DEAD-box proteins (DBPs) are required in gene expression to facilitate changes to ribonucleoprotein complexes, but the cellular mechanisms and regulation of DBPs are not fully defined. Gle1 is a multifunctional regulator of DBPs with roles in mRNA export and translation. In translation, Gle1 modulates Ded1, a DBP required for initiation. However, overexpression causes defects, suggesting that Ded1 can promote or repress translation in different contexts. Here we show that expression suppresses the repressive effects of , and Gle1 counteracts Ded1 in translation assays Furthermore, Ded1 and Gle1 both affect assembly of pre-initiation complexes. Through mutation analysis and binding assays, we show that Gle1 inhibits Ded1 by reducing its affinity for RNA. Our results are consistent with a model wherein active Ded1 promotes translation, but inactive or excess Ded1 leads to translation repression. Gle1 can inhibit either role of Ded1, positioning it as a gatekeeper to optimize Ded1 activity to the appropriate level for translation. This study suggests a paradigm for finely controlling the activity of DEAD-box proteins to optimize their function in RNA-based processes. It also positions the versatile regulator Gle1 as a potential node for the coordination of different steps of gene expression.

Medulloblastoma is the most common pediatric brain cancer, and sequencing studies identified frequent mutations in DDX3X , a DEAD-box RNA helicase primarily implicated in translation. Forty-two different sites were identified, suggesting that the functional effects of the mutations are complex. To investigate how these mutations are affecting DDX3X cellular function, we constructed a full set of equivalent mutant alleles in DED1 , the Saccharomyces cerevisiae ortholog of DDX3X , and characterized their effects in vivo and in vitro . Most of the medulloblastoma-associated mutants in DDX3X/DED1 ( ded1-mam ) showed substantial growth defects, indicating that functional effects are conserved in yeast. Further, while translation was affected in some mutants, translation defects affecting bulk mRNA were neither consistent nor correlated with the growth phenotypes. Likewise, increased formation of stress granules in ded1-mam mutants was common but did not correspond to the severity of the mutants’ growth defects. In contrast, defects in translating mRNAs containing secondary structure in their 5’ untranslated regions (UTRs) were found in almost all ded1-mam mutants and correlated well with growth phenotypes. We thus conclude that these specific translation defects, rather than generalized effects on translation, are responsible for the observed cellular phenotypes and likely contribute to DDX3X -mutant medulloblastoma. Examination of ATPase activity and RNA binding of recombinant mutant proteins also did not reveal a consistent defect, indicating that the translation defects are derived from multiple enzymatic deficiencies. This work suggests that future studies into medulloblastoma pathology should focus on this specific translation defect, while taking into account the wide spectrum of DDX3X mutations.

The activated B-cell cell-of-origin subtype of diffuse large B-cell lymphoma (ABC-DLBCL) requires NF-kB pathway activation to maintain the malignant phenotype. NF-kB activation is downstream of B-cell receptor (BCR) stimulation and can become constitutively turned on in ABC-DLBCL through mutations in multiple different BCR signaling intermediates. Drugs targeting upstream signaling proteins such as Bruton’s Tyrosine Kinase (BTK, ibrutinib) or protein kinase C (PKC, AEB071) have shown promising results in other lymphomas driven by BCR activation and are under evaluation in ABC-DLBCL. Many ABC-DBCL cases, however, have mutations in mediators that are downstream from these targets, particularly coiled-coil domain mutations in CARD11 that stabilize NF-kB activation and A20 loss-of-function alterations that reduce protein turnover of oncogenic NF-kB intermediates. In this study we explore a potential role for PIM kinase inhibition in ABC-DLBCL and find the clinical pan-PIM inhibitor LGH447 has promising activity in particular against cells carrying these downstream mutations. We find some ABC cells were highly sensitive to LGH447 with IC50 < 0.4 µM (OCI-Ly3 and OCI-Ly10), while some others were completely insensitive with IC50 > 10.0 µM (TMD8 and HBL-1). Strikingly, all ABC lines sensitive to LGH447 carry mutations in either CARD11, TNFAIP3 (encoding A20), or both, while insensitive typically lines lack such lesions. Insensitive lines including TMD8 and HBL-1 instead have upstream mutations in CD79B and are highly sensitive to the upstream inhibitors ibrutinib and AEB071. The PIM1-3 kinases inhibited by LGH447 have multiple targets mediating cell growth and survival, including several that activate cap-dependent protein translation activation. We find LGH447 is toxic to sensitive cells due to lost translational activation. Western blots show reduced phosphorylation of ribosomal protein S6 and 4EBP1, indicating loss of mTORC1 activity. In addition, LGH447, in a manner similar to the potent direct cap-dependent translation inhibitor silvestrol, causes knockdown of key translationally regulated oncoproteins, including c-MYC, MCL1, and Cyclin D3. We also directly monitored protein synthesis through O-Propargyl-puromycin (OP-PURO) incorporation and found a direct effect of LGH447 that was similar to silvestrol, although requiring higher concentrations. PIM’s effects on activation of protein translation therefore are required in LGH447-sensitive ABC-DLBCL cells but dispensable in insensitive cells. In conclusion, pan-PIM kinase inhibition provides a strong potential therapeutic opportunity in a subset of ABC-DLBCL. Cases that bypass upstream signaling to turn on NF-kB activation more directly also bypass pathways with redundant activation of cap-dependent translation, making them dependent on the therapeutically targetable PIM kinases to carry out this critical process. Disclosures No relevant conflicts of interest to declare.