R-547

Cell cycle transitions are controlled by multiple cell cycle regulators, especially CDKs. Several CDKs, including CDK1-4 and CDK6, promote cell cycle progression directly. Among them, CDK3 is critically important because it triggers the transitions of G0 to G1 and G1 to S phase through binding to cyclin C and cyclin E1, respectively. In contrast to its highly related homologs, the molecular basis of CDK3 activation remains elusive due to the lack of structural information of CDK3, particularly in cyclin bound form. Here we report the crystal structure of CDK3 in complex with cyclin E1 at 2.25 Å resolution. CDK3 resembles CDK2 in that both adopt a similar fold and bind cyclin E1 in a similar way. The structural discrepancy between CDK3 and CDK2 may reflect their substrate specificity. Profiling a panel of CDK inhibitors reveals that dinaciclib inhibits CDK3-cyclin E1 potently and specifically. The structure of CDK3-cyclin E1 bound to dinaciclib reveals the inhibitory mechanism. The structural and biochemical results uncover the mechanism of CDK3 activation by cyclin E1 and lays a foundation for structural-based drug design.

The discovery of recurrent mutations in subunits and regulators of the vacuolar-type H⁺-translocating ATPase (V-ATPase) in follicular lymphoma (FL) highlights a role for macroautophagy/autophagy, amino-acid, and nutrient-sensing pathways in the pathogenesis of this disease. Here, we report on novel mutations in the ER-resident chaperone VMA21, which is involved in V-ATPase assembly in 12% of FL. Mutations in a novel VMA21 hotspot (p.93X) result in the removal of a C-terminal non-canonical ER retrieval signal thus causing VMA21 mislocalization to lysosomes. The resulting impairment in V-ATPase activity prevents full lysosomal acidification and function, including impaired pH-dependent protein degradation as shown via lysosomal metabolomics and ultimately causes a degree of amino acid depletion in the cytoplasm. These deficiencies result in compensatory autophagy activation, as measured using multiple complementary assays in human and yeast cells. Of translational significance, the compensatory activation of autophagy creates a dependency for survival for VMA21-mutated primary human FL as shown using inhibitors to ULK1, the proximal autophagy-regulating kinase. Using high-throughput microscopy-based screening assays for autophagy-inhibiting compounds, we identify multiple clinical grade cyclin-dependent kinase inhibitors as promising drugs and thus provide new rationale for innovative clinical trials in FL harboring aberrant V-ATPase.Abbreviations: ALs: autolysosomes; APs: autophagosomes; ER: endoplasmic reticulum; FL: follicular lymphoma; GFP: green fluorescent protein; IP: immunoprecipitation; LE/LY: late endosomes/lysosomes; Lyso-IP: lysosomal immunoprecipitation; OST: oligosaccharide transferase; prApe1: precursor aminopeptidase I; SEP: super ecliptic pHluorin; V-ATPase: vacuolar-type H⁺-translocating ATPase.