2025 Hot Topic in Basic & Translational Science: Small Cell Lung Cancer - Posters-PP01.18: Genome-Wide CRISPR/Cas9 Screens Utilizing an Endogenous Reporter for ASCL1 Abundance Identifies Mechanisms of ASCL1 Protein Stability

PP01.18: Genome-Wide CRISPR/Cas9 Screens Utilizing an Endogenous Reporter for ASCL1 Abundance Identifies Mechanisms of ASCL1 Protein Stability

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The study investigates the mechanisms that regulate ASCL1 protein stability in small cell lung cancer (SCLC), a highly lethal cancer. ASCL1 is a crucial oncogenic factor in about 60% of SCLC cases, and while it represents a potential therapeutic target, it is difficult to target directly with drugs. <br /><br />To explore the regulators of ASCL1 stability, the researchers developed an endogenous ASCL1 reporter system in a murine SCLC cell line. They employed CRISPR/Cas9 to create a fusion protein that tagged ASCL1, enabling genome-wide knockout screenings using the Brie sgRNA library. This approach aimed to identify both positive and negative regulators of ASCL1. Subsequent validation was performed in murine and human SCLC cell lines.<br /><br />The screenings identified several E3 ubiquitin ligases, most notably HUWE1, which are involved in ASCL1 protein degradation. Inactivation of HUWE1 through CRISPR or chemical inhibitors was found to robustly stabilize ASCL1 by preventing its polyubiquitination. Using HUWE1 inhibitors and Tandem Mass Tag proteomics, the study confirmed stabilization of ASCL1 through specific on-target mechanisms. Furthermore, protein modeling using AlphaFold suggested a conserved interaction between HUWE1 and the c-terminus of ASCL1. Further experiments demonstrated that HUWE1 mediated ASCL1 protein degradation via this c-terminal degron.<br /><br />The conclusions reveal that HUWE1 serves as a key E3 ubiquitin ligase for ASCL1 degradation in SCLC, specifically targeting the c-terminal degron of ASCL1. This finding provides novel mechanistic insights into ASCL1 regulation and suggests potential strategies to therapeutically modulate ASCL1 levels, potentially offering new approaches for targeting ASCL1 in SCLC treatment.

Asset Subtitle

Keita Masuzawa

Keywords

ASCL1

small cell lung cancer

SCLC

protein stability

CRISPR/Cas9

HUWE1

E3 ubiquitin ligase

polyubiquitination

therapeutic target

c-terminal degron