ALBERT

Description: Introduction: Chronic myeloid leukemia (CML) is a hematological malignancy associated with the BCR-ABL1 oncoprotein, which results in deregulated kinase activity-driven leukemic phenotype. Most CML cases are successfully treated with tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1, but a significant percentage of patients develop drug resistance and/or progress to blast crisis, a disease stage that is refractory to therapy. Furthermore, recent studies have provided evidence that the CML leukemia stem cell is resistant to imatinib treatment. In this sense, the identification of other crucial proteins that cooperate with BCR-ABL1 signaling pathways may represent secondary therapeutic targets optimize treatment strategies. Increased IRS1 mRNA expression has been previously identified as positively correlated with worse overall survival in BCR-ABL1-positive adult acute lymphoblastic leukemia. In K562 cells, IRS1 has been identified as a binding partner of BCR-ABL1 and is capable of activating the PI3K/Akt/mTOR and MAPK pathways. Recently, a pharmacological IRS1/2 inhibitor (NT157) has been developed and has shown promising results in preclinical studies on solid tumors. We have previously shown that NT157 treatment reduces colony formation of primary CML cells but not in normal cells, decreases cell viability and proliferation of K562 cells, and increases apoptosis of K562 cells in a time- and dose-dependent manner (ASH 2015 - Abstract #83876). To further characterize the mechanism of the sensitivity of CML cells to IRS1/2 inhibition, here we performed PCR-based gene expression analyses in CML cells in response to NT157 treatment and investigated the effects of IRS1/2 inhibition on cell proliferation and apoptosis in Ba/F3 cells expressing wild-type or T315I mutant BCR-ABL1. Materials and Methods: K562 cells treated with vehicle or with the IRS1/2 pharmacological inhibitor NT157 (1.6 µM) for 48 hours were submitted to PCR array analysis using the PI3K-AKT Signaling Pathway and CancerPathwayFinder RT2 Profiler PCR Array kit (#PAHS-033A and #PAHS-058A, respectively; SA Biosciences, Frederick, MD, USA). Each cDNA sample was processed in a 96-well plate containing 156 signaling pathway-related genes and 5 endogenous control genes. Ba/F3 parental, BCR-ABL1WT and BCR-ABL1T315I cells were subjected to IRS1/2 pharmacological inhibition using NT157 (0.2, 0.4, 0.8, 1.6 and 3.2 µM) for 24, 48 and 72 hours and were evaluated for cell viability (MTT assay), apoptosis (annexin V/PI), and protein expression/activation (Western blot). Statistical analyses were performed by Student's t-test or Mann-Whitney test, as appropriate. Results: Sixteen genes were found to be differentially expressed (fold change ≥ 2); 5 upregulated genes (FOXO3A, CDKN1A, FOS, JUN, VEGF) and 1 downregulated gene (E2F1) were chosen for validation in a larger number of experiments. Notably, NT157 treatment also resulted in significant dose- and time-dependent decrease in viability in BCR-ABL1-expressing cells regardless of BCR-ABL1 mutation status, compared to parental Ba/F3 cells. NT157 treatment (1.6 µM) for 48 hours reduced cell viability of Ba/F3 BCR-ABL1WT and BCR-ABL1T315I cells to 64% and 76%, respectively (p

Description: Introduction: Chronic myeloid leukemia (CML) is a hematological malignancy associated with the BCR-ABL1 fusion gene, which drives the proliferative disease phenotype by activating multiple signaling pathways. Most CML cases are successfully treated with tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1. However, in some cases, drug resistance limits TKIs efficacy, and the identification of other crucial proteins in the BCR-ABL1 signaling pathways may contribute to optimize anti-CML approaches. IRS1 mRNA expression has been previously identified as positively correlated with overall survival in BCR-ABL1-positive adult acute lymphoblastic leukemia. In K562 cells, IRS1 has been identified as a binding partner of BCR-ABL1 protein and was capable of activating PI3K/Akt/mTOR and MAPK pathways. Recently, a pharmacological IRS1/2 inhibitor (NT157) has been developed and has shown promising results in preclinical studies on solid tumors. We herein aimed to investigate IRS1 and IRS2 expression and the effects of IRS1/2 inhibition on cell