Exploring cell-derived extracellular vesicles in peripheral blood and bone marrow of B-cell acute lymphoblastic leukemia pediatric patients: proof-of-concept study.

Extracellular vesicles (EVs) are heterogeneous, phospholipid membrane enclosed particles that are secreted by healthy and cancerous cells. EVs are present in diverse biological fluids and have been associated with the severity of diseases, which indicates their potential as biomarkers for diagnosis, prognosis and as therapeutic targets. This study investigated the phenotypic characteristics of EVs derived from peripheral blood (PB) and bone marrow (BM) in pediatric patients with B-cell acute lymphoblastic leukemia (B-ALL) during different treatment stages. PB and BM plasma were collected from 20 B-ALL patients at three time points during induction therapy, referred to as: diagnosis baseline (D0), day 15 of induction therapy (D15) and the end of the induction therapy (D35). In addition, PB samples were collected from 10 healthy children at a single time point. The EVs were measured using CytoFLEX S flow cytometer. Calibration beads were employed to ensure accurate size analysis. The following, fluorescent-labeled specific cellular markers were used to label the EVs: Annexin V (phosphatidylserine), CD235a (erythrocyte), CD41a (platelet), CD51 (endothelial cell), CD45 (leukocyte), CD66b (neutrophil), CD14 (monocyte), CD3 (T lymphocyte), CD19, CD34 and CD10 (B lymphoblast/leukemic blast). Our results demonstrate that B-ALL patients had a marked production of EV-CD51/61+, EV-CD10+, EV-CD19+ and EV-CD10+CD19+ (double-positive) with a decrease in EV-CD41a+ on D0. However, the kinetics and signature of production during induction therapy revealed a clear decline in EV-CD10+ and EV-CD19+, with an increase of EV-CD41a+ on D35. Furthermore, B-ALL patients showed a complex biological network, exhibiting distinct profiles on D0 and D35. Interestingly, fold change and ROC curve analysis demonstrated that EV-CD10+CD19+ were associated with B-ALL patients, exhibited excellent clinical performance and standing out as a potential diagnostic biomarker. In conclusion, our data indicate that EVs represent a promising field of investigation in B-ALL, offering the possibility of identifying potential biomarkers and therapeutic targets.

Latex C-serum from Hevea brasiliensis induces apoptotic cell death in a leukemic cell line.

BACKGROUND: Hevea brasiliensis latex is generally cultivated for the use of rubber particles. Previous studies have shown that the antiproliferative activity of C-serum in hepatocellular carcinoma is not induced through the classical apoptotic signaling pathway. However, in a leukemic cell line, the anti-proliferation effect of latex C serum remained unclear. METHODS: Leukemic cell lines (K562 and U937) and human peripheral blood mononuclear cells (PBMCs) were examined for cell viability using the MTT assay. Flow cytometry was used for apoptotic cell detection by annexin V/PI staining. The expression levels of proapoptotic and antiapoptotic marker genes were measured by qRT‒PCR. Moreover, the caspase activities of the extrinsic and intrinsic apoptotic pathways were detected by enzymatic activities. RESULTS: Latex C-serum inhibited cell proliferation in the K562 and U937 leukemic cell lines but did not affect human PBMCs. Latex C-serum significantly induced the percentage of early and late apoptotic cells in the leukemic cell line. The expression levels of the pro-apoptotic marker genes BAD, BAX, and CASPASE3 significantly increased in the leukemic cell line after post-latex C-serum leukemic cell treatment. The extrinsic, intrinsic and common apoptotic pathways were also studied through caspase-8, -9, and -3 activities. Latex C-serum treatment significantly induced caspase-8, -9, and -3 activation in the K562 cell line and U937 cell line compared to the untreated cells. CONCLUSIONS: These results indicate that latex C-serum enhanced anti-proliferation in leukemic cell lines by inducing apoptosis and caspase activation.

In vitro simulation of the acute lymphoblastic leukemia niche: a critical view on the optimal approximation for drug testing.

