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Agradecimentos: This study was supported by Sao Paulo Research Foundation (FAPESP - grants 2017/19674-2, 2017/21801-2), National Council for Scientific and Technological Development (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Brasil (CAPES) - Finance Code 001 Abstract: Culturing bone marrow mesenchymal stem cells (BM-MSCs) is a key point in different fields of research, including tissue engineering and regenerative medicine and studies of the bone marrow microenvironment. However, isolating and expanding murine BM-MSCs in vitro has challenged researchers due to the low purity and yield of obtained cells. In this study, we aimed to evaluate five different protocols to culture murine BMMSCs in vitro. Methods: All protocols were based on the adhesion capacity of BM-MSCs to the tissue culture plastic surface and varied in the types of plate, culture media, serum, additional supplementation and initial cell density. Flow cytometry analysis was used to investigate lineage purity after expansion. Results: The expression of CD45 and CD11b was detected in the cultures generated according to all protocols, indicating low purity with the presence of hematopoietic cells and macrophages. The cellular growth rate and morphology varied between the cultures performed according to each protocol. Cells cultured according to protocol 5 (8 £ 107 cells/plate, Roswell Park Memorial Institute (RPMI) culture medium during first passage and then Iscove's Modified Delbecco's Medium (IMDM) culture medium, both supplemented with 9% fetal bovine serum, 9% horse serum, 12mM L-glutamine) presented the best performance, with a satisfactory growth rate and spindle-shape morphology. Conclusion: Our results point out that the purity and satisfactory growth rate of murine BMMSC cultures are not easily achieved and additional approaches must be tested for a proper cell expansion FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO (FAPESP) CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO (CNPQ) COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR (CAPES) Fechado

Despite great advances that have been made in the understanding of the molecular complexity of acute myeloid leukemia (AML), very little has been translated into new therapies. Here, we set out to investigate the impact of cytoskeleton regulatory genes on clinical outcomes and their potential as therapeutic targets in AML. Gene expression and clinical data were retrieved from The Cancer Genome Atlas (TCGA) AML study and used for survival and functional genomics analyses. For pharmacological tests, AML cells were exposed to ezrin (EZR) inhibitors and submitted to several cellular and molecular assays. High EZR expression was identified as an independent marker of worse outcomes in AML patients from the TCGA cohort (p

Tissue factor (TF) is a procoagulant protein associated with increased risk of thrombotic events in antiphospholipid syndrome (t-APS). The mechanisms by which TF levels are increased in APS have not yet been established. The aim of this study was to investigate whether TF mRNA expression is associated with TF levels and thrombosis in APS. We compared levels of circulating TF and high sensitivity C-reactive protein (hs-CRP) between t-APS and controls (individuals without thrombosis). The association between TF mRNA expression, quantified by real time quantitative polymerase chain reaction, and t-APS was accessed using regression analysis. We included 41 controls and 42 t-APS patients, mean age was 41 years old (SD 14) in both groups. Hs-CRP and TF levels were higher in t-APS patients (mean hs-CRP levels 0.81 mg/dL [SD 1.88] and median TF levels 249.0 pg/mL [IQR 138.77–447.61]) as compared to controls (mean hs-CRP levels 0.24 mg/dL [SD 0.26] and median TF levels 113.0 pg/mL [IQR 81.17–161.53]; P = 0.02 and P

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Chemotherapy with cytotoxic agents is the standard of care, but is associated with a high rate of adverse events. Elderly patients are frequently intolerant to such treatment, presenting a very poor prognosis. The hypomethylating agents (HMA) azacitidine or decitabine represent one of the main therapeutic alternatives for these patients. Isocitrate dehydrogenase inhibitors (IDH) constitute another therapeutic class with DNA methylation effects in AML. In this article, we review the use of first- and second-generation HMA and IDH inhibitors in AML. The data collected demonstrated that HMA are generally considered effective and safe for AML patients who are not eligible for standard chemotherapy. The combination of azacitidine or decitabine with venetoclax was recently approved by the US Food and Drug Administration (FDA) for older AML patients and those unfit for intense chemotherapy. IDH inhibitors also showed encouraging results for relapsed/refractory AML patients harboring an IDH mutation and received FDA approval. Therefore, recent studies have led to the emergence of new therapeutic options using HMA and IDH inhibitors for specific groups of AML patients, representing an important step in the treatment of this aggressive malignancy. New options should emerge from the ongoing studies in the coming years.

Acute myeloid leukemia (AML) is a highly heterogeneous hematological neoplasm with low survival rates. Thus, the investigation of new therapeutic targets is essential. The Rac subfamily of GTPase proteins has been shown to participate in the physiopathology of hematological malignancies. However, their expression and function in AML remain unclear. In this study, we evaluated Rac1, Rac2 and Rac3 gene expressions in AML and their impact on clinical outcomes. We further investigated the effects of the in vitro treatment with a Rac inhibitor (EHT-1864) on AML cell lines. Rac3 expression was increased in AML derived from myelodysplastic syndromes compared to healthy donors. Rac2 expression did not differ between AML patients and healthy donors, but de novo AML patients with higher Rac2 presented lower overall survival. Oncogenic pathway gene-sets related to AKT/mTOR were identified as associated with Rac1, Rac2 and Rac3 expressions. EHT-1864 treatment reduced the viability of OCI-AML3, KG1 and Kasumi-1 cells in a time and dose-dependent manner. In OCI-AML3 cells, treatment with EHT-1864 induced apoptosis, autophagy, and led to the accumulation of cells in the G1 phase of the cell cycle. These changes were concomitant with alterations in p53 and cyclins. Dowregulation of the PI3K/AKT/mTOR pathway was also observed. Interestingly, the combined treatment of EHT-1864 and low doses of daunorubicin enhanced OCI-AML3 cell apoptosis. In conclusion, Rac2 expression is a prognostic factor in AML and our preclinical results suggest that Rac inhibition may be an attractive mechanism to compose the antineoplastic strategy for this disease.

