Hhex induces promyelocyte self-renewal and cooperates with growth factor independence to cause myeloid leukemia in mice

Jacob T Jackson,Ashley P Ng,Benjamin J Shields,Sue Haupt,Ygal Haupt,Matthew P Mccormack

doi:10.1182/bloodadvances.2017013243

Abstract

AbstractThe hematopoietically expressed homeobox (Hhex) transcription factor is overexpressed in human myeloid leukemias. Conditional knockout models of murine acute myeloid leukemia indicate that Hhex maintains leukemia stem cell self-renewal by enabling Polycomb-mediated epigenetic repression of the Cdkn2a tumor suppressor locus, encoding p16Ink4a and p19Arf. However, whether Hhex overexpression also affects hematopoietic differentiation is unknown. To study this, we retrovirally overexpressed Hhex in hematopoietic progenitors. This enabled serial replating of myeloid progenitors, leading to the rapid establishment of interleukin-3 (IL-3)–dependent promyelocytic cell lines. Use of a Hhex-ERT2 fusion protein demonstrated that continuous nuclear Hhex is required for transformation, and structure function analysis demonstrated a requirement of the DNA-binding and N-terminal–repressive domains of Hhex for promyelocytic transformation. This included the N-terminal promyelocytic leukemia protein (Pml) interaction domain, although deletion of Pml failed to prevent Hhex-induced promyelocyte transformation, implying other critical partners. Furthermore, deletion of p16Ink4a or p19Arf did not promote promyelocyte transformation, indicating that repression of distinct Hhex target genes is required for this process. Indeed, transcriptome analysis showed that Hhex overexpression resulted in repression of several myeloid developmental genes. To test the potential for Hhex overexpression to contribute to leukemic transformation, Hhex-transformed promyelocyte lines were rendered growth factor–independent using a constitutively active IL-3 receptor common β subunit (βcV449E). The resultant cell lines resulted in a rapid promyelocytic leukemia in vivo. Thus, Hhex overexpression can contribute to myeloid leukemia via multiple mechanisms including differentiation blockade and enabling epigenetic repression of the Cdkn2a locus.

Highlights

The hematopoietically expressed homeobox gene (Hhex) is an oligomeric, nonclustered homeobox transcription factor that acts primarily as a transcriptional repressor.[1,2] Hhex was first cloned via its expression in hematopoietic tissues, where it is highly expressed in hematopoietic stem cells (HSCs) and progenitors but undergoes transcriptional downregulation during differentiation, in the T-cell lineage.[3,4,5] Consistent with the function of members of the homeobox gene family, Hhex has been shown to have important developmental roles during embryogenesis
Hhex overexpression resulted in a developmental block and subsequent expansion of promyelocytic myeloid progenitors (Figure 1D), which, upon transfer to liquid culture in IL-3 after 4 weeks, gave rise to immortalized cell lines that were dependent on IL-3 or granulocyte-macrophage colony-stimulating factor for growth
We found that sustained Hhex expression enables serial replating of myeloid progenitors and rapid development of myeloid cell lines

Summary

Introduction

The hematopoietically expressed homeobox gene (Hhex) is an oligomeric, nonclustered homeobox transcription factor that acts primarily as a transcriptional repressor.[1,2] Hhex was first cloned via its expression in hematopoietic tissues, where it is highly expressed in hematopoietic stem cells (HSCs) and progenitors but undergoes transcriptional downregulation during differentiation, in the T-cell lineage.[3,4,5] Consistent with the function of members of the homeobox gene family, Hhex has been shown to have important developmental roles during embryogenesis. Hhex-knockout mice die during midgestation with perturbed mesodermal tissue development, resulting in defects in liver, thyroid, and cardiac formation.[6,7,8]. Submitted 13 October 2017; accepted 24 January 2018.

Methods

Results

Conclusion