒π stacking effect with Phe 679 (Fig. 3A). On the other hand, the tetrahydropyran ring did not interact with the protein and was exposed to the solvent region, providing a potential tethering site for linking an E3 ligase ligand for HDAC7 PROTACs design (Fig. 3B). Consequently, we decided to replace the oxygen atom in the tetrahydropyran ring with a nitrogen atom and connect multiple linkers from this nitrogen atom (Fig. 3C). We chose 3- or 4-substituted thalidomide derivatives as E3 ligands and utilized both flexible and rigid linkers in the design of HDAC7 PROTACs. Therefore, we designed HDAC7 PROTACs by tethering derivatives of TMP269 (compound 14a) with the aforementioned E3 ligands and linkers of varying lengths and ring sizes to investigate the HDAC7 enzymatic activities in vitro and the degradation ability in NB4 cells (Fig. 3D, Supporting Information Fig. S2 and Tables 1‒4).

3.3.　 Chemistry

The synthesis of HDAC7 PROTACs is shown in Schemes 1-4. The synthetic route of the Boc-protected POI ligand 8 is outlined in Scheme 1. The commercially available 3-cyano benzoic acid (1) reacted with hydroxylamine hydrochloride to synthesize the intermediate 2 in the presence of isoquinolin-8-ol, Na₂CO₃ in EtOH/H₂O, followed by trifluoroacetic anhydride to afford intermediate 3. Intermediate 5 was synthesized by 2-Cyanothioacetamide (4) and 2-Bromoacetophenone in EtOH, followed by tert-butyl bis (2-chloroethyl) carbamate to obtain Intermediate 6. Intermediate 7 was synthesized from intermediate 6 by reduction reaction. The HDAC ligand 8 was further prepared from intermediate 3 and intermediate 7 through a condensation reaction.

The synthesis of CRBN ligand-conjugated linkers (12a‒b, h‒i, and 13a‒e) is outlined in Scheme 2. Intermediates 11a‒b were synthesized from fluoro-substituted phthalic anhydride (9a and 9b) through ammonolysis (10) in KOAc/AcOH, and then nucleophilic substitution with various amino derivatives to yield 12a‒i. Intermediates 13a‒e were synthesized by the Dess-Martin Oxidation of 12c‒g with Dess-Martin periodinane (DMP) in DCM.

The general synthetic routes of HDAC7 PROTACs A1‒A16 are shown in Scheme 3. Compound 14a was prepared through Boc-deprotection of compound 8 using trifluoroacetic acid (TFA). Condensation reactions of compound 14a with carboxylate derivatives produced 14b‒k. After intermediates 14a‒k and 12a‒b removed the Boc group separately, the condensation reaction was carried out to obtain compound A1‒A13. Compound A14 was synthesized from 14a through Borch reduction with 13e. Compound 14a and tert-Butyl (2-(2-(2-(2-bromoethoxy) ethoxy) ethoxy) ethyl) carbamate were nucleophilic to obtain 15 in the presence of K₂CO₃. After intermediates 15 and 12a‒b removed the Boc group separately, the condensation reaction was carried out to obtain compound A15‒A16.

The general synthetic routes of HDAC7 PROTACs B1–B19 and B14–N are shown in Scheme 4. Condensation reactions of compound 14a with carboxylate derivatives produced 16a‒c. After intermediates 16a‒c removed the Boc group, Borch reduction with 13a‒d was carried out to obtain compound B1–B8. Intermediates 17a‒d were synthesized from 14a through Borch reduction with aldehyde-based derivatives. After intermediates 17a‒d removed the Boc group, Borch reduction with 13a‒d was carried out to obtain compound B9–B13. The nucleophilic substitution of intermediate 18 with tert-butyl piperidine-4-carboxylate resulted in the formation of intermediate 19. After intermediates 17a‒d, 12h‒i, and 19 removed the protecting group separately, the condensation reaction was carried out to obtain compounds B14–B19 and B14–N.

