BCR-ABL mRNA Levels Research Articles

Overcoming imatinib resistance using Src inhibitor CGP76030, Abl inhibitor nilotinib and Abl/Lyn inhibitor INNO‐406 in newly established K562 variants with BCR‐ABL gene amplification

Because imatinib (IM) resistance in chronic myeloid leukemia is primarily caused by the re-establishment of Abl kinase, new inhibitors may be efficacious. We evaluated 3 new agents against 2 new K562 variants, IM-R1 and IM-R2 cells, which were developed having 7- and 27-fold greater IM resistance, respectively, than the parental K562 cells. Both variants possessed BCR-ABL gene amplification along with elevated levels of its transcript and protein. Greater BCR-ABL gene amplification was observed in IM-R2 cells than in IM-R1 cells, which was consistent with the higher mRNA and protein levels of Bcr-Abl, and ultimately correlated with the greater IM resistance in IM-R2 cells. No mutation in the Abl kinase domain was detected in either variant. Despite the absence of Lyn overexpression, the Src kinase inhibitor CGP76030 showed positive cooperability with IM in inhibiting cell growth of not only K562 cells but also these 2 variants. This might be because of the augmented inhibition of Erk1/2 phosphorylation. The new Abl kinase inhibitor nilotinib was 10-fold more potent than IM in inhibiting the growth of K562 cells. Nilotinib inhibited the growth of IM-R1 and IM-R2 cells as potently as K562 cells. The combination of nilotinib with CGP76030 showed little additivity, because the potency of nilotinib masked the efficacy of CGP76030. The new dual Abl/Lyn inhibitor INNO-406 (formerly NS-187) was slightly more potent than nilotinib in inhibiting the growth of all 3 cell lines. Because BCR-ABL gene amplification occurs in blast crisis, these inhibitors might overcome IM resistance in such patients' leukemia.

An Intron-Derived Insertion/Truncation Mutation in the BCR-ABL Kinase Domain in Chronic Myeloid Leukemia Patients Undergoing Kinase Inhibitor Therapy

Although targeted inhibition of BCR-ABL with imatinib is an effective therapy for patients with chronic myeloid leukemia (CML), a minority of patients acquire mutations in the BCR-ABL kinase domain, resulting in imatinib resistance. The spectrum of kinase domain mutations discovered to date is quite heterogeneous, consisting almost exclusively of single nucleotide substitutions affecting key amino acids that regulate drug binding or BCR-ABL function. Here, we describe an alternative kinase domain insertion/truncation mutation in three CML patients undergoing kinase inhibitor therapy. In each of these patients, direct DNA sequencing of BCR-ABL RT-PCR products revealed that the same 35 nucleotides from ABL intron 8 had been inserted at the normal exon 8 to 9 splice junction. This 35-bp intronic sequence was flanked by excellent consensus splice donor and acceptor sequences, suggesting alternative splicing as the likely mutational mechanism. The insertion created a premature translational stop codon after 10 intron-encoded amino acids (amino acid 484). This resulted in truncation of 653 C-terminal amino acids, which included part of the kinase domain and the entire "last exon" region. These findings demonstrate that kinase domain insertions are an alternative (and not entirely uncommon) mutational mechanism in CML patients undergoing kinase inhibitor therapy.

