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Daunorubicin induces apoptosis in myeloid leukemia cells by activation of neutral sphingomyelinase and ceramide generation occurring 4–10 min after daunorubicin addition. We show here that daunorubicin is able to increase the phosphoinositide 3-kinase activity and enhance intracellular phosphoinositide 3-kinase lipid products prior to ceramide generation. Daunorubicin activates Akt, a downstream phosphoinositide 3-kinase effector. Interestingly, the phosphoinositide 3-kinase inhibitors wortmannin and LY294002 accelerate daunorubicin-induced apoptosis in U937 cells. The phosphoinositide 3-kinase/Akt pathway has been involved in cell survival following serum deprivation, tumor necrosis factor α, anti-Fas and UV radiations. Our results suggest that anti-tumor agents such as daunorubicin may also activate anti-apoptotic signals that could contribute to drug resistance.

Abstract 1758 During the last several years, iPS cells have been described as a new powerful tool for disease modeling and drug screening. It became possible to reprogram adult patients cells into specific pluripotent cell lines harboring inherited and/or acquired genetic abnormalities. We have focused our work on modeling Jak2V617F positive MPN patients in iPS cells. By retroviral infection (OCT4, SOX2, KLF4, c-Myc), several iPS cell lines were generated from CD34+ cells of a healthy donor (control), a heterozygous JAK2 V617F patient and a homozygous JAK2 V617F patient. All cell lines expressed pluripotent surface markers (TRA1–81, SSEA4), pluripotent genes (NANOG, OCT4, SOX2) and were capable to induce teratomas in immunodeficient mice. Moreover, they were silenced for the expression of the 4 exogenous transgenes and showed no new clonal karyotypic abnormalities. All the iPS cell lines derived from the JAK2V617F heterozygous and homozygous patients were also heterozygous and homozygous for JAK2V617F. No JAK2 wild type iPS could be obtained from the patients CD34+ cells. Interestingly all the homozygous JAK2V617Fhad no 20q deletion by cytogenetic and CGH analysis demonstrating that this deletion was a secondary event in this MPN. JAK2V617F mutation did not elicit a significant increase in hematopoiesis in comparison to the control iPS. However, marked differences in cytokine sensitivity were found. Homozygous iPS-derived erythroid progenitors had spontaneous growth, hypersensitivity to EPO and gave rise to slightly larger erythroid colonies in methylcellulose assays as compared to the control, whereas erythroid progenitors from JAK2V617F heterozygous iPS has similar Epo-response than those from the control. Interestingly, Megakaryocytes (MK) progenitors from JAK2V617F heterozygous iPS had hypersensitivity to TPO whereas those from homozygous iPS showed a complete TPO independence. Those cytokine-response profiles recapitulate the disease's primary features and validate the iPS as a good tool for JAK2V617F MPN modeling. MK differentiation showed a maturation defect with an excess of immature MKs only in the JAK2 V617Fhomozygous lines, who was derived with a post-PV myelofibrosis, suggesting the existence of intrinsic abnormalities of the MK lineage. Finally, we tested several JAK2, PI3K and ERK inhibitors on the JAK2V617F-mediated erythroid growth and showed that only JAK2 and PI3K inhibitors were able to block the erythroid spontaneous growth. iPS technology recapitulates the cytokine hypersensitivity of JAK2V617F MPNs with marked differences between heterozygous and homozygous mutations, enabling us to screen various pharmaceutical inhibitors. In an addition it can also be used to study the clonal hierarchy in a MPN and in the future to study the synergistic effects of different mutations. Disclosures: No relevant conflicts of interest to declare.

