Letters to the Editor 1197 Acknowledgements patient subgroup characterized by very poor prognosis. J Clin Oncol 1989; 7: 119–125. This study is supported in part by the grants CA 62242, CA 107476 3 San Miguel JF, Garcia-Sanz R, Gonzalez M, Moro MJ, Hernandez JM, and CA 62242 from the National Cancer Institute, National Ortega F et al. A new staging system for multiple myeloma based on the number of S-phase plasma cells. Blood 1995; 85: Institutes of Health, Bethesda, MD and the Eastern Cooperative 448–455. Group, Boston, MA (SVR). 4 Rajkumar SV, Fonseca R, Dewald GW, Therneau TM, Lacy MQ, Kyle RA et al. Cytogenetic abnormalities correlate with the plasma P Kapoor1, S Kumar1, SJ Mandrekar2, KM Laumann2, 1 1 1 1 cell labeling index and extent of bone marrow involvement in A Dispenzieri , MQ Lacy , D Dingli , MA Gertz , myeloma. Cancer Genet Cytogenet 1999; 113: 73–77. 1 1 1 1 RA Kyle , PR Greipp , SV Rajkumar and TE Witzig 5 Garcia-Sanz R, Gonzalez-Fraile MI, Mateo G, Hernandez JM, 1 Division of , Mayo Clinic, Lopez-Berges MC, de las Heras N et al. Proliferative activity of Rochester, MN, USA and plasma cells is the most relevant prognostic factor in elderly 2Division of Biomedical Statistics and Informatics, multiple myeloma patients. Int J Cancer 2004; 112: 884–889. Mayo Clinic, Rochester, MN, USA 6 Lacy MQ, Gertz MA, Dispenzieri A, Hayman SR, Geyer S, E-mail: [email protected] Kabat B et al. Long-term results of response to , time to progression, and survival with lenalidomide plus dexametha- sone in newly diagnosed myeloma. Mayo Clin Proc 2007; 82: 1179–1184. References 7 Rajkumar SV, Hayman S, Gertz MA, Dispenzieri A, Lacy MQ, Greipp PR et al. Combination therapy with thalidomide plus 1 Greipp PR, Lust JA, O’Fallon WM, Katzmann JA, Witzig TE, Kyle RA. dexamethasone for newly diagnosed myeloma. J Clin Oncol Plasma cell labeling index and beta 2-microglobulin predict survival 2002; 20: 4319–4323. independent of thymidine kinase and C-reactive protein in multiple 8 Kapoor P, Kumar S, Fonseca R, Lacy MQ, Witzig TE, Hayman SR myeloma. Blood 1993; 81: 3382–3387. et al. Impact of risk stratification on outcome among patients with 2 Boccadoro M, Marmont F, Tribalto M, Fossati G, Redoglia V, multiple myeloma receiving initial therapy with lenalidomide and Battaglio S et al. Early responder myeloma: kinetic studies identify a dexamethasone. Blood 2009; 114: 518–521.

Inhibition of eIF4E with ribavirin cooperates with common chemotherapies in primary acute myeloid leukemia specimens

