Multi-unit ribozyme-mediated cleavage of bcr-abl mRNA in myeloid leukemias

Chronic myelogenous leukemia is characterized by the Philadelphia chromosome, which at the molecular level results from the fusion of the bcr gene on chromosome 22 and the abl gene on chromosome 9. The bcr-abl fusion gene encodes a novel tyrosine kinase with transforming activity. In this study, we have synthesized a multi-unti ribozyme that targets bcr-abl mRNA. In vitro ribozyme cleavage reactions show increased cleavage efficiency of this multi-unit ribozyme compared with single or double ribozymes. The multiunit ribozyme was then transfected into murine myeloblasts transformed with the bcr-abl gene (32D cells). Ribozyme transfection was accomplished either by liposomes or using follic acid-polylysine as a carrier. Multi-unit ribozyme transfection reduced the level of bcr-abl mRNA 3 logs when transfected via folate receptor-mediated uptake into transformed 32D cells. These results suggest that a multi-unit ribozyme could be an effective therapeutic agent for the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia.

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Identification of human chronic myelogenous leukemia progenitor cells with hemangioblastic characteristics

Blood ◽

10.1182/blood-2004-07-2514 ◽

2005 ◽

Vol 105 (7) ◽

pp. 2733-2740 ◽

Cited By ~ 59

Author(s):

Baijun Fang ◽

Chunmei Zheng ◽

Lianming Liao ◽

Qin Han ◽

Zhao Sun ◽

...

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Progenitor Cells ◽

Chronic Myelogenous Leukemia ◽

Blood Cells ◽

Fusion Gene ◽

Fetal Liver ◽

Philadelphia Chromosome ◽

Myelogenous Leukemia ◽

Leukemic Cells

AbstractOverwhelming evidence from leukemia research has shown that the clonal population of neoplastic cells exhibits marked heterogeneity with respect to proliferation and differentiation. There are rare stem cells within the leukemic population that possess extensive proliferation and self-renewal capacity not found in the majority of the leukemic cells. These leukemic stem cells are necessary and sufficient to maintain the leukemia. Interestingly, the BCR/ABL fusion gene, which is present in chronic myelogenous leukemia (CML), was also detected in the endothelial cells of patients with CML, suggesting that CML might originate from hemangioblastic progenitor cells that can give rise to both blood cells and endothelial cells. Here we isolated fetal liver kinase-1–positive (Flk1+) cells carrying the BCR/ABL fusion gene from the bone marrow of 17 Philadelphia chromosome–positive (Ph+) patients with CML and found that these cells could differentiate into malignant blood cells and phenotypically defined endothelial cells at the single-cell level. These findings provide direct evidence for the first time that rearrangement of the BCR/ABL gene might happen at or even before the level of hemangioblastic progenitor cells, thus resulting in detection of the BCR/ABL fusion gene in both blood and endothelial cells.

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Detection by enzymatic amplification of bcr-abl mRNA in peripheral blood and bone marrow cells of patients with chronic myelogenous leukemia

Blood ◽

10.1182/blood.v73.6.1735.1735 ◽

1989 ◽

Vol 73 (6) ◽

pp. 1735-1741 ◽

Cited By ~ 44

Author(s):

W Lange ◽

DS Snyder ◽

R Castro ◽

JJ Rossi ◽

KG Blume

Keyword(s):

Bone Marrow ◽

Chronic Myelogenous Leukemia ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Philadelphia Chromosome ◽

Chromosome 9 ◽

Myelogenous Leukemia ◽

Enzymatic Amplification ◽

Polymerase Chain ◽

Marrow Cells

Abstract The Philadelphia chromosome of chronic myelogenous leukemia (CML) patients is caused by a translocation of the c-abl gene from chromosome 9 to the breakpoint cluster region (bcr) on chromosome 22. A new bcr- abl mRNA is expressed in these cases. We have developed a modified polymerase chain reaction (PCR) for the detection of this mRNA. The method is extremely sensitive, reliable, and relatively fast. The analysis of peripheral blood or bone marrow cells from CML patients treated with chemotherapy shows that the two possible mRNAs are expressed in various combinations. Our results show that even after myeloablative therapy for bone marrow transplantation bcr-abl mRNAs are still expressed. Further studies, however, are necessary to determine the clinical relevance of a small number of persisting cells expressing the bcr-abl mRNA.

