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

  1. Satoshi Takahashi
  2. Akinori Kanai
  3. Hiroshi Okuda
  4. Ryo Miyamoto
  5. Yosuke Komata
  6. Takeshi Kawamura
  7. Hirotaka Matsui
  8. Toshiya Inaba
  9. Akifumi Takaori-Kondo
  10. Akihiko Yokoyama

  • Curated by eLife

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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. Version published to 10.7554/elife.65872 on eLife
  2. eLife

    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 AF4 and EAF1. Furthermore, co-IP and ChIP experiments with various fragments show that AF4 and EAF1 form two distinct SL1/MED26-containing complexes and likely the AEP/SL1/MED26 complex is competent for transactivation. Series of transformation assays suggest that MLL-ELL transforms hematopoietic progenitors via association with AEP, but not other ELL-associated proteins. Finally, the authors also show that NUP98-HBO1 fusion transforms myeloid progenitors through interaction with MLL. Overall, this is a quite comprehensive study demonstrating that various MLL fusions and NUP98 fusions transform hematopoietic progenitors via HBO1-MLL interaction, which suggests that targeting their interaction might be s new therapeutic approach.

    We appreciate the comments and inputs from the reviewers.

    Reviewer #2:

    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.

    We performed a sgRNA-dropout assay which showed that HBO1 is critically required for the survival of leukemia cell lines, as depicted in Figure 2F and Figure 2-figure supplement 3.

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

    The details of the antibodies are provided in Key Resource Table.

    1. It might be interesting to check whether other JADE proteins and also BRD1 (another partner of HBO1) are involved.

    We agree that it would be very interesting to examine the involvement of other JADE/BRPF family proteins in the future because they share the ING4/5 subunits and BRD1 plays an important role in hematopoiesis (1). This can be addressed in future studies.

    1. The acronym TRX2 may be confusing as some might think that it is thioredoxin.

    As advised, we have changed this to THD2 (TRX homology domain 2).

    Reviewer #3:

    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, with the HBO complex. Drop out experiments confirm that HBO1 knockout is lethal to MLL-rearranged leukemia, nicely confirming recent work (Ay et al., MacPherson et al.).

    ChIP-seq experiments in an ALL with MLL-ENL fusion, and then more extensively in a kidney cancer cell line indicate overlap with some of the HBO complex subunits and MLL, however this does not establish recruitment at these sites. ChIP-qPCR at a few MLL-fusion target genes with MLL depletion supports the recruitment hypothesis somewhat although mixed and modest effect sizes indicate that alternate pathways for HBO1 recruitment are involved, and could also be explained as reduced deposition of marks known to recruit HBO1, rather than direct recruitment. Sadly, the real potential strength of this work goes unrealized, as the recruitment of HBO1 mechanism remains tantalizingly out of reach. More experiments in this space could conclusively establish the molecular mechanism of a seemingly biomedically important recruitment paradigm, and thereby have much more impact.

    As the reviewer pointed out, MLL is not the only element that recruits the HBO1 complex to the target chromatin. MLL is known to deposit H3K4me marks, and the HBO1 complex is known to recognize these marks via ING4/5 subunits. We performed a ChIP-qPCR analysis of H3K4me3 in MLL-knockout cells. At the MYC promoter, the H3K4me3 marks were substantially decreased (Figure 3F). Moreover, recruitment of HBO1 was not recovered by transient expression of an MLL mutant containing THD2, indicating that the presence of H3K4me3 marks is a prerequisite for HBO1 recruitment. In accordance with this, ING5-histone interaction is required for the stable association of MLL with the HBO1 complex (Figure 8A-C). Thus, a more appropriate molecular mechanism would be the cooperative recruitment of the HBO1 complex by ING4/5-mediated chromatin association and MLL-mediated association. Because of the multiple contacts involved in this molecular network, it is not easy to pinpoint the direct contacts as desired, but our biochemical analyses indicate that PHF16 and ING4/5 offer relatively strong binding surfaces (Figure 8A-C). The ING domain of ING5 is the most likely direct binding surface identified thus far.

    At this point the paper shifts to a seemingly distinct line of inquiry, which is not closely related to the HBO1-TRX2 story to the first three figures. The new direction examines the ELL fusion partner in some detail using similar fusion protein chimeras, but a portion of Figure 4, is largely confirmatory of previously established findings about the critical regions of ELL for transformation and its AF4/EAF1 partners, adding only that portions of the MLL fusion protein are dispensable, provided that they are replaced with the PWWP of LEDGF. It is a little bit of a Frankenstein's monster experiment, and does not add much new to the field. Further experiments define potentially two distinct complexes that have already been characterized being recruited by ELL, although there is overlap here again with their previous studies, and the results are a little hard to interpret.