Acute lymphoblastic leukemia with the worst prognosis is related to minimal residual disease. Minimal residual disease not only depends on the individual peculiarities of leukemic clones but also reflects the protective role of the acute lymphoblastic leukemia microenvironment. In this review, we discuss in detail cell-to-cell interactions in the 2 leukemic niches, more explored bone marrow and less studied extramedullary adipose tissue. A special emphasis is given to multiple ways of interactions of acute lymphoblastic leukemia cells with the bone marrow or extramedullary adipose tissue microenvironment, indicating observed differences in B- and T-cell-derived acute lymphoblastic leukemia behavior. This analysis argued for the usage of coculture systems for drug testing. Starting with a review of available sources and characteristics of acute lymphoblastic leukemia cells, mesenchymal stromal cells, endothelial cells, and adipocytes, we have then made an update of the available 2-dimensional and 3-dimensional systems, which bring together cellular elements, components of the extracellular matrix, or its imitation. We discussed the most complex available 3-dimensional systems like "leukemia-on-a-chip," which include either a prefabricated microfluidics platform or, alternatively, the microarchitecture, designed by using the 3-dimensional bioprinting technologies. From our analysis, it follows that for preclinical antileukemic drug testing, in most cases, intermediately complex in vitro cell systems are optimal, such as a "2.5-dimensional" coculture of acute lymphoblastic leukemia cells with niche cells (mesenchymal stromal cells, endothelial cells) plus matrix components or scaffold-free mesenchymal stromal cell organoids, populated by acute lymphoblastic leukemia cells. Due to emerging evidence for the correlation of obesity and poor prognosis, a coculture of adipocytes with acute lymphoblastic leukemia cells as a drug testing system is gaining shape.

Dexamethasone-Induced Fatty Acid Oxidation and Autophagy/Mitophagy Are Essential for T-ALL Glucocorticoid Resistance.

ALL is a highly aggressive subtype of leukemia that affects children and adults. Glucocorticoids (GCs) are a critical component of the chemotherapeutic strategy against T-ALL. Cases of resistance to GC therapy and recurrent disease require novel strategies to overcome them. The present study analyzed the effects of Dex, one of the main GCs used in ALL treatment, on two T-ALL cell lines: resistant Jurkat and unselected CCRF-CEM, representing a mixture of sensitive and resistant clones. In addition to nuclear targeting, we observed a massive accumulation of Dex in mitochondria. Dex-treated leukemic cells suffered metabolic reprogramming from glycolysis and glutaminolysis towards lipolysis and increased FAO, along with increased membrane polarization and ROS production. Dex provoked mitochondrial fragmentation and induced autophagy/mitophagy. Mitophagy preceded cell death in susceptible populations of CCRF-CEM cells while serving as a pro-survival mechanism in resistant Jurkat. Accordingly, preventing FAO or autophagy greatly increased the Dex cytotoxicity and overcame GC resistance. Dex acted synergistically with mitochondria-targeted drugs, curcumin, and cannabidiol. Collectively, our data suggest that GCs treatment should not be neglected even in apparently GC-resistant clinical cases. Co-administration of drugs targeting mitochondria, FAO, or autophagy can help to overcome GC resistance.

Leukemia/lymphoma oral infiltration and its impact on disease outcomes: A Brazilian study.

OBJECTIVE: Oral malignant infiltrations (OMI) are relevant for the diagnosis and prognosis of leukemia/lymphoma. This study analysed the oral health status and OMI of individuals with leukemia/lymphoma. MATERIALS AND METHODS: A retrospective analysis (2010-2021) of data from individuals seen at a specialized hospital-based dental service in Brazil. RESULTS: A total of 781 cases of leukemia/lymphoma were surveyed. Acute lymphoblastic leukemia (30.1%), acute myeloid leukemia (AML; 26.0%), and non-Hodgkin lymphoma (22.2%) were the most common diagnoses. The first (21.3%) and second (19.3%) decades of life were the most affected. Overall, dental caries (36.7%) and periodontal changes (34.6%) were the most frequent oral conditions. OMI occurred in 25 (3.2%) individuals. Lesions mainly involved the gingiva (80%) and patients diagnosed with AML (64%). Death (p < 0.001) and worse periodontal condition (p = 0.036) were more frequent among adults with OMI than among those without OMI. Death (p = 0.002) was more frequent among paediatric individuals with OMI than among those without OMI. When controlling for underlying disease, no association was observed between OMI and these outcomes. CONCLUSION: Oral status of individuals with leukemia, particularly those with acute leukemia or lymphoma, should be closely monitored since one or multiple conditions may occur, including OMI, which may influence disease outcomes.

Cell Contact with Endothelial Cells Favors the In Vitro Maintenance of Human Chronic Myeloid Leukemia Stem and Progenitor Cells.

