Infant acute lymphoblastic leukemia (ALL) is characterized by the presence of the proB phenotype (CD10−/CD19+), poor prognosis and frequent rearrangement of the mixed lineage leukemia (MLL) gene. The most frequent rearrangement is t(4;11)(q21;q23), the role of whose product, the MLL-AF4 fusion transcript, has been extensively studied in leukemogenesis. In a cell line of infant leukemia with MLL rearrangement denoted KP-L-RY, panhandle PCR amplification of cDNA revealed the presence of a fusion transcript, MLL-AF5q31, indicating that AF5q31 is also a partner gene of MLL. In this fusion transcript the MLL exon 6 is fused in frame to the 5′ side of the putative transactivation domain of AF5q31. The AF5q31 protein is a member of the AF4/LAF4/FMR2-related family of proteins, which have been suggested to play a role in hematopoietic cell growth and differentiation. The MLL-AF5q31 fusion transcript, although probably rare, appears to be associated with the pathogenesis of infant ALL like MLL-AF4. Co-expression of HoxA9 and Meis1 genes in the KP-L-RY cell line indicated possible functional similarity between MLL-AF4 and MLL-AF5q31. Further understanding of the function of AF5q31 as well as the specific leukemogenic mechanism of MLL-AF5q31 awaits future studies.
In spite of recent progress in the treatment of hematological malignancies in childhood, infant acute leukemia (AL), and particularly acute lymphoblastic leukemia (ALL), still shows a poor therapeutic outcome.1,2 In order to develop new effective therapeutic measures against infant AL, a number of investigations, involving the analyses of leukemogenic mechanisms, are now in progress. The most important finding so far from cytogenetic and molecular analyses is the close association of rearrangements of the mixed lineage leukemia (MLL) gene on chromosome 11q23 with specific types of leukemia such as infant AL,1,3,4,5 therapy-related acute leukemia and myelodysplastic syndrome (t-AL/MDS).6,7 Rearrangement of the MLL gene is found in approximately 10% of ALL cases, 5% of acute myeloblastic leukemia (AML) cases, and 85% of t-AL cases, which arise as a result of treatment with topoisomerase II (topoII) inhibitors.8 To date, different partner genes at a variety of chromosomal regions, such as AF4,9 AF9,10 ENL11 and CBP,12 have been found to be associated with MLL in the entity of 11q23 abnormality.8,13,14 However, data on the partner genes of MLL in infantile ALL cases are still limited.8 In addition, leukemogenic function and the mechanism of lineage-specific restriction related to the MLL-linked fusion transcript have not been fully clarified. In the present study, we established a cell line, KP-L-RY, from the peripheral blood of an ALL infant with MLL rearrangement. The partner gene of MLL was revealed to be AF5q31 on chromosome 5 by panhandle PCR amplification of cDNA.15 Thereafter, using specific primers, we attempted to determine the frequency of MLL-AF5q31 in several infant ALL samples. To analyze the possible function of MLL-AF5q31, expression of HoxA9 and Meis1 genes was also studied.
Materials and methods
Patient and cell line establishment
A 1-month-old boy, who showed hepatosplenomegaly and petechiae with hyperleukocytosis (WBC 252, 800/μl, of which 94% were abnormal lymphoblasts) on admission to our hospital in May, 1988, was diagnosed with ALL. The leukemic blasts were positive for CD19 and HLA-DR, but negative for CD10, and showed the abnormal karyotype, involving 11q23) [20/20]. The patient died of the disease 18 days after diagnosis. At diagnosis, leukemic blasts from peripheral blood were grown in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37°C in 5% CO2 in air. One month after starting culture, the blast cells showed stable proliferation and gave rise to a continuous cell line (KP-L-RY) within another month. The cell line was then maintained over a number of years in our laboratory.
Control hematopoietic cell lines and clinical infant ALL samples
Several lymphoid (RPMI8402, RPMI1788, Raji, Jurkat, Hut78, H-9, KO-PN-67, KOCL45) and myeloid cell lines (U-937, KP-MO-TS, HL-60, K562) were employed. The majority of these cell lines were commercially available except for KOCL45 with t(4;11)(q11;q23) and KO-PN-67 with t(9;22)(q34;q11), both of which were gifts from Dr Nakazawa, Yamanashi Medical College. KP-MO-TS was established from an acute myelomonocytic leukemia patient in our laboratory. These cell lines were cultured under the same conditions described above, and served as control hematopoietic cell lines for RT-PCR study of AF5q31, HoxA9 and Meis1.
