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Targeting the protein translation factor eIF4E with Ribavirin A Novel Therapeutic Avenue in
Human Cancer
A.
A.
Figure 3. Re-localization of eIF4E correlates with Clinical Response during Ribavirin Treatment
A. Chemical structures of the keto forms of ribavirin and m7G nucleosides. +, positive charge; R, ribose.
B. Ribavirin reduces colony formation of eIF4E-dependent AML-M5 with an apparent IC50 of ≈1μM, and with no effect on M1 and normal bone marrow myeloid progenitor cells at this concentration. Colony formation of primary human CD34+ myeloid progenitors isolated from patients with AML (M1, solid circles; M5, solid squares) and normal bone marrow (BM, open squares), as a function of ribavirin concentration.
Achievable dose in humans
10-3 10-1 100 101 102 1030
30
60
90
120
Normal BMAML-M1
Pri
ma
ry c
olo
nie
s (
% o
f u
ntr
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)
M Ribavirin ( )
AML-M5
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N
N
N
R
O
NH2
H
+m7-guanosine
N
N
N
NH2
OH
R
+ribavirin
Figure 1. Ribavirin competes with the m7G cap and inhibits growth of M4/M5 AML cells
References
1. Topisirovic I, Guzman ML, McConnell MJ, et al. Mol Cell Biol. 2003;23:8992-9002.2. Ruggero D, Pandolfi PP. Nat Rev Cancer. 2003;3:179-192.3. Graff JR, Zimmer SG. Clin Exp Metastasis. 2003;20:265-273.4. Culjkovic B, Topisirovic I, Borden KL. Cell Cycle. 2007;6:65-69.5. Cohen N, Sharma M, Kentsis A, Perez JM, Strudwick S, Borden KL. Embo J. 2001;20:4547-4559.6. Topisirovic I, Culjkovic B, Cohen N, Perez JM, Skrabanek L, Borden KL. Embo J. 2003;22:689-703.7. Kentsis A, Volpon L, Topisirovic I, et al. Rna. 2005;11:1762-1766.8. Kentsis A, Topisirovic I, Culjkovic B, Shao L, Borden KL. Proc Natl Acad Sci U S A. 2004;101:18105- 18110.9. Tan K, Culjkovic B, Amri A, Borden KL. Biochem Biophys Res Commun. 2008;375:341-345.10. Kentsis A, Topisirovic I, Culjkovic B, Shao L, Borden KL. Proc Natl Acad Sci U S A. 2004.11. Culjkovic BT, K., Orolicki, S., Amri, A., Meloche, S. and Borden, K.L.B. The Journal of Cell Biology.
2008;181:51-63.12. Koren G, King S, Knowles S, Phillips E. Cmaj. 2003;168:1289-1292.13. Cheson BD. Bruce D. Cheson. Drugs. 2003;63:2325.14. Topisirovic I, Kentsis A, Perez JM, Guzman ML, Jordan CT, Borden KL. Mol Cell Biol. 2005;25:1100- 1112.15. Graff JR, Konicek BW, Vincent TM, et al. J Clin Invest. 2007;117:2638-2648.16. DeFatta RJ, Nathan CO, De Benedetti A. Laryngoscope. 2000;110:928-933.17. Dong K, Wang R, Wang X, et al. Breast Cancer Res Treat. 2008.18. Smee DF, Matthews TR. Antimicrob Agents Chemother. 1986;30:117-121.19. Streeter DG, Witkowski JT, Khare GP, et al. Proc Natl Acad Sci U S A. 1973;70:1174-1178.
References
1. Topisirovic I, Guzman ML, McConnell MJ, et al. Mol Cell Biol. 2003;23:8992-9002.2. Ruggero D, Pandolfi PP. Nat Rev Cancer. 2003;3:179-192.3. Graff JR, Zimmer SG. Clin Exp Metastasis. 2003;20:265-273.4. Culjkovic B, Topisirovic I, Borden KL. Cell Cycle. 2007;6:65-69.5. Cohen N, Sharma M, Kentsis A, Perez JM, Strudwick S, Borden KL. Embo J. 2001;20:4547-4559.6. Topisirovic I, Culjkovic B, Cohen N, Perez JM, Skrabanek L, Borden KL. Embo J. 2003;22:689-703.7. Kentsis A, Volpon L, Topisirovic I, et al. Rna. 2005;11:1762-1766.8. Kentsis A, Topisirovic I, Culjkovic B, Shao L, Borden KL. Proc Natl Acad Sci U S A. 2004;101:18105- 18110.9. Tan K, Culjkovic B, Amri A, Borden KL. Biochem Biophys Res Commun. 2008;375:341-345.10. Kentsis A, Topisirovic I, Culjkovic B, Shao L, Borden KL. Proc Natl Acad Sci U S A. 2004.11. Culjkovic BT, K., Orolicki, S., Amri, A., Meloche, S. and Borden, K.L.B. The Journal of Cell Biology.