Chronic Myeloid Leukemia (CML) originates in a leukemic stem cell that resides in the bone marrow microenvironment, where they coexist with cellular and non-cellular elements. The vascular microenvironment has been identified as an important element in CML development since an increase in the vascularization has been suggested to be related with poor prognosis; also, using murine models, it has been reported that bone marrow endothelium can regulate the quiescence and proliferation of leukemic stem and progenitor cells. This observation, however, has not been evaluated in primary human cells. In this report, we used a co-culture of primitive (progenitor and stem) CML cells with endothelial colony forming cells (ECFC) as an in vitro model to evaluate the effects of the vascular microenvironment in the leukemic hematopoiesis. Our results show that this interaction allows the in vitro maintenance of primitive CML cells through an inflammatory microenvironment able to regulate the proliferation of progenitor cells and the permanence in a quiescent state of leukemic stem cells.

Evolución temporal de una línea celular leucémica que compite con la hematopoyesis saludable / Temporal evolution of a leukemic line that competes with healthy hematopoiesis

Introducción: La hipótesis del cáncer de las células madre se ha convertido en uno de los paradigmas más importantes en la investigación biomédica. Durante los últimos años se ha ido acumulando evidencia de la existencia de poblaciones similares a células madre en diferentes tipos de cáncer, especialmente en las leucemias. Objetivos: Mostrar mediante la modelación matemática y la simulación computacional cómo los cambios en los parámetros que describen las tasas de proliferación y las propiedades de autorrenovación pueden influir en la dinámica de las poblaciones de células sanas y leucémicas. Métodos: Se utilizó un modelo matemático que es una extensión de los modelos de hematopoyesis sana. Se resolvió el modelo mediante herramientas computacionales basadas en métodos numéricos, lo que permitió realizar simulaciones con diferentes parámetros e intervalos de tiempo. Resultados: Al imponer ciertas condiciones iniciales y resolver matemáticamente el modelo se obtuvo la evolución temporal de las variables de estado del sistema hematopoyético. Partiendo de un estado conocido del sistema hematopoyético se predijo el comportamiento en el tiempo de las variables de estado. Se particularizó para cuatro escenarios clínicamente relevantes. Conclusiones: El análisis del modelo dio como resultado diferentes escenarios de crecimiento de células leucémicas, entre los cuales la proliferación aumentada de células malignas es el más prominente. Sin embargo, diferentes escenarios son posibles, como la inducción de apoptosis o la autorrenovación mejorada(AU)

Introduction: The stem cell cancer hypothesis has become one of the most important paradigms in biomedical research. In recent years, evidence has accumulated for the existence of stem cell-like populations in different types of cancer, especially in leukemias. Objectives: To show, through mathematical modeling and computational simulation, how changes in the parameters that describe proliferation rates and self-renewal properties can influence the dynamics of healthy and leukemic cell populations. Methods: A mathematical model was used which is an extension of the healthy hematopoiesis models. The model was solved using computational tools based on numerical methods, this allowed to carry out countless simulations with different parameters and time intervals. Results: By imposing certain initial conditions and mathematically solving the model, the temporal evolution of the state variables of the hematopoietic system was obtained, that is, starting from a known state of the hematopoietic system, the behavior over time of the state variables of the system was predicted. It was particularized for four clinically relevant scenarios. Conclusions: The analysis of the model results in different growth scenarios of leukemic cells, among which the increased proliferation of malignant cells is the most prominent. However, different scenarios are possible, such as apoptosis induction or enhanced self-renewal(AU)

In Vitro and In Vivo Modeling of Normal and Leukemic Bone Marrow Niches: Cellular Senescence Contribution to Leukemia Induction and Progression.

Cellular senescence is recognized as a dynamic process in which cells evolve and adapt in a context dependent manner; consequently, senescent cells can exert both beneficial and deleterious effects on their surroundings. Specifically, senescent mesenchymal stromal cells (MSC) in the bone marrow (BM) have been linked to the generation of a supporting microenvironment that enhances malignant cell survival. However, the study of MSC's senescence role in leukemia development has been straitened not only by the availability of suitable models that faithfully reflect the structural complexity and biological diversity of the events triggered in the BM, but also by the lack of a universal, standardized method to measure senescence. Despite these constraints, two- and three dimensional in vitro models have been continuously improved in terms of cell culture techniques, support materials and analysis methods; in addition, research on animal models tends to focus on the development of techniques that allow tracking leukemic and senescent cells in the living organism, as well as to modify the available mice strains to generate individuals that mimic human BM characteristics. Here, we present the main advances in leukemic niche modeling, discussing advantages and limitations of the different systems, focusing on the contribution of senescent MSC to leukemia progression.

