Metallothionein 1 Regulates Growth and Survival of DNMT3A;NPM1-Mutant Acute Myeloid Leukemia

Adult de novo acute myeloid leukemia (AML) is a hematologic malignancy with poor prognosis, commonly driven by mutations in genes including the DNA methyltransferase DNMT3A and nucleophosmin NPM1. We previously generated sequentially inducible mouse models of these mutations and observed transformation from clonal hematopoiesis (CH) to myeloproliferative disorder to AML. In transformed AML, leukemia-propagating cells have a myeloid-restricted progenitor cell phenotype (c-Kit+). Here, to identify mechanisms that sustain tumorigenesis, we performed single-cell RNA-seq of c-Kit+ cells from four primary Dnmt3a;Npm1-mutant AML samples. The most primitive subset of c-Kit+ cells, a multipotent progenitor population Multi-Lin-2, had increased expression of the antioxidant and heavy metal chelator metallothionein 1 (Mt1) in all AML samples. Cas9-mediated Mt1 knockout in primary Dnmt3a;Npm1-mutant AML resulted in reduced cell cycling and proliferation and increased pyroptosis in vitro and increased overall survival following transplant into congenic recipient mice. Examining the human DNMT3A;NPM1-mutant AML cell line, OCI-AML3, we found that the metallothionein isoform MT1G was increased in expression relative to normal CD34+ hematopoietic stem and progenitor cells. Cas9-mediated MT1G knockout in OCI-AML3 cells resulted in reduced proliferation and cell growth in vitro. Together, our study reports an essential role for metallothionein in growth and survival of DNMT3A;NPM1-mutant AML cells. Overall design: To understand the molecular impact of malignant cell populations relative to their non-malignant analogues, we leveraged and recently produced healthy hematopoietic cell atlas of 87 cell states, spanning all major bone marrow lineages. This atlas includes rare and transitional cell states, with evidence deriving from cell surface protein or transcriptome evidence (CITE-Seq). As this atlas excludes predominant hematopoietic and stromal cell populations in the spleen, we combined identified distinct cell states from multiple integrated spleen scRNA-Seq for the annotation of our datasets (Methods). The subsequent malignant cell populations were compared to normal equivalents to derive gene expression impacts retained across distinct DNMT3A mouse models.