Introducao As proteinas Rac1, Rac2 e Rac3 pertencem a familia de proteinas Rho GTPases, que e tradicionalmente conhecida pelas suas funcoes na regulacao do citoesqueleto. Estas proteinas estao envolvidas na regulacao de diferentes funcoes de celulas hematopoieticas, como entrada e saida de celulas-tronco hematopoieticas na medula ossea e maturacao das celulas sanguineas. Apesar destas propriedades celulares estarem desreguladas em leucemia mieloide aguda, as funcoes das proteinas Rac1, Rac2 e Rac3 ainda nao foram exploradas nesta doenca. Objetivos Neste estudo avaliamos os efeitos do tratamento com EHT-1864, um inibidor farmacologico de Rac1, Rac2 e Rac3, na via PI3K/AKT/mTOR em linhagem leucemica OCI-AML3. Os efeitos da combinacao de EHT-1864 e daunorrubicina, um dos farmacos ja utilizados no tratamento da LMA, tambem foram avaliados na sobrevivencia destas mesmas celulas. Metodos A expressao de proteinas da via PI3K/AKT/mTOR foi avaliada por western blot em celulas OCI-AML3 tratadas com EHT-1864 (0,5-30μM) por 48 e 72 horas. A expressao de GAPDH foi utilizada como normalizadora. Os efeitos do tratamento combinado de EHT-1864 (5μM e 10μM) com daunorrubicina (1-200M) por 48 e 72 horas foram avaliados na viabilidade e apoptose de celulas OCI-AML3. Para isso, as celulas foram tratadas com os farmacos sozinhos ou em combinacao e avaliadas por ensaio de MTT e marcacao de anexina V e iodeto propideo seguida de analise por citometria de fluxo. Resultados O tratamento com EHT-1864 inibiu a via PI3K/AKT/mTOR, evidenciado pela reducao da fosforilacao de AKT (Ser 473), mTOR (Ser 2448), EBP1 (Thr 70), PDK1, p70s6k (Ser 424 Thr 421). Tambem observamos aumento da ativacao de PTEN (reducao de p-PTEN Ser 380). Interessantemente, o tratamento combinado de EHT-1864 e daunorrubicina foi significativamente mais efetivo que os tratamentos dos farmacos sozinhos na reducao da apoptose e reducao da viabilidade celular. Discussao Resultados anteriores de nosso grupo mostraram que o tratamento com EHT-1864 induz os processos de apoptose e autofagia, bem como parada no ciclo celular de celulas leucemicas. Os achados deste estudo indicam que estes efeitos podem ser pelo menos parcialmente explicados pela inibicao da via PI3K/AKT/mTOR. A potencializacao dos efeitos do tratamento com daunorrubicina em celulas leucemicas sugere que o tratamento combinado e uma opcao terapeutica interessante para ser melhor explorada em estudos futuros. Conclusao A inibicao farmacologica de proteinas Rac atraves de EHT-1864 inibiu a via PI3K/AKT/mTOR em celulas leucemicas. O tratamento in vitro combinado de EHT-1864 e baixas doses de daunorrubicina potencializou a apoptose de celulas OCI-AML3. Financiamento: FAPESP.

Glioblastoma patients have a poor prognosis with a low median survival rate mainly due to temozolomide (TMZ) resistance. NRF2 is an important transcript factor involved in chemotherapy resistance due to its ability to regulate genes related to the antioxidant response and to prevent cell death processes, such as ferroptosis. However, the relation between NRF2 and iron-dependent cell death is contradictory and poorly understood. This study aimed to analyze the role of NRF2 in ferroptosis modulation in glioblastoma cells. To this end, it was analyzed two human glioblastoma cell lines (U251MG and T98G) after treatment with TMZ, ferroptosis inducers (Erastin, RSL3, and Sorafenib), and ferroptosis inhibitor (Ferrostatin-1). Also, we performed gene expression analysis of glioma patients. Our results demonstrated that T98G compared to the U251MG was more resistant to chemotherapy and showed elevated levels of NRF2 expression and its targets xCT, HMOX1, and ABCC1. Interestingly, T98G cells revealed higher sensitivity and lipoperoxidation levels after ferroptotic treatment. Next, we established T98G NRF2 silenced cells and we observed a significant reduction in cellular viability after TMZ treatment when compared to wild-type cells. On the other hand, T98G-shNRF2 was more resistant to ferroptosis induction, indicating that NRF2 plays a key role in the modulation of chemoresistance and ferroptosis. After, we showed that NRF2 controls levels of ABCC1/MRP1 in glioblastoma cells, and ABCC1 silencing promotes sensitivity to TMZ and resistance to Erastin. These results support a possible mechanism of ferroptosis modulation by NRF2 on TMZ-resistant gliomas through ABCC1, which has been recently associated with ferroptosis induction by promoting efflux of glutathione out of the cell. Furthermore, we confirmed that NRF2 has a positive correlation with ABCC1 in glioma patients, and higher ABCC1 expression was associated with tumor aggressiveness. Also, we validated ABCC1 as an independent prognostic factor for poor overall survival on glioma. Finally, we observed that T98G cells have sensitivity to the ferroptosis inducer FDA-approved, sorafenib. Altogether our data suggest that high levels of NRF2 may result in ferroptosis sensitivity on glioblastoma through the high levels of expression of its pro-ferroptotic target ABCC1, once the xCT system is blocked by Erastin. Thus, glioblastoma cell vulnerability to ferroptosis by NRF2 and ABCC1 high expression can be an Achilles’ heel to reverse drug resistance on glioblastoma through the treatment with ferroptosis inducers. Citation Format: Izadora de Souza, Linda Karolynne Monteiro, Camila Banca Guedes, Marcela Latancia, Marina Tomaz Andrade, Matheus Molina Silva, Bruna Contieri, Bruna Felício Milazzotto Ribeiro, Mariana Lazarini, Luciana Gomes, Clarissa Rocha. Temozolomide-resistant glioblastoma cells are collaterally sensitive to ferroptosis through NRF2 high expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5360.

Nifuroxazide has been employed as an anti-diarrheic agent since 1966, but in the last decade has brought to the research spotlight again due to its recently described antitumoral activity through the JAK2 inhibitory potential. Since 2008, more than 70 papers have been published about the issue and more are expected to the following years. Herein we discuss the findings of molecular modelling studies which were performed to elucidate the potential binding mode of this drug into the JAK2 ATP recognition site and also into the allosteric region near the catalytic site. Molecular modelling followed by dynamics simulations indicated the NFZ could bind at both sites, such as a Type II kinase inhibitor since residues from both ATP and modulatory site would exhibit contacts with the drug when in a stable complex. Synthesis of NFZ and its sulfur bioisosteric analogue GPQF-63 were performed and experimental assays against HEL cells indicate the potential of NFZ and, mainly of its analogue GPQF-63 in acting as inhibitors of cell growth. HEL-cells present the JAK2 V617F mutation which leads to an enhanced JAK/STAT pathway and they have never been tested by the NFZ activity before. A mechanistic approach was also performed and revealed that both compounds induce cell apoptosis.Taken together, both the theoretical and experimental approaches point out the N-acylhydrazones as good starting points in the search for JAK2 modulatory small molecules which could then, be studied as promising leads toward new alternatives to control the JAK-STAT pathway related pathologies. This is the first study, as far as we have known, to propose a potential binding mode for NFZ as well as reporting the activity of this drug against HEL cells, which are a usual cellular model to human erythroleukemia and other myeloproliferative diseases.