Bone Marrow Aging and the Leukaemia-Induced Senescence of Mesenchymal Stem/Stromal Cells: Exploring Similarities.

Bone marrow aging is associated with multiple cellular dysfunctions, including perturbed haematopoiesis, the propensity to haematological transformation, and the maintenance of leukaemia. It has been shown that instructive signals from different leukemic cells are delivered to stromal cells to remodel the bone marrow into a supportive leukemic niche. In particular, cellular senescence, a physiological program with both beneficial and deleterious effects on the health of the organisms, may be responsible for the increased incidence of haematological malignancies in the elderly and for the survival of diverse leukemic cells. Here, we will review the connection between BM aging and cellular senescence and the role that these processes play in leukaemia progression. Specifically, we discuss the role of mesenchymal stem cells as a central component of the supportive niche. Due to the specificity of the genetic defects present in leukaemia, one would think that bone marrow alterations would also have particular changes, making it difficult to envisage a shared therapeutic use. We have tried to summarize the coincident features present in BM stromal cells during aging and senescence and in two different leukaemias, acute myeloid leukaemia, with high frequency in the elderly, and B-acute lymphoblastic leukaemia, mainly a childhood disease. We propose that mesenchymal stem cells are similarly affected in these different leukaemias, and that the changes that we observed in terms of cellular function, redox balance, genetics and epigenetics, soluble factor repertoire and stemness are equivalent to those occurring during BM aging and cellular senescence. These coincident features may be used to explore strategies useful to treat various haematological malignancies.

Estudo da infiltração de tecidos orais em pacientes com doenças hematológicas malignas / Study of oral infiltration in patients with malignant haematological diseases

Este trabalho realizou um estudo retrospectivo dos prontuários do Hospital das Clínicas (HC) da Universidade Federal de Minas Gerais (UFMG). Foram incluídos os indivíduos admitidos no período de 2010 a 2021 com diagnóstico de leucemia/linfoma e que foram avaliados pela equipe do Serviço de Odontologia do HC­UFMG. Foram coletados dados demográficos, características clínicas da doença de base e da cavidade bucal e presença de infiltração oral maligna. O teste t não pareado foi utilizado para avaliar os hemogramas e o teste McNemar para comparar indivíduos que desenvolveram infiltração oral maligna e aqueles que não desenvolveram. A significância estatística foi estabelecida como p<0,05. Adicionalmente, uma revisão da literatura de relatos de casos e séries de casos foi realizada em quatro bases de dados eletrônicas (PubMed, Web of Science, Scopus e Embase). Dos 781 prontuários analisados, a leucemia linfocítica aguda (30,1%) foi o diagnóstico mais frequente. Pacientes nas duas primeiras décadas de vida foram mais acometidos pela doença de base. Cárie (36,7%) e alterações periodontais (34,6%) foram as condições bucais mais observadas. Infiltração oral maligna ocorreu em 25 (3,2%) indivíduos, envolvendo principalmente a gengiva (80%) e indivíduos diagnosticados com leucemia mieloide aguda (64%). Comparando os dados de pacientes pediátricos que desenvolveram infiltração maligna e aqueles que não desenvolveram, a proporção de óbitos foi maior naqueles que tiveram infiltração (p=0,002), enquanto em adultos, aqueles que desenvolveram infiltração exibiram pior condição periodontal e maior proporção de óbitos (p<0,001). Dados da revisão da literatura demonstraram que a infiltração oral maligna foi mais frequente na gengiva (37%) e em pacientes com leucemia mieloide aguda (47%). As principais características clínicas e de imagem associadas à infiltração oral foram aumento de volume e lesões osteolíticas. Em conjunto, os dados sugerem a importância do monitoramento clínico odontológico de pacientes com leucemia/linfoma considerando os piores desfechos clínicos relacionados à infiltração dos tecidos orais.

