Germline RUNX1 variation and predisposition to childhood acute lymphoblastic leukemia
Yizhen Li, … , Mignon L. Loh, Jun J. Yang
Yizhen Li, … , Mignon L. Loh, Jun J. Yang
Published June 24, 2021
Citation Information: J Clin Invest. 2021;131(17):e147898. https://doi.org/10.1172/JCI147898.
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Research Article Genetics Oncology

Germline RUNX1 variation and predisposition to childhood acute lymphoblastic leukemia

Abstract

Genetic alterations in the RUNX1 gene are associated with benign and malignant blood disorders, particularly of megakaryocyte and myeloid lineages. The role of RUNX1 in acute lymphoblastic leukemia (ALL) is less clear, particularly in terms of how germline genetic variation influences the predisposition to this type of leukemia. Sequencing DNA of 4836 children with B cell ALL (B-ALL) and 1354 with T cell ALL (T-ALL), we identified 31 and 18 germline RUNX1 variants, respectively. RUNX1 variants in B-ALL consistently showed minimal damaging effects. In contrast, 6 T-ALL–related variants resulted in drastic loss of RUNX1 activity as a transcription activator in vitro. Ectopic expression of dominant-negative RUNX1 variants in human CD34+ cells repressed differentiation into erythroid cells, megakaryocytes, and T cells, while promoting myeloid cell development. Chromatin immunoprecipitation sequencing of T-ALL models showed distinctive patterns of RUNX1 binding by variant proteins. Further whole-genome sequencing identified the JAK3 mutation as the most frequent somatic genomic abnormality in T-ALL with germline RUNX1 variants. Cointroduction of RUNX1 variant and JAK3 mutation in hematopoietic stem and progenitor cells in mice gave rise to T-ALL with the early T cell precursor phenotype. Taken together, these results indicate that RUNX1 is an important predisposition gene for T-ALL and point to biology of RUNX1-mediated leukemogenesis in the lymphoid lineages.

Authors

Yizhen Li, Wentao Yang, Meenakshi Devidas, Stuart S. Winter, Chimene Kesserwan, Wenjian Yang, Kimberly P. Dunsmore, Colton Smith, Maoxiang Qian, Xujie Zhao, Ranran Zhang, Julie M. Gastier-Foster, Elizabeth A. Raetz, William L. Carroll, Chunliang Li, Paul P. Liu, Karen R. Rabin, Takaomi Sanda, Charles G. Mullighan, Kim E. Nichols, William E. Evans, Ching-Hon Pui, Stephen P. Hunger, David T. Teachey, Mary V. Relling, Mignon L. Loh, Jun J. Yang

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Figure 4

RUNX1 variants affect in vitro differentiation of human cord blood CD34+ cells.

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RUNX1 variants affect in vitro differentiation of human cord blood CD34...
(A and B) Schematic showing in vitro hematopoietic differentiation assay. RUNX1 variants (with IRES-GFP) were lentivirally introduced into human cord blood CD34+ cells. Successfully transduced cells were sorted by flow cytometry (B), processed for CFU assays, and assessed for cell proliferation and apoptosis, as appropriate. (C) Western blot was used to confirm RUNX1 expression, with vinculin as the internal control. (D) 1000 RUNX1-expressing CD34+ cells were plated in MethoCult H4034. The y axis shows the count of colonies for each lineage: BFU-E, CFU-macrophage (CFU-M), and CFU-GM. EV, p.R233fs, and p.Y287*, n = 3; p.G365R, n = 6; WT, n = 5. (E) Colony number of CFU assays and CFU-replating assays (n = 4). (F) Proliferation of RUNX1-expressing CD34+ cells was monitored for 5 weeks in IMDM containing 20% BIT9500, 10 ng/mL FLT-3 ligand, TPO, SCF, IL-3, and IL-6. The number of cells was counted every week for 5 weeks. n = 4. (G) Apoptosis of RUNX1-transduced CD34+ cells after 7 (n = 3) and 16 (n = 4) days of culture (same culture medium as in F) was measured by flow cytometry using annexin V and DAPI antibodies. (H and I) CD34+ cells ectopically expressing RUNX1 variants were also subjected to in vitro differentiation assays for megakaryocyte (n = 3) or T cell (n = 4) lineages. Following RUNX1 transduction, cells were cultured in the presence of SFEMII-containing megakaryocyte expansion supplement or T cell progenitor differentiation supplement for 2 weeks. Megakaryocyte (H) was identified as CD41a+CD42b+, and T cells (I) were defined as CD5+CD7+ by flow cytometry. Data are represented as mean ± SEM. P values were estimated by using Dunnett’s test. *P < 0.05; **P < 0.01; ***P < 0.001.