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The Abl/bcr Fusion Gene on Chromosome 9 in Ph-Negative Chronic Myelogenous Leukemia

Cancer Genetics and Cytogenetics ◽

10.1016/s0165-4608(97)00430-5 ◽

1998 ◽

Vol 104 (1) ◽

pp. 57-60 ◽

Cited By ~ 16

Author(s):

Wei-Tong Hsu ◽

Harvey Preisler ◽

Katarina Szego ◽

Rita Sprudzs ◽

Xue-Zhi Gao

Keyword(s):

Chronic Myelogenous Leukemia ◽

Fusion Gene ◽

Chromosome 9 ◽

Myelogenous Leukemia

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Application of FASTTM Fragment-Based Lead Discovery and Structure-Guided Design to Discovery of Small Molecule Inhibitors of BCR-ABL Tyrosine Kinase Active Against the T315I Imatinib-Resistant Mutant.

Blood ◽

10.1182/blood.v106.11.698.698 ◽

2005 ◽

Vol 106 (11) ◽

pp. 698-698 ◽

Cited By ~ 10

Author(s):

Stephen K. Burley

Keyword(s):

Tyrosine Kinase ◽

Fusion Gene ◽

Clinical Success ◽

Philadelphia Chromosome ◽

Myelogenous Leukemia ◽

Wild Type ◽

Lead Discovery ◽

Abl Tyrosine Kinase ◽

Imatinib Resistant

Abstract The Philadelphia chromosome translocation creates a BCR-ABL fusion gene that encodes a constitutively active BCR-ABL tyrosine kinase, which gives rise to chronic myelogenous leukemia (CML). The clinical success of imatinib (Gleevec) demonstrated that BCR-ABL tyrosine kinase inhibitors can provide effective treatment for CML. However, some CML patients treated with imatinib develop resistance leading to disease progression. The majority of resistance is due to point mutations in BCR-ABL, which give rise to active mutant enzymes that are insensitive to imatinib. In all, ~30 imatinib-resistant BCR-ABL mutants have been identified in clinical isolates. The T315I mutant represents ~20% of clinically observed mutations, making it one of the most common causes of resistance. Second-generation BCR-ABL inhibitors, including AMN-107 and BMS-354825, inhibit many of the clinically relevant mutants but not T315I. Mutant T315I BCR-ABL is, therefore, an important and challenging target for discovery of CML therapeutics. We have applied a proprietary X-ray crystallographic fragment-based lead discovery platform (FASTTM) and structure-guided lead optimization to identify potent inhibitors of wild-type BCR-ABL and the four most common mutants, including T315I. Our lead discovery efforts yielded five chemical series that inhibit both wild-type (WT) and T315I BCR-ABL. Compounds in our most advanced lead series potently inhibit proliferation of K562 cells and Ba/F3 cells with WT BCR-ABL and the four major clinically relevant BCR-ABL mutations (T315I, E255K, M351T, Y253F; see below). Further details describing in vitro and in vivo profiling of these novel BCR-ABL T315I inhibitors will be presented. Ba/F3 cell proliferation for BCR-ABL Inhibitors (EC50, nM) BCR-ABL Form Imatinib AMN-107 BMS-354825 SGX-70430 WT 790 33 12 11 T315I > 10000 > 10000 > 10000 21 Y253F 5700 370 8 334 E255K 8300 350 7 77 M351T 2000 38 28 15 Control Assay Ba/F3 (T315I) + IL3 > 10000 > 10000 > 10000 > 10000

