BCL-XL/CRBN PROTAC: Mechanism, Optimization, and Novel Strategies in Cancer Therapy

Abstract

BCL-XL, as a core anti-apoptotic factor, is frequently overexpressed in hematological malignancies and solid tumors, driving chemotherapy resistance and tumor survival. Traditional BCL-XL inhibitors (e.g., ABT-263) are severely limited in clinical applications due to dose-dependent platelet toxicity (on-target toxicity). Proteolysis-targeting chimera (PROTAC) technology provides a disruptive solution to this bottleneck. Among these, BCL-XL/CRBN PROTAC utilizes CRBN as the E3 ubiquitin ligase to selectively degrade BCL-XL through the ubiquitin-proteasome system, retaining potent anti-tumor activity while significantly reducing platelet toxicity, making it a cutting-edge direction for precision tumor therapy. This article systematically elucidates the mechanism of action, molecular optimization, anti-tumor activity, and clinical translation potential of BCL-XL/CRBN PROTAC, providing a reference for related research.

Keywords

BCL-XL; CRBN; PROTAC; protein degradation; tumor therapy; platelet toxicity

1 Introduction

BCL-2 family proteins are key regulators of apoptosis, with BCL-XL as an anti-apoptotic member that maintains tumor cell survival by antagonizing the BAX/BAK-mediated mitochondrial apoptosis pathway. In various tumors such as acute lymphoblastic leukemia (ALL), small cell lung cancer (SCLC), and cholangiocarcinoma, BCL-XL is aberrantly overexpressed and closely associated with chemotherapy resistance and poor prognosis. Traditional small-molecule inhibitors (e.g., ABT-263, A-1331852) bind to the BH3 domain of BCL-XL to block its anti-apoptotic function. However, due to platelets' high dependence on BCL-XL for their lifecycle, these inhibitors induce severe thrombocytopenia, leading to bleeding risks and significantly limiting the therapeutic window.

PROTAC technology employs bifunctional chimera molecules that simultaneously bind to target proteins and E3 ubiquitin ligases, inducing ubiquitination and proteasomal degradation of the target protein. This achieves "catalytic" protein clearance, overcoming the limitations of traditional "occupancy-driven" inhibitors. CRBN, as one of the most commonly used E3 ligases, is highly expressed in tumor cells but low in platelets, providing an ideal scaffold for constructing low-toxicity BCL-XL PROTACs. BCL-XL/CRBN PROTAC can selectively degrade BCL-XL in tumor cells, avoiding platelet toxicity while overcoming resistance, offering a new strategy for tumor therapy.

2 Mechanism of Action of BCL-XL/CRBN PROTAC

BCL-XL/CRBN PROTAC is a bifunctional molecule consisting of three parts: a BCL-XL-targeting ligand (e.g., ABT-263 derivative), a CRBN-recruiting ligand (e.g., thalidomide, pomalidomide), and a flexible linker (e.g., PEG chain). Its mechanism follows the typical PROTAC degradation pathway, with the core being ternary complex-mediated ubiquitination and degradation:

Ternary complex formation: The PROTAC molecule specifically binds intracellular BCL-XL through its targeting ligand while recruiting the CRBN-E3 ubiquitin ligase complex (CRBN-Cul4A-DDB1) via the CRBN ligand, forming a "BCL-XL-PROTAC-CRBN" ternary complex. Flexible linkers (e.g., PEG6) optimize spatial conformation, enhancing ternary complex stability and degradation efficiency.

BCL-XL ubiquitination: The CRBN-E3 complex catalyzes the transfer of ubiquitin molecules from E2 ubiquitin-conjugating enzymes to lysine residues on BCL-XL, forming polyubiquitin chains.

Proteasomal degradation: Ubiquitinated BCL-XL is recognized by the 26S proteasome and degraded into short peptides, releasing the PROTAC molecule for recycling and continuous target protein clearance.

Apoptosis activation and toxicity avoidance: After BCL-XL degradation, pro-apoptotic proteins (BAX/BAK) are released, activating the mitochondrial apoptosis pathway and inducing tumor cell apoptosis. Platelets, due to low expression of CRBN and ubiquitin-proteasome system components, cannot effectively degrade BCL-XL, significantly reducing platelet toxicity.

3 Molecular Optimization and Representative Compounds of BCL-XL/CRBN PROTAC

Based on the combination strategy of ABT-263 skeleton and CRBN ligands, researchers have developed a series of highly active, low-toxicity BCL-XL/CRBN PROTACs through linker modification, ligand optimization, and structural refinement. Representative compounds include XZ739, PZ671, and SIAIS361034.

3.1 XZ739: First-Generation High-Efficiency BCL-XL/CRBN PROTAC

XZ739 was developed by the Zheng Guangrong/Zhou Daohong team, replacing the VHL ligand of DT2216 (a VHL-type BCL-XL PROTAC) with pomalidomide (a CRBN ligand), optimizing the polymethylene linker to a PEG chain, and substituting the piperazine group with N-methylamine, significantly enhancing degradation activity. In MOLT-4 cells, XZ739's anti-proliferative activity (IC50) was 20 times stronger than ABT-263, with a DC50 (half-maximal degradation concentration) below 10 nM. It also greatly reduced toxicity to human platelets, with no significant decrease in platelet count. In vivo experiments showed that XZ739 effectively inhibited the growth of SCLC and ALL xenografts without obvious thrombocytopenia side effects.

