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Introduction Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). These are acquired clonal disorders of hematopoietic stem cells (HSC) leading to the hyperplasia of one or several myeloid lineages. MPN are caused by three main recurrent mutations: JAK2V617F, mutations in the calreticulin (CALR) and thrombopoietin receptor (MPL) genes. Interferon alpha (IFNα) treatment induces not only a hematological response in around 70% of ET, PV and early myelofibrosis, but also a significant molecular response on both JAK2V617F- and CALR-mutated cells. However, a complete molecular response is only achieved in around 20% of patients. Our aim is to predict the long-term efficacy of IFNα in JAK2V617F- and CALR-mutated patients by monitoring the fate of the disease-initiating mutated HSC in order to better stratify the molecular responders. Methods A longitudinal observational study (3-5 years) was performed in 46 IFNα-treated patients. The MPN disease distribution was 42% ET, 47% PV and 11% PMF. We detected 33 patients with JAK2V617F mutation, 11 with CALR mutations (7 type 1/type 1-like and 4 type 2/type 2-like), 1 with both JAK2V617F and CALR mutation and 1 with JAK2V617F, CALR mutation and MPLS505N. At 4-month intervals, the JAK2V617For CALR mutation variant allele frequency was measured in mature cells (granulocytes, platelets). Simultaneously, the clonal architecture was determined by studying the presence of the mutations in colonies derived from the different hematopoietic stem and progenitor cell (HSPC) populations (CD90+CD34+CD38-HSC-enriched, CD90-CD34+CD38- immature and CD34+CD38+committed progenitors). We used a combination of mathematical modeling (Michor et al., Nature, 2005) and Bayesian analysis to infer the long-term behavior of mutated HSC. Results After a median follow-up of 40 months, IFNα targeted more efficiently and more rapidly the HSPC, particularly the HSC-enriched progenitors, than the mature blood cells in JAK2V617Fpatients (p Since it is very difficult to purify true HSC from patients, we used a combination of mathematical and statistical modeling to infer the behavior and the kinetics of IFNα-targeted mutated HSC. The model gave a good fit to the data and indicated that mutated HSC are exhausted slowly (> 1 year) with concomitant increase in mutated HSPC and granulocytes in well-responding patients. We calculated the rate of HSC decrease for each patient. Rates of decrease are very low for heterozygous JAK2V617F and CALR-mutated HSC and greater for homozygous JAK2V617FHSC, but all increase with high IFNα dose (>100 µg/week). Moreover, very low proportion of heterozygous mutated HSC compared to high proportion can be targeted more easily in patients. The associated mutations at diagnosis and at the last timepoint were also investigated using an NGS-targeted myeloid panel. Results indicate that IFNα does not induce any further mutations on additional genes and the mathematical approach predicts that associated mutations have no major impact on the ratio of HSC decrease. Conclusion Altogether, using a rigorous method of statistical inference, our results show that IFNα exhaust the human mutated HSC by differentiation in HSPC and mature cells. This is likely due to IFNα inducing a stronger proliferation of mutated compared to wild-type HSC, as previously shown in a mouse model (Mullally et al., Blood, 2013). Our study predicts that IFNα can slowly eradicate the mutated HSC, but this beneficial effect would be more efficient: i) in patients with homozygous JAK2V617F versus those with heterozygous JAK2V617F or CALR-mutated, ii) with high IFNα dose, iii) in patients with very low proportion of heterozygous JAK2V617F and CALR-mutated HSC. Thus, this study will help to stratify patients for IFNα treatment.These results might also explain the different outcomes in current IFNα clinical trials. Disclosures Constantinescu: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AlsaTech: Other: Co-Founde; AgenDix GmbH: Other: Co-Founder, MyeloPro Research and Diagnostics; Wiley & Sons: Other: Editor in Chief, Journal of Cellular and Molecular Medicine. Kiladjian:AOP Orphan: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Consultancy.

Fusion oncogenes are prevalent in several pediatric cancers, yet little is known about the specific associations between age and phenotype. We observed that fusion oncogenes, such as ETO2-GLIS2, are associated with acute megakaryoblastic or other myeloid leukemia subtypes in an age-dependent manner. Analysis of a novel inducible transgenic mouse model showed that ETO2-GLIS2 expression in fetal hematopoietic stem cells induced rapid megakaryoblastic leukemia whereas expression in adult bone marrow hematopoietic stem cells resulted in a shift toward myeloid transformation with a strikingly delayed in vivo leukemogenic potential. Chromatin accessibility and single-cell transcriptome analyses indicate ontogeny-dependent intrinsic and ETO2-GLIS2-induced differences in the activities of key transcription factors, including ERG, SPI1, GATA1, and CEBPA. Importantly, switching off the fusion oncogene restored terminal differentiation of the leukemic blasts. Together, these data show that aggressiveness and phenotypes in pediatric acute myeloid leukemia result from an ontogeny-related differential susceptibility to transformation by fusion oncogenes. SIGNIFICANCE: This work demonstrates that the clinical phenotype of pediatric acute myeloid leukemia is determined by ontogeny-dependent susceptibility for transformation by oncogenic fusion genes. The phenotype is maintained by potentially reversible alteration of key transcription factors, indicating that targeting of the fusions may overcome the differentiation blockage and revert the leukemic state.See related commentary by Cruz Hernandez and Vyas, p. 1653.This article is highlighted in the In This Issue feature, p. 1631.