Study of rare mutations of the TPO receptor, MPL, in sporadic and hereditary thrombocytosis

Les thrombocytoses correspondent à une élévation du taux de plaquettes au-delà de 450x109/L et sont soit réactionnelles dans la plupart des cas, soit primitives quand il existe une altération génétique sur la voie de la production plaquettaire. Dans ce dernier cas il est habituel de distinguer les thrombocytoses sporadiques des thrombocytoses familiales, qui partagent néanmoins un mécanisme physiopathologique commun à l’image de la mutation MPLS505N résultant en une activation constitutive du récepteur. En effet les thrombocytémies essentielles (TE), les plus fréquentes, présentent une mutation activatrice dans 85% des cas soit de JAK2, de CALR ou MPL. Les thrombocytoses héréditaires quant à elles peuvent être la conséquence de mutations de MPL, de JAK2 ou de la TPO. Nous avons étudié dans un premier temps la mutation MPLP106L décrite chez une famille saoudienne mais dont le mécanisme physiopathologique aboutissant à la thrombocytose était mal compris. En effet les sujets homozygotes présentent une thrombocytose importante et de manière paradoxale un taux de TPO élevé. Nous avons pu montrer qu’il s’agit d’un récepteur fonctionnel sans autonomie de croissance mais qu’il existe un défaut de trafic cellulaire, MPL restant bloqué dans le réticulum endoplasmique et étant adressé faiblement à la membrane. Néanmoins l’expression membranaire de MPL est moins diminuée à la surface des cellules immatures par rapport aux cellules matures expliquant un découplage entre la fonction de prolifération (mégacaryocytes) et de clearance de la TPO (plaquettes). Nous avons également étudié les TE triples négatives pouvant correspondre potentiellement à des mutations de MPL liées à un défaut de trafic cellulaire comme pour MPLP106L. Grâce à un séquençage à haut débit de l’exome entier, nous avons pu mettre en évidence de nouvelles mutations récurrentes comme MPLS204P ou Y591N qui sont des mutations faiblement activatrices ne donnant pas de croissance autonome mais n’altérant pas le trafic cellulaire et nécessitant la présence éventuellement d’autres mutations pour déclencher une thrombocytose.Thrombocytosis is defined as a platelet count exceeding 450x109/L and generally either is a reactive process or is caused by genetic alteration on the way of platelet production. In the latter case sporadic thrombocytosis are usually distinguished from familial thrombocytosis, the boundaries between these two cases becoming less sharp since the mutation first described in familial thrombocytosis (MPLS505N) is also found in sporadic cases. Essential thrombocythemia (ET), the most frequent, sporadic thrombocytosis have an activating mutation in 85% of cases in either JAK2, MPL or CALR genes. Hereditary thrombocytosis may be the result of mutations of MPL, JAK2 or TPO. We studied initially MPLP106L already described in a Saudi family in which the pathophysiologic mechanism leading to thrombocytosis was misunderstood. Indeed homozygous patients exhibit significant thrombocytosis and paradoxically high TPO plasma level. We have shown that MPLP106L is a functional receptor without spontaneous growth but with a trafficking defect and a low cell surface expression. Nevertheless membrane expression of MPL is less reduced on immature cell surface compared to mature cells, explaining an uncoupling between proliferation (megakaryocyte precursors) and TPO clearance (platelets) functions. We also studied triple negative ET, to find potentially MPL mutations linked to a trafficking defect like MPLP106L. Thanks to Whole Exome Sequencing we were able to decipher new MPL mutations as MPLS204P or Y591N, which are weakly activating mutations without autonomous growth and the absence of trafficking defect and requiring the presence of other mutations in most cases to trigger thrombocytosis

Physiopathologie d une forme de thrombocytose familiale associée à une perte de fonction de MPL, le récepteur de la TPO

PARIS7-Xavier Bichat (751182101) / SudocSudocFranceF

Dominance of an UBA1 mutant clone over a CALR mutant clone: from essential thrombocytemia to VEXAS.

International audienc

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

The 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 [(125)I]-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. Stefan N. Constantinescu and William Vainchenker are co-last Autho

Clinical and biological impact of ATP-binding cassette transporter activity in adult acute myeloid leukemia

International audienceChemotherapy resistance is the main cause of treatment failure in acute myeloid leukemia (AML) and has been related to ATP-binding cassette (ABC) transporter activity. However, the links between ABC activity, immunophenotype, and molecular AML parameters have been poorly evaluated. Moreover, the prognostic value of ABC activity, when compared to new molecular markers, is unknown. Here we investigated the links between ABC activity, as evaluated by JC-1 +/- cyclosporine A assay, and immunophenotypic, cytogenetic, molecular, and targeted next-generation sequencing features in 361 AML patients. High ABC activity was found in 164 patients and was significantly associated with less proliferating disease, an immature immunophenotype (expression of CD34, HLA-DR, CD117, CD13), and gene mutations defining AML as belonging to secondary-type ontogenic groups. Low ABC activity was associated with more mature myeloid differentiation (CD34-, cyMPO+, CD15+, CD33+) or monocytic commitment (CD64+, CD4+weak, CD14+), with NPM1 mutations, KMT2A rearrangements, and core-binding factor gene fusions, hallmarks of the de novo-type AML ontogeny. ABC activity was one of the major factors we identified using a random forest model for early prediction of AML ontogeny. In the 230 patients evaluated at diagnosis and intensively treated, high ABC activity was a predictive factor for primary resistance, and in multivariate analysis including full molecular data, an independent factor for event-free survival (P=0.0370). JC-1 +/- cyclosporine A assay could be used at diagnosis to predict AML ontogeny and to complete prognosis evaluation in addition to new molecular markers