This work carried out a retrospective study of the medical records at the Hospital das Clínicas (HC), Universidade Federal de Minas Gerais (UFMG). Individuals admitted in the period from 2010 to 2021 with a diagnosis of leukaemia/lymphoma and who were evaluated by the team of the dental service of HC­UFMG were included. Demographic data, clinical characteristics of the underlying disease and oral cavity, and presence of malignant oral infiltration were collected. The unpaired t test was employed to assess the blood count and the McNemar test to compare individuals who developed malignant oral infiltration and those who did not. Statistical significance was set at p<0.05. Additionally, a literature review of case reports and case series was undertaken in four electronic databases (PubMed, Web of Science, Scopus, and Embase). Of the 781 medical records analysed, acute lymphocytic leukaemia (30.1%) was the most frequent diagnosis. Patients in the first two decades of life were more affected by the underlying disease. Caries (36.7%) and periodontal changes (34.6%) were the most frequently observed oral conditions. Oral malignant infiltration took place in 25 (3.2%) individuals, mainly involving the gingiva (80%) and individuals diagnosed with acute myeloid leukaemia (64%). Comparing data from paediatric patients who developed malignant infiltration and those who did not, the proportion of deaths was higher in those who had infiltration (p=0.002), while in adults, those who developed infiltration had worse periodontal status and a higher proportion of death (p<0.001). Data from the literature review showed that oral malignant infiltration was more frequent in the gingiva (37%) and in patients with acute myeloid leukaemia (47%). The main clinical and imaging features associated with oral infiltration were swelling and osteolytic lesions. Altogether, the data suggest the importance of clinical dental monitoring of patients with leukaemia/lymphoma considering the worst clinical outcomes related to oral tissue infiltration.

γδ T Cells for Leukemia Immunotherapy: New and Expanding Trends.

Recently, many discoveries have elucidated the cellular and molecular diversity in the leukemic microenvironment and improved our knowledge regarding their complex nature. This has allowed the development of new therapeutic strategies against leukemia. Advances in biotechnology and the current understanding of T cell-engineering have led to new approaches in this fight, thus improving cell-mediated immune response against cancer. However, most of the investigations focus only on conventional cytotoxic cells, while ignoring the potential of unconventional T cells that until now have been little studied. γδ T cells are a unique lymphocyte subpopulation that has an extensive repertoire of tumor sensing and may have new immunotherapeutic applications in a wide range of tumors. The ability to respond regardless of human leukocyte antigen (HLA) expression, the secretion of antitumor mediators and high functional plasticity are hallmarks of γδ T cells, and are ones that make them a promising alternative in the field of cell therapy. Despite this situation, in particular cases, the leukemic microenvironment can adopt strategies to circumvent the antitumor response of these lymphocytes, causing their exhaustion or polarization to a tumor-promoting phenotype. Intervening in this crosstalk can improve their capabilities and clinical applications and can make them key components in new therapeutic antileukemic approaches. In this review, we highlight several characteristics of γδ T cells and their interactions in leukemia. Furthermore, we explore strategies for maximizing their antitumor functions, aiming to illustrate the findings destined for a better mobilization of γδ T cells against the tumor. Finally, we outline our perspectives on their therapeutic applicability and indicate outstanding issues for future basic and clinical leukemia research, in the hope of contributing to the advancement of studies on γδ T cells in cancer immunotherapy.

Complete reversal of bilateral optic nerve infiltration from lymphoblastic leukemia using chemotherapy without adjuvant radiotherapy.

BACKGROUND: Leukemic involvement of the eyes is rare, therefore, treatment relies on previous case reports. The treatment of ocular complications poses additional difficulties, because the eye is considered as a pharmacological "sanctuary" for patients with acute lymphoblastic leukemia (ALL). Therefore, radiotherapy is the main therapeutic choice; however, it might lead to many important side effects. To the best of our knowledge, this is the first case report of a bilateral leukemic optic nerve infiltration that remitted with chemotherapy without adjuvant radiotherapy. CASE PRESENTATION: A 30-year-old female patient with previous history of remitted ALL presented with a one-week history of floaters in her right eye. Her ophthalmological exam showed remarkable optic disc swelling, in both eyes. She was diagnosed with ALL relapse presenting as a bilateral optic nerve leukemic infiltration. Local radiotherapy was planned for both eyes, however, due to efficient recovery with chemotherapy, it was cancelled. Allogenic bone marrow transplantation was subsequently performed. The patient is being followed up and ALL remitted. CONCLUSION: Leukemia relapse on central nervous system, despite rare, is a sign of poor prognosis and requires prompt treatment. Its occurrence on ocular tissues is even rarer. It is hypothesized that the blood-brain barrier limits the delivery of chemotherapeutic drugs to the eye and infiltration of the optic nerve by leukemic cells might prejudice the flow of cerebrospinal fluid between the cranial space and the optic disc.