    A portion of Figure 4 was confirmatory to previous results. We have moved this to figure supplements in the revised manuscript (Figure 4-figure supplement 1B,C). The main topic of this paper is the role of the HBO1 complex in MLL-mediated transactivation pathways. The structure/function analysis of MLL fusion proteins demonstrated that MLL-ELL is highly dependent on the HBO1-mediated function in leukemic transformation (Figures 1 and 2). Hence, it was important to clarify the mechanism of gene activation by MLL-ELL in this study to understand why HBO1 association is required for MLL-ELL-mediated transformation. Because MLL-ELL associates with AEP similarly to major MLL fusions such as MLL-AF4 and MLL-ENL, it was speculated that MLL-ELL also activates its target genes via AEP. However, ELL associates with EAF family proteins and MLL-EAF also has transforming ability (3). Thus, EAF1-mediated functions could be more important for MLL-ELL-mediated transformation rather than AEP-mediated functions. To clarify the mechanism of MLL-ELL-mediated transformation, we generated a point mutant that selectively impaired ELL-EAF interaction and demonstrated that EAF1-association is dispensable for MLL-ELL-mediated transformation (Figure 6), thereby indicating that MLL-ELL transforms via AEP-mediated functions, which demands HBO1-mediated functions. We also showed that the presence of THD2 enhances ELL-AEP association to further suggest that one of the roles of the HBO1 complex is to enhance the association of ELL with AEP (Figure 6E). These findings are not reinterpretations of our prior results and are relevant to the main topic of this paper. We believe this part adds new information to the field, and therefore we have included it in the revised manuscript.

    The authors create structure-guided separation of function mutants in the ELL domain that binds both AEP and SL1, permitting them to specifically disrupt EAF1 interactions but not AF4. Further experiments solidify this interpretation, and find that this mutant shows no deficits in hematopoietic progenitor transformation or primary leukemia lethality, although there appears to be some effect upon reimplantation.

    The last figure in the paper tackles the seemingly unrelated Nup98-HBO1 fusion, a rare patient mutation-they demonstrate a requirement for MLL for viability of hematopoietic progenitors transformed by this fusion, connecting back to the TRX2 interaction, and show that menin inhibitors slow growth.

    Strengths:

    The identification of the TRX2 region of the MLL-N protein as the major point of contact (perhaps not direct), to the HBO1 complex adds mechanistic depth to the really important recent discovery (confirmed in this work) the MLL-fusion leukemias rely on HBO1 function. This lab has published a number of technically similar types of papers defining minimal regions of MLL and distinct interacting partners by chimeric fusions, with bone marrow transformation assays, mouse model engrafting studies, IP's, ChIP etc. In my view they are very much under cited, likely because they are similarly so challenging to read.

    Thank you for your pointed feedback. We will try our best to make the necessary improvements so that our papers are widely read and cited.

    The mixture of Co-IP biochemistry, bone marrow transformation assays, and ChIP, to define interactions, minimal requirements for transformation, and their chromatin consequences for a host of different MLL-fusions and HBO1-fusions has the potential to define the key interfaces underlying recruitment.

    Weaknesses:

    The mechanistic inquiry stops short of really defining the critical MLL-HBO1 complex interface. Defining the point of contact on the HBO1 side (even which subunit) and determining whether it is direct, or bridged by some, as yet unidentified factor, as well as conclusively demonstrating that this is the mechanism of HBO1 recruitment remain the major shortcomings.

    To address this criticism, we further investigated the mechanism of complex formation by MLL and the HBO1 complex. As we demonstrated in Figure 8A-C, the association appears to be mediated by multiple contacts mainly through PHF16 and ING4/5. Because this association needs an intact PHD finger of ING5, it likely occurs depending on the context where ING4/5 is bound to histone H3K4me2/3. The ING domain of ING5 was also required for the association, indicating that this portion may contains a point of direct contact. We speculate that HBO1 recruitment is mediated primarily by ING4/5-H3K4me3 interaction and MLL reinforces its chromatin association.

    And the follow-on figures apart from the last one, appear disconnected from this portion of the story and distract from it.

    We depicted a revised model incorporating the above-mentioned aspect in Figure 8D of the revised manuscript.

    The complex nomenclature and density/organization/logic of the presentation of experiments makes this paper difficult to read. Absence of sufficient grounding in the broader literature much beyond their own lab's work further compounds the problem.

    We changed some of the nomenclature and density/organization/logic of the presentation of the experiments to improve the readability.

    There is a lot of overlap, particularly in parts of figure 1 and figure 4 with previously published results. So perhaps re-organizing the display of data, and the organization of presentation, putting confirmatory work in the supplementary figures, would improve accessibility.

    We moved some portions of Figure 4 to figure supplement. The data for MLL-AF10 and MLL-ENL were retained in the Figure 1 as important references.

  3. eLife

    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.)