3.4.　 Structure degradation relationships

To investigate the efficiency of HDAC7 degradation, we initially connected compound 14a (a derivative of TMP269) to CRBN (Thalidomide) using alkyl chains ranging from 3 to 15 carbon atoms and polyethylene glycol (PEG) chains ranging from 9 to 18 carbon atoms, resulting in HDAC7 PROTACs A1‒A9. The in vitro enzymatic activity inhibition of the designed HDAC7 PROTACs A1‒A9 was assessed at concentrations of 0.3 μmol/L and 3 μmol/L (Table 1). It was observed that the in vitro enzyme activities of compounds with alkyl chains (A1‒A5) were lower than those of TMP269 at both concentrations, while the in vitro enzyme activities of compounds with PEG chains (A6-A9) were 2–4 times lower than those of TMP269 at 0.3 μmol/L and similar to those of TMP269 at 3 μmol/L. Overall, the analysis indicated that the in vitro enzymatic activities of the PEG chain compounds A6‒A9 were superior to those of the alkyl chain compounds A1‒A5, suggesting that the PEG chain facilitated binding to HDAC7. Subsequently, we evaluated their ability to induce HDAC7 degradation in NB4 cells at concentrations of 1, 5, and 10 μmol/L after 24 h (Table 1). The data revealed that the degradation rates of compounds with alkyl chains (A1‒A5) were below 50% at 1 μmol/L in NB4 cells, indicating poor degradation efficacy. Among the compounds with PEG chains, compound A8 exhibited the most potent degradation activity, reducing the HDAC7 protein level by 52.28% at 1 μmol/L and 88.54% at 10 μmol/L. This suggested that PEG chains with a length of 15 carbon atoms were advantageous for HDAC7 degradation. Consequently, compound A8 was chosen for further optimization.

To enhance the degradation efficiency of HDAC7 PROTACs, seven HDAC7 PROTACs A10‒A16 were designed and synthesized by refining the linker length or changing the linker attachment method at compound 14a based on compound A8. Compounds A10‒A13 were synthesized to refine the linker length, while compounds A14‒A16 were synthesized by changing the linker attachment method at the HDAC7 ligand. The in vitro enzymatic activity inhibition as well as the degradation efficiency of HDAC7 was performed (Table 2). It was noteworthy that the in vitro enzyme activities of compounds A10‒A16 at 3 μmol/L were similar to those of TMP269. The in vitro enzymatic activities of compounds A10‒A13 with linker at the HDAC7 ligand using carbonyl linkage were significantly better than those of compounds A14‒A16 with methylene linkage at 0.3 μmol/L, indicating that the linker carbonyl-linked compounds were conducive to binding to HDAC7. The degradation rates of compounds A10‒A11 were all below 50% at 1 μmol/L in NB4 cells, and the degradation rates of compounds A12‒A13 were comparable to those of A8 at 1 μmol/L, indicating that the optimal length of the linker was about 15–17 carbon atoms. The degradation rates of HDAC7 at 1 μmol/L for compounds A14 and A16 were 86.90% and 80.20%, respectively, suggesting that the linker is conducive to the degradation efficiency by flexible linkage of HDAC7 ligands via methylene. Further, we validated the efficient degradation of HDAC7 by compound A16 in NB4 cells (Supporting Information Fig. S3A). Overall, it is initially demonstrated that HDAC7 can be effectively degraded by PROTAC.

Studies have shown that PROTACs containing PEG chains generally have poor oral druggability properties. However, the use of a rigid linker can improve these properties^50-52. Therefore, we chose rigid chain fragments containing nitrogen heterocycles as linking chains, and eight HDAC7 PROTACs (B1–B8) were synthesized by linking them to compound 14avia carbonyl groups. The in vitro enzymatic activity inhibition and the degradation efficiency of HDAC7 were performed (Table 3). It was shown that the in vitro enzymatic activities of compounds B1–B8 at 0.3 μmol/L and 3 μmol/L were both significantly weaker than those of compounds A6‒A16 and TMP269, indicating a lower binding to HDAC7. The degradation rates of compound B5–B8 at various concentrations were low. Based on the in vitro enzyme activity and degradation activity data of compounds B5–B8, the degradation effect of compounds B1–B4 was not tested. The results indicate that the use of carbonyl linkage between the linker and the HDAC7 ligand leads to a significant decrease in vitro enzymatic activity and degradation efficiency. The hypothesis proposed that this carbonyl linkage would impede the ligand's ability to maintain its dominant conformation and greatly reduce its binding to HDAC7 protein. This would negatively impact the formation of ternary complexes and result in lower degradation efficiency.

