Abstract
Introduction: A number of oncogenes have been shown to promote the pathogenesis and correlate with poor survival in aggressive lymphoma. For example, deregulation of C-MYC is prevalent in diffuse large B cell lymphoma (DLBCL), chromosome translocations involving C-MYC and BCL2 define "double hit lymphoma" (DHL), and chromosome translocation involving CCND1 is pathognomonic for mantle cell lymphoma (MCL). Despite impressive progress being made in the treatment of these aggressive lymphomas, the treatment options for patients with relapsed aggressive lymphoma, especially DHL, remain inadequate. Interestingly, translation of C-MYC, BCL2, CCND1, and a number of other oncogenes is highly sensitive to inhibition of the eukaryotic translation initiation factor 4F (eIF4F) (Manier et al., 2017; Wolfe et al., 2014), which is comprised of the eIF4E, eIF4A, and eIF4G subunits. However, inhibitors of eIF4F are currently not available for the clinic. eIF4F is negatively regulated by the eIF4E binding protein 1 (4E-BP1), which in the hypo-phosphorylated form sequesters eIF4E (Haghighat et al., 1995). To allow for robust initiation of translation in cancer cells 4E-BP1 is hyper-phosphorylated by upstream signals, such as mechanistic target of rapamycin (mTOR). Our recent results indicate that casein kinase 1 epsilon (CK1ε) positively regulate translation via phosphorylating4E-BP1 (Deng et al., 2016). There may be additional poorly understood signals regulating 4E-BP1. Although no CK1ε inhibitor has been developed for the clinic, we recently discovered that the PI3Kδ inhibitor umbralisib/TGR-1202 acts additionally to inhibit CK1ε. Given that TGR-1202 has demonstrated a favorable safety profile in clinical trials and is capable of targeting both PI3Kδ and CK1ε, the drug may be particularly valuable for the silencing of oncogenes in aggressive lymphoma.Our hypothesis is the following: If hyper-phosphorylation of 4E-BP1 serves as a nexus for upregulating oncogene translation, then co-targeting signals phosphorylating 4E-BP1, such as CK1ε and mTOR, may potently and synergistically silence oncogenes in aggressive lymphomas.
Materials and Methods: Cytotoxicity was studied in lymphoma cell lines and using Cell TiterGlo (Promega®). Screen of drug synergy was performed between pairs of TGR-1202, mTOR inhibitors, proteasome inhibitors, and CK1 inhibitors. The Bliss additivism model was used to determine the expected inhibition of cell growth and the excess over Bliss (EOB) values. EOB values above 0 indicate synergy, with higher values indicating higher levels of synergy. Expression of oncogenes was studied by Western blot and qPCR. 4E-BP1 was studied by Western blot and by 4E-BP1 cap-binding assay using m7GTP Sepharose beads. Translation was determined using a bicistronic reporter. Drug studies were validated by gene knockout of expected molecular targets such as CK1ε and PI3Kδ using CRISPR/Cas9. To further investigate the global effects of targeting translation initiation, we are conducting in parallel proteomic and RNA-Seq studies in 3 cell lines representing DLBCL, DHL, and MCL. Furthermore, the mechanism will be validated by rescuing experiments through over-expression of eIF4E, MYC, or CCND1. The efficacy of TGR-1202-containing regimens is being investigated in xenograft mouse models of MCL.
Results: We investigated the combinatorial effect of TGR-1202 and the proteasome inhibitor carfilzomib. The combination of TGR-1202 and carfilzomib (TC) was highly synergistic in mantle cell lymphoma and double hit lymphoma cell lines (Figure 1A). TC synergistically inhibited phosphorylation of 4E-BP1 at the Ser65 in both MCL and DHL (Figure1B). The TC combination synergistically produced cleavage of poly-ADP ribose polymerase (PARP), indicating apoptosis as its mechanism (Figure 1C). TC reduced the protein expression levels of several oncogenes responsible for malignancies such as c-Myc, cyclin D1, and Bcl-2. In contrast, this combination had no significant effect on the mRNA level of c-Myc and cyclin D1 (Figure 1D).
Conclusion: A novel therapeutic strategy can be developed by combining umbralisib/TGR-1202 and the proteasome inhibitor carfilzomib for the treatment of DLBCL, DHL, and MCL. The combination induces apoptosis by inhibiting the phosphorylation of 4E-BP1, leading to translational silencing and suppressed protein levels of c-Myc, cyclin D1, and Bcl-2.
Ali: Onconova Therapeutics: Consultancy; Kura Oncology: Consultancy. O'Connor: Celgene: Honoraria, Research Funding; Trillium Therapeutics: Research Funding.
