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

Swati Garg, Armando Reyes-Palomares, Lixiazi He, Anne Bergeron, Vincent-Philippe Lavallée, Sébastien Lemieux, Patrick Gendron, Christian Rohde, Jianglong Xia, Prarabdha Jagdhane, Carsten Müller-Tidow, Daniel B. Lipka, Suzan Imren, R. Keith Humphries, Claudia Waskow, Binje Vick, Irmela Jeremias, Guillaume Richard-Carpentier, Josée Hébert, Guy Sauvageau, Judith Zaugg, Frédéric Barabé and Caroline Pabst

Key Points

  • HLF is overexpressed in normal karyotype AML triple-mutated for NPM1, DNMT3A, and FLT3-ITD.

  • Loss of HLF reduces the CD34+GPR56+ compartment, accelerates cell cycle progression and decreases HES1 and CDKN1C expression.

Abstract

FLT3, DNMT3A, and NPM1 are the most frequently mutated genes in cytogenetically normal acute myeloid leukemia (AML), but little is known about how these mutations synergize upon co-occurrence. Here we show that triple-mutated AML is characterized by high leukemia stem cell (LSC) frequency, an aberrant leukemia specific GPR56highCD34low immunophenotype, and synergistic upregulation of Hepatic Leukemia Factor (HLF). Cell sorting based on the LSC marker GPR56 allowed isolation of triple mutated from DNMT3A/NPM1 double-mutated subclones. Moreover, in DNMT3A R882 mutated patients, CpG hypomethylation at the HLF transcription start site correlated with high HLF mRNA expression, which was itself associated with poor survival. Loss of HLF 3 via CRISPR/Cas9 significantly reduced the CD34+GPR56+ LSC compartment of primary human triple-mutated AML cells in serial xenotransplantation assays. HLF knockout cells were more actively cycling when freshly harvested from mice, but rapidly exhausted when re-introduced in culture. RNA-sequencing (RNA-Seq) of primary human triple-mutated AML cells after shRNA mediated HLF knockdown revealed the NOTCH target Hairy And Enhancer Of Split 1 (HES1) and the cyclin-dependent kinase inhibitor CDKN1C/p57 as novel targets of HLF potentially mediating these effects. Overall our data establish HLF as a novel LSC regulator in this genetically defined high-risk AML subgroup.

  • Submitted July 10, 2018.
  • Revision received May 4, 2019.
  • Accepted May 2, 2019.