Seventeen infant ALL samples, which are known to have MLL gene rearrangements, were obtained through the Japan Infant Leukemia Study Group in order to determine the frequency of MLL-AF5q31.
Characterization of KP-L-RY
For the morphological study, air-dried smears were prepared from cell suspensions in culture using cytospin and stained with May–Grünwald-Giemsa. Flow cytometric analysis used the panel of MoAbs reactive for T-, B-, and myelomonocytic-lineage cells.
Cytogenetic analysis was performed with the standard trypsinGiemsa banding procedure. Spectral karyotyping (SKY) analysis followed the procedure previously described by Kakazu et al.16 The karyotypes were described according to the ISCN (1995).17
For exact determination of the karyotype of KP-L-RY, fluorescence in situ hybridization (FISH) analysis was performed as previously described.18 The probes used in this study were two bacterial artificial chromosome (BAC) clones (RP11–115L24 located at chromosome band 5q21.1 and RP11–772D18 containing 5′ region of the AF5q31 gene), yeast artificial chromosome (YAC) clone 13HH4 containing the MLL gene, and α-satellite probe specific for the centromere region of chromosome 11 (D11Z1). Two BAC clones were obtained from the RP11 library. These probes were directly labeled with SpectrumOrange (SO)-dUTP or SpectrumGreen (SG)-dUTP (Vysis, Downers Grove, IL, USA) by nick translation. Two-color FISH images were captured and analyzed with a PowerGene system (Applied Imaging, Santa Clara, CA, USA).
For further characterization, KP-L-RY cells were suspended at 0.3 × 105/ml in growth medium with or without 12–O-tetradecanoyl-phorbol-13-acetate (TPA) at various concentrations (0.1 to 10 ng/ml) for 5 days. All cultures were incubated at 37°C in a humidified atmosphere of 5% CO2 in air. Cultured cells were examined for morphological changes and subjected daily for cell counting, with cell viability being determined by trypan blue exclusion. Surface marker analysis was performed as described above.
Molecular studies of KP-LRY
Determination of MLL rearrangement by Southern blot analysis:
Southern blot analysis was performed as described elsewhere.19 High molecular weight DNA extracted from the cell line KP-L-RY was digested with EcoRV or BamHI, and hybridized with an MLL cDNA fragment spanning exon 7 to exon 10 as probe.
Cloning of partner gene(s) of by cDNA panhandle PCR and sequence analysis:
Total RNA was extracted from the cell line KP-L-RY with Trizol reagent (GIBCO-BRL) according to the manufacturer's specifications. Panhandle PCR for cDNA was performed following the procedures previously described by Megonigal et al.15 cDNA panhandle PCR products were subcloned into Max efficiency DH5α competent cells (Life Technologies) by recombination PCR20,21 with HindIII-linearized pUC19 (Life Technologies). The sequences of all primer sets used in cDNA panhandle and recombination PCR were the same as described elsewhere.15
Amplified PCR products subcloned into pUC19 were purified by the SV miniprep DNA purification system kit (Promega) from DH5α transformants. Sequencing was performed in a mixture containing 500 ng of subcloned DNA, 2 μl each of dNTP reagent (BigDye Terminator Cycle Sequencing FS Ready Reaction Kit; Applied Biosystems) and 0.2 μl of 20 pmol up- or under-stream primers. Reactions were incubated for 30 cycles at 96°C for 10 s, at 50°C for 5 s and at 60°C for 4 min and products were sequenced on an ABI Prism 377 DNA sequencing System (Applied Biosystem).
The presence of MLL-AF5q31 was confirmed by reverse transcription (RT)-PCR and by genomic PCR
Total RNA (5 μg) from the KP-L-RY cell line was reverse transcribed to cDNA in a total volume of 25 μl containing random hexamers. A quarter of the resulting cDNA was amplified by PCR. After 35 cycles of PCR (30 s at 94°C, 30 s at 55°C, and 1 min at 72°C), 10 μl of each PCR product was subjected to 1.5% agarose gel electrophoresis. The primers used were as follows: MLL-411BS, 5′-AAGTGGCTCCCCGCCCAAGTAT-3′; AF5-AS, 5′-CCATCACTGTCTTCACTGCT-3′. Genomic DNA (100 ng) derived from the KP-L-RY cell line was also amplified under the same conditions as for RT-PCR. The primers used were MLL-411BS and AF5-AS2, 5′-GA GGCCATGAATGCGTCAT-3’. The PCR product was sequenced directly.
