Establishment and characterization of a DOT1L inhibitor-sensitive human acute monocytic leukemia cell line YBT-5 with a novel KMT2A-MLLT3 fusion

Abstract

Immortalized cell lines are useful for deciphering the pathogenesis of acute leukemia and developing novel therapeutic agents against this malignancy. In this study, a new human myeloid leukemia cell line YBT-5 was established. After more than 1-year cultivation from the bone marrow of a patient with acute monocytic leukemia, YBT cell line was established. Then a subclone, YBT-5, was isolated from YBT using single cell sorting. Morphological and cytogenetical characterizations of the YBT-5 cell line were determined by cytochemical staining, flow cytometry analysis, and karyotype analysis. Molecular features were identified by transcriptomic analysis and reverse transcription-polymerase chain reaction. To establish a tumor model, 5 × 10⁶ YBT-5 cells were injected subcutaneously in nonobese diabetic/severe combined immune-deficiency (NOD/SCID) mice. DOT1L has been proposed as a potential therapeutic target for KMT2A-related leukemia; therefore, to explore the potential application of this new cell line, its sensitivity to a specific DOT1L inhibitor, EPZ004777 was measured ex vivo. The growth of YBT-5 does not depend on granulocyte-macrophage colony-stimulating factor. Cytochemical staining showed that α-naphthyl acetate esterase staining was positive and partially inhibited by sodium fluoride, while peroxidase staining was negative. Flow cytometry analysis of YBT-5 cells showed positive myeloid and monocytic markers. Karyotype analysis of YBT-5 showed 48,XY,+8,+8. The breakpoints between KMT2A exon 10 and exon 11 (KMT2A exon 10/11) and MLLT3 exon 5 and exon 6 (MLLT3 exon 5/6) were identified, which was different from all known breakpoint locations, and a novel fusion transcript KMT2A exon 10/MLLT3 exon 6 was formed. A tumor model was established successfully in NOD/SCID mice. EPZ004777 could inhibit the proliferation and induce the differentiation of YBT-5 cells. Therefore, a new acute monocytic leukemia cell line with clear biological and molecular features was established and may be used in the research and development of new agents targeting KMT2A-associated leukemia.

Keywords: DOT1L inhibitor; KMT2A-associated leukemia; acute monocytic leukemia; cell line.