The AML HOX-Ome: The Landscape of Developmental Transcription Factors across Pediatric Acute Myeloid Leukemia

Introduction

Acute myeloid leukemia (AML) is a malignancy driven by dysregulated transcription factors. The Homeobox family of genes is an evolutionarily conserved class of transcription factors that are key regulators of developmental processes. Homeobox genes, especially the HOX family of genes, are known to be dysregulated and implicated in AML pathogenesis. We provide a comprehensive evaluation of the Homeobox transcriptome in AML and contrast the HOX-ome of normal hematopoiesis to that of AML and define the unique homeobox expression profile and associations with genomic subtypes of AML.

Methods

We utilized RNA sequencing data from 1,534 diagnostic bone marrow samples from the Target Pediatric AML (TpAML) Initiative as well as 68 normal bone marrow (NBM) and 16 CD34+ PBSC specimens for comparison. A list of 346 genes which contained a homeobox domain was used for this analysis. Patient clustering was performed using R (4.3.0) and Ward method within gene z-score normalized patient gene counts for all homeobox domain containing genes and then subset for HOX family genes.

Results and Discussion

Unsupervised clustering of the transcriptome of 1534 cases across all 346 human homeobox genes demonstrated distinct clustering of NBM and CD34+ cases. NBM displayed a paucity of expression of HOX family of genes. Of note is the distinct and uniform clustering of patients with PML-RARA and CBFA2T3-GLIS2 fusions with lack of expression of HOX family of genes. Those with PML-RARA had enrichment of MYB/SANT genes (EZH, MTA, NCOR, SMARCC, RERE, TERF) and CBFA2T3-GLIS2 fusions were enriched in NKL subclass homeoboxes (HMX3, DLX3, NKX2-8 and EN1).

The most striking feature of the homeobox transcriptome was the distinct expression profile of the HOX family of genes (HOX A, B, C and D). We separately interrogated the expression profile of the 39 HOX family of genes in our AML cohort. Interrogation of the HOX gene family revealed a distinct transcriptome of normal hematopoiesis with over expression of HOX D genes and low/no of expression of HOX A, and B and low expression of HOX C in NBM.

The entirety of the over 1500 cases were clearly separated based on HOX A or HOX B expression where in contrast to NBM, nearly 25% of patients had significant upregulation of HOX A or HOX B family of genes with paucity of expression of HOX C or HOX D genes. The cluster with the highest expression of HOX A genes was highly enriched in KMT2A-r AML in which 88% of patients were KMT2A-r AML. Virtually the entirety of the KMT2A-r AML cases were clustered into 3 different clades based on HOX A gene expression. Clade 1 with the highest expression of all HOX A, clade 2 with paucity of expression of HOX A genes and clade 3 with moderate but distinct HOX A expression. KMT2A-r AML patients constituted 88% of all patients in clade 1 (highest HOX A expression) with an EFS of 23%, significantly worse than those in the other 2 clades or those without KMT2A-r AML (p<0.0001). Those with standard risk KMT2A-r AML within clade 1 had similarly poor EFS of 22% compared to others (p=0.037). More importantly, patients without KMT2A-r AML that clustered within clade 1, had a dismal outcome with EFS of 10%, similar to that in KMT2A-r AML. In contrast to HOX A family and enrichment of KMT2A-r AML, there was distinct clustering of patients with HOX B gene expression. This HOX B cluster was enriched with FLT3-ITD mutations with 63% of all those in HOX B cluster having FLT3-ITD with substantial enrichment of NUP98-NSD1 fusions. Although HOX C and HOX D were not prominently expressed in AML, a small but distinct cohort of patients had high HOX C expression that were enriched in those with ETS family fusions.

Given interest in expression of homeobox TALE family genes including MEIS1 in therapeutic response to menin inhibitors, we interrogated expression of members of this gene family and their association with HOX A expression. TALE family genes did not provide a distinct clustering as seen with the HOX family of genes. However, evaluation of expression of HOX genes and MEIS1 demonstrated clear co-expression where those with HOX A expression also had MEIS1 expression that was enriched in those with KMT2A-r AML.

Our investigation provides a comprehensive evaluation of the landscape of developmental transcription factors across AML, demonstrating clear dysregulation of these developmental genes. We also demonstrate unique association of HOX family of genes with fusion transcripts and associated outcome.