Long non-coding RNAs as novel therapeutic targets in juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) treatment primarily relies on hematopoietic stem cell transplantation and results in long-term overall survival of 50-60%, demonstrating a need to develop novel treatments. Dysregulation of the non-coding RNA transcriptome has been demonstrated before in this rare and unique disorder of early childhood. In this study, we investigated the therapeutic potential of targeting overexpressed long non-coding RNAs (lncRNAs) in JMML. Total RNA sequencing of bone marrow and peripheral blood mononuclear cell preparations from 19 untreated JMML patients and three healthy children revealed 185 differentially expressed lncRNA genes (131 up- and 54 downregulated). LNA GapmeRs were designed for 10 overexpressed and validated lncRNAs. Molecular knockdown (>= 70% compared to mock control) after 24 h of incubation was observed with two or more independent GapmeRs in 6 of them. For three lncRNAs (lnc-THADA-4, lnc-ACOT9-1 and NRIR) knockdown resulted in a significant decrease of cell viability after 72 h of incubation in primary cultures of JMML mononuclear cells, respectively. Importantly, the extent of cellular damage correlated with the expression level of the lncRNA of interest. In conclusion, we demonstrated in primary JMML cell cultures that knockdown of overexpressed lncRNAs such as lnc-THADA-4, lnc-ACOT9-1 and NRIR may be a feasible therapeutic strategy

Despite mutation acquisition in hematopoietic stem cells, JMML-propagating cells are not always restricted to this compartment

Juvenile myelomonocytic leukemia (JMML) is a rare aggressive myelodysplastic/myeloproliferative neoplasm of early childhood, initiated by RAS-activating mutations. Genomic analyses have recently described JMML mutational landscape; however, the nature of JMML-propagating cells (JMML-PCs) and the clonal architecture of the disease remained until now elusive. Combining genomic (exome, RNA-seq), Colony forming assay and xenograft studies, we detect the presence of JMML-PCs that faithfully reproduce JMML features including the complex/nonlinear organization of dominant/minor clones, both at diagnosis and relapse. Further integrated analysis also reveals that although the mutations are acquired in hematopoietic stem cells, JMML-PCs are not always restricted to this compartment, highlighting the heterogeneity of the disease during the initiation steps. We show that the hematopoietic stem/progenitor cell phenotype is globally maintained in JMML despite overexpression of CD90/THY-1 in a subset of patients. This study shed new lights into the ontogeny of JMML, and the identity of JMML-PCs, and provides robust models to monitor the disease and test novel therapeutic approaches

LIN28B overexpression defines a novel fetal-like subgroup of juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is a rare and aggressive stem cell disease of early childhood. RAS activation constitutes the core component of oncogenic signaling. In addition, leukemic blasts in one-fourth of JMML patients present with monosomy 7, and more than half of patients show elevated age-adjusted fetal hemoglobin (HbF) levels. Hematopoietic stem cell transplantation is the current standard of care and results in an event-free survival rate of 50% to 60%, indicating that novel molecular-driven therapeutic options are urgently needed. Using gene expression profiling in a series of 82 patient samples, we aimed at understanding the molecular biology behind JMML and identified a previously unrecognized molecular subgroup characterized by high LIN28B expression. LIN28B over expression was significantly correlated with higher HbF levels, whereas patients with monosomy 7 seldom showed enhanced LIN28B expression. This finding gives a biological explanation of why patients with monosomy7 are rarely diagnosed with high age-adjusted HbF levels. In addition, this new fetal-like JMML subgroup presented with reduced levels of most members of the let-7 microRNA family and showed characteristic overexpression of genes involved in fetal hematopoiesis and stem cell self-renewal. Lastly, high LIN28B expression was associated with poor clinical outcome in our JMML patient series but was not independent from other prognostic factors such as age and age-adjusted HbF levels. In conclusion, we identified elevated LIN28B expression as a hallmark of a novel fetal-like subgroup in JMM

CD200/BTLA deletions in pediatric precursor B-cell acute lymphoblastic leukemia treated according to the EORTC-CLG 58951 protocol

