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TERRA (do ingles telomeric repeat-containing RNA) e um RNA longo nao-codificante transcrito a partir das regioes subtelomericas e telomericas. Os transcritos de TERRA possuem sequencias repetidas em tandem ricas em Guanina oriundas das sequencias telomericas TTAGGG e formam hibridos de DNA:RNA nas regioes telomericas denominados R-loops. A alta expressao de TERRA e a formacao de R-loops promove dano ao DNA telomerico e, consequente, encurtamento telomerico. TERRA participa de um mecanismo de feedback que depende do ciclo celular e do comprimento telomerico. Em telomeros longos, os R-loops sao degradados pelas enzimas RNaseH1 e RNaseH2 antes da replicacao telomerica, o que permite o alongamento telomerico a partir da telomerase. Em tolemeros criticamente curtos, essas enzimas nao sao recrutadas, ocorre acumulo de R-loops, estresse replicativo e ativacao de vias de reparo dirigido por homologia. Essas vias promovem alongamento telomerico por via alternativas a telomerase. As leucemias mieloides agudas (LMAs) sao caracterizadas pela expansao de celulas mieloides imaturas e insuficiencia da medula ossea. Atualmente, as LMAs sao classificadas em 6 grandes grupos: 1) LMAs com alteracoes citogeneticas recorrentes; 2) LMAs relacionadas a terapia; 3) LMAs relacionadas a alteracoes Mielodisplasicas; 4) LMAs sem outra especificacao; 5) Sarcoma mieloide e 6) proliferacao mieloide relacionada a Sindrome de Down. Como exposto anteriormente, o acumulo de TERRA em telomeros curtos desencadeia, indiretamente, vias de reparo homologo que podem promover alongamento telomerico independente de telomerase. Comprimento telomerico curto e uma caracteristica de blastos na LMA. O objetivo do presente estudo foi determinar a contribuicao de TERRA para a leucemogenese na LMA. Para isso, a dinâmica telomerica e a expressao de TERRA foram investigadas em celulas leucemicas de medula ossea de pacientes ao diagnostico e em linhagens de LMA. A RNaseH1 foi superexpressa em linhagens com alta expressao de TERRA e telomero curto para verificar se ha aumento da taxa de apoptose. Oitenta e oito pacientes foram utilizados na analise final. A media de idade foi de 43 anos, sendo a relacao M:F de 1:1. O diagnostico de LMA foi categorizado conforme a atualizacao da OMS de 2016, sendo: Leucemia promielocitica aguda, 23/88 pacientes; LMA com t(8;21)(q22;q22.1), RUNX1-RUNX1T1, 9/88 pacientes; LMA com t(16;16)(p13.1;q22), CBFB-MYH11, 3/88 pacientes; LMA com NPM1 mutado, 11/98 pacientes, LMA com mutacao bialelica do CEBPA, 2/88 pacientes; LMA sem outra especificacoes, 40/88 pacientes. A taxa de resposta completa apos a primeira inducao (IR) foi de 55,6%, de resposta parcial foi de 5,6%. Seis pacientes foram refratarios a IR, 6,8% dos pacientes. Os dados de expressao de TERRA 10q, 15q, 20q, XqYq, XpYp, 17p, ATRX, RNaseH1, RNaseH2, TERT, TRF2 obtidos por RT-qPCR e comprimento telomerico realizados por PCR foram normalizados e utilizados para realizar a hierarquizacao por cluster atraves do calculo por distância euclidiana. As leucemias foram classificadas em 4 clusters, denominados HCL1-4 (figura 1). O grupo 2 destaca-se pela alta expressao de TERRA e baixa expressao de ATRX, RNaseH1, RNaseH2, TERT, TRF2 e alta expressao TERRA 10q, 15, 20q e XqYq. O ensaio com MTT demonstrou que as linhagens nas linhagens NB4 (linhagem de leucemia promielocitica) e THP-1 (linhagem de leucemia mieloide aguda com diferenciacao monocitica) que superexpressao RNaseH1 sao mais susceptiveis a citarabina do que aquelas transfectadas com vetor vazio.

