Etude fonctionnelle de l'inactivation de TET2 au cours de l'hématopoïèse chez la souris.

Des mutations acquises du gène TET2 ont été décrites dans les hémopathies malignes humaines. La fréquence de ces anomalies dans les hémopathies myéloïdes est de 10 à 20%, atteignant 50% dans les échantillons de leucémies myélo-monocytaires chroniques (LMMC). Les mutations observées sont inactivatrices, ce qui suggère que TET2 est un gène de type suppresseur de tumeur et que les mutations retrouvées conduisent à une perte de fonction de la protéine. Ce gène code pour une enzyme capable de modifier les cytosines méthylées. Il participerait ainsi au contrôle de la méthylation de l'ADN et donc à la régulation épigénétique de l expression génique. Afin de mieux comprendre son rôle au cours de l hématopoïèse, deux modèles murins d'inactivation du gène Tet2 ont été développés. Des expériences de greffe de cellules médullaires dans des souris syngéniques montrent que les cellules déficientes pour ce gène présentent un avantage compétitif par rapport aux cellules sauvages. L analyse des souris invalidées pour ce gène montre une amplification des populations hématopoïétiques immatures, ainsi que des anomalies de la différenciation des lignages myéloïdes et également des lignages lymphoïdes. Une fraction des souris invalidées pour Tet2 âgées de plus de six mois développe des hémopathies malignes ressemblant à la LMMC humaine. Des anomalies équivalentes sont retrouvées dans les souris hémizygotes pour Tet2 et dans des souris portant un allèle hypomorphe du gène. L ensemble de ces résultats montre qu une dérégulation de l'activité de Tet2 conduit à des anomalies précoces de l'hématopoïèse, mais n'entraine pas directement la transformation des cellules progénitrices immatures. La latence du développement de ces tumeurs suggère la nécessité d'une coopération avec d'autres évènements oncogéniques, comme des anomalies d autres acteurs épigénétiquesAcquired loss-of-function mutations of TET2 gene are frequently observed in patients with myeloid malignancies, including acute myeloblastic leukemia, myeloproliferative neoplasm, myelodysplastic syndrome, and chronic myelomonocytic leukemia (CMML). The Ten-Eleven-Translocation (TET) family proteins are 2-oxoglutarate/Fe(II)-dependent dioxygenases that catalyze the conversion of 5-methyl-cytosine into 5-hydroxymethyl-cytosine, which is proposed to constitute a first step toward cytosine demethylation. To study the function of Tet2 in murine hematopoiesis, we developed two mouse models in which the catalytic domain of the protein is disrupted. In both models, Tet2 deficiency leads to the progressive expansion of the immature hematopoietic compartment that includes stem cell and multipotent progenitors. In addition, both Tet2-deficient animals display abnormalities of erythroid, megakaryocytic, myelo-monocytic and lymphoid lineages, recapitulated in competitive transplantation assays. With age, Tet2-deficient mice develop bona fide myeloid tumors. All these properties were shown to be cell-autonomous by bone marrow cells transplantation and in vitro assays. Together these data suggest that TET2 activity is essential for normal homeostasis of the hematopoietic system. Its inactivation results in the development of hematologic disorders resembling human CMML myeloid disorders. TET2 deficiency endows the cells with a competitive advantage over wild type cells, induces hematopoietic differentiation abnormalities but is not responsible for full cellular transformation. The latency observed for CMML development in mouse models of Tet2 deficiency suggests a requirement for cooperating mutations, such other epigenetic regulator alterations.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Role of Tet2 inactivation in mouse hematopoiesis

