Introduction: As an oncoprotein and a transcription factor, C-MYC has been extensively validated as a key driver in many cancers such as Diffuse large B cell lymphoma (DLBCL). C-MYC has been intensely investigated as a therapeutic target in preclinical models. However, no drugs have been successfully developed to target c-Myc, and the c-Myc oncoprotein has been recognized as undruggable. Recent data from our lab and others suggest that the translation of C-MYC and some other oncogenes may be preferentially repressed using inhibitors of the translation apparatus. Translation can be divided into three steps, namely translational initiation, elongation and termination. Translational initiation involves various kinases that stimulate phosphorylation of the eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BP) such as 4E-BP1. In its hypo-phosphorylated state 4E-BP1 sequesters eIF4E and acts as a "brake" for translational initiation. mTORC1 has been established as a key activator for hyper-phosphorylation of 4E-BP1. Hyper-phosphorylation of 4E-BP1 allows the release of eIF4E for the assembly of the eukaryotic initiation factor 4F (eIF4F) complex. eIF4F is comprised of 3 subunits, including the mRNA 5ʹ-cap-binding subunit eIF4E, the large scaffolding subunit eIF4G, and the RNA helicase subunit eIF4A. Subsequently, the 40S and 60S ribosomes are assembled on the AUG start codon of mRNA and translational elongation proceeds along the polysomes. Despite the extensive fund of knowledge in translation, there has been limited success in targeting translation as a therapeutic option for cancers. Omacetaxine mepesuccinate (homo-harringtonine) is approved for the treatment of chronic myeloid leukemia (CML), using the mechanism of competing for aminoacyl-tRNA thereby inhibiting translational elongation. No other translation inhibitors have been approved. Insights into novel regulators of translation will be key to successful development of drugs targeting translation for the treatment of cancers. Such drugs may be particularly useful for cancers driven by translationally regulated oncogenes such as C-MYC.
Materials and Methods: Gene knockout of molecular targets such as CK1δ was done using CRISPR/Cas9. Assembly of eIF4F was determined by cap-binding assay using m7GTP Sepharose beads. Global and gene-specific translation was determined using (a) surface sensing of translation (SUnSET) assay and (b) polysome profile followed by Western blotting and qPCR. To further investigate the mode of the action and the effect on the translation, we are conducting an unbiased, proteome-wide experiment and RNA-seq studies in 2 cell lines representing DLBCL and MCL. We will determine the binding partners of CK1δ using co-immunoprecipitation followed by LC-MS. Finally, we determined in vitro the pharmacological activity of CK1δ inhibitors using the Cell Titer Glo assay and Annexin V assay; and their in vivo activity is being studied using mouse models of human lymphoma xenografts.
Results: Knocking down CK1δ led to marked reduction in the phosphorylation of 4E-BP1 and translational initiation. CK1δ inhibitors phenocopied the effects of CK1δ knockdown on 4E-BP1 or eIF4F, and profoundly inhibited mRNA translation, as demonstrated using the polysome profiling and SUnSET assays. CK1δ inhibitors inhibited the translation of important oncogenes including C-MYC, CCND1, and MDM2 in lymphoma cells. CK1δ inhibitors potently induced cell death in a number of human lymphoma cell lines such as DLBCL and mantle cell lymphoma (MCL). We expect to complete and present the in vivo and omics studies by the time of the ASH meeting in December.
Conclusion: Our results suggest that CK1δ is a key regulator of translation and a promising druggable target in lymphoma. CK1δ inhibitors, if successfully developed, will represent a new class of antineoplastic drugs with a completely new mechanism of action.
Figure
Disclosures
Ali: VOR Biopharma: Patents & Royalties. O'Connor:Acetylon Pharma: Other: Travel expenses, Research Funding; Allos Therapeutics: Consultancy; Millenium: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Seattle Genetics, Inc.: Consultancy, Other: Travel expenses, Research Funding; Spectrum Pharma: Consultancy, Other: Travel expenses, Research Funding; Celgene: Research Funding; Novartis: Consultancy, Honoraria; Roche: Research Funding.
Hypusine modification in eukaryotic initiation factor 5A in rodent cells selected for resistance to growth inhibition by ornithine decarboxylase-inhibiting drugs