Detection of unknown genes fused to AF5q31 or MLL at 7p15 by Northern blot analysis:
Total RNA (10 μg) from the KP-L-RY cell line was hybridized with RNA probes labeled with digoxygenin (DIG) (DIG Northern Starter Kit; Roche). RNA labeling was carried out according to the instruction manual. A 0.9-kb BamHI fragment (designated probe X) derived from MLL cDNA and a 0.5-kb AF5q31 cDNA, which covered the region between nucleotides 1150 and 1650 (GenBank accession number XM011230), was transcribed into RNA with DIG labeling for use as an RNA probe.
Molecular studies of other cell lines, comparing with KP-L-RY
Detection of AF5q31 expression in various hematopoietic cell lines and screening for MLL-AF5q31 in infantile leukemia samples:
Total RNA (5 μg) was extracted from each cell line to study AF5q31 gene expression by RT-PCR. The PCR conditions were the same as described above for the KP-L-RY cell line. The primers used were AF5S, 5′-GAGCACTACAGCCATCC-3′, and AF5-AS3, 5′-TCCAGAGCTGCTTTCAGATCC-3′. MLL-AF5q31 was screened for in 17 infantile ALL samples possessing known MLL rearrangements by RT-PCR using the MLL-411BS and AF5-AS primers.
Study of HoxA9 and Meis1 expression in various lymphoid cell lines:
To determine the role of other genes related to t(4;11)(q21;q23) or 11q23 rearranged ALL, HoxA9 and Meis1 gene expression were also examined in KP-L-RY and other lymphoid cell lines. Total RNA (5 μg) was extracted from each lymphoid cell line to determine the expression of HoxA9 and Meis1 genes by RT-PCR. Amplification was carried out for 35 cycles with denaturation at 94°C for 10 s, annealing at 57°C for 30 s, and extension for 1 min at 72°C. The primers used were HoxA9 F1, 5′-AGACGCTGGAACTGGAGAAA-3′, HoxA9 R1, 5′-CTTCATTTTCATCCTGCGGT-3′, Meis1 F1, 5′-AAGGTGATGGCTTGGACAAC-3′ and Meis1 R1, 5′-GGCTGCACTATTCTTCTCCG-3′.
Characterization of the cell line KP-L-RY
KP-L-RY cells showed a lymphoblastic morphology (FAB L1 subtype) with a vacuole-filled cytoplasm (Figure 1a). The cells grew in suspension. By flow cytometric analysis, both original fresh leukemic cells and KP-L-RY cells expressed CD19, and HLA-DR, but not CD4, CD8, CD10, CD14, CD20, CD56, or cyMPO. Although KP-L-RY cells moderately expressed CD13 and CD33, we assumed that they had the proB cell phenotype. In the presence of TPA, the cells became larger, more vacuolated and irregularly shaped, comparable to the findings observed in RS4:11 cell line (Figure 1b). Phenotypically, the expressions of CD13 and CD33 were augmented (from 26.9% to 50.3% and from 29.7% to 38.9%, respectively). However, non-specific esterase activity remained negative. Cell growth and viability were markedly reduced during the culture with high concentration of TPA (>1.0 ng/ml) (data not shown).
Cytogenetic analysis of KP-L-RY showed an abnormal karyotype: 46,XY, ?t(5;11;7)(q1?5;q23;p1?5),add(16)(q24). To confirm the three-way translocation and identify the origin of the material attached to add(16)(q24), we used SKY analysis. SKY analysis showed the presence of t(5;11;7) and identified add(16)(q24) as dup(16)(q?). Reevaluation of the G-banding pattern suggested an inversion in the portion of 5q translocated to band 11q23.
FISH analysis clearly revealed inversion of 5q15-q31 (Figure 2a and c) in der(11) chromosome. Thus, the exact karyotype of KP-L-RY was determined as 46, XY, der(5)t(5;7) (q15p15), der(7)t(7;11)(p15;q23), der(11)t(5;11)(q15q23)inv(5) (q15q31), dup(16)(q?).