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Application of chromosome microdissection probes for elucidation of BCR- ABL fusion and variant Philadelphia chromosome translocations in chronic myelogenous leukemia

Blood ◽

10.1182/blood.v81.12.3365.3365 ◽

1993 ◽

Vol 81 (12) ◽

pp. 3365-3371 ◽

Cited By ~ 32

Author(s):

J Zhang ◽

P Meltzer ◽

R Jenkins ◽

XY Guan ◽

J Trent

Keyword(s):

Chronic Myelogenous Leukemia ◽

Chromosome Rearrangement ◽

Philadelphia Chromosome ◽

Leukemic Cell ◽

Myelogenous Leukemia ◽

Chromosome Microdissection ◽

Chromosome Translocations ◽

Ph Chromosome

Abstract Fluorescence in situ hybridization (FISH) has become an increasingly important method for assessing chromosome rearrangement. The reciprocal translocation constituting the Philadelphia (Ph) chromosome [t(9;22)(q34;q11)] characterizes more than 90% of patients with chronic myelogenous leukemia (CML). However, in the remaining cases the Ph chromosome (genetically characterized by the fusion of the BCR-ABL genes) is thought to arise through complex translocations that are often not readily apparent using routine chromosome-banding analysis. For this reason we have developed unique band-specific probes for two- color FISH that detect unequivocally the Ph chromosome, and its derivatives. Results of the application of these probes are illustrated by analysis of 11 cases of CML (9 of which contain “variant” translocations). The probes were generated by chromosome microdissection and in vitro amplification of the bands involved in the Ph translocation, leading to an extremely fast and sensitive approach to identify this alteration in leukemic cell populations.

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BCR-ABL Is Not an Immunodominant Antigen in CML.

Blood ◽

10.1182/blood.v106.11.2881.2881 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2881-2881

Author(s):

Frank Grünebach ◽

Valbona Mirakaj ◽

Martin R. Müller ◽

Tim Brümmendorf ◽

Peter Brossart

Keyword(s):

Bone Marrow ◽

T Lymphocytes ◽

Philadelphia Chromosome ◽

Chimeric Protein ◽

Genetically Engineered ◽

Chromosome 9 ◽

Myelogenous Leukemia ◽

Target Cells ◽

Specific Ctls

Abstract Chronic myelogenous leukemia (CML) is a myeloproliferative disorder, which originates from pluripotent hematopoietic bone marrow progenitor cells. It is characterized by excessive proliferation of the granulopoiesis in the bone marrow. In approximately 90% of the patients with CML the Philadelphia chromosome (Ph) is the characteristic cytogenetic hallmark. The Ph is a shortened chromosome 22, which arises from a acquired reciprocal translocation between chromosome 9 and 22 (t(9;22) (q34;q11)). This translocation fuses the bcr gene with part of the c-abl gene, which encodes a protein-tyrosine kinase. The chimeric bcr-abl oncogene is translated into a 210-kDa chimeric protein (p210) which exhibits constitutive ABL kinase activity. In the present study, we analysed the involvement of the BCR-ABL protein in the induction of antigen-specific cytotoxic T lymphocytes (CTLs) in order to develop an immunotherapeutic approach targeting this neo-antigen in patients with CML. To accomplish this, we generated dendritic cells (DCs) in vitro and electroporated them with various sources of RNA harbouring the chimeric bcr-abl transcript. These genetically engineered DCs where used as antigen presenting cells (APCs) for the in vitro induction of CTLs. By applying this approach we found that the CTLs induced by DCs transfected with RNA extracted from bcr-abl-positive K-562 cells or CML blasts do not recognize epitopes derived from the chimeric BCR-ABL fusion protein. In contrast, they were able to lyse autologous DCs electroporated with RNA isolated from patients with acute myeloid leukemia (AML), indicating that antigens shared among these malignant cells are involved and recognized by these CTLs. However, we successfully generated BCR-ABL-specific CTLs by DCs electroporated with in vitro transcribed bcr-abl-RNA. In patients with CML in complete cytogenetic remission during IFN-α treatment there was some reactivity against BCR-ABL in IFN-γ ELISPOT assays, which was weaker as compared to proteinase 3 (PR3)- or Prame-directed responses supporting the observations obtained in healthy donors. In summary, BCR-ABL is processed and presented by Ph+ cells but is not the immunodominat antigen that induces CD8+ T lymphocytes in competition with other tumor-associated antigens. Nevertheless, APCs transfected with pure bcr-abl-IVT in vitro are capable of inducing BCR-ABL specific CTLs that efficiently lyse Ph+ target cells.