Leukemia (2011) 25, 1197–1200; doi:10.1038/leu.2011.57; In preclinical studies, it was shown that M4/M5 AML published online 1 April 2011 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 Acute myeloid leukemia (AML) is the most common type of acute In a Phase II trial, ribavirin monotherapy led to clinical leukemia in adults and it is associated with poor outcomes. Typical responses including remissions in poor prognosis M4 and M5 induction strategies involve treatment with cytarabine (Ara-C) AML patients.7 Further, molecular studies showed that ribavirin along with an anthracycline, either idarubicin or daunorubicin, inhibited eIF4E activity in patients.7 Importantly, there was no andisreferredtoas7þ 3 therapy.1 This strategy can result in an therapy-related toxicity observed in these patients.7 These approximately 70% complete remission rate, but of these, about clinical studies suggested that ribavirin could be a new tool 70% will relapse.1 Further confounding the difficulty in inducing for AML treatment. Thus, we set out to investigate whether remissions is the morbidity associated with this treatment regimen. ribavirin treatment enhances the activity of anti-cancer agents Many patients over the age of 60, who are the majority of AML commonly used in the treatment of AML, including Ara-C, patients, cannot tolerate this treatment modality.1 Thus, it is idarubicin, sorafenib and azacytidine. imperative to develop novel strategies to treat AML. The effects of drug combinations were assessed using primary The eukaryotic translation initiation factor eIF4E is over- specimens from AML patients (obtained from the BCLQ tissue expressed in many cancers, including the M4 and M5 FAB bank with ethics committee approval) or healthy volunteers 2,3 subtypes of AML. Elevated eIF4E levels are generally correlated (Supplementary Table 1). We examined whether 1 mM ribavirin 2,3 with poor prognosis in these cancers. eIF4E affects gene expres- potentiated the effects of Ara-C (0.5 nM), idarubicin (0.3 nM)or sion post-transcriptionally by enhancing the mRNA export and/or Ara-C þ idarubicin, using colony growth assays in methylcellu- translation of specific growth promoting transcripts.2–5 Both of lose. Results from AML specimens derived from seven indivi- these activities contribute to the oncogenic potential of eIF4E and duals diagnosed with M4/M5 AML were assessed and these both require the ability of eIF4E to associate with the 50 7-methyl results are amalgamated in Table 1. Although both ribavirin and guanosine cap (m7G) structure found on mRNAs.4,5 Ara-C alone reduced colony number to about 40% of the Ribavirin acts as a competitive inhibitor of the cap, thereby untreated samples, the combination further reduced colonies to inhibiting the mRNA export and translation functions of about 20%. Similarly, idarubicin alone slightly reduced colony eIF4E.2,6 The ability of ribavirin to directly bind eIF4E and number to about 80%, but in combination with ribavirin to inhibit cap binding is based on a battery of biochemical studies 20–30%. Idarubicin with Ara-C reduced colony numbers to including NMR, mass spectrometry, fluorescence, cap chromato- about 20–30%, with the exception of the M4 AML Flt3-WT graphy and 3H ribavirin eIF4E immunoprecipitation.2,6 specimen (see below). Strikingly, the triple combination

Leukemia Letters to the Editor 1198 Table 1 Effects of drugs on colony formation of primary AML specimens

Drug AML-M4 AML-M4 AML-M5 AML-M5 AML-M1 AML-M1 AML-M2 Normal Normal FLT3 MUT FLT3 WT FLT3 MUT FLT3 WT High 4E PBMC CD34+ %Av±s.d. %Av ± s.d. %Av±s.d. %Av±s.d. %Av±s.d. %Av±s.d. %Av±s.d. %Av±s.d. %Av±s.d.

Untreated 100±8a 100±10 100±9b 100±8 100±7 100±8 100±3 100±12a 100±3 Rib 41±8a 41±548±6b 53±445±477±569±580±14a 87±3 Ara-C 49±5a 63±12 39±7b 42±454±559±971±568±11b 82±3 Ara-C+Rib 18±3a 20±726±9b 17±719±453±753±575±8b 70±5 Ida 70±6a 99±13 75±6b 76±684±492±584±386±10a 94±3 Ida+Rib 19±3a 16±10 34±6b 33±529±475±679±362±10a 88±5 Ida+Ara-C 24±3a 60±723±4b 20±421±456±664±363±13b 82±3 Ida+Ara-C+Rib 10±3a 7±513±36±214±247±656±661±10b 74±4 Sor 50±955±10 64±7b 57±667±478±11 70±11 77±5NA Sor+Rib 21±416±323±4b 31±425±365±11 73±362±7NA Aza 77±778±963±867±677±572±478±360±766±7 Aza+Rib 30±67±432±524±628±559±767±349±570±3 Abbreviations: Av, average as a percent of the relevant untreated control; FLT3 MUT, specimens with internal tandem duplication; FLT3 WT, no mutation detected; NA, not available; s.d., standard deviation. Specimens are treated as described in the materials and methods. All experiments were carried out in replicates of five and repeated at least three independent times. Drug abbreviations and dosages were as follows: Rib ¼ ribavirin (1 mM), Ara-C ¼ cytarabine (0.5 nM), Ida ¼ idarubicin (0.3 nM), Sor ¼ sorafenib (2 mM), Aza ¼ azacytidine (3 mM). Note that cross comparison indicated that there was no correlation between NPM status and ribavirin dependent colony inhibition consistent with results in the clinical trial.6 aIndicates results amalgamated from three specimens. bIndicates results amalgamated from two specimens.

