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•AML cells depend on extracellular cysteine to prevent ferroptosis, which cannot be compensated by methionine.•Targeting redox balance by combined inhibiting of cysteine import and antioxidant machinery is a therapeutic vulnerability in AML.

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:In the clinical setting, low KMT2Eexpression has been associated with poor prognosis in both acute myeloid leukemia (Damm et al. J Clin Oncol. 2011 Feb 20;29(6):682-9) and acute promyelocytic leukemia (APL) (Lucena-Araujo et al. Br J Haematol. 2014 Aug;166(4):540-9). Member of the Trithorax-group, the KMT2Eis implicated in the positive transcriptional control of a specific set of genes related to hematopoiesis and differentiation processes in many cell types. Functional analyses showed that the encoded protein is involved in terminal myeloid differentiation and facilitate retinoic-acid induced granulopoiesis in human promyelocytes. Using lentiviral gene transfer, we hypothesized that the forced expression of KMT2Eis implicated in the ATRA-induced granulocytic differentiation in APL. Aims:To investigate the effects of overexpression of KMT2Egene in cell viability and signaling in response to ATRA plus anthracycline or arsenic trioxide (ATO) treatment in APL cell lines. Methods:NB4 and NB4-R2 (resistant to ATRA treatment) cell lines were transduced with pMEG or pMEG- KMT2Elentiviral vectors and cultured with stable selection using puromycin. Before all functional experiments, cells were tested for KMT2Egene and protein expression by qPCR and confocal microscopy, respectively. The cells transduced with KMT2Eand control cells were evaluated for cell viability (MTT assay), proliferation (Ki-67 staining), cell cycle (flow cytometry), mitochondrial reactive oxygen species (ROS) production (flow cytometry) in basal conditions and under treatment with ATRA (1 μM), ATO (0.187, 0.375, 0.75, 1.5 and 3 μM), and the association of ATO (1 μM) plus ATRA (1 μM) or cytarabine (150 nM). The granulocytic differentiation in response to ATRA treatment was evaluated based on the CD11b expression (flow cytometry) plus morphological analysis (Leishman staining). The expression of the main target genes (p21, MDM2, and BMI1) encoding for proteins known to be involved in KMT2Efunction were evaluated by qPCR. Results:Overexpression of KMT2Ewas significantly associated with higher cell proliferation rates in basal or ATO and cytarabine treatment compared to vehicle treated cells (all P<.001). KMT2Eoverexpression significantly increase cell proliferation in NB4, but not in NB4-R2 cells (Ki-67 mean intensity of fluorescence (MFI) value - pMEG: 489±10 vs KMT2E: 1432±25; P<.001). The distribution of cells in the phases of the cell cycle was similar between groups. Using a nonlinear regression analysis, IC50 for ATO was of 2.96 and 6 μM for NB4 pMEG and KMT2Etransduced cells, respectively. NB4- KMT2Ecells showed lower levels of ROS generation upon ATO treatment (1.5 μM) compared to controls (ROS - MFI value - pMEG: 270.6±6 vs KMT2E: 84±11; P<.001). In the attempt to assess the role of KMT2Ein granulocytic differentiation, NB4- KMT2Ecells were treated with ATRA 1 μM. The cells containing an extra copy of KMT2Egene showed more differentiation rate in 72, 96 and 120 hours compared to empty vector transduced cells (all p<.05). Considering the expression of target genes of KMT2E, only p21showed an increase in KMT2Ecells treated with ATRA. Conclusion:In summary, the overexpression of KMT2Eis associated with higher proliferation capacity and increased resistance to ATO treatment, associated with less ROS generation.

INTRODUCTION:

Introduction:The application of the standardized therapy based on anthracyclines and cytarabine for all patients with AML regardless of which molecular aberration is present, results in a heterogeneous response exemplified by a worse prognosis for both young and elderly patients exhibiting TP53mutation. Thus, the treatment of AML requires adjustments in order to improve the patient's therapeutic response. Recently, a new compound - Halofuginone (HF), revealing multitarget effects (antifibrotic, anti-inflammatory and pro-apoptotic) was described as a potential anticancerogenic drug. The diverse targets of HF have been studied in several tumors such as in multiple myeloma, where HF triggers the caspase dependent apoptotic pathway. Our group conducted a study in APL cell lines (NB4 and NB4-R2 - resistant to ATRA treatment), demonstrating HF anti-proliferative and anti-angiogenic effects in vitrosuggesting that this molecule may become a new therapeutic agent.