Ppara Deletion Promotes Monocytic Leukemia Granulocytic Differentiation

Introduction

Acute myeloid leukemia (AML) is a highly aggressive malignancy of myeloid progenitors associated with a poor prognosis. AML is characterized by a block in differentiation and the acquisition of self-renewal capacity. The hematopoietic system balances self-renewal and differentiation in response to various environmental cues. Peroxisome proliferator activated receptors (PPARs) are transcription factors that belong to a subfamily of fatty acid-activated nuclear steroid hormone receptors, which coordinate cellular gene expression programs with environmental cues effecting regulation of multiple cellular processes including proliferation, apoptosis, differentiation and inflammation. We found that PPARA expression levels and signaling signature were upregulated in intermediate and high cytogenetic risk subgroups of AML leading us to hypothesize that PPARA regulates AML maintenance.

Methods

We generated murine Ppara-homozygous and heterozygous-deficient GFP+ MLL-AF9 leukemia through retroviral infection and transplantation into primary wildtype or Rag2-/- recipients. FACS-sorted GFP+ cells of each genotype were isolated for downstream flow cytometric analysis of subpopulations, cytospins and ex-vivo assays. Transcriptomic data was obtained by RNAseq.

Results

Ppara deletion improves monocytic leukemia survival: Secondary recipients of Ppara -/- and +/- leukemia displayed decreased GFP+ bone marrow-mononuclear cells (BM-MNC) and peripheral blood-MNC and spleen infiltration which resulted in a statistically significant survival advantage compared to wildtype leukemia controls [p=0.002]. This was not due to increased apoptosis evaluated by Annexin-V staining or proliferation rate of GFP+ knockout as compared to wildtype leukemia ex-vivo.

Ppara deletion promotes monocytic leukemia granulocytic differentiation: Review of GFP+ BM-MNC revealed differentiation in Ppara-/- leukemia characterized by a decrease in cells with monocytic immunophenotype by flow cytometry employing monocyte and granulocyte markers Ly6C and Ly6G respectively. Notably, a population of cells with a granulocytic immunophenotype was detected in Ppara-/- leukemia but absent in Ppara+/+ leukemia. Cytospins of the Ppara-/- GFP+ FACS-sorted granulocytic population provided morphologic evidence of terminally differentiated neutrophils. Furthermore, bulk RNAseq of FACS-sorted GFP+ granulocytic cells from 4 Ppara-/- leukemia mice and unbiased cell type recognition using SingleR (Bioconductor) was performed. Using two mouse reference datasets, SingleR annotated all 4 samples as neutrophils. By contrast, FACS-sorted Ppara+/+ and Ppara-/- GFP+ monocytic populations were annotated as stem cells, similar to KSL and GMP cells from non-leukemic WT mice.

Ppara deletion regulates adaptive immune response: To understand how PPARA regulates leukemia progression, we undertook global transcriptomics analysis. Most perturbations in Ppara-/- versus Ppara+/+ leukemia involved downregulation of pathways, consistent with PPARA's known role as a transcriptional activator, with multiple pathways noted to converge on metabolism, macromolecule trafficking and processing. Additionally, multiple biological process terms differentially enriched between Ppara+/+ and Ppara-/- leukemia also reflected alterations in immune response. To evaluate this further, we transplanted Ppara-/- leukemia into Rag2-/- recipients, which resulted in a partial collapse of the survival benefit compared to wildtype leukemia. We queried the human relevance of these findings and found positive regulation of adaptive immune response and antigen processing and presentation in upregulated pathways of PPARA_low AML vs. PPARA_high AML cases from the BEAT AML database.

Conclusion

A monocytic AML model with genetic Ppara deletion demonstrates granulocytic differentiation resulting in leukemia-derived terminally differentiated neutrophils, associated with positive regulation of adaptive immune response and with prolonged survival. Future studies on the interaction of PPARA, leukemia cell composition and anti-tumor immunity are warranted.

Mendez:Rigel: Consultancy; Inventiva: Consultancy.