(ribavirin/Ara-C/idarubicin) reduced colonies to 5–10% in all (Supplementary Table 2), modestly reduced colony number (to M4/M5 specimens examined. The triple combination, at these 70%) in M4/M5 and M1 high-4E specimens (Table 1). The concentrations, only slightly affected specimens from healthy addition of ribavirin strikingly reduced this (to 10–30%). M1/M2 volunteers (Table 1, Supplementary Table 2). specimens, or those from healthy volunteers, were sensitive to Our studies show that ribavirin was active in both Flt3 wild treatment with azacytidine (60–70%), but ribavirin did not type and mutant specimens (Table 1), reducing colony numbers substantially augment this. Thus, M4/M5 AML and M1-high4E to about 40% for both Flt3 mutant and wild-type specimens in specimens are more sensitive to the azacytidine-ribavirin the M4 or M5 setting. This suggests, in this limited number of combination than the other groups. specimens, that ribavirin sensitivity is independent of Flt3 status, Our studies indicate that a wide variety of agents cooperate as we observed in our Phase II clinical trial. The only major with ribavirin to reduce colony growth. Thus, we examined difference observed in this context was the lack of sensitivity of the effects of these agents on eIF4E activity in primary AML the M4 AML Flt3-WT for idarubicin alone, or combined with specimens treated for 48 h. We confirmed that ribavirin targeted Ara-C. Clearly, this difference could arise due to specimen the eIF4E gene expression network. For instance, eIF4E mRNA idiosyncrasies. export and translation targets such as c-Myc, the inhibitor of During our analysis, we identified an M1 AML specimen with apoptosis XIAP, Cyclin E and NBS 1 were downregulated elevated eIF4E (M1-high4E; Table 1, Supplementary Table 1, (Figure 1, Supplementary Figure 2), as reported previously in Supplementary Figure 1). Ribavirin responses (alone or in patients treated with ribavirin and in cell lines.2,6,7 We also note combination) in the M1-high4E specimen paralleled the that these changes were not as dramatic in the AML M1 M4/M5 AML specimens, with the most striking reductions in specimen monitored, consistent with the fact that factors other the triple combination (14%). In contrast, ribavirin only had a than eIF4E are driving the expression of these targets, and the minor effect on M1/M2 AML specimens with normal levels of reduced efficacy of ribavirin addition on inhibiting colony eIF4E (Table 1). Thus, in this limited number of specimens, the growth in M1 and M2 AML specimens. main feature driving response to ribavirin (alone or in As we observed previously, ribavirin alone did not affect combination) appears to be elevated eIF4E and not lineage. eIF4E levels after short-term treatment (Figure 1). Downregula- Given recent reports that sorafenib targets the phosphoryla- tion of eIF4E levels was observed in patients after 28–56 days of 8 tion state of eIF4E, we examined the effects of combining 2 mM ribavirin treatment, suggesting that a distinct population sorafenib with 1 mM ribavirin; both of these concentrations are emerges after treatment in heterogenous blast populations found clinically achievable in patients (Supplementary Table 2). For all in patients. In contrast to ribavirin treatment alone, most of the of the high eIF4E specimens, sorafenib reduced colony growth drugs tested did not affect NBS1, c-Myc, Cyclin E or XIAP levels, to about 50–60% and ribavirin further reduced this to 16–30%, suggesting that they do not substantially impair eIF4E activity with no correlation with Flt3 status (Table 1). In specimens from when used as single agents (Figure 1, Supplementary Figure 2). healthy volunteers or M1/M2 AML patients, sorafenib reduced However, generally, ribavirin addition leads to decreased levels colony number to 70–80% and ribavirin addition only modestly of the eIF4E targets examined. augmented this effect. Thus, the combination of sorafenib and Further, we examined phosphorylation status of eIF4E, as ribavirin preferentially limits colony growth in high eIF4E AML we previously determined it to be important to its role in specimens. transformation of fibroblasts.10 With the exception of sorafenib, Azacytidine is a therapy gaining widespread use in AML.9 We none of the drugs alone (including ribavirin), or in combination observed that 3 mM azacytidine, which is clinically achievable with ribavirin, led to alterations in phospho-eIF4E levels under

Leukemia Letters to the Editor 1199

Figure 1 Ribavirin combinations target eIF4E activity. Results from a representative primary AML M5 specimen treated with drugs and analyzed for the expression of eIF4E target genes are shown. Ph eIF4E indicates phosphorylated eIF4E. Ctrl indicates vehicle controls (dimethyl sulfoxide for sorafenib, otherwise phosphate-buffered saline). b-Actin, a-tubulin and glyceraldehyde-3 phosphate dehydrogenase are provided as a loading control. Drug concentrations were as follows: Rib ¼ ribavirin (20 mM); Ara-C ¼ cytarabine (0.5 nM); Ida ¼ idarubicin (0.3 nM); I+A+R ¼ idarubicin, ara-C, ribavirin; Sor ¼ sorafenib (2 mM); Aza ¼ azacytidine (3 mM). Treatments were for 48 h.