Therapeutic Application of Small Interfering RNA Directed Against bcr-abl Transcripts to a Patient with Imatinib-Resistant Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is a relatively well differentiated myeloproliferative disorder originating from transformed hematopoietic stem cells. However, as patients may relapse after allogeneic stem cell transplantation (SCT) or develop imatinib-resistant disease, additional CML therapies are required. RNA interference is referred to as the recently discovered process of post-transcriptional gene silencing that is initiated by double-stranded RNA molecules known as small interfering RNAs (siRNA). We herein present a first report on the in vivo application of targeted non-virally delivered synthetic bcr-abl siRNA in a 47-year-old female patient with recurrent CML (b3a2 translocation for a p210 oncoprotein) resistant to imatinib (Y253F mutation) and chemotherapy (cytosine arabinoside, ARA-C) after an HLA partially matched unrelated SCT by receiving a myeloablative conditioning therapy with fractionated total body irradiation (10 Gray), cyclophosphamide (120mg/kg b.w.) and anti-thymocyte globulin (30mg/kg b.w.). Transplant associated complications included renal insufficiency and acute graft-versus host disease (GVHD) grade II (skin stage 3, gut stage 1), which developed into an extensive chronic GVHD with mainly skin involvement. Prednisone was continuously tapered to a minimum of 12.5 mg/day post-transplant. The patient developed an histologically relapse of CML. SiRNA treatment was started on day +426 after transplant without discontinuing imatinib mesylate, ARA-C treatment or the immunosuppressive medication. The patient received for the first time 10μg/kg b.w. bcr-abl siRNA intravenously in an anionic dispersion lipid solution (DLS) of 20 ml as an accompanying therapy trial. SiRNA was administered over 1–2 minutes as a bolus injection. In addition, 300μg siRNA in 2 ml DLS were injected directly into a subcutaneous CML node. No side effects were observed during the first hours after administration. Two days after siRNA treatment the patient suffered from moderate dizziness (grade 0–1), this resolved completely after three days. The bcr-abl mRNA level decreased from 6.6% to 0.053% nine days after the first siRNA injection. At day 21 after initial siRNA treatment the bcr-abl expression increased again. We repeated the intravenous application of siRNA with 30μg/kg b.w. in 60 ml DLS (injected over 15 minutes) and 7 days later with another 10μg/kg b.w. in 20 ml DLS (injected over 5 minutes). A transient decrease of the bcr-abl amount was detected after the second - and the third siRNA administration as shown in the figure. After the first siRNA administration we found a transient improvement of blood leucocytes with disappearance of immature cells that defined a complete hematological response. FISH analysis showed an absence of the Philadelphia chromosomes of 200 analyzed interphase nuclei. On day +455 were found again 70% blasts in the blood and a palliative chemotherapy with mitoxantrone (flat 20mg) was initiated according to the patient's wish. The patient died at day +473 post transplant. Our findings imply that the clinical application of synthetic siRNA is feasible, safe and has real potential for genetic-based therapies using synthetic non-viral carriers. [Display omitted]

Evaluation of the Cepheid GeneXpert BCR-ABL Assay

Patients with chronic myeloid leukemia harbor the chromosomal translocation t(9;22), which corresponds to fusion of the BCR and ABL genes at the DNA level. The translated fusion product is an oncogenic protein with increased ABL tyrosine kinase activity causing cell transformation. To date, reverse transcriptase-polymerase chain reaction is considered the most sensitive method available for detecting low copy numbers of the BCR-ABL gene fusion. Recently, Cepheid introduced its GeneXpert-based assay for the identification of the BCR-ABL gene fusion in cells from blood samples. This system comprises a walk-away self-contained instrument that combines cartridge-based microfluidic sample preparation with reverse transcriptase-polymerase chain reaction-based fluorescent signal detection and BCR-ABL and ABL Ct (threshold cycle) determination. The difference between the BCR-ABL Ct and ABL Ct (DeltaCt) is expected to represent the ratio of the two populations of mRNAs and ultimately the percentage of neoplastic cells present. We tested whether this BCR-ABL fusion detection system could be used as a clinical diagnostic tool for monitoring patients with minimal residual disease of chronic myelogenous leukemia. We report similar performance characteristics, including limit of detection, specificity, sensitivity, and precision, of this automated BCR-ABL fusion detection system to those of a manual TaqMan reverse transcriptase-polymerase chain reaction-based test.

Plasma RNA as an alternative to cells for monitoring molecular response in patients with chronic myeloid leukemia

Quantitation of BCR-ABL mRNA is emerging as the standard of care to monitor the status of chronic myeloid leukemia (CML). Peripheral blood plasma was analyzed in this study because of previous detection of nucleic acids and proteins from tumor cells in plasma samples. Reverse transcriptase polyemrase chain reaction was used to establish ratios of BCR-ABL:ABL mRNA in peripheral blood cells and plasma, and absolute levels of BCR-ABL mRNA per unit volume of plasma. Samples from 160 CML patients and 180 control individuals without CML were tested. Cells and plasma samples from 93 of the CML patients were re-analyzed 3-12 months after imatinib treatment. Ratios of BCR-ABL:ABL mRNA in paired cell and plasma samples of the 160 CML patients correlated significantly (r=0.83; p<0.001). When results were compared directly using the sign test, the pre-therapy plasma results were significantly different from those from peripheral blood cells (p=0.028), but not bone marrow cells (p=0.119). Absolute levels of BCR-ABL mRNA in plasma strongly correlated with many laboratory characteristics in pre-therapy CML patients. Higher BCR-ABL: ABL ratios were detected in plasma samples at all time points after treatment, although this was significant only at 3 months (p=0.0003). In cases in which results from the assays disagreed, minimal residual disease was detected in plasma samples significantly more frequently than in cell samples (p<0.001). Plasma was a reliable source for monitoring BCR-ABL mRNA levels. Minimal residual disease detection from plasma was more sensitive than from cell samples. Our results suggest that absolute levels of BCR-ABL mRNA per unit volume of plasma may reflect tumor load.

The Recent Thymic Output Function Might Relate with BCR-ABL mRNA Level in Patients with CML.