Objectives: Immune aplastic anemia (AA) is a hematological disease characterized by pancytopenia and bone marrow (BM) hypocellularity. It is caused by an immune attack against hematopoietic stem and progenitor cells (HSPC). Its treatment is based on allogeneic BM transplantation and immunosuppressive therapy. More recently, the addition of eltrombopag showed that the expansion of hematopoietic precursors is a therapeutic alternative. Fares et al. demonstrated that the small molecule UM171 is capable of expanding the most primitive CD34+ cell compartment of umbilical cord blood (UCB) ex vivo. Our research group showed that UM171 is also capable of effectively expanding immature subpopulations of CD34+ cells from patients with immune AA. The aim of this study was to evaluate the engraftment capacity of CD34+ cells from patients with AA expanded with UM171 in a murine xenotransplantation model. Methods: CD34+ cells were purified from either bone marrow of one patient or one UCB unit using immunomagnetic labeling with human CD34 MicroBeads, MS columns, and a magnetic separator. Cells were cultured for seven days in medium supplemented with cytokines and 35 nM UM171 or 0.1% DMSO (control) and then transplanted via retro-orbital injection into pre-conditioned 8-to-12-week-old female NSG-SGM3 (NSGS) mice. The progeny of 3×104UCB CD34+ cells or 8.250 AA patient CD34+ cells was transplanted per mouse (four mice per experimental condition). The conditioning regimen consisted of two intraperitoneal injections of 12.5 mg/kg busulfan administered 48 and 24 hours before transplantation. Human hematopoietic cells were monitored in the murine BM by flow cytometry at week 20 post-transplantation. BM cells were collected from the two femurs and the two tibias of each animal and treated twice with red cell lysis, labeled with APC-Cy7 anti-mouse and PerCP-Cy5.5 anti-human CD45 antibodies, then analyzed on a flow cytometer using the FlowJo V.10 software. Results: We found that both progenies of UCB and AA BM CD34+ cells cultured with UM171 showed an improved reconstitution potential in mice BM 20 weeks after transplant compared to DMSO, which was demonstrated by the detection of higher percentages of human CD45+ cells in the mice BM (UCB: UM171 = 10,07% ±2,29 vs DMSO = 3,73% ±1,99; AA BM: UM171 = 0,095% ±0,075 vs DMSO = 0,042% ±0,002; data represent mean ±Standard Error). Differences were not significant. Discussion: We wondered whether the UM171 treatment would impact the engraftment capacity of the in vitro expanded CD34+ cells. The enhanced engraftment capacity enabled by UM171 could allow for immune AA patients to be treated with a novel strategy, autologous transplantation of their own CD34+ expanded cells. The present study indicates that UM171 could be a promising candidate drug for treating patients with immune AA. More replicates (patients and controls) are required to firmly establish the benefit of UM171 in conferring repopulation potential to ex vivo expanded CD34+ cells from AA patients. Conclusion: In initial experiments, in vitro treatment with UM171 appears to increase the engraftment of CD34+ cells isolated from UCB and from the BM of immune AA patients. These results need to be confirmed in additional experiments.

Eltrombopag (EPAG), a thrombopoietin receptor agonist, has been recently added to first-line immunosuppressive therapy (IST) in immune aplastic anemia (AA), significantly improving hematologic recovery and survival (Townsley et al. NEJM 2017), corroborating preclinical data suggesting a direct effect of EPAG on hematopoietic precursors. Here, we analysed the in vivo effects of EPAG on the composition of the hematopoietic stem and progenitor cell (HSPC) compartment after hematologic recovery in human AA patients treated with IST combined with EPAG. Twelve patients with severe immune aplastic anemia who achieved hematologic response six months after initial treatment were invited to participate in the study. Six patients were treated with EPAG associated with cyclosporine and six were treated with rabbit antithymocyte globulin and cyclosporine, but treatment choice was not determined by this study. 25 mL of marrow aspirate was obtained from each patient for multiparametric analysis in an 8-color flow cytometry. Mononuclear cells were separated by gradient density centrifugation and CD34+ cells were purified using magnetic column beads. We used a 9-antibody panel to identify HSPC subpopulations, as follows: hematopoietic stem cells (HSC), CD34+ CD38- CD45RA- CD90+ CD49f+; multipotent progenitor cells (MPP), CD34+ CD38- CD45RA- CD90- CD49f-; multilymphoid progenitor cells (MLP), CD34+ CD38+ CD45RA+ CD10+ CD7-; common myeloid progenitor cells (CMP), CD34+ CD38+ CD45RA- CD135+ CD10- CD7-; granulocyte-monocyte progenitor cells (GMP), CD34+ CD38+ CD45RA+ CD135+ CD10- CD7-; and megakaryocytic-erythroid progenitor cells (MEP), CD34+ CD38+ CD45RA- CD135- CD10- CD7-. Purified marrow CD34+ cells were also assessed