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Blood, Vol. 114, Issue 2, 257-260, July 9, 2009
Molecular targeting of the oncogene eIF4E in acute myeloid leukemia (AML): a proof-of-principle clinical trial with ribavirin
Blood Assouline et al.
114: 257
Supplementary materials for: Assouline et al
Patients
A total of 13 patients were enrolled in the study in 3 participating centers; Jewish General Hospital, Hamilton Health Sciences, and Hôpital Maisonneuve Rosemont. IRB approval was obtained at all sites (Jewish General Hospital, Hamilton Health Sciences, Hôpital Maisonneuve Rosemont, and University of Montreal). Patients had to receive a diagnosis of primary or secondary AML, French-American-British FAB subtypes M4 or M5 only, relapsed or refractory after at least 1 cycle of conventional chemotherapy; or newly diagnosed but not be candidates for induction chemotherapy. Patients must also have been at least 18 years of age; and must have had an Eastern Cooperative Oncology Group (ECOG) performance status lower than 3; a life expectancy of at least 12 weeks; adequate hepatic and renal function (hepatic transaminase level lower than 2.5 times the institutional upper limit of normal ULN, total bilirubin level less than 1.5 times the ULN, serum creatinine level below 1.5 times the ULN). No concurrent cytoreductive chemotherapy was permitted, except for hydroxyurea. The use of hydroxyurea was permitted throughout the study but investigators were asked not to increase the dose of hydrea, if possible, while patients were taking ribavirin. Patients with central nervous system (CNS) leukemia, active cardiovascular disease, intercurrent illness or medical condition precluding safe administration of the study drug and known human immunodeficiency virus infection were not permitted on study. Female patients were to be postmenopausal, surgically sterile, or taking effective contraception. Dosage and drug administration
Ribavirin, purchased from Three Rivers Pharmaceuticals, was dosed orally at 400 mg every am and 600 mg every pm continuously. Ribavirin was administered daily for each 28-day cycle with no breaks between cycles. We observed no drug-related toxicity requiring either dose reduction or discontinuation. Responding patients could receive up to 6 cycles of treatment, which could be extended for ongoing clinical benefit as determined by the investigator. Dose escalation was permitted for lack of hematological response (no reduction in peripheral blood blast count) at 14 days (3 patients), or for lack of marrow response at day 28 (2 patients), or at any subsequent 28 day cycle (5 patients). Patient 3, 6, and 10 underwent more than one dose escalation. The first dose escalation was to 600 mg every am and 800 mg every pm, a second dose escalation to 1000 mg twice per day was permitted as was a maximum dose of 1400 mg twice daily for lack of response. Pretreatment and follow-up studies
Medical history, performance status of the patient, concurrent medication, vital signs, and routine laboratory evaluation were recorded before treatment was started and were repeated weekly on the first cycle of therapy and every 2 weeks thereafter. Laboratory evaluation included complete blood count, biochemistry studies (including electrolyte levels; renal and hepatic function; pancreatic amylase, and lipase levels). Samples for laboratory analysis were taken prior to treatment for later comparison. Patients were not pre-screened for eIF4E levels. Adverse events were graded according to the NCI CTC, version 3.0 (http://ctep.cancer.gov/forms/CTCAEv3.pdf). Patients experiencing unacceptable toxicity were required to stop therapy and to be withdrawn from the study. Standard criteria were used to evaluate hematologic response.1 A bone marrow aspirate was performed prior to the start of study therapy and on day 28 of each cycle. A complete remission (CR) was defined as absence of leukemic blasts from peripheral blood, fewer than 5% blasts in bone marrow, peripheral level of hemoglobin higher than 90 g/L (higher than 9 g/dL), platelet count greater than 100 × 109/L and absolute neutrophil count greater than 1 × 109/L, without the requirement for transfusion of red cells and platelets. A designation of complete remission with incomplete blood count recovery (CRi) required that all criteria for a CR were met, but that there was either a residual neutropenia (<1 × 109/L) or thrombocytopenia (<100 × 109/L). Partial remission (PR) required the hematologic criteria for CR, and a 50% reduction in bone marrow blasts with a post-treatment blast count between 5 and 25%. Incomplete partial remission (PRi) required the same marrow criteria as PR but allowed for an incomplete recovery or neutrophils and platelets as for CRi. A blast response (BR) required a greater than 2-log decrease in absolute peripheral blood blast count and/or at least a 50% decrease in bone marrow blast percentage sustained for a 28-day period in the absence of fulfilling the criteria for a CR, CRi, PR, or PRi. Progressive disease (PD) was defined as a 50% increase in the absolute number of blasts in the bone marrow relative to baseline, or an increase in the absolute peripheral blast count of at least 10 × 109/L. Stable disease (SD) was defined as failure to achieve a BR, yet not fulfilling the criteria for PD. The best response for each patient was recorded. Pharmacokinetic assessments
