Metallothionein 1 (MT1) Is Selectively Required for DNMT3A;NPM1-Mutant Acute Myeloid Leukemia Proliferation

Adult de novo acute myeloid leukemia (AML) is a hematologic malignancy with poor prognosis and poor treatment options, especially in elderly individuals. The highly heterogeneous nature of AML motivates identification of specific dependencies within genetic subtypes for the development of effective targeted therapies. In cytogenetically normal AML (CN-AML), two of the most common and frequently co-mutated genes are the DNA methyltransferase DNMT3A and nucleophosmin NPM1, which encodes a multifunctional nuclear protein. Understanding the molecular mechanisms by which mutations in these genes cooperate to cause AML is critical for development of effective treatment options with reduced toxicity for new and/or relapsed disease.

To uncover candidate mechanisms, we began by using a mouse model developed in our laboratory with an inducible dual-recombinase system that combines flippase-FRT (Flp-FRT) and Cre-LoxP for sequential induction of a Dnmt3a-R878H hotspot mutation and a Npm1c hotspot mutation, replicating DNMT3A-R882H and NPM1c mutations found in human AML (Loberg et al., Leukemia 2019). Using single-cell RNA-sequencing and cellHarmony analysis, we identified differentially expressed transcripts in clusters of Dnmt3a;Npm1-mutant AML stem and progenitor cells compared to wild-type stem and progenitor cell counterparts. Across four independently derived Dnmt3a;Npm1-mutant AMLs, metallothionein 1 (Mt1) was the top common, significantly upregulated transcript in the most primitive AML stem and progenitor cell cluster. MT1 is a cytosolic protein that plays a vital role in protecting against heavy metal toxicity, oxidative stress, and inflammation, and has been implicated in inflammatory disease pathology and tumor progression. This led us to hypothesize that MT1 upregulation is critical for growth and survival of DNMT3A;NPM1-mutant AML.

First, we validated increased Mt1 expression in additional independent Dnmt3a;Npm1-mutant murine AML samples isolated from the bone marrow and spleen, relative to wild-type tissue samples. Next, we tested the extent to which Dnmt3a;Npm1-mutant murine AML cells depend on Mt1 for their growth and survival. We achieved high efficiency (>95%) knockout of Mt1 in primary Dnmt3a;Npm1-mutant murine AML cells using CRISPR-Cas9. In ex vivo culture, knockout of Mt1 significantly reduced their proliferation relative to control sgRNA-targeted cells. Using the cell cycle marker Ki-67, we observed Mt1 knockout resulted in cell cycle arrest at G1 phase. Further, we transplanted Mt1-knockout Dnmt3a;Npm1-mutant AML cells into recipient mice and observed reduced AML burden at 4 weeks post-transplant compared to control sgRNA-targeted cells, with long-term survival studies ongoing. In parallel to these studies, we interrogated MT1 function in human AML. Using the DNMT3A;NPM1-mutant human AML cell line OCI-AML3, we found that MT1 expression was significantly increased relative the non-DNMT3A;NPM1-mutant human AML cell line OCI-AML2 and other control human cell lines. CRISPR-mediated knockout of MT1 significantly reduced the in vitro growth of OCI-AML3 cells but did not impair growth of control OCI-AML2 cells. Together, our study reports the discovery that MT1 is critical for growth and survival of DNMT3A;NPM1-mutant AML cells and that this is conserved across mouse and human. We nominate MT1 as a target for continued studies towards the goal of treatment and prevention of DNMT3A;NPM1-mutant AML.

Trowbridge:H3 Biomedicine: Research Funding.