Des mutations acquises du gène TET2 ont été décrites dans les hémopathies malignes humaines. La fréquence de ces anomalies dans les hémopathies myéloïdes est de 10 à 20%, atteignant 50% dans les échantillons de leucémies myélo-monocytaires chroniques (LMMC). Les mutations observées sont inactivatrices, ce qui suggère que TET2 est un gène de type suppresseur de tumeur et que les mutations retrouvées conduisent à une perte de fonction de la protéine. Ce gène code pour une enzyme capable de modifier les cytosines méthylées. Il participerait ainsi au contrôle de la méthylation de l'ADN et donc à la régulation épigénétique de l’expression génique. Afin de mieux comprendre son rôle au cours de l’hématopoïèse, deux modèles murins d'inactivation du gène Tet2 ont été développés. Des expériences de greffe de cellules médullaires dans des souris syngéniques montrent que les cellules déficientes pour ce gène présentent un avantage compétitif par rapport aux cellules sauvages. L’analyse des souris invalidées pour ce gène montre une amplification des populations hématopoïétiques immatures, ainsi que des anomalies de la différenciation des lignages myéloïdes et également des lignages lymphoïdes. Une fraction des souris invalidées pour Tet2 âgées de plus de six mois développe des hémopathies malignes ressemblant à la LMMC humaine. Des anomalies équivalentes sont retrouvées dans les souris hémizygotes pour Tet2 et dans des souris portant un allèle hypomorphe du gène. L’ensemble de ces résultats montre qu’une dérégulation de l'activité de Tet2 conduit à des anomalies précoces de l'hématopoïèse, mais n'entraine pas directement la transformation des cellules progénitrices immatures. La latence du développement de ces tumeurs suggère la nécessité d'une coopération avec d'autres évènements oncogéniques, comme des anomalies d’autres acteurs épigénétiquesAcquired loss-of-function mutations of TET2 gene are frequently observed in patients with myeloid malignancies, including acute myeloblastic leukemia, myeloproliferative neoplasm, myelodysplastic syndrome, and chronic myelomonocytic leukemia (CMML). The Ten-Eleven-Translocation (TET) family proteins are 2-oxoglutarate/Fe(II)-dependent dioxygenases that catalyze the conversion of 5-methyl-cytosine into 5-hydroxymethyl-cytosine, which is proposed to constitute a first step toward cytosine demethylation. To study the function of Tet2 in murine hematopoiesis, we developed two mouse models in which the catalytic domain of the protein is disrupted. In both models, Tet2 deficiency leads to the progressive expansion of the immature hematopoietic compartment that includes stem cell and multipotent progenitors. In addition, both Tet2-deficient animals display abnormalities of erythroid, megakaryocytic, myelo-monocytic and lymphoid lineages, recapitulated in competitive transplantation assays. With age, Tet2-deficient mice develop bona fide myeloid tumors. All these properties were shown to be cell-autonomous by bone marrow cells transplantation and in vitro assays. Together these data suggest that TET2 activity is essential for normal homeostasis of the hematopoietic system. Its inactivation results in the development of hematologic disorders resembling human CMML myeloid disorders. TET2 deficiency endows the cells with a competitive advantage over wild type cells, induces hematopoietic differentiation abnormalities but is not responsible for full cellular transformation. The latency observed for CMML development in mouse models of Tet2 deficiency suggests a requirement for cooperating mutations, such other epigenetic regulator alterations

Etude fonctionnelle de l'inactivation de TET2 au cours de l'hématopoïèse chez la souris

