LAUSR

Juvenile Myelomonocytic Leukemia (JMML) is a rare, poor prognosis childhood leukemia characterized by aberrant proliferation of dysplastic myeloid cells and germline or somatic RAS-activating mutations. The only curative treatment is hematopoietic stem cell (HSC) transplantation, however, post-transplant relapse is frequent (~35%) and long-term disease-free survival is ~64%. Approximately half of patients show evidence of clonal evolution with acquisition of additional abnormalities, e.g. mutations of epigenetic/spliceosome components which confer a particularly adverse prognosis. The cellular hierarchy in JMML is poorly characterized with little known about the identity of leukemia stem cells (LSCs), a crucial step towards understanding disease evolution and propagation of relapse. We therefore set out to functionally and molecularly characterize LSCs in a cohort of 16 JMML patients enrolled in the UK NIHR Paediatric MDS/JMML study with samples at diagnosis, post-transplant and relapse. All patients underwent genetic analysis using a novel targeted next generation sequencing (NGS) panel confirming typical RAS pathway mutations in all patients and additional mutations in epigenetic/spliceosome genes in 44% of the patients. Based on adult MDS/MPN, we hypothesized that the CD34+ HSC and progenitor cell compartment (HSPC) would be preserved in JMML and that JMML-LSCs may reside within the phenotypic HSC compartment (Lin-CD34+CD38-CD90+CD45RA-). However, FACS analysis revealed disruption of HSPCs with a 2 fold reduction in phenotypic HSCs (Lin-CD34+CD38-CD90+CD45RA-) and an aberrant Lin-CD34+CD38-CD90+CD45RA+ (90+/45RA+) population not present in normal pediatric bone marrow (BM). In single cell and bulk in vitro assays, JMML-HSPCs had myeloid-biased lineage output with reduced and dysplastic erythroid and severely impaired megakaryocyte and lymphoid potential. Using a novel, integrated single cell genotyping technique combining HSPC index-sorting with clonogenic assays we backtracked patient-specific mutations to HSPC populations, establishing that: 1. all somatic mutations could be backtracked to the phenotypic HSC compartment (492 colonies genotyped for 9 different mutations in 4 patients); and 2. clonal evolution events (eg ASXL1 mutation) in JMML occurred within the HSC compartment with RAS pathway mutations as a "first hit". Moreover, a PTPN11 mutation could be traced back to cells within the HSPC compartment of a post-BMT patient prior to molecular evidence of overt disease relapse in whole BM. We next assessed the stem cell capacity of FACS-purified phenotypic HSPCs in JMML by in vitro long-term culture initiating cell (LTC-IC) assays and in vivo xenotransplantation in NSG mice. LTC-IC assays consistently demonstrated self-renewal capacity in HSCs together with variable LTC potential in the novel 90+45RA+ population. Similarly, transplantation of purified JMML HSCs resulted in higher engraftment at 5, 10, and 16 weeks than 90+45RA+ or GMP populations (16 weeks post BMT: HSC = 91.7% human CD45; 90+/45RA+ = 0.14%; GMP = 0.2%, p Single cell RNA-seq on Lin-CD34+ HSPCs using the Chromium 10X platform (n > 10,000 single cells) showed co-clustering of JMML and normal cells transcriptionally resembling myeloid progenitors expressing MPO and MS4A3, while a distinct cluster of JMML HSPCs uniquely co-expressed fetal genes (e.g. HMGA2), stem cell transcription factors (e.g. GATA2) and myeloid leukemia-associated genes (e.g HEMGN, HOPX, MEF2C). RNA-seq of FACS-purified JMML HSCs also showed upregulation of stem cell and fetal genes (HLF, MEIS1, ABCB1, MPL, and HMGA2) as well as candidate therapeutic targets e.g. MTOR, PIM1, SLC2A1, and CD96 compared to normal HSCs. We confirmed aberrant expression of CD96 in JMML HSCs by flow cytometry, and experiments inhibiting therapeutic targets in vitro and in vivo are underway. In conclusion, we show that JMML HSPCs are organized as a hierarchy, with counterparts of phenotypic HSCs representing disease propagating LSCs. Single cell molecular profiling of JMML HSCs has identified a number of novel potential therapeutic candidates for future clinical development. Disclosures de la Fuente:bluebird bio: Consultancy; HCA: Consultancy; Jazz Pharmaceuticals: Honoraria. Mead:BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau.