Cancer is driven by genetic alterations that disrupt normal cellular regulation, leading to uncontrolled proliferation and evasion of apoptosis. One crucial mechanism in tumor development is oncogene addiction, where malignant cells become highly dependent on a single oncogene for survival. Additionally, synthetic lethality—where the simultaneous loss of two genes results in cell death, while the loss of a gene alone is tolerated—is a common feature in cancer pathology. Targeting either the key oncogene or its synthetic lethal partner can induce tumor regression when cancer-specific mutations are identified. In solid tumors, BCL-xL, an anti-apoptotic BCL-2 family member, is often overexpressed, promoting survival and resistance. The small molecule BCL-2 inhibitor, venetoclax, was approved for the treatment of hematopoietic tumors, highlighting the therapeutic potential of targeting BCL family in cancer. Targeted protein degradation, including BCL-xL degraders, has emerged as a promising strategy to restore apoptosis in cancer cells. A cell-based assay was developed to assess BCL-xL protein degradation, offering a sensitive and reliable tool for studying protein turnover dynamics and to evaluate the potency of BCL-xL degraders in cancer cells.

To enable precise tracking of BCL-xL degradation, a gene editing approach was applied to introduce a HiBiT sequence into the endogenous BCL-xL gene expressed in a relevant cancer cell line. This modification allows for the direct detection of BCL-xL protein levels through a luminescent signal. In the presence of LgBiT, the reconstituted NanoLuc luciferase produces a luminescent signal directly proportional to protein levels, enabling accurate quantification of degradation. The assay was developed in 384-well plate format, allowing for high-throughput screening and scalability.

The assay was validated using DT2216, a well-characterized BCL-xL degrader, which effectively diminished the luminescent signal by promoting target degradation. After optimization for high-throughput screening, the assay demonstrated robust performance in evaluating candidate degraders acting on HiBiT-tagged proteins. Further validation through western blot analysis in cancer cells confirmed that the assay faithfully represents the behavior of the native, untagged protein, underscoring its suitability for extensive screening campaigns.

This study introduces a robust cell-based assay for monitoring targeted protein degradation in cancer cells. The method streamlines the screening process for novel degraders and can be adapted to other oncogenic targets. Ultimately, this approach holds great promise for the development of targeted cancer therapies that selectively eradicate tumor cells while minimizing collateral damage to normal tissues.