ABSTRACT
OBJECTIVE: We investigated the mechanism responsible for imatinib (IM) resistance in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) cell lines. METHODS: We established cell lines from a patient with Ph(+) ALL at the time of first diagnosis and relapsed phase and designated as NPhA1 and NPhA2, respectively. We also derived IM-resistant cells, NPhA2/STIR, from NPhA2 under gradually increasing IM concentrations. RESULTS: NPhA1 was sensitive to IM (IC(50) 0.05 microm) and NPhA2 showed mild IM resistance (IC(50) 0.3 microm). NPhA2/STIR could be maintained in the presence of 10 microm IM. Phosphorylation of MEK and ERK was slightly elevated in NPhA2 and significantly elevated in NPhA2/STIR compared to NPhA1 cells. After treatment with IM, phosphorylation of MEK and ERK was not suppressed but rather increased in NPhA2 and NPhA2/STIR. Active RAS was also increased markedly in NPhA2/STIR after IM treatment. The expression of BCL-2 was increased in NPhA2 compared to NPhA1, but no further increase in NPhA2/STIR. Proliferation of NPhA2/STIR was significantly inhibited by a combination of MEK inhibitor and IM. Analysis of tyrosine phosphorylation status with a protein tyrosine kinase array showed increased phosphorylation of EphB4 in NPhA2/STIR after IM treatment. Although transcription of EphB4 was suppressed in NPhA1 and NPhA2 after IM treatment, it was not suppressed and its ligand, ephrinB2, was increased in NPhA2/STIR. Suppression of EphB4 transcripts by introducing short hairpin RNA into NPhA2/STIR partially restored their sensitivity to IM. CONCLUSIONS: These results suggest a new mechanism of IM resistance mediated by the activation of RAS/MAPK pathway and EphB4.
Subject(s)
Drug Resistance, Neoplasm/physiology , Fusion Proteins, bcr-abl/antagonists & inhibitors , Neoplasm Proteins/physiology , Piperazines/pharmacology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/enzymology , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Receptor, EphB4/physiology , ras Proteins/physiology , Benzamides , Cell Line, Tumor/drug effects , Cell Line, Tumor/enzymology , Enzyme Activation , Enzyme Induction , Ephrin-B2/genetics , Ephrin-B2/physiology , Female , Humans , Imatinib Mesylate , MAP Kinase Signaling System/physiology , Middle Aged , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Phosphorylation/drug effects , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Protein Processing, Post-Translational/drug effects , RNA, Small Interfering/pharmacology , Receptor, EphB4/antagonists & inhibitors , Receptor, EphB4/genetics , RecurrenceABSTRACT
OBJECTIVE: The RUNX1 (also known as AML1) gene is observed frequently as the target of chromosomal rearrangements in human acute leukemia. We describe here a previously unreported rearrangement, t(11;21)(q13;q22), that disrupts the RUNX1 gene in a patient with acute leukemia and the molecular analysis of the fusion gene. METHODS: We have established a monocytic leukemia cell line, ELAM-1, from a patient with acute leukemia evolving from myelodysplastic syndrome (MDS). Translocation (11;21) (q13;q22) was observed in both patient leukemia cells and ELAM-1. RESULTS: The split signal of RUNX1 was detected by fluorescence in situ hybridization and indicated the involvement of RUNX1 in ELAM-1. Using 3'- Rapid amplification of cDNA ends and reverse transcription-Polymerase chain reaction analysis, we detected both RUNX1 (exon 5)-LRP16 and RUNX1 (exon 6)-LRP16 transcripts, suggesting that the RUNX1 breakpoint lies in intron 6 and that alternative fusion splice variants are generated. Reciprocal LRP16-RUNX1 fusion was also detected. CONCLUSIONS: We identified a novel RUNX1 fusion partner, LRP16 on 11q13 involving t(11;21)(q13;q22). Although it was reported that overexpression of LRP16 promotes human breast cancer cell proliferation, the function of LRP16 in leukemia remains to be studied. This fusion gene and cell line may provide a new research tool to investigate the mechanism of leukemogenesis generated by the RUNX1 fusion gene.
Subject(s)
Chromosomes, Human, Pair 11 , Chromosomes, Human, Pair 21 , Core Binding Factor Alpha 2 Subunit/genetics , Leukemia, Monocytic, Acute/genetics , Neoplasm Proteins/genetics , Translocation, Genetic , Base Sequence , Carboxylic Ester Hydrolases , Cell Line, Tumor , DNA Primers , Female , Humans , Karyotyping , Leukemia, Monocytic, Acute/pathology , Middle Aged , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
We developed a method of clone proliferation promoting fusion genes whose proteins were activated by protein oligomerization through the helix-loop-helix region (PNT domain) of TEL. We inserted a cDNA library downstream of the PNT domain with a retrovirus vector. The resulting retrovirus infected cytokine-dependent 32D cells and cells with cytokine-independent growth were analyzed for the inserted cDNA. We cloned 25 independent fusion genes including seven kinds of partner genes. Six of the seven were a fusion of TEL with protein tyrosine kinase, LYN, HCK, FGR, SYK, FLT3, and TYK2. A serine/threonine kinase, ARAF1, was also found to fuse with TEL. These kinase fusion proteins included kinase domains with proper reading frames. These fusions may be a useful model for clarifying the downstream signal transduction of constitutive active kinase and this expression cloning method may provide a new tool with which to study cell proliferation signalling.