Acquired loss-of-function mutations of TET2 gene are frequently observed in patients with myeloid malignancies, including acute myeloblastic leukemia, myeloproliferative neoplasm, myelodysplastic syndrome, and chronic myelomonocytic leukemia (CMML). The Ten-Eleven-Translocation (TET) family proteins are 2-oxoglutarate/Fe(II)-dependent dioxygenases that catalyze the conversion of 5-methyl-cytosine into 5-hydroxymethyl-cytosine, which is proposed to constitute a first step toward cytosine demethylation. To study the function of Tet2 in murine hematopoiesis, we developed two mouse models in which the catalytic domain of the protein is disrupted. In both models, Tet2 deficiency leads to the progressive expansion of the immature hematopoietic compartment that includes stem cell and multipotent progenitors. In addition, both Tet2-deficient animals display abnormalities of erythroid, megakaryocytic, myelo-monocytic and lymphoid lineages, recapitulated in competitive transplantation assays. With age, Tet2-deficient mice develop bona fide myeloid tumors. All these properties were shown to be cell-autonomous by bone marrow cells transplantation and in vitro assays. Together these data suggest that TET2 activity is essential for normal homeostasis of the hematopoietic system. Its inactivation results in the development of hematologic disorders resembling human CMML myeloid disorders. TET2 deficiency endows the cells with a competitive advantage over wild type cells, induces hematopoietic differentiation abnormalities but is not responsible for full cellular transformation. The latency observed for CMML development in mouse models of Tet2 deficiency suggests a requirement for cooperating mutations, such other epigenetic regulator alterations.Des mutations acquises du gène TET2 ont été décrites dans les hémopathies malignes humaines. La fréquence de ces anomalies dans les hémopathies myéloïdes est de 10 à 20%, atteignant 50% dans les échantillons de leucémies myélo-monocytaires chroniques (LMMC). Les mutations observées sont inactivatrices, ce qui suggère que TET2 est un gène de type suppresseur de tumeur et que les mutations retrouvées conduisent à une perte de fonction de la protéine. Ce gène code pour une enzyme capable de modifier les cytosines méthylées. Il participerait ainsi au contrôle de la méthylation de l'ADN et donc à la régulation épigénétique de l’expression génique. Afin de mieux comprendre son rôle au cours de l’hématopoïèse, deux modèles murins d'inactivation du gène Tet2 ont été développés. Des expériences de greffe de cellules médullaires dans des souris syngéniques montrent que les cellules déficientes pour ce gène présentent un avantage compétitif par rapport aux cellules sauvages. L’analyse des souris invalidées pour ce gène montre une amplification des populations hématopoïétiques immatures, ainsi que des anomalies de la différenciation des lignages myéloïdes et également des lignages lymphoïdes. Une fraction des souris invalidées pour Tet2 âgées de plus de six mois développe des hémopathies malignes ressemblant à la LMMC humaine. Des anomalies équivalentes sont retrouvées dans les souris hémizygotes pour Tet2 et dans des souris portant un allèle hypomorphe du gène. L’ensemble de ces résultats montre qu’une dérégulation de l'activité de Tet2 conduit à des anomalies précoces de l'hématopoïèse, mais n'entraine pas directement la transformation des cellules progénitrices immatures. La latence du développement de ces tumeurs suggère la nécessité d'une coopération avec d'autres évènements oncogéniques, comme des anomalies d’autres acteurs épigénétique

Endothelial and hematopoietic hPSCs differentiation via a hematoendothelial progenitor

International audienceAbstract Background hPSC-derived endothelial and hematopoietic cells (ECs and HCs) are an interesting source of cells for tissue engineering. Despite their close spatial and temporal embryonic development, current hPSC differentiation protocols are specialized in only one of these lineages. In this study, we generated a hematoendothelial population that could be further differentiated in vitro to both lineages. Methods Two hESCs and one hiPSC lines were differentiated into a hematoendothelial population, hPSC-ECs and blast colonies (hPSC-BCs) via CD144 + -embryoid bodies (hPSC-EBs). hPSC-ECs were characterized by endothelial colony-forming assay, LDL uptake assay, endothelial activation by TNF-α, nitric oxide detection and Matrigel-based tube formation. Hematopoietic colony-forming cell assay was performed from hPSC-BCs. Interestingly, we identified a hPSC-BC population characterized by the expression of both CD144 and CD45. hPSC-ECs and hPSC-BCs were analyzed by flow cytometry and RT-qPCR; in vivo experiments have been realized by ischemic tissue injury model on a mouse dorsal skinfold chamber and hematopoietic reconstitution in irradiated immunosuppressed mouse from hPSC-ECs and hPSC-EB-CD144 + , respectively. Transcriptomic analyses were performed to confirm the endothelial and hematopoietic identity of hESC-derived cell populations by comparing them against undifferentiated hESC, among each other’s ( e.g. hPSC-ECs vs. hPSC-EB-CD144 + ) and against human embryonic liver (EL) endothelial, hematoendothelial and hematopoietic cell subpopulations. Results A hematoendothelial population was obtained after 84 h of hPSC-EBs formation under serum-free conditions and isolated based on CD144 expression. Intrafemorally injection of hPSC-EB-CD144 + contributed to the generation of CD45 + human cells in immunodeficient mice suggesting the existence of hemogenic ECs within hPSC-EB-CD144 + . Endothelial differentiation of hPSC-EB-CD144 + yields a population of > 95% functional ECs in vitro. hPSC-ECs derived through this protocol participated at the formation of new vessels in vivo in a mouse ischemia model. In vitro, hematopoietic differentiation of hPSC-EB-CD144 + generated an intermediate population of > 90% CD43 + hPSC-BCs capable to generate myeloid and erythroid colonies. Finally, the transcriptomic analyses confirmed the hematoendothelial, endothelial and hematopoietic identity of hPSC-EB-CD144 + , hPSC-ECs and hPSC-BCs, respectively, and the similarities between hPSC-BC-CD144 + CD45 + , a subpopulation of hPSC-BCs, and human EL hematopoietic stem cells/hematopoietic progenitors. Conclusion The present work reports a hPSC differentiation protocol into functional hematopoietic and endothelial cells through a hematoendothelial population. Both lineages were proven to display characteristics of physiological human cells, and therefore, they represent an interesting rapid source of cells for future cell therapy and tissue engineering