Objective In order to analyze the relationship between the content of T-cell receptor excision DNA circles (TRECs) and BCR-ABL mRNA levels, evaluating the prognostic significance of thymic recent output function monitoring in patients with chronic myelogenous leukemia (CML).Methods Quantitative detection of TRECs and BCR-ABL fusion gene transcripts in peripheral blood from 15 CML patients were preformed using real-time PCR technique. And the TREC-number was related to the number of T-cells by determination of the number of CD3-positive cells. The change of BCR-ABL level in 6 CML patients was followed-up for two years.Results There was no significant correlation between TRECs and BCR-ABL mRNA in peripheral blood from CML patients at the first diagnose. Patients who had higher TRECs at diagnosis had a larger reduction of BCR-ABL level after 2 years of follow-up. While in 2 patients who underwent haemopoietic stem cell transplantation(HSCT), BCR-ABL in one patient with higher pre-transplantation TRECs value became undetectable with three consecutive detections during the first year post transplantation, but low level of BCR-ABL could be identified in the other patient.Conclusion High thymic output function in CML patients could be helpful for anti-residual CML cells.

Hematopoietic Stem Cells and Primitive Progenitors Express Leukemia-Associated Antigens That May Be Targets for Graft-Versus-Leukemia Effect or for Vaccine-Based Immunotherapy in Chronic Myeloid Leukemia.

The cure of chronic myeloid leukemia (CML) following allogeneic stem cell transplantation (SCT) is attributed to a graft-versus-leukemia (GvL) effect which eliminates residual leukemia cells that might otherwise have the capacity to regenerate the leukemia. Whether the GvL effect is directed against the most primitive leukemia stem cell or only against a more mature leukemia progenitor cell is unknown. In contrast, although tyrosine kinase inhibitors (TKI), notably imatinib, effectively suppresses at least 2 logs of CML hematopoiesis in most patients, they appear not to affect the leukemia stem cell or the most primitive progenitor and so fail to eradicate the leukemia. To characterize further the GvL reaction and to identify targets that might be exploited for vaccine therapy outside the context of a stem cell transplant, we studied expression of selected proteins in CML progenitors at various levels of commitment. CD34+ progenitor subpopulations were purified by fluorescence activated cell sorting from 10 CML patients (5 chronic phase, 3 accelerated phase and 2 blastic phase) and from 8 healthy individuals. We used real-time quantitative polymerase chain reaction (RQ-PCR) for BCR-ABL, WT1, proteinase 3 (PR3), β-catenin and BMI-1. Hematopoietic stem cells, CD34+CD38-Lin-CD90+ and primitive progenitor subpopulations CD34+CD38- Lin- CD7+, CD34+CD38+ Lin-CD7+, and more differentiated CD34+CD38+ Lin + CD7- and CD34+CD38+ Lin+ CD7+ populations were collected. In addition, CD34+CD38+Lin- cells were sorted into common myeloid progenitor (CMP) CD34+CD38+Lin- IL3Rα+CD45RA- and granulocyte-macrophage progenitor (GMP) CD34+CD38+Lin- IL3Rα+CD45RA+ populations and their CD7+ and CD7- subpopulations collected. Mature myeloid progenitor subpopulations CD34+CD38+Lin-CD33+ were also separated into CD7+ and CD7- groups. All primitive leukemic CD34+ subpopulations expressed similar levels of BCR-ABL mRNA and contained greater than 80% BCR-ABL positive cells by fluorescence in situ hybridization. By RQ-PCR we detected higher expression of the leukemia-associated antigen WT1 in the most primitive CD34+CD38-CD90+Lin- subset of hematopoietic progenitors in CML compared to normal. In more mature CMP and GMP populations, the expression of both WT1 and PR3 was higher in CML than normal. The CD7+ subpopulation of GMPs in CML, which are implicated in disease progression, also have higher expression of leukemia-associated antigens than normal. The expression of β-catenin and BMI-1 was lower in CML CD34+ progenitors in comparison to those from healthy individuals in all subpopulations except in CD7+ GMPs, indicating low self-renewal activity. These results suggest that the most primitive leukemic hematopoietic progenitors, as well as their more differentiated progeny, express WT1 and PR3 at relatively high levels and may thus be targets for peptide-based vaccines. The mechanism that protects primitive leukemic progenitors from the effects of TKI remains undefined but may be related to their quiescent status or to their high levels of BCR-ABL expression.