The previous optimization study indicated that the degradation of HDAC7 was enhanced by using methylene-flexibly conjugated HDAC7 ligands. Therefore, we designed and synthesized eleven HDAC7 PROTACs (B9–B19) with rigid chains containing nitrogen heterocycles linked to HDAC7 ligands via methylene. In vitro enzymatic activity inhibition and degradation efficiency of HDAC7 were then evaluated (Table 4). The results showed that the inhibition activity of compounds B9–B19 exhibited significantly better compared to compounds B1–B8, at both 0.3 μmol/L and 3 μmol/L. This suggests that the HDAC7 ligand linked to the linker via a flexible methylene linkage facilitated the recovery of in vitro enzyme activities. Among the compounds, B14–B19, which contained carbonyl groups in the linker, generally exhibited better inhibition activity than compounds B9–B13, which lacked carbonyl groups in the linker, at 0.3 μmol/L. This indicates that the presence of carbonyl groups in the linker facilitated the binding to HDAC7 protein. Over 50% of HDAC7 protein was degraded at 1 μmol/L, except for B13, in NB4 cells. Further compounds B14, B15, and B16 showed the highest HDAC7 degradation with percentages of 90.01%, 88.48%, and 89.07%, respectively, which were superior to compounds B10, B13, and B12 at 1 μmol/L, where the efficient degradation of HDAC7 by compound B14 in NB4 cells was further verified with a DC₅₀ value of 25.10 nmol/L (Fig. S3B). This suggests that the carbonyl group in the linker enhances HDAC7 degradation. Based on these results, it can be inferred that utilizing methylene flexibly conjugated the HDAC7 ligand is beneficial for restoring the in vitro enzymatic activity and degradation ability of HDAC7. Additionally, the presence of a carbonyl group in the linker is advantageous for enhancing HDAC7 degradation, possibly due to the carbonyl group stabilizing the ternary complex.

Moreover, we focused on selecting PROTACs with superior degradation activity to determine the IC₅₀ value of in vitro enzymatic activity (Supporting Information Table S3). The results revealed that the IC₅₀ value of all class IIa HDACs by PROTACs increased to varying degrees compared to TMP269, implying a decrease in PROTAC binding to class IIa HDAC proteins, which may be caused by the relatively large structure of PROTACs. The IC₅₀ value of B14 for HDAC7 (2.4 μmol/L) was significantly higher than that of TMP269 and other PROTACs, indicating a lower binding affinity of B14 for HDAC7. Despite this, B14 still exhibits high degradation activity towards HDAC7, suggesting that the IC₅₀ may not be directly related to the efficacy of PROTACs, and the overall conformation of POI-PROTAC-E3 may be one of the key factors.

3.5.　 Degradation ability and selectivity examination

Based on preliminary experimental data, compound B14 was chosen for further evaluation (Fig. 4A). We conducted an investigation into the concentration-dependent and time-dependent manner of HDAC7 degradation in DHL6 cells and NB4 cells using B14. Immunoblotting blotting analysis revealed that B14 induced the HDAC7 degradation in a concentration-dependent manner in both DHL6 and NB4 cells (Fig. 4B). The HDAC7 protein level exhibited a reduction starting from 0.1 μmol/L, reaching maximum degradation at 3 μmol/L, with over 90% degradation observed and no ‘hook’ effect—similar results were observed in HL60 cells (Supporting Information Fig. S4A). Additionally, B14 also led to the time-dependent HDAC7 degradation in DHL6 and NB4 cells (Fig. 4C). After 6 h of treatment with B14 (1 μmol/L), a significant reduction in HDAC7 protein level was observed, reaching over 85% degradation at 24 h. In HL60 cells, the degradation followed a time-dependent manner from 0 to 12 h, but degradation was reversed during the 24-h treatment. The maximum degradation occurred after 12 h, resulting in the degradation of over 85% of HDAC7 (Fig. S4B). These different degradation patterns may be attributed to variations in the states of E3 ligase or differences in the expression and production rates of HDAC7 proteins across different cell types.

