Malignant Progression of an MDS-Derived Cell Line Serves As an in Vitro Model for the Leukemic Evolution of MDS

• Published in: Blood 2018-11, Vol.132 (Supplement 1), p.5501-5501
• Main Authors: Kida, Jun-ichiro, Tsujioka, Takayuki, Suemori, Shin-ichiro, Okamoto, Shuichiro, Sakakibara, Kanae, Yamauchi, Takahiro, Kitanaka, Akira, Tohyama, Yumi, Tohyama, Kaoru
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2018-99-110583

Myelodysplastic syndromes (MDS) have a risk of progression to acute myeloid leukemia (AML), but the deterioration mechanisms of MDS and the alteration points still remain to be elucidated. We previously established a myelodysplastic cell line, MDS92 from the bone marrow of an MDS patient, and after a long-term interleukin(IL)-3-containing culture of MDS92, five blastic sublines including MDS-L were isolated. From MDS-L, we obtained two sublines, MDS-L-2007 and MDS-LGF after culture in the presence and absence of IL-3, respectively. To investigate the mechanism of leukemic evolution, we applied a next-generation sequencing (NGS) to the series of cell lines for comprehensive, comparative exome analyses, and searched for the origin of mutations by ultra-deep target sequencing of the original patient bone marrow. Whole exome sequencing and ultra-deep target sequencing demonstrated: (1) TP53 mutation was found in the patient bone marrow and this mutation was inherited by all subsequent cell lines; (2) CEBPA mutation was originally present in a small fraction of the bone marrow; (3) NRAS mutation emerged by chance during IL-3-containing culture; (4) HIST1H3C(K27M) mutation (Histone-H3-K27M) was newly detected at the generation of MDS-L from MDS92. H3-K27M mutation was detected in MDS-L-2007 but not in MDS-LGF. We focused on H3-K27M mutation because it is frequently found in pediatric brain stem tumors and recently found in a small population of AML cases (Lehnertz et al. Blood. 2017). MDS-L cells were a mixture of H3-K27M-mutant and wild-type clones. When MDS-L was cultured in the presence of IL-3, the proportion of H3-K27M-mutant fraction gradually increased. In contrast, when MDS-L was cultured without IL-3, the proportion of H3-K27M-mutant fraction gradually decreased. To investigate the implication of H3-K27M mutation, we tried single cell cloning from MDS-L and secured four wild-type clones and seven H3-K27M-mutant clones. In all H3-K27M-mutant clones, there was a marked reduction in H3-K27me3/2. Expression of a tumor-suppressor molecule p16 was reduced in six of the seven H3-K27M-mutant clones. H3-K27M-mutant clones showed rapid growth in the presence of IL-3, but cell proliferation was suppressed without IL-3. Competitive growth experiment by co-culture of H3-K27-wild-type and H3-K27M-mutant clones in the presence or absence of IL-3 showed that H3-K27M-mutant clones were predominant in the presence of IL-3, whereas wild-type clones were sustained comparat