A serine/threonine kinase largely overlooked in myeloma biology drives autophagy-dependent tumor cell survival and proteasome inhibitor resistance, according to a preclinical study published August 25 in Nature Communications by researchers at Tampa, Florida-based H. Lee Moffitt Cancer Center and Research Institute. The work identifies ULK3 (Unc-51 like kinase 3) as a disease-stage-correlated dependency in multiple myeloma and describes a second-generation small-molecule inhibitor, MA9-060, that reduced tumor burden, suppressed osteolytic bone disease, and improved survival in mouse xenograft models.
Corresponding author Conor Lynch and colleagues analyzed RNA-sequencing data from 813 Moffitt patient samples spanning disease stages from monoclonal gammopathy of undetermined significance (MGUS) through late relapsed/refractory multiple myeloma (LRMM), and validated findings in an independent cohort of 615 patients. ULK3 expression rose progressively across disease stages and was elevated in proteasome inhibitor-resistant cell lines, a pattern not observed for the related kinases ULK1 or ULK2. Tissue microarray analysis confirmed elevated ULK3 protein in bone marrow biopsies from newly diagnosed (n=14) and relapsed/refractory (n=11) patients compared with 15 healthy donor controls.
CRISPR-Cas9 knockdown of ULK3 in U266 and MM.1S cell lines reduced proliferation and impaired autophagic flux, and lentiviral re-expression of ULK3 in knockdown cells restored proliferative capacity, confirming on-target specificity. In NSG mice bearing luciferase-labeled U266 myeloma cells, ULK3 knockdown significantly reduced tumor burden by bioluminescence imaging and improved survival compared with non-targeting controls (n=20 per group). Critically, pharmacologic treatment with MA9-060 provided no additional survival benefit in mice bearing ULK3-knockout tumors, directly confirming on-target activity in vivo.
The mechanistic basis centers on the ULK3–ATG13–FIP200 autophagy initiation complex. Co-immunoprecipitation and LC-MS/MS proteomics confirmed that ULK3 physically associates with ATG13 and FIP200 in myeloma cells but not in healthy B cells, suggesting a disease-specific dependency. ULK3 appears to operate through a stress-induced, mTORC1-independent mechanism, making it relevant under the proteotoxic stress conditions generated by proteasome inhibitor treatment — precisely the context in which myeloma cells upregulate autophagy as an adaptive survival response.
The researchers optimized an initial multikinase tool compound into MA9-060, which inhibited ULK3 with an IC50 of approximately 227 nM and showed around fivefold selectivity over ULK1. X-ray crystallography confirmed binding within the ULK3 ATP-binding pocket, while experiments using selective JAK2 and BRD4 inhibitors supported ULK3 inhibition as the principal driver of the anti-myeloma activity.
In NSG mouse xenograft experiments (n=6–10 per group), MA9-060 administered at 10 mg/kg/day subcutaneously on a Monday-to-Friday schedule significantly reduced bioluminescence tumor signal versus vehicle and improved overall survival, according to the paper. Faxitron X-ray and histological analysis also showed protection against myeloma-associated osteolytic bone loss in treated animals, an effect not observed with JQ1 or chloroquine, suggesting a bone-protective dimension beyond direct tumor cytotoxicity.