Kevin P. Callahan

Architecture of the yeast Rrp44-exosome complex suggests routes of RNA recruitment for 3 end processing

The eukaryotic core exosome (CE) is a conserved nine-subunit protein complex important for 3′ end trimming and degradation of RNA. In yeast, the Rrp44 protein constitutively associates with the CE and provides the sole source of processive 3′-to-5′ exoribonuclease activity. Here we present EM reconstructions of the core and Rrp44-bound exosome complexes. The two-lobed Rrp44 protein binds to the RNase PH domain side of the exosome and buttresses the bottom of the exosome-processing chamber. The Rrp44 C-terminal body part containing an RNase II-type active site is anchored to the exosome through a conserved set of interactions mainly to the Rrp45 and Rrp43 subunit, whereas the Rrp44 N-terminal head part is anchored to the Rrp41 subunit and may function as a roadblock to restrict access of RNA to the active site in the body region. The Rrp44–exosome (RE) architecture suggests an active site sequestration mechanism for strict control of 3′ exoribonuclease activity in the RE complex.

TRAMP Complex Enhances RNA Degradation by the Nuclear Exosome Component Rrp6

The RNA-processing exosome contains ribonucleases that degrade aberrant RNAs in archael and eukaryotic cells. In Saccharomyces cerevisiae, the nuclear/nucleolar 3′–5′ exoribonuclease Rrp6 distinguishes the nuclear exosome from the cytoplasmic exosome. In vivo, the TRAMP complex enhances the ability of the nuclear exosome to destroy some aberrant RNAs. Previous reports showed that purified TRAMP enhanced RNA degradation by the nuclear exosome in vitro. However, the exoribonucleolytic component(s) of the nuclear exosome enhanced by TRAMP remain unidentified. We show that TRAMP does not significantly enhance RNA degradation by purified exosomes lacking Rrp6 in vitro, suggesting that TRAMP activation experiments with nuclear exosome preparations reflect, in part, effects on the activity of Rrp6. Consistent with this, we show that incubation of purified TRAMP with recombinant Rrp6 results in a 10-fold enhancement of the rate of RNA degradation. This increased activity results from enhancement of the hydrolytic activity of Rrp6 because TRAMP cannot enhance the activity of an Rrp6 mutant lacking a key amino acid side chain in its active site. We observed no ATP or polyadenylation dependence for the enhancement of Rrp6 activity by TRAMP, suggesting that neither the poly(A) polymerase activity of Trf4 nor the helicase activity of Mtr4 plays a role in the enhancement. These findings identify TRAMP as an exosome-independent enhancer of Rrp6 activity.

Evidence for core exosome independent function of the nuclear exoribonuclease Rrp6p

The RNA exosome processes and degrades RNAs in archaeal and eukaryotic cells. Exosomes from yeast and humans contain two active exoribonuclease components, Rrp6p and Dis3p/Rrp44p. Rrp6p is concentrated in the nucleus and the dependence of its function on the nine-subunit core exosome and Dis3p remains unclear. We found that cells lacking Rrp6p accumulate poly(A)+ rRNA degradation intermediates distinct from those found in cells depleted of Dis3p, or the core exosome component Rrp43p. Depletion of Dis3p in the absence of Rrp6p causes a synergistic increase in the levels of degradation substrates common to the core exosome and Rrp6p, but has no effect on Rrp6p-specific substrates. Rrp6p lacking a portion of its C-terminal domain no longer co-purifies with the core exosome, but continues to carry out RNA 3′-end processing of 5.8S rRNA and snoRNAs, as well as the degradation of certain truncated Rrp6-specific rRNA intermediates. However, disruption of Rrp6p–core exosome interaction results in the inability of the cell to efficiently degrade certain poly(A)+ rRNA processing products that require the combined activities of Dis3p and Rrp6p. These findings indicate that Rrp6p may carry out some of its critical functions without physical association with the core exosome.

Targeting Aberrant Glutathione Metabolism to Eradicate Human Acute Myelogenous Leukemia Cells

Background: Eradication of primary human leukemia cells represents a major challenge. Therapies have not substantially changed in over 30 years. Results: Using normal versus leukemia specimens enriched for primitive cells, we document aberrant regulation of glutathione metabolism. Conclusion: Aberrant glutathione metabolism is an intrinsic property of human leukemia cells. Significance: Interventions based on modulation of glutathione metabolism represent a powerful means to improve therapy. The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34+) leukemic versus normal specimens. Our data indicate that CD34+ AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34+ AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34+ cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34+ AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34+ cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells.

Flavaglines target primitive leukemia cells and enhance anti-leukemia drug activity

Identification of agents that target human leukemia stem cells is an important consideration for the development of new therapies. The present study demonstrates that rocaglamide and silvestrol, closely related natural products from the flavagline class of compounds, are able to preferentially kill functionally defined leukemia stem cells, while sparing normal stem and progenitor cells. In addition to efficacy as single agents, flavaglines sensitize leukemia cells to several anticancer compounds, including front-line chemotherapeutic drugs used to treat leukemia patients. Mechanistic studies indicate that flavaglines strongly inhibit protein synthesis, leading to the reduction of short-lived antiapoptotic proteins. Notably though, treatment with flavaglines, alone or in combination with other drugs, yields a much stronger cytotoxic activity toward leukemia cells than the translational inhibitor temsirolimus. These results indicate that the underlying cell death mechanism of flavaglines is more complex than simply inhibiting general protein translation. Global gene expression profiling and cell biological assays identified Myc inhibition and the disruption of mitochondrial integrity to be features of flavaglines, which we propose contribute to their efficacy in targeting leukemia cells. Taken together, these findings indicate that rocaglamide and silvestrol are distinct from clinically available translational inhibitors and represent promising candidates for the treatment of leukemia.

Lifting the veil on the transcriptome

Inhibition of the cellular RNA surveillance system in Arabidopsis thaliana results in the accumulation of thousands of transcripts arising from annotated and unannotated regions of the genome. This normally hidden transcriptome is replete with noncoding RNAs with the potential to regulate wide-ranging physiological activities.

Monitoring Response and Resistance to the Novel Arsenical Darinaparsin in an AML Patient.

Acute myeloid leukemia (AML) with inversion of chromosome 3 is characterized by overexpression of EVI1 and carries a dismal prognosis. Arsenic-containing compounds have been described to be efficacious in malignancies overexpressing EVI1. Here, we describe a case of AML with inv(3)(q21q26.2) treated with the organic arsenical darinaparsin. Using a “personalized medicine approach,” two different arsenicals were screened for anti-leukemic effect against the patient’s cells ex vivo. The most promising compound, darinaparsin, was selected for in vivo treatment. Clinical effect was almost immediate, with a normalization of temperature, a stabilization of white blood cell (WBC) counts and an increased quality of life. Longitudinal monitoring of patient response and resistance incorporating significant correlative studies on patient-derived blood samples over the two cycles of darinaparsin given to this patient allowed us to evaluate potential mechanisms of response and resistance. The anti-leukemic effects of darinaparsin correlated with inhibition of the alternative NF-κB pathway and production of the inflammatory cytokine IL-8. Emergence of resistance was suspected during treatment cycle 2 and supported by xenograft studies in nude mice. Darinaparsin resistance correlated with an attenuation of the effect of treatment on the alternative NF-κB pathway. The results from this patient indicate that darinaparsin may be a good treatment option for inv(3) AML and that inhibition of the alternative NF-κB pathway may be predictive of response. Longitudinal monitoring of disease response as well as several correlative parameters allowed for the generation of novel correlations and predictors of response to experimental therapy in a heavily pretreated patient.