The nucleophosmin (NPM1) gene encodes for a multifunctional protein with prominent nucleolar localization that shuttles between nucleus and cytoplasm. NPM1 mutations represent the most common genetic lesion in adult acute myeloid leukemia (AML; about one third of cases), and they act deterministically to cause the aberrant cytoplasmic delocalization of NPM1 mutants. Because of its unique features, NPM1-mutated AML is recognized as a distinct entity in the 2017 World Health Organization (WHO) classification of hematopoietic neoplasms. Here, we focus on recently identified functions of wild-type NPM1 in the nucleolus and address new biological and clinical issues related to NPM1-mutated AML. The relevance of the cooperation between NPM1 and other mutations in driving AML with different outcomes is presented. We also discuss the importance of eradicating NPM1-mutated clones to achieve AML cure and the impact of preleukemic clonal hematopoiesis persistence in predisposing to second AML. The contribution of HOX genes’ expression to the development of NPM1-mutated AML is also highlighted. Clinically, yet unsolved diagnostic issues in the 2017 WHO classification of myeloid neoplasms and the importance of NPM1 mutations in defining the framework of European LeukemiaNet genetic-based risk stratification are discussed. Finally, we address the value and limits of NPM1-based measurable residual disease assessment for treatment guidance and present the results of promising preclinical studies with XPO1 and menin-MLL inhibitors.

Immune checkpoint therapy has resulted in remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible Src homology 2–containing (CIS) protein, a key negative regulator of interleukin 15 (IL-15) signaling, with fourth-generation “armored” chimeric antigen receptor (CAR) engineering of cord blood–derived natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell antitumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that targeting a cytokine checkpoint further enhances the antitumor activity of IL-15–secreting armored CAR-NK cells by promoting their metabolic fitness and antitumor activity. This combined approach represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy.

Our understanding of the mechanisms that regulate hematopoietic stem/progenitor cells (HSPCs) has been advanced by the ability to genetically manipulate mice; however, germline modification is time consuming and expensive. Here, we describe fast, efficient, and cost-effective methods to directly modify the genomes of mouse and human HSPCs using the CRISPR/Cas9 system. Using plasmid and virus-free delivery of guide RNAs alone into Cas9-expressing HSPCs or Cas9-guide RNA ribonucleoprotein (RNP) complexes into wild-type cells, we have achieved extremely efficient gene disruption in primary HSPCs from mouse (>60%) and human (∼75%). These techniques enabled rapid evaluation of the functional effects of gene loss of Eed, Suz12, and DNMT3A. We also achieved homology-directed repair in primary human HSPCs (>20%). These methods will significantly expand applications for CRISPR/Cas9 technologies for studying normal and malignant hematopoiesis.

DNA methylation has widespread effects on gene expression during development. However, our ability to assign specific function to regions of DNA methylation is limited by the poor correlation between global patterns of DNA methylation and gene expression. Here, we utilize nuclease-deactivated Cas9 protein fused to repetitive peptide epitopes (SunTag) recruiting multiple copies of antibody-fused de novo DNA methyltransferase 3A (DNMT3A) (dCas9-SunTag-DNMT3A) to amplify the local DNMT3A concentration to methylate genomic sites of interest. We demonstrate that dCas9-SunTag-DNMT3A dramatically increases CpG methylation at the HOXA5 locus in human embryonic kidney (HEK293T) cells. Furthermore, using a single guide RNA, dCas9-SunTag-DNMT3A is able to methylate a 4.5-kb genomic region and repress HOXA5 gene expression. Reduced representation bisulfite sequencing and RNA-seq show that dCas9-SunTag-DNMT3A methylates regions of interest with minimal impact on the global DNA methylome and transcriptome. This effective and precise tool enables site-specific manipulation of DNA methylation and may be used to address the relationship between DNA methylation and gene expression.

DNA methylation is an epigenetic process involved in development, aging, and cancer. Although the advent of new molecular techniques has enhanced our knowledge of how DNA methylation alters chromatin and subsequently affects gene expression, a direct link between epigenetic marks and tumorigenesis has not been established. DNMT3A is a de novo DNA methyltransferase that has recently gained relevance because of its frequent mutation in a large variety of immature and mature hematologic neoplasms. DNMT3A mutations are early events during cancer development and seem to confer poor prognosis to acute myeloid leukemia (AML) patients making this gene an attractive target for new therapies. Here, we discuss the biology of DNMT3A and its role in controlling hematopoietic stem cell fate decisions. In addition, we review how mutant DNMT3A may contribute to leukemogenesis and the clinical relevance of DNMT3A mutations in hematologic cancers.