proliferation, apoptosis and clonogenicity in BCR-ABL1 positive and normal hematopoietic cells. Materials and Methods: Total bone marrow cells from healthy donors (n=11) and CML patients at the time of diagnosis (n=24) were submitted to gene expression analysis by quantitative PCR with specific primers for IRS1, IRS2 and β-actin. All subjects provided informed written consent and the study was approved by the ethics committee of the Institution. K562 cells were submitted to IRS1/2 pharmacological inhibition using NT157 (0.2, 0.4, 0.8, 1.6, 3.2 and/or 6.4 µM) for 24, 48 and 72 hours and were evaluated for cell viability (MTT assay), proliferation (Ki-67), apoptosis (Annexin V/PI), and protein expression/activation (Western blot). Alternatively, cells were submitted to IRS1 and IRS2 gene silencing using specific shRNA lentiviral delivery, and submitted to functional studies. NT157 effects were analyzed by in vitro hematopoietic colony formation of bone marrow cells from two patients with CML at diagnosis, and of normal cord blood cells from one individual. Cells were seeded at 4.5x104 per well in a culture system for 14 days. Statistical analyses were performed by Student's t-test or Mann-Whitney test, as appropriate. Results: IRS1 and IRS2 mRNA expression was similar between normal donors and CML samples (p ≥.05). NT157 treatment reduced K562 cell viability in a time and dose-dependent manner; using a nonlinear regression analysis, IC50 for cytotoxicity was 9.8, 0.6 and 0.68 µM for 24, 48 and 72 hours, respectively. NT157 0.8 and 3.2 µM reduced cell viability in 14% and 19% at 24 hours, 50% and 61% at 48 hours and in 59% and 68% at 72 hours of treatment (all p

Description: Background Myeloproliferative neoplasms (MPNs) are chronic hematopoietic stem cell disorders, including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (MF). JAK2, MPL, and CALR mutations are considered as "driver mutations" and are directly implicated in the disease pathogenesis by activation of JAK/STAT signaling. However, some patients do not harbor any of these mutations. Since such triple-negative MPNs are very rare, no specific molecular markers were established to use for a precise differential diagnosis yet. So far, the introduction of next generation sequencing (NGS) technologies in research of myeloid neoplasms has provided valuable contributions on the identification of new molecular biomarkers, establishing more accurate risk rating and selection of more specific therapeutic interventions. This study aimed to identify, through targeted deep sequencing, specific genetic variants in patients with triple-negative MPNs. Methods We performed NGS targeted sequencing in 18 Brazilian triple-negative patients (11 MF and 7 ET). The median age at diagnosis was 64 years for primary myelofibrosis (range 42-78), and 52 years for essential thrombocythemia (range 19-79). In 14 cases, we used the Illumina TruSight Myeloid Panel covering 54 genes and in 4 cases we used a custom Sure Select Agilent panel containing more than 300 genes previously reported to be related to myeloid neoplasm. The inclusion criteria for variant filtering was quality score〉30, read count〉50, minor allele frequency

Description: Abstract 1735 One of the models of the pathophysiology of myelodysplastic syndromes (MDS) suggests that the transformation of hematopoietic cells induces an autoimmune response of T cells with bone marrow (BM) becoming the target organ. Evidence has shown that T-cell mediated marrow suppression is the cause of cytopenia in approximately 20–30% of MDS patients. Higher frequencies of cytotoxic CD8+ cells has been shown in low-risk MDS, as compared to high-risk MDS and these cells mediate the cytotoxicity of BM precursors. On the other hand, the suppressed immune response observed in high-risk MDS results from increased numbers of regulatory T (Treg) cells. FMNL1 belongs to a conserved family of formin-related proteins, indispensable for many fundamental actin-dependent processes, including migration, morphogenesis and cytokinesis. FMNL1 is restrictedly expressed in lymphoid hematopoietic-lineage-derived cells and overexpressed in malignant hematopoeitic cells. Depletion of FMNL1 in cytotoxic lymphocytes was recently reported to abrogate cell-mediated killing. The aim of this