Myeloproliferative neoplasms (MPNs) are clonal malignant disorders characterized by the increased production of mature myeloid cells in blood. The classical MPNs include Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). Those pathologies are due to the acquisition of gain-of-function mutations leading to the constitutive activation of the cytokine receptor / JAK2 signaling pathway: JAK2V617F in 70% of cases and mutations in the thrombopoietin receptor (MPL) gene in 5% of cases. More recently, around fifty different mutations in the calreticulin (CALR) gene have been described in 30% of ET and PMF with two more frequent mutations called del52 (type 1) and ins5 (type 2). All the CALR mutations induce a frameshiflt to an alternative reading frame in the exon 9 leading to a new C-term tail of the protein with hydrophobic features, and the loss of the KDEL sequence, which is involved in its endoplasmic reticulum retention. The goal of this work was to understand the role of CALR mutants (del52, del46, del34, ins5, del19, del13) in human hematopoiesis. By studying the variant allele frequency (VAF) in 20 patients, we have shown that the CALR mutations are present in all blood mature cells not only in granulocytes and monocytes (CD14+) with a VAF >30% but also in B cells (CD19+), NK cells (CD56+) and in some cases in T cells (CD3+). Moreover, we have observed that CALR mutations are present in all hematopoietic progenitors including CD34+CD38-CD90+ (HSC), CD34+CD38-CD90- (immature progenitors) and CD34+CD38+ (committed progenitors) cell fractions after investigating the clonal architecture of the progenitors. CALR mutation was detectable in more than 40% of progenitor cells except in 2 patients (15 patients studied) and with, in some cases, no detectable wild type CALR progenitors. Homozygous CALR mutations were rare except in one case associated with disease progression. Whatever the VAF, there was no significant differences among the different progenitor types and granulocytes. Finally, we observed that all the associated mutations studied (TET2, PHF6, SYNE1, SCARA5, PIK3CD, SETD1B) in 6 patients postdated CALR mutations. We could also show in 15 patients samples that CALR mutants give a specific megakaryocytic progenitor (CFU-MK) spontaneous growth mediated both by MPL and JAK2 activation using specific inhibitors and short hairpin RNAs. The CFU-MK spontaneous growth correlated with a constitutive activation of JAK2/STAT3/5 pathway in megakaryocytes derived from in vitro cultures of CD34+ progenitors. In aggregate, these results show that all CALR mutants studied are present in all human hematopoietic cells including myeloid and lymphoid cells, give an early clonal advantage at the level of the HSC compartment and a specific increased growth of the megakaryocytic lineage via MPL/JAK2 activation. Disclosures No relevant conflicts of interest to declare.

Interferon alpha is the only drug that treats patients with MPN in a durable way and gives 20% of Complete Molecular Remission (CMR). However, treatments are long (2 to 5 years) and failures are frequent. Moreover, the mechanism of action of IFNα is poorly understood which limits the improvement of treatment. Our hypothesis is the involvement of PML (promyelocytic leukemia), a tumor suppressor gene controlled at the transcriptional level by IFN. This "Interferon Stimulated Gene" (ISG) is the main organizer of the PML nuclear bodies (PML-NB) which recruit and modify partner proteins in a post-translational manner. In particular, they activate the P53 and Rb pathways with apoptosis, senescence and cell cycle arrest. The biogenesis of PML-NB is enhanced by the oxidative stress and action of arsenic trioxide (ATO). This was done with a mouse model mimicking human MPNs: Jak2V617F Knock-In (KI) mice. This preclinical study shows that ATO increases the efficacy of IFNα on the hematological and molecular response, the disappearance of disease-initiating cells (tested in secondary transplants) and the relapse rate of the disease after stopping the treatment. Treatment of mice transplanted with 50% Jak2V617F Pml GFP- 50% Jak2V617FPml ⁻⁄⁻ show that there is a selection of Pml ⁻⁄⁻ cells that are more resistant to the treatment and that PML is involved in these effects. Overall, these results highlight the involvement of PML and open the prospect of more efficient therapeutic trials in MPN by combining IFN and ATO in the treatment of patients.; L’interféron alpha est le seul médicament qui traite de façon durable les patients atteints de NMP et donne 20% de rémissions moléculaires complètes (RMC). Cependant les traitements sont longs (2 à 5 ans) et les échecs fréquents. De plus, le mécanisme d'action de l'IFNα est mal connu, ce qui limite l'amélioration des traitements. Notre hypothèse de départ est l’implication de PML (promyelocytic leukemia), un gène suppresseur de tumeur contrôlé au niveau transcriptionnel par l’IFN. Ce gène, stimulé par l’interféron (ISG) est le principal organisateur des corps nucléaires PML (CN-PML) qui recrutent et modifient des protéines partenaires d'une façon post traductionnelle. Ils activent notamment les protéines p53 et Rb avec apoptose, sénescence et arrêt du cycle cellulaire. La biogenèse des CN-PML est augmentée par le stress oxydatif et l’action du trioxyde d’arsenic (ATO). Cela a été utilisé sur un modèle murin miman les NMP humaines : les souris Knock-In (KI) Jak2V617F. Cette étude préclinique montre que l’ATO augmente l'efficacité de l'IFNα sur la réponse hématologique et moléculaire, la disparition des cellules initiatrices de la maladie (testée en transplantations secondaires) et le taux de rechure de la maladie après l'arrêt du traitement. Le traitement des souris greffées 50% Jak2V617F Pml GFP-50% Jak2V617F Pml ⁻⁄⁻ montre qu'il y a une sélection des cellules Pml ⁻⁄⁻ qui sont résistantes au traitement. Cela montre que PML est impliquée dans ces effets. De plus, nous montrons qu'il s'agit d'une augmentation de sénescence, une des fonctions principales des CN-PML. Au total, ces résultats mettent en évidence l’implication de PML et ouvrent la perspective d’essais thérapeutiques plus performants dans les NMP.

