Megakaryopoiesis is an original and complex cell process which leads to the formation of platelets. The homeostatic production of platelets is mainly regulated and controlled by thrombopoietin (TPO) and the TPO receptor (MPL)/JAK2 axis. Therefore, any hereditary or acquired abnormality affecting this signaling axis can result in thrombocytosis or thrombocytopenia. Thrombocytosis can be due to genetic alterations that affect either the intrinsic MPL signaling through gain-of-function (GOF) activity (MPL, JAK2, CALR) and loss-of-function (LOF) activity of negative regulators (CBL, LNK) or the extrinsic MPL signaling by THPO GOF mutations leading to increased TPO synthesis. Alternatively, thrombocytosis may paradoxically result from mutations of MPL leading to an abnormal MPL trafficking, inducing increased TPO levels by alteration of its clearance. In contrast, thrombocytopenia can also result from LOF THPO or MPL mutations, which cause a complete defect in MPL trafficking to the cell membrane, impaired MPL signaling or stability, defects in the TPO/MPL interaction, or an absence of TPO production.

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.

Mutations of calreticulin (CALRm) define a subtype of myeloproliferative neoplasms (MPN). We studied the biological and genetic features of CALR-mutated essential thrombocythemia and myelofibrosis patients. In most cases, CALRm were found in granulocytes, monocytes, B and NK cells, but also in T cells. However, the type 1 CALRm spreads more easily than the type 2 CALRm in lymphoid cells. The CALRm were also associated with an early clonal dominance at the level of hematopoietic stem and progenitor cells (HSPC) with no significant increase during granulo/monocytic differentiation in most cases. Moreover, we found that half of type 2 CALRm patients harbors some homozygous progenitors. Those patients were associated with a higher clonal dominance during granulo/monocytic differentiation than patients with only heterozygous type 2 CALRm progenitors. When associated mutations were present, CALRm were the first genetic event suggesting that they are both the initiating and phenotypic event. In blood, type 1 CALRm led to a greater increased number of all types of progenitors compared with the type 2 CALRm. However, both types of CALRm induced an increase in megakaryocytic progenitors associated with a ruxolitinib-sensitive independent growth and with a mild constitutive signaling in megakaryocytes. At the transcriptional level, type 1 CALRm seems to deregulate more pathways than the type 2 CALRm in megakaryocytes. Altogether, our results show that CALRm modify both the HSPC and megakaryocyte biology with a stronger effect for type 1 than for type 2 CALRm.

Heat shock protein 27 (HSP27/HSPB1) is a stress-inducible chaperone that facilitates cancer development by its proliferative and anti-apoptotic functions. The OGX-427 antisense oligonucleotide against HSP27 has been reported to be beneficial against idiopathic pulmonary fibrosis. Here we show that OGX-427 is effective in two murine models of thrombopoietin- and JAKV617F-induced myelofibrosis. OGX-427 limits disease progression and is associated with a reduction in spleen weight, in megakaryocyte expansion and, for the JAKV617F model, in fibrosis. HSP27 regulates the proliferation of JAK2V617F-positive cells and interacts directly with JAK2/STAT5. We also show that its expression is increased in both CD34+ circulating progenitors and in the serum of patients with JAK2-dependent myeloproliferative neoplasms with fibrosis. Our data suggest that HSP27 plays a key role in the pathophysiology of myelofibrosis and represents a new potential therapeutic target for patients with myeloproliferative neoplasms.

The molecular basis of hereditary thrombocytosis is germline mutations affecting the thrombopoietin (TPO)/TPO receptor (MPL)/JAK2 signaling axis. Here, we report one family presenting two cases with a mild thrombocytosis. By sequencing JAK2 and MPL coding exons, we identified a germline MPL R102P heterozygous mutation in the proband and his daughter. Concomitantly, we detected high TPO levels in the serum of these two patients. The mutation was not found in three other unaffected cases from the family except in another proband’s daughter who did not present thrombocytosis but had a high TPO level. The MPL R102P mutation was first described in congenital amegakaryocytic thrombocytopenia in a homozygous state with a loss-of-function activity. It was previously shown that MPL R102P was blocked in the endoplasmic reticulum without being able to translocate to the plasma membrane. Thus, this case report identifies for the first time that MPL R102P mutation can differently impact megakaryopoiesis: thrombocytosis or thrombocytopenia depending on the presence of the heterozygous or homozygous state, respectively. The paradoxical effect associated with heterozygous MPL R102P may be due to subnormal cell-surface expression of wild-type MPL in platelets inducing a defective TPO clearance. As a consequence, increased TPO levels may activate megakaryocyte progenitors that express a lower, but still sufficient level of MPL for the induction of proliferation.

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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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<.05). Moreover, kinetics of response of homozygous JAK2V617FHSPC to IFNα were more rapid than that of heterozygous HSPC and mature cells. This IFNα-specificity towards homozygous HSPC slightly increased after a median follow-up of 51 months. In contrast, during a 40-month median follow-up of CALR-mutated patients, IFNα targeted similarly the HSPC and the mature cells. Moreover, IFNα was less efficient in targeting the CALR-mutated than the JAK2V617FHSPC (p<.05). 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.