220 - CRISPR Screening Identifies PCCB as a Mitochondrial Driver of Radioresistance via Stabilizing Mitochondrial Homeostasis

Purpose/Objective(s):

Resistance to radiotherapy represents a major barrier in cancer management. As an instrumental cancer hallmark, mitochondrial functional remodeling endows tumor cells with treatment resistance. Herein, we aim to identify a core mitochondrial driver of tumor cell radioresistance via CRISPR/Cas9 screening and proteomic analysis, clarify the resistance-mediating mechanisms, and provide novel therapeutic targets for radiosensitization.

Materials/Methods:

CRISPR/Cas9-based loss-of-function screening was performed to identify genes whose loss confers synthetic lethality with radiation. Proteomic analyses of total protein and mitochondrial fractions indicated radiation-induced PCCB translocation, which was further validated by western blot and immunofluorescence. Endogenous PCCB was stably depleted via CRISPR/Cas9-mediated knockout; subsequent cell viability assays and colony formation assays at graded radiation doses were conducted to verify its radiosensitizing effect. Mitochondrial reactive oxygen species (ROS) levels and membrane potential were detected using MitoSOX and JC-10 staining, respectively. The oxygen consumption rate (OCR) of cells was measured with the Seahorse XF system. In vivo therapeutic efficacy was validated in murine models of lung cancer and head and neck squamous cell carcinoma (HNSCC). The tumor immune microenvironment was profiled by single-cell RNA sequencing (scRNA-seq).

Results:

Mitochondrial-focused CRISPR/Cas9 knockout screening identified PCCB gene, which encodes the ß subunit of propionyl-CoA carboxylase as a mediator of radioresistance in lung cancer cells. Loss of PCCB restored radiosensitivity across multiple tumor cell lines. Mechanistically, radiation promoted the mitochondrial translocation of PCCB, and mitochondrial PCCB rather than its cytoplasmic isoform (a truncated variant lacking the N-terminal 1–28 amino acids) acted to stabilize mitochondrial homeostasis. PCCB depletion aggravated radiation-induced mitochondrial damage, leading to disrupted mitochondrial membrane potential and impaired ATP-linked respiration, and thus abolished the adaptive capacity to cope with post-irradiation stress. In cells relying on glycolysis rather than mitochondrial respiration, PCCB depletion no longer exerted a radiosensitizing effect. In vivo, PCCB ablation enhanced the therapeutic efficacy of radiotherapy and activated adaptive anti-tumor immunity, thereby eliciting a systemic abscopal effect that suppressed distal tumor progression in irradiated mice.

Conclusion:

This study identifies PCCB as a key mitochondrial driver of radioresistance through CRISPR/Cas9 screening. PCCB maintains mitochondrial homeostasis to promote tumor cell survival under irradiation. Targeting PCCB enhances radiotherapy efficacy and anti-tumor immunity, providing a promising strategy to overcome treatment resistance.