Down-regulation of p210bcr/abl by curcumin involves disrupting molecular chaperone functions of Hsp901

To investigate the effects of curcumin (Cur) on p210(bcr/abl) level in K562 cells, and the relationship between these effects and the molecular chaperone functions of heat shock protein 90 (Hsp90). Flow cytometry and Western blot were used to examine the abundance of p210(bcr/abl), Hsp90, p23, Hsp70, and p60(Hop) in K562 cells treated with Cur. Reverse transcription polymerase chain reaction (RT-PCR) was used to determine the bcr-abl mRNA level in K562 cells treated with Cur. After co-immunoprecipitation of p210(bcr/abl) and its molecular chaperones, the immunoprecipitate was then subjected to Western blot analysis with anti-Hsp90, anti-Hsp70, anti-p23, and anti- p60(Hop)mAb. An exposure of K562 cells to Cur produced time-dependent down-regulation of p210(bcr/abl), the inhibition rate of p210(bcr/abl) in K562 cells determined by flow cytometry after treatment with Cur 27.2 micromol/L for 1 h, 6 h, 12 h and 24 h was 31.2%, 63.7%, 81.3% and 94.5%, respectively. In contrast, Cur had almost no influence on bcr-abl mRNA level. Treatment with Cur for 24 h reduced the association of p210(bcr/abl) with Hsp90/p23 complex, while increasing the association of p210(bcr/abl) with Hsp70/ p60(Hop) complex; however, the total protein abundance of Hsp90, p23, and p60(Hop) in K562 cells had no apparent change, while Hsp70 increased greatly. Down-regulation of p210(bcr/abl) by Cur involves dissociating the binding of p210(bcr/abl) with Hsp90/p23 complex. In contrast, the association of p210(bcr/abl) with Hsp70/ p60(Hop) complex increased.

BCR-ABL mRNA levels at and after the time of a complete cytogenetic response (CCR) predict the duration of CCR in imatinib mesylate–treated patients with CML

AbstractAlthough most patients with chronic myeloid leukemia (CML) treated with imatinib mesylate achieve a complete cytogenetic response (CCR), some patients will relapse. To determine the potential of real-time quantitative BCR-ABL reverse transcriptase–polymerase chain reaction (RT-PCR) to predict the duration of continued CCR, we monitored 85 patients treated with imatinib mesylate who achieved a CCR. With a median follow-up of 13 months after CCR (29 months after imatinib mesylate; median 6 RQ-PCR assays), 23 patients (27%) had disease progression (predominantly loss of CCR). Compared with the median baseline level of BCR-ABL mRNA, 42% of patients achieved at least a 2-log molecular response at the time of first reaching CCR. Failure to achieve a 2-log response at the time of CCR was an independent predictive marker of subsequent progression-free survival (hazard ratio = 5.8; 95% CI, 1.7-20; P = .005). After CCR, BCR-ABL mRNA levels progressively declined for at least the next 15 months, and 42 patients (49%) ultimately achieved at least a 3-log reduction in BCR-ABL mRNA. Patients failing to achieve this 3-log response, at any time during therapy, had significantly shorter progression-free survival (hazard ratio = 8.1; 95% CI, 3.1-22; P < .001). The achievement of either a 2-log molecular response at the time of CCR or a 3-log response anytime thereafter is a significant and independent prognostic marker of subsequent progression-free survival.

Photodynamic treatment (ALA-PDT) suppresses the expression of the oncogenic Bcr-Abl kinase and affects the cytoskeleton organization in K562 cells

K562 is the chronic myelogenous leukemia (CML)-derived cell line that expresses high levels of chimeric oncoprotein Bcr-Abl. The deregulated (permanent) kinase activity of Bcr-Abl leads to continuous proliferation of K562 cells and their resistance to the apoptosis promotion by conventional drugs. The photodynamic treatment (PDT) based on the application of 5-aminolevulinic acid (ALA) and irradiation with blue light (ALA-PDT) resulted in the suppression of K562 cells proliferation. It was followed by a necrosis-like cell death [K. Kuželová, D. Grebeňová, M. Pluskalová, I. Marinov, Z. Hrkal, J. Photochem. Photobiol. B 73 (2004) 67–78]. ALA-PDT led to the perturbation of the Hsp90/p23 multichaperone complex of which the Bcr-Abl is the client protein. Bcr-Abl protein was suppressed whereas the bcr-abl mRNA level was not affected. Further on, we observed several changes in the cytoskeleton organization. We detected ALA-PDT-mediated disruption of filamental actin structure using FITC-Phalloidin staining. In connection with this we uncovered certain cytoskeleton organizing proteins involved in the cell response to the treatment. Among these proteins, Septin2, which plays a role in maintaining actin bundles, was suppressed. Another one, PDZ-LIM domain protein 1 (CLP36) was altered. This protein acts as an adaptor molecule for LIM-kinase which phosphorylates and thus inactivates cofilin. Cofilin was indeed dephosphorylated and could thus be activated and operate as an actin-depolymerizing factor. We propose the scheme of molecular response of K562 cells to ALA-PDT.

