Pre-malignant extracellular matrix drives leukemia transformation through reprogramming of pre-leukemic stem cells

Abstract Acute myeloid leukemia (AML) often arises from myelodysplasia (MDS), a pre-leukemic condition characterized by dysplasia and ineffective hematopoiesis. Despite extensive genetic and epigenetic profiling of AML, pathogenic mechanisms of disease development from pre-leukemic states remain largely unknown. Generation of mouse models of MDS and AML allowed us to pinpoint a global decrease in nucleoporin expression which was confirmed in public patient databases. shRNA-mediated downregulation of NUPs in mouse MDS HSPCs followed by transplantation led to fully penetrant AML with blasts in blood, bone marrow (BM) and spleen of lethally irradiated recipients. Moreover, NUP downregulation in induced Pluripotent Stem Cell (iPSC) harboring MDS-relevant SRSF2P95L and ASXL1646fs*12 mutations transformed cells as evidenced by the loss of CD34 expression, extended growth in vitro, a 2.5-fold upregulation of the leukemic biomarker CD123 and the acquisition of phenotypic characteristics of AML blasts. More importantly, shNUPs-SA HSPCs were able to engraft in NSG mice, evidencing the oncogenic properties of NUPs downregulation in a humanized in vivo setting. RNAseq analysis in mice, patients and human iPSC models revealed a signature that involves proliferative and anti-apoptotic gene pathways that promote survival of clones and arrest differentiation at the very early myeloid stage. ATAC-seq performed on a cell line with NUPs knockdown identified a significant increase in chromatin accessibility in genes involved in cell cycle, proliferation and negative regulation of apoptosis. Single-cell RNA profiling of human healthy, pre-leukemic and leukemic niche identified a unique cell population termed Fibro-MSCs that is defined by expression of Pdpn. This cell type secretes a matricellular protein, Tenascin X (TNXB), whose downregulation triggers this transformative signature. We confirmed that TNXB levels are significantly decreased in the BM of AML as compared to MDS, both in our mouse models and our patient cohorts. Transcriptomic analysis performed in bone marrow biopsies from patients with low and high blast counts showed a significant downregulation of TNXB with disease progression. These results identify a biological process and the accompanying mechanistic cascade of events within it that impose leukemia development and reveal novel targets to be exploited therapeutically.