Hereditary Thrombocytosis Research Articles

Relative impact of THPO mutation causing hereditary thrombocythemia

Germline mutations of THPO were reported as causes of hereditary thrombocythemia. Six previously reported distinct sites of the mutation were clustered at the 5`-untranslated region or the exon 3 splicing donor site of the THPO gene. Each mutation was identified in an independent pedigree, and the differences between the mutations were not compared. We cloned six distinct THPO mutations (THPO c.-47delG, THPO c.-31G>T, THPO c.13G>A, THPO c.13+1G>A, THPO c.13+2T>C, and THPO c.13+5G>A) and compared the molecular mechanisms that underlie the increased production of THPO protein. At the transcript level, all of the mutations except THPO c.-47delG showed an exon 3 skipping transcript, including two mutations (THPO c.-31G>T and THPO c.13+5G>A) that were distant from the splicing donor site. THPO c.-47delG showed the same full-length transcript as that of the wild-type transcript. At the protein level, all mutations resulted in a higher level of production of thrombopoietin (THPO) protein compared with wild-type THPO. There are only two distinct patterns of mechanisms for increased production of THPO: exon 3 skipping that deleted upstream suppressive open reading frame (ORF)7 and one base deletion that shifted ORF7 to connect to the initial codon of THPO in-frame. The common mechanisms of hereditary thrombocytosis due to THPO mutations are unleashed THPO translations, which are usually suppressed by upstream out-of-frame ORF7.

Germline MPL Mutations May be a Rare Cause of “Triple-Negative” Thrombocytosis

Background Rare cases of primary thrombocytosis are related to inherited genetic abnormalities and are called hereditary thrombocytosis (HT). HT has clinical features resembling sporadic essential thrombocythemia (ET) but has usually an autosomal dominant (AD) inheritance with variable penetrance. To date, germline mutation involving the MPL, THPO, JAK2 genes have been associated with HT. Methods The study included 933 patients evaluated at our Division because of persistent isolated thrombocytosis for whom secondary reactive causes were excluded. Patients were screened for the three canonical somatic driver mutations associated with myeloproliferative neoplasms: JAK2 V617F mutation, CALR exon 9 mutations, MPL exon 10 mutations. Triple-negative cases with unexplained isolated thrombocytosis which also resulted negative for non-canonical exon 10 MPL mutations were analyzed using next generation sequencing (NGS) to test a panel of 80 myeloid genes. Results Of 933 patients with isolated thrombocytosis screened, 567 were JAK2-mutated, 255 CALR-mutated, 41 MPL-mutated, 2 double-mutated and 68 triple-negative. Two MPL-mutated patients carried a non-canonical mutation located in exon 10: MPL W515* and MPL V501A. Of the 68 triple-negative patients, 32 were evaluated by NGS and one of them showed another non-canonical mutation of MPL located outside exon 10: MPL R102P. These 3 non-canonical MPL mutations resulted to be germline as they were detected also in DNA extracted from T lymphocytes and from hair roots. Due to the germline nature of the mutations, a study of the family was carried out. In 2 out of 3 pedigrees at least a second relative carried the same germline mutation discovered in the proband; these 2 pedigrees are shown in the figure. Patient MPC17_15 was an 80 years-old patient with a history of thrombocytosis. She resulted JAK2- and CALR- wild-type while HRM screening was positive for exon 10 MPL mutations; Sanger sequencing identified the MPL W515* mutation in DNA from granulocytes, T-lymphocytes and hair roots, confirming its germline nature. Her children showed a normal complete blood count (CBC) and were MPL wild-type, thus confirming a segregation between mutation and clinical phenotype. Patient MPC14_674 (panel A) was a 36 years-old women with a history of mild thrombocytosis never investigated before. She was referred to our clinic by Pediatricians after the delivery because her baby (MPC17_167) showed persistent isolated thrombocytosis (826 x10 9/L) during the first months of life despite the exclusion of the most common reactive causes of thrombocytosis. Sanger sequencing identified the MPL V501A mutation in granulocyte and in T-lymphocyte in both cases. To evaluate the segregation of MPL V501A with the disease phenotype, we evaluated the CBC and MPL mutation in the other relatives. Both proband's father (MPC23_597) and sister (MPC23_602) carried germline heterozygous MPL V501A mutation associated with a PLT count above or at the upper limit of normal range (570 x10 9/L and 355 x10 9/L, respectively). Conversely, the husband (MPC17_495), the second son (MPC18_43) and the mother (MPC23_603) of the proband showed a normal CBC and were MPL wild-type, confirming an AD inheritance. Patient MPC07_130 (panel B) was a 44 years-old women with triple-negative isolated thrombocytosis lasting since 2007. NGS analysis showed the presence of a heterozygous germline MPL R102P mutation. As this mutation is located in exon 3, it was not detected through HRM and Sanger sequencing which target only exon 10. Her son (MPC13_672), 18 years-old, was completely asymptomatic but the CBC showed mild thrombocytosis (472 x 10 9/L). He tested negative for the 3 driver mutations but Sanger sequencing of MPL exon 3 identified the heterozygous germline MPL R102P mutation. Conclusions We identified 3 non-canonical germline MPL mutations in 3 different unrelated subjects with isolated thrombocytosis: MPL W515*, MPL R102P, and MPL V501A. Germline MPL mutations may underly HT and should be evaluated by sequencing of the whole exon 10 of MPL gene or by NGS in cases with isolated thrombocytosis negative for the three canonical somatic driver mutations. Namely, patients with HT might present as sporadic cases, and in the initial workup they might be inappropriately diagnosed with triple-negative ET. A correct diagnosis is pivotal to avoid unnecessary treatments.

Germline MPL mutations may be a rare cause of “triple-negative” thrombocytosis

Hereditary thrombocytosis (HT) is a rare inherited disorder with clinical features resembling those of sporadic essential thrombocythemia. This study included 933 patients with persistent isolated thrombocytosis for whom secondary reactive causes were excluded. Of 933 patients screened, 567 were JAK2-mutated, 255 CALR-mutated, 41 MPL-mutated, 2 double-mutated, and 68 were triple-negative. Two patients carried germline non-canonical mutations in exon 10: MPL W515* and MPL V501A. One triple-negative patient carried another germline non-canonical MPL mutation outside exon 10: MPL R102P. As germline MPL mutations may be underlying causes of HT, we recommend screening patients with triple-negative isolated thrombocytosis for non-canonical MPL mutations. Although clear evidence concerning HT treatment is still lacking, individuals with HT should probably be excluded from cytoreductive treatment. Thus, an accurate diagnosis is pivotal in avoiding unnecessary treatments.

Identification of putative noncanonical driver mutations in patients with essential thrombocythemia.