DNA copy number analysis has been instrumental for the identification of genetic alterations in B-cell precursor acute lymphoblastic leukemia. Notably, some of these genetic defects have been associated with poor treatment outcome and might be relevant for future risk stratification. In this study, we characterized recurrent deletions of CD200 and BTLA genes, mediated by recombination-activating genes, and used breakpoint-specific polymerase chain reaction assay to screen a cohort of 1154 cases of B-cell precursor acute lymphoblastic leukemia uniformly treated according to the EORTC-CLG 58951 protocol. CD200/BTLA deletions were identified in 56 of the patients (4.8%) and were associated with an inferior 8-year event free survival in this treatment protocol [70.2% +/- 1.2% for patients with deletions versus 83.5% +/- 6.4% for non-deleted cases (hazard ratio 2.02; 95% confidence interval 1.23-3.32; P=0.005)]. Genetically, CD200/BTLA deletions were strongly associated with ETV6-RUNX1-positive leukemias (P<0.0001), but were also identified in patients who did not have any genetic abnormality that is currently used for risk stratification. Within the latter population of patients, the presence of CD200/BTLA deletions was associated with inferior event-free survival and overall survival. Moreover, the multivariate Cox model indicated that these deletions had independent prognostic impact on event-free survival when adjusting for conventional risk criteria. All together, these findings further underscore the rationale for copy number profiling as an important tool for risk stratification in human B-cell precursor acute lymphoblastic leukemia

Differential activation of basal and IL-7-induced PI3K/Akt/mTOR and JAK/STAT5 signaling distinguishes pediatric from adult acute lymphoblastic leukemia

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A predictive classifier of poor prognosis in transplanted patients with juvenile myelomonocytic leukemia: a study on behalf of the Société Francophone de Greffe de Moelle et de Thérapie Cellulaire

Juvenile myelomonocytic leukemia (JMML) is an aggressive pediatric myeloproliferative neoplasm requiring hematopoietic stem cell transplantation (HSCT) in most cases. We retrospectively analyzed 119 JMML patients who underwent first allogeneic HSCT between 2002 and 2021. The majority (97%) carried a RAS-pathway mutation, and 62% exhibited karyotypic alterations or additional mutations in SETBP1, ASXL1, JAK3 and/or the RAS pathway. Relapse was the primary cause of death, with a 5-year cumulative incidence of 24.6% (95%CI: 17.1-32.9). Toxic deaths occurred in 12 patients, resulting in treatmentrelated mortality (TRM) of 9.0% (95%CI: 4.6-15.3). The 5-year overall (OS) and event-free survival were 73.6% (95%CI: 65.7-82.4) and 66.4% (95%CI: 58.2-75.8), respectively. Four independent adverse prognostic factors for OS were identified: age at diagnosis >2 years, time from diagnosis to HSCT >6 months, monocyte count at diagnosis >7.2x109/L, and the presence of additional genetic alterations. Based on these factors, we proposed a predictive classifier. Patients with three or more predictors (21% of the cohort) had a 5-year OS of 34.2%, whereas those with none (7%) had a 5-year OS of 100%. Our study demonstrates improved transplant outcomes compared to prior published data, which can be attributed to the synergistic impacts of a low TRM and a reduced yet still substantial relapse incidence. By integrating genetic information with clinical and hematological features, we have devised a predictive classifier. This classifier effectively identifies a subgroup of patients who are at a heightened risk of unfavorable post-transplant outcomes who would benefit novel therapeutic agents and post-transplant strategies

Germline bi-allelic <i>SH2B3/LNK</i> alteration predisposes to a neonatal juvenile myelomonocytic leukemia-like disorder

