Biljana Culjkovic-Kraljacic

The sonic hedgehog factor GLI1 imparts drug resistance through inducible glucuronidation

Drug resistance is a major hurdle in oncology. Responses of acute myeloid leukaemia (AML) patients to cytarabine (Ara-C)-based therapies are often short lived with a median overall survival of months. Therapies are under development to improve outcomes and include targeting the eukaryotic translation initiation factor (eIF4E) with its inhibitor ribavirin. In a Phase II clinical trial in poor prognosis AML, ribavirin monotherapy yielded promising responses including remissions; however, all patients relapsed. Here we identify a novel form of drug resistance to ribavirin and Ara-C. We observe that the sonic hedgehog transcription factor glioma-associated protein 1 (GLI1) and the UDP glucuronosyltransferase (UGT1A) family of enzymes are elevated in resistant cells. UGT1As add glucuronic acid to many drugs, modifying their activity in diverse tissues. GLI1 alone is sufficient to drive UGT1A-dependent glucuronidation of ribavirin and Ara-C, and thus drug resistance. Resistance is overcome by genetic or pharmacological inhibition of GLI1, revealing a potential strategy to overcome drug resistance in some patients.

Ribavirin as an anti-cancer therapy: Acute Myeloid Leukemia and beyond?

Ribavirin was discovered nearly 40 years ago as a broad-spectrum antiviral drug. Recent data suggest that ribavirin may also be an effective cancer therapy. In this case, ribavirin targets an oncogene, the eukaryotic translation initiation factor eIF4E, elevated in approximately 30% of cancers including many leukemias and lymphomas. Specifically, ribavirin impedes eIF4E mediated oncogenic transformation by acting as an inhibitor of eIF4E. In a phase II clinical trial, ribavirin treatment led to substantial clinical benefit in patients with poor-prognosis acute myeloid leukemia (AML). Here molecular targeting of eIF4E correlated with clinical response. Ribavirin also targets a key enzyme in the guanosine biosynthetic pathway, inosine monophosphate dehydrogenase (IMPDH), and also modulates immunity. Parallels with known antiviral mechanisms could be informative; however, after 40 years, these are not entirely clear. The antiviral effects of ribavirin appear cell-type specific. This variation likely arises for many reasons, including cell specific variations in ribavirin metabolism as well as virus specific factors. Thus, it seems that the mechanisms for ribavirin action in cancer therapy may also vary in terms of the cancer/tissue under study. Here we review the anticancer activities of ribavirin and discuss the possible utility of incorporating ribavirin into diverse cancer therapeutic regimens.

Ribavirin Treatment Effects on Breast Cancers Overexpressing eIF4E, a Biomarker with Prognostic Specificity for Luminal B-Type Breast Cancer

Purpose: We have evaluated the eukaryotic translation initiation factor 4E (eIF4E) as a potential biomarker and therapeutic target in breast cancer. eIF4E facilitates nuclear export and translation of specific, growth-stimulatory mRNAs and is frequently overexpressed in cancer. Experimental Design: Breast cancer cells were treated with ribavirin, an inhibitor of eIF4E, and effects on cell proliferation and on known mRNA targets of eIF4E were determined. eIF4E expression was assessed, at the mRNA and protein level, in breast cancer cell lines and in skin biopsies from patients with metastatic disease. Additionally, pooled microarray data from 621 adjuvant untreated, node-negative breast cancers were analyzed for eIF4E expression levels and correlation with distant metastasis–free survival (DMFS), overall and within each intrinsic breast cancer subtype. Results: At clinically relevant concentrations, ribavirin reduced cell proliferation and suppressed clonogenic potential, correlating with reduced mRNA export and protein expression of important eIF4E targets. This effect was suppressed by knockdown of eIF4E. Although eIF4E expression is elevated in all breast cancer cell lines, variability in ribavirin responsiveness was observed, indicating that other factors contribute to an eIF4E-dependent phenotype. Assessment of the prognostic value of high eIF4E mRNA in patient tumors found that significant discrimination between good and poor outcome groups was observed only in luminal B cases, suggesting that a specific molecular profile may predict response to eIF4E-targeted therapy. Conclusions: Inhibition of eIF4E is a potential breast cancer therapeutic strategy that may be especially promising against specific molecular subtypes and in metastatic as well as primary tumors. Clin Cancer Res; 17(9); 2874–84. ©2011 AACR.