Essential thrombocythemia (ET) cases without canonical JAK2, CALR, or MPL mutations, that is, triple-negative (TN) ET, have been found in 10%-20% of ET cases. Owing to the limited number of TN ET cases, its clinical significance remains unclear. This study evaluated TN ET's clinical characteristics and identified novel driver mutations. Among 119 patients with ET, 20 (16.8%) had no canonical JAK2/CALR/MPL mutations. Patients with TN ET tended to be younger and had lower white blood cell counts and lactate dehydrogenase values. We identified putative driver mutations in 7 (35%): MPL S204P, MPL L265F, JAK2 R683G, and JAK2 T875N were previously reported as candidate driver mutations in ET. Moreover, we identified a THPO splicing site mutation, MPL*636Wext*12, and MPL E237K. Four of the seven identified driver mutations were germline. Functional studies on MPL*636Wext*12 and MPL E237K revealed that they are gain-of-function mutants that increase MPL signaling and confer thrombopoietin hypersensitivity with very low efficiency. Patients with TN ET tended to be younger, although this was thought to be due to the inclusion of germline mutations, hereditary thrombocytosis. Accumulating the genetic and clinical characteristics of noncanonical mutations may help future clinical interventions in TN ET and hereditary thrombocytosis.

JAK: Not Just Another Kinase.

The JAK/STAT axis is implicated in cancer, inflammation, and immunity. Numerous cytokines/growth factors affect JAK/STAT signaling. JAKs (JAK1, JAK2, JAK3, and TYK2) noncovalently associate with cytokine receptors, mediate receptor tyrosine phosphorylation, and recruit ≥1 STAT proteins (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6). Tyrosine-phosphorylated STATs dimerize and are then transported into the nucleus to function as transcription factors. Signaling is attenuated by specific suppressor of cytokine signaling proteins, creating a negative feedback loop. Both germline mutations and polymorphisms of JAK family members correlate with specific diseases: Systemic lupus erythematosus (TYK2 polymorphisms); severe combined immunodeficiency (JAK3 mutations); pediatric acute lymphoblastic leukemia (TYK2 mutations); and hereditary thrombocytosis (JAK2 mutations). Somatic gain-of-function JAK mutations mainly occur in hematologic malignancies, with the activating JAK2 V617F being a myeloproliferative disorder hallmark; it is also seen in clonal hematopoiesis of indeterminate potential. Several T-cell malignancies, as well as B-cell acute lymphoblastic leukemia, and acute megakaryoblastic leukemia also harbor JAK family somatic alterations. On the other hand, JAK2 copy-number loss is associated with immune checkpoint inhibitor resistance. JAK inhibitors (jakinibs) have been deployed in many conditions with JAK activation; they are approved in myeloproliferative disorders, rheumatoid and psoriatic arthritis, atopic dermatitis, ulcerative colitis, graft-versus-host disease, alopecia areata, ankylosing spondylitis, and in patients hospitalized for COVID-19. Clinical trials are investigating jakinibs in multiple other autoimmune/inflammatory conditions. Furthermore, dermatologic and neurologic improvements have been observed in children with Aicardi-Goutieres syndrome (a genetic interferonopathy) treated with JAK inhibitors.

Cell-autonomous megakaryopoiesis associated with polyclonal hematopoiesis in triple-negative essential thrombocythemia

A subset of essential thrombocythemia (ET) cases are negative for disease-defining mutations on JAK2, MPL, and CALR and defined as triple negative (TN). The lack of recurrent mutations in TN-ET patients makes its pathogenesis ambiguous. Here, we screened 483 patients with suspected ET in a single institution, centrally reviewed bone marrow specimens, and identified 23 TN-ET patients. Analysis of clinical records revealed that TN-ET patients were mostly young female, without a history of thrombosis or progression to secondary myelofibrosis and leukemia. Sequencing analysis and human androgen receptor assays revealed that the majority of TN-ET patients exhibited polyclonal hematopoiesis, suggesting a possibility of reactive thrombocytosis in TN-ET. However, the serum levels of thrombopoietin (TPO) and interleukin-6 in TN-ET patients were not significantly different from those in ET patients with canonical mutations and healthy individuals. Rather, CD34-positive cells from TN-ET patients showed a capacity to form megakaryocytic colonies, even in the absence of TPO. No signs of thrombocytosis were observed before TN-ET development, denying the possibility of hereditary thrombocytosis in TN-ET. Overall, these findings indicate that TN-ET is a distinctive disease entity associated with polyclonal hematopoiesis and is paradoxically caused by hematopoietic stem cells harboring a capacity for cell-autonomous megakaryopoiesis.

The S505A thrombopoietin receptor mutation in childhood hereditary thrombocytosis and essential thrombocythemia is S505N: single letter amino acid code matters

The S505A thrombopoietin receptor mutation in childhood hereditary thrombocytosis and essential thrombocythemia is S505N: single letter amino acid code matters

Next-Generation Sequencing (NGS) in Childhood Myeloproliferative Diseases (MPD)

