Kasumi-6

Establishment of the Kasumi-6 Cell Line

The Kasumi-6 cells had been increasing continuously in liquid suspension culture for 22 months, with a doubling time of about 55 hr. The original leukemic cells relapsed leukemic cells, and the Kasumi-6 cells (Fig. 1 A, B, and C) presented features of immature blasts with abundant cytoplasm and fine granules. The leukemic cells at diagnosis were positive for MPO and CD34, but the relapsed leukemic cells and the Kasumi-6 cells were negative for both. Staining for NBE, PAS, and NAP was not tested for the cells at diagnosis or relapse, but tested negative for the Kasumi-6 cells. Both the original leukemic cells and the Kasumi-6 cells were positive for CD33, CD13, CD11b, c-kit, and HLA-DR, but negative for CD3, CD14, CD19, and CD41. Cytoplasmic MPO was positive for the original leukemic cells, but it could not be detected in the Kasumi-6 cell line. Both the relapsed leukemic cells and the Kasumi-6 cells had the same karyotype: 45, XY, 9, add(12)(p11), add(13)(p11).

An identical shifted band was detected by PCR-SSCP analysis in the original leukemic cells at diagnosis, the leukemic cells at relapse, and the Kasumi-6 cells (Fig. 2). On the other hand, Kasumi-1, -3, and -5 cell lines, which were established from other individuals with M2, M0, and L2 FAB-type leukemia, respectively, displayed a normal SSCP pattern (Fig. 2). DNA sequencing analysis of the shifted bands identified a G to C transversion at nucleotide 1,063, resulting in an R305P amino acid change.  Both SSCP and DNA sequencing analyses revealed that the wild-type and mutant alleles were present in all samples, indicating that the mutation was hemizygous. This mutation occurred in the fork or hinge region between the DNA-binding basic domain and the leucine zipper dimerization domain. The presence of a helical-destabilizing residue such as proline is predicted to have an impact on the function of the C/EBPα protein.

Fig. 1 Morphology of the (A) original leukemic cells, (B) relapsed leukemic cells and (C) Kasumi-6 cells. (Asou H, et al., 2003)

Fig. 2 Mutation of the C/EBPα gene in the patient sample and Kasumi-6 cell line. (Asou H, et al., 2003)

Characterization of Kasumi Leukemia Cell Line Genomes

Leukemia genomes are primarily characterized by abnormal karyotypes, which often include the formation of a fusion gene. Ten Kasumi cell lines established in Japan, including five that were previously unknown, have been characterized by SNP microarray and targeted sequencing.

Copy number alterations were identified in nine of the ten cell lines, except for Kasumi-4. Gains and losses were observed in 6 and 8 cell lines, respectively (Table 1). Trisomy 10 was observed in Kasumi-1 and 6, demonstrating a characteristic feature of AML-M2. Loss of heterozygosity (LOH) resulting in one copy of alleles did not occur in a whole chromosome but in partial regions. DNA size compared with normal diploid was calculated from 98.4 to 104.7% for the three AML cell lines and from 98.8 to 99.7% for the five BCP-ALL cell lines (Fig. 3). The largest difference among the 10 cell lines was found in Kasumi-6, which increased its DNA size to 280.2 Mb. SNP profiles revealed that UPD, equivalent to copy neutral LOH consisting of homozygous DNA copies, occurred in 7 cell lines.

Hotspot mutations were identified in 7 cell lines (Table 2). TP53 mutations were detected in Kasumi-1 and 6, exhibiting 17p LOH in the array profiles, resulting in a 100% allele frequency. FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) was detected in Kasumi-6 and -10, derived from elderly AML and infant ALL patients, respectively. Kasumi-1 has been used as a KIT mutant AML model, which was detected at 4q12 with 79.3% variant frequency under 4 copies, indicating duplications of the mutated allele. Fusion transcripts were detected in five cell lines (Table 2). A very high level of TCF3-PBX1 expression was observed in Kasumi-2, compared with other fusions. NUP98-RAP1GDS1 has been identified in Kasumi-5, but it was not covered as a target in the panel.

Table 1. Quantitative assessment of large-scale genomic changes. (Kasai F, et al., 2020)

Fig. 3 Assessment of tumor genomes. (Kasai F, et al., 2020)

Table 2. Genomic features as a reference panel. (Kasai F, et al., 2020)