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Background:Recently, the SLIT/ROBO axis became a therapeutic target for a variety of non-cerebral solid tumors because of its relevance in the regulation of angiogenesis, inflammatory cell chemotaxis, tumor cell migration and metastasis. In acute myeloid leukemia (AML), lower levels of SLIT2expression were associated with poorer prognosis in studies that excluded patients with acute promyelocytic leukemia (APL) of the analysis (Golos et al., Arch Immunol2019). Our group previously reported that higher SLIT2expression was associated with improved overall survival, disease-free survival and decreased cumulative incidence of relapse. Subsequently, our in vitrofunctional studies indicated anti-proliferative effects exercised by exogenous SLIT2 peptide treatment in APL (Weinhauser et al., Blood2018). Although, the expression of SLIT/ROBO was detected on leukemic cells, the fact that in healthy subjects the main source of SLIT2 derives from bone marrow stromal cells (BMSC) (Smith-Berdan et al., Cell Cycle2012) was so far neglected in the context of hematological malignancies. Aims:Here, we expanded our data on the role of SLIT2 using lentivirally knocked down APL cells, and addressed the issue of the importance of BMSC-derived SLIT2 on APL development. Methods:NB4 and NB4R2 (RA-resistant) and primary APL cells (age, 25-47y; n=4) were transduced with shRNA-SLIT2or shCT (control). After synchronization using double-thymidine block, transduced cells were submitted to proliferation and cell cycle assays. For the apoptosis analysis, cells were treated with ATO (1 µM) alone or in combination with RA (1 µM) for 24, 48 and 72 hours. The granulocytic differentiation in response to RA treatment alone (1 µM) or in combination with ATO (1 µM) for 48 and 72 hours, was evaluated based on the CD11b and CD11c levels. To assess the effect of SLIT2silencing in BMSCs, HS5 and HS27A cell lines were transduced with shSLIT2or shCT. Non-transduced NB4, NB4R2 and primary APL cells were co-cultured with engineered BMSCs and proliferation, apoptosis and granulocytic differentiation was performed in response to ATO alone or in combination with RA for 48 and 72 hours. In vivoassessment was performed using four 8-12 week-old C57/BL6PepBoy (CD45.1) lethally irradiated, transplanted with 5 x 106hCG-PMLRARa blasts (CD45.2) transduced with shSlit2-murine and shCT. Results:Silencing SLIT2in primary APL samples resulted in a significant increase in cell growth at the sixth day of culture compared to shCT samples (P<.001). Accordingly, enhanced staining of Ki67 was detected in NB4 and NB4R2-shSLIT2transduced compared to shCT cells. Moreover, NB4-shSLIT2cells exhibited lower ATO-induced apoptosis either when ATO was used alone or in combination with RA. Nevertheless, SLIT2knockdown had no effect on NB4R2 cells suggesting that RARA moiety of PML/RARA may be important to the observed effect. Concomitantly, NB4 cell differentiation rate, was greatly increased in NB4-shSLIT2in the presence of RA after 48 and 72h (P<.05) and 48h when RA was combined with ATO (P<.05). In vivotransplantation of primary murine shSLIT2hCG-PMLRARa blasts, resulted in significant increase of white blood cell count during the course of APL emergence and increased extramedullary hematopoiesis in the spleen, yet with no difference in the BM at the time of sacrifice. Next, we evaluated the number of transcripts and the function of SLIT2in AML derived MSC cells. SLIT2transcript levels were significantly reduced in primary AML-derived BMSC compared to BMSCs obtained from healthy donors (P<.05). In vitroassays co-culturing NB4 and NB4R2 cells on engineered HS5 or HS27A BMSC cells showed no difference in drug-induced apoptosis or differentiation (P>.05). In contrast to shSLIT2APL induced cell growth, co-culture of primary APL blast cells with transduced HS27A cells had no impact on proliferation, while the expression of the neutrophilic differentiation gene ELANE2was significantly decreased in APL cells co-cultured with HS27A-shSLIT2.ConclusionSLIT2is expressed in APL cells and exerts an anti-proliferative effect. On the other hand, SLIT2produced by BMSC seemed to promote APL cellular stemness. Our results reinforce that SLIT2 function is cell type dependent and may be involved in the cross-talk between microenvironment and leukemic cells.

Aims:The active methylation of histones plays an important role in regulation of gene expression. Nuclear SET Domain (NSD)Histone Lysine Methyltransferases family (KMT) is composed of three members: NSD1, 2 and 3 which regulates gene expression through methylation of H3K36. NSD2overexpression, initially reported in multiple myeloma, was recently associate with EZH2mRNA levels through interaction with H3K27me3 which suppresses the expression of miR-203, miR-26a and miR-31 leading to the up regulation of NSD2mRNA levels. This correlation has been observed in various types of cancer, although there is no data in myeloid malignancies. Aim: To evaluate the gene expression profile of the NSDfamily in three de novoAML cohorts: one Brazilian cohort and two public databases addressing its applicability in prediction of treatment outcomes, retrospectively. Methods:As a learning set, samples from 146 subjects (age, 18-87y) were analyzed. Seventy (48%) patients were treated in a University Hospital that serves as reference for northeast of Brazil, while 76 patients were treated in a center of similar characteristics in the southeast of the country. The baseline features distribution was similar between centers. The treatment protocol was described elsewhere (Lima AS et al., Blood, 2015), but was based on anthracycline and cytarabine for induction and intermediate to high doses of cytarabine as consolidation. As controls, sixteen samples of CD34+cells isolated from total bone marrow (BM) of healthy volunteers (age, 18-60y) were analyzed. The external validation cohort composed by 173 patients (age, 18-88y) were obtained from TCGA AML study available online on CBioPortal for Cancer Genomics (Gao et al. 2013). Patients were dichotomized into “low” and “high” NSD1, 2and 3expression groups based on survival receiver operating characteristic (ROC) curve and the C index analyses. To validate our data, NSDtranscript levels from an independent cohort was used (525 patients from Amazonia!- NSD1Probe #219084_at; NSD2Probe #209052_s_at; NSD3Probe #218173_s_at - and five normal CD34+samples included in the same databank). The following parameters were used to evaluate treatment outcome: complete hematological remission (CHR); 5-year Disease-Free Survival (DFS) and 5-year Overall Survival (OS) rates. Results:The median age of the learning set was 46y with 70 males (48%). NSD1, 2and 3expression levels are lower in de novoAML samples compared to CD34+cells (P<.001). These results were not validated in an Amazonia!cohort, which patients with AML had a higher-than-normal expression of NSD1and 3(P=.01). Expression of all NSDwas closely correlated in the learning set cohort (r>0.96) albeit only NSD2and 3had a correlation in the validation cohort (r=0.6). Baseline characteristics (sex, age, leukocyte count, cytogenetic risk, FLT3 and NPM1 mutations)were similar between the cohort of patients with NSDslow and high expression, except for higher blast count in BM, but not in peripheral blood, in high expression group for NSD1and 2(P=.021 and P=.033, respectively). Overall, 78/146 (57%) patients achieved CHR. Patients with a high NSD1expression had a lower CHR rate (44%) compared with those a low expression (56%) (OR: 0.5; 95%CI: 0.2-0.9; P=.04). With a median follow up of 21 months (1-61 months) the estimated 5y DFS rate was 49% (95%CI: 30-65%). The group with high NSD1expression presented a lower 5y DFS rate (13%; 95%CI: 1-42%) than those with low NSD1expression (72%; 95%CI: 47-87%) (P=.003). This result was consistent with the multivariable proportional hazards analysis (HR: 7.3; 95%CI: 1.3-38; P=.02). The estimated 5y OS rate was 24% (95%CI: 14-35%) which patients with high NSD1expression exhibiting lower 5y OS rate (13%; 95%CI: 4-29%). The median age of the external validation cohort was 58y with 107 males (62%). Only NSD2levels had a association with higher diagnosis age (P=.008). With a median follow up of 40 months (1-118 months) the NSDexpression may predict only a higher 5y OS rate in patients with high NSD2expression (30%; 95%CI: 26-49%) retained the result in a multivariable model (HR: 0.53; 95%CI: 0.3-0.9; P=.02). Conclusions: In summary, NSD1and 2transcript levels were independent factors associated with treatment outcomes in two AML cohorts treated with standard protocols based on anthracyclines and cytarabine.