Driver mutations of JAK2, CALR and MPL are found in >90% of adults with BCR-ABL1-negative myeloproliferative neoplasms (MPN). In children, the presence of clonal markers ranges between 22 and 40%, and inherited forms of MPD, such as familial erythrocytosis (FE) and hereditary thrombocytosis (HT), are common. Data on the mutational spectrum and biology of childhood MPD are limited. The aims of this study were: a) to evaluate the ability of a next-generation sequencing (NGS)-based 44-gene analysis to better characterize wild type (WT) MPD, and b) to identify non-canonical and/or non-driver mutations in children and adolescents with MPD.Eighty patients (pts) aged ≤20 years (yrs) at diagnosis of MPD, observed between June 1980 and September 2015, were first investigated with standardized methods for driver mutations of MPN (JAK, MPL, CALR), for genes involved in FE (HRE, EpoR, HIF2α, HIF1α, VHL, PHD1-3, STAT5, LNK, TET2) and HT (THPO, MPL, LNK and TET2). Then, a 44-gene panel providing diagnostic information in myeloid malignancies and in rare inherited erythrocytosis/thrombocytosis (JAK2, CALR, MPL, ASXL1, CBL, C-Kit, CSF3R, CUX1, DNMT3A, ETNK1, EZH2, IDH1, IDH2, IKZF1, KRAS, LNK, NFE2, NRAS, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1, ZRSR2, BPGM, EGLN1 (PHD2), EPAS1 (HIF2A), EPOR, GATA1, GELSOLIN, HBA1, HBA2, HBB, JAK2,MPL, RUNX1, SH2B3, SRC, THPO, VHL, WAS) was employed to better characterize these diseases. Sequencing analyses of DNA from mononuclear peripheral blood cells were performed in 57/80 pts.Eighty pts (M 41, F 39; median age at diagnosis: 149/12 yrs, range 3 months-1911/12 yrs), investigated by standardized methods, were retrospectively classified according to the WHO 2016 criteria as follows: 35 essential thrombocythemia (ET) (10 JAK2V617F, 2 CALR type1, 6 CALR type2, 1 CALR atypical, 16 WT), 9 polycythemia vera (PV) (4 JAK2V617F, 5 WT) and 3 primary myelofibrosis (PMF) (1 JAK2V617F, 2 WT). Twenty-three pts with MPLS505N or MPLV501A mutations and 10 pts with HIF mutations (3 pts) and/or anamnestic criteria of FE (7 WT) were considered HT and FE, respectively. The NGS-based 44-gene panel was applied to 57 MPD pts (11 JAK2V617F, 6 CALR, 12 MPLS505N, 2 MPLV501A, 3 HIF2α and 23 WT). According to the WHO 2016 criteria, 27 pts were ET, 14 HT, 8 FE, 7 PV and 1 PMF. By using the NGS panel, clonal markers were found in 12/23 (52%) pts with MPN WT: HBB and PDH2 in 2 FE, MPLW515_P518>KT in 1 ET pt and non-driver mutations in 9 pts (7 ET, 1 PF and 1 PV). Furthermore, two non-canonical driver mutations, MPLC322G and JAK2G301R were identified in 1 CALR type2 ET and in 1 JAK2V617F PV, respectively. An additional MPLV501M mutation was found in 1 MPLS505N HT. Taken together, among the 57 pts 18 (32%) had one (11/18=68%) or two (7/18=39%) non-driver mutations. Eight of the 34 pts (23.5%) with a clonal marker had additional non-driver mutations, that was single in 6 pts. Within the familial MPD, a single non-driver mutation was found in 3/8 FE pts (37.5%), while no mutations were detected in HT pts. Considering the functional classification of non-driver mutations, we found mutations in signaling (CBL, LNK/SH2B3, CSF3R, KIT, SETBP1) and splicing (U2AF1, ZRSR2) genes in ET and PMF pts, and mutations of epigenetic regulation genes (TET2, ASXL1, DNMT3A) in PV, FE and ET pts (Table 1). The co-occurrence of driver and non-driver mutations in the same individual is illustrated in the circos plot (Figure 1).The use of a NGS-based 44-gene panel in acquired and familial pediatric MPD enabled to identify driver and non-driver mutations, not otherwise detected by conventional methods, with a substantial proportion of MPD pts (81%) showing mutations in the genes analyzed. Interestingly, we found additional neoplastic mutations in some pts with FE. Although the utilized NGS-based panel proved useful to better characterize children and adolescents with MPD, 19% of our pts still remain without any identified clonal marker. Further targeted NGS and whole genome sequencing may enable to better define MPD children without molecular markers. [Display omitted] DisclosuresMalaspina:Sapienza University, Rome: Other: Resident in Hematology. Foà:ABBVIE: Other: ADVISORY BOARD, Speakers Bureau; CELGENE: Other: ADVISORY BOARD, Speakers Bureau; AMGEN: Other: ADVISORY BOARD; INCYTE: Other: ADVISORY BOARD; NOVARTIS: Speakers Bureau; ROCHE: Other: ADVISORY BOARD, Speakers Bureau; GILEAD: Speakers Bureau; JANSSEN: Other: ADVISORY BOARD, Speakers Bureau; CELTRION: Other: ADVISORY BOARD.

An incomplete trafficking defect to the cell-surface leads to paradoxical thrombocytosis for human and murine MPL P106L

AbstractThe mechanisms behind the hereditary thrombocytosis induced by the thrombopoietin (THPO) receptor MPL P106L mutant remain unknown. A complete trafficking defect to the cell surface has been reported, suggesting either weak constitutive activity or nonconventional THPO-dependent mechanisms. Here, we report that the thrombocytosis phenotype induced by MPL P106L belongs to the paradoxical group, where low MPL levels on platelets and mature megakaryocytes (MKs) lead to high serum THPO levels, whereas weak but not absent MPL cell-surface localization in earlier MK progenitors allows response to THPO by signaling and amplification of the platelet lineage. MK progenitors from patients showed no spontaneous growth and responded to THPO, and MKs expressed MPL on their cell surface at low levels, whereas their platelets did not respond to THPO. Transduction of MPL P106L in CD34+ cells showed that this receptor was more efficiently localized at the cell surface on immature than on mature MKs, explaining a proliferative response to THPO of immature cells and a defect in THPO clearance in mature cells. In a retroviral mouse model performed in Mpl−/− mice, MPL P106L could induce a thrombocytosis phenotype with high circulating THPO levels. Furthermore, we could select THPO-dependent cell lines with more cell-surface MPL P106L localization that was detected by flow cytometry and [125I]-THPO binding. Altogether, these results demonstrate that MPL P106L is a receptor with an incomplete defect in trafficking, which induces a low but not absent localization of the receptor on cell surface and a response to THPO in immature MK cells.

Germline Calr Mutation and Thrombocytosis Presenting with Concomitant BCR-ABL1+ CML

CALR mutations are present in 70-84% of JAK2 wild-type myeloproliferative neoplasms (MPN) and 67% and 88% of essential thrombocytopenia (ET) and primary myelofibrosis (PMF) respectively. Most cases of MPN are apparently sporadic, but 7-11% have evidence of familial predisposition. While germline mutations in ET-associated genes, MPL and JAK2, have been described in hereditary thrombocytosis, germline mutations in CALR have not been described in any setting. Two types of CALR mutations are common in MPN: a 52-base pair deletion (bp) and a 5 bp insertion, both in exon 9. With rare exceptions, CALR mutations are generally mutually exclusive with JAK2 or MPL mutations and have very rarely been reported in conjunction with the BCR-ABL1translocation.Here, we report a patient with a germline CALR mutation, thrombocytosis, and subsequent development of BCR-ABL+ CML. A 67-year-old female with no significant medical history presented with severe abdominal pain and nausea. Peripheral blood analysis revealed a marked leukocytosis composed of 66% neutrophils, 16% myelocytes, 6.5% monocytes, 3.5% basophils, 2.5% promyelocytes, 2.5% metamyelocytes, 1.5% lymphocytes, 1.5% blasts, and no eosinophils. The patient was non-anemic and had a normal platelet count (340,000/mm3). Bone marrow biopsy revealed a hypercellular marrow with myeloid predominant trilineage hematopoiesis and 1-2% blasts with morphology consistent with chronic myelogenous leukemia (CML). Fluorescence in-situ hybridization analysis of peripheral blood identified a BCR-ABL1fusion in 98.5% of interphase cells.After 3 months of standard imatinib therapy, quantitative RT-PCR showed a reduction of BCR-ABL1/ABL1 in the peripheral blood, however platelet count was elevated at 539,000/mm3. Thrombocytosis persisted over 2 years with a maximal platelet count of 584,000/mm3. Given the patient's thrombocytosis, her peripheral blood was subjected to a next generation sequencing of JAK2, MPL, and CALR genes. A 52-bp out-of-frame deletion in exon 9 of the CALR gene was detected (52% allele frequency) in peripheral blood. In addition, the same 52-bp CALR deletion (63% allele frequency) was present at the time of diagnosis and within a buccal specimen (47% allele frequency) when the BCR-ABL1 transcript was 1% in the peripheral blood. Immunostain of the buccal sample was strongly positive for cytokeratin (CK) AE1/AE3 but CD45 was not detected indicating no leukocyte contamination. This case reports the first instance of a germline CALR mutation associated with thrombocytosis and is the fourth report of the co-occurrence of BCR-ABL1 and CALR mutation in a single patient. Evolution to BCR-ABL1+ CML suppressed the CALR-mutant thrombocytosis phenotype, emphasizing the effect of these genes on lineage determination in abnormal myeloid proliferation. DisclosuresNo relevant conflicts of interest to declare.

