ONCO-FETAL REPROGRAMMING DRIVES HIGH-RISK JUVENILE MYELOMONOCYTIC LEUKEMIA, WHICH CAN BE TARGETED BY ANTI-CD52 TREATMENT

Background: Juvenile myelomonocytic leukemia (JMML), a myeloproliferative neoplasm of early childhood, is characterized by genetic activation of RAS signaling. Patients with high-risk JMML have a relapse rate over 50% after allogeneic hematopoietic stem cell transplantation, which demonstrates the urgent need for precise diagnostics and novel therapeutic approaches. Recently, DNA methylation subgroups have been described to resolve the clinical heterogeneity observed in this disease (Schönung et al., Clin Cancer Res 2021). In addition, a prenatal origin of JMML has been suggested in a fraction of patients (Behnert et al., Leukemia 2022). Therefore, we hypothesized that JMML DNA methylation subgroups might originate from different stages of human hematopoietic development. Aims: The aim of this study was to decipher the cellular origins of epigenetic subgroups in JMML and the associated molecular programs in order to identify disease-specific aberrations for diagnostic and therapeutic purposes. Methods: We established a multi-omics approach combining genetic profiling, flow cytometry, single-cell RNA-sequencing, and ultra-low input whole-genome bisulfite sequencing (WGBS) to dissect the subgroup-specific molecular programs in primary JMML patient samples. We validated our findings using an inducible Ptpn11-E76K knock-in mouse and a patient-derived xenotransplantation (PDX) model. Results: Multi-modal analysis demonstrated the conservation of epigenetic subgroups in hematopoietic stem cells (HSCs) of JMML patients. DNA methylation changes identified by WGBS affected binding motifs of developmental transcription factors. This epigenomic dysregulation correlated with an ectopic expression of fetal hematopoietic signature genes in HSCs from high-risk patients, including HMGA2 and fetal hemoglobin, whereas low-risk HSCs expressed typical postnatal HSC markers (AVP, CRHBP). Mapping DNA methylomes of JMML HSCs onto the normal developmental trajectory from fetal to adult HSCs determined that the diseased cells correspond to a post-natal HSC state. However, besides the high similarity to postnatal HSCs, high-risk JMML HSCs were epigenetically more immature than low-risk HSCs and presented fetal-like methylation patterns. To experimentally assess whether these fetal-like programs are re-activated fetal signatures or remainders of a fetal cell-of-origin, we employed an HSC-specific JMML mouse model. We found that postnatal induction of the Ptpn11-E76K mutation resulted in reactivation of fetal gene expression programs in HSCs akin to those observed in high-risk JMML. This suggested that high-risk JMML HSCs hijack molecular programs of fetal HSCs which distinguishes these cells from their healthy counterparts. In line with this, an integrative analysis of human and murine multi-omics data identified several subgroup-specific molecular markers which might serve as novel prognostic biomarkers of high-risk JMML. One of those markers, CD52, was both differentially methylated and highly expressed in HSCs from high-risk JMML. Pre-clinical activity of CD52-targeted therapy with alemtuzumab was tested in a JMML PDX mouse model. We found that treated recipients had reduced engraftment of human cells and 2° recipients had significantly increased survival as compared to controls. Summary/Conclusion: In the present study, we identified onco-fetal reprogramming as a hallmark of high-risk JMML. We determined unique molecular programs which can be used to develop new strategies for the treatment of high-risk JMML and provide pre-clinical evidence for the efficacy of alemtuzumab as a targeted treatment for high-risk JMML. Keywords: JMML, DNA methylation, Leukemic stem cell, Alemtuzumab