Disablement of cell death programs in cancer cells contributes to drug resistance and in some cases has been associated with altered translational control. As eukaryotic translation initiation factor 4E (eIF4E) cooperates with c-Myc during lymphomagenesis, induces drug resistance, and is a genetic modifier of the rapamycin response, we have investigated the effect of dysregulation of the ribosome recruitment phase of translation initiation on tumor progression and chemosensitivity. eIF4E is a subunit of eIF4F, a complex that stimulates ribosome recruitment during translation initiation by delivering the DEAD-box RNA helicase eIF4A to the 5′ end of mRNAs. eIF4A is thought to prepare a ribosome landing pad on mRNA templates for incoming 40S ribosomes (and associated factors). Using small molecule screening, we found that cyclopenta[b]benzofuran flavaglines, a class of natural products, modulate eIF4A activity and inhibit translation initiation. One member of this class of compounds, silvestrol, was able to enhance chemosensitivity in a mouse lymphoma model in which carcinogenesis is driven by phosphatase and tensin homolog (PTEN) inactivation or elevated eIF4E levels. These results establish that targeting translation initiation can restore drug sensitivity in vivo and provide an approach to modulating chemosensitivity.

RNA helicases are molecular motors that are involved in virtually all aspects of RNA metabolism. Eukaryotic initiation factor (eIF) 4A is the prototypical member of the DEAD-box family of RNA helicases. It is thought to use energy from ATP hydrolysis to unwind mRNA structure and, in conjunction with other translation factors, it prepares mRNA templates for ribosome recruitment during translation initiation. In screening marine extracts for new eukaryotic translation initiation inhibitors, we identified the natural product hippuristanol. We show here that this compound is a selective and potent inhibitor of eIF4A RNA-binding activity that can be used to distinguish between eIF4A-dependent and -independent modes of translation initiation in vitro and in vivo. We also show that poliovirus replication is delayed when infected cells are exposed to hippuristanol. Our study demonstrates the feasibility of selectively targeting members of the DEAD-box helicase family with small-molecule inhibitors.

RNA helicases represent a large family of proteins implicated in many biological processes including ribosome biogenesis, splicing, translation and mRNA degradation. However, these proteins have little substrate specificity, making inhibition of selected helicases a challenging problem. The prototypical DEAD box RNA helicase, eIF4A, works in conjunction with other translation factors to prepare mRNA templates for ribosome recruitment during translation initiation. Herein, we provide insight into the selectivity of a small molecule inhibitor of eIF4A, hippuristanol. This coral-derived natural product binds to amino acids adjacent to, and overlapping with, two conserved motifs present in the carboxy-terminal domain of eIF4A. Mutagenesis of amino acids within this region allowed us to alter the hippuristanol-sensitivity of eIF4A and undertake structure/function studies. Our results provide an understanding into how selective targeting of RNA helicases for pharmacological intervention can be achieved.

To identify therapeutic targets in acute myeloid leukemia (AML), we chemically interrogated 200 sequenced primary specimens. Mubritinib, a known ERBB2 inhibitor, elicited strong anti-leukemic effects in vitro and in vivo. In the context of AML, mubritinib functions through ubiquinone-dependent inhibition of electron transport chain (ETC) complex I activity. Resistance to mubritinib characterized normal CD34 + hematopoietic cells and chemotherapy-sensitive AMLs, which displayed transcriptomic hallmarks of hypoxia. Conversely, sensitivity correlated with mitochondrial function-related gene expression levels and characterized a large subset of chemotherapy-resistant AMLs with oxidative phosphorylation (OXPHOS) hyperactivity. Altogether, our work thus identifies an ETC complex I inhibitor and reveals the genetic landscape of OXPHOS dependency in AML.

Eukaryotic initiation factor 4A (eIF4A) is a member of the DEAD-box family of putative RNA helicases whose members are involved in many aspects of RNA metabolism. eIF4A is thought to facilitate binding of 43S preinitiation complexes to mRNAs by unwinding secondary structures present in the 5' untranslated region. Pateamine A, a small-molecule inhibitor of translation initiation, acts in an unusual manner by stimulating eIF4A activity. Herein, we report the elucidation of pateamine's mode of action. We demonstrate that Pateamine A is a chemical inducer of dimerization that forces an engagement between eIF4A and RNA and prevents eIF4A from participating in the ribosome-recruitment step of translation initiation.

No abstract

RUNX1 is mutated in ∼10% of adult acute myeloid leukemia (AML). Although most RUNX1 mutations in this disease are believed to be acquired, they can also be germline. Indeed, germline RUNX1 mutations result in the well-described autosomal-dominant familial platelet disorder with predisposition to hematologic malignancies (RUNX1-FPD, FPD/AML, FPDMM); ∼44% of affected individuals progress to AML or myelodysplastic syndromes. Using the Leucegene RUNX1 AML patient group, we sought to investigate the proportion of germline vs acquired RUNX1 mutations in this cohort. Our results showed that 30% of RUNX1 mutations in our AML cohort are germline. Molecular profiling revealed higher frequencies of NRAS mutations and other mutations known to activate various signaling pathways in these patients with RUNX1 germline–mutated AML. Moreover, 2 patients (mother and son) had co-occurrence of RUNX1 and CEBPA germline mutations, with variable AML disease onset at 59 and 27 years, respectively. Together, these data suggest a higher than anticipated frequency of germline RUNX1 mutations in the Leucegene cohort and further highlight the importance of testing for RUNX1 mutations in instances in which allogeneic stem cell transplantation using a related donor is envisioned.

Graphical AbstractHighlights d ITGA3 + cells positively identify LT-repopulating HSCs in cultured cord blood cells d ITGA3 + cells display durable multipotentiality d ITGA3 + is functionally required for the LT-engraftment ability of CB cells SUMMARY Transplantation of expanded hematopoietic stem cells (HSCs) and gene therapy based on HSC engineering have emerged as promising approaches for the treatment of hematological diseases. Nevertheless, the immunophenotype of cultured HSCs remains poorly defined. Here, we identify Integrin-a3 (ITGA3) as a marker of cultured human HSCs. Exploiting the pyrimidoindole derivative UM171 to expand cord blood (CB) cells, we show that ITGA3 expression is sufficient to separate the primitive EPCR + CD90 + CD133 + CD34 + CD45RA À HSC population into two functionally distinct fractions presenting mostly short-term (ITGA3 À ) and both short-term and long-term (ITGA3 + ) repopulating potential. ITGA3 + cells exhibit robust multilineage differentiation potential, serial reconstitution ability in immunocompromised mice, and an HSC-specific transcriptomic signature. Moreover, ITGA3 expression is functionally required for the long-term engraftment of CB cells. Altogether, our results indicate that ITGA3 is a reliable marker of cultured human long-term repopulating HSCs (LT-HSCs) and represents an important tool to improve the accuracy of prospective HSC identification in culture.