these conditions. However, the reduction in phospho-eIF4E of AML treatment, Ara-C and idarubicin. On the basis of these levels by sorafenib did not correlate with better reduction in studies, a Phase I/II clinical trial in M4/M5 AML patients with colony numbers relative to the other treatments. ribavirin plus low-dose Ara-C is ongoing (Clinicaltrials.gov Further, we observed similar inhibition of eIF4E activity by NTC01056523). Future clinical studies with other combinations ribavirin, alone or in combination, but not by the other agents will enable the development of strategies that best take alone in THP-1 cells (Supplementary Figure 3). advantage of targeting eIF4E with ribavirin. Thus, ribavirin In summary, ribavirin cooperated with each agent to target could be used as a potent adjuvant in a variety of clinical eIF4E activity and colony growth in primary AML M4 and M5 modalities, perhaps even in frontline therapy. 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 Conflict of interest many standard treatment modalities. The most effective combination in terms of growth inhibition was obtained with The authors declare no conflict of interest. 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 concentra- Acknowledgements tions of Ara-C and idarubicin we used were much lower than normal plasma levels found in patients undergoing traditional We are grateful for help and advice from Jose´e Hebert. We thank 7 þ 3 therapy (Supplementary Table 2). We observe effects with Christian Charbonneau for technical expertize in confocal Ara-C at levels that are approximately 1000-fold lower than microscopy. KLBB holds a Canada Research Chair and this work typically used in 7 þ 3 and 100-fold less than in low dose Ara-C was supported by a Translational Research Program grant from the regimens. Treatment with 0.3 nM idarubicin (3–12-fold lower Leukemia and Lymphoma Society. IRIC receives infrastructure than typical concentrations) did not have a substantial effect on support from the FRSQ and CIHR. the M4/M5 AML specimens alone, but the combination with BC Kraljacic, M Arguello, A Amri, G Cormack and K Borden ribavirin reduced colony numbers to 20%. This suggests that Institute of Research in and Cancer (IRIC), ribavirin enhances the effects of drugs commonly used in AML Department of and Cell Biology, to the extent that these could be used at much lower Universite´ de Montre´al, Montreal, Canada. concentrations than typically achieved. In this way, ribavirin E-mail: [email protected] could potentially increase efficacy and reduce the toxicity of these agents, consistent with studies that show the down- regulation of eIF4E increases chemosensitivity of breast cancer References cell lines.11,12 In conclusion, we demonstrate that ribavirin cooperates with 1 Martin MG, Abboud CN. Induction therapy for elderly patients established and novel AML including the cornerstones with acute myeloid leukemia. Blood Rev 2008; 22: 311–320.

Leukemia Letters to the Editor 1200 2 Borden KL, Culjkovic-Kraljacic B. Ribavirin as an anti-cancer acute myeloid leukemia (AML): a proof-of-principle clinical trial therapy: acute myeloid leukemia and beyond? Leuk Lymphoma with ribavirin. Blood 2009; 114: 257–260. 2010; 51: 1805–1815. 8 Zhang W, Konopleva M, Shi YX, McQueen T, Harris D, Ling X 3 Topisirovic I, Guzman ML, McConnell MJ, Licht JD, Culjkovic B, et al. Mutant FLT3: a direct target of sorafenib in acute Neering SJ et al. Aberrant eukaryotic translation initiation factor 4E- myelogenous leukemia. J Natl Cancer Inst 2008; 100: 184–198. dependent mRNA transport impedes hematopoietic differentiation 9 Abdel-Wahab O, Levine RL. Recent advances in the treatment of and contributes to leukemogenesis. MolCellBiol2003; 23: acute myeloid leukemia. F1000 Med Rep 2010; 2: 55. 8992–9002. 10 Topisirovic I, Ruiz-Gutierrez M, Borden KL. Phosphorylation of 4 Culjkovic B, Topisirovic I, Borden KL. Controlling gene expression the eukaryotic translation initiation factor eIF4E contributes to its through RNA regulons: the role of the eukaryotic translation transformation and mRNA transport activities. Cancer Res 2004; initiation factor eIF4E. Cell Cycle 2007; 6: 65–69. 64: 8639–8642. 5 Cohen N, Sharma M, Kentsis A, Perez JM, Strudwick S, Borden KL. 11 Dong K, Wang R, Wang X, Lin F, Shen JJ, Gao P et al. Tumor- PML RING suppresses oncogenic transformation by reducing the specific RNAi targeting eIF4E suppresses tumor growth, induces affinity of eIF4E for mRNA. EMBO J 2001; 20: 4547–4559. apoptosis and enhances cisplatin cytotoxicity in human breast 6 Kentsis A, Topisirovic I, Culjkovic B, Shao L, Borden KL. Ribavirin carcinoma cells. Breast Cancer Res Treat 2009; 113: 443–456. suppresses eIF4E-mediated oncogenic transformation by physical 12 Zhou FF, Yan M, Guo GF, Wang F, Qiu HJ, Zheng FM et al. mimicry of the 7-methyl guanosine mRNA cap. Proc Natl Acad Sci Knockdown of eIF4E suppresses cell growth and migration, USA 2004; 101: 18105–18110. enhances chemosensitivity and correlates with increase in Bax/ 7 Assouline S, Culjkovic B, Cocolakis E, Rousseau C, Beslu N, Bcl-2 ratio in triple-negative breast cancer cells. Med Oncol 2010. Amri A et al. Molecular targeting of the oncogene eIF4E in E-pub ahead of print 22 July 2010.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