  4. eLife

    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 and EAF1. Furthermore, co-IP and ChIP experiments with various fragments show that AF4 and EAF1 form two distinct SL1/MED26-containing complexes and likely the AEP/SL1/MED26 complex is competent for transactivation. Series of transformation assays suggest that MLL-ELL transforms hematopoietic progenitors via association with AEP, but not other ELL-associated proteins. Finally, the authors also show that NUP98-HBO1 fusion transforms myeloid progenitors through interaction with MLL. Overall, this is a quite comprehensive study demonstrating that various MLL fusions and NUP98 fusions transform hematopoietic progenitors via HBO1-MLL interaction, which suggests that targeting their interaction might be s new therapeutic approach.

  5. eLife

    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 (another partner of HBO1) are involved.

    4. The acronym TRX2 may be confusing as some might think that it is thioredoxin.

  6. eLife

    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, with the HBO complex. Drop out experiments confirm that HBO1 knockout is lethal to MLL-rearranged leukemia, nicely confirming recent work (Ay et al., MacPherson et al.).

    ChIP-seq experiments in an ALL with MLL-ENL fusion, and then more extensively in a kidney cancer cell line indicate overlap with some of the HBO complex subunits and MLL, however this does not establish recruitment at these sites. ChIP-qPCR at a few MLL-fusion target genes with MLL depletion supports the recruitment hypothesis somewhat although mixed and modest effect sizes indicate that alternate pathways for HBO1 recruitment are involved, and could also be explained as reduced deposition of marks known to recruit HBO1, rather than direct recruitment. Sadly, the real potential strength of this work goes unrealized, as the recruitment of HBO1 mechanism remains tantalizingly out of reach. More experiments in this space could conclusively establish the molecular mechanism of a seemingly biomedically important recruitment paradigm, and thereby have much more impact.

    At this point the paper shifts to a seemingly distinct line of inquiry, which is not closely related to the HBO1-TRX2 story to the first three figures. The new direction examines the ELL fusion partner in some detail using similar fusion protein chimeras, but a portion of Figure 4, is largely confirmatory of previously established findings about the critical regions of ELL for transformation and its AF4/EAF1 partners, adding only that portions of the MLL fusion protein are dispensable, provided that they are replaced with the PWWP of LEDGF. It is a little bit of a Frankenstein's monster experiment, and does not add much new to the field. Further experiments define potentially two distinct complexes that have already been characterized being recruited by ELL, although there is overlap here again with their previous studies, and the results are a little hard to interpret.

    The authors create structure-guided separation of function mutants in the ELL domain that binds both AEP and SL1, permitting them to specifically disrupt EAF1 interactions but not AF4. Further experiments solidify this interpretation, and find that this mutant shows no deficits in hematopoietic progenitor transformation or primary leukemia lethality, although there appears to be some effect upon reimplantation.

    The last figure in the paper tackles the seemingly unrelated Nup98-HBO1 fusion, a rare patient mutation-they demonstrate a requirement for MLL for viability of hematopoietic progenitors transformed by this fusion, connecting back to the TRX2 interaction, and show that menin inhibitors slow growth.

    Strengths:

    The identification of the TRX2 region of the MLL-N protein as the major point of contact (perhaps not direct), to the HBO1 complex adds mechanistic depth to the really important recent discovery (confirmed in this work) the MLL-fusion leukemias rely on HBO1 function. This lab has published a number of technically similar types of papers defining minimal regions of MLL and distinct interacting partners by chimeric fusions, with bone marrow transformation assays, mouse model engrafting studies, IP's, ChIP etc. In my view they are very much under cited, likely because they are similarly so challenging to read.

    The mixture of Co-IP biochemistry, bone marrow transformation assays, and ChIP, to define interactions, minimal requirements for transformation, and their chromatin consequences for a host of different MLL-fusions and HBO1-fusions has the potential to define the key interfaces underlying recruitment.

    Weaknesses:

    The mechanistic inquiry stops short of really defining the critical MLL-HBO1 complex interface. Defining the point of contact on the HBO1 side (even which subunit) and determining whether it is direct, or bridged by some, as yet unidentified factor, as well as conclusively demonstrating that this is the mechanism of HBO1 recruitment remain the major shortcomings.

    And the follow-on figures apart from the last one, appear disconnected from this portion of the story and distract from it.

    The complex nomenclature and density/organization/logic of the presentation of experiments makes this paper difficult to read. Absence of sufficient grounding in the broader literature much beyond their own lab's work further compounds the problem.

    There is a lot of overlap, particularly in parts of figure 1 and figure 4 with previously published results. So perhaps re-organizing the display of data, and the organization of presentation, putting confirmatory work in the supplementary figures, would improve accessibility.

    Impact:

    In its present state, this is an incremental, but important advance for the field. With more mechanistic depth, particularly on the HBO1-MLL interface would substantially increase general interest.

  7. Version published to 10.1101/2021.01.08.425834v1 on bioRxiv