Cooperative gene activation by AF4 and DOT1L drives MLL-rearranged leukemia
Hiroshi Okuda, Boban Stanojevic, Akinori Kanai, Takeshi Kawamura, Satoshi Takahashi, Hirotaka Matsui, Akifumi Takaori-Kondo, Akihiko Yokoyama
Hiroshi Okuda, Boban Stanojevic, Akinori Kanai, Takeshi Kawamura, Satoshi Takahashi, Hirotaka Matsui, Akifumi Takaori-Kondo, Akihiko Yokoyama
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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

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Figure 7

Combined antileukemic effects of the MENIN-MLL interaction inhibitor and the DOT1L HMT inhibitor.

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Combined antileukemic effects of the MENIN-MLL interaction inhibitor and...
(A) Knockdown of WT MLL in NOMO-1 cells. NOMO-1 cells transduced with shRNA for WT MLL were analyzed by WB. MLLN, the amino-terminal MLL fragment; MLLC, the carboxyl-terminal MLL fragment. (B) Effect of MI-2-2 in NOMO-1 cells. NOMO-1 cells in A were cultured in the presence of MI-2-2 (10 μM) for 1 day and analyzed by RT-qPCR. The expression level, normalized to ACTB (representative of 2 independent experiments), is shown as the value relative to that of the vector/vehicle control (set at 100%). Error bars represent the SD of PCRs performed in triplicate. **P ≤ 0.01 and #P ≤ 0.0001, by ordinary 1-way ANOVA, comparing each sample with the vector/vehicle control. (C) Combined effects of MI-2-2 and EPZ-5676 on MLL-ENL leukemia cells. Murine MLL-ENL leukemia cells were cultured in the presence of the drugs at the indicated concentrations ex vivo. CFU relative to those of the vehicle control (set at 100%) are shown with error bars (SD of >3 independent experiments). *P ≤ 0.05, **P ≤ 0.01, §P ≤ 0.001, and #P ≤ 0.0001, by ordinary 1-way ANOVA, comparing each sample with the vehicle control. Images of colonies on day 6 are shown. Scale bars: 100 μm. (D) Combined effects of MI-2-2 and EPZ-5676 on various immortalized progenitors. Murine myeloid progenitors immortalized by various transgenes were analyzed, as in C. **P ≤ 0.01 and #P ≤ 0.0001, by ordinary 1-way ANOVA, comparing each sample with the vehicle control. (E) Gene expression after exposure to MI-2-2 and/or EPZ-5676. RT-qPCR analysis was performed on MLL-ENL leukemia cells after 3 days of drug treatment. The expression level normalized to Gapdh (representative of 2 independent experiments) is shown as the value relative to that of the vehicle control (set at 100%). Error bars represent the SD of PCRs performed in triplicate. **P ≤ 0.01 and #P ≤ 0.0001, by ordinary 1-way ANOVA, comparing each sample with the vehicle control. (F) Combined effects of MI-2-2 and EPZ-5676 on the leukemia stem cell potential. MLL-ENL leukemia cells treated with the drugs for 3 days were transplanted into sublethally irradiated syngeneic mice. *P ≤ 0.05 and **P ≤ 0.01, by log-rank test for the indicated pairs.