2008;181:51-63.12. Koren G, King S, Knowles S, Phillips E. Cmaj. 2003;168:1289-1292.13. Cheson BD. Bruce D. Cheson. Drugs. 2003;63:2325.14. Topisirovic I, Kentsis A, Perez JM, Guzman ML, Jordan CT, Borden KL. Mol Cell Biol. 2005;25:1100- 1112.15. Graff JR, Konicek BW, Vincent TM, et al. J Clin Invest. 2007;117:2638-2648.16. DeFatta RJ, Nathan CO, De Benedetti A. Laryngoscope. 2000;110:928-933.17. Dong K, Wang R, Wang X, et al. Breast Cancer Res Treat. 2008.18. Smee DF, Matthews TR. Antimicrob Agents Chemother. 1986;30:117-121.19. Streeter DG, Witkowski JT, Khare GP, et al. Proc Natl Acad Sci U S A. 1973;70:1174-1178.
AcknowledgementsWe are grateful for helpful discussions and encouragement from Serafin Pinol Roma, Claude Perreault and Craig Jordan. We thank the nursing staff and coordinators at all three sites and the histology (Dr. Louis Gaboury) and flow cytometry platforms at IRIC for all of their assistance. This project was funded by a Translational Research Program grant from the Leukemia and Lymphoma Society and a BCRF-AACR Grants for Translational Breast Cancer Research.
Re-localization of eIF4E during ribavirin treatment correlates with clinical response. Immunohistochemistry was carried out as described where cells were stained for eIF4E and DAPI. Micrographs were collected on a laser scanning confocal microscope (LSM510 Carl Zeiss,Inc) using a 100X objective, with no further zoom at room temperature. Note that patient 12 was only treated for 19 days on ribavirin, not the full 28 days.
CONCLUSIONS• Ribavirin shows significant single agent activity in
patients with poor prognosis AML.
• Ribavirin does not affect normal hematopoietic cells or cause significant treatment related toxicity.
• Ribavirin effectively targets eIF4E in humans.
• Re-localization of eIF4E from the nucleus to cytoplasm correlates with hematological improvement.
• Re-entry of eIF4E into the nucleus correlates with loss of response.
• Ribavirin activity is potentiated by ara-C, idarubicin and sorafenib.
• Ribavirin, at clinically achievable concentrations, inhibits growth and colony formation of breast cancer cells.
• eIF4E is highly expressed in a breast cancer patient’s skin metastasis
INTRODUCTION
The eukaryotic translation factor (eIF4E) is overexpressed in many human malignancies, including acute myelogenous leukemia (AML) and breast cancer (BC), and is associated with poor prognosis as well as clinical progression. eIF4E regulates mRNA export by associating with the m7G cap on the 5’ end of mRNAs. In the cytoplasm, binding of eIF4E to the m7G cap of mRNAs allows translation initiation. In the nucleus, eIF4E acts in nuclear export of growth promoting mRNAs, thereby increasing their availability to the translation machinery and allowing increased protein production in the absence of increased transcription. Ribavirin, an anti-viral molecule, is classically used in the treatment of hepatitis C (with interferon), SARS, RSV, Lassa fever and influenza. Ribavirin competes with the m(7)G cap of mRNA, thus inhibiting eIF4E-induced export and translation of sensitive transcripts.