Cooperation of germ line JAK2 mutations E846D and R1063H in hereditary erythrocytosis with megakaryocytic atypia

AbstractThe role of somatic JAK2 mutations in clonal myeloproliferative neoplasms (MPNs) is well established. Recently, germ line JAK2 mutations were associated with polyclonal hereditary thrombocytosis and triple-negative MPNs. We studied a patient who inherited 2 heterozygous JAK2 mutations, E846D from the mother and R1063H from the father, and exhibited erythrocytosis and megakaryocytic atypia but normal platelet number. Culture of erythroid progenitors from the patient and his parents revealed hypersensitivity to erythropoietin (EPO). Using cellular models, we show that both E846D and R1063H variants lead to constitutive signaling (albeit much weaker than JAK2 V617F), and both weakly hyperactivate JAK2/STAT5 signaling only in the specific context of the EPO receptor (EPOR). JAK2 E846D exhibited slightly stronger effects than JAK2 R1063H and caused prolonged EPO-induced phosphorylation of JAK2/STAT5 via EPOR. We propose that JAK2 E846D predominantly contributes to erythrocytosis, but is not sufficient for the full pathological phenotype to develop. JAK2 R1063H, with very weak effect on JAK2/STAT5 signaling, is necessary to augment JAK2 activity caused by E846D above a threshold level leading to erythrocytosis with megakaryocyte abnormalities. Both mutations were detected in the germ line of rare polycythemia vera, as well as certain leukemia patients, suggesting that they might predispose to hematological malignancy.

In Vitro Functional Rescue of a Double MPL K39N/W272R Mutant Associated with Congenital Amegakaryocytic Thrombocytopenia (CAMT) Using Crispr/Cas9

Introduction: Thrombopoietin (Tpo) and its receptor, Mpl, are the principal regulators of early and late thrombopoiesis. Mutations in MPL can drastically impair its function and be a contributing factor in chronic thrombocytosis and in congenital amegakaryocytic thrombocytopenia (CAMT). CAMT is a rare inherited syndrome characterized by thrombocytopenia at birth, progressing to bone marrow failure and pancytopenia. The functional impact of CAMT mutations on Mpl signaling/trafficking is yet to be determined and could be relevant to multiple hematologic malignancies. Here we report unique familial cases of CAMT presenting with a previously unreported MPL mutation: T814C (W272R) in the background of the MPL K39N (Baltimore mutation), known to cause hereditary thrombocytosis (HT).Patients: Consanguineous parents and their eldest daughter, all heterozygous for Mpl K39N/W272R, do not present any signs of disease. Their monozygotic twin daughters presented at birth with severe thrombocytopenia (platelet counts: 12,000/L and 14,000/L), low hemoglobin levels (10.4 mg/dL and 7.8 mg/dL) and very high Tpo levels (3,650 pg/mL and 3115 pg/mL). Bone marrow smears performed 19 days after birth showed severe megakaryocytopenia (only 1 megakaryocyte (MK) seen). Bone marrow colony formation assays yielded 3 and 9 MK colonies vs 84 MK colonies/105 mononuclear cells for the control, leading to a diagnosis of CAMT type I. Whole blood sequencing revealed the presence of a homozygous double MPL K39N/W272R mutation. One of the twins died after bone marrow transplant. A younger male sibling, homozygous for MPL K39N/W272R mutation, has also been diagnosed with CAMT type I.Objectives: This study focuses on the functional characterization of the novel MPL W272R and K39N/W272R mutations and in vitro genetic engineering as a potential therapeutic option for CAMT.Methods: Human megakaryoblastic UT-7 and murine Ba/F3 cells stably expressing human wild-type (WT) Mpl or K39N, W272R or doubly mutated K39N/W272R Mpl fused to mNeonGreen were used as models. Confocal microscopy, proliferation and surface biotinylation assays, as well as co-immunoprecipitation (co-IP) and western blotting analysis, were used to elucidate the function and trafficking of Mpl mutants. CRISPR/Cas9 genetic engineering was used to repair mutant MPL and rescue its function.Results: Confocal microscopy shows that a significant fraction of chimeric WT Mpl protein reaches the cell surface. Significant surface expression is also noted for Mpl K39N. In contrast, the chimeric Mpl protein bearing the W272R mutation, alone or together with the K39N mutation, showed no detectable surface expression of the Tpo receptor. These results were confirmed by surface biotinylation assay. Co-expression of WT CALR fused to RFP, used as an ER marker, showed significantly higher co-localization (Pearson's R value) with mutant Mpl than with WT Mpl, evidence that the large majority of receptors were retained within the ER. We also evaluated Tpo signaling through the JAK/STAT, MAPK and PI3K pathways and Tpo-induced proliferation. Both WT and K39N-mutated Mpl were competent for signaling, while single or double mutants bearing W272R were unresponsive to Tpo. Tpo-induced signaling was partially restored via GRASP55 over-expression (forcing ER-trapped Mpl to traffic to the cell surface). Genetic engineering performed on cells carrying the W272R mutation restored the WT sequence and the response to Tpo, with similar cell proliferation as WT Mpl cells. In addition, co-IP studies indicate that Jak2 associates strongly with WT Mpl and Mpl K39N but not with Mpl W272R.Conclusion: We report a new mutation of Mpl (W272R) present in cis with HT-causing K39N mutation in the context of CAMT. The absence of symptoms in the Mpl K39N/W272R-mutated parents (found to be heterozygous) can be explained by the opposite (apparently neutralizing) effects of the two mutations on the trafficking and signaling of Mpl, as shown by confocal microscopy, western blotting and proliferation assays. In children homozygous for Mpl K39N/W272R, the W272R mutation prevented Mpl binding of Jak2 and expression at cell surface, rendering Tpo signaling impossible despite the presence of the K39N mutation. Function of the deficient Mpl receptor could be rescued using two separate approaches: CRISPR/Cas9 genetic engineering and GRASP55 over-expression. Cell-permeable Tpo analogs will also be tested. DisclosuresNo relevant conflicts of interest to declare.