Concomitant analysis of EZH2 and ASXL1 mutations in myelofibrosis, chronic myelomonocytic leukemia and blast-phase myeloproliferative neoplasms

Leukemia (2011) 25, 1200–1202; doi:10.1038/leu.2011.58; nor determined to be somatic in paired samples or in recently published online 1 April 2011 reported data were censored. Analysis of germ line DNA allowed us to distinguish between somatic missense mutations and previously unannotated SNPs in ASXL1 (Table 1); all unannotated SNPs were observed in Somatic mutations in EZH2 and ASXL1 have recently been matched normal tissue in at least two samples. All frameshift reported in myelofibrosis (MF) and chronic myelomono- and nonsense mutations were not present in matched normal cytic leukemia (CMML).1–4 The current study examines the tissue. This strategy allowed us to identify nine novel somatic frequency of these mutations in a larger cohort of patients missense mutations in ASXL1 that were not previously reported. with these diseases and their correlation with other myelo- proliferative neoplasm (MPN)-associated mutations and disease phenotype. Given that many earlier studies of mutations in ASXL1 did not utilize paired normal tissue, and have Table 1 Novel unannotated single nucleotide polymorphisms largely reported mutations occurring in exon 12 (refs 3,5–7), (SNPs) in the coding region of ASXL1 we also present previously unpublished/unannotated data regarding novel somatic missense mutations/single-nucleotide Nucleotide change Amino acid change polymorphisms (SNPs) throughout the coding region of 317 C-T S25L ASXL1. 328 A-G M29V Study samples from 143 patients were recruited from the 332 C-T A95V Mayo Clinic, Rochester, MN, USA (n ¼ 94; 46 primary MF, 380 G-A C111Y 22 post-polycythemia vera/essential thrombocythemia myelo- 836 C-T R541W - fibrosis (post-PV/ET MF), 11 blast-phase MPN and 15 CMML), 997 A G T806A - Harvard Institutes (n ¼ 25 primary myelofibrosis (PMF) with 1095 G T K838R 1517 A-G D97G paired normal tissue) and MD Anderson Cancer Center (n ¼ 24 1887 G-T E1102Da CMML with paired normal tissue). High throughput DNA 2062 A-G M1161V resequencing was used to screen bone marrow (Mayo and 2326 A-G M1249Vb MD Anderson samples) or granulocyte (Harvard MPD cohort)- 2720 C-T A1380V - derived DNA coding regions of EZH2 (NM_015338), ASXL1 2785 A C M1402L - (NM_004456) and TET2 (NM_017628), as well as the regions of 3062 C T T1494I 3068 G-A S1496N known mutations in IDH1, IDH2, JAK2 and MPL in all samples. 3070 A-G S1497G CMML samples were also screened for known FLT3, K Ras and N Ras mutations. Nonsynonymous alterations not found in SNP All listed single nucleotide variants were present in tumor and normal tissue of at least two samples. databases (dbSNP) were annotated as somatic mutations or aReported previously as SNP by Perez et al.6 SNPs on the basis of sequence analysis of matched-germ line bReported previously as single nucleotide variant of unclear signifi- buccal swab DNA. Nonsynonymous alterations not in dbSNP cance by Boultwood et al.4

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