The Oncogene eIF4E Reprograms the Nuclear Pore Complex to Promote mRNA Export and Oncogenic Transformation

The eukaryotic translation initiation factor eIF4E is a potent oncogene that promotes the nuclear export and translation of specific transcripts. Here, we have discovered that eIF4E alters the cytoplasmic face of the nuclear pore complex (NPC), which leads to enhanced mRNA export of eIF4E target mRNAs. Specifically, eIF4E substantially reduces the major component of the cytoplasmic fibrils of the NPC, RanBP2, relocalizes an associated nucleoporin, Nup214, and elevates RanBP1 and the RNA export factors, Gle1 and DDX19. Genetic or pharmacological inhibition of eIF4E impedes these effects. RanBP2 overexpression specifically inhibits the eIF4E mRNA export pathway and impairs oncogenic transformation by eIF4E. The RanBP2 cytoplasmic fibrils most likely slow the release and/or recycling of critical export factors to the nucleus. eIF4E overcomes this inhibitory mechanism by indirectly reducing levels of RanBP2. More generally, these results suggest that reprogramming the NPC is a means by which oncogenes can harness the proliferative capacity of the cell.

eIF4E3 acts as a tumor suppressor by utilizing an atypical mode of methyl-7-guanosine cap recognition

Recognition of the methyl-7-guanosine (m7G) cap structure on mRNA is an essential feature of mRNA metabolism and thus gene expression. Eukaryotic translation initiation factor 4E (eIF4E) promotes translation, mRNA export, proliferation, and oncogenic transformation dependent on this cap-binding activity. eIF4E–cap recognition is mediated via complementary charge interactions of the positively charged m7G cap between the negative π-electron clouds from two aromatic residues. Here, we demonstrate that a variant subfamily, eIF4E3, specifically binds the m7G cap in the absence of an aromatic sandwich, using instead a different spatial arrangement of residues to provide the necessary electrostatic and van der Waals contacts. Contacts are much more extensive between eIF4E3–cap than other family members. Structural analyses of other cap-binding proteins indicate this recognition mode is atypical. We demonstrate that eIF4E3 relies on this cap-binding activity to act as a tumor suppressor, competing with the growth-promoting functions of eIF4E. In fact, reduced eIF4E3 in high eIF4E cancers suggests that eIF4E3 underlies a clinically relevant inhibitory mechanism that is lost in some malignancies. Taken together, there is more structural plasticity in cap recognition than previously thought, and this is physiologically relevant.

Aiding and abetting cancer: mRNA export and the nuclear pore

mRNA export is a critical step in gene expression. Export of transcripts can be modulated in response to cellular signaling or stress. Consistently, mRNA export is dysregulated in primary human specimens derived from many different forms of cancer. Aberrant expression of export factors can alter the export of specific transcripts encoding proteins involved in proliferation, survival, and oncogenesis. These specific factors, which are not used for bulk mRNA export, are obvious therapeutic targets. Indeed, given the emerging role of mRNA export in cancer, it is not surprising that efforts to target different aspects of this pathway have reached the clinical trial stage. Thus, like transcription and translation, mRNA export may also play a critical role in cancer genesis and maintenance.

A Phase I trial of ribavirin and low-dose cytarabine for the treatment of relapsed and refractory acute myeloid leukemia with elevated eIF4E

The molecular heterogeneity of acute myeloid leukemia (AML) underlies the wide variation in responses to standard therapy. This heterogeneity occurs at multiple regulatory steps affecting cell survival and proliferation.1 We identified overexpression of the eukaryotic translation initiation factor 4E (eIF4E) as a targetable aberrancy in all examined cases of M4 and M5 FAB (French, American and British classification) AML subtypes, as well as in some M0, M1, and M2 subtypes.2 eIF4E is both over-expressed and highly enriched in the nucleus of these specimens. eIF4E acts in nuclear mRNA export and translation of specific transcripts necessary for the promotion of proliferation, survival and metastases.3,4 These eIF4E functions depend on its binding the m7G cap on the 5′ end of mRNAs.3,4 Use of ribavirin, a competitive inhibitor of the m7G cap, impairs the biochemical and oncogenic functions of eIF4E.5,6 The first clinical trial to directly target eIF4E activity used ribavirin in AML patients with elevated eIF4E who were unfit for induction chemotherapy or who had relapsed disease.7 Complete and partial responses were observed, and responding patients demonstrated a reduction in overall levels of eIF4E, loss of its nuclear localization, and impaired production of eIF4E targets.7

Combinatorial targeting of nuclear export and translation of RNA inhibits aggressive B-cell lymphomas

