Cytogenetic and molecular analysis of the acute monocytic leukemia cell line THP-1 with an MLL-AF9 translocation

Cell lines derived from patients with leukemia are used in many molecular biology studies. Here we report the cytogenetic analysis of the THP-1 cell line using G-banding, fluorescence in situ hybridization (FISH), and spectral karyotyping (SKY), and the molecular characterization of the MLL-AF9 rearrangement by RT-PCR. The THP-1 cell line was established from the peripheral blood of a 1-year-old boy with acute monocytic leukemia (AML-M5). THP-1 is near-diploid and consists of two related subclones with a number of aberrations, including the t(9;11), associated with AML M5. The use of FISH allowed us to identify and characterize otherwise hidden cytogenetic rearrangements, which include duplication of the 3' portion of MLL in the derivative 9 chromosome and a deletion of the 5' portion of the AF9 gene involved in the translocation. In addition to confirming the FISH results, SKY allowed for a more precise characterization of the karyotype of THP-1 and allowed us to identify other abnormalities in this cell line, including der(1)t(1;12), der(20)t(1;20), deletions 6p, 12p, and 17p, trisomy 8, and monosomy 10. Sequencing of the RT-PCR product showed a direct in-frame fusion product on the derivative chromosome 11 between exon 6 (exon 9) of MLL and exon 5 of AF9, which is most commonly involved in MLL-AF9 translocations. This study demonstrates that combining different techniques to achieve a more precise characterization of the THP-1 cell line provides important information that will be valuable for understanding the critical events required for leukemogenesis

Further characterization of complex chromosomal rearrangements in myeloid malignancies: spectral karyotyping adds precision in defining abnormalities associated with poor prognosis

The mixed lineage leukemia gene (MLL, also known as HRX, ALL-1 and Htrx) located at 11q23 is involved in translocations with over 40 different chromosomal bands in a variety of leukemia subtypes. Here we report our analysis of a rare but recurring translocation, t(11;15)(q23;q14). This translocation has been described in a small subset of cases with both acute myeloblastic leukemia and ALL. Recent studies have shown that MLL is fused to AF15q14 in the t(11;15). Here we analyse a sample from another patient with this translocation and confirm the presence of an MLL-AF15q14 fusion. However, we have also identified and cloned another fusion transcript from the same patient sample. In this fusion transcript, MLL is fused to a novel gene, MLL partner containing FYVE domain (MPFYVE). Both MLL-AF15q14 and MLL-MPFYVE are in-frame fusion transcripts with the potential to code for novel fusion proteins. MPFYVE is also located on chromosome 15, approximately 170 kb telomeric to AF15q14. MPFYVE contains a highly conserved motif, the FYVE domain which, in other proteins, has been shown to bind to phosphotidyl-inositol-3 phosphate (PtdIns(3)P). The MLL-MPFYVE fusion may be functionally important in the leukemia process in at least some patients containing this translocation

Molecular cytogenetic characterization of breakpoints in 19 patients with hematologic malignancies and 12p unbalanced translocations

Structural rearrangements of the short arm of chromosome 12 are frequent cytogenetic findings in various hematologic malignancies. The ETV6 gene is the most common target for rearrangements in 12p13. Fluorescence in situ hybridization (FISH) investigations have shown that translocations of 12p other than t(12;21) are frequently accompanied by small interstitial deletions that include ETV6. Unbalanced translocations involving ETV6 have rarely been described, and breakpoints outside ETV6 appear to be strongly associated with complex karyotypes. We studied bone marrow samples from 19 patients known to have 12p unbalanced translocations and complex karyotypes, using FISH and spectral karyotyping. FISH analysis confirmed the hemizygous deletion of the ETV6 and CDKN1B genes in 74% of cases. We found four cases with interstitial deletions. In these four cases and in two others (6/19, 31.5%), the fusion with the partner chromosome was in the subtelomeric region of 12p13.3, confirming that there is a recurrent breakpoint in this region

Identification of new translocations involving ETV6 in hematologic malignancies by fluorescence in situ hybridization and spectral karyotyping

TEL/ETV6 is the first transcription factor identified that is specifically required for hematopoiesis within the bone marrow. This gene has been found to have multiple fusion partners; 35 different chromosome bands have been involved in ETV6 translocations, of which 13 have been cloned. To identify additional ETV6 partner genes and to characterize the chromosomal abnormalities more fully, we studied bone marrow samples from patients known to have rearrangements of 12p, using fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY). FISH analysis was done with 14 probes located on 12p12.1 to 12p13.3. Nine ETV6 rearrangements were identified using FISH. The aberrations include t(1;12)(p36;p13), t(4;12)(q12;p13) (two patients), t(4;12)(q22;p13), t(6;12)(p21;p13), der(6)t(6;21)(q15;q?)t(12;21)(p13;q22), t(6;12)(q25;p13), inv(12)(p13q24), and t(2;2;5;12;17)(p25;q23;q31;p13;q12). Six new ETV6 partner bands were identified: 1p36, 4q22, 6p21, 6q25, 12q24, and 17q12. Our present data as well previous data from us and from other researchers suggest that ETV6 is involved in 41 translocations. The breakpoints in ETV6 were upstream from the exons coding for the HLH (helix-loop-helix) domain in six cases. Although cytogenetic analysis identified 12p abnormalities in all cases, FISH and SKY detected new and unexpected chromosomal rearrangements in many of them. Thus, complete characterization of the samples was achieved by using all three techniques in combination