METHODS
For the AML clinical trials, eligible patients had AML M4 or M5 or high eIF4E and relapsed or refractory disease or were unable/unwilling to undergo induction chemotherapy. For the ribavirin monotherapy in BC clinical trial, eligible patients had metastatic breast cancer with easily accessible lesions for biopsy who had progressed on at least two prior regimens. Written informed consent was obtained according to the Declaration of Helsinki and these studies received IRB and Health Canada approval. All trials are registered at ClinicalTrials.gov (NCT00559091, NCT01056523, and NCT01056757). S.A., W.H.M., C.F., E.C. and C.R. had access to and analyzed clinical data. Clinical response was assessed using the Cheson or RECIST criteria. eIF4E subcellular distribution was monitored by confocal microscopy. RNA and protein isolations from leukemic blasts and analysis were as described. Ribavirin plasma levels were assessed by mass spectrometry.
eIF4E Impact on Survival Signaling
eIF4E
PI3K-P
Akt-P
GSK3 mTOR
BP1-P & S6-P
Skp2
&
+
NBS1/nibrin
Cyclin B1 mdm2
Cyclin D1
c-myc p27
Cyclin E1
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orafe
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ARibav
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Normal PBMC M4 AML M5 AML
Pt17 Pt18
A. Wright Giemsa staining of bone marrow from patient 8 and 11 using a 60X objective before and after 28 days of ribavirin treatment (1000 mg/day).
B. Plot of percentage of peripheral (blue) or bone marrow (red) blasts versus treatment day for patient 6. Arrows indicate times of dose escalation. The starting dose was 1000 mg/day, followed by 1400 mg/day, 2000 mg/day and 2800 mg/day. (bottom panel) Immunohistochemistry was carried out as described where cells were stained for eIF4E and DAPI1,14. Micrographs were collected on a laser scanning confocal microscope (LSM510 Carl Zeiss,Inc) using a 100X objective, with no further zoom at room temperature.
C. eIF4E activity and levels were reduced by ribavirin treatment in patients. Western blot analysis using the antibodies indicated: Antibodies for immunoblotting were obtained fro mCell Signaling unless otherwise mentioned: mAb anti-eIF4E (BD Bioscience), pAb anti-NBS; mAb anti-cyclin D1 (BD Bioscience); pAb anti-Akt, anti-phospho Thr308 Akt and mAb anti--actin (AC-15 Sigma Aldrich).
A. Wright Giemsa staining of bone marrow from patient 8 and 11 using a 60X objective before and after 28 days of ribavirin treatment (1000 mg/day).
B. Plot of percentage of peripheral (blue) or bone marrow (red) blasts versus treatment day for patient 6. Arrows indicate times of dose escalation. The starting dose was 1000 mg/day, followed by 1400 mg/day, 2000 mg/day and 2800 mg/day. (bottom panel) Immunohistochemistry was carried out as described where cells were stained for eIF4E and DAPI1,14. Micrographs were collected on a laser scanning confocal microscope (LSM510 Carl Zeiss,Inc) using a 100X objective, with no further zoom at room temperature.
C. eIF4E activity and levels were reduced by ribavirin treatment in patients. Western blot analysis using the antibodies indicated: Antibodies for immunoblotting were obtained fro mCell Signaling unless otherwise mentioned: mAb anti-eIF4E (BD Bioscience), pAb anti-NBS; mAb anti-cyclin D1 (BD Bioscience); pAb anti-Akt, anti-phospho Thr308 Akt and mAb anti--actin (AC-15 Sigma Aldrich).
Figure 2. Ribavirin treatment targets eIF4E in patients
before 28 days 56 days 84 days
B.
B.
Patient 8 Patient 11
before
28 days
Patient 8
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PlateletsHemoglobin
WBC
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A.
Figure 6. Ribavirin inhibits proliferation and clonogenic survival of breast cancer cell lines
eIF4E was detected by immunohistochemistry Blue staining in represents collagen and brown stain represents eIF4E. Normal skin was used a calibrator (relative quantity =1).
Figure 4. Improved reduction in colony growth of AML specimens when Ribavirin is combined with Ara-C and
Idarubicin
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Normal PBMC M4 AML M5 AML
Figure 5. Sorafenib potentiates Ribavirin induced reduction in colony growth of AML specimens
Colony forming assays on primary AML specimens or primary cells obtained from healthy individuals were conducted to assess their sensitivity to various drugs combined with ribavirin. Cells were treated with 1M ribavirin, 0.3nM idarubicin and 0.5nM ara-C. Error bars indicate standard deviations and each experiment represents a replicate of five.
Colony forming assays on primary AML specimens or primary cells obtained from healthy individuals were conducted to assess their sensitivity to various drugs combined with ribavirin. Cells were treated with 1M ribavirin and 5M of sorafenib. AML specimens tested with sorafenib had flt3 mutations. Error bars indicate standard deviations and each experiment represents a replicate of five.