work was to study the role of FMNL1 in the immune system of MDS. For this, we characterized FMNL1 expression in peripheral blood CD3+ cells of patients with MDS and normal donor and we evaluated the CD4: CD8 T-cell ratios and molecular markers for Treg cells. We also assessed FMNL1 expression in MDS and normal BM cells, and during hematopoietic cell differentiation, using cell line models. A total of eighty patients with a diagnosis of MDS, receiving no treatment, and forty-seven samples from normal donors were included in the study, which was approved by the National Ethical Committee Board. FMNL1 expression levels were determined by quantitative PCR (q-PCR) or Western blotting in cell lines, CD3+ cells (obtained by Ficoll-Hypaque followed by magnetic selection), or total BM cells. CD3+ cell counts and CD4: CD8 T-cell ratios were determined by flow cytometry. Q-PCR was performed to determine IL-10, TGFB1, and CTLA4 expression in CD3+ cells. Megakaryocytic differentiation was obtained by treating K562 with 20nM of PMA for 4 days. For granulocytic differentiation, NB4 was treated with 10−6 M of ATRA for 4 days. FMNL1 expression was significantly higher in CD3+ cells of the low-risk MDS group (according to FAB, WHO and IPSS classification), compared to normal donors (P

Description: Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells (HSC) characterized by ineffective hematopoiesis. In addition to HSC defects, an important role is also played by the hematopoietic microenvironment niche that has as key component the mesenchymal stromal cells (MSC). The MSC of MDS patients have morphological and functional abnormalities. Recently, our group identified new possible target genes involved in MDS pathophysiology through microarray analysis of MSC from MDS patients. An interesting underexpressed gene found was SPINT2, a gene that encodes a transmembrane protein which inhibits the hepatocyte growth factor activator (HGFA), the enzyme responsible for the conversion of hepatocyte growth factor (HGF) into its active form. SPINT2 is downregulated in some types of solid cancer and correlated with their prognostic and progression; however, the functional role of SPINT2 in MDS remains unknown. We herein investigated the role of SPINT2 in MSC, studying HGF and SDF1 secretion and cell adhesion with normal CD34+, P39 and U937 cells by silencing SPINT2 gene in HS5 and HS-27a stromal cell lines. We also investigated, using qPCR, SPINT2 and HGF mRNA expression in MSC and total bone marrow (BM) cells from 56 untreated MDS patients (WHO 35 low-risk, 21 high-risk) and 28 healthy donors. To inhibit SPINT2, specific shRNA expressing lentiviral vectors targeting SPINT2 gene or no specific sequence were used. The HGF and SDF1 secretion in cell supernatant from the cells silenced or not for SPINT2 was measured by BioPlex after 6, 12, 24 and 48h. The cell-cell adhesion of CD34+, P39 or U937 cells onto transduced stromal cells and the adhesion molecule profile were analyzed by flow cytometry. We observed a significant decrease in SPINT2 mRNA expression of MDS MSC (P=0.006) and MDS BM cells (P=0.03) compared to normal cells. Further, HGF mRNA expression of MDS MSC was significantly increased (P=0.01) compared to normal cells. Spearman analysis showed a negative significant correlation between SPINT2 and HGF expressions (P=0.01;r2=0.60). In both stromal cell lines, SPINT2 inhibition resulted in a significant increase in HGF secretion after 24 and 48h and a significant increased secretion of SDF1 after 48h. Moreover, SPINT2 silencing induced a significant increased adherence of CD34+, P39 and U937 cells onto stromal cells probably due to the alteration in integrin expression, since an increase in CD49b and CD49d and decrease in CD49e expressions were also observed in cells silenced for SPINT2. Considering that SPINT2 limits signaling via HGF pathway by inhibiting HGF activation through HGFA, the SPINT2 underexpression in MDS would allow the conversion of the inactive HGF monomer to an active heterodimer. Interestingly, a significant decrease of SPINT2 and increase of HGF expressions were observed in MSC of MDS patients, compared to normal cells. Moreover, recent studies have demonstrated that HGF serum levels are significantly increased in MDS patients and dependent