Mpl Gain-of-Function Mutations Can be Classified By Differential Subcellular Processing, Molecular Mechanisms, Mode of Inheritance and Clinical Impact

The interaction between the c-Mpl receptor and its ligand thrombopoietin (TPO) on the cell surface is crucial for the regulation of thrombopoiesis. Several mutations in the c-Mpl receptor gene have been linked to a gain-of-function resulting in thrombocytosis. We have analyzed the known gain-of-function mutations in the extracellular part of the Mpl receptor, K39N and P106L, as well as the S505N, W515K and W515L mutations in the transmembrane and juxtamembrane region, respectively. Interestingly, the latter mutations can occur as autosomal dominant and/or as somatic mutations and are known to be associated with myeloproliferative malignancies and AML, whereas the abundant K39N and the P106L mutations are the cause of autosomal recessive hereditary thrombocytosis without a known predisposition to hematologic malignancies. To date, these differences in clinical impact and mode of inheritance are poorly understood.Starting from these clinical observations, we have performed functional analyses of the described gain-of-function mutations to address the key functional properties that might explain the observed clinical differences. Three crucial stages of the c-Mpl receptor life cycle were addressed: (1) post-translational processing of the immature receptor protein and its subcellular distribution, (2) membranous expression of the mature receptor and (3) receptor internalization upon stimulation with its ligand TPO.We first analyzed the post-translational processing of the normal, the K39N and the P106L mutated receptor in comparison with receptors carrying the S505N, the W515K and W515L mutations in a HeLa cell culture model. The normal, the K39N, S505N, W515K and W515L mutated c-Mpl receptors were properly glycosylated during their transport through the Golgi apparatus, whereas the P106L mutated receptor did not enter the Golgi and was not fully glycosylated. The K39N mutant was fully glycosylated but did show different running behavior on the SDS Gel, most likely caused by post-translational modifications different from glycosylation. The S505N, the W515K and the W515L mutated receptors displayed stable surface expression in confocal microscopy and FACS analysis, whereas the P106L mutated receptor was not detectable on the cell surface. After stimulation with TPO, a decrease in mean receptor surface protein could be observed for the wild type and all mutants that were expressed on the surface, namely S505N, W515K and W515L, however not significant (p>0.05). Interestingly, our functional analyses of the TPO/c-Mpl signaling pathways in TPO stimulated c-Mpl transfected BA/F3 cells showed activation of the ERK1/2 pathway in all mutants but only weaker activation of the PI3K/m-TOR and Stat3/5 signaling pathways for the P106L mutant. By contrast, cells transfected with the wild type, the S505N, W515K and W515L c-Mpl mutants showed predominant up-regulation of the PI3K/m-TOR and Stat3/5 pathways.These results show that first, both impaired and regular receptor glycosylation and correlating subcellular distribution may occur in c-Mpl gain-of function mutants. Second, the c-Mpl gain-of-function mutants differ substantially in surface expression levels. Third, our results suggest differences in the maintenance of the TPO negative feedback loop across c-Mpl gain-of-function mutants. Indeed, in contrast to P106L, it seems likely that the TPO negative feedback-loop is preserved in the S505N, the W515K and the W515L mutants. In line with this, highly elevated TPO serum levels have only been described for P106L, but not for the other gain-of-function mutations. We hypothesize that maintenance of the TPO negative feedback-loop is sufficient to prevent dysregulation of TPO levels but not transmission of a harmful c-Mpl gain-of-function effect. Instead, the predominant activation of the PI3K/m-TOR and Stat3/5 pathways might explain the different propensity to induce hematopoietic malignancy.In summary, our findings suggest the existence of different disease causing molecular mechanisms behind the mutations' respective clinical correlates and provide the basis for an important extension to the current classification of c-Mpl mutations that is primordially based on clinical observations. DisclosuresNo relevant conflicts of interest to declare.

JAK2V617I results in cytokine hypersensitivity without causing an overt myeloproliferative disorder in a mouse transduction–transplantation model

A germline JAK2(V617I)point mutation results in hereditary thrombocytosis and shares some phenotypic features with myeloproliferative neoplasm, a hematologic malignancy associated with a somatically acquired JAK2(V617F) mutation. We established a mouse transduction-transplantation model of JAK2(V617I) that recapitulated the phenotype of humans with germline JAK2(V617I). We directly compared the phenotypes of JAK2(V617I) and JAK2(V617F) mice. The JAK2(V617I) mice had increased marrow cellularity with expanded myeloid progenitor and megakaryocyte populations, but this phenotype was less severe than that of JAK2(V617F) mice. JAK2(V617I) resulted in cytokine hyperresponsiveness without constitutive activation in the absence of ligand, whereas JAK2(V617F) resulted in constitutive activation. This may explain why JAK2(V617I) produces a mild myeloproliferative phenotype in the mouse model, as well as in humans with germline JAK2(V617I) mutations.

Primary Trombocythemia in Children and Adolescents Includes Different Subtypes Compared to Adult Essential Thrombocythemia

