Targeting Ribosome Biogenesis as a Non-Genotoxic Chemotherapy Strategy: Mechanistic Characterization of NVL Inhibition by Benzothiazepinones

Conventional chemotherapies remain the backbone of treatment for many cancers but often act through genotoxic mechanisms that damage DNA in both tumor and healthy cells, leading to serious side effects and dose-limiting toxicity. We have identified MM17, a small-molecule benzothiazepinone that inhibits NVL, an essential AAA+ ATPase required for large ribosomal (60S) subunit assembly. NVL functions as a remodeling enzyme that extracts ribosome biogenesis factors from pre-60S particles during nucleolar maturation. MM17 binding disrupts this activity, leading to stalled ribosome assembly, nucleolar stress, and potent cell cycle arrest in cancer cells. Notably, MM017 elicits both p53-dependent and p53-independent responses without inducing DNA damage, making it a promising prototype for a new class of nongenotoxic anticancer agents. This proposal aims to define the catalytic mechanism of NVL and characterize how benzothiazepinones modulate its structure and function. Using cryo-EM, we will determine high- resolution structures of wild-type and resistant NVL constructs bound to inhibitors, revealing how these compounds perturb ATPase activity and oligomer formation. Mass photometry experiments will quantify how these compounds affect NVL hexamerization, substrate recognition, and nucleotide engagement. Structural studies will also explore how full-length NVL captures its physiological substrate, the conserved C-terminal tail of MAK16, and how this process is disrupted by inhibitors. In parallel, we will test how NVL inhibition leads to the accumulation of free 5S ribonucleoprotein (5S rRNP) complexes that inhibit the E3 ligase MDM2 and activate p53. We will use heterochronic fluorescent labeling to determine whether free 5S rRNPs are newly synthesized or released from stalled pre-60S particles. Finally, we will investigate whether NVL serves a dual role by recruiting the TRAMP– exosome machinery to degrade stalled intermediates. Together, these studies will provide foundational insight into ribosome assembly inhibition as a cancer-selective strategy and establish MM017 as a mechanistically novel, nongenotoxic therapeutic candidate. Project Number: 1R01CA311928-01 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Jan Erzberger (+1 co-PI) | Institution: UT SOUTHWESTERN MEDICAL CENTER, DALLAS, TX | Award Amount: $639,514 | Activity Code: R01 | Study Section: Macromolecular Structure and Function C Study Section[MSFC] View on NIH RePORTER: https://reporter.nih.gov/project-details/11343774