Sensitization to Drug Treatment in Precursor B-Cell Acute Lymphoblastic Leukemia Is Not Achieved by Stromal NF-κB Inhibition of Cell Adhesion but by Stromal PKC-Dependent Inhibition of ABC Transporters Activity.

Cell adhesion to stromal support and the associated intracellular signaling are central to drug resistance, therefore blocking both has been effective in increasing drug sensitization in leukemia. The stromal Ser/Thr protein kinase C (PKC) has been found to be important for conferring protection to leukemic cells. We aimed at elucidating the intracellular signals connected to cell adhesion and to stromal PKC. We found that NF-κB and Akt were up-regulated in mesenchymal stem cells (MSC) after binding of B-cell acute lymphoblastic leukemia (B-ALL) cells. Nevertheless, Akt inhibition did not induce B-ALL cell detachment. In spite of a clear activation of the NF-κB signaling pathway after B-ALL cell binding (up-regulation NF-κB1/2, and down-regulation of the IKBε and IKBα inhibitors) and an important reduction in cell adhesion after NF-κB inhibition, sensitization to the drug treatment was not observed. This was opposite to the PKC inhibitors Enzastaurin and HKPS, a novel chimeric peptide inhibitor, that were able to increase sensitization to dexamethasone, methotrexate, and vincristine. PLCγ1, Erk1/2, and CREB appear to be related to PKC signaling and PKC effect on drug sensitization since they were contra-regulated by HKPS when compared to dexamethasone-treated cells. Additionally, PKC inhibition by HKPS, but not by Enzastaurin, in MSC reduced the activity of three ABC transporters in leukemic cells treated with dexamethasone, a new indirect mechanism to increase sensitization to drug treatment in B-ALL cells. Our results show the validity of targeting the functional characteristic acquired and modulated during cell-to-cell interactions occurring in the leukemic niche.

Leukemia-Induced Cellular Senescence and Stemness Alterations in Mesenchymal Stem Cells Are Reversible upon Withdrawal of B-Cell Acute Lymphoblastic Leukemia Cells.

Leukemic cell growth in the bone marrow (BM) induces a very stressful condition. Mesenchymal stem cells (MSC), a key component of this BM niche, are affected in several ways with unfavorable consequences on hematopoietic stem cells favoring leukemic cells. These alterations in MSC during B-cell acute lymphoblastic leukemia (B-ALL) have not been fully studied. In this work, we have compared the modifications that occur in an in vitro leukemic niche (LN) with those observed in MSC isolated from B-ALL patients. MSC in this LN niche showed features of a senescence process, i.e., altered morphology, increased senescence-associated ß-Galactosidase (SA-ßGAL) activity, and upregulation of p53 and p21 (without p16 expression), cell-cycle arrest, reduced clonogenicity, and some moderated changes in stemness properties. Importantly, almost all of these features were found in MSC isolated from B-ALL patients. These alterations rendered B-ALL cells susceptible to the chemotherapeutic agent dexamethasone. The senescent process seems to be transient since when leukemic cells are removed, normal MSC morphology is re-established, SA-ßGAL expression is diminished, and MSC are capable of re-entering cell cycle. In addition, few cells showed low γH2AX phosphorylation that was reduced to basal levels upon cultivation. The reversibility of the senescent process in MSC must impinge important biological and clinical significance depending on cell interactions in the bone marrow at different stages of disease progression in B-ALL.

Cell signaling pathways as molecular targets to eliminate AML stem cells.

Acute myeloid leukemia (AML) remains the most lethal of leukemias and a small population of cells called leukemic stem cells (LSCs) has been associated with disease relapses. Some cell signaling pathways play an important role in AML survival, proliferation and self-renewal properties and are abnormally activated or suppressed in LSCs. This includes the NF-κB, Wnt/ß-catenin, Hedgehog, Notch, EGFR, JAK/STAT, PI3K/AKT/mTOR, TGF/SMAD and PPAR pathways. This review aimed to discuss these pathways as molecular targets for eliminating AML LSCs. Herein, inhibitors/activators of these pathways were summarized as a potential new anti-AML therapy capable of eliminating LSCs to guide future researches. The clinical use of cell signaling pathways data can be useful to enhance the anti-AML therapy.