The recent discovery of various mutations of the CALR gene that are mutually exclusive with JAK2 and MPL mutations has allowed a correct diagnosis in about 90% of adult cases of essential thrombocythemia (ET). Moreover, the mutation status of JAK2 and CARL defines subtypes of ET in adults with a substantially different clinical course and outcome. Based on our experience, we suggested that primary thrombocythemia (PT) in children is characterized by subtypes that differ from those found in adult ET. The present study was carried out in children and adolescents with PT in order to (a) characterize the various subtypes of the disease and (b) analyze their clinical and biologic features, treatment approach and outcome.PT patients aged <20 years (yrs) at diagnosis (dx) were evaluated for mutations of JAK2, thrombopoietin (TPO) and its receptor (MPL) and CALR genes, and for clonal hematopoiesis (females). The presence of MPLS505A (confirmed on DNA from buccal swabs) defined a hereditary thrombocytosis (HT). ET was diagnosed according to WHO 2008 criteria. For wild type patients, an additional inclusion criteria was a follow-up >24 months.Among 58 PT patients (males: 23; females: 35; median age at dx: 14.4 yrs), 21 (36%) had HT due to MPLS505A, 14 were JAK2V617F-mutated (24%), 9 (16%) harbored CALR mutations and 14 (24%) were wild type for JAK2, CALR and MPL (Fig 1).JAK2- and CALR-mutated were older than those with wild type ET or with HT (median age, 17.6 and 16.1 vs 10.4 and 13.7 yrs, p .028). As to the hematologic findings, HT patients showed both hematocrit values (median, 36.3%) and leukocytes counts (median, 9.53 x109/L) significantly lower than ET patients, whatever the subtypes (median, 41.2% and 11.2 x109/L, p .006 and p .029, respectively). No differences were found with regard to platelets both between HT and ET and among the different ET subtypes. JAK2-mutated patients exhibited more frequently symptoms (69%) compared to CALR-mutated (22%), wild-type ET (14%) and HT (14%) patients (p. 0057). Splenomegaly at diagnosis was recorded more frequently in JAK2-mutated than in CALR-mutated or wild type-ET or HT (50%, 33% 21% and 14% , respectively, p .122).Antiplatelet agents, mostly acetylsalicylic acid (ASA), were started less frequently in HT than in ET patients, irrespective of the subtypes (57% vs 81%, p .05). The use of ASA progressively decreased over the time; at the last follow-up, 2 patients with HT, 2 CALR-mutated and 1 JAK2-mutated patients were still receiving ASA, while no wild type ET patient was on treatment. Cytoreductive agents, hydroxyurea and/or interferon and/or anagrelide, were used in a minority of HT patients (19%) in comparison with ET patients (65%), p .001, mainly with those wild-type (78%, p <.001). At the last observation, one HT patient was still receiving cytoreductive agents compared to 30% of ET patients whatever the subtypes (p .024). After a median follow-up of 196 months (similar in the different subtypes), all patients are alive. On the whole, 5 thrombotic events were recorded in 3 patients with HT and in 2 ET patients (1 JAK2-mutated and 1 JAK2 and CALR wild-type), without any significant thrombophilic abnormalities during treatment with ASA and/or cytoreductive agents. A progressive splenomegaly was recorded in 9 (15%) patients (2 HT, 4 JAK2-mutated, 3 CALR-mutated) and it was combined with grade ≥2 medullar fibrosis in 2/4 JAK2-mutated and in 2/3 CALR-mutated patients. None of the JAK2 and CALR wild-type patients had spleen enlargement or reticulin fibrosis (p .022). Two untreated patients (1 HT and JAK2 and CALR wild-type) developed malignancies.On the whole, these data emphasize that in young patients with PT, hereditary forms can be frequently observed. Thrombotic events, recorded mainly in HT patients despite treatment with ASA, were probably due to a MRP4 protein overexpression that was found in our MPLS505A HT. Moreover, our observations highlight that, in contrast to adult ET, more than one third of young ET patients have no JAK2 or CALR mutations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

JAK2 E846D Germline Mutation Associated with Erythrocytosis Causes Increased and Prolonged Epo-Induced Activation of STAT5

Until now, disease-causing JAK2 mutations have been associated with clonal myeloproliferative neoplasms (acquired mutations) or with polyclonal hereditary thrombocytosis (congenital mutations). Here we present the first example of a JAK2 mutation associated with congenital erythrocytosis.We studied a young patient with congenital erythrocytosis who inherited two heterozygous mutations, an E846D mutation in JAK2 from the mother and a Q157H in PHD2 from the father. Each mutation apparently sufficed to induce EPO-hypersensitivity in BFU-E progenitors, since BFU-Es from both parents were hypersensitive to EPO, as were those of the patient. Strikingly, only the patient, but not his parents exhibited an erythrocytosis phenotype.Expression analysis of granulocyte mRNA revealed markers of inappropriately increased signaling mediated by hypoxia sensing pathway (HIF) (Kapralova, Blood, 2014,16;123:391-4) in the propositus and his father. This result suggested that the polycythemia phenotype of the propositus could result from a combined impact of otherwise clinically silent defects co-inherited from both parents and affecting EPOR/JAK2 and HIF signaling pathways.Using a Ba/F3-EPOR cellular model we have characterized the impact of JAK2 E846D on EPO-induced JAK2-mediated signaling, cell cycle progression and cell responsiveness to limited concentrations of EPO. We demonstrated prolonged, EPO-dependent JAK2 and STAT5 activation caused by JAK2 E846D, which was mitigated by a JAK2 inhibitor, AZ-960. However, in contrast to the oncogenic V617F mutation, E846D did not trigger factor-independent constitutive signaling. Also unlike the oncogenic V617F or the JAK2 germline mutations associated with hereditary thrombocytosis, the E846D did not activate STAT1, in agreement with differential signaling of STATs in different clinical phenotypes associated with activating JAK2 mutations. The thrombocytosis phenotype (ET for acquired and hereditary thrombocytosis for germline) is promoted by STAT1 signaling, while STAT5 signaling results in erythroid hyper-proliferation.Using BrdU incorporation assay and growth factor starved Ba/F3 cells we also demonstrated that JAK2 E846D - when compared with JAK2 wild type - stimulates more rapid re-entry of the transfectants into the S phase in response to low concentration of EPO. In agreement with immunoblot analyses JAK2 E846D did not support growth factor-independent proliferation in cytokine-free media; however it provided improved survival in limiting concentrations of EPO. The proliferation advantage of JAK2 E846D transfectants was diminished by AZ-960. These results showed that E846D substitution lacks features of a constitutive gain-of-function mutation and were consistent with its germline transmission and with non-clonal hematopoiesis observed in the JAK2 E846D-positive mother.Structural modeling based on the X-ray crystal structures of inactive and active kinase domain explain the mechanism of hypersensitivity and prolonged signaling. The homologous residue of E846 in the JAK2 pseudokinase domain (JH2) is N542, a residue that is targeted by the exon 12 activating mutations in polycythemia vera, and a close residue to this homologous position in JH2 is R564 recently found to be mutated in hereditary thrombocytosis. Thus the E846D mutation belongs to a conformational hotspot in kinases, but this mutation is only inducing hypersensitivity and prolonged action and not constitutive activation.In conclusion, JAK2 E846D mutation associated with congenital erythrocytosis confers hypersensitivity to EPO, prolonged and selective but not constitutive STAT5 activation, and requires additional cooperating event, likely affecting the HIF signaling, for its full clinical expression.(KK, MH, JK contributed equally to this work.)Funding: Czech Science Foundation P301/12/1503, Czech Ministry of Health NT13587-4, Education for Competitiveness Operational Program CZ.1.07/2.3.00/20.0164 and Palacky University (IGA_LF_2014_011). JK was supported by CZ.1.07/2.3.00/30.0041, EL by a FRIA PhD fellowship. SNC was supported from the F.R.S.-F.N.R.S., Belgium, the Salus Sanguinis Foundation, the Action de Recherche Concertée projects MEXP31C1 and ARC10/15-027 of the University catholique de Louvain, Brussels, the Fondation contre le Cancer, Brussels, the PAI Programs BCHM61B5 and Belgian Medical Genetics Initiative. DisclosuresConstantinescu:Shire: Consultancy, Speakers Bureau; Novartis: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Personal Genetics: Membership on an entity’s Board of Directors or advisory committees; Amgen: Speakers Bureau.