Introduction: Acute Myeloid Leukemia (AML) is a hematologic malignancy with poor prognosis that can occur from myelodysplastic syndrome (MDS) progression. RHOC is a Rho GTPase protein involved in cancer progression, but its expression and function have not been explored in these diseases. Objective: To investigate the expression and signaling pathways related to RHOC in AML and MDS. Materials and methods: RHOC gene expression was analyzed by quantitative PCR in bone marrow samples from patients with MDS (n = 47) or AML (n = 58), and heathy donors (n = 14), attended at University of Campinas. The study was approved by the local Ethical Committee on Human Research. Clinical and genomic data of AML patients from The Cancer Genome Atlas (TCGA, n = 173) were also analyzed. Risk factors impacting overall survival (OS) and disease-free survival (DFS) were estimated using a Cox regression model. SRC expression was evaluated by western blot in U937 leukemia cell line stably silenced for RHOC using lentiviral vector-mediated shRNA. Results: RHOC expression was increased in AML patients with previous MDS (n = 15) and de novo AML (n = 43), compared to normal hematopoietic cells (all p < 0.05). No significant differences in RHOC expression were observed in MDS patients. Bone marrow samples from four MDS patients of our cohort were analyzed before and after progression to AML. Interestingly, these patients presented increased RHOC expression after the progression. Analysis from TCGA study revealed that RHOC was upregulated in patients with adverse cytogenetic risk compared to AML with intermediate cytogenetic risk. Univariate analysis showed that higher levels of RHOC (above median) impacted OS and DFS (all p < 0.05). Multivariate analysis confirmed that higher RHOC was an independent prognostic factor for inferior OS (HR = 1.378, CI= 1.194-1.699, p < 0.001) and DFS (HR = 1.421, CI=1.086-1.860, p < 0.010). Higher RHOC expression was associated with the presence of TP53 mutations, and lower RHOC was associated with the presence of CEBPA or NPM1 or WT1 mutations (Fisher's exact test). Gene set enrichment analysis revealed that RHOC is associated with signaling pathways such as regulation of actin cytoskeleton, apoptosis, and DNA damage responses. In addition, RHOC expression positively correlated with the expression of SRC oncogene (R = 0,61, Spearman; p = 4,63e-19). Notably, leukemia cells silenced for RHOC presented decreased SRC protein expression. Conclusion: RHOC expression is increased in AML and negatively impacts patient survival. These results may be possibly explained by the RHOC relation with SRC and participation in signaling pathways such as migration, apoptosis, and DNA damage responses. Funding: FAPESP, and CAPES.

Arhgap21 is a member of the Rho GTPase activating protein (RhoGAP) family, which function as negative regulators of Rho GTPases. Arhgap21 has been implicated in adhesion and migration of cancer cells. However, the role of Arhgap21 has never been investigated in hematopoietic cells. Herein, we evaluated functional aspects of hematopoietic stem and progenitor cells (HSPC) using a haploinsufficient (Arhgap21+/−) mouse. Our results show that Arhgap21+/− mice have an increased frequency of phenotypic HSC, impaired ability to form progenitor colonies in vitro and decreased hematopoietic engraftment in vivo, along with a decrease in LSK cell frequency during serial bone marrow transplantation. Arhgap21+/− hematopoietic progenitor cells have impaired adhesion and enhanced mobilization of immature LSK and myeloid progenitors. Arhgap21+/− mice also exhibit reduced erythroid commitment and differentiation, which was recapitulated in human primary cells, in which knockdown of ARHGAP21 in CMP and MEP resulted in decreased erythroid commitment. Finally, we observed enhanced RhoC activity in the bone marrow cells of Arhgap21+/− mice, indicating that Arhgap21 functions in hematopoiesis may be at least partially mediated by RhoC inactivation. Keywords: Arhgap21, Hematopoiesis, Erythroid cells, Hematopoietic stem and progenitor cells, Fate decision

SIVA is a transcriptional target of p53 that plays a potential role in the development and progression of cancer. In this study, we analyzed SIVA1 and SIVA2 expression, and its association with clinical features and TP53 and MDM2 expression in bone marrow cells from healthy donors and myelodysplastic syndrome (MDS) patients. Fifty-five untreated patients with MDS and 22 healthy donors were included. Gene expression was evaluated by quantitative PCR. For statistical analysis, Mann–Whitney test, Spearman correlation analysis and Log-rank (Mantel-Cox) were used, as appropriate. A p value

Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell-based disorders characterized by ineffective hematopoiesis, increased genomic instability and a tendency to progress toward acute myeloid leukemia (AML). MDS and AML cells present genetic and epigenetic abnormalities and, due to the heterogeneity of these molecular alterations, the current treatment options remain unsatisfactory. Hypomethylating agents (HMA), especially azacitidine, are the mainstay of treatment for high-risk MDS patients and HMA are used in treating elderly AML. The aim of this study was to investigate the potential role of the epigenetic reader bromodomain-containing protein-4 (BRD4) in MDS and AML patients. We identified the upregulation of the short variant BRD4 in MDS and AML patients, which was associated with a worse outcome of MDS. Furthermore, the inhibition of BRD4 in vitro with JQ1 or shRNA induced leukemia cell apoptosis, especially when combined to azacitidine, and triggered the activation of the DNA damage response pathway. JQ1 and AZD6738 (a specific ATR inhibitor) also synergized to induce apoptosis in leukemia cells. Our results indicate that the BRD4-dependent transcriptional program is a defective pathway in MDS and AML pathogenesis and its inhibition induces apoptosis of leukemia cells, which is enhanced in combination with HMA or an ATR inhibitor.

Background Microtubules, actin and Rho GTPase proteins are essential players in the megakaryocyte biology for platelet formation and function. Objectives To investigate the role of ARHGAP21, a RhoGAP protein, in megakaryocytic differentiation and platelet function. Methods Cytoskeletal proteins were investigated in HEL cells silenced for ARHGAP21 and submitted to megakaryocyte differentiation. The role of Arhgap21 in platelet function was accessed using haploinsufficient (Arhgap21+/−) mice. Arhgap21+/− platelet aggregation and p-selectin exposure were evaluated in response to thrombin. Vessel occlusion time and thrombus formation were detected after injury of the carotid artery. Platelet morphology was accessed by electronic microscopy. Results ARHGAP21 was upregulated during megakaryocytic differentiation of the cell line and primary mouse cells. In the HEL cell model, ARHGAP21 was detected in the cytoplasmic protrusions, colocalized and associated with α-tubulin and was mostly detected in the protein cell fraction containing the polymerized tubulin. Silencing of ARHGAP21 decreased the expression of Glu-tubulin, suggesting microtubule instability, and enhanced cell spreading. Platelets from Arhgap21+/− mice presented enhanced thrombin-induced aggregation and p-selectin exposure associated with increased size of α-granules. Arhgap21+/− mice also showed increased CDC42 and RHOA activities, shorter tail-bleeding and accelerated thrombus formation. Conclusions Our results indicate that ARHGAP21 may be a critical protein in the regulation of platelet production and function through the control of cytoskeletal rearrangement.

Objective Articular cartilage is an avascular tissue with limited ability of self-regeneration and the current clinical treatments have restricted capacity to restore damages induced by trauma or diseases. Therefore, new techniques are being tested for cartilage repair, using scaffolds and/or stem cells. Although type II collagen hydrogel, fibrin sealant, and adipose-derived stem cells (ASCs) represent suitable alternatives for cartilage formation, their combination has not yet been investigated in vivo for focal articular cartilage defects. We performed a simple experimental procedure using the combination of these 3 compounds on cartilage lesions of rabbit knees. Design The hydrogel was developed in house and was first tested in vitro for chondrogenic differentiation. Next, implants were performed in chondral defects with or without ASCs and the degree of regeneration was macroscopically and microscopically evaluated. Results Production of proteoglycans and the increased expression of collagen type II (COL2α1), aggrecan (ACAN), and sex-determining region Y-box 9 (SOX9) confirmed the chondrogenic character of ASCs in the hydrogel in vitro. Importantly, the addition of ASC induced a higher overall repair of the chondral lesions and a better cellular organization and collagen fiber alignment compared with the same treatment without ASCs. This regenerating tissue also presented the expression of cartilage glycosaminoglycan and type II collagen. Conclusions Our results indicate that the combination of the 3 compounds is effective for articular cartilage repair and may be of future clinical interest.