A new lectin from the floral capitula of Egletes viscosa (EgviL): Biochemical and biophysical characterization and cytotoxicity to human cancer cells.

Egletes viscosa is a plant with therapeutic value due to its antibacterial, antinociceptive and gastroprotective properties. This study aimed to purify, characterize, and evaluate the cytotoxicity of a lectin (EgviL) from the floral capitula of E. viscosa. The lectin was isolated from saline extract through precipitation with ammonium sulfate followed by Sephadex G-75 chromatography. The molecular mass and isoelectric point (pI) of EgviL were determined as well as its temperature and pH stability. Physical-chemical parameters of interaction between EgviL and carbohydrates were investigated by fluorescence quenching and 1H nuclear magnetic resonance (NMR). Cytotoxicity was investigated against human peripheral blood mononuclear cells (PBMCs) and neoplastic cells. EgviL (28.8 kDa, pI 5.4) showed hemagglutinating activity stable towards heating until 60 °C and at the pH range 5.0-7.0. This lectin is able to interact through hydrophobic and electrostatic bonds with galactose and glucose, respectively. EgviL reduced the viability of PBMCs only at the highest concentration tested (100 µg/mL) while was toxic to Jurkat E6-1 cells with IC50 of 24.1 µg/mL,inducing apoptosis. In summary, EgviL is a galactose/glucose-binding protein with acidic character, stable to heating and with cytotoxic effect on leukemic cells.

Bilateral macular detachment: Choroid as a sanctuary of acute lymphoblastic leukemia.

PURPOSE: To describe a bilateral macular detachment as the only sign of acute lymphoblastic leukemia relapse and prompt reversal with total body irradiation without ocular protection. OBSERVATIONS: We present the case of a 20-year-old patient, diagnosed with a high-risk phy-negative, pre-B acute lymphoblastic leukemia (ALL), with a positive MLL gene rearrangement. After a Berlin-Frankfurt-Munster-like regimen chemotherapy protocol and a first complete remission, ALL relapse was diagnosed, so he was commenced on a FlaG-Ida protocol (fludarabine, idarubicin, granulocyte-colony stimulating factor, and high-dose cytarabine). He achieved a second complete remission with positive minimal residual disease and was scheduled for urgent allogeneic bone marrow transplant.Five days before the conditioning regimen was initiated, the patient complained of visual loss in the left eye and then in the right eye. Ophthalmological evaluation showed a best corrected visual acuity of the right eye (OD) of 20/100 and of the left eye (OS) of 20/400. Optical coherence tomography (OCT) showed a bilateral serous sub-foveal detachment. The sub-foveal choroidal thickness was measured by enhanced depth imaging (EDI-OCT) and showed a significant increase (OD 836 µm and OS 1036 µm) compared with normal (average 310 µm). This choroidal thickness increase, associated with the serous macular detachment, was interpreted as a choroidal leukemic infiltration.A lumbar puncture with cytologic studies and flow cytometry was performed, showing no evidence of central nervous system (CNS) involvement of leukemia. CNS and orbital magnetic nuclear resonance imaging showed no pathology. No extramedullary involvement could be confirmed.Retinal fluorescein angiography showed multiple and diffuse leakage points (pinpoint pattern) within the macular area. This pattern reinforced our presumptive diagnosis, even though the lumbar puncture and flow cytometry were negative.The hematologist decided to proceed with the bone marrow transplant. A myeloablative conditioning regimen was delivered, based on total body irradiation (TBI) with a total dose of 12 Gy plus fludarabine 30 mg/m2 for five days. No ocular protection was used during TBI.Only 2 h after TBI commenced, the patient reported a significant improvement in his visual acuity. We confirmed 20/20 in both eyes. The OCT showed a dramatic decrease in the choroidal thickness measurement (OD 387 µm and OS 408 µm compared with 836 µm and 1036 µm measured before radiotherapy). CONCLUSIONS AND IMPORTANCE: Complete ophthalmological evaluation and EDI-OCT choroidal thickness measurement could be fundamental tools necessary to determine CNS involvement of ALL, even in cases with negative cerebrospinal fluid and brain imaging.

