The Marine Natural Compound Elatol Is a Novel Protein Translation Inhibitor with Potent Activity Against Aggressive Non-Hodgkin Lymphomas

Prognosis remains poor for patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) and other aggressive non-Hodgkin lymphomas. Targeted small-molecule signaling inhibitors have been poorly active in this setting due to a heterogeneous pathogenesis in which multiple different but overlapping signals may drive lymphomagenesis. Activation of cap-dependent protein translation is a convergence point of multiple lymphoma drivers, including B-cell receptor (BCR) signaling pathways. Cap-dependent protein translation begins with formation of the eIF4F complex, a heterotrimeric complex comprised of the cap-binding protein eIF4E, the scaffold protein eIF4G, and the ATP-hydrolyzing RNA helicase eIF4A, which is required for expression of many oncogenes, including the common lymphoma driver MYC. Despite evidence that disruption of eIF4F function is toxic to lymphoma cell lines and sensitizes mouse models of lymphoma to chemotherapy, eIF4A inhibitors have not progressed to clinical evaluation. We designed a screen to identify novel eIF4A inhibitors by measuring ATP hydrolysis and screened a collection of ~1,200 natural compounds and purified extracts. The marine natural product elatol was the top hit in our screen, showing selectivity for eIF4A over other DEAD-box family helicases and a panel of 97 common kinases. Elatol inhibits eIF4A's helicase activity in cell-free in vitro assays and binds to the target at a 2:1 ratio through interaction with two key lysine residues that fold together in its RNA-binding groove. It also induced apoptosis in a broad range of cancer cell lines from the NCI-60 (average LD₅₀= 1.9 μM; LD50<1μM in 33/60) cancer cell line and CCLE panels, but our collection of non-Hodgkin lymphoma lines showed the greatest sensitivity as a group (average LD₅₀= 1.3 μM; 9/12 <1μM; range 213-5749nM). In DLBCL cell lines, Elatol treatment reduced global protein translation measured through polysome profiling and O-propargyl puromycin incorporation as well as cap-dependent translation measured by luciferase reporter and western blotting. Elatol also blocked translational activation downstream of BCR stimulation in primary chronic lymphocytic leukemia (CLL) cells. The effects of elatol are similar to the rocaglate silvestrol, which inhibits eIF4A though through a different mechanism, but elatol requires higher concentrations to achieve these effects. Elatol's reduced in vitro potency, however, is off-set by a maximum tolerated in vivo dose to mice of 65mg/kg, compared to silvestrol's typical dosing at 0.2-05 mg/kg. Elatol significantly reduced the growth of DLBCL tumors in a xenograft model. Mechanistically, elatol and silvestrol both clearly function downstream of mTOR for translational inhibition, but unlike silvestrol, elatol causes strong and rapid induction of the stress response transcription factor ATF4. Assessment of drug activity in murine embryonal fibroblasts lacking ATF4 and those with an unphosphorylatable mutant allele of its upstream activator eIF2α shows elatol's anti-lymphoma activities are not dependent on ATF4 induction. In addition, an inhibitor of the eIF2α kinase PERK and other evidence demonstrate elatol does not produce an unfolded protein response, but the reason for ATF4 induction is not yet clear and additional eIF2α kinases are under investigation. The novel mechanism of inhibition of ATP hydrolysis may be driving these unique observations compared to other eIF4A inhibitors, but unexpected off targets are also under evaluation. In sum we report a new natural inhibitor of protein translation with potent anti-lymphoma activities. Elatol provides a novel starting point for development of eIF4A inhibitors in cancer therapeutics.