Identifying Regulators of Hepatic Leukemia Factor (HLF) Expression in Hematopoietic Cells Using a Fluorescent Reporter Transgene

Hepatic leukemia factor (HLF) is a transcription factor and one of the most selectively expressed genes in human and mouse hematopoietic stem cells (HSCs). HLF identifies functional HSC activity and is a key "stemness" gene that maintains quiescence and self-renewal. Upregulation of HLF has been observed in NPM1c/FLT3-ITD-mutated acute myeloid leukemia (AML) and is thought to protect cells from death and chemotoxic insults. However, the pathways which cause HLF expression in NPM1c-mutated leukemia are unknown. Since HLF expression is associated with HSC activity and decreases upon differentiation, we hypothesized that an HLF-ZsG fluorescent reporter in a leukemia cell line (IMS-M2) could be used as a readout to identify genes that regulate HLF in normal HSCs. The NPM1 protein resides in the nucleus and regulates DNA repair, cell growth and proliferation. Mutations which result in its cytoplasmic localization (NPM1c) have been observed in AML and lead to aberrant cell proliferation and differentiation. Nuclear relocalization of NPM1c results in the downregulation of homeobox genes in IMS-M2 cells. We validated the HLF-ZsG reporter in IMS-M2 cells and confirmed the dependency of HLF expression on NPM1c. We then identified MEIS1, HOXA10 and NKX2-3 as potential regulators of HLF. We have identified a preliminary network of HLF expression downstream of NPM1c, which may also help us understand HLF regulation in normal HSCs.