Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults and it is associated with poor outcomes. Typical induction strategies involve treatment with cytarabine (Ara-C) along with an anthracycline, either idarubicin or daunorubicin, and is referred to as 7+3 therapy.1 This strategy can result in an approximately 70% complete remission rate, but of these, about 70% will relapse.1 Further confounding the difficulty in inducing remissions is the morbidity associated with this treatment regimen. Many patients over the age of 60, who are the majority of AML patients, cannot tolerate this treatment modality.1 Thus, it is imperative to develop novel strategies to treat AML.
The eukaryotic translation initiation factor eIF4E is overexpressed in many cancers, including the M4 and M5 FAB subtypes of AML.2, 3 Elevated eIF4E levels are generally correlated with poor prognosis in these cancers.2, 3 eIF4E affects gene expression post-transcriptionally by enhancing the mRNA export and/or translation of specific growth promoting transcripts.2, 3, 4, 5 Both of these activities contribute to the oncogenic potential of eIF4E and both require the ability of eIF4E to associate with the 5′ 7-methyl guanosine cap (m7G) structure found on mRNAs.4, 5
Ribavirin acts as a competitive inhibitor of the cap, thereby inhibiting the mRNA export and translation functions of eIF4E.2, 6 The ability of ribavirin to directly bind eIF4E and inhibit cap binding is based on a battery of biochemical studies including NMR, mass spectrometry, fluorescence, cap chromatography and 3H ribavirin eIF4E immunoprecipitation.2, 6
In preclinical studies, it was shown that M4/M5 AML specimens were more sensitive to ribavirin than normal controls or M1/M2 AML specimens with normal eIF4E levels.6 This strongly suggests that cells with elevated eIF4E have developed an oncogene addiction to this protein.2, 6
In a Phase II trial, ribavirin monotherapy led to clinical responses including remissions in poor prognosis M4 and M5 AML patients.7 Further, molecular studies showed that ribavirin inhibited eIF4E activity in patients.7 Importantly, there was no therapy-related toxicity observed in these patients.7 These clinical studies suggested that ribavirin could be a new tool for AML treatment. Thus, we set out to investigate whether ribavirin treatment enhances the activity of anti-cancer agents commonly used in the treatment of AML, including Ara-C, idarubicin, sorafenib and azacytidine.
The effects of drug combinations were assessed using primary specimens from AML patients (obtained from the BCLQ tissue bank with ethics committee approval) or healthy volunteers (Supplementary Table 1). We examined whether 1 μM ribavirin potentiated the effects of Ara-C (0.5 nM), idarubicin (0.3 nM) or Ara-C+idarubicin, using colony growth assays in methylcellulose. Results from AML specimens derived from seven individuals diagnosed with M4/M5 AML were assessed and these results are amalgamated in Table 1. Although both ribavirin and Ara-C alone reduced colony number to about 40% of the untreated samples, the combination further reduced colonies to about 20%. Similarly, idarubicin alone slightly reduced colony number to about 80%, but in combination with ribavirin to 20–30%. Idarubicin with Ara-C reduced colony numbers to about 20–30%, with the exception of the M4 AML Flt3-WT specimen (see below). Strikingly, the triple combination (ribavirin/Ara-C/idarubicin) reduced colonies to 5–10% in all M4/M5 specimens examined. The triple combination, at these concentrations, only slightly affected specimens from healthy volunteers (Table 1, Supplementary Table 2).
Our studies show that ribavirin was active in both Flt3 wild type and mutant specimens (Table 1), reducing colony numbers to about 40% for both Flt3 mutant and wild-type specimens in the M4 or M5 setting. This suggests, in this limited number of specimens, that ribavirin sensitivity is independent of Flt3 status, as we observed in our Phase II clinical trial. The only major difference observed in this context was the lack of sensitivity of the M4 AML Flt3-WT for idarubicin alone, or combined with Ara-C. Clearly, this difference could arise due to specimen idiosyncrasies.
During our analysis, we identified an M1 AML specimen with elevated eIF4E (M1-high4E; Table 1, Supplementary Table 1, Supplementary Figure 1). Ribavirin responses (alone or in combination) in the M1-high4E specimen paralleled the M4/M5 AML specimens, with the most striking reductions in the triple combination (14%). In contrast, ribavirin only had a minor effect on M1/M2 AML specimens with normal levels of eIF4E (Table 1). Thus, in this limited number of specimens, the main feature driving response to ribavirin (alone or in combination) appears to be elevated eIF4E and not lineage.
Given recent reports that sorafenib targets the phosphorylation state of eIF4E,8 we examined the effects of combining 2 μM sorafenib with 1 μM ribavirin; both of these concentrations are clinically achievable in patients (Supplementary Table 2). For all of the high eIF4E specimens, sorafenib reduced colony growth to about 50–60% and ribavirin further reduced this to 16–30%, with no correlation with Flt3 status (Table 1). In specimens from healthy volunteers or M1/M2 AML patients, sorafenib reduced colony number to 70–80% and ribavirin addition only modestly augmented this effect. Thus, the combination of sorafenib and ribavirin preferentially limits colony growth in high eIF4E AML specimens.