Megakaryocyte differentiation and platelet biogenesis require profound cytoskeleton rearrangement regulated by the Rho family of GTPases. ARHGAP21 is a RhoGAP protein that has been shown to negatively regulate the activity of RhoA, RhoC and Cdc42. We have previously demonstrated that ARHGAP21 knockdown in human common myeloid progenitors and in bipotent megakaryocyte-erythrocyte progenitors may bias the fate decision toward the megakaryocyte lineage. In addition, a mouse model with reduction of Arhgap21 expression (Arhgap21+/-) present a slight reduction in platelet number and increased platelet volume. However, the participation of ARHGAP21 in platelet biogenesis and hemostatic response has never been investigated. We studied the role of ARHGAP21 on cytoskeletal changes during megakaryocyte differentiation in HEL cell line. Differentiation was induced with 20ηM phorbol-13 myristate-12 acetate (PMA) for four days. ARHGAP21 protein expression was increased during the differentiation and was mostly detected in the protein cell fraction containing polymerized tubulin, in comparison with cell extracts containing soluble tubulin. ARHGAP21 co-localized (R ≥ 0.86 in all days of differentiation) and interacted with α-tubulin on day 2 of megakaryocyte differentiation, when ARHGAP21 expression was the highest. Silencing of ARHGAP21 with siRNA decreased the expression of Glu-tubulin and enhanced CDC42 activity on days 2 and 3 of differentiation. Increased cell size and spreading and alteration of the adhesion proteins p-p130Cas, vinculin, p-zyxin and p-FAK925 were also observed upon ARHGAP21 inhibition. There was no change in the acquisition of CD61, CD41 and CD42 megakaryocytic markers, neither in the polyploidy of HEL cells during differentiation. We further investigated the effects of ARHGAP21 inhibition on platelet morphology and on the hemostatic response in vivo, using the C57BL/6 Arhgap21 heterozygous mouse model (Arhgap21+/-). The study was approved by the Ethical Committee of our Institution. No differences were observed in CD61+CD41+ nor in CD41+CD42+ bone marrow cells from Arhgap21+/- and wild type (WT) mice. However, transmission electron microscopy revealed that Arhgap21+/- platelets presented increased alpha-granule size when compared to wild-type (WT). Tail bleeding time of Arhgap21+/- mice was decreased compared to WT (P= 0.0008). Intravital microscopy of carotid artery injured by FeCl3 showed increased adhesion of platelet and white blood cells on the vessel wall of Arhgap21+/-, which reflected in accelerated occlusion time (twice as fast) compared to WT (P= 0.0150). In conclusion, ARHGAP21 silencing may alter cell morphology and lead to increased microtubule dynamic instability during megakaryocyte differentiation in vitro, without compromising the acquisition of differentiation markers. In vivo, deficiency of Arhgap21 increases platelet granule size and accelerates hemostatic response. Together, these results indicate that ARHGAP21 may be a critical protein in the regulation of platelet production and function through the control of cytoskeletal rearrangement. This study was supported by São Paulo Research Foundation (FAPESP) National Council for Scientific and Technological Development (CNPq) and Coordination for the Improvement of Higher Education Personnel (CAPES). Disclosures No relevant conflicts of interest to declare.

RhoA and RhoC contribute to the regulation of glutamine metabolism, which is a crucial determinant of cell growth in some types of cancer. Here we investigated the participation of RhoA and RhoC in the response of prostate cancer cells to glutamine deprivation. We found that RhoA and RhoC activities were up- or downregulated by glutamine reduction in PC3 and LNCaP cell lines, which was concomitant to a reduction in cell number and proliferation. Stable overexpression of wild type RhoA or RhoC did not alter the sensitivity to glutamine deprivation. However, PC3 cells expressing dominant negative RhoA(N19) or RhoC(N19) mutants were more resistant to glutamine deprivation. Our results indicate that RhoA and RhoC activities could affect cancer treatments targeting the glutamine pathway.

Acute myeloid leukemia (AML) cells exhibit altered glucose metabolism that is associated with poor prognosis and chemotherapy resistance [1]. Nuclear receptor subfamily four group A member 1 (NR4A1...

ANKHD1 is a multiple ankyrin repeat containing protein, recently identified as a novel member of the Hippo signaling pathway. The present study aimed to investigate the role of ANKHD1 in DU145 and LNCaP prostate cancer cells. ANKHD1 and YAP1 were found to be highly expressed in prostate cancer cells, and ANKHD1 silencing decreased cell growth, delayed cell cycle progression at the S phase, and reduced tumor xenograft growth. Moreover, ANKHD1 knockdown downregulated YAP1 expression and activation, and reduced the expression of CCNA2, a YAP1 target gene. These findings indicate that ANKHD1 is a positive regulator of YAP1 and promotes cell growth and cell cycle progression through Cyclin A upregulation.

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 Disclosures: No relevant conflicts of interest to declare.

Background: Several Rho GTPase-activating proteins (RhoGAPs) are implicated in tumor progression through their effects on Rho GTPase activity. ARHGAP21 is a RhoGAP with increased expression in head and neck squamous cell carcinoma and with a possible role in glioblastoma tumor progression, yet little is known about the function of ARHGAP21 in cancer cells. Here we studied the role of ARHGAP21 in two prostate adenocarcinoma cell lines, LNCaP and PC3, which respectively represent initial and advanced stages of prostate carcinogenesis. Results: ARHGAP21 is located in the nucleus and cytoplasm of both cell lines and its depletion resulted in decreased proliferation and increased migration of PC3 cells but not LNCaP cells. In PC3 cells, ARHGAP21 presented GAP activity for RhoA and RhoC and induced changes in cell morphology. Moreover, its silencing altered the expression of genes involved in cell proliferation and cytoskeleton organization, as well as the endothelin-1 canonical pathway. Conclusions: Our results reveal new functions and signaling pathways regulated by ARHGAP21, and indicate that it could contribute to prostate cancer progression.

Insulin receptor substrate 2 (IRS2) is an adaptor protein that associates with the receptor of erythropoietin, insulin-like growth factor 1 and thrombopoietin; however, its role is not known in myelodysplasia. We, herein, report a significantly lower IRS2 expression in MDS cells, compared to normal cells. IRS2 expression was reduced in high-risk, compared to low-risk disease, and positively correlated with neutrophil and platelet counts. IRS2 was upregulated during erythroid differentiation of CD34(+) cells from normal donors and low-risk MDS patients and also during erythroid, granulocytic and megakaryocytic differentiation in cell lines. These results suggest that defective IRS2 expression plays a role in the impaired hematopoietic cell differentiation in MDS.

