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

, and 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 CD34 immunophenotype, and synergistic upregulation of Hepatic Leukemia Factor (). Cell sorting based on the LSC marker GPR56 allowed isolation of triple-mutated from double-mutated subclones. Moreover, in R882-mutated patients, CpG hypomethylation at the transcription start site correlated with high mRNA expression, which was itself associated with poor survival. Loss of via CRISPR/Cas9 significantly reduced the CD34GPR56 LSC compartment of primary human triple-mutated AML cells in serial xenotransplantation assays. 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 knockdown revealed the NOTCH target Hairy and Enhancer of Split 1 () and the cyclin-dependent kinase inhibitor as novel targets of HLF, potentially mediating these effects. Overall, our data establish as a novel LSC regulator in this genetically defined high-risk AML subgroup.