Molecular Landscape of Therapy-related Myeloid Neoplasms in Patients Previously Treated for Gynecologic and Breast Cancers

International audienceDefinition of therapy-related myeloid neoplasms (TRMN) is only based on clinical history of exposure to leukemogenic therapy. No specific molecular classification combining therapy-related acute myeloid leukemia and therapy-related myelodysplastic syndromes has been proposed. We aimed to describe the molecular landscape of TRMN at diagnosis, among 77 patients with previous gynecologic and breast cancer with a dedicated next-generation sequencing panel covering 74 genes. We investigated the impact of clonal hematopoiesis of indeterminate potential-associated mutations (CHIP-AMs defined as presence at TRMN stage of mutations described in CHIP with a frequency >1%) on overall survival (OS) and the clinical relevance of a modified genetic ontogeny-based classifier that categorized patients in 3 subgroups. The most frequently mutated genes were TP53 (31%), DNMT3A (19%), IDH1/2 (13%), NRAS (13%), TET2 (12%), NPM1 (10%), PPM1D (9%), and PTPN11 (9%). CHIP-AMs were detected in 66% of TRMN patients, with no impact on OS. Yet, patients with CHIP-AM were older and had a longer time interval between solid tumor diagnosis and TRMN. According to our modified ontogeny-based classifier, we observed that the patients with TP53 or PPM1D mutations had more treatment lines and complex karyotypes, the "MDS-like" patients were older with more gene mutations, while patients with "De novo/pan-AML" mutations were younger with more balanced chromosomal translocations. Median OS within each subgroup was 7.5, 14.5, and 25.2 months, respectively, with statistically significant difference in multivariate analysis. These results support the integration of cytogenetic and molecular markers into the future TRMN classification to reflect the biological diversity of TRMN and its impact on outcomes

Mutational Profile and Dynamics of PPM1D -Mutant Clones in the Spectrum of Myeloid Disorders

