👤 Jerry L Workman

EBV-induced gene 3 (Ebi3) is a β subunit within the IL-12 cytokine family that canonically binds to α subunits p19, p28, or p35 to form the heterodimeric cytokines IL-39, IL-27, and IL-35, respectively. In the last decade, the binding partners for Ebi3 have continued to expand to include IL-6 and the other IL-12 family β subunit p40, revealing the possibility that Ebi3 may be able to bind to other cytokines and have distinct functions. We first explored this possibility utilizing an in vivo mouse model of regulatory T cell-restricted deletions of the subunits composing the cytokine IL-35, p35, and Ebi3, and we observed a differential impact on CD8+ T cell inhibitory receptor expression despite comparable reduction in tumor growth. We then screened the ability of Ebi3 to bind to different cytokines with varying structural resemblance to the IL-12 family α subunits. These in vitro screens revealed extracellular binding of Ebi3 to both IFN-γ and IL-10. Ebi3 bound to IFN-γ and IL-10 abrogated signal transduction and downstream functions of both cytokines. Lastly, we validated that extracellular complex formation after mixing native proteins resulted in loss of function. These data suggest that secreted partnerless Ebi3 may bind to cytokines within the extracellular microenvironment and act as a cytokine sink, further expanding the potential immunological impact of Ebi3. Show less

Deregulation of the cyclin-dependent kinases (CDKs) has been implicated in the pathogenesis of multiple cancer types. Consequently, CDKs have garnered intense interest as therapeutic targets for the treatment of cancer. We describe herein the molecular and cellular effects of CCT068127, a novel inhibitor of CDK2 and CDK9. Optimized from the purine template of seliciclib, CCT068127 exhibits greater potency and selectivity against purified CDK2 and CDK9 and superior antiproliferative activity against human colon cancer and melanoma cell lines. X-ray crystallography studies reveal that hydrogen bonding with the DFG motif of CDK2 is the likely mechanism of greater enzymatic potency. Commensurate with inhibition of CDK activity, CCT068127 treatment results in decreased retinoblastoma protein (RB) phosphorylation, reduced phosphorylation of RNA polymerase II, and induction of cell cycle arrest and apoptosis. The transcriptional signature of CCT068127 shows greatest similarity to other small-molecule CDK and also HDAC inhibitors. CCT068127 caused a dramatic loss in expression of DUSP6 phosphatase, alongside elevated ERK phosphorylation and activation of MAPK pathway target genes. MCL1 protein levels are rapidly decreased by CCT068127 treatment and this associates with synergistic antiproliferative activity after combined treatment with CCT068127 and ABT263, a BCL2 family inhibitor. These findings support the rational combination of this series of CDK2/9 inhibitors and BCL2 family inhibitors for the treatment of human cancer. Show less

Proteins that respond to DNA damage play critical roles in normal and diseased states in human biology. Studies have suggested that the S. cerevisiae protein CMR1/YDL156w is associated with histones and is possibly associated with DNA repair and replication processes. Through a quantitative proteomic analysis of affinity purifications here we show that the human homologue of this protein, WDR76, shares multiple protein associations with the histones H2A, H2B, and H4. Furthermore, our quantitative proteomic analysis of WDR76 associated proteins demonstrated links to proteins in the DNA damage response like PARP1 and XRCC5 and heterochromatin related proteins like CBX1, CBX3, and CBX5. Co-immunoprecipitation studies validated these interactions. Next, quantitative imaging studies demonstrated that WDR76 was recruited to laser induced DNA damage immediately after induction, and we compared the recruitment of WDR76 to laser induced DNA damage to known DNA damage proteins like PARP1, XRCC5, and RPA1. In addition, WDR76 co-localizes to puncta with the heterochromatin proteins CBX1 and CBX5, which are also recruited to DNA damage but much less intensely than WDR76. This work demonstrates the chromatin and DNA damage protein associations of WDR76 and demonstrates the rapid response of WDR76 to laser induced DNA damage. Show less