on MDS severity. In addition, SPINT2 inhibition in stromal cells significantly increased HGF secretion by these cells. The increased HGF secretion can result in an autocrine regulation which induces the production and secretion of SDF1 by stromal cells themselves. Cytokines secretions provided by MSC are required for adhesion, survival and proliferation of HSC cells. In this way, in MDS MSC, SPINT2 underexpression and, consequently, increased HGF and SDF1 secretion, may lead to an increased adhesion between MSC and normal or malignant HSC cells. Corroborating our hypotheses, we found overexpression of integrins CD49b and CD49d, which mediate cell-MSC interaction, and a decrease in CD49e expression, an integrin that promotes interaction with extracellular matrix. The interaction onto MSC contributes to the maintenance of the stem and malignant cell properties, such as self-renewal, survival and proliferation. Cytokine secretion and cell adhesion onto MSC is important for MDS physiology. Hence, we demonstrate for the first time that, in MDS, SPINT2 plays a role in the HGF and SDF1 secretion by MSC, resulting in an alteration in cell-cell adhesion and molecule adhesion profile. In view of these data, the SPINT2 expression alteration in MDS MSC may constitute a particular mechanism of MDS pathophysiology and maintenance of self-renewal, homing and proliferation of HSC and malignant clones in MDS.Support: FAPESP, CNPq Disclosures: No relevant conflicts of interest to declare.

Description: Background: Palliative care (PC) is an approach that improves the quality of life of patients facing problems associated with any disease that leads to multiple or hard to manage symptoms, through the prevention and relief of suffering by early identification and impeccable assessment of physical, psychosocial and spiritual problems. Sickle cell diseases (SCD) consist of a group of congenital diseases characterized by the presence of the sickle hemoglobin (HbS), which can polymerize and predispose to hemolytic and ischemic events. In addition to the acute pain events, sometimes recurrent and hard to control, lesions in target organs commonly occur, leading to multiple comorbidities and serious decrease in quality of life. Aims: To evaluate clinical and demographic factors that could influence physical, psychosocial and spiritual symptom burden and confer eligibility criteria to PC in a group of SCD patients. Methods: Clinical and demographic data of SCD patients were collected by interviews using a standardized questionnaire: diagnosis, time from diagnosis, number of comorbidities and of medical specialties on regular follow up, infectious episodes, need for seeking the emergency and for hospitalization during the last 12 months, delirium events, wounds, dysphagia, recurrent falls, adverse events to medication, quality of communication with the medical team, fears regarding the disease and its complications, religious support, age, gender, monthly household income (MI), level of schooling (SCH) and profession. Specific PC scores were also applied: Edmonton Symptom Assessment Scale (ESAS) and Palliative Performance Scale (PPS). Statistical univariate and multivariate analysis were performed. P value

Description: Introduction : Stathmin 1, also known as Oncoprotein 18 (OP18) or Leukemia-associated phosphoprotein p18 (LAP18), is an important cytoplasmic microtubule-destabilizing protein that plays a critical role in the process of mitosis, proliferation and accurate chromosome segregation through regulation of microtubule dynamics. High levels of Stathmin 1 have been reported in solid tumors and have been associated with poor prognosis in various types of cancers. The identification of overactive proteins in leukemia cells, compared to normal hematopoietic cells, as well as understanding the molecular and cellular basis of the disease may provide new therapeutic opportunities. Aims: To evaluate Stathmin 1 expression in proliferating and non-proliferating hematopoietic cells, in bone marrow cells from healthy donors and from patients with myelodysplastic syndromes (MDS), acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). In addition, we evaluated the effect of Stathmin 1 silencing on proliferation and apoptosis in the U937 acute myeloid leukemia cell