Minimal Reduction of PU.1 Is Sufficient to Induce a Preleukemic State and Promote Development of Acute Myeloid Leukemia
Authors: Will Britta , Vogler Thomas O. , Narayanagari Swathi-rao , Bartholdy Boris , Todorova Tihomira I. , Chen Jiahao , Yu Yiting , Mayer Jillian , da Silva Ferreira Mariana , Barreyro Laura , Carvajal Luis , Roth Michael , van Oers Johanna , Schaetzlein Sonja , McMahon Christine , Edelmann Winifried , Verma Amit , Steidl Ulrich 
Published: October 2019

Genomic studies have shown that human cancer is rarely associated with a complete loss of transcripts; instead, acquired DNA alterations often occur within the non-coding part of the genome, are enriched in gene-regulatory regions, and cause only moderate transcriptional changes. It is currently not well understood how such moderate gene expression changes impact normal tissue function and how they contribute to malignant transformation.

Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) develop through a multi-step transformation process originating in hematopoietic stem cells (HSCs) and mainly present in the elderly (median age of >65 years at diagnosis). Although loss or near-complete loss of the hematopoietic transcription factor PU.1 induces AML in mice, a similar degree of PU.1 impairment is exceedingly rare in human AML; yet moderate PU.1 inhibition is common in AML patients. At the example of the Ets-family transcription factor PU.1, which is indispensable for HSC function and the differentiation of cells within the myeloid as well as lymphoid lineages, we tested the hypothesis that even moderate gene expression alterations of key regulators can drive malignant transformation. We assessed the effects of minimal PU.1 inhibition on hematopoiesis in a novel mouse model that co-models the genomic context found in aging human individuals and patients with MDS/AML.

Mice lacking Msh2, the key component of the MutSα and MutSβ complexes mediating DNA mismatch repair, accumulate elevated numbers of point mutations, in particular C/G>T/A transitions and small insertions/deletions resembling the mutation spectrum acquired in HSCs in aging human individuals and patients with MDS and AML. We crossed Msh2-/- mice with animals carrying a heterozygous deletion of an upstream regulatory element of PU.1 (UREΔ/+). UREΔ/+Msh2-/- mice exhibited a significant, but very modest reduction of PU.1 expression on average by 26-37% in fractionated hematopoietic multipotent stem and myeloid progenitor cells. Strikingly, this minimal reduction of PU.1 led to the emergence of an aggressive, transplantable AML in more than two thirds of UREΔ/+Msh2-/- mice which was never observed in URE+/+Msh2-/- mice. Overt leukemia was preceded by a preleukemic phase hallmarked by an expanded population of multipotent murine hematopoietic stem cell enriched cells (HSPCs) that was myeloid-biased and less quiescent than their wild type counterpart. Longitudinal monitoring of preleukemic UREΔ/+Msh2-/- mice revealed a progressive increase in immature myeloid cells along with a gradual decrease in mature myeloid cells, as well as expansion of phenotypic HSPC compartments and multi-lineage dysplasia resembling human MDS. AML progression was accompanied by additional inhibition of a PU.1-cooperating factor, interferon responsive factor 8 (Irf8). Irf8 expression restoration rescued impaired expression of genes harboring PU.1/IRF consensus binding sites, led to the loss of aberrant self-renewal, promoted myeloid differentiation, and induced apoptosis in leukemic UREhetMsh2-/- cells demonstrating that Irf8 impairment functionally cooperates with minimally reduced PU.1 expression in our model. We also found evidence of disease-relevant joint PU.1/IRF8 inhibition in human myeloid leukemogenesis: (1) patients with MDS with a higher risk for the progression to AML had lower IRF8 levels; (2) lower IRF8 expression was detected specifically in AML patients with reduced PU.1 levels; (3) restoration of IRF8 expression induced differentiation in IRF8 low expressing AML cells, and (4) a positive correlation of PU.1 and IRF8 expression was found in human leukemia stem cells, but not in healthy HSCs. Strikingly, comparative pathway analysis revealed a genome-wide molecular resemblance of preleukemic and leukemic UREΔ/+Msh2-/- mice with gene expression profiles from human MDS and AML patients, respectively.

Our study demonstrates that minimal reduction of a key lineage-specific transcription factor that commonly occurs in human disease is sufficient to initiate cancer development and provides mechanistic insight into the formation and progression of preleukemic stem cells in MDS and AML.

Disclosures

No relevant conflicts of interest to declare.