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Variable breakpoints on the Philadelphia chromosome in chronic myelogenous leukemia

Blood ◽

10.1182/blood.v66.1.243.243 ◽

1985 ◽

Vol 66 (1) ◽

pp. 243-245 ◽

Cited By ~ 31

Author(s):

D Leibowitz ◽

K Schaefer-Rego ◽

DW Popenoe ◽

JG Mears ◽

A Bank

Keyword(s):

Chronic Myelogenous Leukemia ◽

Southern Blotting ◽

Philadelphia Chromosome ◽

Dna Probes ◽

Chromosome 9 ◽

Myelogenous Leukemia ◽

Chromosome 22 ◽

Human Dna ◽

Translocation Breakpoints ◽

The Creation

Abstract The abl oncogene is translocated from chromosome 9 to 22 in the creation of the Philadelphia (Ph1) chromosome. This article describes new translocation breakpoints identified in two patients with chronic myelogenous leukemia using Southern blotting and cloned human DNA probes from chromosome 9. The translocation breakpoints on chromosome 9 in both of these patients lie closer to the human cellular abl (c-abl) gene, and the chromosome 22 breakpoints are distributed more widely than previously reported. These data help to define more clearly the chromosomal span of the breakpoints and indicate that some translocations include very little chromosome 9 sequence located 5′ to the c-abl gene.

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Relationship of bcr breakpoint to chronic phase duration, survival, and blast crisis lineage in chronic myelogenous leukemia patients presenting in early chronic phase [see comments]

Blood ◽

10.1182/blood.v75.10.2035.bloodjournal75102035 ◽

1990 ◽

Vol 75 (10) ◽

pp. 2035-2041

Author(s):

SW Morris ◽

L Daniel ◽

CM Ahmed ◽

A Elias ◽

P Lebowitz

Keyword(s):

Chronic Myelogenous Leukemia ◽

Fusion Gene ◽

Blast Crisis ◽

Cell Lineage ◽

Chimeric Protein ◽

Chronic Phase ◽

Chromosome 9 ◽

Myelogenous Leukemia ◽

Phase Duration ◽

Total Survival

Strong evidence implicates fusion of control elements and 5′ sequences of the bcr gene of chromosome 22 with 3′ sequences of the c-abl gene of chromosome 9 in the pathogenesis of Ph-positive and certain cases of Ph- negative chronic myelogenous leukemia (CML). Since this fusion gene gives rise to a chimeric tyrosine protein kinase with transforming potential, and since the bcr exon contribution to this chimeric protein is variable, the question has arisen as to whether bcr breakpoint location and bcr exon contribution could influence the clinical course of CML. Prior studies have yielded conflicting results on this point. Here we have looked, in a manner approximating a prospective analysis, at the relation of bcr breakpoint localization to the duration of chronic phase, total survival, and blast crisis phenotype in 81 patients presenting in the chronic phase of CML. We have found no significant differences in chronic phase duration or total survival among patients with breakpoints in the three major subregions of a breakpoint cluster region within the bcr gene. These findings indicate that chronic phase duration and total survival cannot be predicted from bcr breakpoint for CML patients presenting in chronic phase and suggest that unknown oncogenic events determining the onset of blast crisis are the prime determinants of prognosis. Combined analysis of blast crisis cell lineage in our patients and patients presented in a previous study has revealed an overall ratio of myeloid:lymphoid (M:L) crisis of 3.4:1, but a striking predominance of myeloid crisis in patients with breakpoints in subregion 2 (M:L of 9:1), and a lower than expected M:L ratio (1.6:1) among patients with breakpoints in subregion 3 (P for subregion 2 versus 3 = .012; subregions 0,1,2 versus 3 = .012; subregions 0,1,3 versus 2 = .032). The molecular basis for this divergence from the anticipated M:L ratio in patients with breakpoints in bcr subregions 2 and 3 is unknown.