line. Materials and Methods: A panel of human leukemia cell lines that included myeloid (K562, KU812, NB4, HL60, P39, HEL, U937, KG1 and THP1) and lymphoid cells (Jurkat, MOLT4, Daudi, Raji, Namalwa and Karpas 422) in exponential growth was used. Peripheral blood lymphocytes (PBL) were induced, or not, to proliferate upon PHA stimulation for 72 hours. A total of 30 healthy donors and 117 patients at diagnosis (MDS=52 [low-risk=36, high-risk=16], AML=49, and ALL=16) were included in the study. Stathmin 1 gene and protein expression was evaluated by qPCR and Western blot. Stathmin 1 was stably knocked down with specific shRNA-expressing lentiviral vector and cell growth was examined by MTT assay, clonogenicity by colony formation and apoptosis by AnnexinV/PI. Appropriate statistical analyses were performed; results are expressed as median (minimum- maximum). Results: A higher expression of Stathmin 1 was observed in all leukemia cell lines, when compared with normal non-proliferating hematopoietic cells. We also observed a marked increase in Stathmin 1 expression in PBL induced to proliferate with PHA after 72 hours. Stathmin 1 transcripts were significantly increased in total bone marrow cells from patients with AML (2.01 [0.35-8.88]; p=.0009) and ALL (2.94 [1.16-10.82]; p=.0004), compared with healthy donors (1.01 [0.38-4.08]). No difference in Stathmin 1 expression was observed between healthy donors and MDS patients. When the MDS group was stratified by the WHO classification into low and high-risk MDS, Stathmin 1 expression was significantly higher in the high-risk, when compared with low-risk MDS (1.62 [0.42–3.28] vs. 1.13 [0.36–2.61], p=.03). Similar results were found in isolated CD34+ bone marrow cells, Stathmin 1 transcripts were significantly increased in CD34+ AML cells compared with CD34+ normal cells, and in high-risk compared with low-risk MDS (all p≤.02). Interestingly, 3 out of 5 MDS patients showed a significant increase in Stathmin 1 transcripts after disease progression. Also, a significant positive correlation was observed between percentage of bone marrow blasts and Stathmin 1 expression in MDS patients (p=.03; r=.31). In U937 leukemia cells, Stathmin 1 silencing significantly reduced cell proliferation (p=.02) and clonal growth (p

Description: Introduction: The advent of whole transcriptome sequencing technologies revealed that most of the human genome is transcribed, producing a large repertoire of long noncoding RNAs (lncRNAs). They are known to participate in epigenetic regulation at different levels and alter mRNA stability of coding genes, emerging as key players in carcinogenesis. In the present study, we identified a novel unspliced long noncoding RNA that is transcribed from the opposite strand on the NR4A1 gene locus. The tumor suppressor NR4A1 has been associated with the regulation of apoptosis and proliferation in various tumors and shows a reduced expression in myelodysplastic syndrome (MDS). Additionally, the abrogation of this locus in murine models caused the development of acute myeloid leukemia (AML). We hypothesized that lncNR4A1 plays an important role in hematopoietic malignancy pathogenesis and possibly regulates its coding counterpart. Methods: Expression of lncNR4A1 and its sense gene NR4A1 were evaluated in CD34+ cells from bone marrow of normal donors (n=7) and patients (n= 27; MDS= 14 [low‐risk=8, high‐risk=6], AML with myelodysplastic related changes [AML‐MRC]=4 and de novo AML=9) by qRT-PCR. Results were expressed as mean (minimum-maximum) after appropriate statistical analysis. The orientation and full-length of lncNR4A1 were confirmed by strandspecific RT-PCR, RACE-PCR and sequencing techniques. For functional analysis, lncNR4A1 was inhibited with a lentiviral vector system in U937 cells. Apoptosis was evaluated by Annexin‐V/PI and the cell cycle analysis was performed applying PI incorporation by flow cytometer analysis. Mitochondrial activity was evaluated with MTT assays. Western blot was used to measure the NR4A1 protein expression and for monitoring key proteins in the cell cycle signaling pathway. Results: NR4A1 mRNA was significantly decreased in bone marrow/CD34+ from patients with MDS and AML compared to healthy donors (p

