The p27 protein is a canonical negative regulator of cell proliferation and acts primarily by inhibiting cyclin-dependent kinases (CDKs). Under some circumstances, p27 is associated with active CDK4, but no mechanism for activation has been described. We found that p27, when phosphorylated by tyrosine kinases, allosterically activated CDK4 in complex with cyclin D1 (CDK4-CycD1). Structural and biochemical data revealed that binding of phosphorylated p27 (phosp27) to CDK4 altered the kinase adenosine triphosphate site to promote phosphorylation of the retinoblastoma tumor suppressor protein (Rb) and other substrates. Surprisingly, purified and endogenous phosp27-CDK4-CycD1 complexes were insensitive to the CDK4-targeting drug palbociclib. Palbociclib instead primarily targeted monomeric CDK4 and CDK6 (CDK4/6) in breast tumor cells. Our data characterize phosp27-CDK4-CycD1 as an active Rb kinase that is refractory to clinically relevant CDK4/6 inhibitors.

Dicer is a multi-domain enzyme that generates small RNAs for gene silencing in eukaryotes. Current understanding of Dicer structure is restricted to simple forms of the enzyme, while that of the large and complex Dicer, widespread in eukarya, is unknown. Here, we describe a novel domain localization strategy developed to determine the structure of human Dicer by electron microscopy. A rearrangement of the nuclease core, compared to the archetypal Giardia Dicer, explains how metazoan Dicers generate 21–23 nucleotide products. The helicase domains form a clamp-like structure adjacent to the RNase III active site, facilitating recognition of pre-miRNA loops or translocation on long dsRNAs. Drosophila Dicer-2 displays similar features, revealing that the three-dimensional architecture is conserved. These results illuminate the structural basis for small RNA production in eukaryotes and provide a versatile new tool for determining structures of large molecular machines.

The DREAM complex represses cell cycle genes during quiescence through scaffolding MuvB proteins with E2F4/5 and the Rb tumor suppressor paralog p107 or p130. Upon cell cycle entry, MuvB dissociates from p107/p130 and recruits B-Myb and FoxM1 for up-regulating mitotic gene expression. To understand the biochemical mechanisms underpinning DREAM function and regulation, we investigated the structural basis for DREAM assembly. We identified a sequence in the MuvB component LIN52 that binds directly to the pocket domains of p107 and p130 when phosphorylated on the DYRK1A kinase site S28. A crystal structure of the LIN52-p107 complex reveals that LIN52 uses a suboptimal LxSxExL sequence together with the phosphate at nearby S28 to bind the LxCxE cleft of the pocket domain with high affinity. The structure explains the specificity for p107/p130 over Rb in the DREAM complex and how the complex is disrupted by viral oncoproteins. Based on insights from the structure, we addressed how DREAM is disassembled upon cell cycle entry. We found that p130 and B-Myb can both bind the core MuvB complex simultaneously but that cyclin-dependent kinase phosphorylation of p130 weakens its association. Together, our data inform a novel target interface for studying MuvB and p130 function and the design of inhibitors that prevent tumor escape in quiescence.

Overexpression of the oncogene MYBL2 (B-Myb) is associated with increased cell proliferation and serves as a marker of poor prognosis in cancer. However, the mechanism by which B-Myb alters the cell cycle is not fully understood. In proliferating cells, B-Myb interacts with the MuvB core complex including LIN9, LIN37, LIN52, RBBP4, and LIN54, forming the MMB (Myb-MuvB) complex, and promotes transcription of genes required for mitosis. Alternatively, the MuvB core interacts with Rb-like protein p130 and E2F4-DP1 to form the DREAM complex that mediates global repression of cell cycle genes in G0/G1, including a subset of MMB target genes. Here, we show that overexpression of B-Myb disrupts the DREAM complex in human cells, and this activity depends on the intact MuvB-binding domain in B-Myb. Furthermore, we found that B-Myb regulates the protein expression levels of the MuvB core subunit LIN52, a key adapter for assembly of both the DREAM and MMB complexes, by a mechanism that requires S28 phosphorylation site in LIN52. Given that high expression of B-Myb correlates with global loss of repression of DREAM target genes in breast and ovarian cancer, our findings offer mechanistic insights for aggressiveness of cancers with MYBL2 amplification, and establish the rationale for targeting B-Myb to restore cell cycle control.

The MuvB transcriptional regulatory complex, which controls cell-cycle-dependent gene expression, cooperates with B-Myb to activate genes required for the G2 and M phases of the cell cycle. We have identified the domain in B-Myb that is essential for the assembly of the Myb-MuvB (MMB) complex. We determined a crystal structure that reveals how this B-Myb domain binds MuvB through the adaptor protein LIN52 and the scaffold protein LIN9. The structure and biochemical analysis provide an understanding of how oncogenic B-Myb is recruited to regulate genes required for cell-cycle progression, and the MMB interface presents a potential therapeutic target to inhibit cancer cell proliferation.

Drugs that directly impede the function of driver oncogenes offer exceptional efficacy and a therapeutic window. The recently approved mutant selective small-molecule cysteine-reactive covalent inhibitor of the G12C mutant of K-Ras, sotorasib, provides a case in point. KRAS is the most frequently mutated proto-oncogene in human cancer, yet despite success targeting the G12C allele, targeted therapy for other hotspot mutants of KRAS has not been described. Here we report the discovery of small molecules that covalently target a G12S somatic mutation in K-Ras and suppress its oncogenic signaling. We show that these molecules are active in cells expressing K-Ras(G12S) but spare the wild-type protein. Our results provide a path to targeting a second somatic mutation in the oncogene KRAS by overcoming the weak nucleophilicity of an acquired serine residue. The chemistry we describe may serve as a basis for the selective targeting of other unactivated serines.

KRAS mutations are one of the most common oncogenic drivers in human cancer. While small molecule inhibitors for the G12C mutant have been successfully developed, allele-specific inhibition for other KRAS hotspot mutants remains challenging. Here we report the discovery of covalent chemical ligands for the common oncogenic mutant K-Ras(G12R). These ligands bind in the Switch II pocket and irreversibly react with the mutant arginine residue. An X-ray crystal structure reveals an imidazolium condensation product formed between the α,β-diketoamide ligand and the εand η-nitrogens of arginine 12. Our results show that arginine residues can be selectively targeted with small molecule electrophiles despite their weak nucleophilicity and provide the basis for the development of mutant-specific therapies for K-Ras(G12R)-driven cancer.

The transcription factor and tumor suppressor protein p53 is the most frequently mutated and inactivated gene in cancer. Mutations in p53 result in deregulated cell proliferation and genomic instability, both hallmarks of cancer. There are currently no therapies available that directly target mutant p53 to rescue wild-type function. In this study we identify covalent compounds that selectively react with the p53 somatic mutant cysteine Y220C and restore wild-type thermal stability.