New fusion transcripts identified in normal karyotype acute myeloid leukemia

Genetic aberrations contribute to acute myeloid leukemia (AML). However, half of AML cases do not contain the well-known aberrations detectable mostly by cytogenetic analysis, and these cases are classified as normal karyotype AML. Different outcomes of normal karyotype AML suggest that this subgroup of AML could be genetically heterogeneous. But lack of genetic markers makes it difficult to further study this subgroup of AML. Using paired-end RNAseq method, we performed a transcriptome analysis in 45 AML cases including 29 normal karyotype AML, 8 abnormal karyotype AML and 8 AML without karyotype informaiton. Our study identified 134 fusion transcripts, all of which were formed between the partner genes adjacent in the same chromosome and distributed at different frequencies in the AML cases. Seven fusions are exclusively present in normal karyotype AML, and the rest fusions are shared between the normal karyotype AML and abnormal karyotype AML. CIITA, a master regulator of MHC class II gene expression and truncated in B-cell lymphoma and Hodgkin disease, is found to fuse with DEXI in 48% of normal karyotype AML cases. The fusion transcripts formed between adjacent genes highlight the possibility that certain such fusions could be involved in oncological process in AML, and provide a new source to identify genetic markers for normal karyotype AML

A novel gene, MDS2, is fused to ETV6/TEL in a t(1;12)(p36.1;p13) in a patient with myelodysplastic syndrome

ETV6/TEL is the first transcription factor identified that is specifically required for hematopoiesis within the bone marrow. This gene has been found to have multiple fusion partners of which 16 have been cloned. Fluorescence in situ hybridization (FISH) analysis in a patient with myelodysplastic syndrome (MDS) revealed a t(1;12)(p36;p13) involving ETV6, with the breakpoint in this gene between exon 2 and exon 3. We report here the cloning of a novel ETV6 partner located on 1p36.1, involved in the t(1;12). 3' RACE-PCR from RNA identified a novel sequence fused to exon 2 of ETV6. Database searches localized this sequence in a bacterial artificial chromosome (BAC) mapped to 1p36 by fingerprint analysis. This result was confirmed by FISH using this BAC as probe. 5' and 3' RACE experiments with primers from this novel sequence were carried out on RNA from a healthy donor and identified a novel full-length mRNA, which we named MDS2 (myelodysplastic syndrome 2). RT-PCR experiments were performed on a panel of human cDNAs to analyze the expression pattern of this gene and they revealed four splicing variants. RT-PCR analysis showed that ETV6-MDS2, but not the reciprocal MDS2-ETV6 fusion transcript, was expressed in the bone marrow of the patient. The product of the ETV6-MDS2 fusion transcript predicts a short ETV6 protein containing the first 54 amino acids of ETV6 plus four novel amino acids, lacking both the PTN and the DNA-binding domains. Possible mechanisms to account for the development of MDS in this patient are discussed

Mimicry of a constitutively active pre–B cell receptor in acute lymphoblastic leukemia cells

Pre–B cells undergo apoptosis unless they are rescued by pre–B cell receptor–dependent survival signals. We previously showed that the BCR-ABL1 kinase that is expressed in pre–B lymphoblastic leukemia bypasses selection for pre–B cell receptor–dependent survival signals. Investigating possible interference of BCR-ABL1 with pre–B cell receptor signaling, we found that neither SYK nor SLP65 can be phosphorylated in response to pre–B cell receptor engagement. Instead, Bruton's tyrosine kinase (BTK) is constitutively phosphorylated by BCR-ABL1. Activated BTK is essential for survival signals that otherwise would arise from the pre–B cell receptor, including activation of PLCγ1, autonomous Ca(2+) signaling, STAT5-phosphorylation, and up-regulation of BCLX (L). Inhibition of BTK activity specifically induces apoptosis in BCR-ABL1 (+) leukemia cells to a similar extent as inhibition of BCR-ABL1 kinase activity itself. However, BCR-ABL1 cannot directly bind to full-length BTK. Instead, BCR-ABL1 induces the expression of a truncated splice variant of BTK that acts as a linker between the two kinases. As opposed to full-length BTK, truncated BTK lacks kinase activity yet can bind to BCR-ABL1 through its SRC-homology domain 3. Acting as a linker, truncated BTK enables BCR-ABL1–dependent activation of full-length BTK, which initiates downstream survival signals and mimics a constitutively active pre–B cell receptor