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Native Stem Cell Transcriptional Circuits Define Cardinal Features of High-Risk Early T-Cell Precursor Acute Lymphoblastic Leukemia
Mark Y. Chiang1, Qing Wang1, Francesco Boccalatte2-4, Giovanni Gambi2, Bettina Nadorp2,5, Jason Xu6, Fatema Akter1, Carea Mullin1, Ashley F. Melnick7, Elizabeth Choe8, Anna C. McCarter9, Nicole Dean10, Siyi Chen1, Karena Lin7, Rohan Kodgule11, Javier Rodriguez-Hernaez2, Aristotelis Tsirigos2, Sonali Narang2, Kleopatra Avrampou2, Bryan King2, Russell J.H. Ryan11, David T. Teachey12, Kai Tan12, and Iannis Aifantis2,41Division of Hematology-Oncology, Department of Internal Medicine, University of Michigan School of Medicine, Ann Arbor, MI, USA
2Department of Pathology, NYU Grossman School of Medicine, New York, NY, USA
3Department of Women’s and Children’s Health, University of Padua, Italy
4Perlmutter Cancer Center, NYU Langone Health, New York, NY, USA
5Division of Precision Medicine, Department of Medicine, New York University Grossman School of Medicine, New York, NY, USA
6Graduate Group in Genomics and Computational Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
7Cellular and Molecular Biology Program, University of Michigan School of Medicine, Ann Arbor, MI, USA
8Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA
9Department of Anesthesiology, Perioperative and Pain Medicine, Stanford University, Stanford, CA, USA
10Cancer Biology Program, University of Michigan School of Medicine, Ann Arbor, MI, USA
11Department of Pathology, University of Michigan, Ann Arbor, MI, USA
12Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA, USA
Several years ago, early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) was identified as a new clinically aggressive blood cancer based on similarities to mouse ETP cells, the most primitive stem cells in the thymus (Coustan-Smith, Lancet Oncology, 2009). Since then, extensive investigations revealed that these tumors possess a diverse mutational landscape. However, establishing a common framework that activates the stem cell gene expression characteristic of these tumors remains elusive. Given this, we raised the question – Is there a co-opted stem cell transcriptional network that drives the acquisition of clinically aggressive behavior and other cardinal features of ETP-ALL? This question is important as we recently reported that the “bone marrow progenitor-like” (BMP-like) gene expression program identifies a high-risk ETP-ALL subgroup based on treatment resistance and BCL2 inhibitor sensitivity in the landmark Children’s Oncology Group AALL0434 clinical trial.
Previously, we showed that the PIAS-like coactivator Zmiz1 is a direct Notch1 cofactor in committed T-cell precursors and mature T-ALL (Pinnell, Immunity, 2015). However, in the ETP context, ZMIZ1 switches to a Notch antagonistic role to maintain ETPs in an undifferentiated state and is overexpressed in ETP-ALL. These observations led us to consider the possibility that ZMIZ1 induces an essential transcriptional network in ETP-ALL that is distinct from conventional T-ALL networks. To test this possibility, we knocked down Zmiz1 in normal and malignant ETPs. Zmiz1 deletion in normal ETPs impaired cell proliferation, reduced myeloid potential, and increased Notch-induced T-cell commitment. Conversely, overexpression of ZMIZ1 had the opposite effect, promoting ETP cell proliferation and inhibiting T-cell commitment. ZMIZ1 knockdown in BMP-like cell lines and patient-derived xenografts and a genetically engineered ETP-ALL mouse model reduced cell proliferation by 4-100-fold and extended median survival by >200% respectively. These data show that Zmiz1 is essential for maintenance of the undifferentiated ETP state.
To determine mechanism, we performed RNA-seq of high-risk BMP-like cells after ZMIZ1 knockdown. Among the top-25 regulated genes were characteristic oncogenes of the BMP-like subgroup -- BCL2, MYCN, MYB, and MEF2C. Zmiz1 deletion or overexpression in normal ETPs showed repression or induction of these genes respectively, suggesting native circuits. Pathway analysis showed that MYC was the top enriched gene set. To investigate the role of MYCN, we overexpressed MYCN or deleted Mycn in normal ETPs using conditional knockout mice. Like ZMIZ1 overexpression, MYCN overexpression induced robust ETP proliferation, antagonized Notch, increased myeloid fate specification, and rescued Zmiz1-deficient ETP cells. Like Zmiz1 deletion, Mycn deletion impaired ETP proliferation while promoting Notch-induced T-cell commitment. Next, we integrated our ZMIZ1 ChIP-seq and ATAC-seq datasets in BMP-like cells with 3D genome maps followed by validation with CRISPR interference assays. These studies revealed essential ZMIZ1-bound stem cell regulatory elements that induce expression of BMP-like oncogenes. ZMIZ1-bound elements were highly enriched for co-occupancy by MYB (~75%) and MEF2C (~30%), suggesting feedforward circuits. Consistently, RNA-seq showed that MYB regulates ~70% of ZMIZ1 target genes, of which 95% were strikingly in cooperative direction including all BMP-like oncogenes. One of these elements is a MYCN enhancer, which we named “N-Myc Regulating Enhancer” (NMRE). The NMRE showed strong MYCN interactivity, lncRNA expression, and chromatin accessibility in ETP and ETP-ALL cells but not in mature T cells or T-ALL. Importantly, NMRE-deficient mice showed no defect in hematopoiesis while repression of NMRE impaired proliferation of BMP-like cells by 3-5-fold. These data identify co-opted native feedforward circuits involving teams of transcription factors and enhancers that are essential for high-risk ETP-ALL but not for stem cells, other blood cells, or conventional T-ALL.
Regarding significance, our study illustrates how stem cell gene expression programs are co-opted by a primitive cancer (ETP-ALL) but not a related, slightly more differentiated cancer (conventional T-ALL) to acquire high-risk features. Thus, the current strategy of treating ETP-ALL with the same chemotherapy as conventional T-ALL seems outmoded. To improve clinical outcomes, we propose blocking the ZMIZ1 stem cell network as it can mechanistically explain several cardinal features of high-risk ETP-ALL – BCL2 inhibitor sensitivity, Notch antagonism, myeloid gene expression, rare genetic alterations in MYCN or MYB, and aggressive clinical behavior through the synergistic combination of MYC+BCL2. Since Zmiz1 is dispensable for postnatal health, targeting it might safely disable the stem cell gene expression program that is responsible for high-risk features of ETP-ALL.