Comparable Distribution of Minimal Residual Diesease between CD34 Positive Hematopoietic Progenitors, CD34 Negative Cells and Total White Cells in Patients with Chronic Myeloid Leukemia (CML) in Molecular Response.

Objectives: Most patients with chronic phase CML attain a complete cytogenetic response (CCR) to imatinib (IM) but BCR-ABL remains detectable by RT-PCR, indicating persistence of residual disease. One small study (Bhatia et al. Blood 101(12):4701–7, 2003) suggested that the levels of minimal residual disease (MRD) in CCR patients may be highest in the CD34+ cells but it is not clear whether the cohort under study was truly representative of patients with stable CCR. To address this question, we compared the level of MRD in total white cells (TWC), CD34-positive (CD34+), CD34-negative (CD34−) and CFU-GM colonies in patients with a molecular response (MR, defined as a BCR-ABL / ABL ratio below 0.12% as suggested by Paschka et al. Blood 104 (11): #1013, 2004) to IM.Patients and Methods: Twenty-two bone marrow samples were obtained from 13 CML patients with CCR [7 male/6 female, median age 57 (range 23–68) years]. At IM start all patients were in chronic phase (8 newly diagnosed, 5 refractory or intolerant to interferon alpha). The median time on IM at the date of the first analysis was 39 (range 10–50) month, the median duration of CCR and MR was 32 (range 4–49) and 20 (range 0–36) month, respectively. The frequency of CD34+ cells in unmanipulated BM as determined by FACS was compared to 33 patients with newly diagnosed CML and 9 healthy individuals. CD34+ cells were selected over immunomagnetic columns. At least 100 CD34+, CD34− and TWC were analyzed for BCR-ABL by FISH (Vysis LSI BCR-ABL ES probe, lab specific false positive rate of < 2%). CD34+ cells were plated in triplicate at 104 cells/ml and CFU-GM were counted on day 14. Quantitative PCR (qPCR) was performed on CD34+, CD34-, TWC and pools of 10 CFU-GM colonies. PCR-negative samples with less than 1000 copies of the ABL gene / μl cDNA were excluded.Results: The median frequencies of CD34+ cells in patients with MR, newly diagnosed patients and healthy controls were 0.30 (range 0.10–1.56)%, 3.81 (range 0.04–17.77)% and 0.88 (range 0.46 to 1.47)%, respectively (p<0.0005, Kruskal-Wallis-Test). BCR-ABL was undetectable by interphase FISH in all three compartments. Eleven (50%) and 16 (72%) out of 22 samples derived from the CD34+ and CD34− cells contained sufficient c-DNA for PCR. No significant differences were observed between the levels of BCR-ABL mRNA in CD34+, CD34− and TWC [median 0.023 (range, 0.001 to 0.149)%, 0.020 (range 0,005– 0,134)% and 0.011 (range 0.0005 to 0.257)%], respectively (p= ns, Wilcoxon-test). In the patients with more than one sample analyzed, the distribution of MRD between the compartments did not change significantly over time. C-DNA from colonies was available for 14 patients (63%). Again, no significant difference in BCR-ABL mRNA in comparison to CD34+, CD34− and TWC was detected [median 0.001 (range 0,001 to 0.0425)%].Conclusion:In CML with MR to IM CD34+ cells are consistently BCR-ABL-negative.Comparable levels of BCR-ABL mRNA residual disease are present in total white cells, CD34+, CD34- and CFU-GM colonies, suggesting that residual disease is not limited to immature cells.

Transient or long-term silencing of BCR-ABL alone induces cell cycle and proliferation arrest, apoptosis and differentiation

BCR-ABL fusion protein, a t(9;22) translocation product is indispensable for generation, maintenance and progression of chronic myeloid leukemia. RNA interference is an approach to silence gene at post-transcriptional level. We show that dsRNA targeted against the translocation region leads to more than 90% inhibition of BCR-ABL mRNA and protein expression levels using K562 as a model. Lack of BCR-ABL leads to cell cycle arrest in G1 phase as observed by decrease in cyclin D1 and increase in p21 and p27 cdk inhibitors mRNA. Apoptosis resistance imparted by BCR-ABL is lost in these cells in caspase-dependent or independent manner. Decrease in Bcl-XL is observed along with decrease in mitochondrial membrane integrity. Transient removal of BCR-ABL expression has a profound effect on proliferation and clonogenic capacity also confirmed by long-term silencing using lentiviral vectors. Interestingly, low level of BCR-ABL message leads to enhanced erythroid differentiation and reduced expression of megakaryocytic markers. Importantly, in six CML patient samples studied, silencing BCR-ABL in the lineage depleted enriched stem cell population leads to a decrease in colony-forming capacity. Thus, long-term silencing of BCR-ABL might prove to be a promising alternative approach in CML patients especially for those who do not respond to any other drug treatment.