Expression of JAK2V617I Does Not Result in Overt Myeloproliferative Disorder, but Results in Cytokine Hypersensitivity

BACKGROUNDA germline JAK2V617I mutation has recently been identified in a family with hereditary thrombocytosis (Mead et al, NEJM 2012). Like acquired MPN, family members with JAK2V617I have thrombocytosis and megakaryocytic hyperplasia in the marrow with increased risk of thrombosis. But unlike acquired MPN, individuals with this germline mutation do not develop a fibrotic bone marrow, splenomegaly, or transform to acute leukemia. Why germline JAK2V617I recapitulates some aspects but not others of the MPN phenotype in humans is unclear. To delineate the differences between JAK2V617F and JAK2V617I we compared the phenotype of mice with hematopoietic cells expressing JAK2V617F or JAK2V617I. METHODS AND RESULTSLethally irradiated C57B/6 mice were transplanted with bone marrow cells infected with retrovirus expressing JAK2V617F, JAK2V617I, or empty MSCV-IRES-GFP (MIG) vector. As expected, mice transplanted with JAK2V617F-expressing cells developed erythrocytosis and leukocytosis, whereas mice transplanted with JAK2V617I-expressing cells had peripheral blood counts similar to empty vector mice. Humans with germline JAK2V617I do not display constitutive activation of the kinase, but they do demonstrate cytokine hyper-responsiveness as evidenced by increased phosphorylation of STATs at low concentrations of ligand. We compared phosphorylated STAT5 in peripheral blood of mice transplanted with JAK2V617I, JAK2V617F, and MIG empty vector following stimulation with increasing concentrations of GM-CSF. At all concentrations of GM-CSF tested JAK2V617I and JAK2V617F-expressing cells had exaggerated phosphorylation of STAT5 as compared to MIG empty vector mice. We also measured phospho-STAT3 and STAT5 in unstimulated bone marrow and spleen from each mouse at time of euthanasia, there was no difference between JAK2V617I and MIG empty vector mice. JAK2V617F mice did demonstrate phosphorylation of STAT3 and STAT5 in the absence of GM-CSF, confirming the ability of JAK2V617F but not JAK2V617I to constitutively activate downstream signaling pathways. Next, to evaluate for histologic evidence of MPN and assess spleen size, all mice were euthanized at 120 days post-transplant. JAK2V617F mice had splenomegaly as expected, spleens from JAK2V617I mice appeared larger than empty vector mice, but spleen weight was not statistically different (p>0.05). While JAKV617I mice had increased cellularity of their marrow with increased numbers of megakaryocytes as compared to empty vector mice, this was not nearly to the extent of JAK2V617F mice. Mild fibrosis was seen in JAK2V617I mice, JAK2V617F mice had severe reticulin fibrosis in the marrow as expected. In the spleen architecture was preserved in the JAK2V617I mice, whereas in the JAK2V617F mice splenic architecture was disrupted by invasion of myeloid cells including megakaryocytes.To identify whether JAK2V617I affects the frequency of stem and progenitor cells or expands mature myeloid lineage cells we measured the frequency of hematopoietic stem cells, myeloid progenitors, and mature myeloid populations in JAK2V617F, JAK2V617I, and MIG empty vector. The bone marrow of JAK2V617I mice contained an increased percentage of GMP and MEP populations as compared to both the MIG empty vector and the JAK2V617F mice. Mature granulocyte (Gr-1+CD11b+) and erythroid (Ter119+) populations were expanded in the bone marrow and spleen of JAK2V617F but not JAK2V617I mice. CONCLUSIONSThese data demonstrate that the JAK2V617I mouse model recapitulates the effect of germline expression of JAK2V617I seen in humans: it results in cytokine hyper-responsiveness without the ability to constitutively activate downstream signals in the absence of ligand. Why JAK2V617F is so exquisitely conserved in acquired MPN is still unknown. DisclosuresNo relevant conflicts of interest to declare.

The Predominant Activation of Alternative Signaling Pathways Explains the Differential Propensity of Different c-Mpl Gain-of-Function Mutations to Promote Myeloproliferative Malignancies

Thrombopoiesis is tightly regulated by the binding of thrombopoietin (THPO) to its receptor c-Mpl that leads to the clearance of THPO from the plasma thus establishing a negative feedback loop. Several mutations in the c-Mpl receptor gene have been linked to a gain-of-function resulting in thrombocytosis. We focused on a comparison of gain-of-function mutation in the extracellular part of the c-Mpl receptor, where ligand binding and receptor dimerization occur, with the S505N, W515K and W515L mutations in the transmembrane and juxtamembrane region, respectively. Interestingly, the latter mutations are known to also promote myeloproliferative malignancies and AML, whereas the P106L mutation causes hereditary thrombocytosis without a known predisposition to hematologic malignancies.We have now performed functional analyses of these gain-of-function mutations to address the question of how the different propensity to induce malignancy can be explained. We first analyzed the post-translational processing of the normal and the P106L mutated receptor in comparison to the receptors carrying the S505N, W515K and W515L mutations in transfected HeLa and BA/F3 cells. The normal and the S505N, W515K and W515L mutated c-Mpl receptors were properly glycosylated during their transport through the Golgi apparatus, whereas the P106L mutated receptor did not enter the Golgi and was not fully glycosylated. The plasma membrane expression, assayed by confocal microscopy and FACS, of the S515N, W515K and W515L mutated receptors was comparable to the normal receptor, whereas the P106L mutated receptor was not detectable on the cell surface. Functional analyses of the THPO/c-Mpl signaling pathways in THPO stimulated c-Mpl transfected BA/F3 cells showed activation of the ERK1/2 pathway in all mutants but only weaker activation of the PI3K/m-TOR and Stat3/5 signaling pathways for the P106L mutant. By contrast, cells transfected with the normal receptor gene and the S505N, W515K and W515L c-Mpl mutants showed predominant up-regulation of the PI3K/m-TOR and Stat3/5 pathways.These results show that (1) the activation of c-Mpl by THPO does not absolutely require surface expression of the receptor (2) different c-Mpl gain-of-function mutations activate separable downstream pathways and (3) the predominant activation of the PI3K/m-TOR and Stat3/5 pathways correlates with the propensity to induce hematopoietic malignancy.Further, it is interesting to note that the c-Mpl P106L gain-of-function mutant is known to cause 10 to 20-fold elevated thrombopoietin (THPO) plasma levels in patients and is shown here not to be properly glycosylated and transported to the cell surface. By contrast, patients with the other gain-of-function mutations analyzed here show normal or even reduced THPO plasma concentrations and normal post-translational processing and cell surface expression.In conclusion, we propose that c-Mpl gain-of-function mutations exert their effect by predominantly activating either the PI3K/m-TOR and Stat3/5 or the ERK signaling pathways and that the predominant activation of PI3K/m-TOR and Stat3/5 correlates with the propensity to induce malignancy. Furthermore, THPO clearance and maintenance of the negative feedback loop regulating THPO plasma levels, but not signaling activity of the c-Mpl receptor necessarily require proper glycosylation, intracellular trafficking and cell surface expression. DisclosuresNo relevant conflicts of interest to declare.

