Natural killer (NK) cells are a subset of lymphocytes that play a critical role in both innate and adaptive immune responses and provide defense against microbial infections and malignant transformation. When stimulated through their activating or cytokine receptors, NK cells rapidly produce cytokines and chemokines, including IFNγ, TNFα, TGFβ, GM-CSF, MIP1α, MIP1β, IL-10, and others, which can affect the function of hematopoietic stem cells (HSC)s. We hypothesized that NK-mediated cytokine production could regulate HSC function and that its deregulation may favor HSC malignant transformation and proliferation. In this regard, a few studies have shown that NK cells are defective in stem cell-derived diseases such as chronic myeloid leukemia (CML), for example, raising the hypothesis that immunological dysfunctions may be involved in initiating or contributing to the pathogenesis of myeloproliferative diseases. In contrast to CML, the role of NK cells in BCR-ABL1 negative-myeloproliferative neoplasms (MPN) is currently unexplored. Therefore, we aimed to study NK cell activity in murine and human primary cells from MPN. NK cells and receptors were quantified by flow cytometry by the use of spleen cells from a conditional knockin Jak2V617F (Jak2VF) murine model, that faithfully resemble the main clinical and laboratorial characteristics of human Polycytemia Vera (PV), and from MPN patients diagnosed and followed at the Clinical Hospital of the Medical School of Ribeirao Preto, University of Sao Paulo. For immunophenotyping of murine lymphocytes, spleen cells were isolated and stained with fluorescence-conjugated antibodies against murine Ter119, CD19, CD4, CD8, CD3, NK1.1 (for NK cells quantification) and NKG2A, NKG2D, Ly49H and Ly49D (for receptors analysis). For analysis of human lymphocytes, peripheral blood mononuclear cells were purified by density gradient centrifugation, resuspended in serum-enriched phosphate buffered saline and stained with fluorescence-labeled anti-human antibodies against the following molecules: CD45, CD19, CD3, CD16, CD56. All the stainings were performed for 20 minutes at 40 C and cells were acquired using a FACScalibur flow cytometer (BD Biosciences). Data was analyzed by the use of the Flojo software version X and the statistical analyses were performed by the use of SPSS version 20. When compared to Jak2WT controls, Jak2VF mice had lower numbers of NK cells (2.09% versus 22.7% of the spleen cells, P = 0.022) and NKG2A (0.57% versus 2.47% of NK cells, P = 0.037) and NKG2D (0.17% versus 0.46% of NK cells, P = 0.020) receptors. For the study of human NK cells, 38 patients diagnosed with MPN and who were not in use of cytoreductive therapy were included and classified according to the WHO 2008 criteria as Polycytemia Vera (PV, n = 11), Essential Thrombocytemia (ET, n = 11), Primary Myelofibrosis (PMF, n = 11) or Unclassified MPN (uMPN, n = 5). Blood samples from 34 healthy donors served as controls. JAK2V617F (JAK2VF) mutated patients had lower numbers of CD16+ CD56dim NK cells as compared to JAK2VF negative and healthy controls (0.73% versus 1.06% versus 1.54%, P = 0.038, respectively). Among the JAK2 mutated patients, those with PV and PMF presented lower numbers of NK cells than ET patients. In agreement, NK cells were reduced in JAK2VF PV patients when compared to JAK2VF negative and controls (0.39% versus 1.42% and 1.54%, P = 0.047). PMF patients had lower NK cell numbers than controls, regardless of JAK2VF mutation (0.35%, 0.49% and 1.54% for JAK2VF positive, negative and controls, respectively, P = 0.025). No significant differences were observed in ET or uMPN. In summary, NK cells were reduced among lymphocytes in MPN patients, especially in the JAK2VF mutated cases, as shown in PV patients and Jak2VF mice. In addition, the expression of NK receptors was reduced in Jak2VF mice, thus suggesting that these cells are defective in MPN. New insights into the pathogenesis of this group of diseases are of great relevance considering that MPN treatment remains non-curative with the strategies available so far despite the development of drugs that inhibit JAK2. Thus, identifying the mechanisms of NK-cell deficiency in MPN and the detailed interaction between these cells and the malignant stem cell may help to establish combination therapeutic strategies that may improve therapy responses in MPN. Disclosures No relevant conflicts of interest to declare.

The incidence and mortality of malignant melanoma is increasing. Its

Several lines of research suggest that Bcl-xL-mediated anti-apoptotic effects may contribute to the pathogenesis of myeloproliferative neoplasms driven by JAK2V617F and serve as therapeutic target. Here, we used a knock-in JAK2V617F mouse model and confirmed that Bcl-xL was overexpressed in erythroid progenitors. The myeloproliferative neoplasm (MPN)-induced phenotype in the peripheral blood by conditional knock-in of JAK2V617F was abrogated by conditional knockout of Bcl2l1, which presented anemia and thrombocytopenia independently of JAK2 mutation status.Mx1-Cre Jak2V617 W/VF /Bcl2l1 f/f mice presented persistent splenomegaly as a result of extramedullary hematopoiesis and pro-apoptotic stimuli in terminally differentiated erythroid progenitors. The pan-BH3 mimetic inhibitor obatoclax showed superior cytotoxicity in JAK2V617F cell models, and reduced clonogenic capacity in ex vivo

Primeiramente, agradeço à Deus. À minha família, em especial meus pais e meu esposo, Thiago, pelo apoio incondicional e paciência. A minha filha amada, Laura. À meus irmãos; Wilme, Adalgiza, Karol e Camila pelo carinho sempre. À minha orientadora, Dra. Lorena, pela oportunidade e confiança. Obrigada pelos conhecimentos, paciência e tolerância durante todo o desenvolvimento deste projeto. À Adriana, pelos incontáveis momentos de ajuda e companheirismo na execução deste projeto. Sem você, Dri, teria sido muito mais difícil. Aos demais colegas de pós graduação, em especial Izabela, pela disponibilidade e carinho de sempre; Mariana, João, Ana Paula, Bruna, Jaqueline pelos ensinamentos compartilhados. À toda a equipe de laboratório de hematologia do HC, às queridas Aglair, Denise, Ana Silvia, Bárbara, Adriana. À Patrícia e Priscila pela disponibilidade e carinho de sempre na realização dos experimentos de citometria de fluxo. À Amélia e Josi pelos ensinamentos e ajuda na realização do PCR. À Cleide pela disponibilidade e carinho no manejo com os animais. Aos residentes que ajudaram na obtenção das amostras de medula óssea dos pacientes. Aos docentes e professores pelos ensinamentos constantes. Aos meus queridos amigos do hemocentro,