Functional interrogation of HOXA9 regulome in MLLr leukemia via reporter-based CRISPR/Cas9 screen
- University of Alabama at Birmingham, United States
- St Jude Children's Research Hospital, United States
- 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.
-
Acute deletion of CTCF disrupted enhancer-promoter regulation of MYC in human cancer cellsNCBI Gene Expression Omnibus, GSE120781.
-
Microarray Innovations in LEukemia (MILE) study: Stage 1 dataNCBI Gene Expression Omnibus, GSE13159.
-
Acute deletion of CTCF disrupted enhancer-promoter regulation of MYC in human cancer cellsNCBI Gene Expression Omnibus, GSE126619.
-
Therapeutic targeting MLL degradation pathways in MLL-rearranged leukemiaNCBI Gene Expression Omnibus, GSE89485.
-
PAX5-driven Subtypes of B-cell Acute Lymphoblastic LeukemiaEuropean Genome-phenome Archive, EGAS00001003266.
-
PAX5-driven Subtypes of B-cell Acute Lymphoblastic LeukemiaEuropean Genome-phenome Archive, EGAS00001000654.
-
PAX5-driven Subtypes of B-cell Acute Lymphoblastic LeukemiaEuropean Genome-phenome Archive, EGAS00001001952.
-
PAX5-driven Subtypes of B-cell Acute Lymphoblastic LeukemiaEuropean Genome-phenome Archive, EGAS00001001923.
-
PAX5-driven Subtypes of B-cell Acute Lymphoblastic LeukemiaEuropean Genome-phenome Archive, EGAS00001002217.
-
PAX5-driven Subtypes of B-cell Acute Lymphoblastic LeukemiaEuropean Genome-phenome Archive, EGAS00001000447.
Article and author information
Author details
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.
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.
Metrics
-
- 5,036
- views
-
- 703
- downloads
-
- 29
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Functional interrogation of HOXA9 regulome in MLLr leukemia via reporter-based CRISPR/Cas9 screen
eLife 9:e57858.
https://doi.org/10.7554/eLife.57858
Further reading
-
- Cancer Biology
- Computational and Systems Biology
Effects from aging in single cells are heterogenous, whereas at the organ- and tissue-levels aging phenotypes tend to appear as stereotypical changes. The mammary epithelium is a bilayer of two major phenotypically and functionally distinct cell lineages: luminal epithelial and myoepithelial cells. Mammary luminal epithelia exhibit substantial stereotypical changes with age that merit attention because these cells are the putative cells-of-origin for breast cancers. We hypothesize that effects from aging that impinge upon maintenance of lineage fidelity increase susceptibility to cancer initiation. We generated and analyzed transcriptomes from primary luminal epithelial and myoepithelial cells from younger <30 (y)ears old and older >55 y women. In addition to age-dependent directional changes in gene expression, we observed increased transcriptional variance with age that contributed to genome-wide loss of lineage fidelity. Age-dependent variant responses were common to both lineages, whereas directional changes were almost exclusively detected in luminal epithelia and involved altered regulation of chromatin and genome organizers such as SATB1. Epithelial expression variance of gap junction protein GJB6 increased with age, and modulation of GJB6 expression in heterochronous co-cultures revealed that it provided a communication conduit from myoepithelial cells that drove directional change in luminal cells. Age-dependent luminal transcriptomes comprised a prominent signal that could be detected in bulk tissue during aging and transition into cancers. A machine learning classifier based on luminal-specific aging distinguished normal from cancer tissue and was highly predictive of breast cancer subtype. We speculate that luminal epithelia are the ultimate site of integration of the variant responses to aging in their surrounding tissue, and that their emergent phenotype both endows cells with the ability to become cancer-cells-of-origin and represents a biosensor that presages cancer susceptibility.
-
- Cancer Biology
Chemotherapy is widely used to treat lung adenocarcinoma (LUAD) patients comprehensively. Considering the limitations of chemotherapy due to drug resistance and other issues, it is crucial to explore the impact of chemotherapy and immunotherapy on these aspects. In this study, tumor samples from nine LUAD patients, of which four only received surgery and five received neoadjuvant chemotherapy, were subjected to scRNA-seq analysis. In vitro and in vivo assays, including flow cytometry, immunofluorescence, Seahorse assay, and tumor xenograft models, were carried out to validate our findings. A total of 83,622 cells were enrolled for subsequent analyses. The composition of cell types exhibited high heterogeneity across different groups. Functional enrichment analysis revealed that chemotherapy drove significant metabolic reprogramming in tumor cells and macrophages. We identified two subtypes of macrophages: Anti-mac cells (CD45+CD11b+CD86+) and Pro-mac cells (CD45+CD11b+ARG +) and sorted them by flow cytometry. The proportion of Pro-mac cells in LUAD tissues increased significantly after neoadjuvant chemotherapy. Pro-mac cells promote tumor growth and angiogenesis and also suppress tumor immunity. Moreover, by analyzing the remodeling of T and B cells induced by neoadjuvant therapy, we noted that chemotherapy ignited a relatively more robust immune cytotoxic response toward tumor cells. Our study demonstrates that chemotherapy induces metabolic reprogramming within the tumor microenvironment of LUAD, particularly affecting the function and composition of immune cells such as macrophages and T cells. We believe our findings will offer insight into the mechanisms of drug resistance and provide novel therapeutic targets for LUAD in the future.