The Nonviral Episomal Replicating Vector pEPI-1 Allows Long-Term Inhibition of bcr-abl Expression by shRNA

The inhibition of gene expression by RNA interference harbors a high potential for application in the therapy of human diseases. However, while exogenous application of siRNAs efficiently inhibits gene expression, these effects are only transient in mammalian cells. We designed a short hairpin RNA-expression cassette to target the bcr-abl oncogene that was then introduced into the nonviral vector system pEPI-1, which replicates episomally in the absence of selection in the bcr-abl-positive cell line K562. Forty-two days after transfection the bcr-abl- but not the cytokine-dependent growth rate was found to be drastically reduced in K562 cells. Western analysis revealed a more than 90% reduction in the expression of the fusion protein bcr-abl while the expression of the bcr protein remained unaffected. In addition, we show that the level of bcr-abl mRNA was specifically reduced in these cells for more than 90%. These results demonstrate that the vector system pEPI-1 allows specific and efficient long term gene suppression by using a short hairpin RNA transcription unit.

Detection of BCR-ABL kinase mutations in CD34+ cells from chronic myelogenous leukemia patients in complete cytogenetic remission on imatinib mesylate treatment

AbstractThe BCR-ABL kinase inhibitor imatinib mesylate induces complete cytogenetic response (CCR) in a high proportion of chronic myelogenous leukemia (CML) patients. However, patients in CCR usually demonstrate evidence of residual BCR-ABL–positive progenitors. The mechanisms underlying persistence of small numbers of malignant progenitors in imatinib-sensitive patients are unclear. BCR-ABL kinase domain mutations affecting drug binding can lead to secondary resistance to imatinib. We show here that kinase mutations could be detected in CD34+ cells isolated from CML patients in CCR on imatinib. Most mutations seen have not been reported in previous clinical studies. Interestingly, several of the involved amino acid positions have been implicated in an in vitro mutagenesis screen. These BCR-ABL mutations were associated with varying levels of imatinib resistance. Two of 5 patients in whom mutations were detected on initial evaluation have relapsed. In addition, 4 patients in whom mutations were not initially detected, but with rising BCR-ABL mRNA levels on quantitative polymerase chain reaction (Q-PCR) analysis, had mutations detected on follow-up evaluation. We conclude that BCR-ABL kinase mutations can be detected in CD34+ cells from CML patients in CCR on imatinib, may contribute to persistence of small populations of malignant progenitors, and could be a potential source of relapse.

Stable RNA interference (RNAi) as an option for anti-bcr-abl therapy

RNA interference (RNAi) has recently been used for sequence-specific gene silencing of disease-related genes including oncogenes in hematopoietic cells. To characterize its potential therapeutic value, we analyzed different modes to activate RNAi as well as some pharmacokinetic aspects of gene silencing in bcr-abl+ cells. Using lentiviral gene transfer of transcription cassettes for anti-bcr-abl shRNAs and red fluorescence protein (RFP) as a quantitative reporter, we demonstrate that stable but not transient RNAi can efficiently deplete bcr-abl+ K562 and murine TonB cells from suspension cultures. Importantly, depletion of bcr-abl+ cells depends on the dose of lentivirus used for transduction and correlates with the RFP-expression level of transduced target cells: RFP-high K562 cells are eradicated, whereas RFP-low or -intermediate cells may recover after prolonged cell culture. Interestingly, these cells still show reduced bcr-abl mRNA levels, aberrant proliferation kinetics, and enhanced sensitivity to the Bcr-Abl-kinase inhibitor STI571. Quantitative PCR from genomic DNA suggests that more than three lentiviral integrations are required for effective depletion of K562 cells. Finally, we demonstrate that lentivirus-mediated anti-bcr-abl RNAi can inhibit colony formation of primary CD34+ cells from chronic myeloid leukemia patients. These data demonstrate dose-dependent gene silencing by lentivirus-mediated RNAi in bcr-abl+ cells and suggest that stable RNAi may indeed be therapeutically useful in primary hematopoietic cells.

Synergism between 17-AAG and Imatinib in Imatinib-Resistant CML Cells: Inhibition of P-Glycoprotein by 17-AAG as a New Mechanism of Increasing Imatinib Activity.

