Cooperative gene activation by AF4 and DOT1L drives MLL-rearranged leukemia
Hiroshi Okuda, … , Akifumi Takaori-Kondo, Akihiko Yokoyama
Hiroshi Okuda, … , Akifumi Takaori-Kondo, Akihiko Yokoyama
Published April 10, 2017
Citation Information: J Clin Invest. 2017;127(5):1918-1931. https://doi.org/10.1172/JCI91406.
View: Text | PDF
Research Article Hematology Oncology

Cooperative gene activation by AF4 and DOT1L drives MLL-rearranged leukemia

Abstract

The eleven-nineteen leukemia (ENL) protein family, composed of ENL and AF9, is a common component of 3 transcriptional modulators: AF4–ENL–P-TEFb complex (AEP), DOT1L-AF10-ENL complex (referred to as the DOT1L complex) and polycomb-repressive complex 1 (PRC1). Each complex associates with chromatin via distinct mechanisms, conferring different transcriptional properties including activation, maintenance, and repression. The mixed-lineage leukemia (MLL) gene often fuses with ENL and AF10 family genes in leukemia. However, the functional interrelationship among those 3 complexes in leukemic transformation remains largely elusive. Here, we have shown that MLL-ENL and MLL-AF10 constitutively activate transcription by aberrantly inducing both AEP-dependent transcriptional activation and DOT1L-dependent transcriptional maintenance, mostly in the absence of PRC1, to fully transform hematopoietic progenitors. These results reveal a cooperative transcriptional activation mechanism of AEP and DOT1L and suggest a molecular rationale for the simultaneous inhibition of the MLL fusion–AF4 complex and DOT1L for more effective treatment of MLL-rearranged leukemia.

Authors

Hiroshi Okuda, Boban Stanojevic, Akinori Kanai, Takeshi Kawamura, Satoshi Takahashi, Hirotaka Matsui, Akifumi Takaori-Kondo, Akihiko Yokoyama

×

Figure 5

Mechanism of MLL-AF10–dependent transformation.

Options: View larger image (or click on image) Download as PowerPoint
Mechanism of MLL-AF10–dependent transformation. (A) Structural requireme...
(A) Structural requirement of MLL-AF10 for leukemic transformation. Various MLL-AF10 constructs were analyzed by the myeloid progenitor transformation assay, as in Figure 4D. P′, PWWP. (B) Association between MLL and AF4 on chromatin. 293T cells transiently expressing various FLAG-tagged MLL mutants and Xpress-tagged AF4 or AF5Q31 were analyzed by fanChIP-WB using an anti-FLAG antibody. (C) Effect of Dot1l knockdown on various MLL fusion–transformed cells. Transduced cells were selected for puromycin resistance for 2 days. RNA was extracted 3 days after transduction of shRNAs. The expression levels of Dot1l and Hoxa9 normalized to Gapdh (representative of 2 independent experiments) are shown as the value relative to that of the vector control (set at 100%). Error bars represent the SD of PCRs performed in triplicate. CFU relative to those of the vector control in the same experiment (set at 100%) is shown with error bars (SD for >3 independent experiments). *P ≤ 0.05, **P ≤ 0.01, §P ≤ 0.001, and #P ≤ 0.0001, by unpaired, 2-tailed t test, comparing each sample with the vector control. (D) Effect of Enl knockdown on various MLL fusion–transformed cells. Experiments were performed using shRNA for Enl as shown in panel C. **P ≤ 0.01, §P ≤ 0.001, and #P ≤ 0.0001, by by unpaired, 2-tailed t test, comparing each sample with the vector control.