Description: Background: Primary Myelofibrosis (PMF) is a chronic myeloproliferative neoplasm (MPN) characterized by increased myeloid proliferation and associated with mutations that induce tyrosine-kinase activation mainly via JAK-STAT pathway, culminating in extensive bone marrow (BM) fibrosis in the course of disease progression. In contrast to the monoclonal origin of hematopoietic cells, fibroblasts proliferation is polyclonal, and mediators involved in fibrosis, neoangiogenesis and osteosclerosis seem to be involved in disease progression. Metformin (MTF) is a biguanide that exerts selective antineoplastic activity in a variety of malignancies, through its action on nutrients privation and hypoxia, leading to apoptosis. In JAK2-mutated cell lines, MTF reduced cell viability, proliferation and clonogenicity, while in Jak2V617F knock-in-induced mice, MTF reduced Ba/F3 JAK2V617F tumor burden and splenomegaly. These data suggest that MTF could have a therapeutic effect in PMF patients. Aims: To conduct an open label phase II study to evaluate MTF effects on BM fibrosis, inflammation mediators, JAK-STAT pathway activation and disease progression in PMF patients. Methods: PMF non-diabetic adults were eligible. Subjects with severe renal function impairment were not included. Patients received MTF (Glifage XR®) in rising doses until a maximum of 2500mg PO daily, according to tolerance. Primary endpoint was BM fibrosis reversion. Secondary endpoints included reduction of inflammation and downregulation of the JAK-STAT pathway. Clinical data was systematically compiled. Blood and BM samples were collected at the time points: pretreatment (0), 3 mo and 6 mo. Collagen was evaluated in BM biopsy specimens by Masson's trichrome stain: three representative areas from each slide were analyzed and the collagen/sample area was quantified using Image J software; the mean percentage of each slide was used for statistics. IL-6, IL-8 and TNF-α levels were analyzed in BM samples using multiplex assay. Phosphorylation status of intracellular proteins STAT3 and STAT5 was analyzed by flow cytometry and the percentage of cells was recorded using FlowJo software. In order to evaluate gene modulation following MTF exposure, samples at time points 0 and 6 mo were analyzed by PCR array for insulin signaling genes (PAHS-030Z, Qiagen). Genes with ±1.5 fold-change in both directions were selected for validation. For each experiment, statistical analysis was performed and a p value

Description: Introduction:Platelet counts in peripheral blood could possibly be used as prognostic markers in sickle cell disease (SCD), since these blood elements play an important role in the phenomena of vessel occlusion. In this context, the mean platelet volume (MPV) may also reflect the level of platelet activation, a parameter that is already used in the stratification of thrombotic processes in situations such as acute coronary syndromes. Thus, the aim of this study was to evaluate platelet counts and MPV in SCD as possible prognostic markers for the occurrence of clinical complications and their relation with laboratory markers of inflammation and hemolysis. Methods:A retrospective chart review was performed on two hundred and sixty-six adult SCD patients (median age 40 years, ranging from 14 to 66), followed from 2002 to 2019, distributed as follows: 156 HbSS, 16 HbSβ0, 15 HbSβ+ and 79 HbSC. Medical records were reviewed for laboratory data at baseline (first visit); clinical data and hospital admissions were recorded for sickle related complications throughout follow-up: acute chest syndrome, retinopathy, avascular bone necrosis, stroke, priapism, leg ulcers and venous thromboembolism. Results:The median platelet count was significantly different between the different genotypes (Kruskal-Wallis rank sum test, p 〈 0.001), with SS: 420 (95-1043) x 103/uL, Sβ0: 406.5 (206 -713), Sβ+: 362 (87-925) and SC: 295 (84 -927). This difference may be related to the degrees of inflammation and hyposplenia in each genotype. Regardless of the genotypes, platelet counts in peripheral blood tend to decrease with age (Rô = -0.201, p 〈 0.001). Interestingly, higher platelet counts are associated with the presence of leg ulcers (Wilcoxon rank sum test with continuity correction, p = 0.03), acute chest syndrome (p= 0.047), higher levels of microalbuminuria (p