Azacytidine is a therapy gaining widespread use in AML.9 We observed that 3 μM azacytidine, which is clinically achievable (Supplementary Table 2), modestly reduced colony number (to 70%) in M4/M5 and M1 high-4E specimens (Table 1). The addition of ribavirin strikingly reduced this (to 10–30%). M1/M2 specimens, or those from healthy volunteers, were sensitive to treatment with azacytidine (60–70%), but ribavirin did not substantially augment this. Thus, M4/M5 AML and M1-high4E specimens are more sensitive to the azacytidine-ribavirin combination than the other groups.
Our studies indicate that a wide variety of agents cooperate with ribavirin to reduce colony growth. Thus, we examined the effects of these agents on eIF4E activity in primary AML specimens treated for 48 h. We confirmed that ribavirin targeted the eIF4E gene expression network. For instance, eIF4E mRNA export and translation targets such as c-Myc, the inhibitor of apoptosis XIAP, Cyclin E and NBS 1 were downregulated (Figure 1, Supplementary Figure 2), as reported previously in patients treated with ribavirin and in cell lines.2, 6, 7 We also note that these changes were not as dramatic in the AML M1 specimen monitored, consistent with the fact that factors other than eIF4E are driving the expression of these targets, and the reduced efficacy of ribavirin addition on inhibiting colony growth in M1 and M2 AML specimens.
As we observed previously, ribavirin alone did not affect eIF4E levels after short-term treatment (Figure 1). Downregulation of eIF4E levels was observed in patients after 28–56 days of ribavirin treatment, suggesting that a distinct population emerges after treatment in heterogenous blast populations found in patients. In contrast to ribavirin treatment alone, most of the drugs tested did not affect NBS1, c-Myc, Cyclin E or XIAP levels, suggesting that they do not substantially impair eIF4E activity when used as single agents (Figure 1, Supplementary Figure 2). However, generally, ribavirin addition leads to decreased levels of the eIF4E targets examined.
Further, we examined phosphorylation status of eIF4E, as we previously determined it to be important to its role in transformation of fibroblasts.10 With the exception of sorafenib, none of the drugs alone (including ribavirin), or in combination with ribavirin, led to alterations in phospho-eIF4E levels under these conditions. However, the reduction in phospho-eIF4E levels by sorafenib did not correlate with better reduction in colony numbers relative to the other treatments.
Further, we observed similar inhibition of eIF4E activity by ribavirin, alone or in combination, but not by the other agents alone in THP-1 cells (Supplementary Figure 3).
In summary, ribavirin cooperated with each agent to target eIF4E activity and colony growth in primary AML M4 and M5 specimens, the M1-high4E specimen and in THP-1 cells. None of the agents seem to substantially modulate eIF4E activity alone, suggesting that this pathway is going largely untargeted in many standard treatment modalities. The most effective combination in terms of growth inhibition was obtained with Idarubicin/Ara-C/ribavirin triple combination where specimens with high eIF4E responded similarly (10% or less of the untreated controls). It is important to note that the concentrations of Ara-C and idarubicin we used were much lower than normal plasma levels found in patients undergoing traditional 7+3 therapy (Supplementary Table 2). We observe effects with Ara-C at levels that are approximately 1000-fold lower than typically used in 7+3 and 100-fold less than in low dose Ara-C regimens. Treatment with 0.3 nM idarubicin (3–12-fold lower than typical concentrations) did not have a substantial effect on the M4/M5 AML specimens alone, but the combination with ribavirin reduced colony numbers to 20%. This suggests that ribavirin enhances the effects of drugs commonly used in AML to the extent that these could be used at much lower concentrations than typically achieved. In this way, ribavirin could potentially increase efficacy and reduce the toxicity of these agents, consistent with studies that show the downregulation of eIF4E increases chemosensitivity of breast cancer cell lines.11, 12
In conclusion, we demonstrate that ribavirin cooperates with established and novel AML therapies including the cornerstones of AML treatment, Ara-C and idarubicin. On the basis of these studies, a Phase I/II clinical trial in M4/M5 AML patients with ribavirin plus low-dose Ara-C is ongoing (Clinicaltrials.gov NTC01056523). Future clinical studies with other combinations will enable the development of strategies that best take advantage of targeting eIF4E with ribavirin. Thus, ribavirin could be used as a potent adjuvant in a variety of clinical modalities, perhaps even in frontline therapy.
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We are grateful for help and advice from Josée Hebert. We thank Christian Charbonneau for technical expertize in confocal microscopy. KLBB holds a Canada Research Chair and this work was supported by a Translational Research Program grant from the Leukemia and Lymphoma Society. IRIC receives infrastructure support from the FRSQ and CIHR.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Leukemia website
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Kraljacic, B., Arguello, M., Amri, A. et al. Inhibition of eIF4E with ribavirin cooperates with common chemotherapies in primary acute myeloid leukemia specimens. Leukemia 25, 1197–1200 (2011). https://doi.org/10.1038/leu.2011.57
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