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 calreticulin (CALR) and thrombopoietin receptor (MPL). Interferon alpha (IFNα) is the only curative treatment that induces not only a hematological response of ET, PV and early MF but also a molecular response both on JAK2V617F or CALR mutated cells. In this study, we wanted to know how and with what kinetics IFN impacts the different mutated hematopoietic compartments. Thus, we have performed a prospective study with a cohort of 50 patients treated by IFNα for 3-5 years. The MPN diseases distribution was 44% ET, 45% 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 variant allele frequency was measured in mature cells (granulocytes, platelets). Simultaneously, we have also determined the clonal architecture 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). After a median follow-up of 30 months, we observed that IFNα targets more efficiently and rapidly the HSPC particularly in HSC-enriched progenitors, than the mature blood cells in JAK2V617F patients (p

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 calreticulin (CALR) and thrombopoietin receptor (MPL). Interferon alpha (IFNα) is the only curative treatment that induces not only a hematological response of ET, PV and early MF but also a molecular response both on JAK2V617F or CALR mutated cells. In this study, we wanted to know how and with what kinetics IFN impacts the different mutated hematopoietic compartments. Thus, we have performed a prospective study with a cohort of 50 patients treated by IFNα for 3-5 years. The MPN diseases distribution was 44% ET, 45% 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 variant allele frequency was measured in mature cells (granulocytes, platelets). Simultaneously, we have also determined the clonal architecture 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). After a median follow-up of 30 months, we observed that IFNα targets more efficiently and rapidly the HSPC particularly in HSC-enriched progenitors, than the mature blood cells in JAK2V617F patients (p, Les néoplasmes myéloprolifératifs classiques sont des maladies clonales dues à des mutations acquises de JAK2V617F ou de la Calréticuline (CALRm) au niveau des cellules souches hématopoïétiques (CSH) et conduisant à une surproduction de cellules myéloïdes. L’interféron alpha (IFNa) est le seul traitement curatif qui induit une réponse hématologique et moléculaire. Le but de notre projet est de comprendre son mécanisme d’action en utilisant une cohorte de 47 patients, des lignées cellulaires et des modèles de souris JAK2V617F. Ainsi, nous avons constaté que l’IFNa cible plus rapidement les CSH que les cellules matures chez les patients JAK2V617F, ce qui semble différent des patients CALRm. Grâce aux lignées cellulaires et aux souris, nous avons montré que JAK2V617F induit une pré-activation des voies de l’IFNa et une inhibition du cycle cellulaire des CSH. La découverte complète de ce mécanisme d’action conduira à l’amélioration du traitement.