Precision and prognostic value of clone-specific minimal residual disease in acute myeloid leukemia

The genetic landscape of adult acute myeloid leukemias (AML) has been recently unraveled. However, due to their genetic heterogeneity, only a handful of markers are currently used for the evaluation of minimal residual disease (MRD). Recent studies using multi-target strategies indicate that detection of residual mutations in less than 5% of cells in complete remission is associated with a better survival. Here, in a series of 69 AMLs with known clonal architecture, we design a clone-specific strategy based on fluorescent in situ hybridization and high-sensitivity next generation sequencing to detect chromosomal aberrations and mutations, respectively, in follow-up samples. The combination of these techniques allows tracking chromosomal and genomic lesions down to 0.5-0.4% of the cell population in remission samples. By testing all lesions in follow-up samples from 65 of 69 evaluable patients, we find that initiating events often persist and appear to be, on their own, inappropriate markers to predict short-term relapse. In contrast, the persistence of two or more lesions in more than 0.4% of the cells from remission samples is strongly associated with lower leukemia-free and overall survivals in univariate and multivariate analyses. Although larger prospective studies are needed to extend these results, our data show that a personalized, clone-specific, MRD follow up strategy is feasible in the vast majority of AML cases

Presence of atypical thrombopoietin receptor (MPL) mutations in triple negative essential thrombocythemia patients.

International audienceMutations in signaling molecules of the cytokine receptor axis play a central role in myeloproliferative neoplasm (MPN) pathogenesis. Polycythemia Vera is mainly related to JAK2 mutations, whereas a wider mutational spectrum is detected in Essential Thrombocythemia (ET) with mutations in JAK2, the thrombopoietin receptor (MPL) and the calreticulin (CALR) genes. Here, we studied the mutational profile of 17 ET patients negative for JAK2V617F, MPLW515K/L and CALR mutations, using Whole Exome Sequencing and Next Generation Sequencing (NGS) targeted on JAK2 and MPL. We found several signaling mutations including JAK2V617F at very low allele frequency, one homozygous SH2B3 mutation, one MPLS505N, one MPLW515R and two MPLS204P mutations. In the remaining patients, four presented a clonal and seven a polyclonal hematopoiesis, suggesting that certain triple negative ETs are not MPNs. NGS on 26 additional triple negative ETs detected only one MPLY591N mutation. Functional studies on MPLS204P and MPLY591N revealed that they are weak gain-of-function mutants increasing MPL signaling and conferring either TPO hypersensitivity or independence to expressing cells, but with a low efficiency. Further studies should be performed to precisely determine the frequency of MPLS204 and MPLY591 mutants in a bigger cohort of MPN

Erythrocytosis associated with IgA nephropathy

International audienceBackground Erythrocytosis is a hematological disorder usually related to hematopoietic stem cell somatic muta-tions. However, unexplained erythrocytosis remains frequent. In this study, we evaluated the involvement of IgA1, a regulator of erythropoiesis also implicated in IgA nephropathy (IgAN) pathophysiology, in unexplained polycythe-mia/erythrocytosis (PE) of IgAN patients.Methods IgAN-PE patients’ serum was collected, analyzed and used to study IgA1 effect on proliferation and differ-entiation of erythroid progenitors. Hematological parameters of transgenic mice for human alpha1 heavy chain were studied. Multicentric observational cohorts of chronic kidney disease (CKD) patients, including both native kidney diseases and renal transplants, were studied to analyze patient hemoglobin levels.Findings We retrospectively identified 6 patients with IgAN and unexplained PE. In large CKD cohorts, IgAN was associated with PE in 3.5% of patients (p<0.001 compared to other nephropathies). IgAN was an independent factor associated with higher hemoglobin levels (13.1g/dL vs 12.2 g/dL, p=0.01). During post-transplant anemia, anemia recovery was faster in IgAN patients. Elevated polymeric/monomeric IgA1 ratio as well as high Gd-IgA1 rate were observed in circulating IgA1 of the 6 IgAN-PE patients as compared with control or IgAN patients without PE. IgA1 from these patients increased the sensitivity of erythroid progenitors to Epo. In mice, we also observed an elevation of hematocrit in alpha1 knock-in mice compared to wild type controls.Interpretation These data identify a new etiology of erythrocytosis and demonstrate the role of pIgA1 in human erythropoiesis. This syndrome of IgA-related erythrocytosis should be investigated in case of unexplained erythrocy-tosis and renal disease

Different impact of calreticulin mutations on human hematopoiesis in myeloproliferative neoplasms.

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