NK-2 Homeobox Transcription Factor NKX2-3 Confers Differentiation Block in NPM1-Mutated in Acute Myeloid Leukaemia
Authors: Chadwick John A , Wingelhofer Bettina , Somervaille Tim CP 
Published: December 2023

Dysregulation of HOX homeobox family transcription factor (TF) gene expression is important in the pathogenesis of AML in particular in NPM1-mutated and MLL rearranged molecular subtypes. However, little is known about the role of NK2-homeobox (NKX2) family TF genes. NKX2 genes have critical roles in normal development of the central nervous system, thyroid, lung and pancreas. We observed high expression of NKX2-3 in patient AML samples and hypothesised the transcription factor might have a functional role in the disease.
By qPCR, we found that NKX2-3 is highly expressed in normal HSCs but is rapidly down regulated as cells differentiate. In primary AML samples, it is highly expressed in 21-37% cases, including within the immunophenotypic LSC compartment, in particular in cases with NPM1 and/or FLT3-ITD mutations.
Forced expression of NKX2-3 in normal murine KIT + bone marrow (BM) HSPCs transiently enhanced their clonogenic activity and impaired differentiation: NKX2-3-expressing HSPCs expressed higher levels of E2F or MYC target genes, and leukemia stem cell (LSC) maintenance genes. Congenic transplant experiments revealed that forced expression of NKX2-3 in normal BM HSPCs was sufficient to enhance multilineage engraftment across four months of follow up without inducing leukemia. In keeping with this, transplantation of Nkx2-3-/- knockout BM cells led to significantly inferior engraftment compared with BM cells from litter mate control mice. Thus, while increased expression of Nkx2-3 enhances engraftment of normal stem cells in transplantation assays, loss of Nkx2-3 impairs it.
Analysis of NKX2-3high primary human AML cases demonstrated that HOXA9 was the most highly upregulated transcription factor gene compared with NKX2-3low cases. We expressed both factors singly and together in murine KIT+ BM HSPCs: Hoxa9/NKX2-3 cells exhibited significantly higher clonogenic activity and reduced morphologic and immunophenotypic differentiation compared with Hoxa9/MTV cells. Congenic BM transplant experiments demonstrated that Hoxa9/NKX2-3 recipients developed leukemias significantly earlier than Hoxa9/MTV recipients (median 82 vs 155 days). BM morphology and immunophenotyping confirmed that Hoxa9/NKX2-3AMLs exhibited a greater degree of differentiation block than Hoxa9/MTV AMLs. Leukemia cells from Hoxa9/NKX2-3 recipients coordinately expressed higher levels of E2F or MYC target genes, and LSC maintenance genes.
NKX2-3 KD to ~30% of control values reduced proliferation and induced morphologic and immunophenotypic differentiation in primary patient NPM1-mutated AML cells. NKX2-3 KD led to down regulation of E2F and MYC target genes and normal human CD34 + stem cell genes; and up regulation of myeloid differentiation genes. Using a CRISPR ribonucleofection approach to relocate NPM1c to the nucleus we confirmed that NKX2-3 expression in NPM1-mutant primary AML cells is sustained by mutant NPM1c.
ChIPseq in NPM1-mutated primary patient and KO52 AML cells identified NKX2-3 binding peaks. As expected, DNA sequences under peak apices were strongly enriched for NKX2-3 binding motifs; there was also significant enrichment for RUNX and ETS family motifs, but not HOX/MEIS/PBX motifs. To identify genes potentially regulated by NKX2-3, genomic coordinates of strong binding peaks were mapped to the single nearest gene. Remarkably, among genes up regulated following NKX2-3 knockdown in primary human AML cells there was a highly significant enrichment of genes with putative regulatory elements bound by NKX2-3. Likewise, among genes down regulated in NKX2-3-expressing KIT+ murine HSPCs, there was a similar significant enrichment of genes with putative regulatory elements bound by NKX2-3. These data suggest that NKX2-3 functions as a transcription repressor at bound regulatory elements in primary AML cells.
In summary, NKX2-3 is highly expressed in NPM1 & FLT3 mutated molecular subtypes of AML and serves to enhance self-renewal and the level of differentiation block through binding to and repressing regulatory elements of myeloid differentiation genes.