Mechanistic Target of Rapamycin (mTOR) Inhibition Synergizes with Reduced Internal Ribosome Entry Site (IRES)-mediated Translation of Cyclin D1 and c-MYC mRNAs to Treat Glioblastoma

Abstract

Our previous work has demonstrated an intrinsic mRNAspecific protein synthesis salvage pathway operative in glioblastoma (GBM) tumor cells that is resistant to mechanistic target of rapamycin (mTOR) inhibitors. The activation of this internal ribosome entry site (IRES)-dependent mRNA translation initiation pathway results in continued translation of critical transcripts involved in cell cycle progression in the face of global eIF-4E-mediated translation inhibition. Recently we identified compound 11 (C11), a small molecule capable of inhibiting c-MYC IRES translation as a consequence of blocking the interaction of a requisite c-MYC IRES trans-acting factor, heterogeneous nuclear ribonucleoprotein A1, with its IRES. Here we demonstrate that C11 also blocks cyclin D1 IRES-dependent initiation and demonstrates synergistic anti-GBM properties when combined with the mechanistic target of rapamycin kinase inhibitor PP242. The structure-activity relationship of C11 was investigated and resulted in the identification of IRES-J007, which displayed improved IRES-dependent initiation blockade and synergistic anti-GBM effects with PP242. Mechanistic studies with C11 and IRES-J007 revealed binding of the inhibitors within the UP1 fragment of heterogeneous nuclear ribonucleoprotein A1, and docking analysis suggested a small pocket within close proximity to RRM2 as the potential binding site. We further demonstrate that co-therapy with IRES-J007 and PP242 significantly reduces tumor growth of GBM xenografts in mice and that combined inhibitor treatments markedly reduce the mRNA translational state of cyclin D1 and c-MYC transcripts in these tumors. These data support the combined use of IRES-J007 and PP242 to achieve synergistic antitumor responses in GBM.

Repurposing Potential of Riluzole as an ITAF Inhibitor in mTOR Therapy Resistant Glioblastoma

“…In this study, we utilized a molecular docking strategy to identify potential inhibitors which were predicted to bind to the ITAF hnRNP A1. Previously, we had identified a class of inhibitors via a yeast three-hybrid screen in which the tool compound C11, shown in Figure 1A, bound to a small pocket structure close to RRM2 within the UP1 fragment of hnRNP A1 [24]. C11 was subsequently used in structure-activity relationship studies to derive an improved active analog IRES-J007.…”

“…The docking results were then ranked by the binding free energy (see supplementary Table S1) and then the top 10 candidates filtered as potential hnRNP A1 inhibitors (summarized in supplementary Table S2). We subsequently tested these 10 candidates for their ability to affect basal cyclin D1 or c-myc IRES activity in 293T cells which express high levels of hnRNP A1 and show elevated IRES activity [24]. As shown in supplementary Table S2 the benzothiazole CNS compound, riluzole was the most effective inhibitor of IRES activity, markedly blocking both cyclin D1 and c-myc IRES activity and was chosen for further study.…”

“…Recently, we identified a class of small molecule inhibitors which blocks the ability of the ITAF hnRNP A1 from associating with both of the cyclin D1 and c-myc IRES RNAs leading to reduced translation of these determinants and resulting in mTOR inhibitor sensitivity of GBM cells [24].…”

Selective Synthesis in Microdroplets of 2‐Phenyl‐2,3‐dihydrophthalazine‐1,4‐dione from Phenyl Hydrazine with Phthalic Anhydride or Phthalic Acid

“…Pyridazine derivatives, a fascinating class of six‐membered heterocyclic compounds with two adjacent nitrogen atoms, have been found to possess many biological properties, such as anti‐inflammatory, antibacterial, anticancer, cardiovascular diseases, and agrochemical activities . Owing to their biological importance, these chemicals are favored by both synthetic and medicinal chemists, among which, an important six‐membered pyridazine derivative, 2‐phenyl‐2,3‐dihydrophthalazine‐1,4‐dione, was reported to have very significant biological activities against osteosarcoma, glioma, hypolipidemic, and bacteria . In addition, 2‐phenyl‐2,3‐dihydrophthalazine‐1,4‐dione can be used as important optical intermediates .…”

mTORC2/AKT/HSF1/HuR constitute a feed-forward loop regulating Rictor expression and tumor growth in glioblastoma

“…Cells growth was determined via XTT assays (Roche). Cell migration assays were performed using modified Boyden chambers (Chemicon) as previously described 49 Polysome analyses of Luc-Rictor 3' UTR containing reporter mRNAs. U138 cells expressing the indicated reporter mRNAs, native Rictor 3' UTR (Luc-Rictor), mutant 1-2 (Luc-Rictor∆2) in which HuR binding motifs 1 and 2 were mutated, and mutant 1-4 (Luc-Rictor∆4) in which all four HuR binding motifs were mutated (4 unpaired bases were changed to C within the loop structures of the predicted hairpin loop motifs, see Fig.…”