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Application of chromosome microdissection probes for elucidation of BCR- ABL fusion and variant Philadelphia chromosome translocations in chronic myelogenous leukemia

Blood ◽

10.1182/blood.v81.12.3365.bloodjournal81123365 ◽

1993 ◽

Vol 81 (12) ◽

pp. 3365-3371

Author(s):

J Zhang ◽

P Meltzer ◽

R Jenkins ◽

XY Guan ◽

J Trent

Keyword(s):

Chronic Myelogenous Leukemia ◽

Chromosome Rearrangement ◽

Philadelphia Chromosome ◽

Leukemic Cell ◽

Myelogenous Leukemia ◽

Chromosome Microdissection ◽

Chromosome Translocations ◽

Ph Chromosome

Fluorescence in situ hybridization (FISH) has become an increasingly important method for assessing chromosome rearrangement. The reciprocal translocation constituting the Philadelphia (Ph) chromosome [t(9;22)(q34;q11)] characterizes more than 90% of patients with chronic myelogenous leukemia (CML). However, in the remaining cases the Ph chromosome (genetically characterized by the fusion of the BCR-ABL genes) is thought to arise through complex translocations that are often not readily apparent using routine chromosome-banding analysis. For this reason we have developed unique band-specific probes for two- color FISH that detect unequivocally the Ph chromosome, and its derivatives. Results of the application of these probes are illustrated by analysis of 11 cases of CML (9 of which contain “variant” translocations). The probes were generated by chromosome microdissection and in vitro amplification of the bands involved in the Ph translocation, leading to an extremely fast and sensitive approach to identify this alteration in leukemic cell populations.

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C-sis and C-abl expression in chronic myelogenous leukemia and other hematologic malignancies

Blood ◽

10.1182/blood.v67.3.839.bloodjournal673839 ◽

1986 ◽

Vol 67 (3) ◽

pp. 839-841

Author(s):

P Romero ◽

M Blick ◽

M Talpaz ◽

E Murphy ◽

J Hester ◽

...

Keyword(s):

Chronic Myelogenous Leukemia ◽

Chromosomal Rearrangements ◽

Philadelphia Chromosome ◽

Chronic Phase ◽

Hematologic Malignancies ◽

Chromosome 9 ◽

Myelogenous Leukemia ◽

Chromosome 22 ◽

Aberrant Expression

Cellular oncogenes have been localized at the breakpoints of characteristic chromosomal rearrangements occurring in certain hematologic malignancies. This has been reported to result in aberrant expression of the involved oncogenes. Over 90% of chronic myelogenous leukemia (CML) is characterized by a reciprocal translocation that brings c-abl from chromosome 9 to chromosome 22, and c-sis from chromosome 22 to chromosome 9. To investigate the possible role of these two oncogenes in the leukemic process, we studied their expression in a number of fresh samples obtained from patients with various forms of leukemia, by Northern blot analysis using c-onc probes. Seven of 24 samples obtained from patients with either CML or chronic myelomonocytic leukemia expressed a normal 4.0-kilobase (kb) c- sis transcript. C-sis expression was found only in the accelerated/blast phases but not in the chronic phase of CML. All of the CML Philadelphia chromosome-positive (Ph1+) samples expressed an aberrant 8-kb c-abl transcript. The expression of c-sis in

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