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Most patients with acute promyelocytic leukemia (APL) can be cured with combined all-trans retinoic acid (ATRA) and arsenic trioxide therapy, which induces the destruction of PML-RARA, the initiating fusion protein for this disease. However, the underlying mechanisms by which PML-RARAinitiates and maintains APL cells are still not clear. Therefore, we identified genes that are dysregulated by PML-RARAin mouse and human APL cells and prioritized GATA2for functional studies because it is highly expressed in preleukemic cells expressing PML-RARA, its high expression persists in transformed APL cells, and spontaneous somatic mutations of GATA2occur during APL progression in mice and humans. These and other findings suggested that GATA2may be upregulated to thwart the proliferative signal generated by PML-RARAand that its inactivation by mutation (and/or epigenetic silencing) may accelerate disease progression in APL and other forms of acute myeloid leukemia (AML). Indeed, biallelic knockout of Gata2with CRISPR/Cas9-mediated gene editing increased the serial replating efficiency of PML-RARA–expressing myeloid progenitors (as well as progenitors expressing RUNX1-RUNX1T1, or deficient for Cebpa), increased mouse APL penetrance, and decreased latency. Restoration of Gata2expression suppressed PML-RARA–driven aberrant self-renewal and leukemogenesis. Conversely, addback of a mutant GATA2R362Gprotein associated with APL and AML minimally suppressed PML-RARA–induced aberrant self-renewal, suggesting that it is a loss-of-function mutation. These studies reveal a potential role for Gata2as a tumor suppressor in AML and suggest that restoration of its function (when inactivated) may provide benefit for AML patients.

Self-renewal is a key feature of the cells that initiate acute myeloid leukemia. To identify the mechanisms involved in PML-RARA (PR)-driven self-renewal, we made use of Ctsg-PRmice, which have PRknocked into the UTR of the first exon of Ctsg. Ctsg-PRdrives the expression of PRin myeloid progenitor cells and gives these cells the ability to serially replate in methylcellulose-based colony assays. Most Ctsg-PRmice develop acute promyelocytic leukemia (APL) with an average latency of ~300 days. To identify target genes regulated by Ctsg-PR, we performed single cell RNA-seq (scRNA-seq) on whole bone marrow from young, preleukemic Ctsg-PRmice or age-matched littermates. We identified 959 differentially expressed genes (DEGs) within myeloid progenitors (546 upregulated by PR,and 413 downregulated). Gata2was identified as a DEG in this analysis, and we confirmed this phenotype with bulk RNA-seq of purified promyelocytes from young, preleukemic WT vs. Ctsg-PRmice. All APLs derived from Ctsg-PRmice also expressed high levels of Gata2.