BCR-ABL kinase activates downstream signaling pathways, including the PI3K-Akt/mTOR and the MAPK pathway. IRS1 has been previously described as constitutively phosphorylated and associated with BCR-ABL in K562 cells, suggesting that IRS1 has role in the BCR-ABL signaling pathways. In this study, we analyzed the effect of IRS1 silencing, by shRNA-lentiviral delivery, in K562 cells, a CML cell line that presents the BCR-ABL. IRS1 silencing decreased cell proliferation and colony formation in K562 cells, which correlates with the delay of these cells at the G0/G1 phase and a decrease in the S phase of the cell cycle. Furthermore, IRS1 silencing in K562 cells resulted in a decrease of Akt, P70S6K and ERK1/2 phosphorylation. Nevertheless, apoptosis was unaffected by IRS1 knockdown and no alterations were found in the phosphorylation of BAD and in the expression of BCL2 and BAX. BCR-ABL and CRKL phosphorylation levels remained unaffected upon IRS1 silencing, and no synergistic effect was observed with imatinib treatment and IRS1 knockdown, indicating that IRS1 is downstream from BCR-ABL. In conclusion, we demonstrated that inhibition of IRS1 is capable of inducing the downregulation of Akt/mTOR and MAPK pathways and further decreasing proliferation, and clonogenicity and induces to cell cycle delay at G0/G1 phase in BCR-ABL cells.

ANKHD1 is highly expressed in human acute leukemia cells and potentially regulates multiple cellular functions through its ankyrin-repeat domains. In order to identify interaction partners of the ANKHD1 protein and its role in leukemia cells, we performed a yeast two-hybrid system screen and identified SIVA, a cellular protein known to be involved in proapoptotic signaling pathways. The interaction between ANKHD1 and SIVA was confirmed by co-imunoprecipitation assays. Using human leukemia cell models and lentivirus-mediated shRNA approaches, we showed that ANKHD1 and SIVA proteins have opposing effects. While it is known that SIVA silencing promotes Stathmin 1 activation, increased cell migration and xenograft tumor growth, we showed that ANKHD1 silencing leads to Stathmin 1 inactivation, reduced cell migration and xenograft tumor growth, likely through the inhibition of SIVA/Stathmin 1 association. In addition, we observed that ANKHD1 knockdown decreases cell proliferation, without modulating apoptosis of leukemia cells, while SIVA has a proapoptotic function in U937 cells, but does not modulate proliferation in vitro. Results indicate that ANKHD1 binds to SIVA and has an important role in inducing leukemia cell proliferation and migration via the Stathmin 1 pathway. ANKHD1 may be an oncogene and participate in the leukemia cell phenotype.

OBJECTIVE: The aim of this study was to evaluate the expression of protein tyrosine kinase 2 and protein tyrosine phosphatase non-receptor type 11, which respectively encode focal adhesion kinase protein and src homology 2 domain-containing protein-tyrosine phosphatase 2, in hematopoietic cells from patients with myelodysplastic syndromes. METHODS: Protein tyrosine kinase 2 and tyrosine phosphatase non-receptor type 11 expressions were analyzed by quantitative polymerase chain reaction in bone marrow cells from patients with myelodysplastic syndromes and healthy donors. RESULTS: Protein tyrosine kinase 2 and tyrosine phosphatase non-receptor type 11 expressions did not significantly differ between normal cells and myelodysplastic cells. CONCLUSIONS: Our data suggest that despite the relevance of focal adhesion kinase and src homology 2 domain-containing protein-tyrosine phosphatase 2 in hematopoietic disorders, their mRNA expression do not significantly differ between total bone marrow cells from patients with myelodysplastic syndromes and healthy donors.

The human FMNL1 is expressed predominantly in hematopoietic cells and has been described previously as overexpressed in hematopoietic malignancies. However, it is not known whether FMNL1 contributes to leukemogenesis. Here, we investigate the FMNL1 function using two different human leukemia models: Namalwa and K562 cell lines. FMNL1 depletion reduced cell proliferation and colony formation in both leukemic cell types, as well as a decrease in the tumor growth of FMNL1-depleted Namalwa cell xenografts. In addition, there was a decrease in migration and in TEM in FMNL1-depleted Namalwa cells. FMNL1 endogenously associates with Rac1, and FMNL1 silencing resulted in an increased Rac1 activity. The reduced migration observed in FMNL1-depleted cells was restored by inhibiting Rac activity. Our results indicate that FMNL1 stimulates leukemia cell proliferation as well as migration. This suggests that FMNL1 contributes to leukemogenesis and could act in part through Rac1 regulation.

Cell-cell adhesions and the cytoskeletons play important and coordinated roles in cell biology, including cell differentiation, development, and migration. Adhesion and cytoskeletal dynamics are regulated by Rho-GTPases. ARHGAP21 is a negative regulator of Rho-GTPases, particularly Cdc42. Here we assess the function of ARHGAP21 in cell-cell adhesion, cell migration, and scattering. We find that ARHGAP21 is localized in the nucleus, cytoplasm, or perinuclear region but is transiently redistributed to cell-cell junctions 4 h after initiation of cell-cell adhesion. ARHGAP21 interacts with Cdc42, and decreased Cdc42 activity coincides with the appearance of ARHGAP21 at the cell-cell junctions. Cells lacking ARHGAP21 expression show weaker cell-cell adhesions, increased cell migration, and a diminished ability to undergo hepatocyte growth factor-induced epithelial-mesenchymal transition (EMT). In addition, ARHGAP21 interacts with α-tubulin, and it is essential for α-tubulin acetylation in EMT. Our findings indicate that ARHGAP21 is a Rho-GAP involved in cell-cell junction remodeling and that ARHGAP21 affects migration and EMT through α-tubulin interaction and acetylation.

Leukocytes play a primary role in vascular inflammation, and thus an understanding of the pathways involved in the activation of these cells and means to inhibit their consequent adhesion to the vessel wall is of significant interest. This study aimed to determine whether statins have a direct effect upon neutrophil adhesive properties under inflammatory conditions.Neutrophils from healthy individuals were subjected to adhesion assays (with fibronectin as ligand) and flow cytometry.In the presence of a TNF-α inflammatory stimulus, neutrophils displayed a rapid and substantial enhancement in their adhesive properties that was abrogated by preincubation of cells with simvastatin. Neutrophil surface expression of the Mac-1 integrin subunit, CD11b, was augmented by TNF-α, and this increased expression was also inhibited by simvastatin. TNF-α also induced neutrophil LFA-1 and Mac-1 activation, but this activation was not blocked by simvastatin. Interestingly, while addition of the isoprenoids, geranygerayl pyrophosphate and farnesyl pyrophosphate, to cells did not alter the effect of simvastatin on TNF-α-stimulated adhesion, concurrent incubation of cells with the Rho kinase (ROCK) inhibitor reversed the effects of simvastatin on neutrophil adhesion and CD11b expression.Simvastatin appears to have direct anti-inflammatory effects in neutrophils that may be mediated by modulation of ROCK activity.