Identification of the MLL-AF5q31 fusion transcript by panhandle PCR of cDNA
Southern blot analysis of KP-L-RY cells revealed the rearrangement of MLL (Figure 3). Subsequently, we performed cDNA panhandle PCR to clone the partner gene in the 5q15 region that was presumed to be fused to MLL in this cell line. Among the various size products generated (Figure 4a), recombination PCR produced six subclones, which were all sequenced (Figure 4b). In one 476 bp clone (clone 4), derived from an in-frame chimeric transcript, we found that exon 6 of MLL was fused to exon 5 of AF5q31 (cDNA sequence; GenBank accession number XM 011230), which was located in the 5q31 region as illustrated in Figure 4c. This was confirmed by FISH, showing the fusion signal of the MLL and the AF5q31 genes on der(11) chromosome (Figure 2b).
Characterization of the genomic junction at the MLL-AF5q31 breakpoint
Using RT-PCR to confirm the expression of the MLL-AF5q31 fusion transcript in the KP-L-RY cell line, we obtained the expected 266 bp fusion product (Figure 5). Furthermore, by cloning the genomic junction of the breakpoint by genomic PCR, we obtained a 1650 bp product and confirmed the genomic rearrangement between MLL and AF5q31 (Figure 6a). Sequence analysis revealed that the breakpoint is located in intron 6 of MLL, which is fused to intron 4 of AF5 (Figure 6b). Intron 6 of MLL contains an Alu sequence.
Detection of other transcript(s) in KP-L-RY and screening for the MLL-AF5q31 fusion transcript in 17 infant ALL cases with MLL rearrangement
As described, KP-L-RY cell line had three-way translocation involving chromosomes 5, 7 and 11. In addition, molecular studies revealed that breakpoint of MLL exists in intron 6, with the 3′ portion of the MLL bcr retained. Thus, we suspected the possible expression of unknown genes at 7p15 fused to AF5q31 or to MLL. However, we could not detect any such fusion transcripts except for MLL-AF5q31 by Northern blot analysis (data not shown). Considering the reported deletion of the MLL portion on the telomeric side of the breakpoint in about 20–30% of cases with 11q23 abnormalities,14 further studies are in progress to identify other abnormalities in the MLL region.
Because the MLL-AF5q31 transcript was identified in an infantile case of ALL, we suspected that it might also be expressed in other leukemias with the lymphoid phenotype. Testing of AF5q31 expression by RT-PCR revealed that it was positive in all lymphoid (RPMI8402, RPMI1788, Hut78, H-9, KOCL45, KP-L-RY) as well as myeloid (U937, KP-MO-TS, HL60, K562) cell lines (data not shown). To clarify whether any other infantile cases of ALL express MLL-AF5q31, we performed RT-PCR analysis for this fusion transcript in 17 infantile ALL samples known to have the MLL rearrangement; however, none were positive for MLL-AF5q31 (data not shown).
Co-expression of HoxA9 and MeisI genes in KP-L-RY
Considering the close link of co-expression of HoxA9 and Meis1 genes in cases of ALL with t(4;11)(q21;q23),22 we studied whether they were also co-expressed in our KP-L-RY cell line. As shown in Figure 7, we confirmed that HoxA9 and Meis1 genes were co-expressed in the KP-L-RY cells, along with KOCL45 cells possessing t(4;11)(q21;q23).
Although several leukemic cell lines harboring MLL rearrangement have been reported,23,24 the cell line KP-L-RY established by us, expressing the MLL-AF5q31 fusion transcript, is novel. The KP-L-RY presents morphologically L1 blast, and dual-lineage surface antigen expression pattern typical for ALL associated with MLL rearrangement. The character changes of KP-L-RY in the presence of TPA were compatible with those of the RS4;11 cell line, described by Strong et al.23
The non-random association of chromosomal translocations with leukemia suggests that certain specific genes play an important role in leukemogenesis.25,26 A typical chromosomal translocation associated with infant ALL is t(4;11)(q21;q23), whose product, the MLL-AF4 fusion transcript, plays an important role in leukemogenesis and in expressing the specific lymphoid lineage.27 MLL is one of a number of highly characterized genes that are known to be involved in the development of infant AL and t-AML.3,4,5,6,7 Many partner genes that form fusion products with MLL have been identified;8,9 however, those involved in infantile ALL have attracted relatively little attention.8 In order to identify unknown partner gene(s) of MLL, Megonigal et al15 first developed the method of cDNA panhandle PCR. This procedure allowed us to successfully identify the MLL-AF5q31 fusion transcript in the KP-L-RY cell line established from a case of infant ALL with MLL rearrangement.
