Haplodeficiency of the 9p21 Tumor Suppressor Locus Causes Myeloid Disorders Driven by Bone Marrow Microenvironment

The chromosome 9p21 locus comprises several tumor suppressor genes including MTAP, CDKN2A and CDKN2B, and its homo- or heterozygous deletions are associated with reduced survival in multiple cancer types. We report that mice with germline monoallelic deletion or induced biallelic deletion of the 9p21-syntenic locus (9p21s) developed fatal myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN)-like disease associated with aberrant trabecular bone formation and/or fibrosis in the bone marrow (BM). Reciprocal BM transfers and conditional targeting of 9p21s suggested that the disease originates in the BM stroma. Single-cell analysis of 9p21s-deficient BM stroma revealed the expansion of chondrocyte and osteogenic precursors, reflected in increased osteogenic differentiation in vitro. It also showed reduced expression of factors maintaining hematopoietic stem/progenitor cells, including Cxcl12. Accordingly, 9p21s-deficient mice showed reduced levels of circulating Cxcl12 and a concomitant upregulation of the pro-fibrotic chemokine Cxcl13. Our study highlights the potential of mutations in the BM microenvironment to drive MDS/MPN-like disease. We then performed gene expression profiling analysis using data obtained from RNA-seq of 4 different cells at two time points. For Bulk RNA-seq, comparative gene expression profiling analysis of bulk RNA-seq data for 9p21s+/- moribund and age-matched wild type mice bone marrow-derived mesenchymal stromal cells (BM-MSCs) at 0 and 7days after osteogenic diffrentiation. For CITE-seq, BM cells were flushed from two 9p21s+/- moribund and age-matched wild type mice bones including two tibias, two femurs and two humerus. The left bones were digested with Liberase TM (0.2 mg/ml) and DnaseI (200U/ml) at 37°C for 45 mins at 550 rpm shaking. BM cells from the digested bones were combined with the flushed BM cells and treated with 1X RBC lysis buffer to remove red blood cells. Cells were stained with biotinylated antibodies to lineage markers cocktail (CD3, CD11b, CD19, B220, Gr1, TCRb and Ter119) at 4°C for 15 mins and then negatively selected using selection cocktail from EasySep Mouse Mesenchymal Stem/Progenitor enrichment Kit (STEMCELL Technologies). Each sample of lineage-depleted cells was incubated with Fc blocking antibody and stained with sample-specific cell hashing reagent and a cocktail of FACS antibodies (lineage cocktail described above, CD71, CD31) at 4°C for 30 mins. All mice samples were washed and Lin-CD71-CD31- cells were sorted for CITE-seq analysis.