Juvenile myelomonocytic leukemia (JMML) is a rare, generally aggressive myeloproliferative neoplasm affecting young children. It is characterized by granulomonocytic expansion, with monocytosis infiltrating peripheral tissues. JMML is initiated by mutations upregulating RAS signaling. Approximately 10% of cases remain without an identified driver event. Exome sequencing of 2 unrelated cases of familial JMML of unknown genetics and analysis of the French JMML cohort identified 11 patients with variants in SH2B3, encoding LNK, a negative regulator of the JAK-STAT pathway. All variants were absent from healthy population databases, and mutation spectrum was consistent with a loss of function of the LNK protein. A stoploss variant was shown to affect both protein synthesis and stability. The other variants were either truncating or missense, the latter affecting the SH2 domain that interacts with activated JAK. Of the 11 patients, 8 from 5 families inherited pathogenic bi-allelic SH2B3 germline variants from their unaffected heterozygous parents. These children represent half of the cases with no identified causal mutation in the French cohort. They displayed typical clinical and hematological JMML features with neonatal onset and marked thrombocytopenia. They were characterized by absence of additional genetic alterations and a hypomethylated DNA profile with fetal characteristics. All patients showed partial or complete spontaneous clinical resolution. However, progression to thrombocythemia and immunity-related pathologies may be of concern later in life. Bi-allelic SH2B3 germline mutations thus define a new condition predisposing to a JMML-like disorder, suggesting that the JAK pathway deregulation is capable of initiating JMML, and opening new therapeutic options