Dual Targeting of Stromal Cell Support and Leukemic Cell Growth by a Peptidic PKC Inhibitor Shows Effectiveness against B-ALL.

Mesenchymal stem cells (MSC) favour a scenario where leukemic cells survive. The protein kinase C (PKC) is essential to confer MSC support to leukemic cells and may be responsible for the intrinsic leukemic cell growth. Here we have evaluated the capacity of a chimeric peptide (HKPS), directed against classical PKC isoforms, to inhibit leukemic cell growth. HKPS was able to strongly inhibit viability of different leukemic cell lines, while control HK and PS peptides had no effect. Further testing showed that 30% of primary samples from paediatric B-cell acute lymphoblastic leukaemia (B-ALL) were also strongly affected by HKPS. We showed that HKPS disrupted the supportive effect of MSC that promote leukemic cell survival. Interestingly, ICAM-1 and VLA-5 expression increased in MSC during the co-cultures with B-ALL cells, and we found that HKPS inhibited the interaction between MSC and B-ALL cells due to a reduction in the expression of these adhesion molecules. Of note, the susceptibility of B-ALL cells to dexamethasone increased when MSC were treated with HKPS. These results show the relevance of these molecular interactions in the leukemic niche. The use of HKPS may be a new strategy to disrupt intercellular communications, increasing susceptibility to therapy, and at the same time, directly affecting the growth of PKC-dependent leukemic cells.

Leukemia stem cell immunophenotyping tool for diagnostic, prognosis, and therapeutics.

The existence of cancer stem cells is debatable in numerous solid tumors, yet in leukemia, there is compelling evidence of this cell population. Leukemic stem cells (LSCs) are altered cells in which accumulating genetic and/or epigenetic alterations occur, resulting in the transition between the normal, preleukemic, and leukemic status. These cells do not follow the normal differentiation program; they are arrested in a primitive state but with high proliferation potential, generating undifferentiated blast accumulation and a lack of a mature cell population. The identification of LSCs might guide stem cell biology research and provide key points of distinction between these cells and their normal counterparts. The identification and characterization of the main features of LSCs can be useful as tools for diagnosis and treatment. In this context, the aim of the present review was to connect immunophenotype data in the main types of leukemia to further guide technical improvements.

miRNome profiling of LSC-enriched CD34+CD38-CD26+ fraction in Ph+ CML-CP samples from Argentinean patients: a potential new pharmacogenomic tool.

Chronic myeloid leukemia (CML) is a myeloid stem cell neoplasm characterized by an expansion of myeloid progenitor cells and the presence of BCR-ABL1 oncoprotein. Since the introduction of specific BCR-ABL1 tyrosine kinase inhibitors (TKI), overall survival has improved significantly. However, under long-term therapy patients may have residual disease that originates from TKI-resistant leukemic stem cells (LSC). In this work, we analyzed the miRNome of LSC-enriched CD34+CD38-CD26+ and normal hematopoietic stem cells (HSC) fractions obtained from the same chronic phase (CP) CML patients, and stem and progenitor cells obtained from healthy donors (HD) by next-generation sequencing. We detected a global decrease of microRNA levels in LSC-enriched CD34+CD38-CD26+ and HSC fractions from CML-CP patients, and decreased levels of microRNAs and snoRNAs from a genomic cluster in chromosome 14, suggesting a mechanism of silencing of multiple non-coding RNAs. Surprisingly, HSC from CML-CP patients, despite the absence of BCR-ABL1 expression, showed an altered miRNome. We confirmed by RT-qPCR that the levels of miR-196a-5p were increased more than nine-fold in CD26+ (BCR-ABL1 + ) vs. CD26- (BCR-ABL1 -) CD34+CD38- fractions from CML-CP patients at diagnosis, and in silico analysis revealed a significant association to lipid metabolism and hematopoiesis functions. In the light of recent descriptions of increased oxidative metabolism in CML LSC-enriched fractions, these results serve as a guide for future functional studies that evaluate the role of microRNAs in this process. Metabolic vulnerabilities in LSCs open the road for new therapeutic strategies. This is the first report of the miRNome of CML-CP CD34+CD38- fractions that distinguishes between CD26+ (BCR-ABL1 + ) and their CD26- (BCR-ABL1 - ) counterparts, providing valuable data for future studies.