Introduction: During differentiation, the megakaryocyte goes through profound changes in the cytoskeleton of actin and tubulin through Rho GTPase activity. Microtubules provide the elongation of proplatelets, whereas actin microfilaments mediate force to increase branching and release of platelets. ARHGAP21 is a RhoGAP for RhoA, RhoC and Cdc42, which has been shown to interact with α-tubulin in cancer cells. Moreover, arhgap21+/- mice exhibit significant reduction in platelet number and increased platelet volume. Aim: To evaluate ARHGAP21 function in the activity of Rho GTPase and their effectors during megakaryocytic differentiation. Materials and Methods: Megakaryocyte differentiation was stimulated in HEL cells through treatment with 20 nM of phorbol myristate acetate -13 -12 (PMA) for 4 days and was confirmed by the expression of CD41a, CD42b and CD61 and polyploidy using flow cytometry. Morphological changes were observed by optical microscopy. The localization of ARHGAP21, F-Actin and α-Tubulin cytoskeletal proteins was assessed by confocal microscopy. The expression of ARHGAP21, and the Rho GTPases RhoA, RhoC, Cdc42 and downstream proteins Rock1 e2, phospho-MLC2, MLC2, phospho-cofilin, cofilin and mDia1 were analyzed by western blotting. Rho GTPases activity was determined through pull down assays using Rhotekin-GST (RhoA, RhoB and RhoC) and WASP-GST (Cdc42) constructions. Tubulin polymerization was evaluated by soluble and insoluble tubulin precipitation assay. ARHGAP21 silencing was performed by siRNA, after PMA treatment for 2 and 3 days and was followed by the analysis of the expression and activity of Rho GTPases and their effectors, ploidy and differentiation markers. Results: Megakaryocytic differentiation of HEL cells was accompanied by intense rearrangement of the cytoskeleton, increased cell size, polyploidy and increased expression of the membrane receptors CD61, CD41a and CD42b. Interestingly, a gradual upregulation of ARHGAP21 was observed during differentiation, especially on days 2 and 3 of treatment (both 9.33-fold increase) and mainly in extracts containing polymerized tubulin. ARHGAP21 upregulation was concomitant with the reduction of RhoA and Cdc42 activities (92% decreased and 52% decreased, respectively), but not in RhoC. Silencing of ARHGAP21 by siRNA was confirmed by western blot. Downregulation of ARHGAP21 in HEL cells trigged increased phosphorylation on serine 19 of myosin light chain2 (MLC2) on the day 2. Moreover, mDia1, a common effector of RhoA and Cdc42, was also increased at the same point. ARHGAP21 silencing induced an increase in CD42b on day 3 (5% increased, P Conclusion: Our results suggest that the upregulation of ARHGAP21 during megakaryocytic differentiation is important to control the dynamics of the cytoskeleton through the regulation of RhoA and Cdc42. Silencing of ARHGAP21 induces increased phosphorylation of MLC2 and the expression of mDia1, which may impair megakaryocytic differentiation. Furthermore, ARHGAP21 appears to regulate the acquisition of CD42b receptor, participating in the final stages of megakaryopoiesis. Disclosures No relevant conflicts of interest to declare.

The purpose of this study was to investigate the in vitro effects of thalidomide on long-term bone marrow cultures from patients with myelodysplastic syndrome. We demonstrated that thalidomide induced an increase in granulocyte–macrophage colony forming unit numbers and in IL-10 expression. Thalidomide also promoted a slight increase in IL-6, IL-1β and TNF-α expression in the stromal layers. The numbers of erythroid burst forming units, the apoptosis rate of hematopoietic cells, and VEGF and TNF-α expression levels in culture supernatants were not modulated. Our results indicate a participation of thalidomide upon the hematopoietic microenvironment of patients with myelodysplastic syndromes, especially in the up regulation of IL-10.

Introduction: The JAK/STAT pathway is constitutively activated in the myeloproliferative neoplasms (MPN), including essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). JAK/STAT pathway has been targeted by Ruxolitinib, a selective JAK1/2 inhibitor approved by FDA for intermediate and high-risk PMF treatment. Results from phase III clinical trials demonstrated that Ruxolitinib is well-tolerated, reduces inflammatory cytokines and splenomegaly, ameliorated constitutional symptoms; however, Ruxolitinib did not reverse bone marrow fibrosis, suggesting that the development of better approaches are necessary. In addition to the STAT3 transcriptional activity in the nucleus, activated STAT3 binds to and inhibits Stathmin 1 in the cytoplasm, resulting in microtubule stability. Stathmin 1 is an important cytoplasmic microtubule-destabilizing protein that plays critical roles in proliferation and accurate chromosome segregation through regulation of microtubule dynamics. Paclitaxel, an anti-neoplastic compound, is a microtubule-targeted drug that induces microtubule polymerization, cell-cycle block at the metaphase-anaphase transition and cell death, and inhibits Stathmin 1 activity. We hypothesized that an undesirable effect of Ruxolitinib treatment could be Stathmin 1 activation as a consequence of STAT3 inhibition; hence targeting Stathmin 1 in addition to JAK2 might be a reasonable approach in MPN. Aims: To investigate Stathmin 1 expression in CD34+ cells from MPN patients and the effects of Stathmin 1 silencing or Paclitaxel-induced microtubule stability on Ruxolitinib JAK2V617F-treated cells. Materials and Methods: CD34+ cells were obtained from a total of 104 peripheral blood (PB) samples collected from healthy donors (n=22) and patients with MPN (n=82; ET=34, PV=23, PMF=25; JAK2V617F=52, JAK2wt=30, CALRmut=19 and CALRwt=54). Gene and protein expressions were evaluated by qPCR and Western blot. In JAK2V617F-positive HEL cells, Stathmin 1 was stably knocked down with specific shRNA-expressing lentiviral vector, and cell growth, clonogenicity and apoptosis were evaluated by MTT assay, colony formation, and AnnexinV/PI and Caspase 3 activation by flow cytrometry, respectively. Alternatively, HEL cells were submitted or not to Paclitaxel treatment (5 and 10 nM). All conditions were evaluated in the presence or not of Ruxolitinib (100 and 300 nM). Statistical analyses were performed by Student’s t-test or Mann-Whitney test, as appropriate. Results: Stathmin 1 transcripts were significantly increased in PB CD34+ cells from PMF patients (2.73 [0.49-8.32]; p=.005) compared with healthy donors (1.08 [0.08-7.17]). No difference in Stathmin 1 expression was observed between healthy donors, ET and PV PB CD34+ cells. Stathmin 1 levels did not significantly differ between JAK2wtvs. JAK2V617F (p=.20); and CALRwtvs. CALRmut patients (p=.63). In HEL cells, Stathmin 1 silencing significantly reduced cell proliferation (p Conclusions : Stathmin 1 is highly expressed in PMF. Our study adds new insights for Stathmin 1 in hematological malignancies and indicates a possible role of Stathmin 1 in the aberrant JAK2V617F signaling pathway. Targeting Stathmin 1 or microtubule instability may improve Ruxolitinib response in JAK2V617F positive MPN patients. Disclosures No relevant conflicts of interest to declare.