Introduction: The recurrent gain-of-function JAK2V617Fmutation confers growth factor-independent proliferation for hematopoietic cells and is a major contributor to the pathogenesis of myeloproliferative neoplasms (MPN). Metformin (1,1-dimethylbiguanide) is a biguanide widely prescribed for the treatment of type II diabetes and metabolic syndromes. In recent years, studies using cancer cell lines and murine models have provided evidence for potential anticancer activity of metformin. Previous results from our research group indicate that metformin reduces cell viability, proliferation, clonogenicity and JAK2/STAT activation in human JAK2V617Fcell lines and spontaneous erythroid colony formation in primary cells from patients with polycythemia vera (Machado-Neto et al. ASH Annual Meeting Abstracts #1960, 2016). Objectives: To investigate the effects of metformin treatment in tumor formation and MPN phenotype using Ba/F3 JAK2V617Fcells and Jak2V617Fknockin-induced MPN mice. Methods: For tumor formation, experimental groups consisted of 8-10 week-old NSG female mice that received a 100 µL (50 µL PBS plus 50 µL matrigel) dorsal subcutaneous injection of 2×106Ba/F3 JAK2V617Fcells and daily treatment by intraperitoneal injection of vehicle (PBS, n=9) or metformin (125 mg/kg, n=9) for 13 days after initial tumor growth (7 days). Tumor volume (V) was obtained using the formula (V = W2× L × 0.52), where W and L are the smaller and larger diameters, respectively. For MPN phenotype induction, 5×106bone marrow cells from Jak2V617Fknockin (Jak2WT/V617F) mice were transplanted into lethally irradiated Pep boy mice. After 4 weeks, chimerism was evaluated by CD45.1 and CD45.2 markers (Becton-Dickinson) by flow cytrometry in peripheral blood. Mice with >70% CD45.2 cells were randomized and treated daily by intraperitoneal injection of vehicle (PBS, n=4) or metformin (125 mg/kg, n=5) for 6 weeks. At the conclusion of the experiment, animals were harvested and subjected to analysis of spleen and hematological parameters. Erythroid progenitors in the spleen were evaluated by CD71 and Ter119 markers (Becton-Dickinson) by flow cytometry. All experiments were approved by the Animal Ethics Committee. For comparisons, Mann-Whitney test or ANOVA test and Bonferroni post-test was used. A pvalue <0.05 was considered as statistically significant. Results: In NSG mice inoculated with Ba/F3 JAK2V617F, metformin treatment (125 mg/kg/day) was able to significantly reduce the tumor burden after 13 days of treatment. At the endpoint, the mean±SD of tumor volume for vehicle (n=4) and metformin (n=4) treated mice was 1120.7±289.1 mm3and 510.3±244.4 mm3, respectively (p<0.001). A second independent experiment including five mice in each group presented similar results (vehicle versusmetformin: 1307.7±511.9 mm3versus725.1±380.6 mm3; p<0.01). In Jak2V617Fknockin-induced MPN animals, metformin treatment (125 mg/kg/day) was well-tolerated, reduced splenomegaly (p=0.02) and improved splenic architecture. Erythoid progenitors in the spleen and bone marrow did not differ between control and metformin-treated groups (p>0.05), but the smaller spleens presented with a very low percentage of erythroid progenitors. Hemoglobin and hematocrit were not modulated by metformin treatment. Conclusions: Metformin treatment at a dose of 125 mg/kg/day effectively reduced tumor burden in Ba/F3 JAK2V617Fallograft and splenomegaly in Jak2V617Fknockin-induced MPN murine models. In humans, this dose represents a pharmacological dose similar to that which is used for type II diabetes treatment. Our results provide additional insights for metformin in preclinical models of MPN.