Chronic myelogenous leukemia (CML) has gained outstanding importance for targeted cancer therapy. Inhibition of the BCR-ABL tyrosine kinase by imatinib (STI571, Glivec, Gleevec) leads to reduced proliferation of CML cells in vitro and sustained hematological and cytogenetic responses in vivo. However, resistance has been observed after variable periods of imatinib monotherapy especially in advanced stages of disease and overexpression of the BCR-ABL protein is one of the mechanisms of imatinib resistance. As combination therapy may allow to overcome drug resistance, we were interested in the effect of combination treatment with imatinib and 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of the heat shock protein 90 (Hsp90) chaperone complex. Furthermore, a new mechanism of action of the heat Hsp90 inhibitor 17-AAG is brought to light giving implications for an additional benefit of a combination treatment of imatinb-resistant chronic myelogenous leukemia. In imatinib-sensitive CML cell lines, combination index values (CI) obtained using the method of Chou and Talalay indicated additive (CI = 1) or slightly antagonistic (CI > 1) effects following simultaneous treatment with imatinib and 17-AAG. In contrast, the agents acted synergistically in imatinib-resistant BCR-ABL overexpressing LAMA84-R cells (CI = 0.6 at 75 % growth inhibition level). Growth inhibition of CFU-GM colonies of primary CML cells obtained from 3 patients is stronger after combination treatment than after monotherapy and annexin V / propidium iodide staining showed a strong increase of the apoptotic cell fraction in CML cells treated for 48 hrs by the combination treatment as compared to treatment with each drug alone. In imatinib-resistant cells BCR-ABL mRNA levels and protein expression were increased compared to the imatinib-sensitive parental cell line which is consistent to our cytogenetic and multicolor FISH analyses revealing multiple genomic BCR-ABL copies in imatinib-resistant cells. Furthermore, in imatinib-resistant cells P-glycoprotein activity was increased. Single treatment with 17-AAG lowered BCR-ABL and increased Hsp70 protein levels in both cell lines as expected whereas combination treatment was even more effective in this respect. Interestingly, single treatment with 17-AAG also decreased P-glycoprotein activity in a dose-dependent fashion as confirmed by a rhodamine-123 exclusion assay. The synergistic effect of both drugs in imatinib-resistant cells may thus be explained by increased intracellular levels of imatinib following 17-AAG treatment. The relevance of this additional mechanism warrants further exploration in clinical studies.

633. Dose-Dependent Gene Silencing by Lentivirus-Mediated RNA Interference (RNAi) in bcr-abl Positive Cells

RNA interference (RNAi) describes a process of double-stranded RNA-dependent post-transcriptional gene silencing. This gene silencing is mediated by 21–23 nt small interfering RNAs (siRNAs) which induce sequence-specific mRNA degradation. Fusion-transcripts encoding oncogenic proteins may represent potential targets for a tumor-specific RNAi-approach. Recently, siRNAs have been shown to specifically inhibit expression of the bcr-abl oncogene essential for chronic myeloid and bcr-abl positive acute lymphoblastic leukemia (CML, ALL) in hematopoietic cell lines. Since RNAi triggered by a single siRNA application is only transient in mammalian cells potential anti-tumor effects are limited in the models studied so far. Gene transfer of transcription cassettes for suitable RNAi-triggers, however, can induce long-term gene silencing even in primary hematopoietic cells. Using lentiviral gene transfer to transcribe different shRNAs targeting the bcr-abl oncogene and to express red fluorescence protein (RFP) as a quantitative reporter to track cellular transduction we demonstrate that stable but not transient RNAi can efficiently deplete bcr-abl+human K562 and murine TonB cells. Importantly, killing of bcr-abl+cells depends on the dose of lentivirus used for transduction and correlates with the RFP-expression level of transduced cells. Furthermore, we show that lentiviral transduction of homogenous cell lines results in heterogenous cell populations in terms of lentiviral integrations, RFP-and siRNA expression, target gene silencing, and functional phenotype. Whereas transduced K562 cells with high RFP-expression are eradicated, a limited number of cells with lower RFP-expression and smaller number of lentiviral integrations may outgrow after longer periods of cell culture. Interestingly, these cells still show some reduction in bcr-abl mRNA levels, aberrant proliferation kinetics, and higher sensitivity to the Bcr-Abl kinase inhibitor STI571 as compared to controls. Furthermore, lentiviral gene transfer of anti bcr-abl shRNA into primary CML cells can inhibit colony formation of transduced CD34+progenitor cells by about 50%. These data demonstrate that lentivirus-mediated RNAi can induce high efficient and stable gene silencing depending on the number of lentiviral integrations in cell lines and in primary cells. Selection processes of gene modified cells, however, have implications for any potential therapeutic application and for functional genomic analysis based on gene transfer strategies to induce stable RNAi in mammalian cells.

Quantitative real-time reverse-transcription polymerase chain reaction for diagnosis of BCR-ABL positive leukemias and molecular monitoring following allogeneic stem cell transplantation.

