Identification of IRF8 As a Potent Tumor Suppressor in Murine Acute Promyelocytic Leukemia
Authors: Surianarayanan Sangeetha , Gaillard Coline M , Bentley Trevor , Warr Matthew R. , Fitch Briana , Geng Huimin , Passegue Emmanuelle , de Thé Hugues , Kogan Scott C. 
Published: October 2019

Abstract
The classical paradigm suggests that PML/RARA fusion protein is the main driver of pathogenesis in APL. It is believed that the fusion oncogenic protein mediates this effect by potentially repressing key myeloid maturation genes involved in differentiation processes. However, the underlying mechanism is not completely understood. We recently challenged this model re-opening fundamental questions as to understand the precise contribution of the fusion protein to leukemic transformation. This knowledge on the mechanistic pathways can lead to better tailored combinatorial therapeutics.
To understand the role of the PML/RARA fusion protein in leukemogenesis, we initially did a transcriptome analysis in our murine MRP8-PML/RARA APL model. Interestingly, we observed only moderate alterations in gene expression pattern of the key myeloid genes that we thought to be actively involved in differentiation processes. Of particular note, we found significant downregulation of the Irf8 in our promyelocyte compartments.
IRF8 is a known regulator of hematopoiesis. The IRF8 myeloid transcription factor (TF) is expressed in several lineages of the hematopoietic tree and plays an important role in orchestrating specification and differentiation of B cells, dendritic cells and monocytes. Herein, we speculate lower levels of IRF8 could potentially impact tumorigenesis in the context of PML/RARA.
In order to address this question, technically, we employed stringent staining and sorting strategy to distinctly differentiate early and late promyelocytes and looked at the expression pattern of Irf8 gene both at the transcript and protein levels. Results from qRT-PCR demonstrated 4.8 fold decrease in Irf8 expression compared to wildtype controls both in preleukemic promyelocytes and fully differentiated leukemic cells suggesting PML/RARA could be a target of IRF8 and this association could potentially be involved in the emergence and maintenance of leukemia. We next asked whether these changes are reflective at the protein levels and performed a Western blot analysis in our highly purified promyelocyte population and found a dramatic decrease in IRF8 levels in comparison to wild type controls again suggesting a possible protein-protein interaction under normal conditions that may provide an advantage for the cells from turning oncogenic.
In order to study how low levels of IRF8 impact promyelocyte expansion, we generated double knock-outs of mice harboring both PML/RARA Irf8-/- mutations and compared their phenotype with mice harboring single mutations in either PML/RARA or Irf8 gene. As previously observed, young PML/RARA mice had a substantially increased number of marrow promyelocytes in comparison to wild-type mice. Fascinatingly, loss of Irf8 alone resulted in an essentially identical expansion of promyelocytes (as well as a loss of earlier myeloid progenitors in the bone marrow, not seen in PML/RARA mice) and a combination of PML/RARA expression and IRF8 loss did not result in a statistically significant further expansion of promyelocytes. These results suggest an epistatic relationship between PML/RARA and IRF8, compatible with downregulation of IRF8 by PML/RARA as being a key mechanism by which t(15;17) expands promyelocytes in the initiation of APL.
Furthermore, in order to assess the impact of single/double genetic alterations on the overall and leukemia free survival we transplanted lethally irradiated mice with bone marrow cells derived from PML/RARA, Irf8-/- and PML/RARA Irf8-/-double knock outs and followed these mice over a period of one year. We observed there is no difference in their overall survival rate among the different groups of mice. However, looking specifically at the acute leukemic deaths, we observed a reduced latency in our PML/RARA Irf8-/- cohorts compared to mice carrying single mutation at PML/RARA loci. We also noticed that all the acute leukemias in the PML/RARA Irf8-/- cohort occurred prior to the first appearance of acute leukemia in the PML/RARA cohorts.
Altogether, these data support a model of APL leukemogenesis in which the translocation of chromosomes 15 and 17 initiates leukemia development, in part by downregulating IRF8, and in which the resulting expansion of the promyelocyte compartment contributes to acquisition of additional cooperating events (e.g. trisomy of chromosome 8, mutation of FLT3) that complete leukemic transformation.

Disclosures
No relevant conflicts of interest to declare.