Aggressive double- and triple-hit (DH/TH) diffuse large B-cell lymphomas (DLBCLs) feature activation of Hsp90 stress pathways. Herein, we show that Hsp90 controls posttranscriptional dynamics of key messenger RNA (mRNA) species including those encoding BCL6, MYC, and BCL2. Using a proteomics approach, we found that Hsp90 binds to and maintains activity of eIF4E. eIF4E drives nuclear export and translation of BCL6, MYC, and BCL2 mRNA. eIF4E RNA-immunoprecipitation sequencing in DLBCL suggests that nuclear eIF4E controls an extended program that includes B-cell receptor signaling, cellular metabolism, and epigenetic regulation. Accordingly, eIF4E was required for survival of DLBCL including the most aggressive subtypes, DH/TH lymphomas. Indeed, eIF4E inhibition induces tumor regression in cell line and patient-derived tumorgrafts of TH-DLBCL, even in the presence of elevated Hsp90 activity. Targeting Hsp90 is typically limited by counterregulatory elevation of Hsp70B, which induces resistance to Hsp90 inhibitors. Surprisingly, we identify Hsp70 mRNA as an eIF4E target. In this way, eIF4E inhibition can overcome drug resistance to Hsp90 inhibitors. Accordingly, rational combinatorial inhibition of eIF4E and Hsp90 inhibitors resulted in cooperative antilymphoma activity in DH/TH DLBCL in vitro and in vivo.

The eukaryotic translation initiation factor eIF4E is a direct transcriptional target of NF-κB and is aberrantly regulated in acute myeloid leukemia

The eukaryotic translation initiation factor eIF4E is a potent oncogene elevated in many cancers, including the M4 and M5 subtypes of acute myeloid leukemia (AML). Although eIF4E RNA levels are elevated 3- to 10-fold in M4/M5 AML, the molecular underpinnings of this dysregulation were unknown. Here, we demonstrate that EIF4E is a direct transcriptional target of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) that is dysregulated preferentially in M4 and M5 AML. In primary hematopoietic cells and in cell lines, eIF4E levels are induced by NF-κB activating stimuli. Pharmacological or genetic inhibition of NF-κB represses this activation. The endogenous human EIF4E promoter recruits p65 and cRel to evolutionarily conserved κB sites in vitro and in vivo following NF-κB activation. Transcriptional activation is demonstrated by recruitment of p300 to the κB sites and phosphorylated Pol II to the coding region. In primary AML specimens, generally we observe that substantially more NF-κB complexes associate with eIF4E promoter elements in M4 and M5 AML specimens examined than in other subtypes or unstimulated normal primary hematopoietic cells. Consistently, genetic inhibition of NF-κB abrogates eIF4E RNA levels in this same population. These findings provide novel insights into the transcriptional control of eIF4E and a novel molecular basis for its dysregulation in at least a subset of M4/M5 AML specimens.

Importin 8 mediates m7G cap-sensitive nuclear import of the eukaryotic translation initiation factor eIF4E

Significance Dysregulated nuclear trafficking of oncoproteins contributes to cancer. Here, we study the trafficking of eukaryotic translation initiation factor 4E (eIF4E) in acute myeloid leukemia (AML), where eIF4E is highly elevated and accumulates in the nucleus. Nuclear eIF4E promotes export of networks of transcripts encoding oncoproteins. During clinical responses to eIF4E-targeted therapy in patients who have AML, the distribution of eIF4E becomes almost entirely cytoplasmic. At relapse, eIF4E is again mainly nuclear. Our studies provide a molecular understanding for eIF4E trafficking, through association with importin 8. We provide a molecular basis for importin 8 selectivity for certain forms of eIF4E and demonstrate the relevance of its nuclear localization to its oncogenic potential, thereby positioning the importin 8–eIF4E interaction as a novel therapeutic target. Regulation of nuclear-cytoplasmic trafficking of oncoproteins is critical for growth homeostasis. Dysregulated trafficking contributes to malignancy, whereas understanding the process can reveal unique therapeutic opportunities. Here, we focus on eukaryotic translation initiation factor 4E (eIF4E), a prooncogenic protein highly elevated in many cancers, including acute myeloid leukemia (AML). Typically, eIF4E is localized to both the nucleus and cytoplasm, where it acts in export and translation of specific methyl 7-guanosine (m7G)–capped mRNAs, respectively. Nuclear accumulation of eIF4E in patients who have AML is correlated with increased eIF4E-dependent export of transcripts encoding oncoproteins. The subcellular localization of eIF4E closely correlates with patients’ responses. During clinical responses to the m7G-cap competitor ribavirin, eIF4E is mainly cytoplasmic. At relapse, eIF4E reaccumulates in the nucleus, leading to elevated eIF4E-dependent mRNA export. We have identified importin 8 as a factor that directly imports eIF4E into the nucleus. We found that importin 8 is highly elevated in untreated patients with AML, leading to eIF4E nuclear accumulation. Importin 8 only imports cap-free eIF4E. Cap-dependent changes to the structure of eIF4E underpin this selectivity. Indeed, m7G cap analogs or ribavirin prevents nuclear entry of eIF4E, which mirrors the trafficking phenotypes observed in patients with AML. Our studies also suggest that nuclear entry is important for the prooncogenic activity of eIF4E, at least in this context. These findings position nuclear trafficking of eIF4E as a critical step in its regulation and position the importin 8–eIF4E complex as a novel therapeutic target.