Two tumor suppresors, TET2 and P53, play an important role in the homeostasis of hematopoietic stem cells and have been found mutated in hematological malignancies. They are also involved in cell cycle control and DNA repair mechanisms, including the base excision repair pathway (BER). In the first part of this work, we showed that TET2 overexpression and the consequent increase of 5hmC, inhibit cell cycle progression particularly G1/S transition and induces centrosome instability associated with chromosomal instability in Ba/F3 cellular model. In addition, overexpression of TET2 induces increased mutagenesis particularly transitions C->T at CpG sites in a context deficient in thymidine DNA glycosylase (TDG), a protein initiating BER. In the second part of this work, we have shown that p53 activation by MDM2 antagonists has deleterious effect on all haematopoietic progenitors. These antagonists also induce cytotoxicity not only in the early stages of megakaryopoiesis but also mainly in the late stages. This cytotoxicity is not reversible, in contrast to what is observed in clinic, and can not be restored by increasing doses thrombopoietin. To conclude, TET2 and P53 must be strictly controlled to ensure homeostasis and genetic stability of the hematopoietic cells.; TET2 et P53, deux suppresseurs de tumeurs, jouent un rôle important dans l’homéostasie des cellules souches hématopoïétiques et sont trouvés mutés dans les hémopathies malignes. Ils sont aussi impliqués dans le contrôle du cycle cellulaire et les mécanismes de réparation des dommages de l’ADN, notamment la voie de réparation par excision de base (BER). Dans la première partie de ce travail, nous avons montré que la surexpression de TET2 et l’augmentation consécutive des 5hmC, ralentit la progression du cycle cellulaire et la transition G1/S et induit une instabilité centrosomique associée à une instabilité chromosomique dans un modèle cellulaire Ba/F3. De plus, la surexpression de TET2 induit l’augmentation de la mutagenèse particulièrement des transitions C->T dans les sites CpG dans un contexte déficient en thymidine DNA glycosylase (TDG), une protéine initiatrice du BER. Dans la seconde partie de ce travail, nous avons montré que l’activation de P53, par des antagonistes de MDM2, a un effet délétère sur tous les progéniteurs hématopoïétiques. Ces antagonistes induisent aussi une cytotoxicité non seulement dans les stades précoces de la mégacaryopoïèse mais surtout dans les stades tardifs. Cette cytotoxicité n’est pas réversible, contrairement à ce qui est observé en clinique, et ne peut pas être restaurée par des doses croissantes de thrombopoïétine. Au total, TET2 et P53 doivent être strictement régulés pour assurer l’homéostasie et la stabilité génétique des cellules hématopoïétiques.

EPOR mutations are observed in Primary familial and congenital polycythaemia (PFCP) while MPL mutations are found in myeloproliferative neoplasms (MPN). PFCP is an inherited disorder of erythroid progenitor cells resulting in elevated erythrocyte mass. Several mutations of the erythropoietin receptor (EPOR) gene have been associated with PFCP. They are all leading to a premature STOP codon and the truncation of the cytoplasmic COOH-terminal of EPOR. To examine the role of EPOR mutations in the pathogenesis of PFCP, we studied a new EPOR mutation, c.1300dupC (p.Gln434Profs*11). This mutation induced, in primary cells and cell lines, a major hypersensitivity to EPO. This phenotype was not due to the loss of negative regulation domains or an internalisation default, contrary to the current hypothesis, but rather due to conformational modification inducing the stabilisation of the mutant at the cell membrane.In the second part of this work, an EPOR mutation, Pro488Ser, was studied in a myeloproliferative neoplasms (MPN) family. Only a mild spontaneous STAT5 activation was observed in cell lines. Murine models and/or iPSC will be developed in order to test the hypothesis of a cooperation between EPOR P488S and JAK2 V617F. In the last part of this project, 2 rare MPL mutants, Tyr591Asn and Ser204Pro, identified in 3 triple negative ET patients were functionally studied. A weak gain of function was observed, suggesting that these mutants have to be associated to other genetic abnormalities to develop a phenotype.; Le récepteur à l’érythropoïétine (EPOR) peut être muté dans les érythrocytoses congénitales tandis que des mutations de MPL, récepteur à la thrombopoiétine, sont observées dans certains néoplasmes myéloprolifératifs (NMP). L’érythrocytose congénitale touche exclusivement les progéniteurs érythroïdes et se traduit par une polyglobulie isolée. Des mutations d’EPOR sont décrites dans environ 12% des cas. La première partie de cette thèse reposait sur l’étude fonctionnelle d’une mutation d’EPOR, jamais décrite, c.1300dupC (p.Gln434Profs*11). Ce mutant est responsable, dans les cellules primaires et les lignées cellulaires, d’une hypersensibilité majeure à l’EPO qui n’est pas due à la perte de sites de régulation négative du signal ou à un défaut d’internalisation du récepteur, contrairement aux données de la littérature, mais à une stabilisation d’EPOR à la membrane cellulaire par probable changement conformationnel. Dans la seconde partie, un variant d’EPOR Pro488Ser a été étudié au sein d’une famille de NMP. Seule une activation spontanée faible de STAT5 a pu être mise en évidence dans les lignées cellulaires. Des modèles murins et/ou d’iPSC seront développés dans l’hypothèse d’une coopération entre EPOR P488S et JAK2V617F. Enfin, dans la dernière partie de ce travail, nous avons réalisé une étude fonctionnelle de 2 mutations rares de MPL, Tyr591Asn et Ser204Pro, identifiées chez 3 patients TE triples négatifs. Seul un gain de fonction faible a été mis en évidence, suggérant que ces mutations doivent être associées à d’autres anomalies génétiques pour entrainer l’apparition d’un phénotype.