INTRODUCTION: Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. BRD4 is a BET member of bromodomain-containing proteins, known as epigenome readers, exerting key roles in chromatin remodeling and transcriptional regulation. BRD4 gene encodes two major isoforms, short (BRD4S) and long (BRD4L). Recently, Floyd SR et al (Nature, 2013) described BRD4S as an endogenous inhibitor of the DNA damage response (DDR). Under normal conditions, damaged DNA induces histone phosphorylation of H2AX at Ser 139 (γ-H2AX) and activates a protective signaling network that blocks cell cycle and recruits DNA repair factors. Despite having been described as a therapeutic target in acute myeloid leukemia (AML) by Zuber J et al (Nature, 2011), BRD4 has never been studied in myelodysplastic syndrome (MDS) and its role in the pathogenesis is currently unknown. AIMS: To evaluate BRD4L and BRD4S expression in MDS and AML patients, correlating with clinical data, progression and survival. We also explored the BRD4 role in DDR signaling using human leukemia cell line models. MATERIAL AND METHODS: Diagnostic total bone marrow (BM) samples from 24 healthy donors (HD) and 99 patients, including 48 MDS (22 higher-risk MDS and 36 lower-risk MDS) and 51 AML (16 of them with MDS-related changes, AML-MRC), were collected. We also isolated CD34+ cells from 7 HD, 5 de novo AML, 4 AML-MRC and 14 MDS (6 higher-risk and 8 lower-risk MDS). BRD4L and BRD4S gene expressions were assessed through q-PCR and expressed as median (minimum-maximum). MDS patients were stratified according IPSS, WHO classification, R-IPSS and cytogenetic risk. Samples were age-adjusted when significant differences were observed, using ANOVA and Tukey’s test. Progression-free and overall survival curves were estimated by Kaplan-Meier method and were analyzed by the Wilcoxon´s test and Cox regression. JQ1, a specific BRD4 inhibitor, was kindly provided by James Bradner. A panel of human myeloid leukemia cell lines (KG1a, HEL, HL60, U937) in exponential growth was treated with increasing doses of JQ1 for 48 hours and cell growth (MTT colorimetric assay), apoptosis (annexin-V/PI) and cell cycle (flow cytofluorometric analysis detecting nuclear PI incorporation) were evaluated. We also determined the expression of p-γH2AX (DDR signaling) by western blot, after 12 hours of JQ1 treatment. RESULTS: A higher expression of BRD4S was observed in total BM cells from AML (4.01 [0.33-2.58], P=.01) and MDS patients (4.21 [0.01-56.17], P=.01) compared with HD (2.11 [0.04-10.32], P=.01). When stratified according WHO classification, AML-MRC (4.5 [0.33-25.22], P=.04) and higher-risk MDS (4.66 [0.17-56.17], P=.04) subgroups showed higher BRD4S expression. In CD34+ cells, BRD4S expression was increased in de novo AML (0.28 [0.21-0.45]) compared with lower-risk MDS (0.02 [0.00-0.44], P=.01). BRD4L mRNA expression was not modulated in total BM and CD34+ cells from any subgroup. With median follow-up time of 34.4 months, we found that higher BRD4S gene expression was a worse prognostic factor for MDS transformation and survival, along with IPSS, R-IPSS, low hemoglobin (less than 10g/dL) and higher BM blast percentage. After multivariate analysis, BRD4S gene expression and higher-risk (very high, high and intermediate) R-IPSS remained as independent prognostic factors for MDS progression and overall survival. KG1a and U937 cells showed greater resistance to JQ1, with lower apoptosis rate and proliferation (IC50 not reached, over 3000nM), whereas HEL and HL60 were more responsive (IC50 under 800nM in both cell lines). JQ1 suppressed cell proliferation, induced G0/G1 cell cycle arrest and apoptosis and caused a progressive increase in histone phosphorylation of γ-H2AX, indicating activation of DDR signaling. CONCLUSIONS: MDS is a clonal myeloid neoplasm characterized by profound epigenetic modifications. Our data establishes BRD4S as a novel MDS prognostic factor, related to aggressive phenotype, higher progression rate and shorter survival. Biologically, BRD4S plays a role in the inappropriate DNA Damage Response of MDS, favoring the disease towards genetic instability and clonal evolution. Disclosures No relevant conflicts of interest to declare.

Background: In myelodysplastic syndromes (MDS), changes in the balance between pro- and anti-apoptotic proteins are associated with disease progression and evolution towards acute myeloid leukemia (AML). NIX is a target of HIF-1 alpha and a member of the BH3-only subfamily proteins with a unique role in cell death and survival, through the regulation of both apoptosis and autophagy. NIX is also essential during maturation of reticulocytes and has been shown to be upregulated by decitabine (DAC) treatment in mantle cell lymphoma. Despite of being related to the pathogenesis of several diseases, NIX has never been studied in MDS and AML. Aims: We aimed to characterize the expression of NIX in bone marrow samples from healthy controls and patients with MDS and AML and the effects of NIX silencing upon DAC treatment in U937 cells. Patients and Methods: Bone marrow aspirates were obtained from 17 healthy controls, 56 patients at diagnosis of MDS, 12 patients with secondary AML/MDS and 47 patients at diagnosis of de novo AML. MDS patients were grouped in low-risk and high-risk according to WHO (RAUD/RCMD/del5q/=46; RAEB1/RAEB2=17) and IPSS (low/INT1=51; INT2/high=5). NIX expression was evaluated by quantitative PCR. Appropriated statistical analysis was performed and the data is showed as median [max-min]. NIX knockdown was performed in U937 cells with specific shRNA-expressing lentiviral vector. Colony formation was carried out in semisolid methylcellulose medium. Cell viability was evaluated by MTT assays. Apoptosis and autophagy were accessed by flow cytometry. Expression of NIX, Bcl-2, Caspase 3, p62, Beclin and LC3 were investigated by western blot. All assays were performed in lentiviral transduced cells treated or not with 1 µM or 5µM DAC for 72 hours. Results: NIX expression was significantly decreased in high-risk MDS, AML/MDS and patients with de novo AML, compared to healthy donors (high-risk MDS= 1.28 [2.24-0.69]; AML/MDS=0.52 [2.16-0.19]; AML=1.12 [6.15-0.19] versus healthy donors=2.57 [4.39-0.65]; WHO; P Conclusions: NIX is downregulated in high-risk MDS and AML and after disease progression. Moreover, our findings indicate that NIX has a pro-survival function in U937 cells and is upregulated by decitabine. Therefore, we hypothesize that the combination of NIX inhibition and decitabine would be a more effective strategy to induce apoptosis of malignant cells. Supported by Fapesp and CNPq. Disclosures No relevant conflicts of interest to declare.

