Functional interrogation of HOXA9 regulome in MLLr leukemia via reporter-based CRISPR/Cas9 screen

  1. Hao Zhang
  2. Yang Zhang
  3. Xinyue Zhou
  4. Shaela Wright
  5. Judith Hyle
  6. Lianzhong Zhao
  7. Jie An
  8. Xujie Zhao
  9. Ying Shao
  10. Beisi Xu
  11. Hyeong-Min Lee
  12. Taosheng Chen
  13. Yang Zhou
  14. Xiang Chen
  15. Rui Lu  Is a corresponding author
  16. Chunliang Li  Is a corresponding author
  1. University of Alabama at Birmingham, United States
  2. St Jude Children's Research Hospital, United States
  3. St Jude Children's Research Hospital, United States [US]

Abstract

Aberrant HOXA9 expression is a hallmark of most aggressive acute leukemias, notably those with KMT2A (MLL) gene rearrangements. HOXA9 overexpression not only predicts poor diagnosis and outcome but also plays a critical role in leukemia transformation and maintenance. However, our current understanding of HOXA9 regulation in leukemia is limited, hindering development of therapeutic strategies. Here, we generated the HOXA9-mCherry knock-in reporter cell lines to dissect HOXA9 regulation. By utilizing the reporter and CRISPR/Cas9 screens, we identified transcription factors controlling HOXA9 expression, including a novel regulator, USF2, whose depletion significantly down-regulated HOXA9 expression and impaired MLLr leukemia cell proliferation. Ectopic expression of Hoxa9 rescued impaired leukemia cell proliferation upon USF2 loss. Cut&Run analysis revealed the direct occupancy of USF2 at HOXA9 promoter in MLLr leukemia cells. Collectively, the HOXA9 reporter facilitated the functional interrogation of the HOXA9 regulome and has advanced our understanding of the molecular regulation network in HOXA9-driven leukemia.

Data availability

All plasmids created in this study will be deposited to Addgene. Raw data collected from Cut&Run were deposited at NCBI GEO (GSE140664). Raw data collected from CRISPR screening were included in Supplementary File 2. Publicly available dataset used in this study were cited accordingly including Figures 1E and S5D: GSE120781; Figure 1-supplement 1A-C: GSE13159; Figure 3-supplement 2C: GSE126619, GSE74812, GSE89485; Figure 3-supplement 3A: ENCODE (HCT116); Figure 5-supplement 3A-C: European Genome-phenome Archive (EGA) under accession number EGAS00001003266, EGAS00001000654, EGAS00001001952, EGAS00001001923, EGAS00001002217 and EGAS00001000447.

The following previously published data sets were used

Article and author information

Author details

  1. Hao Zhang

    Department of Medicine - Hematology & Oncology, University of Alabama at Birmingham, Birmingham, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Yang Zhang

    Department of Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1712-7211
  3. Xinyue Zhou

    Department of Medicine - Hematology & Oncology, University of Alabama at Birmingham, Birmingham, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Shaela Wright

    Department of Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Judith Hyle

    Department of Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Lianzhong Zhao

    Department of Medicine - Hematology & Oncology, University of Alabama at Birmingham, Birmingham, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Jie An

    Department of Medicine - Hematology & Oncology, University of Alabama at Birmingham, Birmingham, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Xujie Zhao

    Department of Pharmaceutical Sciences, St Jude Children's Research Hospital, Memphis, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Ying Shao

    Department of Computational Biology, St Jude Children's Research Hospital, Memphis, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Beisi Xu

    Center for Applied Bioinformatics (CAB), St Jude Children's Research Hospital, Memphis, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0099-858X
  11. Hyeong-Min Lee

    Department of Chemical Biology and Therapeutics, St Jude Children's Research Hospital, Memphis, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9381-3611
  12. Taosheng Chen

    St Jude Children's Research Hospital, Memphis, United States [US]
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6420-3809
  13. Yang Zhou

    School of Engineering, University of Alabama at Birmingham, Birmingham, United States
    Competing interests
    The authors declare that no competing interests exist.
  14. Xiang Chen

    Department of Computational Biology, St Jude Children's Research Hospital, Memphis, United States
    Competing interests
    The authors declare that no competing interests exist.
  15. Rui Lu

    Department of Medicine - Hematology & Oncology, University of Alabama at Birmingham, Birmingham, United States
    For correspondence
    ruilu1@uabmc.edu
    Competing interests
    The authors declare that no competing interests exist.
  16. Chunliang Li

    Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, United States
    For correspondence
    chunliang.li@stjude.org
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5938-5510

Funding

Leukemia Research Foundation

  • Rui Lu

American Cancer Society (IRG15-59-IRG)

  • Rui Lu

National Cancer Insititute (P30CA021765-37)

  • Chunliang Li

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Xiaobing Shi, Van Andel Institute, United States

Publication history

  1. Received: April 15, 2020
  2. Accepted: September 30, 2020
  3. Accepted Manuscript published: October 1, 2020 (version 1)
  4. Version of Record published: October 30, 2020 (version 2)

Copyright

© 2020, Zhang et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Hao Zhang
  2. Yang Zhang
  3. Xinyue Zhou
  4. Shaela Wright
  5. Judith Hyle
  6. Lianzhong Zhao
  7. Jie An
  8. Xujie Zhao
  9. Ying Shao
  10. Beisi Xu
  11. Hyeong-Min Lee
  12. Taosheng Chen
  13. Yang Zhou
  14. Xiang Chen
  15. Rui Lu
  16. Chunliang Li
(2020)
Functional interrogation of HOXA9 regulome in MLLr leukemia via reporter-based CRISPR/Cas9 screen
eLife 9:e57858.
https://doi.org/10.7554/eLife.57858

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