Blood samples were collected in a 4.0 mL EDTA (ethylenediaminetetraacetic acid) vacutainer prior to the first dosing with ribavirin, on Cycle 1 Day 15, on Cycle 2 Day 1, two weeks following any dose change and at the End-of-Treatment visit. Blood samples were centrifuged at 4°C for 10 minutes; plasma was separated, split into two 1.5 mL Nalgene (Rochester, NY, USA) cryovials, and stored at −70°C protected from light until analysis. Plasma levels of ribavirin were measured by Apredica Pharmaceuticals (Watertown, MA, USA) using LC-MS methods. Whenever possible, specimens were divided into 2 or 3 samples and ribavirin levels analyzed in duplicate or triplicate. Pure ribavirin (Kemoprotec, Middlesborough, United Kingdom) was used as a standard. In no instance was ribavirin observed in the samples obtained prior to the start of treatment. Molecular Response Leukemic blast, protein, and RNA isolation
White blood cells were isolated from peripheral blood or bone marrow using Ficoll Gradient. Leukemic blasts were then isolated using CD45dim side-scatter population as was described for M4 and M5 AML previously.2 Cells were sorted on a Becton Dickson BD FACSAria flow cytometer. For patient 3, blasts were always higher than 80%, thus cells were not sorted after Ficoll gradient isolation. However, cells were sorted for all other patients. For controls, normal peripheral blood mononuclear cells and normal CD34+ cells were obtained from StemCell Technologies (Vancouver, BC, Canada). Protein and RNA were isolated as described (see below).3,4 Western analysis was performed as described5 using a lysis buffer (40 mM Hepes, pH 7.5, 120 mM NaCl, 1 mM EDTA, 10 mM β-glycerophosphate, 50 mM NaF, 0.5 uM NaVO3, and 1% (vol/vol) Triton X-100 supplemented with protease inhibitors, all were purchased from Sigma Aldrich (St Louis, MO, USA). In addition, blots for immunophosphoprotein detection were blocked in BSA blocking solution 2% wt/vol BSA (Sigma Aldrich) in TBS-Tween 20 and primary antibodies were blocked in BSA blocking solution. Antibodies for immunoblotting were obtained from Cell Signalling (Beverly, MA, USA) unless otherwise mentioned: mAb anti-eIF4E (BD Bioscience), pAb anti-NBS; pAb anti-cyclin D1 (Santa Cruz, Santa Cruz, CA, USA); pAb anti Akt, anti-phospho Thr308 or S473 Akt and mAb anti–β–actin (ACz15 Sigma Aldrich). Quantitative real time PCR (qRT-PCR) was done using Sybr Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) in a thermal cycler (Mx3000P;Strategene, Wilmington, DE, USA). All conditions and primers were described elsewhere.5 All calculations were performed using the relative standard curve method described in the Applied Biosystems User Bulletin #2.5 RNAs were normalized to multiple housekeeping genes to ensure that there were no unanticipated alterations in the normalisers upon ribavirin treatment.6,7 These include RPL13a, actin, TBP, G6PDH and PP1A.6,7 Screening for FLT3 and NPM 1 status
Screening of FLT3 ITDs and TDKs as well as NPM1 mutations were done from RNAs by RT-PCR using primers previously described.8,9 Confocal microscopy
Immunohistochemistry was carried out as described3,4 where cells were stained for eIF4E and mounted in Vectasheild with DAP (Vector Laboratories). Fluorescence from several fields were observed at 100× optical magnification and with two times digital zoom on a laser scanning confocal microscope (LSM510 META, Carl Zeiss, Inc, Jena, Germany) exciting at 405 nm and 488 nm at RT. Confocal micrographs were collected using a 100× objective and numerical aperture of 1.4. The confocal micrograph represents a single optical section through the plane of the cell. Images were obtained from LSM510 software version 3.2 (Carl Zeiss, Inc) and displayed using Adobe Photoshop CS2 (Adobe). Histology
Skin lesions were collected at the appropriate hospital and forwarded for use in the study as part of the clinical trial protocol. Biopsies were sectioned, stained with anti-eIF4E antibody (BD Bioscience) and analyzed by a board certified pathologist as part of the Histology Platform service at IRIC. Bone marrow smears were stained with Wright Giemsa and photographed as described in the figure legend.