In this regard, FISH analysis revealed that an inversion of 5q15-q31 in der(11) chromosome exists. Thus, we confirmed that 5q31 fused to 11q23, which then generated the MLL-AF5q31 fusion.
Previously, AF5q31 was first shown to be fused to MLL by Taki et al28 in the bone marrow cells of an infant ALL case that showed the abnormal ins(5;11)(q31;q13q23) karyotype. However, to our knowledge there have been no further reports. Because we could not detect MLL-AF5q31 expression in 17 infant ALL cases with MLL rearrangement,and only one case of invins(5;11)(q31;q23q13) has been reported among 50 infant ALL cases with MLL rearrangement,29 infant ALL expressing this chimeric transcript seems to be rare. Although its incidence is low, we suspect that the MLL-AF5q31 fusion transcript contributes to leukemogenesis in infancy causing AL with pro-B phenotype (CD10−/19+) and hyperleukocytosis,27 through a similar mechanism to that of MLL-AF4. Interestingly, GRAF gene (for GTPase regulatory associated with the focal adhesion kinase pp125FAK), located at the telomeric side of AF5q31, was described as being involved in AML/myelodysplastic syndrome (MDS) cases.30 According to Borkhardt et al,30 GRAF gene was fused to MLL in a case of juvenile myelomonocytic leukemia with t(5;11)(q31;q23), and both alleles were disrupted in 3/13 AML/MDS cases with del(5q). Considering these and other findings, partner genes of MLL, such as ELL and ENL, and GRAF and AF5q31, may be closely linked to the lineage restriction of various types of leukemia.
In terms of AF5q31, it is an AF4-related gene, and shares three homologous regions with the latter.28 AF4 protein displays homology to an AF4-related lymphoid nuclear protein (LAF4), which makes AF5q31 a member of the AF4/LAF4/FMR2 family.31,32 In human and mouse cell lines, LAF4 expression was highest in pre-B cells, intermediate in mature B cells, and absent in plasma cells, suggesting that LAF4 plays a potential regulatory role in early lymphoid development.31 Our results showed that AF5q31 expression was not limited to lymphoid cells but was also widespread in myeloid leukemic cell lines.
Previous findings on MLL-AF4 may help to understand the function of MLL-AF5q31. The breakpoint of AF5q31 in the KP-L-RY, which is different from that described by Taki et al,28 was located on the far 5′ side of the putative transactivation domain. Furthermore, in accordance with previous reports, the MLL-AF5q31 fusion transcript in the KP-L-RY cell line retains AT hook and methyltransferase domains of MLL as well as the transactivation domain of AF5q31. Thus, the MLL-AF5q31 fusion protein possesses DNA binding and transactivation activity, indicating that it may still act as a transcriptional factor (Figure 4c). Previous observations that the breakpoints of AF4 are clustered on the 5′ side of the transactivation domain in cases of MLL-AF4,28,31 strongly suggest that the transactivation domain of AF5q31 is important for the transforming activity of MLL-AF5q31 in leukemogenesis.
Data suggesting a similarity between MLL-AF4 and MLL-AF5q31 were obtained by studying the altered expression of related genes to MLL rearrangement. Previously, ALL cases expressing MLL-AF4 were reported to show co-expression of HoxA9 and Meis1 genes.22 Our study confirmed that the KP-L-RY cell line also co-expressed these two genes which are also expressed in another infant ALL cell line (KOCL45) carrying MLL-AF4 fusion transcript. This indicates that these two fusion transcripts may be functionally related.The cell line KP-L-RY is expected to provide a useful model for the further clarification of the biological and leukemogenic activity of MLL-AF5q31.
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The authors are grateful to the Japan Infant Leukemia Study Group for providing the leukemic specimens and to Yasuko Hashimoto for her secretarial assistance.
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Imamura, T., Morimoto, A., Ikushima, S. et al. A novel infant acute lymphoblastic leukemia cell line with MLL-AF5q31 fusion transcript. Leukemia 16, 2302–2308 (2002). https://doi.org/10.1038/sj.leu.2402665
- infant acute lymphoblastic leukemia
- panhandle PCR for cDNA
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