International audienceIntroduction Mutations in PPM1D a regulator of DNA damage response, are enriched in patients with clonal hematopoiesis (CH) exposed to cytotoxic treatment or with therapy-related myeloid neoplasm. However, their role in leukemic transformation is unclear. Here, we describe a large cohort of patients with PPM1D mutations from CH to acute myeloid leukemia (AML) to understand the molecular landscape of mutant PPM1D related disease and the longitudinal dynamics of CH under treatment. Methods We included, in a non-sequential cohort, 96 PPM1D mutated patients treated at Gustave Roussy with CH, clonal cytopenia of unknown significance (CCUS), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN) or AML. A 77 gene panel NGS analysis was performed. An additional 16 PPM1D mutated AML patients from ALFA trials (1200, 0701,0702) were included. 10 patients with ovarian cancer from this cohort had sequential blood samples (OvBIOMark trial) available prior to hematologic evaluation, from the diagnosis or first relapse of their cancer through the course of therapy. Those samples were analyzed with a UMI based 18 gene panel (HaloplexHS, Agilent). Overall survival (OS) analyses were performed with the Kaplan-Meier method from the time of diagnosis until death from any cause. Results Among the 112 patients with PPM1D mutations, 23 (21%) had CH, 36 (32%) CCUS, 19 (17%) MDS, 25 (22%) AML (including 4 relapses), and 9 (8%) MPN. Median age was 65 [range, 21-88] years with 67% of females. Only 18% of the patients had no previous cancer history. Most frequent primary cancers were gynecological cancer (27%), lymphoid malignancies (22%), and breast cancer (17%). Median time between primary cancer diagnosis and hematologic assessment was 5.3 [range, 1.2-8.5] years. Eighty six percent of patients had one PPM1D mutation, 9% had 2, and 5% had 3 or more (restricted to CH and CCUS). Median variant allele frequency (VAF) was 3% [0.2-38], 3% [1-31], 2.4% [1-41], 23% [1-50], and 4% [1-42] in CH, CCUS, MDS, AML and MPN, respectively. Among CH/CCUS patients, PPM1D was the sole detected somatic mutation in 39% (23/59), compared to 9% (5/53) in MDS/AML/MPN patients (odds ratio=6, p=0.0004); 3/5 of the latter had complex karyotype. The most frequently co-mutated genes were DNMT3A (29%) and TP53 (25%), uniformly across all conditions ( Fig 1A). MDS-related gene mutations, RUNX1 (7%), ASXL1 (4%), SF3B1 (4%), SRSF2 (4%), U2AF1 (4%), were specific to AML/MDS/MPN. Among 28 patients with both PPM1D and TP53 mutations, PPM1D mutations were dominant or co-dominant in 64% (18/28), and secondary in 36% (10/28). IPSS-M risk of evaluable MDS was high/very high in 54% (7/13) of patients. ELN 2022 classification of AML was adverse in 68% (17/25) of patients. AML treatment options included best supportive care for 8% of patients (2), 5-Azacytidine for 32% (8), intensive chemotherapy for 60% (15). With a median follow up of 2.4 years, the median OS was 4.6 (CI95%; 4.1-NA), 1.31 (CI95%; 0.53-NA), 1.27 (0.43-NA), 0.66 (CI95%; 0.42-1.26) and 20.4 (CI95%; 20.4-NA) years for CH, CCUS, MDS, AML and MPN, respectively. TP53 mutation status did not stratify OS. Four ovarian cancer patients with CH/CCUS transformed to MDS/AML with a median of 5.3 [1-12] years. At transformation, 3/4 had a stable PPM1D mutation, 1/4 an increase in PPM1D VAF (2 to 28%), and 3/4 had TP53 mutations before and at transformation. We analyzed 67 timepoints from 10 ovarian cancer patients during therapy (median 7 per patient). 10/10 received alkylating agents and 5/10 PARPi. The median number of mutations per patient was 4 and 6 at baseline and last follow-up, respectively. PPM1D-mutated clone size increased from 0.4% [0.1-3] at baseline to 7% [2-30] at last follow-up. Beyond clonal expansion, PPM1D VAF dynamics showed non-linear changes related to alkylating agents exposure: 2/10 patients had a continuous expansion, 2/10 had expansion then contraction (GR-1/3, Fig 1B), and 6/10 had expansion then stabilization (GR-2, Fig 1B). Conclusion We described a large cohort of PPM1D mutated patients from CH to AML. Their prognosis was poor independently of TP53 mutation at AML/MDS stage. PPM1D mutations were frequently part of the dominant clone. To confirm the clonal architecture, single cell sequencing analysis in 8 AML/MDS patients are ongoing. Trajectory of PPM1D mutations in ovarian cancer patients revealed a non-linear alkylating agent dependency which warrants further investigation