M male; F female; tAML therapy related AML; RGA AML with recurrent genetic abnormality; NOC AML not otherwise categorized; MLD AML with multilineage dysplagia; Del deletion; inv inversion; t translocation; ITD internal tandem duplication; TKD tyrosine kinase domain; CR complete remission; SD stable disease; PD progressive disease; BR blast response; PRi partial remission with incomplete blood count recovery; PR* disappearance/regression of skin lesions; n/a: not available; + continuing; Y yes; N no.
It is noteworthy that patient 3 entered the trial with a diagnosis of M4 AML, but this was later modified to M1. Patient 3 continued treatment because he had elevated eIF4E levels (figure 1C), which is unusual for M1 AML14. Patient 3 weighed 160 kg, and thus even at 2800mg/day ribavirin, only achieved plasma concentrations of 5 μM. In contrast, other patients achieved higher concentrations of ribavirin at lower doses.
Table 1. Summary of clinical responses observed with ribavirin treatment
Wilson Miller Jr1, Sarit Assouline1, Eftihia Cocolakis1, Caroline Rousseau1, Biljana Culjkovic2, Abdellatif Amri2, Cristianno Ferrario1, Filippa Pettersson1, and Katherine L.B. Borden2
1Segal Cancer Center, Sir Mortimer B. Davis - Jewish General Hospital, McGill University, Montreal, QC, Canada. 2Institute for Research in Immunology and Cancer & Dept of Pathology and Cell Biology, Université de Montréal, Montréal, QC, Canada
Figure 7. Elevated expression of eIF4E in a breast cancer patient
Cell viability assay (7 days) Clonogenic assay (14 days)
A. Breast cancer cell lines were cultured with increasing concentrations of ribavirin and cell viability was measured using the TiterGlo Assay (Promega). B. Anchorage dependent clonogenicity was measured after 14 days of ribavirin treatment.
A. B.
Peripheral Absolute Blast
Count (x109/L)
Bone Marrow Blast (%)
Age/ Sex
WHO Class.
Cytogenetics FLT3 Mut.
NPM1 Mut.
Prior Tx Treatment Duration
(days)
Overall Survival (days)
Best Response
Pre- Tx
At Best
Response
Pre- Tx
At Best
Response
Max. Dose Level
Ribavirin (mg)
Plasma Levels
Ribavirin at max.
dose (M)
69F tAML complex n/a n/a none 60 83 SD 1.73 0.51 30 20 1400 n/a
46M NOC normal ITD N 2 x ind;
4 x cons, reind 137 137 SD 47.4 15.5 94 n/a 2800 5
54M RGA 46,XY, t(6;11)
(q27;q23) n/a n/a
2 x ind; 1 x cons; haploT
27 27 PD 0.04 3.6 90 n/a 1000 n/a
77F NOC normal ITD Y none 142 351 BR 23.3 0 95 29 2000 26.2
60M NOC normal ITD N 1 x ind; 1 x cons
104 120 PR* 0 0 1 1 1000 9.3
70M NOC 45, X, -Y ITD Y 1 x ind;
1 x cons; autPBSCT
93 235 PRi 0.06 0 65 10 2000 17
44M NOC n/a TKD Y 1 x ind;
4 x cons; 2 x reind
33 33 PD 4.76 3.46 n/a n/a 1400 36
56F NOC normal ITD N 1 x ind; 4 x
cons 119 148+ BR 12.1 0.6 88 46 2000 14.8
77M MLD normal ITD N none 263 354+ CR 0.06 0 72 4 1000 14.7
79F RGA 46, XX, inv(16)
(p13;q22) ITD N none 19 347+ SD 0.78 n/a 85 90 1000 15.3
72M MLD normal ITD Y Low dose AraC
+ sorafenib 32 39 SD 26.1 32.6 95 95 2000 14.2
67F NOC complex none 19 n/a SD 0 0 45 n/a 1000 14.3
81F NOC n/a none 56 81 SD 1 1 55 65 2000 17.4
85M NOC normal none 42+ 42+ SD 11.9 14.2 75 57 2000 16.4
47F NOC normal 1 x ind; 49+ 49+ SD 0.2 0.55 60 71 2000 34.3
Age/ Sex
WHO Class.
69F tAML
46M NOC
54M RGA
77F NOC
60M NOC
70M NOC
44M NOC
56F NOC
77M MLD
79F RGA
72M MLD
67F NOC
81F NOC
85M NOC
47F NOC
Pt no
1
3
5
6
7
8
9
10
11
12
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15
17
18
N
N
N
Y 1x Ind; 2 x cons
ITD
n/a
n/a
n/a
