Elucidating the Importance of DOT1L Recruitment in MLL-AF9 Leukemia and Hematopoiesis.
Elucidating the Importance of DOT1L Recruitment in MLL-AF9 Leukemia and Hematopoiesis.
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阐明DOT 1 L募集在MLL-AF 9白血病和造血中的重要性。
DOI：
10.3390/cancers13040642
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发表时间：
2021-02-05
期刊：
影响因子：
5.2
通讯作者：
Nikolovska-Coleska Z
中科院分区：
文献类型：
--
作者：
Grigsby SM;Friedman A;Chase J;Waas B;Ropa J;Serio J;Shen C;Muntean AG;Maillard I;Nikolovska-Coleska Z
由MLL融合蛋白驱动的MLL重排白血病是一种侵袭性、治疗耐药的白血病，见于>60%的婴儿白血病和约10%的成人白血病。研究表明，抑制DOT 1 L酶活性可阻断白血病发生。然而，DOT 1 L对各种正常细胞功能至关重要，包括造血。本研究旨在表明，靶向MLL-AF 9融合体和DOT 1 L之间的相互作用将抑制白血病发生，同时保留非白血病造血。我们发现，破坏AF 9-DOT 1 L的相互作用与一个单一的点突变足以削弱白血病的发生。我们还证明，导致非白血病细胞中DOT 1 L酶活性丧失的遗传干预在7-10天内迅速耗尽造血干细胞和祖细胞;然而，当AF 9-DOT 1 L相互作用被破坏时，造血得以保留，使酶功能保持完整。这些研究是概念验证，证明了抑制AF 9-DOT 1 L相互作用和破坏MLL-融合复合物的完整性的潜在治疗优势。MLL 1（KMT 2a）基因重排是侵袭性MLL-driven急性白血病发病机制的基础AF 9是最常见的MLL融合伴侣之一，将组蛋白H3 K79甲基转移酶DOT 1 L募集到MLL靶基因，组成性激活促白血病靶点的转录。DOT 1 L已成为MLL驱动白血病患者的治疗靶点。然而，整体DOT 1 L酶抑制可能导致非白血病细胞中的脱靶毒性，这可能降低DOT 1 L抑制剂的治疗指数。为了绕过这个问题，我们开发了一种新的方法，靶向特定的蛋白质-蛋白质相互作用（PPIs）介导DOT 1 L募集到MLL靶基因，并比较了酶和PPIs抑制对白血病和非白血病造血的影响。MLL-AF 9细胞系经工程改造以携带具有缺陷性AF 9相互作用位点或缺乏酶活性的突变DOT 1 L构建体。在表达DOT 1 L突变体与缺陷AF 9结合的细胞系中，我们观察到DOT 1 L募集到关键靶基因的完全破坏和白血病细胞生长的抑制。为了评估DOT 1 L缺失对非白血病造血的总体影响，我们首先评估了成年小鼠骨髓中急性Dot 1 L失活的影响。我们观察到骨髓祖细胞数量在7天内迅速减少，随后是长期造血干细胞的损失。此外，WT和PPI缺陷的DOT 1 L突变体，但不是一个酶失活的DOT 1 L突变体能够拯救持续的造血。这些数据表明，AF 9-DOT 1 L相互作用在非白血病造血中是无效的。我们的研究结果支持靶向MLL-AF 9-DOT 1 L相互作用作为一种有前途的治疗策略，对MLL驱动的白血病细胞具有选择性毒性。
MLL-rearranged leukemia, driven by MLL-fusion proteins, is an aggressive, therapy-resistant leukemia found in >60% of infant leukemia and ~10% of adult leukemia. Studies have shown that inhibiting DOT1L enzymatic activity blocks leukemogenesis. However, DOT1L is critical for various normal cellular functions, including hematopoiesis. This study aimed to show that targeting the interaction between the MLL-AF9 fusion and DOT1L would inhibit leukemogenesis while sparing non-leukemic hematopoiesis. We found that disrupting the AF9-DOT1L interaction with a single point mutation was sufficient to impair leukemogenesis. We also demonstrate that genetic interventions that result in loss of DOT1L enzymatic activity in non-leukemic cells rapidly depletes hematopoietic stem and progenitor cells within 7–10 days; however, hematopoiesis was preserved when the AF9-DOT1L interaction was disrupted, leaving the enzymatic function intact. These studies are a proof of concept demonstrating the potential therapeutic advantage of inhibiting the AF9-DOT1L interaction and disrupting the integrity of the MLL-fusion complex. MLL1 (KMT2a) gene rearrangements underlie the pathogenesis of aggressive MLL-driven acute leukemia. AF9, one of the most common MLL-fusion partners, recruits the histone H3K79 methyltransferase DOT1L to MLL target genes, constitutively activating transcription of pro-leukemic targets. DOT1L has emerged as a therapeutic target in patients with MLL-driven leukemia. However, global DOT1L enzymatic inhibition may lead to off-target toxicities in non-leukemic cells that could decrease the therapeutic index of DOT1L inhibitors. To bypass this problem, we developed a novel approach targeting specific protein-protein interactions (PPIs) that mediate DOT1L recruitment to MLL target genes, and compared the effects of enzymatic and PPIs inhibition on leukemic and non-leukemic hematopoiesis. MLL-AF9 cell lines were engineered to carry mutant DOT1L constructs with a defective AF9 interaction site or lacking enzymatic activity. In cell lines expressing a DOT1L mutant with defective AF9 binding, we observed complete disruption of DOT1L recruitment to critical target genes and inhibition of leukemic cell growth. To evaluate the overall impact of DOT1L loss in non-leukemic hematopoiesis, we first assessed the impact of acute Dot1l inactivation in adult mouse bone marrow. We observed a rapid reduction in myeloid progenitor cell numbers within 7 days, followed by a loss of long-term hematopoietic stem cells. Furthermore, WT and PPI-deficient DOT1L mutants but not an enzymatically inactive DOT1L mutant were able to rescue sustained hematopoiesis. These data show that the AF9-DOT1L interaction is dispensable in non-leukemic hematopoiesis. Our findings support targeting of the MLL-AF9–DOT1L interaction as a promising therapeutic strategy that is selectively toxic to MLL-driven leukemic cells.
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影响因子：
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