Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) are clonal disorders of hematopoietic stem cells (HSC) caused mainly by recurrent mutations in genes encoding JAK2 (JAK2), calreticulin (CALR), or the thrombopoietin receptor (MPL). Interferon alpha (IFNα) has demonstrated some efficacy in inducing molecular remission in MPN. In order to determine factors that influence molecular response rate, we evaluated the long-term molecular efficacy of IFNα in MPN patients by monitoring the fate of cells carrying driver mutations in a prospective observational and longitudinal study of 48 patients over more than 5 years. We measured several times per year the clonal architecture of early and late hematopoietic progenitors (84,845 measurements) and the global variant allele frequency in mature cells (409 measurements). Using mathematical modeling and hierarchical Bayesian inference, we further inferred the dynamics of IFNα-targeted mutated HSC. Our data support the hypothesis that IFNα targets JAK2V617F HSC by inducing their exit from quiescence and differentiation into progenitors. Our observations indicate that treatment efficacy is higher in homozygous than heterozygous JAK2V617F HSC and increases with high IFNα dosage in heterozygous JAK2V617F HSC. Besides, we found that the molecular responses of CALRm HSC to IFNα were heterogeneous, varying between type 1 and type 2 CALRm, and high dosage of IFNα correlates with worse outcomes. Together, our work indicates that the long-term molecular efficacy of IFNα implies an HSC exhaustion mechanism and depends on both the driver mutation type and IFNα dosage.

Introduction: Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocytemia (ET) and Primary Myelofibrosis (PMF). They are acquired clonal disorders of hematopoietic stem cells (HSC) leading to the hyperplasia of one or several myeloid lineages. They are due to three main recurrent mutations affecting the JAK/STAT signaling pathway: JAK2V617F and mutations in the calreticulin (CALR) and thrombopoietin receptor (MPL). Interferon alpha (IFNα) is the only drug that not only induces a hematological response in ET, PV and early MF, but also a significant molecular response on both JAK2V617F or CALR-mutated cells. Our broad aim was to understand the mechanism of action of IFNα. Previously, our group and others have shown that IFNα specifically targets JAK2V617F HSC in a chimeric JAK2V617F knock-in mouse model. In this study, we wanted to know how and how fast IFNα impacts the different mutated human hematopoietic compartments. Methods: A prospective study was performed with a cohort of 47 patients treated by IFNα for 3-5 years. The MPN disease distribution was 40% ET, 49% PV and 11% MF. This cohort included 33 JAK2V617F-mutated patients, 11 CALR-mutated patients (7 type 1/type 1-like and 4 type 2/type 2-like), 2 both JAK2V617F- and CALR-mutated patients and 1 MPLW515K-mutated patient. At 4-month intervals, the JAK2V617F or/and CALR mutation allele frequency was measured in mature cells (granulocytes, platelets). Simultaneously, the clonal architecture was also determined by studying the presence of the JAK2V617F or CALR mutations in colonies derived from the different hematopoietic stem and progenitor cell (HSPC) populations (CD90+CD34+CD38- HSC-enriched progenitors, CD90-CD34+CD38- immature progenitors and CD90- CD34+CD38+ committed progenitors). Results: After a median follow-up of 33 months, IFNα targets more efficiently and rapidly the HSPC particularly in HSC-enriched progenitors, than the mature blood cells in JAK2V617F patients (p<.05). Moreover, homozygous JAK2V617F clones responded more rapidly than heterozygous clones in all hematopoietic cell compartments showing that the intensity of JAK2V617F signaling is correlated with the efficacy of IFNα. This efficacy was slightly increased after a median follow-up of 51 months. In contrast, during a median follow-up of 33 months for CALR-mutated patients, IFNα targeted similarly the HSPC and the mature cells. Moreover, IFNα induced a slower response in targeting CALR-mutated HSPC than the JAK2V617F HSPC (p<.05) (see Figure). The role of associated mutations at diagnosis was also investigated in the IFNα-mediated HSPC molecular responses using a NGS targeted myeloid panel. In JAK2V617F-mutated patients, the number of associated mutations did not impact the HSPC molecular response. In contrast, in CALR-mutated patients, the only molecular responders were not associated with other mutations, although the lower number of cases should be expanded. Using Ba/F3-MPL cellular models and primary cells, we observed that JAK2V617F was more prone to sensitize to IFNα signaling (increased Phospho-STAT1 and IFN-stimulating genes (ISGs)) compared to controls or CALRdel52 mutated cells. Conclusion: Altogether, our results show that IFNα targets more efficiently the human JAK2V617F-HSPCthan the mature cells. Moreover, IFNα has a greater efficacy on JAK2V617F HSPC thanCALR-mutated HSPC. This former result was associated with a greater priming of the IFNα signaling by JAK2V617F than by CALRdel52. The molecular response was dependent not only on mutational status, but also on the presence of other associated mutations for the CALR-mutated HSPC. Patient data are currently incorporated into a mathematical model taking into account clonal architecture and associated mutations to develop an algorythm able to predict patient response. Figure. Figure. Disclosures Kiladjian: Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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