To evaluate the selectivity of compound B14 against class IIa HDACs, immunoblotting was conducted in DHL6, NB4, and HL60 cells, targeting HDAC4, HDAC5, HDAC7, and HDAC9. B14 exhibited specific degradation selectivity for HDAC7 over other class IIa HDACs in tested cell lines (Fig. 4D and Fig. S4C). Quantitative proteomic and immunoblotting experiments revealed that B14 didn't affect the expression of Class I HDACs (HDAC1, HDAC2, HDAC3, HDAC8), while Class IIb HDACs (HDAC6, HDAC10) showed minimal impact in B14-treated DHL6 and NB4 cells. The result of the proteomics finding was consistent with the immunoblotting, showing a significant downregulation of HDAC7 in DHL6 cells (HDAC7 was not detected in NB4 cells). Regrettably, the isoforms of class IIa HDACs (HDAC4, HDAC5, HDAC9) were not identified in either DHL6 or NB4 cells in the quantitative proteomics data (Fig. 4E and Fig. S4D‒S4F), which may be attributed to their expression falling below the detection limit of quantitative proteomics. Furthermore, an evaluation of potential degradation substrates for the E3 ligand of B14 through quantitative proteomics and immunoblotting analyses revealed that B14 had limited influence on the degradation of neo-substrates such as SALL4, IKZF1/3 (IKZF3 was not detected in NB4 cells in both proteomics and immunoblotting analyses), and GSPT1 in DHL6 and NB4 cells (Fig. S4G‒S4H). In conclusion, these findings highlight the pronounced selectivity of B14 for HDAC7.

The pharmacokinetic properties of B14 were then evaluated in mice through oral administration at a dosage of 30 mg/kg. The results indicated that B14 exhibited oral absorption in mice, with C_max and AUC_0‒t values of 238.37 ng/mL and 825.53 ng/mL·h, respectively. Additionally, B14 demonstrated a short half-life and a high clearance rate, being completely metabolized in mice within 8 h (Supporting Information Table S4). Meanwhile, B14 exhibited suitable pharmacokinetic properties when administered intraperitoneally at a dosage of 30 mg/kg in mice (Supporting Information Table S5). In conclusion, B14 was identified as a selective, potent, and orally absorbed HDAC7 degrader.

3.6.　 Degradation mechanism examination

To validate that the downregulation of HDAC7 results from protein degradation, we investigated the downregulation of HDAC7 by B14 through a proteasome degradation pathway. Cells were preincubated with cycloheximide (CHX) alone (10 μg/mL) or plus with B14 (1 μmol/L). Results showed that B14 effectively accelerated CHX-induced HDAC7 protein degradation in both DHL6 and NB4 cells (Fig. 5A). Examining HDAC7 mRNA expression in DHL6 and NB4 cells post-B14 preincubation (Fig. 5B) revealed no reduction compared to the control group. This suggests that the decrease in HDAC7 protein expression is primarily due to protein degradation rather than reduced HDAC7 mRNA levels. To confirm that B14 mediated the HDAC7 degradation by recruiting a specific E3 ligase, we genetically depleted the CRBN-KO cells by CRISPR-Cas9 approach, B14 degraded HDAC7 in wild-type HEK 293T cells, but not in CRBN knockout HEK 293T cells (Fig. 5C), a conclusion that was further validated in CRBN-KO cells of Jeko-1 (Supporting Information Table S6).

To further verify whether HDAC7 degradation was dependent on the recruitment of the ternary complex, a series of experiments were carried out (Fig. 5D). Co-treatment with increasing dose of TMP269 or CRBN ligands resecured the B14-induced HDAC7 degradation. Meanwhile, the presence of B14 significantly enhanced HDAC7 ubiquitination levels in HEK 293T cells (Fig. 5E). Furthermore, the co-administration of B14 (1 μmol/L) with the proteasome inhibitorMG132 (10 μmol/L) or the neddylation inhibitor MLN4924 (10 μmol/L) would rescue the HDAC7 degradation induced by B14 in HEK 293T cells (Fig. 5F and Fig. S4I). Experiments have demonstrated that the degradation of HDAC7 induced by B14 is consistent with the degradation mechanism observed in PROTACs, which bind both target protein and an E3 ligase to induce the degradation through a ubiquitin-proteasome system (UPS)-dependent pathway.