Real-time reverse-transcription polymerase chain reaction (RT-PCR) (qPCR) of the BCR-ABL mRNA is a suitable technique to measure the amount of circulating leukemic cells in chronic myelogenous leukemia (CML). In this study, we evaluated a BCR-ABL-specific qPCR method using the LightCycler technology in 95 patients with Philadelphia chromosome positive acute leukemia (n = 7) or CML in different stages (n = 88). Primers and hybridization probes were chosen to detect the most prevalent variants of BCR-ABL (b2a2, b3a2, b2a3, b3a3, e19a2, e1a2) with a sensitivity of 10-5 for b2a2 and b3a2. With median BCR-ABL/G6PDH ratios of 10.7% in the untreated chronic phase, 43.2% in the newly diagnosed accelerated phase, and 131.4% in newly diagnosed blast crisis the BCR-ABL mRNA levels varied significantly between different stages of CML whereas no difference was found between blast crisis and untreated acute leukemias (136.9%). There was a strong relationship between qPCR results and cytogenetics in patients treated with imatinib, interferon-alpha, or following allografting. Thirteen patients with CML were sequentially examined by qPCR following myeloablative or non-myeloablative allogeneic peripheral blood stem cell transplantation. Five patients received donor lymphocytes and became BCR-ABL negative as confirmed by nested RT-PCR. The gradual disappearance of BCR-ABL positive cells could be monitored by qPCR following non-myeloablative transplantation. Comparison of BCR-ABL levels with the degree of donor chimerism showed that 91% of samples with complete donor chimerism were BCR-ABL negative. In 22% of BCR-ABL negative samples chimerism between 71% and 98% was observed, indicating the persistence of normal recipient's hematopoietic cells. In conclusion, the qPCR protocol used in this study is a reliable and fast method for monitoring molecular response in CML.

Purging of chronic myelogenous leukemia cells by retrovirally expressed anti-bcr-abl ribozymes with specific cellular compartmentalization.

In patients with chronic myelogenous leukemia (CML), abnormal expansion of myeloid cells is maintained by expression of the p210(bcr-abl) fusion protein. Thus, this protein and its mRNA represent primary targets to inhibit proliferation of these cells. Here we describe the properties of a ribozyme against the bcr-abl mRNA, expressed as a fusion transcript with the human U1 small nuclear RNA or the adenovirus VA1 RNA and delivered to the cells through retroviral vectors. These fusion ribozymes are specifically localized in the nucleus or in the cytoplasm, respectively. Transduction of 32D-LG7 myeloid cells, whose growth is IL-3 independent thanks to deregulated bcr-abl expression, imposed strong negative selective pressure on cell growth and induced restoration of an IL-3-dependent phenotype. Although expressed at a level similar to that of the U1-fusion ribozyme, the cytoplasmic VA1 ribozyme was a more powerful inhibitor of p210(bcr-abl) gene expression. In cells transduced with the vector expressing this ribozyme, the levels of the bcr-abl transcript were reduced up to 10(4)-fold, the p210(bcr-abl) protein became undetectable, and the cells underwent massive apoptosis when cultured in the absence of IL-3. Transduction of primary hematopoietic cells obtained from bone marrow of patients with CML resulted in remarkable reduction of bcr-abl mRNA levels, starting a few days after transduction. These results show the feasibility and efficacy of vector-expressed anti-bcr-abl ribozymes for purging of CML cells.

Monitoring chronic myeloid leukaemia therapy by real-time quantitative PCR in blood is a reliable alternative to bone marrow cytogenetics.

We have developed a rapid real-time quantitative PCR method for measuring BCR-ABL mRNA levels in peripheral blood in chronic myeloid leukaemia (CML). The technique was used to monitor minimal residual disease for the early detection of relapse and as an assessment of treatment response. Normal BCR mRNA was quantitated to control for RNA degradation and the results reported as a percentage of BCR-ABL/BCR. Every patient measured at diagnosis (n = 21) had increased expression of BCR-ABL of up to 5-fold above the normal BCR levels. With effective treatment the BCR-ABL levels decreased. The molecular data was correlated with Philadelphia chromosome levels in bone marrow and a good correlation was found when treatment induced a cytogenetic response (Spearman correlation = 0.94, P < 0.0001, n = 67 samples). In patients receiving interferon-alpha therapy we found a significant difference in the BCR-ABL levels between cytogenetic response groups. The method was sensitive, reproducible, and readily detected a change in BCR-ABL transcript levels in serial blood samples. Sample throughput was high because post PCR processing was unnecessary. We conclude that real-time quantitative PCR monitoring of peripheral blood can be used to reliably monitor disease response in CML.