PU.1-Activated Genomic Regions Define Low-risk MDS Subsets Characterized by Immune Dysregulation and Disease Progression [MDS-L RNA-seq]

Myelodysplastic syndromes (MDS) are heterogeneous myeloid neoplasms with an increased risk of progression to secondary acute myeloid leukemia (sAML). This study investigates the genomic correlates of disease progression in MDS by profiling active genomic regulatory regions and their transcriptional impact through H3K27ac ChIP-seq and RNA-seq analysis on CD34+ bone marrow progenitors cells isolated from a prospective cohort of 86 and 357 patients, respectively. Our analysis revealed distinct patterns of genomic region activation and transcriptional regulation across different disease stages (low-risk MDS, high-risk MDS and sAML). Unexpectedly, unsupervised clustering revealed a subset of low-risk MDS patients displaying regulatory and transcriptional profiles similar to those of high-risk MDS and sAML, highlighting early molecular events that may predispose patients to disease progression. This subset is characterized by PU.1 genomic occupancy in regions linked to immune and inflammatory responses, increased T-cell and NK activation, and a higher frequency of SRSF2 mutations. Clinically, patients in this group exhibit greater susceptibility to infections and cardiovascular events, along with an elevated risk of disease progression, resulting in a significantly reduced overall survival. Functional studies demonstrate that PU.1 inhibition suppresses MDS cell proliferation and clonogenicity, as impaired PU.1 binding inhibits the activation of key transcriptional programs involved in disease advancement. Collectively, these findings identify epigenetic factors that predispose low-risk MDS patients to progression into high-risk MDS and, ultimately, sAML. Overall design: To comprehensively characterize the transcriptional alterations occurring during disease progression, we analysed bone marrow progenitors from a prospective, unselected cohort of 357 patients. Our analyses were exclusively performed on isolated CD34+ bone marrow cells, which include the disease-initiating population in MDS from a cohort of 209 MDS patients plus another set of 148 sAML patients. To investigate the functional role of PU.1 in regulating gene expression, we pharmacologically inhibited its activity by treating MDS-L cells with the PU.1 inhibitor DB2115. To assess the functional role of PU.1, we employed CRISPR/Cas9-mediated depletion of PU.1 in CD34? MDS-L cells after testing five different sgRNAs targeting PU.1 were tested in bulk cell populations.