Session: 603. Lymphoid Oncogenesis: Basic: Mechanisms in Leukemogenesis
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Lymphoid Leukemias, ALL, hematopoiesis, Diseases, immunology, Lymphoid Malignancies, Biological Processes, molecular biology, pathogenesis
To determine mechanism, we performed RNA-Seq of ETP-ALL cells after ZMIZ1 knockdown. Among the top-25 regulated genes were BCL2, MYCN, MYB, and MEF2C, which are known ETP-ALL drivers. Out of deference to the ETP literature, we refer to these genes as “Phase I oncogenes”. Zmiz1 deletion or overexpression in normal ETPs showed repression or induction of these genes respectively. Pathway analysis showed that MYC was the top enriched gene set. To investigate the role of MYCN in ETP-ALL, we overexpressed MYCN or deleted Mycn in normal ETPs using conditional knockout mice. Like ZMIZ1 overexpression, MYCN overexpression induced robust ETP proliferation, skewed differentiation to the myeloid fate, and rescued Zmiz1-deficient ETP cells. Like Zmiz1 deletion, Mycn deletion impaired ETP proliferation while promoting differentiation. Next, we integrated our ZMIZ1 ChIP-Seq and ATAC-Seq datasets with 3D genome maps followed by validation with CRISPR interference and reporter assays. These studies identified essential ZMIZ1-bound developmental regulatory elements that induce expression of Phase I oncogenes. These 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 Phase I oncogenes. Motif analysis showed that ETS motifs were the most significantly enriched at ZMIZ1-bound sites. Accordingly, ETS motif mutation impaired ZMIZ1 occupancy. Next, we sought to understand what triggers the ZMIZ1-centered circuit. MEF2C is a major initiator of ETP-ALL transformation downstream of multiple chromosomal alterations. Consistently, MEF2C occupied the ZMIZ1 enhancer. Further, MEF2C knockdown or inactivation with MARK inhibitors (which dephosphorylate MEF2C) impaired ZMIZ1 expression and cell proliferation. These data identify native ZMIZ1-dependent feedforward circuits involving teams of transcription factors that are hijacked to drive ETP oncogenesis.
There is no established framework that mechanistically explains how stem cell gene expression is activated by disparate mutational landscapes across ETP-ALL patients. Here we contribute by identifying a ZMIZ1-centered network of enhancers and transcription factors acting in feedforward circuits that are distinct from those in conventional T-ALL and are amplified from roots in normal ETPs. This network helps mechanistically explain why malignant ETPs show sensitivity to BCL2 inhibitors like venetoclax and rarely show genetic alterations of oncogenes like MYCN or MYB. ZMIZ1 induction of MYCN and BCL2 in combination might also explain the aggressive clinical behavior of ETP-ALL. Importantly, Zmiz1 is dispensable for postnatal health. Thus, the ZMIZ1-centered ETP-ALL network might guide development of safe therapies against this aggressive cancer.
Disclosures: Ott: Effector Therapeutics: Research Funding; Gilead: Research Funding; Scorpion Therapeutics: Research Funding.
See more of: Oral and Poster Abstracts