MRP4 Expression and Platelet Activation in Children and Adolescents with Different Subtypes of Primary Thrombocythemia

Background and aims. Essential thrombocythemia (ET) rarely occurs in children and adolescents. In our experience, 40% of pediatric patients with primary thrombocythemia (PT) have JAK2 V617F or CALR mutations, 24% have a diagnosis of ET without any known molecular markers while 36% have hereditary thrombocytosis (HT) with a MPLS505A mutation. Thrombotic events, frequent in adult ET presenting high-risk factors and rare in pediatric PT, have been observed in pediatric HT patients with MPLS505A mutation in treatment with aspirin (ASA). The multidrug resistance protein-4 (MRP4) is an ATP-binding cassette transporter involved in the efflux of several pharmacological and physiological compounds. MRP4 has been identified as a modulator of ASA action in platelets; in addition, it also influences platelet activation. Recent studies have shown that MRP4 over-expression has a role in reducing the effect of ASA in patients who have undergone a bypass surgical procedure and that ASA induces platelet MRP4 upregulation. Aims of this study were to evaluate and correlate MRP4 expression and platelet function in children and adolescents aged <20 years at diagnosis with different subtypes of PT.Methods. MRP4 protein and mRNA expressions were evaluated in platelets obtained from healthy volunteers (HV) and from 41 PT patients: M/F ratio: 0.78; median age at diagnosis: 14.5 years; median platelet count at study: 671x109/L. Ten patients had MPLS505A mutation (HT), 10 were JAK2V617F-mutated (ET) and 6 harbored CALR mutations (ET) while 15 cases were JAK2, CALR and MPL wilde-type (ET). Expression of MRP4 protein and mRNA were analyzed by Western blot and RT-PCR, respectively, and the results were reported as ET/HV or HT/HV expression values. Platelet aggregation, using ADP at different concentration (0.8 to 2 μM) and collagen (1 μg/ml) as agonists, and platelet secretion, expressed as ATP release after U46619 + epinephrine (1 + 20 μM), using the luciferin-luciferase assay, were utilized as platelet function tests. This study was conducted in accordance with the Declaration of Helsinki.Results. Protein MRP4 expression was higher both in HT (4.23+/-1.88) and in ET patients with CALR mutations (4.27+/-2.60) compared to the values found in wild-type ET patients (3.55+/-1.52). In JAK2V617F-mutated ET patients, the MRP4 protein expression (2.03+/-1.46) was significantly lower compared to the values observed in all other ET and HT patients (p .05), whereas the MRP4 mRNA expression was significantly higher (ΔΔCt 0.021+0.003) compared both to HT patients (0.009+/-0.0039 ΔΔCt) (p .01) and CALR-mutated patients (ΔΔCt 0.014+/- 0.006). Patients with HT showed a significantly higher response to ADP 0.8 µM (83+/-29 Mx%) compared to all subgroups of ET patients who showed a similar response (60+/-34 Mx%; p .01). A significantly shorter lag-phase in response to collagen (1 µg/ml) was observed in HT compared to ET patients (33+/-3 sec vs 51+/-4 sec, p .005). Among the ET population, JAK2-mutated patients showed a significantly shorter lag-phase in response to collagen compared to wild-type patients (39+/-12.5 sec vs 50+/-15 sec), p .011. Platelet secretion was significantly higher in HT compared to ET patients, p .001.Conclusions. This study for the first time provides evidence that children and adolescents with MPLS505A-mutated HT show a higher platelet reactivity compared to age-matched patients with ET. Moreover, platelet reactivity correlates with MRP4 protein overexpression. These findings help to shed light into the thrombotic events observed in HT MPLS505A patients despite treatment with ASA. DisclosuresNo relevant conflicts of interest to declare.

Myeloproliferative neoplasms: JAK2 signaling pathway as a central target for therapy.

The discovery of the JAK2V617F mutation followed by the discovery of other genetic abnormalities allowed important progress in the understanding of the pathogenesis and management of myeloproliferative neoplasms (MPN)s. Classical Breakpoint cluster region-Abelson (BCR-ABL)-negative neoplasms include 3 main disorders: essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). Genomic studies have shown that these disorders are more heterogeneous than previously thought with 3 main entities corresponding to different gene mutations: the JAK2 disorder, essentially due to JAK2V617F mutation, which includes nearly all PVs and a majority of ETs and PMFs with a continuum between these diseases and the myeloproliferative leukemia (MPL) and calreticulin (CALR) disorders, which include a fraction of ET and PMF. All of these mutations lead to a JAK2 constitutive activation. Murine models either with JAK2V617F or MPLW515L, but also with JAK2 or MPL germ line mutations found in hereditary thrombocytosis, have demonstrated that they are drivers of myeloproliferation. However, the myeloproliferative driver mutation is still unknown in approximately 15% of ET and PMF, but appears to also target the JAK/Signal Transducer and Activator of Transcription (STAT) pathway. However, other mutations in genes involved in epigenetics or splicing also can be present and can predate or follow mutations in signaling. They are involved either in clonal dominance or in phenotypic changes, more particularly in PMF. They can be associated with leukemic progression and might have an important prognostic value such as additional sex comb-like 1 mutations. Despite this heterogeneity, it is tempting to target JAK2 and its signaling for therapy. However in PMF, Adenosine Tri-Phosphate (ATP)-competitive JAK2 inhibitors have shown their interest, but also their important limitations. Thus, other approaches are required, which are discussed in this review.