Methods to Elucidate Hematopoietic Clonal Organization in Bone Marrow

Every tissue in the human body becomes a mosaic of somatic mutations throughout life. Each cell in the body contains a record of the genetic alterations occurring from conception until that individual cell came into existence through cell division. Most of these changes are benign, but cells with alterations that endow a selective advantage can preferentially divide and expand. Already, this process has been described in a diverse set of phenotypically normal tissues including skin, esophagus and colon (Colom et al., 2021; Lee-Six et al., 2019; Martincorena et al., 2015). In these solid organs, clonal mutations are geographically constrained within contiguous regions of tissue. In the blood, these somatic mutations are termed clonal hematopoiesis (CH) (Steensma et al., 2015). Previous work has extensively characterized the burden of CH in humans, showing an increased prevalence with age and increased risk for hematologic malignancy and all-cause mortality (Genovese et al., 2014; Jaiswal et al., 2014; Young et al., 2016; Young et al., 2019). Despite our detailed understanding of CH in the blood, little is known about how these hematopoietic clones exist in the bone marrow (BM) and how they interact with the BM niche. We hypothesize that the spatial restriction of clones observed in solid organs also exists in the BM. To characterize the spatial and clonal organization within the BM niche, we combined single cell proteogenomics with novel methods for spatially aware variant detection. We characterized the BM clonal organization of a patient with polycythemia vera (PV)-a myeloproliferative neoplasm (MPN). MPNs are blood disorders characterized by abnormal hematopoiesis and an increased risk of transformation to aggressive leukemia. Studying a patient with PV was ideal as this is an oligoclonal disease, and we had prior knowledge that the patient's disease was driven by a large JAK2 V617F mutated clone and a presumed DNMT3A R882Cmutated subclone. Single BM cells were prepared for proteogenomics using the Tapestri platform (Mission Bio). From the BM aspirate, 1,486 individual live BM cells were assayed using the TotalSeq-D Heme Oncology Cocktail V1.0 (BioLegend) for immunophenotyping and the 45 gene myeloid panel (Mission Bio) for genomic characterization. Analysis identified the known JAK2 V617F and DNMT3A R882C mutations, and previously unknown TET2 L1081* and GNAS R844H mutations. The single cell assay disentangled the clonal complexity of this patient's dis