3.2 PZ671: Next-Generation High-Activity BCL-XL Degrader

PZ671 is an optimized derivative of XZ739, further improving degradation efficiency and anti-tumor activity through structural modifications. In MOLT-4 cells, PZ671's IC50 reached 1.3 nM, with a DC50 of 0.9 nM, showing 6-fold higher degradation activity than XZ739. It exhibited potent killing effects in multiple SCLC cell lines. In vivo studies demonstrated that PZ671 significantly inhibited the growth of MOLT-4 xenografts, causing only moderate and transient platelet count reductions, with superior safety.

3.3 SIAIS361034: Dual-Effect BCL-XL/CRBN PROTAC

SIAIS361034 uses a BCL-XL-specific inhibitor developed by the Shanghai Institute of Materia Medica as the targeting ligand and thalidomide as the CRBN ligand. It selectively degrades BCL-XL (DC50 < 10 nM) while inhibiting the Hedgehog pathway, showing effects against both Hh-dependent and -independent tumors. In hematological malignancies and SCLC models, SIAIS361034 potently killed BCL-XL-dependent tumor cells with minimal impact on platelets.

4 Anti-Tumor Activity and Combination Therapy of BCL-XL/CRBN PROTAC

4.1 Single-Agent Anti-Tumor Activity

BCL-XL/CRBN PROTAC exhibits potent single-agent activity in various BCL-XL-dependent tumors: In ALL (MOLT-4, RS4;11), SCLC, and cholangiocarcinoma cell lines, it efficiently degrades BCL-XL, inducing cell cycle arrest and apoptosis, and inhibiting tumor cell proliferation and colony formation. In xenograft models, it significantly reduced tumor volume and prolonged survival in tumor-bearing mice without notable weight loss or organ toxicity.

4.2 Synergistic Effects in Combination Therapy

BCL-XL/CRBN PROTAC shows significant synergistic effects when combined with chemotherapy or targeted drugs, reversing resistance:

Combination with chemotherapy: XZ739 combined with gemcitabine, cisplatin, or paclitaxel synergistically inhibited tumor growth in cholangiocarcinoma and SCLC models (combination index, CI < 1) without increasing platelet toxicity. PZ671 combined with chemotherapy enhanced anti-ALL activity, outperforming monotherapy.

Combination with BCL-2 inhibitors: BCL-XL/CRBN PROTAC combined with ABT-199 (a BCL-2 inhibitor) simultaneously blocks the BCL-2/BCL-XL anti-apoptotic pathway, producing potent killing effects against dual-dependent tumor cells and overcoming single-agent resistance.

Combination with pathway inhibitors: SIAIS361034 combined with Hedgehog pathway inhibitors synergistically inhibits Hh-dependent tumors, enhancing anti-tumor effects.

5 Safety and Clinical Translation Prospects

5.1 Significantly Reduced Platelet Toxicity

Traditional BCL-XL inhibitors (e.g., ABT-263) cause severe thrombocytopenia due to high BCL-XL expression in platelets. In contrast, BCL-XL/CRBN PROTAC relies on CRBN and the ubiquitin-proteasome system to degrade BCL-XL. Platelets' low expression of CRBN and UPS components prevents effective BCL-XL degradation, significantly reducing platelet toxicity. In vivo experiments show that compounds like XZ739 and PZ671 cause only transient, moderate platelet count reductions without severe bleeding risks, markedly expanding the therapeutic window.

5.2 Clinical Translation Potential

Currently, BCL-XL/CRBN PROTAC remains in preclinical research but shows immense translation potential. Compounds like XZ739 and PZ671 exhibit both high anti-tumor activity and low toxicity in preclinical models, laying the foundation for future clinical trials. For refractory tumors such as cholangiocarcinoma and SCLC, the strategy of combining BCL-XL/CRBN PROTAC with chemotherapy may break through current treatment bottlenecks, offering new options for patients. Additionally, organ-targeting modifications (e.g., conjugation with targeting carriers) could further enhance tumor accumulation and reduce systemic toxicity, advancing precision therapy.

6 Challenges and Future Directions

Despite significant progress, BCL-XL/CRBN PROTAC faces several challenges: ① Resistance: Long-term use may lead to CRBN mutations, target protein mutations, or autophagy activation, causing degradation resistance. ② Pharmacokinetic optimization: PROTAC molecules have relatively large molecular weights, posing issues such as poor solubility, metabolic instability, and low oral bioavailability. ③ Off-target effects: High concentrations may degrade non-specific proteins, triggering potential toxicity.

Future research directions focus on: ① Molecular structure optimization: Improving solubility, metabolic stability, and oral bioavailability through linker modification, ligand screening, and salt formation. ② Resistance mechanism analysis: In-depth study of CRBN mutations, autophagy activation, and other resistance mechanisms to develop combination strategies. ③ Targeted delivery systems: Constructing tumor microenvironment-responsive and organ-targeted PROTAC delivery systems to enhance tumor accumulation and reduce systemic toxicity. ④ Clinical research advancement: Accelerating IND applications for high-activity, low-toxicity candidate compounds and conducting Phase I/II clinical trials to evaluate safety and efficacy.

7 Conclusion

BCL-XL/CRBN PROTAC selectively degrades BCL-XL through the CRBN-mediated ubiquitin-proteasome system, overcoming the platelet toxicity bottleneck of traditional BCL-XL inhibitors. It exhibits potent anti-tumor activity and favorable safety in hematological malignancies and solid tumors, offering a new strategy for precision tumor therapy. With ongoing molecular optimization, delivery system development, and clinical research, BCL-XL/CRBN PROTAC is poised to become an important tool for treating refractory tumors, benefiting more patients.