in methylcellulose medium and colonies were counted 14 days after triplicate plating. Cell populations and colony formation assay were compared between the two groups using a two-tailed non-parametric Mann-Whitney test. The percentage of CD34+ of total nucleated bone marrow cells was 1.2% (range, 0.28-2.9%) in patients who received IST/EPAG and 0.2% (0.11-0.65%) in those who received IST alone (P Townsley et al. (NEJM 2017) reported on the CD34+ compartment reconstitution three and six months after initial therapy, but did not compare this recovery to patients not receiving EPAG. Additionally, they did not indicate which primitive cells are directly affected by EPAG. It was speculated that EPAG may act on HSPC proliferation via the THPO-MPL signalling pathway, supported by previously evidence that loss-of-function mutations in the THPO gene result in aplastic anemia (Dasouki et al. Blood 2013). Here, we compared for the first time the HSPC compartment reconstitution in AA patients treated with either IST alone or EPAG added to IST. We found that marrow CD34+ cell numbers were higher when EPAG was added to IST, indicating a positive effect on the HSPC compartment. More importantly, we found that MPPs were the only HSPC subset to be significantly increased when patients were treated with EPAG. The role of the THPO-MPL signalling pathway in HSPCs is not entirely clear, but our findings are in agreement with recent observations. In xenotransplant models, Matsuoka et al (Cell Transplantation 2017) found that human CD34+ MPL- cells are able to sustain long-term engraftment, whereas MPL+ cells showed deficient repopulation capacity. Likewise, Cocault et al (Experimental Hematology 2016) found that in human CD34+CD38- cells, MPL is preferentially expressed in CD90-CD49f- cells. Recent observations in refractory AA patients suggest that EPAG may expand quiescent preexisting abnormal clones in the marrow (Winkler et al. Blood 2019). However, in our cohort, we did not observe any aberrant HSPC immunophenotype suggesting the expansion of abnormal clones. In conclusion, we found that in patients with immune aplastic anemia, EPAG significantly expands CD34+ cells and specifically expands hematopoietic multipotent progenitor cells. Our findings support the hypothesis that in vivo the THPO-MPL signalling pathway plays a role in human MPP proliferation, but not in HSCs. Disclosures Scheinberg: Novartis Brasil S.A.: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Calado:Novartis Brasil S.A.: Speakers Bureau.

Telomeropathies are a group of phenotypically heterogeneous diseases molecularly unified by pathogenic mutations in telomere-maintenance genes causing critically short telomeres. X-linked dyskeratosis congenita (DC), the prototypical telomere disease, manifested with ectodermal dysplasia, cancer predisposition, and severe bone marrow failure, is caused by mutations in DKC1, encoding a protein responsible for telomerase holoenzyme complex stability. To investigate the effects of pathogenic DKC1 mutations on telomere repair and hematopoietic development, we derived induced pluripotent stem cells (iPSCs) from fibroblasts of a DC patient carrying the most frequent mutation: DKC1 p.A353V. Telomeres eroded immediately after reprogramming in DKC1-mutant iPSCs but stabilized in later passages. The telomerase activity of mutant iPSCs was comparable to that observed in human embryonic stem cells, and no evidence of alternative lengthening of telomere pathways was detected. Hematopoietic differentiation was carried out in DKC1-mutant iPSC clones that resulted in increased capacity to generate hematopoietic colony-forming units compared to controls. Our study indicates that telomerase-dependent telomere maintenance is defective in pluripotent stem cells harboring DKC1 mutation and unable to elongate telomeres, but sufficient to maintain cell proliferation and self-renewal, as well as to support the primitive hematopoiesis, the program that is recapitulated with our differentiation protocol. Keywords: Induced pluripotent stem cells (iPSCs), Telomeres, Telomerase, X-linked dyskeratosis congenita, DKC1, Hematopoietic differentiation

A anemia aplástica (AA) é uma doença hematológica rara caracterizada pela hipocelularidade da medula óssea, o que provoca pancitopenia. Esta pode ser de origem genética (associada a encurtamento telomérico) ou adquirida (não-associada a desgaste excessivo dos telômeros). Na forma adquirida, a ativação anormal de linfócitos T provoca a destruição das células hematopoéticas. O mecanismo que leva a essa destruição ainda não foi elucidado. Um dos tratamentos mais eficazes para repovoar a medula óssea hipocelular é o transplante com célulastronco hematopoéticas (CTHs). Porém, uma grande porcentagem de pacientes não se beneficia de nenhum tratamento, fazendo-se necessário o desenvolvimento de novas alternativas para terapia. A geração de células-tronco pluripotentes induzidas (iPSCs) a partir de células somáticas (reprogramação) representa uma ferramenta promissora para o estudo de doenças e para o desenvolvimento de possíveis terapias paciente-especificas, como transplantes autólogos. Neste trabalho, avaliamos a capacidade de reprogramação de fibroblastos e eritroblastos de pacientes com AA adquirida. Metodologias de reprogramação utilizando lentivírus ou plasmídeos epissomais não integrativos foram testadas em células de quatro pacientes e de um controle saudável. Eritroblastos dos quatro pacientes e do controle foram reprogramados utilizando os plasmídeos não integrativos. As iPSCs geradas apresentaram-se similares a células-tronco embrionárias quanto à morfologia, expressão dos marcadores de pluripotência OCT4, SOX2, NANOG, SSEA-4, Tra-1-60 e Tra-1-81, e capacidade de diferenciação in vitro em corpos embrioides (EBs). A dinâmica telomérica das células pré- e pós-reprogramação foi avaliada em diferentes passagens utilizando a técnica de flow-FISH. O comprimento telomérico foi aumentado nas iPSCs quando comparado às células parentais o que indica que a célula foi completamente reprogramada. No presente trabalho, células de pacientes com AA adquirida foram reprogramadas a um estado de pluripotência por meio de um método não integrativo. As iPSCs geradas serão essenciais para futuros ensaios de diferenciação hematopoética, o que poderá contribuir para o entendimento dos mecanismos envolvidos no desenvolvimento dessa doença. Além disso, a diferenciação dessas células livres de transgenes poderá servir como uma alternativa terapêutica para os pacientes com AA como, por exemplo, em transplantes autólogos Aplastic anemia (AA) is a rare hematological disease characterized by bone marrow hypocellularity that leads to pancytopenia. Its origin can be genetic (associated with telomere shortening) or acquired (non-associated with telomere shortening). The acquired form exhibit T lymphocytes abnormal activation, which leads to hematopoietic cells destruction. The mechanisms behind this phenomenon are still unclear. One of the most effective treatments for hypocelullar bone marrow repopulation is hematopoietic stem cell (HSCs) transplantation. However, a large percentage of patients do not benefit from any of the available treatments. This highlights the need to develop new therapeutic strategies. The generation of induced pluripotent stem cells (iPSCs) from somatic cells (reprogramming) represents a powerful tool for disease modeling and for the development of patient-specific therapies such as autologous transplants. In this study, we evaluate the capacity of reprogramming acquired AA patients\' fibroblasts and erythroblasts. Reprogramming methods using lentivirus or non-integrative episomal plasmids were tested in four patients\' cells and in cells from one healthy donor. Erythroblasts from these four patients and healthy donor were reprogrammed using non-integrative plasmids. The iPSCs resembled human embryonic stem cells in morphology, in the expression of pluripotent markers such as OCT4, SOX2, NANOG, SSEA-4, Tra-1-60 and Tra-1-81, and in in vitro differentiation (capacity to form embryoid bodies). The telomere dynamics of the cells before and after reprogramming was assessed along passaging using flow-FISH. The telomere length in the iPSCs was increased when compared to the parental cells. Thus, acquire AA patients\' cells could be reprogrammed to a pluripotent state by a nonintegrative method. The iPSCs will be essential for future hematopoietic differentiation assays that could contribute to the understanding of the mechanisms involved in the disease development. Furthermore, the differentiation of transgene-free cells may serve as an alternative therapy for patients with AA such as autologous transplants

Background aims For patients with aplastic anemia (AA) who are refractory to anti-thymocyte globulin (ATG) and cyclosporine, a second course of immunosuppression is successful in only one-fourth to one-third of cases. Methods We conducted a phase 1/2 study to evaluate the addition of two to five weekly intravenous infusions of allogeneic unrelated non–human leukocyte antigen–matched bone marrow–derived mesenchymal stromal cells (MSCs) (median, 2.7 × 106 cells/kg/infusion; range, 1.3–4.5) to standard rabbit ATG and cyclosporine in nine patients with refractory or relapsed AA. Results After a median follow-up of 20 months, no infusion-related adverse event was observed, but four deaths occurred as the result of heart failure and bacterial or invasive fungal infections; only two patients achieved partial hematologic responses at 6 months. We failed to demonstrate by fluorescence in situ hybridization or variable number tandem repeat any MSC engraftment in patient marrow 30, 90 or 180 days after infusions. Conclusions Infusion of allogeneic MSCs in AA is safe but does not improve clinical hematologic response or engraft in recipient bone marrow. This study was registered at clinicaltrials.gov , identifier: NCT01297972 .