Natural killer (NK) cells are lymphocytes that play a critical role in both innate and adaptive immune responses. It has been shown that CD27 and CD11b are antigens expressed in the last stage of NK cells development and allow the classification of NK cells into three functional subsets. Tolerant NK cells are double negative (DN) for CD27 and CD11b, have an immature phenotype and present low secretory and cytotoxic potential. The secretory NK cells are CD27 positive, have a great ability to release cytokines, but do not exhibit cytotoxicity properties. The cytotoxic NK cells express CD11b, release granzymes and perforins, but are less capable of secreting cytokines. While cytotoxic NK cells are essential for the natural defenses against infected or malignant cells, secretory NK cells, when activated, produce different cytokines, including IFNγ, TNFα, TGFβ, GM-CSF, MIP1α, MIP1β, IL-10 and others, which may modify the functions or other hematopoietic cells including the hematopoietic stem cells (HSC). We hypothesized that deregulation of the balance between secretory or cytotoxic subtypes of NK cells may favor HSC malignant transformation and contribute to stem cell-derived diseases. In this regard, few studies have shown that NK cells are defective in chronic myeloid leukemia (CML). In contrast to CML, the role of NK cells in BCR-ABL1 negative-myeloproliferative neoplasms (MPN) is currently unexplored. We aimed to study the activity of NK cells and their subsets in murine primary cells from MPN. NK cells subsets were quantified by flow cytometry by the use of spleen cells from a pre-established conditional vavCre knockin Jak2V617F(Jak2VF) murine model. Besides genotyping to identify controls (Jak2 wt/wt vavCre+, or Jak2WT) and Jak2-mutated mice (Jak2 wt/V617FvavCre+, Jak2VF), the presence of polycythemia and splenomegaly was confirmed in the animals with MPN. Mice (n=5 per group) were euthanized between 8-12 weeks of age. For immunophenotyping, spleen cells were isolated, submitted to red cell lyse, and stained with fluorescence-conjugated antibodies against Ter119, CD19, CD4, CD8, CD3, NK1.1 and CD27 and CD11b. All the stainings were performed at 4°C for 20 minutes and acquisition was performed in a FACSCanto™II flow cytometer (BD Biosciences). Data was analyzed by the use of the Flowjo software version X. When compared to Jak2WT controls, the Jak2VF mice had lower numbers of NK cells (1,447 x 106versus 0,381 x 106p<0.05) and T cells (0,638 x 106versus 0,158 x 106of the spleen; p<0.05) among the total splenocytes. The analysis of NK subsets showed an increase in the percentages of DN NK cells (CD27-CD11b-) in the spleen of Jak2VF mice as compared to controls (14.84% versus 4.97%; p<0.05). CD27+ immature and mature secretory NK (CD27+CD11b-/+) cells also seemed to be increased in Jak2-mutated mice but this data did not reach statistical significance (37.43% versus 17.65%; p=0.1). On the other hand, when compared to Jak2WT controls, the Jak2VF mice presented lower percentages of CD11b+ cytotoxic NK cells (77.07% versus 47.80% of the spleen cells; p<0.05). In agreement, when the expression of NK receptors was analyzed by flow cytometry, the inhibitory NKG2A receptor was found to be higher expressed in Jak2VF NK cells as compared to non-mutated cells, while the expression of the activatory receptors Ly49H and Ly49D was reduced in Jak2-mutated NK cells. Of note, low levels of the Jak2V617Fmutation were detected in sorted NK cells isolated from Jak2VF mice as compared to controls by Real Time quantitative RT-PCR, thus suggesting that the distribution between NK cell subtypes observed here is associated with the JAK2 mutational status. In summary, when compared to controls, Jak2-mutated mice presented an increased proportion of tolerant NK cells, previously described as immature cells with low secretory and cytotolytic potential. To date, CD11b-/CD27-tolerant cells have been observed among solid tumor-infiltrating NK cells, but have not been previously associated with MPN. Moreover, an inverted ratio between the secretory and cytotoxic NK subsets was also detected in Jak2VF animals, suggesting that a cytotoxic deficiency occurs and may contribute to the leukemic stem cell expansion. The expansion of the NK secretory potential deserves further elucidation. Our study provided new insights into the distribution of JAK2V617FNK functional subsets that may be used as future therapeutic targets for MPN.