The MLL recombinome of acute leukemias in 2017

Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients

Genetics and clonal architecture of Juvenile Myelomonocytic Leukemia

La LMMJ est un syndrome myéloprolifératif et myélodysplasique rare du jeune enfant, initiée par des mutations classiquement décrites comme mutuellement exclusives de RAS (NRAS, KRAS) ou de régulateurs de la voie RAS (PTPN11, NF1 ou CBL). Ces mutations, somatiques ou constitutionnelles, entraînent l’hyperactivation de cette voie de signalisation et une hypersensibilité spécifique au GM-CSF. La LMMJ est une hémopathie sévère dont le seul traitement est l’allogreffe de moelle osseuse. Cependant sa présentation et son évolution sont particulièrement hétérogènes puisqu’une transformation en leucémie aiguë myéloide survient chez un tiers des patients quand d’autres présentent des formes plus indolentes, voire des rémissions spontanées en l’absence de greffe. Cette hétérogénéité n’est que partiellement liée à la mutation initiatrice et pourrait s’expliquer par la présence de mutations additionnelles et/ou par une variabilité dans la cellule initiatrice de la leucémie, qui n’a jamais été précisément caractérisée.La caractérisation génétique de 118 LMMJ nous a permis de montrer que les anomalies génétiques additionnelles sont peu nombreuses dans les LMMJ sporadiques, et exceptionnelles dans les LMMJ syndromiques sauf en cas de neurofibromatose de type-1. Ces anomalies se concentrent sur deux grands systèmes, la voie RAS et le PRC2, et leur présence s’accompagne d’un pronostic défavorable (particulièrement en cas de mutations multiples de la voie RAS). L’absence d’anomalie additionnelle permet à l’inverse de distinguer un sous-groupe de patients qui présentent une forte probabilité de survie à long terme sans greffe et pour lesquels une soultion attentiste serait à privilégier. Une collaboration avec l’équipe de D Bonnet (Crick Institute) nous a ensuite permis d’établir un modèle murin de xénotransplantation dans des souris immunodéficientes de type NSG ou NSG-S et de montrer que la capacité de propagation de la leucémie est bien portée par la fraction souche, mais s’étend aussi chez certains patients à des fractions plus différenciées. Le profil génétique des 15 xénogreffes étudiées reproduit fidèlement l’architecture clonale des LMMJ natives, tant dans les souris NSG que NSG-S. L’architecture clonale des LMMJ est dans la majorité des cas compatible avec une acquisition linéaire des altérations, mais une architecture complexe est parfois observée, avec coexistence de clones distincts, dont les plus faiblement représentés sont susceptibles de devenir dominants lors de la rechute. Le séquençage de sous-populations isolées a montré que l’ensemble des mutations (initiatrice et additionnelles) est présent dès les fractions les plus immatures (HSC/MPP/MLP). Le séquençage de colonies obtenues par culture des progéniteurs en méthylcellulose révèle que les mutations coexistent dans les mêmes cellules, sans qu’il soit possible de hiérarchiser leur ordre de survenue, témoignant d’un avantage sélectif majeur de leur association dès la cellule souche. Au total, nos résultats remettent en cause le dogme de l’exclusivité mutuelle des mutations activant RAS dans les LMMJ, confirment le rôle central et initiateur de cette voie oncogénique dans la leucémogénèse et suggérent un effet-dose de l’activation de RAS, en particulier en cas de mutation de NRAS. La présence d’altérations multiples ciblant la voie RAS marque des LMMJ agressives et rapidement évolutives. La mise en évidence d’une fréquente dérégulation du PRC2 offre de nouvelles perspectives therapeutiques (comme l’utilisation des inhibiteurs de bromodomaine). La mise en place d’un modèle de souris xénotransplantée devrait de plus faciliter les études biologiques et la mise en place d’évaluations précliniques.JMML is a rare myeloproliferative and myelodysplastic neoplasm of early childhood, initiated by mutations classically described as mutually exclusive of RAS (NRAS, KRAS) or RAS pathway regulators (PTPN11, NF1 or CBL). These mutations, either germline or somatically aquired, lead to an hyperactivation of the RAS signalling pathway and a to a specific hypersensitivity to GM-CSF. JMML is a severe hemopathy, and the only curative treatment is allogenic bone marrow transplantation. However, its presentation and evolution are particularly heterogeneous since transformation into acute myeloid leukaemia occurs in about one third of patients, when others present more indolent forms, or even spontaneous remissions in the absence of transplantation. This heterogeneity is only partially accounted for by the initiating mutation and could be related to the presence of additional mutations, or some variability in the leukemia initiating cell, which has never been precisely characterized so far.Establishing the genetic landscape of 118 LMMJ allowed us to show that additional genetic abnormalities are scarse in sporadic JMML and exceptional in syndromic JMML, except in the case of type-1 neurofibromatosis. These additional abnormalities mainly target two major biologic components, the RAS pathway and the PRC2, and their presence is associated with an unfavourable prognosis (particularly in the case of multiple mutations targeting the RAS pathway). On the other hand, the absence of any additional abnormality allows to delineate a subgroup of patients who have a high probability of long-term survival in the absence of bone marrow transplantation, and for whom a wait-and-see approach would be preferable. A collaboration with D. Bonnet’s group (Crick Institute) allowed us to establish a mouse model of xenotransplantation in immunodeficient NSG or NSG-S mice and to demonstrate that the leukemia propagating cell is present in the stem cell fractions (HSC, CD34+/CD38-…) but also extends in certain patients to more differentiated fractions, such as CMP. The genetic profile of xenografts established from 15 JMML faithfully reproduced the clonal architecture of the native leukemia, either in NSG or NSG-S mice. The clonal architecture of JMML is linear in the great majority of cases, with linear acquisition of alterations, but a complex architecture is sometimes observed, with coexistence of distinct clones, the weakest of which being susceptible to become dominant at relapse. Sequencing of sorted cell populations showed that all mutations (initiating and additional) are present in the most immature fractions (HSC/MPP/MLP). The sequencing of colonies obtained by culturing progenitors into methylcellulose revealed that mutations coexist in the same cells, their order of appearance being often impossible to determine, showing a major selective advantage of their association from the most immature compartment. In conclusion, our findings confirm the central role of RAS activation in JMML leukemogenesis. The identification of multiple alterations targeting the RAS pathway challenges the dogma of the mutual exclusivity of these mutations and defines a subset of aggressive and rapidly evolving JMML, suggesting a dose-effect of RAS activation, particularly in case with NRAS mutation. Recurrent deregulation of PRC2 in JMML may offer new therapeutic approaches, such as bromodomain inhibitors. The implementation of a xenotransplanted mouse model should also facilitate biological studies and the implementation of preclinical evaluations