3.7.　 Cell apoptosis and cell cycle changes in DLBCL and AML cells induced by B14

To investigate the biological function of HDAC7 in DLBCL and AML, we employed compound B14 as a selective HDAC7 degrader to probe HDAC7's role in DLBCL and AML. To explore the correlation between HDAC7 degradation and cell proliferation inhibition, compound B14–N which methylated on the N of the E3 ligand of B14 was synthesized and its structural formula was illustrated in Fig. 4A. In DHL6 and NB4 cells, B14 markedly inhibited cell proliferation (IC₅₀: 1.00 and 0.84 μmol/L, respectively), surpassing the cell proliferation inhibition effects of TMP269, B14–N, and pomalidomide (Fig. 6A). Similarly, the proliferation inhibitory effect of B14 was significantly superior to that of the inhibitor TMP269 in various DLBCL (DHL4, DHL2, TMD8, and OCI-ly10) and AML (HL60) cells (Supporting Information Table S7), suggesting a crucial regulatory role of HDAC7's non-enzymatic function in cell proliferation. Further, an immunoblotting assay revealed that B14 promoted apoptosis and upregulated the apoptosis-related proteins cleaved PARP and cleaved caspase 3 while downregulating uncleaved PARP and uncleaved caspase 3 in DHL6 and NB4 cells (Fig. 6B). The impact of B14 on the cell cycle was examined by flow cytometry, B14 induced a concentration-dependent cell cycle arrest in the G0 phase in DHL6 and NB4 cells (Fig. 6C–D). In DHL6 cells, concentrations from 0 to 3 μmol/L led to a maximum block rate of 80.6% at 3 μmol/L, while a time-dependent arrest within 0–48 h reached a peak block rate of 99.3% after 48 h. In NB4 cells, concentrations from 0 to 5 μmol/L led to a maximum block rate of 43.4% at 5 μmol/L. Nevertheless, at 24–48 h, all time points significantly contributed to the cell cycle arrest in the G0 phase compared to the control.

The in vivo antitumor efficacy of compound B14 was further assessed in the HL60 and DHL6 xenograft models (Supporting Information Fig. S5). In the HL60 model, mice received either vehicle or B14 (15 mg/kg, qod) intratumorally for 16 days, resulting in significant inhibition of tumor growth without affecting body weight (Fig. S5A‒S5E). Western blot analysis confirmed B14-induced HDAC7 degradation in tumor tissues (Fig. S5F). Similarly, in the DHL6 model, intraperitoneal injection of B14 significantly inhibited tumor growth without impacting body weight, collectively demonstrating significant in vivo anti-cancer activity (Fig. S5G‒S5I).

3.8.　 Studies on the regulation of BCL6 by HDAC7 in DHL6 cells

To delve deeper into the biological function of HDAC7, we first performed the transcriptome sequencing analysis of DHL6 cells after B14 incubation. Differential genes were identified following the degradation of HDAC7 by B14 and the inhibition of HDAC7 by TMP269. The results revealed that TMP269 incubation led to significant changes in 240 genes (210 upregulated and 30 downregulated), while B14 incubation resulted in significant changes in 1831 genes (442 upregulated and 1389 downregulated). Additionally, 71 genes (57 upregulated and 14 downregulated) exhibited similar changes under TMP269 and B14 incubation, respectively (Fig. 7A). These findings suggest that HDAC7 degradation can regulate a greater number of genes compared to HDAC7 inhibition, implying the presence of a regulatory mechanism in DHL6 cells that is independent of HDAC7's enzymatic function and relies on its non-enzymatic function. A similar phenomenon was also observed in NB4 cells (Supporting Information Fig. S6A). Furthermore, Gene Set Enrichment Analysis (GSEA) demonstrated significant inhibition of the mismatch repair and DNA replication pathways (Fig. 7B and Fig. S6B). This suggests that the apoptosis in DHL6 and NB4 cells, after the degradation of HDAC7, may be primarily caused by these pathways.

Previous studies have demonstrated that BCL6 is one of the binding proteins for HDAC7^53,54. Building on this knowledge, we hypothesized that the HDAC7 degradation could potentially impede the function of BCL6, thus providing a therapeutic effect in DLBCL cells. Bioinformatics analysis revealed a significant positive correlation between HDAC7 and BCL6 in DLBCL (Fig. 7C). To validate the HDAC7-BCL6 relationship, we analyzed transcriptome sequencing data and measured the mRNA levels by quantitative real-time PCR (qRT-PCR) for expression changes in BCL6 downstream factors in both B14-treated and TMP269-treated cells. Results showed significant upregulation of BCL6 downstream genes (CXCR4, CD69, CD80, TP53, CDKN1A, and PDRM1) after B14 incubation, with no significant changes observed in TMP269-treated DHL6 cells (Fig. 7D–F and Supporting Information Fig. S7A), and a similar phenomenon was also observed in DHL4 cells (Fig. S7B‒S7C). Given mSIN3A's pivotal role in BCL6 transcriptional suppression⁵⁴, we next examined whether HDAC7 degradation could impact the formation of mSIN3A and BCL6 complexes. The ability of HDAC7 to form complexes with mSIN3A and BCL6 was confirmed through CO-IP experiments, and the interaction between BCL6 and mSIN3A was weakened when HDAC7 was degraded (Fig. 7G‒H). It indicated that HDAC7 potentially regulates the transcriptional repression function of BCL6 in the form of complexes through its non-enzymatic function.

