HBO1-MLL interaction promotes AF4/ENL/P-TEFb-mediated leukemogenesis

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    Evaluation Summary:

    This manuscript describes the identification and characterization of the interaction between MLL fusion proteins with the HBO1 histone acetyltransferase complex and its role in leukemogenesis. This study adds mechanistic depth into the important recent discovery of HBO1 functions in MLL-fusion leukemias and opens possibilities for a new therapeutic approach.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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Abstract

Leukemic oncoproteins cause uncontrolled self-renewal of hematopoietic progenitors by aberrant gene activation, eventually causing leukemia. However, the molecular mechanism underlying aberrant gene activation remains elusive. Here, we showed that leukemic MLL fusion proteins associate with the HBO1 histone acetyltransferase (HAT) complex through their trithorax homology domain 2 (THD2) in various human cell lines. MLL proteins associated with the HBO1 complex through multiple contacts mediated mainly by the ING4/5 and PHF16 subunits in a chromatin-bound context where histone H3 lysine 4 tri-methylation marks were present. Of the many MLL fusions, MLL-ELL particularly depended on the THD2-mediated association with the HBO1 complex for leukemic transformation. The C-terminal portion of ELL provided a binding platform for multiple factors including AF4, EAF1, and p53. MLL-ELL activated gene expression in murine hematopoietic progenitors by loading an AF4/ENL/P-TEFb (AEP) complex onto the target promoters wherein the HBO1 complex promoted the association with AEP complex over EAF1 and p53. Moreover, the NUP98-HBO1 fusion protein exerted its oncogenic properties via interaction with MLL but not its intrinsic HAT activity. Thus, the interaction between the HBO1 complex and MLL is an important nexus in leukemic transformation, which may serve as a therapeutic target for drug development.

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  1. Author Response:

    Reviewer #1:

    The manuscript by Takahashi et al describes the interaction between MLL fusion proteins with HBO1 and its role in leukemogenesis. Myeloid progenitor transformation assays using various MLL fusion proteins reveal that MLL fusion proteins requires the TRX2 domain of MLL for effective leukemic transformation. IP-MS identifies HBO1 as a bona fide binding partner of the MLL TRX2 domain. ChIP-seq experiments show genome-wide colocalization of HBO1 complex with MLL-ENL and the WT MLL in MLL-fusion leukemia cells and MLL WT cells, respectively. ChIP-qPCR in MLL-deficient cells suggest that recruitment of HBO1 to MLL target genes (such as MYC and CDKN2C) depends on MLL. Truncation analysis of the ELL part of the MLL-ELL fusion reveal that MLL-ELL transformation activity requires OHD domain-mediated recruitment of …

  2. Evaluation Summary:

    This manuscript describes the identification and characterization of the interaction between MLL fusion proteins with the HBO1 histone acetyltransferase complex and its role in leukemogenesis. This study adds mechanistic depth into the important recent discovery of HBO1 functions in MLL-fusion leukemias and opens possibilities for a new therapeutic approach.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  3. Reviewer #1 (Public Review):

    The manuscript by Takahashi et al describes the interaction between MLL fusion proteins with HBO1 and its role in leukemogenesis. Myeloid progenitor transformation assays using various MLL fusion proteins reveal that MLL fusion proteins requires the TRX2 domain of MLL for effective leukemic transformation. IP-MS identifies HBO1 as a bona fide binding partner of the MLL TRX2 domain. ChIP-seq experiments show genome-wide colocalization of HBO1 complex with MLL-ENL and the WT MLL in MLL-fusion leukemia cells and MLL WT cells, respectively. ChIP-qPCR in MLL-deficient cells suggest that recruitment of HBO1 to MLL target genes (such as MYC and CDKN2C) depends on MLL. Truncation analysis of the ELL part of the MLL-ELL fusion reveal that MLL-ELL transformation activity requires OHD domain-mediated recruitment of AF4 …

  4. Reviewer #2 (Public Review):

    In this manuscript, the authors identified an interesting interaction of MLL (a methyltransferase) with an HBO1-JADE complex (an acetyltransferase) and investigated the functional impact in leukemogenesis by fusion proteins containing MLL or HBO1. The data is clear and the connection between MLL and HBO1 is unexpected. The manuscript is also well organized and relatively easy to follow.

    Comments:

    1. The functional relevance of the interaction between MLL and HBO1 is still correlative. It would be important to know whether there are any results directly about the impact of the loss of the HBO1 complex on the function of MLL.

    2. It is important to show the source and specificity of the antibodies that were used for ChIP of the HBO1 complex.

    3. It might be interesting to check whether other JADE proteins and also BRD1 …

  5. Reviewer #3 (Public Review):

    This paper starts with a series of bone marrow transformation assays comparing MLL fusions and domain-deletion mutants thereof to define the minimal elements for robust leukemic transformation and surveying growth and attendant common fusion targets HoxA9, Meis1 in colony replanting assays. Here they discover that a region of the MLL-N portion just upstream of the well-studied CXXC domain, termed in their previous work the "TRX2 domain" is important for the transformation capacity for several different MLL-fusions (and more minimal chimeras of key modules). A small region of the MLL-N protein encompassing the TRX2 domain and the CXXC module are subjected to complex purification, it is clear from comparison to number of controls that the TRX2 domain is an important mediator of association, perhaps indirect, …