Files in this Data Supplement:
- Table S1. eIF4E levels before and after ribavirin treatment (PDF, 25.0 KB)
- Figure S1. eIF4E levels and activity reduced upon ribavirin treatment (JPG, 211 KB)
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(A) eIF4E levels in patient 7 pre- and post-ribavirin treatment. A biopsy from a normal individual is shown for comparison. Formalin-fixed paraffin-embedded skin biopsy from a normal person or from skin lesions isolated from patient 7 prior to and at 100 days of ribavirin treatment were stained using standard protocols and the anti-eIF4E antibody (BD Bioscience; 1/50 dilution) and the Universal Secondary Antibody (Ventana Medical Systems, Tuscon, AZ, USA). Streptavidin-horseradish peroxidase, and 3,3-diaminobenzidine detection kit were used according to the manufacturer’s instructions (Ventana Medical Systems). The sections were counterstained with hematoxylin and a bluing reagent was applied for post-counterstaining. Immunohistochemical detection was done with a Discovery XT system (Ventana Medical Systems) performing deparaffinization and Ag retrieval with proprietary reagents (Cell Conditioning 1). Sections were scanned at 40× magnification using the C9600 NanoZoomer System (Hamamatsu Photonics K.K., Iwata City, Japan) which is capable of adjusting focus on any part of the slide. NDP Scan ver. 2.0 software was used to visualize virtual slides and take pictures. Images were acquired at room temperature. Staining of the sections was done at the same time with the identical solution of antibody, thus staining differences are due to differences in eIF4E protein levels. Black lines indicate the reduced depth of the skin lesion after ribavirin treatment. (B) eIF4E dependent mRNA export of NBS1 transcripts was impaired by ribavirin treatment. Similar results were observed for cyclin D1 mRNA (data not shown). Patient 10 was the only patient for which there was sufficient material to analyze mRNA export directly; however, other patients consistently had reduced NBS1 and cyclin D1 protein levels. Cells were fractionated into nuclear and cytoplasmic compartments and the levels of NBS1 mRNA were measured using RT-qPCR. Cytoplasmic/nuclear RNA ratios represent relative fold + ∕− SD normalized to actin. Actin mRNA export does not change upon ribavirin treatment. Fractionation controls indicated that the fractionation was of good quality (data not shown). Here, tRNAlys and U6snRNA were used as cytoplasmic and nuclear markers as we have carried out previously10 (data not shown).
- Figure S2. Re-localization of eIF4E during ribavirin treatment coincides with clinical response (JPG, 229 KB)
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For comparison, cells from a normal specimen are shown in the inset. Immunohistochemistry was carried out as described where cells were stained for eIF4E and DAPI.1,13 Micrographs were collected on a laser scanning confocal microscope (LSM510 Carl Zeiss, Inc) using a 100× objective with a numerical aperture of 1.4, with further two times digital zoom at room temperature. See supplemental methods and figure 1 legend for further information. Note that patient 12 was only treated for 19 days on ribavirin, not the full 28 days. All micrographs were taken at the same magnification and digital zoom. The possibility that the observed re-localization of eIF4E during treatment is a result of the re-emergence of normal progenitor cells cannot be excluded. However, in the sorted population used, one expects to find only about 1% of this population in normal marrow. In most cases, we have a much higher fraction in this compartment, suggesting that normal progenitors would be greatly out-numbered by leukemic cells.
- Figure S3. Quantification of cells with primarily nuclear or cytoplasmic eIF4E localization at baseline and during treatment with ribavirin (JPG, 95.2 KB)
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Cells were defined as being primarily nuclear or primarily cytoplasmic. Generally, 50–100 cells were analyzed for each time-point. Error bars indicate standard deviations. D indicates days of treatment; PT patient.
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