The inflammasome is a protein complex activated by signals of cellular danger to trigger the innate immune defenses through the maturation of proinflammatory cytokines such as interleukin (IL)1β and IL18. Inflammasomes consist of pattern-recognition sensors (NLR family), adaptors (ASC), and effectors (caspase 1). IL1β initiates feedback loops through the IL1R-MyD88-NFκB pathway. Inflammasome activation promotes adaptive responses and limits reactive oxygen species (ROS) production. Autophagy is a cytoprotective pathway by which the cell sequesters damaged proteins and organelles for lysosomal degradation. The inflammasome is negatively regulated by autophagy and inflammasome activates autophagy. Inflammasome activity may play an important role in several nonmicrobial diseases with chronic inflammation, such as obesity, type 2 diabetes and cancer. We collected a cohort of diagnostic samples of total bone marrow (BM) from 31 MM patients, 3 plasma cell leukemia (PCL) patients and 9 healthy bone marrow donors (HD), together with clinical data. Four smoldering MM, 27 symptomatic MM and 3 PCL (2 primary and 1 secondary) were included. According to ISS, six patients were ISS I, 9 patients were ISS II and 16 were ISS III. We also stratified 18 of our symptomatic MM into groups according to response (CR/VGPR, PR, SD/PD). The offered treatment was thalidomide-based triplets. Essential genes from inflammasome (NLRP3, PYCARD, CASP1), pro-inflammatory interleukins (IL1B, IL18) and autophagy (BECN1, SQSTM1, MAP1LC3B) were verified by q-PCR. Gene expression was compared among subgroups and correlated with clinical data. CASP1 and PYCARD were increased in MM compared with HD, without differences among ISS subgroups. In PCL cases, CASP1 and PYCARD had lower expression when compared with MM, but did not differ from HD, confirming that PCL did not have inflammasome activation as MM did. NLRP3 was not different among all diagnostic subgroups. IL18 had decreased expression in PCL patients, compared with HD and MM. IL1B was not different among subgroups. PCL and MM had higher BECN1 expression compared with HD and these differences were also highlighted among ISS subgroups. SQSTM1 had increased expression in PCL, compared with HD and MM, but MM did not differ from HD. MAP1LC3B gene expression was similar among groups. A positive correlation was observed between CASP1 and PYCARD, IL1B and IL18, and between the three autophagic genes (BECN1, SQSTM1 and MAP1LC3B) as expected. BECN1 also correlated with IL1B and with CASP1; CASP1 and PYCARD correlated with BECN1 and SQSTM1, reinforcing the relation between autophagy and inflammasome. When the clinical data were analyzed, monocyte counts correlated with CASP1 and PYCARD; beta-2-microglobulin levels correlated with CASP1 and with NLRP3 and, finally, leukocyte and neutrophil counts correlated with IL1B, BECN1 and SQSTM1 BM expression levels. Analyzing response to thalidomide-based first line chemotherapy, VGPR/CR responders showed higher diagnostic NLRP3 expression (unpaired t test, P=0.03). The intricate interplay between autophagy and inflammasome has only recently been elucidated. Multiple myeloma is a B-cell neoplasm with great dependence of BM microenvironment. Inflammatory cells, especially monocytes and macrophages, are the main inflammasome activators, reducing ROS in the BM microenvironment. Autophagy is essential for plasmocytes, due to their high levels of intracellular proteins. Basal levels of autophagy in myeloma are high and are upregulated in response to ER stress and proteasome inhibition, protecting myeloma cells against apoptosis. Our results confirm the essential role of autophagy activation in myeloma. Interestingly, inflammasome activation in our cohort predicted a better response to thalidomide-based regimens and could be a novel response biomarker. Reinforcing our theory, recently thalidomide was described as an inhibitor of caspase 1 activation. Chronic inflammation is an established cancer hallmark and its role in myeloma pathogenesis seems to be important through inflammasome and autophagy dysfunction during disease progression, possibly creating dependency loops between inflammatory monocytes and neoplastic plasmocyte within the BM microenvironment. Disclosures: No relevant conflicts of interest to declare.

Dynamic protein turnover regulated through protein synthesis and degradation ensures cellular growth, proliferation and differentiation and has growing importance in cancer cells. Ndfip1 and Ndfip2 are known activators of the Nedd4 family of E3 ligases and regulate PTEN/Akt and MAP kinase signaling pathways. Autophagy is a major intracellular lysosomal degradation system, serving as a dynamic recycling platform, frequently deregulated in cancer. Acute myeloid leukemia (AML) has a constitutive Akt activation and autophagy suppression. Acute promyelocytic leukemia (APL) has increased protein accumulation and constitutive autophagy activation. Arsenic trioxide (ATO) is an effective treatment for APL and an established autophagy inductor. The crosstalks between ubiquitin proteasome system (UPS) and autophagy are an ongoing work and mostly unknown. We sought to investigate the expression levels of NDFIP1 and NDFIP2 in AML patients and to evaluate the role of both genes in autophagy. Bone marrow diagnostic samples from 27 patients with AML, 5 patients with APL and 15 healthy donors were analyzed, using qPCR. Functional studies were performed in vitro using the leukemic cell line U937 silenced for NDFIP1 (shNDFIP1) or NDFIP2 (shNDFIP2) through lentiviral particles. After puromycin selection and confirmation of transduction efficiency, cells were cultured upon ATO treatment 1µM or vehicle in the same concentration for 24h in order to induce autophagy. Acridine orange staining was used to quantify the percentage of acid vesicle organelles (AVO) and mitochondrial mass was estimated with Mitotracker Green, analyzed with flow cytometry. The expression of autophagic proteins (PIK3C3, Sqstm1, LC3 I/II) was investigated with western blot. NDFIP2 expression was significantly decreased in AML (P=0.04) and was increased in APL (P=0.018), compared with healthy donors. NDFIP2 expression also differed between AML and APL (P=0.002). NDFIP1 expression showed no differences among the subgroups analyzed. shNDFIP1 cells showed increased AVO percentage compared with the control. A slight AVO increase also occurred in shNDFIP2 cells. PIK3C3 expression was increased in shNDFIP1 and shNDFIP2 untreated cells. However, the mitochondrial mass was increased in both silenced cells. Moreover, shNDFIP1 and shNDFIP2 cells do not have modifications in LC3II or Sqstm1 expression. Interestingly, ATO treatment induced a significant increase of AVO only in control cells (P=0.01), but not in shNDFIP1 or shNDFIP2 cells. Increased AVO in control cells was accompanied by upregulation of PIK3C3 and LC3II and a decrease in Sqstm1. Conversely, ATO induced a downregulation of PIK3C3 and LC3II in silenced cells and upregulation of Sqstm1 in shNDFIP1 cells. The interplay between autophagy machinery and UPS is important under physiological conditions and has critical role in cancer. Autophagy is an essential adaptive response and modulates leukemic stemness. NDFIP is an endosomal protein with a role in vesicular trafficking. Better knowledge of UPS and autophagy intersections could lead to novel therapeutic targets for AML. Our results suggest that NDFIP is a novel autophagy regulator, as its silencing induces AVO accumulation, but without canonical autophagy activation, since the effector autophagy protein LC3II is decreased. NDFIP could be essential to autophagosome-lysosome fusion and its inhibition would block terminal stages of autophagy

Myelodysplastic syndrome (MDS) encompasses a heterogeneous group of clonal hematopoietic stem cell disorders, characterized by ineffective hematopoiesis and a tendency to progress towards acute myeloid leukemia (AML). The autophagy process has been implicated in the pathophysiology of both disorders. MDS treatment usually focuses on reducing or preventing complications, and single-target drugs are ineffective. The identification of novel compounds with anti-leukemia activity, as well as understanding the molecular and cellular basis of the disease may provide new therapeutic opportunities for MDS. In this concern, natural compounds such as Rhodiola rosea are considered interesting for the development of drugs against various molecular targets. Phytochemicals analyses of R. rosea extracts (RRE) revealed the presence of several components including polyphenols. The aim of this study was to study RRE effects using human tumor xenograft model. A total of 2x106 P39 cells (AML transformed from MDS), were subcutaneously injected in dorsal region of NOD.CB17-Prkdcscid/J mice. Tumor volume was measured once every 7 days and RRE treatment initiated when tumors reached 100-200 mm3 (n=6). The dose of 250mg/Kg body was given once every day by oral route (gavage). Control group received vehicle only (n=6). After 14 days, mice were sacrificed; tumors were removed and autophagy-related protein expression was evaluated by Western blot. After 14 days treatment, there was reduction of approximately 30% in tumor volume compared to controls. We found increased expression of beclin-1 and STSQM1/p62, and also Bcl-2 expression. In conclusion, Rhodiola rosea extract treatment in AML transformed from MDS tumor xenograft model reduces tumor growth, with pronounced inhibition of autophagy process. Disclosures: No relevant conflicts of interest to declare.