Overall, one possible explanation for the superior ability of B14, a selective HDAC7 degrader, to inhibit cell proliferation in DHL6 cells compared to TMP269 may be attributed to the non-enzymatic regulation of BCL6 by HDAC7.

3.9.　 Ternary complex simulation

To investigate the potential binding patterns of ternary complexes, a molecular docking simulation was performed on the HDAC7-B14-CRBN complexes (Fig. 8). The results demonstrated that B14 binds to both HDAC7 and E3 ligase through the corresponding POI ligand and E3 ligand, respectively. In the E3 ligand-CRBN part of the ternary complex docking model, the two carbonyl groups of the glutamine formed hydrogen bonds with His380 and Trp382 from CRBN, while the amino group formed a hydrogen bond with His380 from CRBN. Additionally, a carbonyl group of the succinimide formed a hydrogen bond with Trp402 from CRBN. In the POI ligand-HDAC7 part of the ternary complex docking model, trifluoromethyl oxadiazole formed a metal interaction with Zn, and the nitrogen atom in trifluoromethyl oxadiazole formed a hydrogen bond with His670 from HDAC7. Furthermore, the nitrogen atom in benzamide formed a hydrogen bond with Asp626 from HDAC7. The carbonyl group in the linker may contribute to maintaining the dominant conformation of the ternary complex by increasing the rigidity of B14, particularly for the formation of the dominant conformation of the E3 ligand moiety, which promotes tight binding to CRBN.

4.　Conclusions

Less

HDAC7 plays a crucial regulatory role in lymphoma, solid tumors, and various inflammatory diseases. However, the limited selectivity and inhibitory activity of inhibitor-based agents in this field have impeded the development and application of HDAC7 drugs. In our study, we employed bioinformatics-related methods to illustrate the regulatory role of HDAC7 in DLBCL and AML, emphasizing its potential as a novel target for treating lymphoma. Through multiple rounds of optimization using the PROTAC technique, we focused on varying the linker type and linker linkage, ultimately identifying compound B14 as a selective and potent HDAC7 degrader with oral bioavailability. Significantly, B14 exhibited robust HDAC7 degradation potency and high selectivity in DHL6, NB4, and HL60 cells. It effectively induced HDAC7 degradation in a concentration- and time-dependent manner, while not significantly affecting the degradation of other class IIa HDACs, class I HDACs, and class IIb HDACs, as well as the neo-substrates of the E3 ligand. Mechanistic studies revealed that B14 reduces HDAC7 expression through protein degradation via the CRBN E3 ubiquitin pathway, and the formation of a ternary complex is necessary. Intriguingly, B14 exhibited superior cell proliferation inhibitory potency compared to TMP269 in various DLBCL and AML cells, which can upregulate apoptosis-associated proteins cleaved PARP and cleaved caspase 3, and significantly block the G0 phase of the cell cycle. Encouragingly, the non-enzymatic function of B14 was found to regulate the physiological function of BCL6 in DHL6 by interfering with BCL6 to form a transcriptional inhibition complex by degrading HDAC7, while TMP269 had no such effect. Ultimately, the potential binding patterns of HDAC7-B14-CRBN complexes were investigated to explain its degradative effects. Collectively, this study introduces the noteworthy B14 as the first selective and potent HDAC7 PROTAC degrader with oral bioavailability. Its potential is not only a chemical tool to study the non-enzymatic function of the HDAC7 but also a valuable treatment option for lymphoma, especially in DLBCL and AML, which underscores its significance. Furthermore, this study underscores the importance and advantages of developing degraders, and B14 can be a valuable chemical tool for further exploring the non-enzymatic roles and associated biological mechanisms of HDAC7 in other indications such as solid tumors and immune diseases.

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Year 2025 volume 15 Issue 3

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doi: 10.1016/j.apsb.2025.01.021

Receive Date：2024-09-08
Online Date：2026-09-17

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Received：2024-09-08
Revised：2024-11-10
Accepted：2024-12-18

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