Hepatic leukemia factor is a novel leukemic stem cell regulator in DNMT3A, NPM1, and FLT3-ITD triple-mutated AML

Abstract

<i>FLT3, DNMT3A</i>, and <i>NPM1</i> are the most frequently mutated genes in cytogenetically normal acute myeloid leukemia (AML), but little is known about how these mutations synergize upon cooccurrence. Here we show that triple-mutated AML is characterized by high leukemia stem cell (LSC) frequency, an aberrant leukemia-specific <i>GPR56</i> ^highCD34^low immunophenotype, and synergistic upregulation of Hepatic Leukemia Factor (<i>HLF</i>). Cell sorting based on the LSC marker GPR56 allowed isolation of triple-mutated from <i>DNMT3A/NPM1</i> double-mutated subclones. Moreover, in <i>DNMT3A</i> R882-mutated patients, CpG hypomethylation at the <i>HLF</i> transcription start site correlated with high <i>HLF</i> mRNA expression, which was itself associated with poor survival. Loss of <i>HLF</i> via CRISPR/Cas9 significantly reduced the CD34⁺GPR56⁺ LSC compartment of primary human triple-mutated AML cells in serial xenotransplantation assays. <i>HLF</i> knockout cells were more actively cycling when freshly harvested from mice, but rapidly exhausted when reintroduced in culture. RNA sequencing of primary human triple-mutated AML cells after shRNA-mediated <i>HLF</i> knockdown revealed the NOTCH target Hairy and Enhancer of Split 1 (<i>HES1</i>) and the cyclin-dependent kinase inhibitor <i>CDKN1C/p57</i> as novel targets of HLF, potentially mediating these effects. Overall, our data establish <i>HLF</i> as a novel LSC regulator in this genetically defined high-risk AML subgroup.